ECOLOGÍA DE MACROALGAS CYLINDRACEA EN EL Jaime … · definitivamente la sensación de...

ECOLOGÍA DE MACROALGAS MARINAS EXÓTICAS:

APROXIMACIÓN A LOS FACTORES QUE REGULA LA

COLONIZACIÓN DE CAULERPA CYLINDRACEA EN EL MEDITERRÁNEO Y SU

INTERACCIÓN CON LOS HÁBITAS BENTÓNICOS

(PRADERAS DE POSIDONEA OCEÁNICA)

Jaime Bernardeau Esteller

http://www.ua.es/

http://www.eltallerdigital.com/

OCT · 2015

JAIME BERNARDEAU ESTELLER

Ecología de Macroalgas Marinas Exóticas:

aproximación a los factores que regulan la

colonización de Caulerpa cylindracea en el Mediterráneo

y su interacción con los hábitats bentónicos

(praderas de Posidonia Oceanica).

TESIS DOCTORAL

DEPARTAMENTO DE CIENCIAS DEL MAR Y BIOLOGÍA APLICADA

FACULTAD DE CIENCIASGRUPO DE ECOLOGÍA DE ANGIOSPERMAS MARINAS (GEAM)

CENTRO OCEANOGRÁFICO DE MURCIA. INSTITUTO ESPAÑOL DE OCEANOGRAFÍA

Ecología de Macroalgas Marinas Exóticas: aproximación a los factores que regulan la colonización

de Caulerpa cylindracea en el Mediterráneo y su interacción con los hábitats bentónicos

(praderas de Posidonia Oceanica).


Memoria presentada para aspirar al grado de:

DOCTOR POR LA UNIVERSIDAD DE ALICANTE

MENCIÓN DE DOCTOR INTERNACIONAL

DOCTORADO EN CIENCIAS DEL MAR

Dirigida por:

Juan Manuel Ruiz Fernández

Investigador Titular del Instituto Español de Oceanografía

Codirigida por:

José Luis Sánchez Lizaso

Profesor Titular de Universidad de Alicante

Lázaro Marín Guirao

Investigador Titular del Instituto Español de Oceanografía

Exotic Marine Macroalgae Ecology:approach to factors that regulate colonization

of Caulerpa cylindracea in the Mediterranean Sea and interaction with benthic habitats

(Posidonia oceanica meadows)

A mi Luca y a mi amore

Al Mediterráneo

A la red de Posidonia oceanica de la región de Murcia

Y al Windsurf!

Quién podría vivir en la tierra

si no fuera por el mar

Luis Cernuda. El joven marino

Cuando Hans Reiter vio por primera vez un bosque de

algas se emocionó tanto que se puso a llorar debajo del

agua. Esto parece difícil, que un ser humano llore mien-

tras bucea con los ojos abiertos (…)

Roberto Bolaño. 2666

¿Por qué nos gusta el mar? Es porque tiene una podero-

sa capacidad para hacernos pensar cosas que nos gusta

pensar.

Robert Henri

Life is passing time as gracefully as possible

Miki Dora

Los ecosistemas reflejan el ambiente físico en el que se

han desarrollado y los ecólogos reflejan las propiedades

de los ecosistemas en que han crecido y madurado.

Ramón Margalef. Perspectivas de la teoría ecológica.

· Un punto indefinido entre Villaricos y San Juan de Terreros. 12:00 am (aproximadamente). Agosto de 1991.

Mi padre apaga el motor mientras el tío Pepe termina de afianzar el ancla. Un sol radiante y el mar

hecho una balsica. Otro día de otro verano eterno. Mientras mis primos y hermanos revoletean por la

zodiac, se tiran al agua y juguetean, mi padre y yo lo tenemos claro. Gafas, aletas y nuestros maltrechos

fusiles de pescasub amateurs. Por delante un par de horas buenas de buceo. De ese pausado y sin pre-

tensiones, seguros de que al día siguiente y al otro habrá más. Sigo a mi padre desde el claro de arena

donde hemos fondeado hacia unas rocas que quedan más hacia costa. Se mueve tranquilo, acostum-

brado a ese medio en el que ha pasado ya media vida, y como siempre con su fusil sin cargar, ya llegara

el momento, si es que tiene que llegar. Conforme nos acercamos a las rocas comienzo a ver como

todo el fondo está recubierto por un “alga” de hojas verduzcas alargadas. En mi corta vida de apneista,

acostumbrado a los fondos más desnudos y rocosos de la zona de Mojacar o al cascajo infinito marme-

roniense, no había visto algo igual. El “alga” forma un denso tapete que lo cubre todo y se mece al son

del mar, de forma embriagadora, en un baile sin fin que aleja tu mente de la realidad y la hace fluir.

· Facultad de biología de la Universidad de Murcia. Noviembre de 2000.

Deambulo por el hall de la facultad. Con la mente espesa después de una noche un poco pasada me

resisto a tirar para clase. Pierdo el tiempo entre charla y charla, como buscando una excusa que diluya

definitivamente la sensación de culpabilidad. Un cartel en la pared llama mi atención, es un anuncio

de la lectura de una tesis de un tal Juan Manuel Ruiz Fernández. No sé qué historias sobre Posidonia

oceanica. En el cartel una foto de una pradera y diversos artefactos que parecen sacados de una peli de

ciencia ficción de serie B (bueno, incluso C). Que disparate es este, pienso. Definitivamente es la excusa

perfecta que andaba buscando. La sala de grados está a tope y me acurruco en una butaca buscando

pasar desapercibido entre tanto fruto seco con estética al más puro estilo biólogo desarrapado. Al rato

aparece el Juan Manuel este, con unas gafas de pasta escandalosas, y comienza a hablar. Su discurso

me atrapa rápido, durante algo menos de una hora se suceden imagines de experimentos imposibles,

sombreros gigantes, graficas y mas graficas, términos y cosas que apenas soy capaz de entender. Foto-

síntesis, eutrofización, balances energéticos, carbohidratos, impactos. Cuando acaba, mi cuerpo apenas

sobresale de la butaca. Ha sido apabullante, brutal. Buf, esto debe ser ciencia, pienso.

· AP-7, a la altura de Villajoyosa. Marzo o quizás abril de 2005.

Otro día duro de agua. Es media tarde, el sol aun aprieta y la hora y pico de vuelta a Murcia desde Altae

se hace larga. De no ser por la conversación incesante y sincopada de Lázaro sería del todo insoporta-

ble. Sin embargo hoy hay algo distinto, su voz esta algo más apagada y fija la mirada al frente mientras

sujeta con fuerza el volante de su Ford Sierra. Por un momento hay un silencio que se lo come todo, y

entonces explota. Mientras alguna lagrima se escapa me cuenta que no puede seguir con este trabajo,

que por las noches no puede dormir bien, que aquello se aleja absolutamente de las razones que lo

han llevado a la investigación y que ser consecuente con los principios y valores de cada uno es muy

importante y al final es lo único que nos queda. Sus palabras golpean con fuerza en mi tierno cerebro

pseudocientífico, y ya nunca saldrán.

· Murcia. Diciembre de 2008.

Cena en familia en un restaurante de Murcia. A mitad de cena una llamada rompe la cordialidad. En la

pantalla del movil pone Rossss, y me da un cierto sobresalto por la hora (más allá de las 9 de la noche) y

el día (viernes). Por un momento pienso en no contestar, y seguir entre vinos y quesos, pero al final me

pueden los remordimientos y me levanto teléfono en mano. Rocío me cuenta que hay un contrato de un

año para un experimento que llevan en marcha en estos momentos sobre salinidad y Posidonia, y que

había pensado en mí. Por un momento pienso en mi trabajo actual, en mis horarios y en mi seguridad,

en mi edad un poco pasada para meterme en estos jaleos. Se me pasa por la cabeza decirle que nece-

sito un poco de tiempo para darle vueltas, tiempo para buscarme las excusas necesarias para pensar

que estoy mejor así. Pero ese día hay algo distinto, de mi interior nace una necesidad de aprovechar

esa oportunidad que durante tanto tiempo busqué. Este es mi momento, me digo, y entre risas (sobre

todo la de Rosss, que como siempre se lo come todo) me sorprendo a mi mismo diciéndole que cuenten

conmigo. Al volver a la mesa ataco sin miramientos, les cuento a todos las novedades, mi madre se

queda muda durante un momento, pero rápidamente fagocita sus miedos protectores y me dice, “si es

lo que te gusta y tu quieres, adelante”.

· Zulo del Geam. Un viernes cualquiera de 2010.

Hoy es viernes y toca Vangelis. El Chemi me pregunta las contraseñas del spotify. Esta mañana se ha

levantado innovador y duda entre la BSO de The Bounty o la de 1492. Le paro los pies, “Chemilín, déjate

de rollos y pon Blade Runner”. Por los altavoces suena ya imponente “Love theme” y nuestras mentes

despiertan. Diseños experimentales, análisis estadísticos, revisiones de textos, diseño y construcción de

estructuras, bandejas, filtros y cualquier otra cosa que sea necesaria. No hay desafío que pueda achan-

tar la mente de un molinero.

· Dirección general del Geam. Enero 2015.

- ¿Cómo se llama este grupo?

- Aranzazu, no me lo puedo creer. ¿Otra vez? si lo oímos ayer, y antes de ayer

- ¿Si? Uf, no me acuerdo, ¿Cómo se llama?

- Son de the Windy Hills

- Ah, es verdad, si lo tengo apuntado en mi lista. Este es el grupo del surfista, director de docus y demás,

¿no?

- Exactamente

- Oye

- Dime

- Luego pon el disco ese de Neil Young, ese tan tranquilito

-Jejeje, vale. Pero me tienes que hacer un mapa.

- ¿Otra vez?

- Si, pero esta vez c

· Una noche entre 2011 y 2015. 4 a.m.

Me despierto. Sobresaltado. Todavía es noche cerrada. Mi cabeza rápidamente se activa, llena de temo-

res, inseguridades. Vuelta hacia un lado, vuelta hacia al otro. La almohada estirada, con un doblez, con

dos. Boca arriba, boca abajo, de lado. Por un momento pienso en saltar de la cama. En ese momento

Maria me abraza, me susurra algo que no soy capaz de entender y me besa en la mejilla. Respiro pro-

fundamente. El sueño vuelve.

· Playa de las Cañas, Calblanque. 21 de junio de 2015.

Al final todo llega a su fin, y es hora de mirar atrás y agradecer a todos los que habéis estado ahí apo-

yándome, de una forma o de otra. Ya sabéis que mi memoria nos es muy buena, y algún nombre se me

pasará, pero no puedo dejar de acordarme especialmente de algunos de vosotros:

De mi familia, por supuesto, por todo.

Del Geam, al completo (eso va también por ti, Maridolis), mi otra familia, bueno, mi familia también.

De Jose Luis, por su confianza. Eternamente agradecido.

Del Charton, por ponerme en vereda y contribuir de una manera u otra que todo esto empezase.

De Juan y Tamara, me hubiera encantado compartir todo esto con vosotros hasta al final, mil gracias

por todos los momentos que pasamos juntos y por toda vuestra ayuda.

De Fiona, por su infinita paciencia.

Del equipo de la Universidad de Sassari, Giulia, Stefania, Prof. Cosu. ¡Que grandes días!.

Y de toda la plantilla del IEO, Fina, Colache, Iñaki , Vera, Julio, Fernando, Ricardo, Silverio, los pesqueros

de solera (Javi, Antonio, Angelopoulos), los pesqueros gastronómicos (Miguel, Ester, Encarni), los pes-

queros del más allá (Belli, la piccolina, Lolo, Iosu) y las pesqueras del más acá (Elena y Cris), la cúpula

dorada (Paco, Jorge, Mr. Rocamora, Lola, Geli), los contaminantes contaminados (Victor G y Victor L,

Juan Antonio, Juliana, Cristóbal, Pencho, Cristina, David, Emily, Carlos, Nané, Concha, Ines, Juanjo), los

molusquistas hermanos (Marina, Esmeralda, Paco, Diana, Carmen), Dani y sus mejunjes levanta espíri-

tus, las pobrecicas que han visto dar con sus huesos en la recepción (Alejandra, Encarna, Juani, Paqui,

Marina, Esther y la terremoto de San Javier, la mismísima Maria Antonia), los becarios (ufa, cuantos

han sido en estos años, ¡grandes!) super Rocio informatica (y su Nayara) y por supuesto mi queridísima

Mara (que ha tenido que soportar mi inmundicia día si y día también). Muchísimas gracias a todos por

vuestras sonrisas y compañía durante todos estos años. Espero que sigamos juntos muchos más.

Por último agradecer al Servicio de Pesca de la región de Murcia su confianza total en el proyecto “Red

de Seguimiento de las praderas de P. oceánica de la Región de Murcia”, marco en el que se engloban

todos los trabajos de esta tesis.

Introducción general

Chapter 1. Recent spread of the invasive alga Caulerpa

cylindracea (Bryopsidales, Chlorophyta) along the Medi-

terranean coast of the Murcia Region (SE Spain)

Chapter 2. Photosynthesis and daily metabolic carbon

balance of the invasive Caulerpa cylindracea (Chloro-

phyta:Bryopsidales) along a depth gradient

Chapter 3. Resistance of Posidonia oceanica seagrass

meadows to the spread of the introduced green alga

Caulerpa cylindracea: assessment of the role of light

Chapter 4. Photoacclimation of Caulerpa cylindracea:

light as a limiting factor in the invasion of native Medite-

rranean seagrass meadows

Discusión general

Conclusiones

Anexo. Assessment of long-term interaction between

the endemic seagrass Posidonia oceanica and Caulerpa

cylindracea in the Mediterranean Sea

Bibliografía

INDICE

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135

INTRODUCCIÓNGENERAL

I N T R O D U C T I O N

p. 03

1. La introducción de especies: un fenómeno global

La dispersión de los organismos es un proceso natural implicado en los fenómenos de distribución y de-

sarrollo de la biodiversidad en el planeta. Sin embargo, la existencia de barreras naturales (p.e. geográ-

ficas), condiciona su capacidad colonizadora y determina la composición de la flora y fauna específicas

de cada región. La intervención humana en los ecosistemas ha permitido a muchas especies superar

estas barreras que impiden su dispersión, acelerando e intensificando los procesos de introducción de

especies a escala global. Los sistemas de transporte humanos, ya sea de forma voluntaria o involun-

taria, han favorecido la dispersión de cientos de especies fuera de sus áreas naturales de distribución,

fenómeno que se ha visto acelerado en los últimos siglos como consecuencia del importante desarrollo

tecnológico (Di Castri 1989). Como resultado de esta situación la biota del planeta se encuentra some-

tida a un proceso de cambio y homogenización sin precedentes (Crooks and Suarez 2006).

Una especie introducida o exótica puede ser definida como aquella que cumple las siguientes caracte-

rísticas, a saber, (i) coloniza nuevas áreas donde previamente no estaba presente, (ii) este nuevo rango

de distribución esta relacionado de manera directa o indirecta a la actividad humana, (iii) presentan

una discontinuidad geográfica con el área natural de distribución de la especie, y (iv) son capaces de

reproducirse dentro de estas nuevas áreas de distribución sin la ayuda del hombre (Carlton 1985, Bou-

douresque and Verlaque 2002). Cuando estas especies introducidas son capaces de transformar signi-

ficativamente la estructura y función de los ecosistemas receptores, amenazar su biodiversidad, y tener

incluso consecuencias a nivel socioeconómico y de la propia salud humana, se les considera Especies

Exóticas Invasoras (EEI) o simplemente especies invasoras (NISC 2006).

Las invasiones biológicas se manifiestan como el crecimiento masivo de las especies una vez han sido

introducidas. De todas las especies introducidas solo una pequeña fracción tiene potencial invasor y

puede ser considerada una amenaza real para la biodiversidad y el funcionamiento de los ecosistemas

afectados (Norse 1993, Carlton, 2000, Primack 2004, Mooney et al. 2005; ver revisión en Mack et al.

2000).

La identificación de las especies introducidas, la determinación de su potencial invasor, así como el

análisis de los patrones de propagación y de los mecanismos y factores que determinan su éxito en los

ecosistemas invadidos son un tema de interés central en ecología, no solo por sus implicaciones en la

gestión de los ecosistemas y recursos marinos (Rejmaneck 2000), sino también porque son una oportu-

nidad única para el estudio de procesos fundamentales relacionados con el funcionamiento de los eco-

sistemas. (ver revisión en Sax et al. 2005 y Cadotte et al. 2006). En efecto, el estudio de la capacidad de

las especies invasoras para establecerse en una comunidad y sus efectos sobre la biota autóctona han

proporcionado información sobre aspectos fundamentales de la Ecología, tales como el conocimiento

de los factores que limitan la distribución de especies (Richardson y De Bonos 1991) o la importancia

de la identidad de las especies y el papel que desempeñan en el funcionamiento de los ecosistemas

(Vitousek y Walker 1989).

p. 04 p. 05

INTRODUCCIÓN GENERAL

TESIS DOCTORAL

2. Los macrófitos marinos como objeto de estudio en los fenómenos de introducción de especies

Los ecosistemas marinos costeros están considerados como uno de los ambientes más afectados por

la introducción de especies (Carlton 1996). La presente tesis doctoral centra su interés en el estudio de

macroalgas introducidas que han demostrado un gran potencial invasor en las nuevas áreas marinas

costeras colonizadas (Schaffelke et al. 2006), como es el caso del clorófito Caulerpa cylindracea (Son-

der) (en adelante C. cylindracea) en el Mediterráneo.

En los ecosistemas marinos costeros los macrófitos (angiospermas marinas y macroalgas) son un com-

ponente clave de la estructura y funcionamiento de las comunidades bentónicas que integran dichos

sistemas. Este grupo taxonómico y funcional integra un elevado número de especies introducidas o

exóticas, entre las que destacan varias especies de algas verdes sifonales como Caulerpa taxifolia, Cau-

lerpa cylindracea o Codium fragile, con un potencial invasor muy elevado (Williamson y Smith, 2007).

La capacidad de estas especies de macroalgas invasoras de transformar los paisajes colonizados, y sus

posibles consecuencias ambientales y socio-económicas, ha motivado una especial preocupación por

parte de gestores, científicos y público general en las áreas costeras invadidas (p.e. Mediterráneo). A

pesar de ello, los estudios que analizan su impacto en los ecosistemas marinos costeros son hasta la

fecha escasos, están realizados a una escala espacial y temporal reducida, y abarcan un número muy

reducido de especies (Grosholz 2002, McQuaid and Arenas 2006).

También es muy limitado el conocimiento de los mecanismos y factores que determinan y controlan la

capacidad invasora de las macroalgas. Diversas características de estas especies de macroalgas invaso-

ras, como su elevada capacidad de propagación, tasas de crecimiento o plasticidad fenotípica, parecen

explicar dicho potencial y, por tanto, su habilidad para competir y desplazar las especies nativas (In-

derjit et al. 2006, Schaeffelke et al. 2006). Por otro lado, la capacidad de algunas de estas especies de

actuar como especies ingenieras (sensu Crooks 2002) puede provocar la aparición de profundos cam-

bios en las características de los ecosistemas receptores derivados de la alteración en mayor o menor

medida de los regímenes sedimentarios, las condiciones oceanográficas, la estructura del hábitat y/o

de la red trófica de dichos ecosistemas (Dukes y Mooney, 2004, Wallentinus y Nyberg 2007, Deudero

et al. 2011).

3. Dinámica de la introducción de algas invasoras

Igual que se ha descrito para la mayoría de las introducciones documentadas, en la dinámica de intro-

ducción de macrófitos marinos bentónicos se pueden definir cuatro fases diferenciadas según la escala

temporal y espacial en la que se desarrollan y las barreras y vectores implicados (Theoarides y Dukes

2007, Blackburn et al. 2011; Fig. 1):

(i) Fase de transporte: Esta fase implica el transporte interregional de la especie, asociado a deter-

minadas actividades humanas de forma accidental, involuntaria o deliberada, a través de largas

distancias, salvando barreras geográficas de diversa naturaleza. En el caso concreto de los macró-

fitos marinos las vías o vectores de entrada en esta fase están principalmente relacionadas con el

(i) transporte marítimo, ya sea formando parte del fouling en los cascos de embarcaciones y otras

estructuras marítimas o en aguas de lastre y, (ii) la acuicultura (Williams y Smith 2007). Los grandes

puertos son por tanto considerados una de las principales vías de introducción de este tipo de orga-

nismos marinos (Ruiz et al. 2000, Hewitt et al. 2004).

(ii) Fase de introducción: La fase de introducción afecta exclusivamente a especies transportadas de

forma deliberada y que son cultivadas o mantenidas en cautividad fuera de sus rangos naturales de

distribución. Estas especies se enfrentan a barreras físicas asociadas a sus formas de confinamiento

que limitan su introducción en la nueva región.

(iii) Fase de establecimiento: una vez la especie llega a una nueva región debe ser capaz de sobrevi-

vir y desarrollar tasas positivas de crecimiento que le permitan desarrollar poblaciones viables que

perduren en el tiempo. En la mayoría de los casos, estas nuevas especies no consiguen superar esta

fase y desaparecen (especies ocasionales), mientras que un número muy pequeño de especies in-

troducidas (ca. 10%, Williamson and Filter 1996) son capaces de desarrollar poblaciones viables y

naturalizarse (especie naturalizada/establecida). Las barreras a las que se enfrenta la nueva especie

y que pueden determinar su éxito de supervivencia y/o reproductivo son de naturaleza muy diversa

y están asociadas a factores relacionados con las propias características de la especie (p.e. tasas de

crecimiento o reproductivas), y propiedades abióticas y bióticas de la nueva zona (p.e. característi-

cas ambientales o fenómenos de resistencia biótica).

(iv) Fase de propagación: tras el establecimiento, la especie puede comenzar un proceso de dispersión

hacia regiones adyacentes a la zona de introducción, ocupando una mayor o menor variedad de

hábitats y condiciones ambientales y desarrollando en algunos casos un carácter invasor. Durante

esta fase, en el caso de los macrofitos y otros organismos marinos, además de los vectores comen-

tados en la primera fase, intervienen otros de carácter más local como por ejemplo las embarcacio-

nes deportivas, las redes de pesca o las corrientes marinas. En esta fase o etapa, la especie puede

extinguirse de forma natural tras un periodo de expansión muy activo, o bien puede persistir con

fluctuaciones más o menos intensas y regulares de su abundancia.

Fig. 1. Dinámica de la introducción de especies exóticas (adaptado de Blackburn et al. 2011)

p. 06 p. 07


TESIS DOCTORAL

4. Factores que afectan a la introducción y al éxito invasor

La introducción de especies es un proceso multifactorial cuyo éxito depende de la interacción entre el

organismo introducido y el ecosistema receptor a lo largo de una escala espacial y temporal amplia

(Londsale 1999, Shea y Chesson 2002). A pesar de que han sido identificados como fenómenos al-

tamente idiosincrásicos y dependientes de las condiciones locales donde se producen (Meiners et al.

2004, McQuaid y Arenas 2009), se pueden diferenciar tres grandes grupos de factores que controlan el

éxito de los procesos de introducción e invasión (Londsale 1999, Theoarides y Dukes, 2007):

(i) el numero de eventos y tasas de introducción de organismos, denominado de forma general

como “esfuerzo de introducción”,

(ii) las características y atributos de las especies que son introducidas,

(iii) la susceptibilidad (o resistencia) de los hábitats a ser colonizados por la nueva especie.

En el caso particular de los macrófitos marinos, los principales atributos a los que se atribuye el éxito

de su introducción son una elevada capacidad de crecimiento, el desarrollo de fases microscópicas y

de resistencia, la presencia de mecanismos de reproducción sexual y/o asexual (esporas y propágulos)

que facilitan su dispersión, ciclos vitales poliploides y contenidos genómicos reducidos, así como una

elevada capacidad para mantener un estatus biológico adecuado en un amplio rango de condiciones

ambientales, o lo que es lo mismo, una amplia tolerancia ambiental (Smith y Walters 1999, Nyberg y

Wallentinus 2005, Inderjit y Drake 2006, Schaeffelke et al. 2006, Varela-Álvarez et al. 2012).

Las especies exóticas con una mayor tolerancia ambiental serán capaces de resistir condiciones cam-

biantes y/o desfavorables durante su transporte desde las zonas de origen (Hewitt y Hayes 2002, Hewitt

et al.2007). Por otro lado, esta mayor tolerancia puede dotar a la nueva especie de una mayor capaci-

dad de aclimatación a las condiciones ambientales de la nueva zona así como facilitar la colonización

de una gran variedad de hábitats y gradientes ambientales. Así, por ejemplo, la capacidad de crecer en

un amplio rango de temperatura e irradiancia ha sido identificado como uno de los factores determi-

nantes en la introducción de Caulerpa taxifolia en el Mar Mediterráneo (Meinesz et al. 1993), alga que

a pesar de su origen tropical ha sido capaz de colonizar las aguas costeras mediterráneas de marcado

carácter templado (al menos en sus fases iniciales). El grado de tolerancia ambiental y la capacidad de

aclimatación están condicionados por la variabilidad genética, o polimorfismo genético de la pobla-

ción, y por la plasticidad fenotípica individual del organismo, es decir, la capacidad para modificar sus

características fisiológicas, morfológicas o de su ciclo vital en respuesta a señales ambientales y dentro

de un periodo de tiempo inferior a una generación (Harvell 1986, Schlichting y Pigliucci 1998, DeWitt

y Scheiner 2004). Aunque la relación entre plasticidad fenotípica y éxito en el proceso de introducción

no ha sido todavía estudiada en profundidad en macrófitos marinos, existen numerosas evidencias de

su importancia en ecosistemas terrestres (Parker et al. 2003) y en ecosistemas acuáticos no marinos

(Hastwell et al. 2008, Hyldgaard y Brix 2012).

La mayor o menor susceptibilidad (o resistencia) de un hábitat o biocenosis a ser colonizado por una

especie exótica es el resultado de la acción simultánea de las condiciones abióticas del medio, del ré-

gimen de perturbaciones y las interacciones bióticas con las especies nativas (Londsale 1999, Davis et

al. 2000). Las perturbaciones del medio, tanto de origen natural como antrópico, pueden modificar la

resistencia de las comunidades nativas a la colonización de una especie introducida (Planty-Tabacchi et

al. 1996, Burke y Grime 1996, Arrontes 2002, Valentine y Johnson 2003, Bulleri et al. 2011). Esto puede

suceder, por ejemplo, como consecuencia de la alteración de la cantidad de recursos disponibles (p.e. a

través de la eliminación de posibles competidores o por el enriquecimiento del medio), o por los efectos

de las perturbaciones físicas sobre la estructura del hábitat (p.e. fragmentación de la vegetación bentó-

nica por la influencia de temporales) (Davis et al. 2000, Sánchez y Fernández 2006).

Respecto a las interacciones de la especie introducida con la biota nativa, éstas pueden actuar tan-

to inhibiendo como facilitando el proceso de introducción, a través de mecanismos diversos como la

competencia, la depredación, los daños derivados de patógenos, el mutualismo o la facilitación. Dos

grandes hipótesis han sido postuladas para explicar el efecto de las interacciones bióticas sobre la sus-

ceptibilidad de las comunidades nativas a la introducción de nuevas especies:

1. The Enemy Releases Hipotesis (ERH) según la cual la ausencia de enemigos naturales,

tales como predadores o patógenos permite el desarrollo de las especies introducídas en su

nuevo rango de distribución. Esta teoría fue formulada por primera vez por Darwin (1859) para

explicar como algunas especies que son consideraras raras o poco abundantes en sus áreas

originales son especialmente abundantes en otras áreas donde son introducidas.

2. The Biotic Resistance Hipotesis (BRH), propuesta por primera vez por Elton en 1958 es-

tablece, a grandes rasgos, que los fenómenos de competencia desarrollados por las especies

nativas pueden impedir la introducción de especies. Esta teoría esta fundamentada sobre la

idea de que los factores bióticos del medio interaccionan con las condicionas abióticas restrin-

giendo el nicho ecológico de las especies (Hutchinson 1957).

La teoría definida por Elton asume que aquellas comunidades con una mayor diversidad taxonómica

serán menos susceptibles a la introducción de especies como consecuencia de una utilización de los

recursos disponibles más eficiente y completa (hipótesis del uso complementario de los recursos según

Hooper (1998)). Sin embargo, a pesar de que estudios posteriores han corroborado esta idea (Naeem

et al. 2000, Stachowicz et al. 2007), en las últimas décadas diversas investigaciones no solo no han

encontrado una clara relación positiva entre diversidad especifica y resistencia a la invasión, sino que

en algunos casos identifican una relación negativa entre ambos factores (Dukes 2001, Stohlgren et al.

1999, Capers et al. 2007). Estos resultados contradictorios han llevado a sugerir la existencia de otros

factores clave que afectan a la vulnerabilidad de las comunidades nativas a la introducción dentro del

contexto de la interacción entre comunidades nativas y especies exóticas. Algunos investigadores han

postulado que son los atributos funcionales de una o pocas especies clave dentro de la comunidad,

y el estricto control que establecen sobre los recursos y factores ambientales, los que condicionan la

p. 08 p. 09


TESIS DOCTORAL

disponibilidad de recursos a la especie exótica y que hace a las comunidades nativas competidores

superiores por dichos recursos. (Prieur-Richard and Lavorel 2000, Symstad 2000). Diversos estudios rea-

lizados en comunidades de algas bentónicas parecen apoyar esta hipótesis; dichos estudios sugieren

que el control que ejercen determinadas especies de macrófitos formadores de doseles vegetales (ca-

nopy-forming species) sobre factores primarios como la luz o el sustrato, constituyen un mecanismo

clave en el control de los procesos de introducción (Arenas 2006, Britton-Simmons 2006). Este tipo de

comunidades bentónicas actuarían, por tanto, como barreras naturales efectivas contra la introducción

y propagación de especies invasoras. El conocimiento del funcionamiento de estos hábitats nativos, y

la medida en la que transforman el medio es, por tanto, un aspecto clave para determinar la resistencia

de los ecosistemas receptores a la introducción de especies exóticas.

5. Macrófitos invasores del Mediterráneo

El Mediterráneo representa el 0,82% de la superficie total de los océanos del planeta, pero es considera-

do uno de los “puntos calientes” de la biodiversidad marina, siendo su biodiversidad global un 6,3% de

la estimada a nivel mundial (Coll et al. 2010, Costello et al. 2010). De todas las especies macroscópicas

marinas conocidas del Mediterráneo, un 8,9% son macrófitos, repartidos en 277 especies de algas par-

das, 657 algas rojas, 190 algas verdes y 5 angiospermas marinas, siendo un 22% de ellas endémicas de

este mar. En contraste con esta excepcional biodiversidad, estudios recientes indican que desde princi-

pios del siglo XX el número de especies exóticas se ha ido duplicando cada 20 años aproximadamente,

lo que sitúa al Mediterráneo como una de las regiones con mayores tasas de introducción a nivel global

(Boudouresque y Verlaque 2002). Teniendo en cuenta solo el número de especies de macrófitos intro-

ducidos, Williams y Smith (2007) identifican el Mediterráneo como la región más invadida del planeta.

Esta situación se explica en buena medida por la confluencia de múltiples vías de introducción como la

acuicultura, el denso tráfico marítimo y la conexión con el Mar Rojo (región Indopacífica en general) a

través del Canal de Suez (Galil 2009). De las más de 90 especies de macrófitos catalogadas como intro-

ducidas en el Mediterráneo, 10 han sido descritas como invasoras (según Otero et al. 2013 y Rodríguez

Prieto et al. 2013). Éstas son las algas verdes Caulerpa taxifolia, Caulerpa cylindracea, y Codium fragile

subsp. fragile, las algas rojas Acrothamnion preissii, Lophocladia lallemandii, Asparagopsis taxiformis,

Asparagopsis armata y Womersleyella setacea, las algas pardas Sargassum muticum y Stypopodium

schimperi y la angiosperma Halophila stipulacea.

6. Invasión de Caulerpa cylindracea en el Mar Mediterráneo

El género Caulerpa, incluido en la familia Caulerpaceae del orden Bryopsidales (Clase Ulvophyceae,

phylum Clorophycophyta) incluye 86 especies (Guiry y Guiry 2007) de las cuales 6 han sido descritas en

el Mar Mediterráneo:

(i) Caulerpa prolifera (Forsskål) Lamouroux, única especie del genero nativa del Mediterrá-

neo

(ii) Caulerpa scalpelliformis R. Brown ex (Turner), especie introducida detectada por primera

vez en las costas de Israel y Libia (Ryass 1941)

(iii) Caulerpa mexicana Sonder ex. (Kützing), especie introducida de origen lessepsiano (Ma-

yhoub 1976)

(iv) Caulerpa sertularioides (SG Gmelin), especie introducida proveniente del Mar Rojo (Ol-

sen et al. 1998)

(v) Caulerpa taxifolia (M. Vahl) C. Agardh, especie tropical introducida en las costas de Fran-

cia en los años 90 procedente de su cultivo en acuario (Jousson et al. 1998) y protago-

nista de episodios invasivos en zonas del Mediterráneo occidental (Boudoruesque 1995).

Recientemente, Jongma et al. (2012) han identificado en aguas del Mediterráneo una

nueva variedad procedente del suroeste de Australia y que ha sido denominada como

Caulerpa taxifolia var. Distichophylla

(vi) Caulerpa chemnitizia (Esper) J.V. Lamouroux . En este taxón se incluye un hibrido en-

tre las variedades conocidas anteriormente como Caulerpa racemosa var turbinata (J.

Agardh) Eubank y Caulerpa racemosa var. uvifera (C. Agardh) J. Agardh, presente al me-

nos desde 1926.

(vi) Caulerpa lamourouxii (Turner) C. Agardh (Forsskål) J. Agardh, especie ampliamente

distribuida en regiones tropicales y templadas de todo el planeta y cuya presencia se

conoce desde los años 50.

(vii) Caulerpa cylindracea (Sonder) [anteriormente conocida como Caulerpa racemosa (For-

sskål) J. Agardh var cylindracea (Sonder) Verlaque, Huisman et Boudouresque (de aquí

en adelante C. cylindracea)], taxón que ha demostrado un un fuerte carácter invasor en

el Mediterráneo.

C. cylindracea es una especie de aguas templadas y subtropicales procedente probablemente del su-

roeste de Australia (Verlaque et al. 2003, Belton et al. 2014)) cuyo importante desarrollo como espe-

cie invasora justifica su catalogación entre las “100 peores especies invasoras del Mar Mediterráneo“

(Streftaris y Zeneteos 2006). En los siguientes apartados se profundiza sobre diversos aspectos de la

invasión protagonizada por esta especie en el Mediterráneo y las características biológicas y ecológicas

que parecen explicar su elevado potencial invasor.

6.1. Introducción y dispersión Los mecanismos a través de los cuales se produjo la introducción en el Mediterráneo son todavía objeto

de especulación, si bien el tráfico marítimo (a través de las aguas de lastre) y el comercio asociado a

la acuariofilia han sido considerados como posibles vectores de entrada (Klein y Verlaque 2008) C.

cylindracea fue detectada por primera vez en las costas de Libia en 1990 (Nizamudin, 1991). A partir de

esa primera observación se registra una primera fase de dispersión sobre la cuenca oriental, en la que

alcanza de forma sucesiva las costas de Grecia, Albania y Chipre. Posteriormente se registra una segun-

p. 10 p. 11


TESIS DOCTORAL

da fase en la que el alga se desplaza en sentido oeste hacia la cubeta occidental a través del estrecho

de Sicilia, colonizando progresivamente las costas italianas, francesas y españolas, así como algunos

países de la ribera africana como Túnez y Argelia (ver revisión sobre este tema en Piazzi et al. 2005b y en

Klein y Verlaque 2008). En la actualidad se considera presente prácticamente en todo el Mediterráneo,

si bien la escasez de citas sobre su desarrollo en las costas mediterráneas africanas está posiblemente

relacionada con un menor esfuerzo de muestreo en estas zonas. Así mismo, también ha sido detectada

en aguas de las Islas Canarias (Verlaque et al. 2004), lo que evidencia también una posible dispersión

del alga en el océano Atlántico.

La primera observación en las costas españolas se produce en las islas Baleares en 1998 (Ballesteros et

al. 1999) alcanzando en 1999 la costa este peninsular, más concretamente las aguas de la Comunidad

Valenciana (Aranda et al. 1999) desde donde se inicia un proceso de dispersión en sentido suroeste que

alcanza la Región de Murcia en 2005 (Ruiz et al. 2011, Capítulo 1de esta tesis) y continua por las costas

de Andalucía y Ceuta en 2007 y 2008 (Rivera-Ingraham et. al. 2010). En este mismo año es identificada

en aguas de Cataluña, lo que determina su presencia en todo el litoral español (no existen referencias).

Los patrones de distribución del alga observados en el Mediterráneo se caracterizan por la aparición de

poblaciones aisladas y separadas entre sí por distancias relativamente largas, lo que refleja la influencia

de la actividad humana en su dispersión a gran escala (Ould-Amhed y Meisnez 2007, Flagella et al.

2008, Papini et al. 2013).

6.2. Morfología y biología Al igual que el resto de especies de este orden, C. cylindracea se caracteriza por ser una alga de natura-

leza cenocítica y por tanto con estructura sifonal. Presenta un desarrollo característico a través de esto-

lones horizontales de 1-2 mm de diámetro de los que surgen múltiples y delgados rizoides, que permiten

el anclaje del alga al sustrato, y frondes aislados de tamaño pequeño (inferiores a 15cm normalmente

aunque se han detectado ejemplares con longitudes próximas a los 20 cm) divididos en pinnas de as-

pecto vesicular o claviformes denominadas ramuli con una disposición radial o dística y orientados ha-

cia arriba. La longitud de estos ramuli oscila entre 1,5 y los 7 mm mientras que su diámetro varía entre 1

y 3mm. En cualquier caso, los diferentes estudios realizados en el Mediterráneo indican una importante

variación y plasticidad morfológica asociada a factores como la batimetría, los cambios estacionales o

la localización geográfica (ver revisión en Klein y Verlaque 2008).

En relación a la biología de esta variedad en aguas del Mediterráneo, C. cylindracea presenta un ciclo de

vida endopoliploide con una presencia dominante de clones haplofásicos capaces de producir gametos

y un contenido genómico reducido (Varela-Álvarez et al. 2012). Los fenómenos de producción de ga-

metos tanto masculinos como femeninos indican la posibilidad de que existan eventos de reproducción

sexual, si bien la formación de zigotos solo ha sido descrita en laboratorio (Panayotidis y Žuljević 2001).

La producción de estos gametos es holocárpica (Panayotidis y Žuljević 2001), lo que determina que

todo el citoplasma esté implicado en la formación de dichos gametos y por tanto una vez expulsados se

produce la degradación del estolón. La colonización y dispersión de la especie se produce principalmen-

te mediante reproducción vegetativa o asexual, mediante tres mecanismos distintos relacionados con

su constitución sifonal: (i) estolonización, (ii) fragmentación y (iii) formación de propágulos (Ceccherelli

y Piazzi et al. 2001a, Renoncourt y Meinesz 2002).

6.3. Ecología En el Mediterráneo, C. cylindracea no ha mostrado unos requerimientos ecológicos demasiado estric-

tos, lo que le ha permitido colonizar una amplia variedad de sustratos y profundidades y tolerar las

marcadas variaciones estacionales de las condiciones ambientales (Klein y Verlaque 2008). Así, por

ejemplo, en algunas zonas ha sido observada hasta una profundidad de 70 metros y en regiones como

el golfo de León llega a soportar variaciones de entre 8 y 28ºC de temperatura. Esta elevada tolerancia

ambiental también se muestra en relación a otros factores abióticos como la salinidad, siendo capaz

de colonizar lagunas litorales caracterizadas por importantes fluctuaciones en este parámetro (Mastro-

totaro et al. 2003).

Las tasas de crecimiento, estimadas como velocidad de elongación del estolón, pueden alcanzar valo-

res que oscilan entre los 1,3 y 2 cm día -1 (Piazzi y Cinelli 1999; datos propios). Esta elevada capacidad

explica que el alga sea potencialmente capaz de formar densos tapices sobre el sustrato (y las especies

nativas que lo colonizan) en los que los estolones forman estructuras tridimensionales de hasta 15 cm

de grosor (Klein y Verlaque 2008). En estos casos la longitud de estolón por unidad de superficie puede

llegar a alcanzar valores de hasta 2600 m m-2 (Žuljević et al. 2003) y niveles de biomasa que pueden

rondar los 1.260 g PS m-2 (Iveša y Devescovi 2006).

Diversos estudios han identificado un periodo máximo de abundancia y crecimiento entre finales de

verano y principios de otoño, y un periodo de crecimiento mínimo en invierno, asociado s ha impor-

tantes fenómenos de regresión poblacional, (Ruitton et al. 2005b, Lenzi et al. 2007). Durante la época

favorable. Este aparente patrón estacional de crecimiento mostrado en algunas zonas del Mediterráneo

es coherente con la estrecha correlación positiva observada entre la temperatura y el metabolismo y

Fig. 2.C. cylindracea en aguas de la Región de Murcia.

p. 12 p. 13


TESIS DOCTORAL

crecimiento del alga en laboratorio (Flagella et al. 2008). Esto sugiere que el alga desarrolla mecanis-

mos de anticipación como estrategia de aclimatación fisiológica a los cambios estacionales del medio

(“seasonal anticipator species” sensu Kain (1989); Flagella et al. 2008). Sin embargo, la ausencia de

patrones estacionales de abundancia observada en diversos estudios (Giaccone y Di Martino 1995,

Cebrian y Ballesteros 2009) indica a su vez que otros factores abióticos y bióticos del medio como el

hidrodinamismo (Klein y Verlaque 2008), la herbivoría (Tomas et al. 2011) o las perturbaciones de

origen antrópico (Bulleri et al. 2011, Gennaro y Piazzi 2013) pueden jugar un papel determinante en el

desarrollo del alga a escala local.

C. cylindracea ha mostrado tener una elevada capacidad de aclimatarse a las importantes variaciones

espacio-temporales de la irradiancia submarina, como las que tienen lugar entre épocas del año y a lo

largo de gradientes de profundidad. Las poblaciones del alga muestran un mayor desarrollo entre los 5

y los 30m (ver revisión en Klein y Verlaque 2008), aunque se ha llegado a observar a profundidades tan

extremas como 70 metros. Desde un punto de vista fisiológico, los escasos estudios realizados indican

una elevada plasticidad para fotoaclimatarse a ambientes con condiciones lumínicas muy diferentes.

Así, por ejemplo, se ha observado que es capaz de reorganizar su aparato fotosintético en respuesta a

variaciones de la luz asociadas a gradientes de profundidad, a las causadas por el sombreado producido

por el dosel vegetal de otros macrófitos (p.e. Cymodocea nodosa), y a las que tienen lugar a lo largo de

ciclos diarios y estacionales (Raniello et al. 2004, 2006).

El tipo de sustrato es un factor determinante para la colonización del alga, prefiriendo sustratos esta-

bles, duros o con cierto grado de consolidación, frente a los sedimentos arenosos inestables. De esta for-

ma, las comunidades bentónicas más invadidas por el alga son los fondos rocosos fotófilos dominados

por macroalgas autóctonas cespitosas, los fondos detríticos con o sin presencia de rodolitos calcáreos

(maërl), y la mata muerta de Posidonia oceanica (L.) Delile (ver revision en Klein y Verlaque 2008) . Sin

embargo, determinadas biocenosis dominadas por algas de porte erecto y las praderas de angiosper-

mas marinas, en especial las de P. oceanica, a pesar de ofrecer sustratos de colonización relativamente

estables, han mostrado una mayor resistencia a ser colonizados por el alga invasora (Ceccherelli et al.

2000, Piazzi et al. 2001a, Ceccherelli et al. 2002, Ceccherelli y Campo 2002, Bulleri et al. 2010, Katsa-

nevakis et al. 2010, Infantes el al 2011).

En cualquier caso, se desconocen los factores o mecanismos que determinan la interacción entre el alga

invasora y las comunidades nativas y la mayor o menor resistencia de estas últimas a ser invadidas. Cec-

cherellli et al. (2002b) sugieren que las comunidades con especies de porte erecto, formadoras de es-

tructuras tridimensionales o doseles foliares, son las que muestran mayor resistencia a la colonización,

a pesar de no identificar los mecanismos y factores que determinan dicha resistencia. De hecho, las

praderas de P. oceanica han mostrado, como se comentaba anteriormente, ser una de las comunidades

con una mayor resistencia a ser penetradas por los estolones del alga, que normalmente está ausente

en el interior de los densos doseles foliares de dichas praderas. Mediante experimentos manipulativos in

situ, se ha comprobado que la reducción de la densidad de haces favorece el desarrollo del alga dentro

de la pradera (Ceccherelli et al. 2000), lo que sugiere la existencia de una serie de factores asociados

a la estructura del dosel vegetal que limitan el crecimiento de C. cylindracea dentro de las praderas de

P. oceanica, o lo que es lo mismo la existencia de algún tipo de interacción competitiva a favor de la an-

giosperma. De forma similar, praderas que han experimentado algún tipo de alteración de su estructura

a consecuencia de impactos antrópicos parecen ser más vulnerables a la invasión por C. cylindracea

que aquellas en las que su estructura permanece en buen estado de conservación (Montefalcone et

al. 2010, Lenzi et al. 2013). Ceccherelli et al. (2000) plantean la hipótesis de que la disponibilidad de

sustrato dentro de la pradera es uno de dichos factores. Sin embargo, el papel de otros factores prima-

rios clave para el crecimiento y supervivencia algal como la disponibilidad de luz no han sido todavía

investigados, a pesar de la dramática reducción de la luz que los doseles foliares de P. oceanica causan

sobre el fondo (Dalla Via et al. 1998).

6.4. Impactos sobre las comunidades nativasLos fondos colonizados por C. cylindracea pueden llegar a experimentar profundas transformaciones de

sus características físico-químicas y biológicas. Los densos tapices que el alga puede desarrollar tienen

una elevada capacidad de retención de partículas capaz de modificar profundamente sus característi-

cas biogeoquímicas (Holmer et al. 2009, Hendriks et al. 2010). Los efectos asociados a esta alteración

han sido comparados con los generados por el incremento de las tasas de sedimentación sobre las co-

munidades de macroalgas bentónicas (Piazzi et al. 2005a) e incluyen reducciones en la diversidad y en

la cobertura de especies nativas (Piazzi et al. 2001b, Balata et al. 2004, Piazzi et al. 2005a). La presencia

del alga ha sido también relacionada con cambios en la diversidad funcional de estas comunidades,

favoreciendo el desarrollo de especies cespitosas en detrimento de otras de porte erecto o mayor com-

plejidad estructural (Bulleri et al. 2010).

En relación al impacto sobre las angiospermas marinas, se han detectado cambios en las tasas de

floración y producción de Zoostera noltii Hornem. y Cymodoecea nodosa (Ucria) Aschers. en praderas

mixtas colonizadas por el alga (Ceccherelli y Campo 2002). En el caso de P. oceanica, como ya ha sido

comentado, el alga parece ser incapaz de colonizar el interior de sus densos doseles foliares y parece

que su expansión se encuentra limitada a los bordes de la pradera en contacto con poblaciones del alga

desarrolladas sobre otros tipos de habitas adyacentes (observaciones personales). En cualquier caso,

Dumay et al. (2002a) identifican cambios en el ciclo vegetativo de praderas de P. oceanica invadidas

por el alga que incluyen reducciones en la longitud foliar e índice de área foliar así como un aumento

de la tasa de recambio foliar. Estos efectos han sido relacionados con fenómenos de interacción entre

ambas especies asociados a la producción de sustancias alelopáticas (caulerpenina) por parte del alga.

De hecho, experimentos realizados con extractos del alga han demostrado la actividad fitotóxica de la

caulerpenina sobre el rendimiento fotosintético de la fanerógama marina Cymodocea nodosa (Raniello

et al. 2007). Si bien, se trata de un aspecto que todavía no ha sido estudiado en profundidad, especial-

mente en relación a su repercusión a largo plazo sobre las praderas (reducción de la resiliencia de las

praderas de áreas colonizadas a largo plazo y, por tanto, aumento de su vulnerabilidad).

Los estudios realizados sobre los efectos de C. cylindracea en las comunidades de invertebrados bentó-

nicos muestran resultados contradictorios en relación a índices de diversidad y abundancia de especies,

p. 14 p. 15


TESIS DOCTORAL

aunque todos coinciden en la elevada capacidad del alga de cambiar su estructura y dinámica (Buia

et al. 2001, Vázquez-Luis et al. 2008, Pacciardi et al. 2011). Aunque la información disponible sobre

el impacto causado por el alga en niveles tróficos superiores es muy reducida, C. cylindracea ha sido

también relacionada con la disminución en la abundancia de especies de macrofauna como esponjas

(Baldacconi y Corriero 2009) y gorgonias (Cebrian et al. 2012). Por otro lado, algunos estudios han do-

cumentado el consumo activo del alga por parte de especies de peces, sugiriendo la posibilidad de ge-

nerarse cambios estructurales en la cadena trófica derivados de los efectos negativos a nivel fisiológico

del consumo de caulerpenina contenido en el alga (Terlizzi et al. 2011, Deudero et al. 2011, Felline et al.

2012) y que explicarían los cambios detectados en la comunidad íctica en zonas con una alta presencia

de C. cylindracea (Bernardeau-Esteller y Martínez-Garrido 2010).

Por último, y aunque no hay estudios concretos sobre este aspecto, cabe indicar que la capacidad de

C. cylindracea de desarrollar praderas monospecíficas mas o menos continuas puede ser considerada

como una fuente potencial de impacto sobre las características a gran escala de los paisajes en algunos

ecosistemas marinos afectados por la introducción de alga.

7. Justificación de la tesis

A pesar del importante número de estudios realizados sobre la biología y ecología de C. cylindracea en

aguas del Mediterráneo existen todavía importantes carencias sobre el conocimiento de los factores

implicados en su éxito invasor, lo que dificulta en último término un análisis global y riguroso sobre

el alcance y consecuencias de la invasión así como el desarrollo de estrategias adecuadas de gestión

destinadas a limitar y controlar el impacto del alga en esta región (Klein y Verlaque 2008).

Como se ha descrito en los primeros apartados de esta introducción, el éxito invasor de una especie

introducida está relacionado con multitud de factores, como la capacidad de aclimatarse a las nuevas

condiciones ambientales y la interacción con las comunidades nativas que juegan un papel determi-

nante.

Aunque existen evidencias de que C. cylindracea presenta una elevada plasticidad fisiológica en res-

puesta a variaciones en las condiciones ambientales, no ha sido todavía evaluado el grado en el que los

mecanismos de aclimatación desarrollados por el alga ante factores abióticos clave (p.e. luz) inciden so-

bre su capacidad productiva y por tanto, sobre su crecimiento. A su vez, las investigaciones sobre la inte-

racción entre el alga y las comunidades nativas han sido en general desarrolladas en escalas temporales

cortas (inferiores a dos años) dificultando la evaluación de los fenómenos competitivos desarrollados

por el alga. En el caso concreto de las praderas de P. oceanica, los resultados obtenidos hasta la fecha

parecen evidenciar una elevada resistencia de esta comunidad a la invasión, sin embargo no existen

estudios que analicen la interacción entre ambas especies en un marco temporal amplio en el que otros

fenómenos ya descritos para el alga, como el deterioro de las condiciones del sustrato o efectos por fito-

toxicidad alelopática, pueden jugar un papel determinante. A su vez, el grado de conocimiento sobre los

factores que pueden estar relacionados con dicha resistencia sigue siendo muy reducido. Estos aspectos

son especialmente relevantes para poder establecer posibles impactos y escenarios futuros en base a la

evolución de las propias praderas en el Mediterráneo y a su relación con otros fenómenos potenciales de

perturbación sobre estas comunidades (p.e. cambio global, impactos de origen antrópico).

7.1. Objetivo y estructura de la tesis. El objetivo general de la presente tesis doctoral es analizar el establecimiento y ecología de C. cylin-

dracea en el Mar Mediterráneo bajo la perspectiva de su potencial colonizador, profundizando en el

estudio de la interacción del alga con las praderas de P. oceanica y en el análisis del papel de la luz como

factor determinante en los procesos de invasión del alga en esta región.

Los objetivos específicos de la tesis y que constituyen los cuatro capítulos que la conforman son los

siguientes:

Fig. 3.Colonización de C. cylindracea sobre fondos rocosos.

Fig. 4.C. cylindracea en el limite de una pradera de Posidonia oceanica.

p. 16


TESIS DOCTORAL

1. Análisis del establecimiento y dispersión de C. cylindracea en litoral de la región de Murcia

Los mecanismos usados por C. cylindracea para su dispersión en aguas del Mediterráneo han sido am-

pliamente discutidos y relacionados con su éxito invasor. Sin embargo, la dinámica de propagación del

alga a escala local y regional y su capacidad de expansión una vez asentada en una determinada zona

ha sido escasamente documentada y los resultados existentes están, en general, restringidos a unas po-

cas áreas del Mediterráneo. Los objetivos del capítulo 1 son (i) documentar la presencia y dispersión de

C. cylindracea en las costas de la Región de Murcia y (ii) analizar de manera cuantitativa la capacidad

de expansión y grado de desarrollo del alga tras su introducción. Este capítulo define además el marco

general en el que se establecen los estudios de la presente tesis doctoral, describiendo las poblaciones

del alga y los hábitats sobre los que se desarrollan los trabajos que la integran. Para conseguir los ob-

jetivos de este capítulo se investigo la presencia y superficie de ocupación del alga en 42 localidades

localizadas a lo largo de la costa murciana entre los años 2005, año de primera detección, y 2007.

Además, en algunas de las localidades seleccionadas se analizó la abundancia en términos de biomasa

y características biométricas con el fin de caracterizar y definir el grado de desarrollo vegetativo de las

poblaciones.

2. Estudio de la capacidad fotosintética de C. cylindracea a lo largo de un gradiente de profundidad

y su repercusión sobre el metabolismo del carbono

El objetivo de este capítulo es determinar la repercusión que los mecanismos de fotoaclimatación de-

sarrollados por C. cylindracea ante variaciones naturales en los regímenes lumínicos tienen sobre la

capacidad productiva del alga. Con este fin se evaluó la capacidad fotosintética y productiva (mediante

una aproximación basada en la estima del balance de carbono diario) del alga en tres poblaciones

naturales desarrolladas a diferente profundidad bajo regimenes lumínicos significativamente distintos

que representan, por tanto, una aproximación a las condiciones determinadas por un gradiente de pro-

fundidad. La hipótesis analizada es que la plasticidad fotosintética del alga constituye un mecanismo

efectivo para optimizar su capacidad productiva ante las variaciones lumínicas definidas por gradientes

de profundidad.

3. Valoración del papel de luz en la resistencia de las praderas de P. oceanica a la invasión de

C. cylindracea

El objetivo general de este capítulo es identificar y evaluar los mecanismos y factores implicados en la

resistencia a la invasión mostrada por P. oceanica. Esta angiosperma marina genera un dosel vegetal

de elevada complejidad cuya estructura tridimensional modifica intensamente las condiciones ambien-

tales en su interior. Esta modificación, como ya ha sido comentado anteriormente en esta introducción,

es especialmente relevante en el caso del ambiente lumínico, que sufre una profunda alteración tanto

a nivel cuantitativo (se ha estimado que la reducción en la disponibilidad puede llegar a ser de hasta el

5% de la irradiancia superficial) como cualitativo (perdida de longitudes de onda de bajo rango ener-

gético). Con el fin de evaluar la posible implicación de la disponibilidad lumínica dentro de las praderas

de P. oceanica en su alta resistencia a la colonización se llevó a cabo un análisis comparativo de los

regímenes de luz, abundancia, características fotosintéticas y capacidad productiva entre poblaciones

de C. cylindracea desarrolladas dentro y fuera de praderas de P. oceanica. La hipótesis planteada es

que las condiciones de luz bajo el dosel vegetal de P. oceanica constituye un factor determinante para

el crecimiento y supervivencia del alga y por lo tanto para el desarrollo de su potencial invasor en este

valioso ecosistema mediterráneo.

4. Estudio experimental del efecto de la disponibilidad de luz sobre la fotosíntesis, metabolismo del

carbono y crecimiento de C. cylindracea: la luz como factor limitante en la colonización de praderas

de P. oceanica

Los resultados obtenidos en el estudio previo evidenciaron la implicación de la luz en los fenómenos

de resistencia a la colonización de las praderas de P. oceanica. Sin embargo, le metodología aplicada

en dicho estudio, basada en una análisis de poblaciones in situ, dificultaba la capacidad de diferenciar

el efecto de este factor del generado por otros factores ambientales relacionados las características

del dosel vegetal (pe. el hidrodinamismo o la disponibilidad de nutrientes) y que también pudiesen

estar implicados en el proceso de colonización. El objetivo de este capítulo es por tanto establecer si

las condiciones de luz dentro de la pradera son capaces de explicar de forma aislada los fenómenos

de resistencia observados y parte de una hipótesis similar a la del capitulo anterior. Con este fin se ha

estudiado la respuesta fotoaclimatativa y la capacidad de producción y crecimiento de C. cylindracea

en dos experimentos manipulativos complementarios, desarrollados en condiciones de mesocosmos y

campo, en los que las condiciones lumínicas fueron controladas.

Ademas se incorporan en una anexo los resultados sobre la Evaluación de la interacción a largo plazo

entre C. cylindracea y las praderas de P. oceanica, estudio que ha sido recientemente enviado para

su publicación y que por su relación con los aspectos tratados en la presente tesis se ha considerado

oportuno su inclusión.

En este Anexo se presentan los resultados (2007-2014) obtenidos en el estudio sobre la interacción a

largo plazo entre ambas especies que se está desarrollando en aguas del litoral de Murcia en el contexto

de la Red de Seguimiento de las Praderas de P. oceanica de esta región. La hipótesis planteada en este

estudio es que, a pesar de la alta resistencia a la invasión mostrada por las praderas de P. oceanica, C.

cylindracea es capaz de competir con la angiosperma marina, de manera que puede provocar cambios

estructurales en la pradera, reducir su resiliencia ante otros fenómenos de perturbación e incrementar

su capacidad colonizadora. El seguimiento de la interacción entre ambas especies se está realizando

en tres zonas en las que los fondos colonizados por C. cylindracea están en contacto con praderas de

P. oceanica, llegando a colonizar los primeros centímetros de dichas praderas.

CHAPTER 1Recent spread of the invasive alga

Caulerpa cylindracea (Bryopsidales,

Chlorophyta) along the Mediterranean

coast of the Murcia Region (SE Spain)

p. 21

Recent Spread of the Invasive Alga Caulerpa cylindra-cea (Bryopsidales, Chlorophyta) along the Mediterra-nean coast of the Murcia Region (SE Spain).

Abstract

The aim of this paper is to document the recent

appearance and spread of the green alga Cau-

lerpa cylindracea along the coast of Murcia in

south-east Spain. This is the westernmost sigh-

ting of the invasive alga in the Mediterranean

Sea. It was found for the first time in the area in

2005 and over the next two years the number of

new sightings increased almost exponentially. At

some of the invaded stations the alga increased

its surface area 6.5- to 44-fold in one year. In the

period 2005–2007 the total surface area coloni-

sed by the alga in the region was estimated to be

at least 265 ha. Benthic assemblages colonised

by the alga were rocky photophilic algae, dead P.

oceanica rhizomes, infralittoral and circumlittoral

soft bottoms and maërl beds. No penetration of

the alga was observed in P. oceanica meadows,

except in one locality. Biometric analysis indica-

ted high vegetative development in the establi-

shed colonies in comparison to those described

in other Mediterranean areas. The results of this

study reveal that the rapid spreading dynamics

of C. cylindracea in the region of Murcia are a po-

tential threat for the native benthic communities.

Introduction

The biological characteristics of Caulerpa cylin-

dracea Sonder. (hereinafter C. cylindracea) (high

rates of vegetative dispersal, production of alle-

lopathic substances, etc.) determine its high colo

nisation potential and its extraordinary ability to

outcompete and alter native benthic assembla-

ges, which make this species a particular poten-

tial threat for the Mediterranean coastal ecosys-

tem (Piazzi et al. 2005b).

C. cylindracea was observed in the Eastern Medi-

terranean Sea for the first time along the coast

of Libya in 1990 (Nizamuddin 1991), being the

origin of this invasive variety still unknown (Verla-

que et al. 2003, Durand et al. 2002, Panayotidis

2006). Since then, the species has spread rapidly,

gradually invading the Mediterranean Sea. This

has been well-documented in the western basin

along the coasts of Italy, France and North Afri-

ca (Piazzi et al. 2005b). Along the Mediterranean

coast of Spain, the species was first sighted in the

Balearic Islands in 1998 (Ballesteros et al. 1999).

It reached the east coast of the Iberian Peninsula

(Castellón) in 1999 (Aranda et al. 1999) and be-

gan to spread quickly southward, being sighted in

Alicante (SE Spain) in 2000 (Aranda et al. 2003).

At that point, the algal spread seemed to stabili-

se (Fig. 1), but its presence was confirmed in the

Murcia region in 2005, indicating that the coloni-

sing process was continuing southward.

Precise studies documenting the presence of the

alga in newly colonised areas (i.e. colony size,

depth range, substrate type, morphometric data

and invaded native communities) are fundamen-

Publicado en: Ruiz JM, Marín-Guirao L, Bernardeau-Esteller J, Ramos-Segura A, García-Muñoz R, Sandoval-Gil JM

(2011) Spread of the invasive alga Caulerpa racemosa var. cylindracea (Caulerpales, Chlorophyta)

along the Mediterranean coast of the Murcia region (SE Spain). Anim Biod Conserv 34(1): 73-82.

p. 23p. 22

CHAPTER 1

TESIS DOCTORAL

tal to elucidate its colonising potential, spreading

dynamics and mechanisms (vectors) at local and

large spatial scales (Klein and Verlaque 2008).

Cartographic methods make it possible to mea-

sure the extent of the spread and can assist in

helping to predict potential impacts and future

scenarios (Meinesz 2007). Detailed informa-

tion on the spreading dynamics and extent of

C. cylindracea is available for a limited number

of Mediterranean regions (Piazzi et al. 1997b;

Ruitton et al. 2005a). The goals of the present

study were: (1) to document the spreading dyna-

mics of C. cylindracea along the coast of Murcia

(SE Spain) from its appearance in 2005 to 2007,

both at regional and local scales; (2) to provide

some quantitative estimate of the colonised sur-

face area. Furthermore, the work includes several

characteristics of the invaded sites (colonised as-

semblages, colonization depth) together with the

vegetative development of several colinies in this

geographical area.

Material and Methods

Study Area and Field Sampling Programme

This study was carried out on the Mediterranean

coast of Murcia, SE Spain (Fig. 1). After C. cylin-

dracea was first sighted in the region in 2005, an

active detection programme was established to

map the distribution of the alga and its spreading

dynamics over time (Meinesz 2007, Ruitton et al.

2005a). To this end we initially selected 42 sam-

pling stations uneven distributed along 224 km

of the Murcia coastline through a depth range

from 2 to 30 m (Fig. 1). These stations were selec-

ted from different long-term sampling program-

mes that had already been initiated in the region

for different purposes (scientific monitoring of

P. oceanica meadows, environmental impact as-

sessments and scientific projects), but since they

were visited at least once a year by specialised

divers this ensured reliable information about the

date of appearance of the alga. Of course, this

sampling strategy resulted in a non-systematic

sampling design, but it allowed us an insight into

the colonisation process in a representative area

of the Murcia coast. The period covered by this

sampling programme was 2005–2007 i.e. the

first three years of the colonisation process of C.

cylindracea in the Murcia region.

Once the alga was detected at a given station, di-

vers from our research team surveyed a total sur-

face area of 0.5 ha to characterise the colonised

area (depth range, types of colonised substrate

and benthic assemblages) and to estimate its

surface area (i.e. colonisation levels sensu Ruitton

et al. 2005a). Based on the field data obtained,

invaded localities were assigned to one of the

following five categories of colonisation level:

(I) one or few small colonies covering a surface

area of less than 10 m2; (II) colonies of varying

sizes covering a total surface area between 10

and 104 m2; and (III) meadows covering surfa-

ce areas between 104 and 105 m2, (IV) 105 and

106 m2 and (V) greater than 106 m2. For cate-

gories I and II, the surface area was estimated in

a single survey within the sampling station using

quadrats and transects. For cases belonging to

categories III–V, where the colonised area ex-

tended beyond the area surveyed by divers at a

single station, additional dives were necessary to

determine the limits of the total colonised area.

These additional dives were performed at nei-

ghbouring points separated from the sampling

station by several hundreds of metres and at di-

fferent depths and directions (a specific sampling

design was established in each case). Once the

limits of the invaded area were identified these

were determined by GPS and input into a Geo-

graphic Information System (Arcview microcom-

puter programme Version 9.0, Esri ©) to estimate

the surface area of the polygon thus generated.

Biometric Analysis

Biometric analysis of C. cylindracea colonies was

performed using data from summer 2007 (June

28th to August 9th), a season in which the ve-

getative development of the alga was close to

its annual maximum (Klein and Verlaque, 2008).

Samples were collected at three of the most in-

vaded stations: station 1 (–10 m), station 14

(–25 m), and station 25 (–22 m) (Fig. 1). Fronds,

stolons and rhizoids were carefully collected by

hand within six replicated 1,600 cm 2 quadrats

that were randomly placed within fully colonised

areas (i.e. 100% cover) along a 50 m transect.

Samples were processed in the laboratory to de-

termine the following biometric variables as des-

cribed by Capiomont et al. (2005) and Ruitton et

al. (2005b): the total length of stolons (m m-2),

number of stolon apices (no. apices m-2), number

of fronds (no. fronds m-2) and frond height (cm).

Total biomass (g dw m-2) was determined by dr-

ying the samples at 70 ºC until constant weight.

Results

Distribution and Estimated Colonised Area

Field data obtained at the invaded localities are

Fig. 1.

Recent spread of C. cylindracea in the

Western Mediterranean basin (A). Dis-

tribution of sampling stations on the

Murcia coast (B), and in the Marine

Reserve Cabo de Palos-Islas Hormigas

(C). Invaded stations are indicated by

black circles, the size of which corres-

ponds to one of the five categories of

colonisation level (see legend and me-

thods section). Information related to

the 42 sampling stations is included

in Appendix 1.

p. 25p. 24

CHAPTER 1

TESIS DOCTORAL

summarised in Figure 1 and Table 1. C. cylindra-

cea was first detected in station 14 (locality of

Cablanque) in 2005 as dispersed patches cove-

ring a total surface area of more than 104 m2. By

2007, the colony had formed a more homoge-

nous meadow of at least 2.5·106 m2. After 2005,

the number of new invaded localities increased

almost exponentially: two in 2006 and six in 2007

(Table 1). In 2006, the population of station 4 (lo-

cality of Isla Grosa) was first found as a few small

patches over a total surface area of 221 m2 that

increased to 104 m2 in 2007 (Figs. 1b and 2). In

the station 25 (locality of Cabo Tiñoso) the initial

surface area in 2006 was estimated as 13,724 m2

and this increased to 89,187 m2 in 2007. In 2007,

all new sightings were concentrated along the

easternmost coast of the region (stations 1, 6, 9,

12, 13 and 19) with very different colonisation

levels, ranging between categories I and III (Fig.

1b and c, Table 1). The cumulated field data gave

a gross estimation of the total invaded area of

265 ha in 2007, which is probably an underesti-

mation of the real colonised area since informa-

tion on areas deeper than 30 m was not available

and some coastal zones were excluded from the

survey.

Characteristics of the Colonised Areas

The depth of invaded areas ranged from 2–30 m,

but the maximum colonised depth was greater

than 30 m since deeper stands continued fur-

ther into this isobath (Table 1). Shallow colonies

(<10m) were the least frequent while most of the

studied colonies fell within 10–30 m. The alga

colonised a wide range of substrates and native

assemblages: rocky photophilic algae (boulders

and vertical walls), infralittoral and circumlitto-

ral soft-bottoms, dead mats of P. oceanica, and

mäerl beds (Table 1). In most localities, C. cylin-

dracea formed compact multilayered mats up to

12 cm thick over the substrate. Cabo Tiñoso was

the only locality where the P. oceanica meadow

was partially invaded by the alga, but no penetra-

tion of the seagrass canopy was observed at the

other localities.

Biometric Characterization of the Colonies

Table 2 summarises the biometric characteris-

tics of C. cylindracea colonies at three selected

stations: 4, 14 and 25. The total biomass varied

between 9.4 and 135.9 g dw·m-2. The number of

stolon apices ranged from 150 to 3,756 m-2, while

the total number of fronds ranged from 937 to

9,018.7 m-2. In addition, the total length of sto-

lons ranged from 1,684 to 5,777 m·m-2, while the

height of fronds varied between 0.3 and 9.5 cm.

Discussion

C. cylindracea was observed for the first time on

the coast of Murcia as an isolated colony at the

locality of Calblanque (station 14) in 2005. The

origin and the introducing vector of the alga in

the region is unknown, but two hypothesis can

be advanced: (1) dispersion from the nearest

colonies, located in the province of Alicante 90

km to the north, and (2) introduction through the

nearby harbour at Cartagena, which is a crucial

point for the very dense maritime traffic suppor-

ted by this part of the Mediterranean Sea (Fig.

1b). Further regional dispersion in subsequent

years occurred in an almost exponential manner

and new colonies appeared without a clear spa-

Station no.

41425

Locality

Isla GrosaCalblanque

Cabo Tiñoso

Depth (m)

102522

Total biomass

(g dw · m-2)62.7 ± 42.716.9 ± 7.3

49.7 ± 21.0

Number of apices

(No. apices · m-2)1.133 ± 958323 ± 187

2.238 ± 832

Number of fronds

(No. fronds · m-2)5.401 ± 2.6311.260 ± 589

6.256 ± 1.316

Total stolon length

(m · m-2)3.487 ± 1.0733.913 ± 1.1334.528 ± 798

Frond height

(cm)1.6 ± 0.43.1 ± 0.31.9 ± 0.7

Station no.

1469

1213141925

Locality

La MangaIsla Grosa

Piles IIsla Hormiga

La BarraLos Punchosos

CalblanqueCabo Negrete

C. Tiñoso

colonization

year

200720062007200720072007200520072006

2005

(m2)

000000

30000

2006

(m2)

0221

0000

NA0

13,724

2007

(m2)

10104

11

104

4.5·104

2.5·106

104 - 105

9·105

Level of

colonization

IIIIIIIIIIV

IIIIV

Table 1.

Sampling stations colonised by C. cylindracea along the coast of Murcia. Stations correspond to Figure 1 and Appendix 1. Level of

colonisation as explained in the methods section.

Table 2. Biometric analysis of the C. cylindracea populations studied. Mean values ± SD.

Fig. 2.

Distribution and estimated surface area colo-

nised by C. cylindracea in station 4 (locality of

Isla Grosa) and station 25 (locality of Cabo Ti-

ñoso) in 2006 and 2007. Black arrows indicate

the presence of new small patches of the alga.

p. 27p. 26

CHAPTER 1

TESIS DOCTORAL

tial pattern in localities separated by hundreds

of metres to tens of kilometres. This rapid and

discontinuous regional spread is similar to that

described by Langar et al. (2002) on the Tuni-

sian coast and by Ruitton et al. (2005a) along

the French Mediterranean coast. This pattern of

spread has been attributed to the efficient repro-

ductive mechanisms reported for C. cylindracea,

both sexual (Panayotidis and Zulevic 2001) and

vegetative (Renoncourt and Meinesz 2002), that

determine its higher colonisation potential relati-

ve to other invasive Caulerpales (e.g. C. taxifolia,

Meinesz 2007).

In 2007 the most widespread population of C.

cylindracea was found in station 14 at the loca-

lity of Calblanque, the site where it was first sigh-

ted. However, there was no relationship between

the actual colony size and the time elapsed since

it was first observed, as indicated by the large

variation in the estimated surface area between

new colonies detected in 2006 and 2007 (1 to

105 m2). This is because at some localities the

alga appeared before the date of its first sigh-

ting, but was not detected in the preceding year.

The alga was probably already present as one or

a few small inconspicuous patches that would

be difficult to find, even by trained divers, but

this implies that C. cylindracea is able to spread

over a surface area of at least 1 ha in a 1-year

period, which represents a very fast colonisation

rate. Station 25 at the locality of Cabo Tiñoso is

a good example: the invaded area increased 44-

fold in 1 year (i.e. 7.5 ha year-1). Similary, the alga

colonised a surface area of almost 1 ha in 2007 in

station 4 from only few small patches found one

year earlier (Fig. 2).

Similar spreading dynamics have been reported

from other Mediterranean localities (Piazzi et

al. 1997b, Piazzi and Cinelli 1999, Piazzi et al.

2001a, Ruitton et al. 2005b), showing that once

the alga arrives at a locality, substrate colonisa-

tion can be a very rapid process. This could be

due to the high stolon elongation rate (up to 2

cm·day-1) and reproductive capacity of the alga

(Panayotidis and Zuljevic 2001, Ceccherelli and

Piazzi 2001, Renoncourt and Meinesz 2002, Ruit-

ton et al. 2005b), but habitat characteristics such

as substrate type and the complexity of native

communities are also determinant. As described

for other Mediterranean localities (Ruitton et al.

2005a, Zuljevic et al. 2003, Piazzi et al. 2005b,

Piazzi and Balatta 2009), the alga fully colonised

a variety of substrates and biocenoses present

throughout its depth distributional range (i.e.

rocky photophilic algae, dead rhizomes of P. oce-

anica, detritic soft bottoms and maërl beds), with

the exception of P. oceanica meadows. Similarly,

penetration of C. cylindracea in P. oceanica mea-

dows has rarely been reported and only occurs in

low density canopies (Piazzi et al. 1997a, Piazzi

and Cinelli 1999; Ceccherelli et al. 2000, Monte-

falcone et al. 2007).

Biometric analysis of C. cylindracea meadows

indicated a high degree of vegetative develop-

ment in the studied colonies. The mean values of

biometric descriptors were within the ranges ob-

served in other invaded localities of the Western

Mediterranean at a similar depth range (Buia et

al. 2001, Ruitton et al. 2005b, Capiomont et al.

2005, Klein and Verlaque 2008). However, the

mean values of total stolon length found in this

study (3–4.5 km m-2) were higher than the maxi-

mum values reported elsewhere in the season of

maximum algal development (summer–autumn:

1–2.6 km m-2; Capiomont et al. 2005, Ruitton et

al. 2005b, Zuljevic et al. 2003). This extensive

stolon development indicates overgrowth over

the colonised substrate (Capiomont et al. 2005)

and hence, a high potential impact on the native

assemblages, particularly in those with lower ver-

tical stratification (Balata et al. 2004, Piazzi and

Balata 2009) such as the maërl beds observed in

deeper waters of our study area.

The recent appearance of C. cylindracea on the

coast of Murcia and its appearance in Algeria

in 2006 (Ould-Ahmed and Meinesz 2007) re-

presents the most recent spread of the invasive

alga documented in the Western Mediterranean

Sea and indicates that its geographical propa-

gation is still continuing in a westerly direction.

In fact, at the time of writing (2008–2009), new

sightings of the alga confirm its presence along

the west coast of Murcia and in the neighbou-

ring province of Almeria. The estimated rate of

spread of the alga in the Murcia region (at least

265 ha in three years) is higher than that repor-

ted in the Marseille region (France) over six years

(350% increase; Ruitton et al. 2005a), but is si-

milar to the highest values reported for southern

latitudes (coast of Tuscany, Italy) for a similar

time period (1993–1997; Piazzi et al. 1997b).

This evidence, together with the high degree of

stolon development reported in this study, sug-

gest that the highly invasive nature of C. cylin-

dracea on the SE coast of Spain may be favou-

red by the higher temperatures and irradiance

characteristic of these southern Mediterranean

latitudes. However, interpretation of these regio-

nal variations must be made with caution due to

the low number of studied cases, and the use of

different methodologies and experimental condi-

tions (Klein and Verlaque, 2008). It is clear that

the invasion by this and other introduced species

represents a serious potential threat for nati-

ve marine communities, but the real ecological

consequences, and their economic impact (local

fisheries, tourism), are subjects that need to be

addressed further.

p. 28

CHAPTER 1

TESIS DOCTORAL

Station no.

123456789

101112131415161718192021222324252627282930313233343536373839404142

Locality

La MangaTomás Maestre

Isla GrosaIsla GrosaCala Túnez

Piles IPiles II

Bajo de en medioIs. Hormiga

C. Escalera-someraC. Escalera-profunda

La BarraLos Punchosos

CalblanqueCalblanqueCalblanqueCalblanqueCalblanque

Cabo NegreteCabo Negrete

El GorguelCabo de Agua

Punta del AguilónIsla de las Palomas

C. TiñosoC. TiñosoC. TiñosoLa AzohíaMazarronMazarrónBolnuevoBolnuevoBolnuevoCalnegreCalnegreCalnegre

CalabardinaIsla del FrailePunta PardaPunta PardaPunta PardaPunta Parda

Depth (m)

26-275-74-8

4-125-8

18-22 (20)10-2010-2010-2510-1220-24

2-45-13 (7)25-26

272529272727

5-175-175-17

19-225-300-12

21-2517-2117-2117-21

2124-2517-1816-1719-2024-257-15

14-1630303030

X703019700822701766701985703513704710704991706008707082703946703966702954702866700052693773694689695176697283697404697569687416683908682494673129664394663125663172661074656472655306646505645186644435643447642439641492632933629651622786622625621972621615

RF

C

CNCNCNCNC

CC

NCNCNC

CNC

NC

RS

NC

C

NC

IS

NC

NC

NC

NCNC

DC

NC

NC

NCNCC

NC

NCNCNCNCNCNCNCNC

DM

C

CNCNCNC

CNC

C

NCNCNCNC

P

NCNC

NCNC

NC

C

NC

NCNC

PU

NCNC

NCNC

NC

PDNC

NCNCNCNCNCNCNC

NC

NC

NCNCNCNCNCNCNCNC

NCNCNCNC

PM

C

NC

Y418244441794424178400417794641681614168591416875641684094170103416762941675434167457416676641618474160728416020841594984159339415936041595194160406415852841589274160784415652041567044156648415799941595104159294415727341561164155026415459441533624151921414298641415824137131413698141361364136042

UTM Habitat/Biocenosis

Appendix 1.

Summary of the information related to the 42 sampling stations: localities, coordinates UTM, surveyed depth (in brakets colonized

depth) and habitat/Biocenosis present (C = colonised, NC = not colonised).

CHAPTER 2Photosynthesis and daily metabolic

carbon balance of the invasive Caulerpa

cylindracea (Chlorophyta:Bryopsidales)

along a depth gradient

p. 33

Photosynthesis and daily metabolic carbon balance of the invasive Caulerpa cylindracea (Chlorophyta:Bryop-sidales) along a depth gradient.

Abstrac

The photosynthetic plasticity of the invasive

green alga Caulerpa cylindracea has been pro-

posed as a relevant mechanism determining its

successful performance on Mediterranean ben-

thic assemblages over broad depth gradients. In

the present study, the photosynthetic performan-

ce of C. cylindracea was evaluated through a car-

bon balance approach in three invaded sites with

contrasting depths (11, 18 and 26 m) and light

regimes. At each sampling depth, photosynthesis

vs irradiance (P vs E) curves were performed on C.

cylindracea fronds and daily net productivity va-

lues were obtained by the numerical integration

of P vs E models with continuous recording of irra-

diance measured on the sea floor. Photosynthe-

tic responses were consistent with those typically

exhibited by shade-adapted macroalgal species

and other Mediterranean populations of C. cylin-

dracea: a significant reduction in maximum pho-

tosynthesis (Pmax

) occurring at an intermediate

depth (18 m) and a higher photosynthetic effi-

ciency (α) and lower dark respiration rate (Rd) at

the deepest sampling depth. Mean values of dai-

ly net C balance obtained in the deepest site were

only 15% lower than that of the shallower depth,

despite the severe reduction in light availability.

Mean daily net carbon balances obtained from

the deepest site were only 15% lower

than those obtained from the shallower depth,

despite the severe reduction in light availability.

This daily net carbon gain was ca. 29% higher

than what would be expected if photosynthetic

adjustments did not occur in the deepest algal

population. The evidence provided by this data

support the hypothesis of photoacclimation in C.

cylindracea as an effective mechanism to opti-

mise algal productivity across depth gradients in

the Mediterranean Sea.

Introduction

In the colonized sites Caulerpa cylindracea

Sonder. (hereafter C. cylindracea) is able to de-

velop high biomasses over different substrate

types, constraining the diversity of native ben-

thic assemblages (Argyrou et al. 1999, Piazzi et

al. 2001b, Balata et al. 2004, Piazzi and Balatta

2008, Vázquez-Luis et al. 2008, Klein and Verla-

que 2009).

Whilst many studies have focused on spatial pat-

terns and temporal dynamics of the distribution,

phenology and biomass of C. cylindracea, only

a few have dealt with the potential competitive

mechanisms responsible for its ecological success

in sublittoral Mediterranean environments (see

Klein and Verlaque, 2008 for a review). Among

Publicado en:Bernardeau-Esteller J, Marín-Guirao L, Sandoval-Gil JM, Ruiz, JM (2011) Photosynthesis and daily me-

tabolic carbon balance of the invasive Caulerpa racemosa var. cylindracea (Chlorophyta: Caulerpales)

along a depth gradient. Sci Mar 75(4): 803-810.

p. 34 p. 35

CHAPTER 2

TESIS DOCTORAL

other plant traits (e.g. vegetative and sexual re-

productive success, production of allelopathic

substances, physiological resistance to stress),

morphological and physiological plasticity has

been suggested as a likely adaptive feature ena-

bling acclimation to a wide range of environmen-

tal conditions in this (Klein and Verlaque, 2008)

and other C. cylindracea varieties (Peterson 1972,

Riechert and Dawes 1986, Ohba et al. 1992). The

capacity of the alga to photoacclimate to varying

light regimes has special relevance in this con-

text, since C. cylindracea has been shown to be

able to develop down to 70 m depth (Klein and

Verlaque 2008), colonize the understory of ma-

crophyte canopies (Cecherelli and Campo 2002)

and maintain biomass through time - even during

conditions of severe light limitation (e.g. deep

populations in winter: Cebrian and Ballesteros

2009). However, our knowledge of the photo-ac-

climative capacity of Mediterranean populations

of C. cylindracea is sparse at best (Raniello et al.

2004, 2006). Raniello et al. (2004, 2006) repor-

ted interesting data showing how C. cylindracea

is able to re-organize its photosynthetic pigment

system in response to varying light conditions

caused by depth gradients, seagrass canopies,

as well as daily and seasonal cycles. Regarding

depth (Raniello et al. 2006), changes in pigment

composition were thought to represent algal

photoacclimation responses in order to optimize

light capture (increase in α) and photosynthetic

performance (decrease in Ek) as light becomes

limiting. These are common responses seen in

some macroalgae species able to develop over

broad depth gradients (Ramus et al. 1977, Mar-

kager and Sand-Jensen 1992, Gómez et al. 1997,

Johansson and Snoeijs 2002). Nonetheless, the

extent to which the ability of Mediterranean

populations of C. cylindracea to photoacclima-

te effectively is responsible for productivity and

potential colonization success remains unknown.

In the present study we analyzed the phosynthe-

tic responses of C. cylindracea in order to assess

the pattern of algal productivity along a depth

gradient. To this end a carbon balance approach

was taken, based on the numerical integration

of Photosynthesis vs Irradiance (P vs E) models

throughout continuous measurement of instan-

taneous irradiance recorded at the sea floor. This

mechanistic approach has been previously de-

monstrated to provide reliable estimates of pri-

mary productivity in marine macrophytes (Matta

and Chapman 1991, Zimmerman et al. 1994).

Photosynthesis and respiration rates of C. cylin-

dracea fronds, together with continuous irradian-

ce field data, were measured at three different

locations of contrasting depth and light on the

coast of the Murcia Region of SE Spain, a part of

the Spanish Mediterranean coast invaded by the

alga since 2005 (Ruiz et al. 2011).


Study area

The present study was performed at three sam-

pling stations, each located at three separate

locations at different depths on the coast of the

Murcia Region of SE Spain: shallow station (S,

11m, Isla Grosa; N37º43’, E00º42’), interme-

diate station (I, 18 m, Cabo Tiñoso; N37º32’,

E00º44’) and deep station (D, 26 m, Calblanque;

N37º32’N, E1º07’) (Fig. 1). These depths are re-

presentative of the current bathymetric range of

C. cylindracea on the Murcian coast (10-30 m,

Ruiz et al. 2011). At the time of sampling, the se-

lected stations were located in the most invaded

areas (in terms of colonized surface area) of the

Murcian coast, with the alga present at stations

I and D since 2005 and at station S since 2006.

The most commonly invaded benthic communi-

ties are the unvegetated sediments and photo-

philic macroalgal assemblages on hard substra-

tes found at station S and the coastal detritic

sediments found at stations I and D, the latter

being dominated by rhodoliths.

Field measurements:

PAR irradiance and algal biomass

All field work was performed by SCUBA divers at

the end of summer (August 2008), a time of year

in which the development of the alga is close to

maximum in many areas of Murcia (personal

observation) and other Mediterranean regions

(Klein and Verlaque 2008, but see Cebrian and

Ballesteros 2009). Incident irradiance on the

sea floor was determined at each sampling sta-

tion from continuous recording of instantaneous

PAR irradiance (E, μmol quanta m-2 s-1, 400-700

nm; Kirk 1994), obtained via the deployment of

a spherical 4π quantum sensor (Alec Electronics,

MDS MK5, Japan) for a period of 21 days (12th

August-1st September). All sensors were placed at

a height of 5 cm from the sea floor. During the

measuring period, the accumulation of epiphytes

or particles on the sensor surface was observed

to be negligible and no associated decline in in-

cident light with time was observed. Quantum

sensors were programmed to record at 10 mi-

nute intervals (n = 144 measurements per day).

A mean daily light cycle was obtained for each

sampling station, characterized by maximum

instantaneous irradiance at noon and the inte-

grated daily irradiance (mol quanta m-2 s-1). In

addition, measurement of sub-surface instanta-

neous irradiance (Eo) was performed at noon on

five standard days (i.e. those with full sunlight, a

cloudless sky and calm weather) using a flat, co-

sine-corrected 2π sensor connected to a LI-COR

quantum meter (model LI-190SA). Mean Eo and

noon instantaneous irradiance values measured

at the sea floor on the same days were used to

Fig. 1.

Location of sampling stations: shallow (-11 m, S), intermediate (-18 m, I) and deep (-26 m, D).

p. 36 p. 37

CHAPTER 2

TESIS DOCTORAL

calculate both the percentage of Eo reaching

the seabed and the water-column attenuation

coefficient (Kd, m-1; Kirk 1994) for each sampling

station. Measurements of both sensor types were

intercalibrated in the laboratory and showed a

very strong linear relationship (R2=0.998) with a

constant factor of 1.176.

At each sampling station, the abundance of C.

cylindracea was determined by measuring its to-

tal biomass (fronds, stolons and rhizoids) in six re-

plicate quadrats of 400 cm2, randomly positioned

within a surface area of 25 m2 colonized by the

alga. Plant material of each sample was placed

in dark plastic bags before being sorted, dried at

70ºC (24 h) and weighed for biomass quantifica-

tion (g dw m-2) in the laboratory.

Measurement of photosynthesis and dark res-

piration rates

Samples of C. cylindracea collected at each sta-

tion were transported to the laboratory in cooled

and aerated containers for the measurement of

photosynthetic and respiration rates and the de-

termination of photosynthetic parameters. Prior

to photosynthetic measurement, plants were

held overnight in dark conditions and at a contro-

lled temperature. Photosynthesis vs irradiance (P

vs E) curves of C. cylindracea assimilatory fronds

were generated by following the incubation me-

thods described by Walker (1985) and Cayabyab

and Enríquez (2007). Photosynthetic and respi-

ration rates were obtained by measuring oxygen

flux evolution with a Clark-type O2 electrode ins-

talled in a DW3 chamber (Hansatech, UK) con-

nected to a controlled temperature circulating

bath. The incubation medium was filtered seawa-

ter at a temperature equal to that measured in

the field during plant collection: 23ºC for stations

S and I and 18ºC for station D. Dark respiration

rate was calculated after an initial incubation in-

terval of 15 min in darkness (initial Rd), with net

oxygen production (P) subsequently determined

for 13 light levels between of 14 and 1500 μmol

quanta m-2 s-1 provided by a LS2 tungsten-halo-

gen light source (Hansatech, UK). After exposure

to the final level of light intensity, the frond was

returned to darkness and the final dark respira-

tion rate determined (final Rd). No significant

differences were found between initial and final

respiration rates in all incubations performed.

Four C. cylindracea frond replicates of ca. 2 cm

length were incubated for each sampling station.

Prior to incubations, NaCO3 was added to the in-

cubation chamber up to a concentration of 5 mM

to prevent carbon limitation and N2 bubbled into

water to maintain oxygen concentrations within

a saturation range of 20-80%.

Rates of oxygen flux were normalized to biomass

(fresh weight, fw) and plotted against E values to

construct the P vs E curve, from which the pho-

tosynthetic parameters were then derived. The

maximum rate of net photosynthesis (net-Pmax

)

was estimated by averaging the maximum P

values obtained at saturating irradiances, with

gross photosynthesis (gross-Pmax

) then obtained

by adding Rd to net-P

max. Photosynthetic efficien-

cy (α) was estimated from the slope of the regres-

sion line fitted to the initial linear part of the cur-

ve. The point of compensation irradiance (Ec) was

calculated as the intercept on the irradiance axis

and the saturation irradiance (Ek) as the quotient

between and net-Pmax

. Mean values of c and Ek

obtained at each sampling station were then em-

ployed to calculate the light-compensation (Hc)

and light-saturation (Hk) periods, respectively, for

each daily light curve obtained from continuous

light measurement at the sea floor.

Daily metabolic carbon balance.

For the calculation of sampling station carbon

balance, the net photosynthetic rate (P) was de-

rived for each irradiance value of the entire light

time series obtained for that station using the fo-

llowing Michaelis-Menten function fitted to each

P vs E data set:

P = [gross-Pmax

·E/(E+Ek)] + R

d (Baly, 1935)

where gross-Pmax

, Ek and R

d are mean maximum

gross photosynthesis, saturation irradiance and

dark respiration rate obtained from the P vs E cur-

ves, respectively. This model is the best of a num-

ber of functions that have been previously found

to fit well with P-E plots obtained for Caulerpa

species (Gatusso and Jaubert 1985, Chisholm

and Jaubert 1997). The accuracy of this appli-

cation was evaluated by non-linear least-squa-

res regression at 95% probability (Sigma-Plot,

Jandel Scientific). Daily net photosynthesis was

obtained by numerical integration of calculated

P values over 24 h periods, before being trans-

formed into equivalent carbon units (daily net C

gain, mg C g-1 fw d-1) using the ratio g C:g O2 = 0.3,

assuming a photosynthetic quotient of 1.0 (Ma-

tta and Chapman 1991, Rosenberg et al. 1995).

If photoacclimation does occur, the photoac-

climation efficiency can be estimated as the

proportion of daily net photosynthesis due to

changes in photosynthetic parameters (Ruiz and

Romero 2001, Cayabyab and Enríquez, 2007).

For this purpose, we considered that if photoac-

climation does not occur at the deeper stations

(I and D), then the P vs E curve obtained from

these stations must be equal to that obtained for

plants from the shallower station, S. Therefore,

by integration of the P vs E curve from station S

with light data from stations I and D, we can si-

mulate the daily net photosynthesis that would

be expected for plants from these deeper sites if

photoacclimation did not occur. The photoaccli-

mation efficiency can be then calculated as the

relative percent difference between the simula-

ted values and real values, which were previously

obtained using the respective P vs E curves.

Data analysis

A one-way ANOVA (Quinn and Keough 2002) was

used to assess the significance of differences in

irradiance, algal biomass and photosynthetic pa-

rameters between sampling depths. Prior to this,

data were transformed when the assumption of

homoscedasticity was not fulfilled. A post-hoc

multiple pairwise comparison of means - the

Student-Newman-Keuls test (SNK; Quinn and

Keough 2002) - was used when ANOVA revealed

significant differences between depths. All signi-

ficant effects were tested at a probability level of

p = 0.05.

Results

The irradiance measurements of the studied

stations present a well-defined gradient related

to depth (Fig. 2, Table 1). Relative to their mean

value obtained at station S, both the noon ins-

tantaneous irradiance and the integral of daily

irradiance decreased significantly at the other

two stations: by 20 and 19%, respectively, at sta-

tion I and by 42 and 36% at station D (1-way

ANOVA, p<0.05; Table 1). Both the mean noon

sub-surface irradiance (Eo) and the water-column

attenuation coefficient (Kd) showed very similar

values between sampling stations, with no signi-

ficant differences observed (Table 1). Mean noon

instantaneous irradiance at the sea floor repre-

sented 33.6, 26.2 and 21.6% of Eo for stations S,

I and D, respectively.

Total algal biomass showed significant differen-

ces between sampling depths (F=8.3, P<0.05; Fig.

3). The highest mean value was obtained at the

shallowest depth (station S; 62.6 ± 17.4 g dw m-2),

decreasing by 20% at station I (49.7 ± 8.5 g dw

m-2) and by 73% at the deepest station (D, 16.9

± 2.9 g dw m-2).

The P vs E curves of C. cylindracea fronds ob-

tained at each station are presented in Figure

4, with mean values of the photosynthetic pa-

rameters summarized in Table 2. All photosyn-

p. 38 p. 39

CHAPTER 2

TESIS DOCTORAL

thetic parameters showed significant variation

between sampling depths. Mean net and gross-

Pmax

and Ek showed a substantial and significant

decrease with depth, although no significant

differences were found between mean values

of these parameters at stations I and D (SNK,

p>0.05), which were significantly lower than tho-

se obtained at the shallowest station (S) (Table

2). These differences were greater for the deepest

station (D) (38% for net-Pmax

, 37% for gross-Pmax

and 57% for Ek) than for station I (28, 25 and

38%, respectively). A similar general pattern was

found for Rd and E

c, but here the significant di-

fferences were caused only by the low mean va-

lues of these parameters obtained at the deepest

station (D), which were both ca. 33% lower than

those obtained at stations S and I. This variation

in mean photosynthetic and respiration rates

resulted in the gross-Pmax

:Rd ratios measured at

stations I (6.25) and D (7.39) being very close to

that obtained at station S (7.78). Photosynthe-

tic efficiency (α) was significantly higher at the

deepest station (D) than at the shallower two.

The average daily periods of light compensation

(Hc) and saturation (H

k) obtained by intercepting

the mean Ec and E

k derived from the P vs E curves

(Table 2) with the daily light curves (Fig. 2) are

also shown in Table 2. While mean Hc was rather

similar at each sampling depth, mean Hk showed

a slight but significant increase with depth, with

the absolute difference between stations S and

D being almost 2 hours. The daily metabolic car-

bon balance was highly positive at all sampling

depths, although the mean net C gain was 26%

and 16% lower at stations I and D, respectively,

relative to the mean values calculated for the

shallowest station S (Table 2). When considering

a scenario of no photoacclimation (Table 2), the

mean Hc values of stations I and D were very si-

milar to those obtained via their respective P-E

models. However mean Hk values recalculated at

these stations were ca. 27 and 52% lower, res-

pectively. The mean daily net C gain calculated

for station I under the assumption of no photoac-

climation was similar to that obtained using the

P-E model for this station, but was ca. 29% lower

at station D.

Discussion

Photosynthetic responses observed along the

studied depth gradient essentially consisted of:

a) an increase in photosynthetic efficiency at

sub-saturating irradiances (α); b) a decrease in

the maximum photosynthetic rates at saturating

irradiances (gross- and net-Pmax

); and c) a reduc-

tion of the respiratory demand (Rd). Clearly, the

simplicity of the sampling design employed in

this study (i.e. a comparison between the three,

single sampling sites) precludes interpretation

of the reported photosynthetic behaviour of C.

cylindracea across sampling depths exclusively

in terms of photoacclimation. In fact, despite the

clear pattern of light reduction (Fig. 2 and Table

1), other local factors not controlled by the ex-

perimental design can also vary with depth and

influence photosynthetic performance. However,

the photosynthetic responses observed in this

study are in good agreement with general meta-

bolic strategies used by sublittoral macroalgae to

optimise light use and productivity under varia-

ble light conditions (Markager and Sand-Jensen

1992, Kirk 1994, Pérez-Lloréns et al. 1996, Gómez

et al. 1997, Lobban and Harrison 1997, Gómez

2001). Furthermore, our results are highly consis-

tent with differences in photosynthetic behaviour

reported by other studied Mediterranean popula-

tions of C. cylindracea between shallow and deep

habitats (Raniello et al. 2006). Importantly, these

authors also interpreted there results as due to

photoacclimation.

The pattern of variation of photosynthetic para-

meters was not uniform between sampling dep-

ths. The decline in photosynthetic rates (net and

gross Pmax

) and Ek represents the most significant

photosynthetic response of fronds at the interme-

diate depth (I, 18 m). No further decline in pho-

tosynthetic rate was observed at the deepest sta-

tion (D, 26 m), but the lowest light requirements

(Ek and E

c mean values) were recorded here. This

is likely due to the increase in photosynthetic effi-

ciency (α) and decrease in respiration activity Rd,

both considered clear adaptations for the growth

and survival of macroalgae under limiting light

conditions (Markager and Sand Jensen 1992, Gó-

mez 2001).

Table 1.

Summary of irradiance measurements at each sampling station, determined from continuous light measu-

rement at the sea floor during a 21 day period (i.e. daily light curves; Fig. 2) and instantaneous irradiance

just below the surface measured at noon on standard sunny days (n = 5). Data are presented as means ±

standard error. MS = means squares, F = F statistic, *p<0.05, **p<0.01, n.s. = not significant.

Measurement:Daily light curves at sea floor:Noon instantaneous irradiance(μmol quanta m-2 s-1)Integrated daily irradiance(mol quanta m-2 d-1)Sub-surface irradiance:Noon instantaneous irradianceE

o (μmol quanta m-2 s-1)

Water-column attenuation coefficientK

d (m-1)

% Eo

Sampling station One way ANOVA

S (- 11 m)

442 ± 28

11.74 ± 0.56

1710 ± 78

0.098 ± 0.01

36.68

I (-18 m)

354 ± 21

9.47 ± 0.30

1677 ± 224

0.082 ± 0.01

26.24

D (-26 m)

254 ± 17

7.52 ± 0.24

1659 ± 219

0.095 ± 0.01

21.6

MS

40686

0.047

42432

0.000007

-

F

146.86**

19.56**

0.44 n.s.

0.25 n.s.

-

Fig. 2.

Daily variation in sea floor irradiance at

each sampling station. Data are presen-

ted as the average of 21 d (see methods

section). These curves were used to esti-

mate the daily period of light saturation

(Hk) and compensation (H

c) from satura-

tion (Ek) and compensation (E

c) irradian-

ces obtained in the P vs E curves (see

Table 2).

p. 40 p. 41

CHAPTER 2

TESIS DOCTORAL

Higher photosynthetic efficiencies (α) is a com-

mon feature of macroalgae to reduce light

requirements for photosynthesis in low light

environments and is usually achieved through

adjustment of light-harvesting pigments; i.e. the

size of the absorption cross section (Gómez et al.

1997, Lobban and Harrison 1997, Falkowski and

Raven 2007). Accordingly, high ratios of both

chlorophyll b and siphonoxantin to chlorophyll

a have been reported for a number of Caulerpa

species living in deep water environments (Yoko-

hama and Misonou 1980, Riechert and Dawes

1986, Williams and Dennison 1990, Raniello et

al. 2006), although no changes in α and pigment

contents was found for the other Mediterranean

invader C. taxifolia across a depth range simi-

lar to that of this study (Chisholm and Jaubert

1997). Studying a Mediterranean C. cylindracea

population, Raniello et al. (2006) found increased

concentrations of these accessory pigments with

depth parallel to an increment in α and a decline

in Ek derived from ETR curves, which is consistent

with the photosynthetic behaviour of the alga

reported in this study for the deepest station (D)

using P vs E curves. All these evidence suggest

that the plasticity of α can be an important pho-

toacclimatory mechanism of C. cylindracea to

cope with light reduction with depth.

A low respiration rate is also considered a com-

mon strategy to minimize light requirements and

carbon losses in macroalgal species growing in

deep water habitats (Littler et al. 1986, Marka-

ger and Sand-Jensen 1992, Gómez et al. 1997,

Johanson and Snoeijs 2002). In our study, the

reduced dark respiration observed at the deepest

station (D) enabled net-Pmax

and the P:Rd ratio

to be maintained at levels similar to those ob-

served at the shallower intermediate station (I).

This reduction in dark respiration has not been

previously documented in Mediterranean popu-

lations of C. cylindracea although it is consistent

with that observed in a tropical population of C.

racemosa across a similar depth gradient (Rie-

chert and Dawes 19867). The lower ambient

temperature of the deepest station (D; 18ºC) in

relation to the shallower intermediate station (I;

21ºC) could also have contributed to the decrea-

se in respiratory activity (e.g. Terrados and Ros

1992). However, previous experimental evidence

for this (Flagella et al. 2008) and other species

of this genus (Gattuso and Jaubert 1985) indi-

cates a low sensitivity of Rd to this factor within

this narrow temperature range. Similarly, plants

of the congeneric C. taxifolia collected in summer

showed very constant dark respiration rates when

incubated across the temperature range between

Table 2.

Photosynthetic parameters derived from P vs E curves and daily net carbon gain calculated for each sampling station. Data are presented

as mean ± standard error. Different letters indicate groups of homogeneous means obtained in the post-hoc test SNK (p<0.05). MS = means

squares, F = F statistic, *p<0.05, **p<0.01. Mean values of Hk, H

c and daily C gains calculated under the assumption of no photoacclimation are

indicated in the lower part of the table, as well as the photoacclimation efficiency (see the Materials and Methods section).

Variablenet-P

max

(μmol O2 g fw-1 h-1)

Rd

(μmol O2 g fw-1 h-1)

gross-Pmax

(μmol O

2 g fw-1 h-1)

α(μmol O

2 g fw-1 h-1/μmol quanta m-2 s-1)

Ek

(μmol quanta m-2 s-1)E

c

(μmol quanta m-2 s-1)H

k

(h)H

c

(h)Daily net C gain(mg C g FW-1 d-1)

Hk

(h)H

c

(h)Daily net C gain(mg C g FW-1 d-1)

Sampling station (depth) One way ANOVA

S (11 m)

12.00 ± 0.8 a

1.76 ± 0.15 a

13.77±0.92 a

0.05 ± 0.01a

229.50 ± 17.9 a

22.60 ± 3.4 a,b

6.4 ± 0.4 a

11.9 ± 0.1a

0.45 ± 0.03 a

I (18 m)

8.60 ± 0,1b

1.64 ± 0.04 a

10.25 ± 0.16 b

0.05 ± 0.01a

141.70 ± 35.0 b

37.30. ± 8.8 a

7.8 ± 0.2 b

10.8 ± 0.1 b

0.33 ± 0.01 b

I (18 m)

5.7 ± 0.3

11.5 ± 0.05

0.36 ± 0.02

I (18 m)

-26.9%

-3.6

+9%

D (26 m)

7.50 ± 0.4 b

1.17 ± 0.41 b

8.65 ± 0.50 b

0.08 ± 0.01 b

98.90 ± 4.8 b

15.20 ± 7.0 b

8.3 ±0.2 b

11.9 ± 0.1 a

0.38 ± 0.01 b

D (26 m)

4.0 ± 0.3

11.5 ± 0.01

0.27 ± 0.01

D (26 m)

-52%

-3.6

- 28.9%

MS

0.04

0.01

24.35

0.004

0.48

0.25

20.50

8.70

0.07

F

27.0**

5.80*

13.72**

7.20*

12.7**

5.00*

12.4**

69.8**

9.80**

simulation of no photoacclimation: photoacclimation efficiency:

Fig. 3.

Total biomass (g DW m-2) of C. cylindra-cea at each sampling station. Data are

presented as means and standard errors

(n = 6). Different letters indicate groups

of homogeneous means obtained in the

post-hoc test SNK (p<0.05).

Fig. 4.

Photosynthesis vs Irradiance (P vs E)

curves determined from C. cylindracea fronds at sampling stations S (full circles),

I (full squares) and D (empty triangles).

Points are means ± SE (n = 4). The solid

line represents the curve model fitted to

experimental data (see methods section).

p. 42 p. 43

CHAPTER 2

TESIS DOCTORAL

15 and 25ºC (Gacia et al. 1996b). Thus, reduction

in metabolic demands could effectively reflect a

strategy of the alga to maximize carbon gains

under more limiting light conditions.

The photosynthetic responses reported in this

study support the hypothesis that photosynthe-

tic plasticity can be an important mechanism

accounting for the success of C. cylindracea

across the depth gradients found in Mediterra-

nean coastal waters (Raniello et al. 2006), and

that this can be achieved through optimisation

of carbon fixation as light becomes more limiting

(Markager and Sand Jensen, 1992, Markager and

Sand-Jensen 1994, Gómez 2001). Carbon balan-

ce calculations support this hypothesis for the

studied C. cylindracea populations. In the inter-

mediate station I, reductions in incident light and

daily net productivity were similar in magnitude

(28% and 26%, respectively, relative to station

S). However, at the deeper station D, where there

is a more severe light reduction (41%), the daily

net C gain was only 15% lower than that esti-

mated for the shallower station S. This result can

only be explained by reduced light requirements

and respiratory losses (and higher photosynthe-

tic efficiency) in C. cylindracea fronds from the

deepest population, which yielded mean P:R ra-

tios and Hk and H

c periods very close to (or even

higher than) those obtained for the shallow sta-

tion S. In the absence of photoacclimation, Hk va-

lues should be considerably shorter (4 h) and me-

tabolic carbon balance ca. 29% lower than the

values actually measured in fronds from station

D. Therefore, the reported photosynthetic res-

ponses effectively reverted during optimisation

of C. cylindracea productivity in the deepest site.

In conclusion, our results provide evidence su-

pporting the capacity of C. cylindracea to cope

with changes in the light regime caused by depth

variations through photosynthetic adjustments.

Furthermore, this coping mechanism enables

deep-water C. cylindracea populations to achieve

positive carbon balances close to those quantified

for shallower populations: at least during a period

of the year when growth conditions are optimal

(i.e. the end summer, see for example Ruitton et

al. 2005b). However, generalization of this con-

clusion must be made with caution until confir-

mation by similar studies at broader spatial and

temporal scales. Other questions arise from our

results that should be addressed by further future

research. For example, the lower respiration rates

observed for C. cylindracea fronds from the dee-

pest station (D) suggest reduced growth rates at

this depth (Gómez 2001), which would allow for

carbon storage to support algal growth and bio-

mass under situations of severe light limitation in

these deeper waters, such as during the winter.

Although evidence for this metabolic adaptation

has been reported in Caulerpa species (Robledo

and Freile-Pelegrín, 2005), and has been sugges-

ted for macroalgae species which display nega-

tive carbon balances in winter (see Dunton and

Shell 1986, Gómez 2001), this metabolic adap-

tation has not yet been demonstrated for Medi-

terranean C. cylindracea populations. Moreover,

the existence of such adaptive mechanisms could

limit the capacity of this species to accumulate

biomass in deep assemblages (as suggested by

the lower algal abundance measured in station

D). In this sense, much more research is neces-

sary to determine which factor(s) are responsible

for uncoupling C. cylindracea net productivity

and biomass. In particular, attention must be

paid to the multiple environmental (abiotic and

biotic) factors that can influence vertical patterns

of algal abundance (Piazzi et al. 2001a, Ruitton

et al. 2005a,Bulleri and Benedetti-Cecchi 2008,

Cebrian and Ballesteros 2009, Klein and Verlaque

2008, 2009, Tomas et al. 2011), as well as the an-

nual carbon balance of the algae.

CHAPTER 3Resistance of Posidonia oceanica

seagrass meadows to the spread of the

introduced green alga Caulerpa cylin-

dracea: assessment of the role of light

p. 47

Resistance of Posidonia oceanica seagrass meadows to the spread of the introduced green alga Caulerpa cylin-dracea: assessment of the role of light.

Abstract

Posidonia oceanica seagrass meadows are one

of most resistant Mediterranean habitats to in-

vasion by the green alga Caulerpa cylindracea.

We evaluated the hypothesis that light reduction

caused by the seagrass canopy can limit algal

photosynthesis and growth and hence potentia-

lly explain this resistance. To this end, we analy-

sed light regimes and C. cylindracea biomass and

photoacclimative variables measured outside

and within P. oceanica meadows at different si-

tes and during contrasting times. The success of

photoacclimatory responses was assessed using

an ecophysiological, carbon balance approach.

C. cylindracea abundance significantly varied

depending on the sampling site and time, but its

biomass was always 10- to 50-fold higher out-

side the meadow. Outside the canopy, C. cylin-

dracea showed characteristic morphological and

photosynthetic plasticity closely related to the

spatio-temporal variation in light regimes, which

varied as expected with depth and season. Under

these conditions, the alga was able to perform

successful photoacclimation, although some

degree of light limitation was observed at the

deepest sites and in winter conditions, as indica-

ted by near-zero carbon balance and lower algal

abundances. Within the P. oceanica canopy, light

was reduced by 60–89% relative to that outside

and was at its lowest levels recorded (1–7% of

the sub-surface irradiance), close to the minimum

light requirements for growth. Light limitation

was evident inside the canopy in the winter sam-

pling, when the photosynthetic plasticity of the

alga appears to be exceeded and when carbon

balances were clearly negative. Therefore, light

appears to play a key role in the apparent inca-

pacity of C. cylindracea to penetrate within P.

oceanica meadow edges.

Introduction

The green alga Caulerpa cylindracea (Sonder)

(hereinafter, C. cylindracea), described as one

of the most successful invaders of the Medite-

rranean Sea (Streftaris and Zenetos 2006) has

successfully colonized a wide variety of soft and

hard substrata, including dead Posidonia oceani-

ca rhizomes or ‘‘matte’’ (i.e. the compact bioge-

nic structure resulting from growth of rhizomes

intertwined with roots and autochthonous and

allochthonous detritus; Boudouresque and Meis-

nez, 1982). It is found at depths between 0 to

60 m, being most abundant between 0 and 30

m (Klein and Verlaque 2008). Overgrowth by C.

cylindracea on Mediterranean benthic commu-

nities can alter biodiversity (Argyrou et al. 1999,

Piazzi et al. 2001b, Balata et al. 2004, Piazzi and

Publicado en:Marín-Guirao L, Bernardeau-Esteller J, Ruiz JM, Sandoval-Gil JM (2015) Resistance of Posidonia oceani-

ca seagrass meadows to the spread of the introduced green alga Caulerpa cylindracea: assessment of

the role of light. Biol Inv 17 (7): 1989-2009.

p. 48 p. 49

CHAPTER 3

TESIS DOCTORAL

Balatta 2008, Vázquez-Luis et al. 2008, Klein and

Verlaque 2009), but the degree and extent of this

impact depends on many factors, including the

type of assemblage (Lonsdale 1999, Arenas et al.

2006).

At present, little is known about the variation in

the resistance of natural Mediterranean commu-

nities to C. cylindracea invasion, but it has been

proposed that benthic assemblages dominated

by canopy-forming species are more resistant to

invasion since the canopy might limit resources,

especially light and space (Piazzi et al. 2001a,

Ceccherelli et al. 2002, Klein and Verlaque 2008,

Bulleri and Benedetti 2008, Bulleri et al. 2010).

However, the mechanisms underlying the resis-

tance to invasion of benthic assemblages have

rarely been investigated and are poorly unders-

tood (Lonsdale 1999, Arenas et al. 2006, Brit-

ton-Simmons 2006). Meadows of P. oceanica are

one of the Mediterranean infralittoral biocenoses

that is more resistant to invasion by C. cylindra-

cea (Klein and Verlaque 2008). The invasive alga

is not usually found within P. oceanica meadows,

whereas it has often been found at the edges of

meadows or in very sparse or patchy meadows

(Occhipinti-Ambrogi and Savini 2003, Piazzi et

al. 1997a, b, Piazzi and Cinelli 1999, Ceccherelli

et al. 2000, Montefalcone et al. 2007, Katsane-

vakis et al. 2010, Infantes et al 2011, Ruiz et al.

2011). This resistance to the invasion of the alga

has been related to P. oceanica shoot density,

suggesting that some factors correlated with the

canopy structure must be involved in the reduced

capacity of C. cylindracea to penetrate the mea-

dows, such as space limitation, water motion, nu-

trient supply or canopy shading (Ceccherelli et al.

2000). In the present study we examine the role

that light may play in determining the resilience

of P. oceanica to this highly invasive alga.

P. oceanica is an ecosystem engineer (Koch

2001) that forms conspicuous and extensive

meadows from near the surface, to depths of

30–40 m and its ecological importance is widely

recognised (e.g. Pergent et al. 2012). P. oceani-

ca is a clonal plant consisting of a basal rooted

rhizome, with shoots of vertical and horizontal

growth, bearing 5 to 10 blade-like leaves, 12 mm

broad and more than 1 m long. This large shoot

size, together with the high shoot densities of P.

oceanica meadows (400–1,000 shoots m-2; Ba-

lestri et al. 2003, Procaccini et al. 2003), creates

a highly complex canopy structure. In fact, the

mean leaf area index (LAI) of P. oceanica mea-

dows can reach values as high as 13 m2.m-2 (e.g.

Romero 1985, Balestri et al. 2003), which is com-

parable with the maximum values measured in

terrestrial forest canopies (Scurlock et al. 2001).

These LAI values are also very high when com-

pared with those obtained for other seagrass

species of similar architecture (e.g. Thalassia tes-

tudinum, LAI = 0.65–4.34 m2 m-2; Enríquez and

Pantoja-Reyes 2005). These seagrass meadows

thus strongly modify the environmental condi-

tions within their leaf canopies, particularly the

light climate (Enríquez et al. 1992, Dalla Via et

al. 1998, Zimmerman 2006). As previously shown

in other canopy-forming plant communities, such

as terrestrial (Canhan et al. 1990) and kelp (Clark

et al. 2004) forests, modification of the light envi-

ronment has been shown to be involved in the de-

termination of the structure of the understorey.

Accordingly, most algal species that inhabit the

basal part of P. oceanica meadows are sciaphi-

lic, and a large number of them are undeveloped

and do not grow beyond juvenile stages (Templa-

do et al. 2004).

Previous studies have demonstrated the high

photosynthetic plasticity of Mediterranean po-

pulations of C. cylindracea, which could allow

the alga to acclimate to reduced light conditions

(Bernardeau-Esteller et al. 2011, Raniello et al.

2004, 2006). C. cylindracea has been shown to

be able to colonise and photoacclimate the ba-

sal substratum of C. nodosa meadows, another

common Mediterranean seagrass, the canopy of

which is less complex than that of P. oceanica re-

ducing any shading effect (Raniello et al. 2004).

However, the capacity of the alga to photoaccli-

mate to the more severe light reductions created

by P. oceanica leaf canopies has not yet been in-

vestigated. Furthermore, the extent to which the

photoacclimatory responses elicited by the alga

effectively compensate for imbalances of the

metabolic carbon budget, which ultimately de-

termines the availability of resources for survival

and growth under reduced-light conditions (Mal-

ta and Chapman 1991), has also been neglected.

The general aim of the present study was to con-

tribute to the understanding of the mechanisms

underlying the resistance by native P. oceanica

meadows to the spread of C. cylindracea. We

specifically examined the hypothesis that the

light regime within P. oceanica leaf canopies

might limit C. cylindracea growth and survival

under canopies of this seagrass species. To this

end, we performed a comparative analysis of li-

ght regimes and C. cylindracea variables related

to its abundance (total biomass) and photoac-

climative capacities (frond length, pigments and

photosynthetic parameters), characterised outsi-

de and within P. oceanica meadows of different

highly-invaded sites of the southeastern coast of

Spain (Ruiz et al. 2011). We used an ecophysio-

logical, carbon balance approach by integrating

daily light curves and photosynthesis-irradiance

(P-E) models to obtain the average daily net pro-

ductivity of the alga (Bernardeau et al 2011).


Experimental design

The present study was conducted in 2009 on the

Mediterranean coast of Murcia (SE Spain), where

the invasive alga C. cylindracea was observed for

the first time in 2005 (Ruiz et al. 2011). Canopy

properties, light regimes and algal abundance

were sampled within and outside P. oceanica

leaf canopies of highly-invaded areas identified

in this region (Ruiz et al. 2011; Bernardeau et al

2011). Within this area, sampling was done at

different sites and times to assess a variety of en-

vironmental situations and encompass as much

as possible the spatio-temporal variability of this

habitat. The three sites, Isla Grosa (IG; 37º43’N,

00º42’E), Cabo Tiñoso (CT; 37º32’N, 00º44’E)

and Calblanque (CB;37º32’N, 01º07’E), had con-

trasting depths (11, 18 and 26 m, respectively),

with a range that encompassed most of the verti-

cal distribution of P. oceanica and C. cylindracea

in this region. The three sites were in an area with

similar climate and oceanography (Vargas-Yáñez

et al. 2010) and substratum type (i.e. detritic soft

bottoms mixed with dead P. oceanica “matte”;

Calvín-Calvo et al. 1998, Ruiz et al. 2011). At all

sites, the substratum outside the P. oceanica

meadow was almost totally covered by dense

C. cylindracea stands, from which some stolons

penetrated inside the seagrass leaf canopy, al-

though only up to the first 25–40 cm from the

meadow edge. As for the outside, within the P.

oceanica canopy, the stolons of the alga coloni-

sed both sediments and basal seagrass rhizomes

(dead and alive). There was no apparent discon-

tinuity in the nature of the substratum or any

other environmental feature that could be rela-

ted to the position of the transition between the

meadow and the algal stand. To assess temporal

variation, sampling of all three sites was done in

both January and July, times that represented

the two extremes of the seasonal environmental

variation: winter (T1) and summer (T2). In order

to avoid temporal resampling of the sites, the

sampling of the selected variables at each time

p. 50 p. 51

CHAPTER 3

TESIS DOCTORAL

and site was done in a randomly-selected mea-

dow area 50 meters in length and 10 meters in

width (i.e. a sampling area of 500 m2), with the

long axis of the rectangle centered on the sea-

grass meadow edge.

For each sampling site and time, algal variables

and light regimes were determined at two posi-

tions relative to the edge of the seagrass mea-

dow: i) an outer position (OUT), on the adjacent

densely-invaded detritic sediments within 1-2 m

from the meadow edge and ii) at an inner posi-

tion (IN), 25–40 cm from the meadow edge. In

addition, general descriptive data of the P. ocea-

nica meadow structure were also collected.

Caulerpa cylindracea biomass, frond height and

pigments

Samples of C. cylindracea were gathered in five

randomly-selected sampling locations separated

by 10 m at each site, time and position. In each

sampling location, fronds, stolons and rhizoids of

C. cylindracea were carefully collected by hand

within three 400 cm2 square frames randomly

distributed in the area (Ruitton et al. 2005b).

Samples were transported to the laboratory in

plastic bags together with seawater, in chilled

containers. After removing the sediment, debris

and other algal species, total C. cylindracea bio-

mass (g DW m-2) was determined by drying the

samples at 70°C to constant weight. The three

biomass values determined in each sampling lo-

cation were then averaged to obtain five replica-

tes (n = 5) for each site, time and position combi-

nation. The frond height (cm) was determined by

measuring the height of 10 algal fronds rando-

mly selected from each sample. Measurements

were averaged per sample and then per sampling

location to constitute one of the five replicates (n

= 5) of each site, time and position combination.

The pigment content was analysed in 2 rando-

mly-selected healthy and non-epiphytised C.

cylindracea fronds of approximately 3 cm in hei-

ght from each C. cylindracea sample. Results from

the analyses were averaged per sample and sam-

pling location so the final number of replicates

was five (n = 5) for each site, time and position.

The analysis was conducted spectrophotometri-

cally after manual extraction of a homogenised

suspension using 90% acetone (Dennison 1990),

with MgCO3 added as a chlorophyll stabiliser. The

acetone extracts (10 ml) were stored at 4°C in the

dark for 24 h and centrifuged. The chlorophyll a

and b content was computed using the equations

of Lichtenthaler & Wellburn (1983).

Posidonia oceanica meadow and leaf canopy

structure

To characterise the structure of the three selected

meadows, the shoot density and the percentage

of meadow cover were measured at all sampling

sites and times, following standard methods adop-

ted for this seagrass species (Ruiz et al. 2010 a,b).

Shoot density (shoots m-2) was estimated in six

randomly-selected locations in each meadow by

counting the number of shoots within two 400-

cm2 quadrats randomly placed within each loca-

tion. The average of each pair of measurements

was the individual, independent replicate (n = 6).

The percentage of meadow cover was visually es-

timated as the percentage of the bottom covered

by seagrass patches within 1,600-cm2 square fra-

mes subdivided into four 20×20 cm squares. Visual

estimations were performed every meter along

three (n = 3), 10-m linear transects randomly selec-

ted within the meadow at each visit.

The leaf area index (LAI) and the canopy height

were used to characterise the leaf canopy. In

each site and time, 5 shoots were collected in

four randomly-selected locations. The total leaf

surface area (based on one side) was calculated

for each shoot by measuring the length and wid-

th of all leaves per shoot and averaged for each

sampling location. LAI (m2 m-2), as a descriptor

of the degree of leaf packing within the canopy

(Enriquez and Pantoja-Reyes 2005), was then cal-

culated by multiplying the total leaf area of each

location by the averaged shoot density of each

meadow, so the total number of replicates was

four (n = 4) in each site and time. The canopy

height (cm) was estimated in situ by divers, using

a ruler and taking two measurements at six di-

fferent locations along the meadow edges. The

average value of the two measurements in each

location constituted each one of the replicates

per site and time (n = 6).

Irradiance measurements

The light field at each site, time and position was

characterised 5 cm above the bottom using sphe-

rical quantum sensors (Alec MDS MK5). Sensors

were programmed to record irradiance values

every 10 min and recorded data for at least two

weeks in each season. Maximum instantaneous

irradiance at noon (Emax

, μmol quanta m-2 s-1) and

total daily irradiance values (Etotal

, mol quanta m-2

d-1) were obtained from the diurnal irradiance cy-

cles.

Light attenuation coefficients were determined

for both the water column (water-Kd, m-1) and

the meadow canopy (canopy-Kd, m-1) under stan-

dard conditions (i.e. between 12:00 h and 14:00

h on standard sunny days with minimal water

movement and sediment resuspension; Enrí-

quez and Pantoja-Reyes 2005). Water column

down-welling irradiance was measured using a

cosine-corrected quantum sensor (LI-190SA; LI-

COR). Irradiance data were recorded for each m

from the sea subsurface (E0) to the sea bottom

(Ez), integrating the values obtained over 10 s on

three different days in both summer and winter.

Irradiance within the seagrass canopy was me-

asured using spherical quantum sensors (Alec

MDS MK5) at every 5 cm from the base of the

meadow to the top of the canopy using a marked

vertical bar for reference. Data were averaged for

a 10-s period (one measurement s-1) at each hei-

ght. Light attenuation coefficients (water-Kd and

canopy-Kd) were estimated using the Beer-Lam-

bert equation: Ez=E

0e-Kdz; where E

z and E

0 are the

irradiance values at a given depth (z in m) and at

the sea subsurface/top canopy, respectively; and

Kd is the light attenuation coefficient (Kirk 1994).

The percentage of subsurface irradiance that

reached the sea bottom was calculated from E0

(subsurface irradiance) measured at noon on

calm sunny days using the LI-COR quantum sen-

sor and the corresponding sea floor values within

and outside the meadows measured using the

spherical quantum sensors. Both sensor types

were intercalibrated in the laboratory, showing a

strong linear relationship with a constant factor

of 1.02.

C. cylindracea photosynthetic variables

Prior to photosynthetic measurements, C. cylin-

dracea samples were kept overnight in the dark

under controlled temperature in natural seawa-

ter taken from the collection site. Photosynthesis

and dark respiration rates were measured using

a polarographic oxygen electrode and a mag-

netic stirrer (DW3, Hansatech Instruments Ltd)

under controlled temperature (Bernardeau et al.

2011). Incubation was carried out at the same

temperature as that measured in the field during

sampling: 13°C in winter for all three sites; and

24°C at IG and 22°C at CT and CB in summer.

Apical segments of non-epiphytised C. cylindra-

cea fronds of approximately 2 cm in length were

used for the measurements. Two replicated algal

segments from three of the five sampling loca-

tions (see above) were randomly selected and

p. 52 p. 53

CHAPTER 3

TESIS DOCTORAL

employed for the photosynthetic measurements

(n = 3). Dark respiration rates were measured by

maintaining the fronds in the dark for 15 min. Net

oxygen production was then determined at 13 di-

fferent light intensities (from 14 to 2,271 μmol

quanta m-2 s-1) using a high-intensity light sour-

ce (LS2, Hansatech Instruments Ltd). Net pho-

tosynthetic rates were plotted against the light

intensities (P-E curves), and the photosynthetic

parameters were calculated as follows: the maxi-

mum rate of net photosynthesis (net-Pmax

, μmol

O2 g-1 FW h-1) was determined by averaging the

maximum values above the saturating irradian-

ce (Ek). The photosynthetic efficiency (α, μmol O

2

g-1 FW h-1/μmol quanta m-2 s-1) was calculated as

the slope of the regression line fitted to the initial

linear part of the P-E curve, and the compensa-

tion irradiance (Ec) as the intercept on the X-axis.

Ek was calculated as the ratio P

max/α. Mean daily

compensation (Hc) and saturation (H

k) periods

were calculated from each daily light curve as the

number of h per day that irradiance values excee-

ded Ec and E

k mean values, respectively.

Daily metabolic carbon balances

Daily carbon balance, as a predictor of plant li-

ght limitation (Dennison and Alberte 1985), was

calculated according to the Michaelis-Menten

function (P = [gross-Pmax

E/(E+Ek)] + R [Baly 1935])

previously applied to C. cylindracea (Gatusso and

Jaubert 1985, Bernardeau et al. 2011), where P

is net photosynthesis, gross-Pmax

is the maximum

gross photosynthetic rate, E is the irradiance me-

asured in the field, Ek is the saturation irradiance,

and R is the respiration rate. Photosynthetic para-

meters obtained from P-E curves and continuous

recordings of field irradiance measurements were

entered into the function to generate estimates

of net production, which were integrated across

24 h periods to yield daily net production values

(n = 3). If the photosynthetic quotient is assumed

to equal unity, and the ratio g C:g O2 = 0.3 (Matta

and Chapman 1991), then the net productivity

in oxygen units can be multiplied by 0.012 to ob-

tain the equivalent carbon units (mg C g FW-1).

This calculation presumes constant dark respira-

tion throughout the day and does not consider

other carbon losses (exudation, grazing) or gains

(light-independent carbon fixation).

Data analyses

The spatio-temporal variation of P. oceanica

meadow structure descriptors was evaluated

using a two-way ANOVA with sampling sites

(three levels: IG, CT and CB) and times (two levels:

T1, in winter, and T2, in summer) as fixed factors.

For the analysis of C. cylindracea variables a ran-

domized-block design was applied for each sam-

pling time separately, defining sites as blocks and

position (two levels: IN vs OUT) as main factor.

For both designs, prior to carrying out the ANO-

VA, the data were tested for heterogeneity of

variance using Cochran’s C-test and transformed

when necessary. Where variance remained hete-

rogeneous, untransformed data were analysed,

as ANOVA is a robust statistical test and is rela-

tively unaffected by the heterogeneity of varian-

ces, particularly in balanced designs (Underwood

1997). The Student-Newman-Keuls (SNK) test

was used for a posteriori pairwise comparisons of

means. A probability level of 0.05 was regarded

as significant except when data transformation

was not possible. In such cases, the level of signi-

ficance was reduced to P < 0.01 to minimize type

I errors. Randomized block analysis also assumes

that there are no interactions between blocks

and the main factor. To test this assumption plots

of dependent variables versus blocks were exami-

ned (Quinn and Keough 2002). Regression analy-

sis was used in order to describe the relationship

between irradiance and plant variables with the

depth of the site. Univariate statistical analysis

was performed using the statistical package STA-

TISTICA (StatSoft Inc. 2001, version 6.0).

We also employed a multivariate approach to

explore photoacclimatative response patterns

between sites, times and positions, but based on

the integration of the multiple univariate respon-

ses obtained in each case. Principal Components

Analysis (PCA) was carried out on the correlation

matrix of photoaclimatation variables, following

fourth square transformation of the data. This

analysis provides a measure of association be-

tween each original variable and the resulting

principal components. Multivariate analysis was

conducted using the software package CANOCO

version 4.5 for Windows (Ter Braack and Šmilauer

2002).

Results

Light regimes

At both sampling times, mean noon subsurface

irradiance (E0) was similar between sites, but with

higher values in the summer sampling (T2) than

in the winter sampling (T1) (Table 1). The water

column attenuation coefficients (water-Kd) were

also similar between sites and times (two-way

ANOVA, P > 0.05), with mean values ranging from

0.082 to 0.124 m-1.

At T1, the mean total daily irradiance (Etotal

) and

the noon maximum irradiance (Emax

) obtained at

the bottom in the OUT-position varied by an or-

der of magnitude between sites, with mean Emax

values representing 4 to 27% of E0. Irradiance

showed a high and negative exponential relation

with the depth (z) of the sampling site (e.g. Etotal

= 1149.5·e-0.13z , R2 = -0.994), with highest mean

values recorded at the shallowest site IG, and the

lowest at the deepest, CB. At T2, these irradian-

ce mean values were 13–41% of Eo and 2–5 fold

higher than at T1, and also showed a similar, but

linear negative relation with site depth (Etotal

=

939- 27.9·z ; R2 = -0.992).

At both sampling times, Etotal

and Emax

mean va-

lues obtained inside the seagrass canopy (IN-po-

sition) at the three sites were reduced (82–88%

at T1 and 60–89% at T2), relative to those deter-

mined in the OUT-position. At T1, between-site

variation of irradiance mean values in the IN-po-

sition reflected that of external light availability

(i.e. OUT-position mean values), but not in T2

where mean values determined at the IN-posi-

tion showed very small variation between sites

(1.59–2.30 mol quanta m-2 s-1). The sharp light

extinction associated with the seagrass canopy

shelf-shading corresponded to canopy-Kd mean

values that ranged between 5.8 and 8.5 m-1. Sites

showed significant differences between mean ca-

nopy-Kd values (two-way ANOVA, P < 0.001), with

the deepest meadow CB always showing the

lowest values (Table 1).

Seagrass meadow structure

Meadow structure descriptors showed significant

variation between sampling sites and times, ex-

cept in the case of meadow cover, for which diffe-

rences were only significant among sites (Fig. 1,

Table 2). Spatial variation was the major source

of variation of shoot density (54.7%) and mea-

dow cover (97.6%), and temporal variation in the

case of LAI (70.0%) and canopy height (87.2%).

For shoot density, LAI and canopy height, the pa-

ttern of variation among sites differed between

sampling times, as indicated by the significant

effect of the ANOVA interaction term (Table 2).

At both sampling times, spatial variation showed

a high significant negative correlation with the

depth of the sampling sites in the case of the

shoot density (R2 = 0.58 – 0.72, β = -0.76 – -0.85,

P < 0.001, N = 18), meadow cover (R2 = 0.78 –

0.88, β = -0.88 – -0.94, P < 0.01, N = 9) and LAI

(R2 = 0. 53 – 0.93, β = -0.73 – -0.96, P < 0.01, N =

12). In all these cases, maximum values of the

variable were found at the shallowest site and

minimum values at the deeper one.

p. 54 p. 55

CHAPTER 3

TESIS DOCTORAL

Total algal biomass

The position of the alga relative to the meadow

edge showed a significant effect on the total

alga biomass at both times (Fig. 2, Table 3). The

stands of C. cylindracea growing outside the sea-

grass meadow showed up to a 50-fold higher bio-

mass than that of stands growing beneath the

leaf canopy, except at T1 at the shallower IG site

where this variable was similar in OUT and IN-po-

sitions. Algal biomass was also significantly affec-

ted by sites at both times (Table 3) and showed a

strong negative correlation with the depth of the

sampling site (R2 = 0.64, β = -0.80, P < 0.001, N =

15), but only at T2 at the OUT-position.

Caulerpa cylindracea frond variables

In relation to the height of the fronds, the posi-

tion of C. cylindracea stands was the factor that

explained the major component of its total va-

riance in both sampling times (59-66%), and on

average, fronds growing within the meadows (i.e.

IN-position) were almost twice as tall as those

growing in the OUT-position. The blocking factor

‘site’ had also a significant effect on this variable

at both sampling times (Fig. 3, Table 3), which

showed a significant positive correlation with the

depth of the sampling sites outside the leaf ca-

nopy at T2 (R2 = 0. 0.90, β = 0.95, P < 0.001, N

= 15).

Position also had significant effects on the pig-

ment content (Chl a, Chl b and the molar Chl b/a

ratio) of algal fronds in both sampling times, with

higher percentages of total explained variance

in T2 (Fig. 3, Table 3). In general, those fronds

growing within the meadows (i.e. IN-position)

had significantly higher content in chlorophyll

a and b, as well as higher Chl b/a molar ratios,

than those growing in the OUT-position. The

chlorophyll content also significantly differed

among sites at both sampling times and showed

a negative correlation with the depth of the sam-

pling site for the Chl a and b content at T1 at the

OUT-position, and for Chl b and Chl a/b ratio at

T2 at the IN-position due to the significantly hi-

gher mean values observed for these variables at

the shallower sites relative to the deeper ones.

In the T2 sampling time all photosynthetic va-

riables derived from P-E curves were significantly

affected by the position of C. cylindracea stands,

except for maximum photosynthetic rate (Pmax

),

which in addition was the only photosynthetic va-

riable that significantly differed among positions

in T1 (Fig. 4, Table 3). In the summer sampling

(T2), the significant lower respiratory demands

(65% in average) exhibited by C. cylindracea at

the IN-positions significantly increased their pho-

tosynthetic efficiency (i.e. α) and reduced their Ec

and Ek values with respect to fronds growing at

the OUT-positions. The factor ‘site’ also signifi-

cantly affected the photosynthetic parameters

Pmax

, Rd, and α at the winter T1 sampling and only

α at the summer T2 (Fig. 4, Table 3). For Pmax

and

Rd this spatial variability showed a close, negative

and significant correlation with the depth of the

sampling site at T1 in both positions (R2 = 0.54 –

0.82, β = -0.69 – -0.82, P < 0.05, N = 9). Whereas,

between-site differences in photosynthetic effi-

ciency (α) inside the leaf canopy had a negative

and significant correlation with the depth of the

sampling site at both sampling times (R2 = 0.50 –

0.61, β = -0.71 – -0.78, P < 0.05, N = 9).

The mean daily period of photosynthetic com-

pensation (Hc) and the mean daily period of

photosynthetic saturation (Hk) were both signi-

ficantly affected by the factors ‘position’ and

‘site’ in both sampling times; the former factor

explaining in general the higher percentages of

the total variance (Fig. 5, Table 3). Under full illu-

mination conditions (i.e. at the outside positions)

CB (26m)

1084 ± 171506 ± 138

0.106 ± 0.0120.095 ± 0.010

40 ± 6 (3.7%E0)204 ± 15 (13.5%E0)

0.63 ± 0.104.75 ± 0.34

5.79 ± 0.546.37 ± 0.70

7 ± 1 (0.7%E0)82 ± 7 (5.4%E0)

0.11 ± 0.021.59 ± 0.15

Variablea) water column:

noon subsurface irradiance(E

0, μmol quanta m-2 s-1)

water column Kd (m-1)

b) seabed OUT-position:

max. noon bottom irradiance–OUT(E

max, μmol quanta m-2 s-1)

total daily bottom irradiance–OUT(E

total, mol quanta m-2 d-1)

c) seabed IN-position

canopy Kd (m-1)

max. noon bottom irradiance–IN(E

max, μmol quanta m-2 s-1)

total daily bottom irradiance–IN(E

total, mol quanta m-2 d-1)

Site

Time

T1T2

T1T2

T1T2

T1T2

T1T2

T1T2

T1T2

IG (11m)

1059 ±1131496 ± 63

0.122 ± 0.0080.124 ± 0.020

286 ± 17 (27.0%E0)622 ± 51 (41.5%E0)

5.06 ± 0.3115.74 ± 0.92

8.43 ± 0.566.69 ± 0.51

51 ± 4 (4.8%E0)101 ± 9 (6.7%E0)

0.71 ± 0.061.78 ± 0.13

CT (18m)

1017 ± 341305 ± 124

0.082 ± 0.0100.082 ± 0.010

100 ± 13 (9.8%E0)459 ± 13 (35.2%E0)

1.61 ± 0.2310.50 ± 0.30

8.52 ± 0.717.96 ± 0.83

17 ± 3 (1.7%E0)90 ± 16 (6.9%E0)

0.20 ± 0.042.30 ± 0.26

Table 1.

Characteristics of light regimes determined at sampling sites, times and positions from irradiance measurements performed across

vertical profiles of the water column and the Posidonia oceanica seagrass canopy and from continuous light measurements perfor-

med on the seabed (see methods). Data are presented as means ± standard error.

T1 = winter sampling time, T2 = summer sampling time, IN = inside canopy position, OUT = outside canopy position, Kd = light atte-

nuation coefficient, %Eo = proportion of the subsurface irradiance reaching the seabed.

Fig. 1.

Mean and standard error

of Posidonia oceanica

meadow structure varia-

bles obtained for each

combination of sampling

site (IG, CT and CB) and

time (T1 and T2). Di-

fferent letters indicate

groups of homogeneous

means obtained in the

post-hoc SNK test (P <

0.05).

p. 56 p. 57

CHAPTER 3

TESIS DOCTORAL

the highest light levels (i.e. at T2, in the outside

position) are positioned on the right extreme of

the axis (the most positive values), whereas those

exposed to the lowest irradiances (i.e. T1, inside

position) are at the opposite position (the most

negative values). Moreover, the position of the

objects on the PC1 axis showed a high and positi-

ve significant correlation (r = 0.82, P < 0.001) with

the mean total daily irradiance (Etotal

, Table 1; Fig.

6B); the lineal regression model fitted to these

data revealed that this factor explained 68% of

the total variation along the PC1 axis (Fig. 6B).

The vectors depicted in the plot (Fig. 6A), with the

arrow pointing to the higher values of the varia-

ble, indicate that this first axis had strong positive

correlations (scores > 0.8) with Ek, E

c, and R

d, and

negative correlations (scores < -0.8) with the con-

centration of chlorophyll a and b (Table 4). These

strong correlations identified the importance of

this set of responses in the photoacclimative res-

ponse of C. cylindracea to cope with the shady

conditions found within the meadows.

Discussion

Outside the seagrass leaf canopy, between-site

variability of the light regime showed a high ne-

gative correlation with the depth of the site that

was consistent with the characteristic pattern

of light extinction that occurred together with

water column vertical profiles (Kirk, 1994). Sites

had similar mean water-Kd values and were also

very similar in many other climatic, geological

and oceanographic features (Marín-Guirao et al

pers. obs.; Vargas-Yáñez et al. 2010). Therefore,

the possibility that the reported spatial variation

in light regimes was caused by other local factors

apart from depth, is assumed to be very low. Si-

milarly, the pronounced variation in irradiance

between sampling times (about one order of

magnitude), matched typical differences in un-

derwater irradiance between winter and summer

at similar depths and latitudes (e.g. Enríquez et

al. 2004, Raniello et al. 2004, Vargas et al. 2010).

Therefore, it can be considered that the reported

differences in light regime are representative

of the typical spatio-temporal variation of this

factor associated with depth and seasonality,

at least in benthic macroalgal assemblages wi-

thin the depth range and region considered in

this study. The invasive C. cylindracea has been

shown to be able to photoacclimate and persist

across the environmental light gradient asso-

ciated with depth and season in sublittoral Me-

diterranean environments (Raniello et al. 2004,

2006; Bernardeau et al. 2011), which has been

invoked as one of the key mechanisms involved

in its colonisation success in native habitats. In

support of this, many of the significant effects

associated with sampling sites and times ob-

served in the analysed variables of algal stands

Hc ranged between 7 and 13 h and H

k varied be-

tween 1 to 10 h; in contrast, these daily periods

were significantly and consistently shortened by

43% and 72%, respectively, at the inside posi-

tions (Fig. 5). Minimum mean values (1–7 h for Hc

and 0–1.1 h for Hk) were usually found in C. cylin-

dracea fronds of the inside position at the winter

T1 sampling, but also at T2 at the deepest site CB

(Fig.5). Regarding the variation among sites, Hc

and Hk periods were in general shorter at deeper

sites than at shallower ones (Fig. 5).

According to these results, daily metabolic carbon

balances showed significant differences among

positions only for the winter T1 sampling, when

carbon balances within the meadow canopy (i.e.

IN-position) were consistently negative in all sites

(-0.14 – -0.20 mg C g-1 FW d-1) but positive (site

IG: 0.52 mg C g-1 FW d-1) or close to zero (sites CT

and CB: -0.01 mg C g-1 FW d-1) at the OUT-posi-

tion (Fig. 5, Table 3). C. cylindracea carbon balan-

ces were also significantly affected by the factor

‘site’, with the shallowest site IG showing signifi-

cantly higher carbon balances than the deepest

ones CT and CB.

Multivariate analysis

The PCA performed using the selected C. cylin-

dracea photoacclimative variables (frond height,

pigment content and photosynthetic parame-

ters) yielded eigenvalues of 0.759 and 0.132

for the PCI and PC2 axes, respectively (Table 4).

The first PCA axis (PC1) explained 75.9% of the

variance in the original data set. The ordination

of the objects along this axis (Fig. 6A) appears

to relate to the reported differences in light re-

gime, since those cases that were exposed to

Table 2.

Summary of the two-way ANOVA test per-

formed to assess the effect of sampling sites

and times on Posidonia oceanica meadow

structure variables.

df = degrees of freedom, MS = Mean Squares,

%Var. = percentage of explained variance,

F = F-statistics, P = P value; ns = not signifi-

cant, *P < 0.05, **P < 0.01, ***P < 0.001.

Shoot density

Site (S)Time (T)SxTResidual

LAI


Meadow cover


Canopy height


df

2

1

2

30

df

2

1

2

18

df

2

1

2

12

df

2

1

2

30

MS

167066

104275

30087

4085

MS

261.9

835.1

91.4

5.0

MS

621.2

2.2

6.7

6.6

MS

372.6

3700.7

146.3

26.1

%Var.

54.7

34.1

9.8

1.3

%Var.

21.9

70.0

7.7

0.4

%Var.

97.6

0.3

1.1

1.0

%Var.

8.8

87.2

3.4

0.6

F

40.9

25.5

7.4

F

52.2

166.6

18.2

F

93.8

0.3

1.0

F

14.3

141.6

5.6

P

***

***

**

P

***

***

***

P

***

ns

ns

P

***

***

**

Fig. 2.

Mean and standard error of Caulerpa

cylindracea total biomass determined in-

side (IN-position, black bars) and outside

(OUT-position, grey bars) Posidonia ocea-

nica seagrass meadows for each combina-

tion of sampling site (IG, CT and CB) and

time (T1 and T2).

p. 58 p. 59

CHAPTER 3

TESIS DOCTORAL

growing outside the seagrass canopy reflected

photoacclimatory responses previously observed

in response to variability in the light regime.

During the summer sampling (T2), C. cylindracea

stands growing outside the canopy showed in ge-

neral highest Pmax

and Rd rates, which is consistent

with the highest irradiance levels (well above Ec

and Ek mean values) and day-length recorded in

this time. This high light availability for photosyn-

thesis and growth could explain the lack of signi-

ficant differences in most of individual photosyn-

thetic characteristics (P-I curves) between sites,

as it was also reflected in the integrative multiva-

riate analysis (PCA, Fig. 6B), with cases belonging

to T2-OUT occupying a very close position in the

PCI axis despite their differences in light climate

(Etotal

, Fig. 6B). Accordingly, the alga showed high

Hk and carbon balance mean values except in the

deepest site (CB) with the lowest light availabili-

ty and total biomass. This suggest the existence

of some degree of light limitation at this site in

summer, which is supported by the considerable

enhancement of frond height, a typical morpho-

logical adaptation of this and other macroalgal

species to light-limiting conditions (Calvert 1976,

Ohba and Enomoto 1987, Kirk 1994). In addition,

these results were also consistent with those ob-

tained in a previous study performed at the same

sampling sites in a similar sampling time (Bernar-

deau-Esteller et al. 2011), confirming a more limi-

ted capacity of colonization by the alga in these

deepest areas.

Effect Biomass (g FW m-2) Position Site error Frond height (cm) Position Site error Chlorophyll a (mg g-1 FW) Position Site error Chlorophyll b (mg g-1 FW) Position Site error Chlorophyll b/a (molar ratio) Position Site errorPmax (mmol O2 g

-1 FW h-1) Position Site error Rd (mmol O2 g

-1 FW h-1) Position Site error α (mmol O2 g

-1 FW h-1/mmol quanta m-2 s-1) Position Site error Ek (mmol quanta m-2 s-1) Position Site error Ec (mmol quanta m-2 s-1) Position Site error Hc (h) Position Site error Hk (h) Position Site error Carbon balance (mg C g-1 FW d-1) Position Site error

df

12

26 12

26 12

26 12

26 12

26 12

14 12

14 12

14 12

14

12

14 12

14 12

14

12

14

MS

2193.22553.9

98.1

19.1625.3730.108

3183.54994.1664.0

1996.1950.4184.7

0.0090.0010.000

42.2576.625.75

0.653.810.20

0.0020.0240.002

1118.6369.6137.2

21.424.28.4

77.327.20.5

44.719.71.5

0.4960.1540.024

MS

12401.61241.1128.8

56.2557.1590.559

13670.21021.9128.3

9910.501649.28238.153

0.0580.0110.000

31.222.910.6

30.540.021.53

0.0200.0090.002

12068.8

986.1741.6

2345.0

28.752.7

78.253.45.8

112.725.11.6

0.3830.7020.125

%Var

22.351.825.9

58.632.98.6

10.532.856.7

22.921.955.2

56.76.6

36.7

15.355.529.2

5.9

69.125.1

2.0

63.234.8

29.619.650.8

11.425.762.9

55.539.15.47

42.637.719.7

43.627.129.4

%Var

68.013.618.4

66.116.817.1

71.810.717.5

81.210.68.13

69.926.43.7

13.920.465.8

58.70.1

41.2

32.429.338.4

49.48.1

42.5

74.71.8

23.5

29.440.230.4

61.127.211.8

10.839.749.5

F

22.3526.02

177.749.8

4.7957.521

10.8095.147

40.2282.342

7.3413.32

3.2819.29

0.81612.73

8.152.69

2.532.87

141.950.0

30.313.4

20.76.4

F

96.309.64

100.5812.80

106.68.0

259.817.0

490.592.4

2.952.17

19.960.014

11.805.34

16.31.3

44.50.5

13.69.3

72.616.1

3.075.61

P

<0.001<0.001

<0.001<0.001

<0.05<0.01

<0.01<0.05

<0.001n.s.

<0.05<0.001

n.s.<0.001

n.s.<0.001

n.s. (0.012)n.s.

n.s.n.s.

<0.001<0.001

<0.001<0.001

<0.001<0.05

P

<0.001<0.001

<0.001<0.001

<0.001<0.01

<0.001<0.001

<0.001<0.001

n.s.n.s.

<0.001n.s.

<0.01<0.05

<0.01n.s.

<0.001n.s.

<0.01<0.01

<0.001<0.001

n.s.<0.05

SNK-test

IN<OUTIG=CB<CT

IN>OUTIG<CT<CB

IN>OUTIG>CT=CB

IN>OUTIG>CT=CB

IN>OUT - -

IN<OUTIG>CT=CB

- -IG>CT=CB

- -IG>CT=CB

- - - -

- - - -

IN<OUTIG>CT=CB

IN<OUTIG>CT=CB

IN<OUTIG>CT=CB

SNK-test

IN<OUTIG=CT>CB

IN>OUTIG=CT<CB

IN>OUTIG=CT>CB

IN>OUTIG>CT>B

IN>OUTIG>CT=CB

- - - -

IN<OUT - -

IN>OUTIG=CT>CB

IN<OUT - -

IN<OUT - -

IN<OUTIG=CT>CB

IN<OUTIG=CT>CB

- -

IG=CT>CB

T1 (Winter) T1 (Summer)Table 3.

Summary ANOVA test performed to assess the effect of the position (P) and sampling site (S) on all Caulerpa cylindracea variables at

the two studied sampling times (T1 & T2). df = degrees of freedom, MS = Mean Squares, %Var. = percentage of explained variance,

F = F-statistics, P = P value, SNK-test = SNK post-hoc analysis. ns = not significant.

Fig. 3.

Mean and standard

error the height

and pigment con-

tent (chlorophyll a,

b and b/a) of Cau-

lerpa cylindracea

fronds determined

inside (IN-position,

black bars) and out-

side (OUT-position,

grey bars) Posido-

nia oceanica sea-

grass meadows for

each combination

of sampling site

(IG, CT and CB) and

time (T1 and T2).

p. 60 p. 61

CHAPTER 3

TESIS DOCTORAL

Fig. 4.

Mean and standard error of photosynthetic characteristics deri-

ved from P-E curves obtained from Caulerpa cylindracea fronds

inside (IN-position, black bars) and outside (OUT-position, grey

bars) Posidonia oceanica seagrass meadows for each combina-

tion of sampling site (IG, CT and CB) and time (T1 and T2).

Fig. 5.

Mean and standard error of light compensation (Hc) and sa-

turation (Hk) periods and daily carbon balance estimated for

Caulerpa cylindracea fronds inside (IN-position, black bars) and

outside (OUT-position, grey bars) Posidonia oceanica seagrass

meadows for each combination of sampling site (IG, CT and CB)

and time (T1 and T2).

p. 62 p. 63

CHAPTER 3

TESIS DOCTORAL

During the winter sampling (T1), physiological

variables of C. cylindracea growing outside the

seagrass canopy reflected a major photoacclima-

tory effort (relative to T2) according to the more

reduced light availability characteristic of this

season, particularly in the deepest sites. Thus,

photosynthesis and respiration rates, as well as

Ec and E

k, were in general lower than in T2 and

showed significantly lower mean values in the

deepest sites (CT and CB), where frond height

was significantly higher than in the shallower site

IG. All these are characteristic photoacclimatory

responses of marine macrophytes to overcome

light limitation (Falkowski and Raven 2007, Lob-

ban and Harrison 1997, Littler et al. 1986, Lüning

1990) that allows the lengthening of Hc and H

k

periods and counterbalances the metabolic car-

bon budget (Dennison and Alberte 1982, 1985,

Dunton 1986, Gomez et al. 1997). Other res-

ponses were opposite to those expected under a

situation of light limitation, such as the signifi-

cant reduction in photosynthetic efficiency and

pigment content reported in the deepest sites.

However, in this case, the adjustments of the

photosynthetic metabolism (particularly in respi-

ration) probably avoided further reductions in Hc

and Hk and allowed the average carbon balance

to remain close to zero at those sites with greater

depths. Under such situation the alga can main-

tain the standing biomass but with a very limited

growth. The capacity of C. cylindracea to main-

tain biomass during winter in these deeper areas

has been reported at other sites at similar latitu-

des (Giaccone and Di Martino 1995; Cebrián and

Ballesteros 2009), but not in colder areas where

a winter decline occurs (e.g. Piazzi et al. 1997b,

Piazzi and Cinelli 1999, Buia et al. 2001, Capio-

mont et al. 2005, Ruitton et al. 2005b, Lenzi et

al. 2007). At the shallowest site (IG), a clear un-

coupling between biomass and carbon balance

was evident, and we attribute this to the effect

of abiotic factors other than light that might in-

fluence the pattern of vertical distribution of the

alga, such as winter storms (e.g. Cebrián and Ba-

llesteros 2009; Marín-Guirao et al. pers. obs.).

Within the P. oceanica leaf canopy, light availabi-

lity was drastically reduced up to levels that were

always 3.0–8.8 times lower than those recorded

outside. Such low irradiance levels are typically

measured inside P. oceanica meadows (1–7% of

Eo) and reflect the elevated K

d values associated

with the strong self-shading caused by complex

canopies formed by this seagrass species (Dalla

Via et al. 1998). The complexity of the leaf ca-

nopy showed significant spatio-temporal varia-

tion characteristic of P. oceanica meadows el-

sewhere (Romero 1989, Buia et al. 1992, Pergent

et al. 1995, Dalla Via et al. 1998, Olensen et al.

2002). On one hand, shoot density, meadow co-

ver and LAI decreased with depth, which explains

the lowest mean Kd values observed in the dee-

pest site CB; on the other hand, LAI and canopy

height were lower in the winter time, accordingly

with the seasonal variation of the seagrass pro-

duction, but Kd values were equal or even higher

than those in the summer time, contributing to

the explanation of the considerably low light le-

vels within the canopy at that time. In fact, this

variation in the canopy structure is considered

the main adaptive mechanisms of this and other

Posidonia species to offset depth-related light

reductions (Olesen et al 2002, Ralph et al. 2007,

Collier et al 2008).

Evidence provided by this study strongly suggests

that the low light levels reported within the sea-

grass canopy are more limiting for C. cylindracea

growth and survival than those recorded outside.

In general, fronds growing inside the meadow

margin, showed photoacclimatory responses al-

ready described for plants growing outside toge-

ther with other typical acclimation adjustments

Fig. 6.

A) Ordination diagram of the principal com-

ponents analysis with all selected photoac-

climative variables. B) Lineal relationship be-

tween X-axis positions and mean total daily

irradiance (Etotal

, mol quanta m-2 d-1; Table 1),

indicating the lineal regression model, slope

(i.e. regression coefficient; p< 0.05) and coe-

fficient of determination (r2). Sampling sites

= IG, CT and CB. Fh = frond height.

Table 4.

Eigenvalues of the first two axes of the PCA

(PC1 and PC2) and the scores of selected

photoacclimative variables.

Variable / axisEigenvaluesEcEkRdChl b Chl a Pmax Frond heightChl b/aα

PC1

0.759

0.957

0.940

0.887

-0.731

-0.769

0.759

-0.252

-0.214

-0.006

PC2

0.132

-0.017

-0.075

0.403

0.641

0.570

0.581

-0.183

0.455

0.939

p. 64 p. 65

CHAPTER 3

TESIS DOCTORAL

of marine macrophytes to low light conditions,

such as an increase in chlorophyll content and

in the chlorophyll b/a ratio, aimed at enhancing

the efficiency of light absorption (e.g. Kirk 1994,

Falkowski and Raven 2007, Raniello et al. 2004).

Raniello et al. (2004) reported increments in Chl

b and other complementary pigments (e.g. sipho-

noxantin) in C. cylindracea growing under dense

C. nodosa canopies, which could be linked to a

more efficient exploitation of green light, which is

the dominant light under seagrass canopies (e.g.

P. oceanica; Dalla Via et al 1998). In the summer

sampling (T2), the increment in pigment content

and Chl b/a ratio could explain the maintenance

of photosynthetic efficiencies (α) very similar to

those plants growing outside the seagrass ca-

nopy. Further to this, the inhibition of respiration

(up to 89% with respect to the fronds outside)

likely allowed C. cylindracea to attain positive car-

bon balances within the seagrass canopy at the

three sites, despite the fact that the daily satura-

tion periods were less than 4 h (i.e. 64–72% lower

than for plants growing outside the canopy). The

high correlation of this variable with the first PCA

axis (Fig. 4) suggests that the inhibition of respi-

ratory rates represent one of the most important

photoacclimatory mechanisms of C. cylindracea,

not only within the severe shading created by

the canopy, but also (as explained above) outsi-

de the canopy in winter. Although this response

might also reflect low temperature effects on

algal metabolism in winter conditions (Flagella

et al. 2008, Robledo and Freile-Pelegrín 2005, Te-

rrados and Ros 1992), it has been recognized to

be a common physiological strategy to minimize

carbon losses and allow seaweed survival under

low light regimes (Littler et al. 1986, Lüning et

al. 1990, Markager and Sand-Jensen 1994, Pé-

rez-Lloréns et al. 1996, Bernardeau et al. 2011).

In addition, low respiration rates are indicative

of limited growth (Kirk 1994, Pérez-Lloréns et al.

1996), which could further explain the large di-

fferences in algal biomass between the inner and

outer stands observed at the deepest sites (CT

and CB) during the summer, despite their almost

identical carbon balances.

Light climate within the canopy in the winter

represented the most extreme condition for the

alga, since it showed a very limited photoaccli-

matory capacity unable to maintain Hc values

and achieve Hk daily periods longer than 1.1 h at

the shallower site (IG) and of zero h at the deeper

sites. As a result, carbon balances were negative

and hence light availability in these conditions

must be below the minimum requirements for

algal growth and survival (Dennison and Alber-

te 1982, Gómez et al. 1997). In agreement with

this, and based only on water-Kd values (Table 1)

and the Beer-Lambert equation (Kirk 1994), light

levels recorded within the canopy are equivalent

to the range of maximum distributional depths

reported in the Western Mediterranean basin

(35–60 m: Piazzi et al. 2005b; Klein and Verlaque

2008; Ruiz et al. 2011). These results are also con-

sistent with those obtained in the PCA analysis

(Fig. 4), in which the ordination of the objects (i.e.

measurements obtained in each combination of

position, location and time) along the PC1 axis

was highly correlated with light availability and

mainly represented the integration of all pho-

toacclimatative responses in each case. With

respect to the objects during the winter time, it

should be noted that measurement made at the

inner position at the shallow site were very clo-

se to those at the outer position of the deepest

site. This simple observation suggests that the

extreme low light conditions observed beneath

the seagrass meadow in winter have overcome,

or are close to, the limit of the photosynthetic

plasticity of C. cylindracea. For instance, it can be

seen that, particularly in deepest sites, values of

Pmax

and Rd values (and E

c and E

k) of C. cylindracea

plants growing inside the seagrass canopy did not

differ from those showed by plants growing out-

side, suggesting a limit for the plasticity of these

variables under more limiting light conditions. In

such a situation, the maintenance of algal bio-

mass observed underneath the seagrass canopy

in winter is only possible by using carbon storage

reserves during summer, when the carbon balan-

ce was shown to be positive and growth arrested.

This is a strategy to survive light-limiting periods

previously reported for this (e.g. Terrados and

Ros 1992, Robledo and Freile-Pelegrín 2005) and

other seaweed species (e.g. Rosemberg and Ra-

mus 1982, Gagne et al. 1982, Dunton and Schell

1986, Gómez and Wiencke 1998, Lobban and

Harrison 1997). Other possible mechanisms of C.

cylindracea survival beyond its photoautotrophic

limits might be carbon acquisition by heterotro-

phy, as reported for the congenerous C. taxifolia

(Chisholm and Jaubert 1997), or sharing of re-

sources between shaded and illuminated parts of

the coenocytic stolons (Collado-Vides and Roble-

do 1999, De Senerpont Domis et al. 2003).

Our results show that the development of C.

cylindracea biomass is consistently limited inside

the P. oceanica canopy, irrespective the sampling

site and time considered in this study. A monito-

ring study performed at the same sampling sites

(unpubl. data) has demonstrated that this sharp

biomass gradient is stable over years (i.e. 2007–

2013) without any symptoms of seagrass mea-

dow deterioration. In fact, this is consistent with

the observation that P. oceanica meadows are

one of the least-invaded habitats elsewhere and

the idea that P. oceanica can be considered as an

effective “ecological barrier” against the spread

of this highly invasive alien species. Nonetheless,

these types of generalizations must be subjec-

ted to future evaluations of possible long-term

interactions between the alga and the seagrass

through, for instance, phytotoxic allelochemical

effects (Dumay et al. 2002a, Raniello et al. 2007)

or deterioration of substrate conditions (Holmer

et al. 2009). This study provides extensive and

consistent evidence supporting the hypothesis

that light plays a key role in explaining the high

resilience of the P. oceanica meadow with regard

to the C. cylindracea bioinvasion. As reported in

this and other studies using similar ecophysio-

logical approaches, C. cylindracea has a great

physiological and vegetative plasticity allowing it

to adapt to a wide range of environmental con-

ditions and light climates (Raniello et al. 2004,

2006; Bernardeau-Esteller et al 2011), which in

turn is one of the traits contributing to explain

its highly invasive character (Klein and Verlaque

2008). However, results obtained in this study has

shown that the extremely low light levels within

P. oceanica meadows can be below the minimum

light requirements for C. cylindracea growth, sur-

passing its plastic capacity to acclimate to further

light reductions. However, much more research

must be done before attaining some robust con-

clusions about this topic. First of all, other factors

could be involved, or interact with light availabili-

ty, that should be investigated. In the case of this

particular study, the substrate type was the same

at both sides of the seagrass meadow edge (i.e.

P. oceanica “matte”), and hence other kind of fac-

tors such as nutrients, sedimentation, water mo-

vement or space limitation should be considered.

Secondly, given the experimental design used in

this study, results obtained here must be corro-

borated with similar studies in other regions and

using complementary experimental work in the

field and in the laboratory. Furthermore, other

basic aspects should be addressed, such as the

potential of early recruitment phases (e.g. spo-

res) to colonize the seagrass meadows, in addi-

tion to the acclimation capacity of the adult sta-

p. 66 p. 67

CHAPTER 3

TESIS DOCTORAL

ges. Regardless the factors involved, the apparent

ecological resistance of P. oceanica meadows

seem to be linked to its complex canopy struc-

ture. Therefore, and considering that recovery

of damaged P. oceanica meadows is a very slow

process (Duarte et al. 2006), the conservation of

its integrity against anthropogenic disturbances

must be a priority of environmental policies con-

cerned with the control of bioinvasions in the Me-

diterranean Sea, such as the Marine Strategy EU

Directive or the Ecosystem Approach.

CHAPTER 4Photoacclimation of Caulerpa cylindracea: light

as a limiting factor in the invasion of native

Mediterranean seagrass meadows

p. 71

Photoacclimation of Caulerpa cylindracea: light as a li-miting factor in the invasion of native Mediterranean seagrass meadows.

Abstract

Reduction in light availability caused by the ca-

nopy of the Mediterranean seagrass Posidonia

oceanica has been suggested as a critical me-

chanism to resist the invasion of the exotic ma-

croalga Caulerpa cylindracea. We experimentally

evaluated the role of light as a limiting factor

on the capacity of colonization and spread of

this invasive seaweed in P. oceanica meadows

by assessing photoacclimation responses and

productivity and growth capacity of C. cylindra-

cea in mesocosm and in situ light manipulation

experiments. Despite the high photoacclimative

plasticity developed by the alga, the light regime

within the seagrass meadow during the study pe-

riod was close to the minimum light requirements

for growth, restricting the development capacity

of this species. In addition, while increases in li-

ght availability resulting from canopy alteration

also enhanced the productive capacity of the

invasive seaweed in the field, such increase was

not followed by gains in biomass production. Our

results thus support the hypothesis that light

availability has a major role in the underlying

resistance of seagrass meadows to the invasion

by C. cylindracea, but also indicate that there are

additional factors related to the canopy of P. oce-

anica that further hinder the growth and coloni-

zation capacity of the alga.

1. Introduction

A main goal for ecologists is to understand the

factors and mechanisms that determine inva-

sive success of introduced species. Phenotypic

plasticity has been recognized as an important

mechanism related to successful invasion proces-

ses. Plasticity enhances ecological niche breadth

and allows organisms to express advantageous

phenotypes in a broader range of environmen-

tal conditions, which contributes to maintain

positive population growth and increases the

likelihood of invasiveness (Richards et al. 2006).

In addition, native communities strongly differ in

their resistance to invasions (Londsale 1999). Di-

fferences in susceptibility to invasion have been

linked, among other processes, to biotic resis-

tance derived from interspecific competition for

resources between native and introduced species

(Theoharides and Dukes 2007; Branch and Ste-

ffani 2004).

Exotic seaweeds are a major threat to coastal

marine habitats worldwide as they often have

negative effects on the structure and diversity of

native communities (Williams and Smith 2007).

Among all the factors influencing macrophyte

communities, light is key in regulating produc-

tivity, abundance and distribution (Lobban and

Harrison 2004, Kirk 1994, Breeman 1988). The

Publicado en:Bernardeau-Esteller J, Ruiz JM, Tomas F, Marín-Guirao L (2015) Photoacclimation of Caulerpa cylindra-

cea: Light as a limiting factor in the invasion of native Mediterranean seagrass meadows. J Exp Mar Biol

Ecol 465:130-141.

p. 72 p. 73

CHAPTER 4

TESIS DOCTORAL

minimum light requirement for algal growth is

reached when captured light allows to balan-

ce loss processes within the tissue (e.g. respira-

tion and exudation, Markager and Sand-Jensen

1992). If available light in a habitat is below or

close to those minimum requirements, develop-

ment capacity and therefore invasive potential of

exotic macrophytes will be hampered. This occurs

for example in the case of the tropical red alga

Womersleyella setacea in the Mediterranean Sea,

as light requirements of this species reduce dra-

matically its invasion capacity at depths greater

than 35 meters (Cebrian and Rodriquez-Prieto

2012). A high plasticity in photoacclimation me-

chanisms allows an exotic alga to develop an effi-

cient photosynthetic response (that determine

an efficient use of light) in a wide range of light

conditions, that could enhance its competitive

capacity and colonizing potential in new habitats

(Raniello et al. 2006, Bernardeau-Esteller et al.

2011;, Marín-Guirao et al. 2015).

Interspecific competition for light is especially

relevant in structured communities dominated

by large-sized species (canopy formers) since

they generate intense changes in the quality and

quantity of light available at the understory la-

yers (Middelboe and Binzer 2004; Reed and Fos-

ter 1984). Such changes in light availability may

be an important mechanism underlying invasion

resistance of these communities if shading by

the canopy creates light conditions near the mi-

nimum light requirements of the introduced spe-

cies (Arenas et al. 2006). For instance, a decrease

in survival of the invasive japanese seaweed Sar-

gassum muticum in nearshore marine communi-

ties in the western coast of USA has been linked

to shading effects produced by the native canopy

species (Britton-Simmons 2006). The structure of

macrophyte assemblages can be modified due

to the action of natural (high water movements,

herbivory) and human-induced (pollution, fishe-

ries) stressors (Lobban and Harrison 1996). A

reduction in abundance of canopy-forming spe-

cies following these disturbances will result in an

increase in the available light in the lower layers

of the community, which can promote growth of

species constrained by limited light levels (Reed

and Foster 1984). Therefore, it can be assumed

that disturbance of canopy species could promo-

te success of introduced species whose growth

capacity is limited by light conditions.

The Mediterranean Sea, recognized as a hotspot

of biodiversity, is one of the seas most affected

by species introductions (Coll et al. 2010). To

date, 3.3% of total described species in the Me-

diterranean Sea (more than 900, Zenetos et al.

2010) are considered exotic, of which 85 are ma-

crophytes. Within this functional group, the green

alga Caulerpa cylindracea (Sonder) has a strong

invasive character, rapidly colonizing most of the

Mediterranean Sea (Piazzi et al. 2005b, Klein and

Verlaque 2008). Under certain conditions, the

alga is able to develop large biomass, which has

been linked with significant changes in physico-

chemical and community characteristics of recei-

ving habitats (see Klein and Verlaque 2008 and

literature cited therein). The ecological success of

this species in invading the Mediterranean Sea

has been linked, in addition to other traits (e.g.

vegetative and sexual reproductive success, high

growth rates) to high morphological and physio-

logical plasticity (Gacia et al. 1996a, for a review

see Klein and Verlaque 2008). The alga has been

described in a wide range of depths (between

0-60m), suggesting a large capacity of photoac-

climation. In this regard, previous studies have

reported stable populations of the alga in depths

close to 30 meters as well as under the leaf ca-

nopy of macrophytes, indicating a high toleran-

ce of the species to low light regimes. However,

C. cylindracea has shown a reduced capacity to

colonize healthy meadows of the dominant me-

diterranean seagrass Posidonia oceanica (Katsa-

nevakis et al. 2010, Bulleri et al. 2011, Ceccherelli

et al., 2014), the greater structural complexity of

which determines more intense shading condi-

tions (Enriquez et al. 1992, Dalla Via et al. 1998 ).

Recently, Marín-Guirao et al. (2015) analyzed the

photosynthetic and productive characteristics of

natural C. cylindracea populations growing insi-

de and outside of leaf canopies of P. oceanica

in a highly invaded area. Results obtained in this

study suggest that light availability inside the

meadow exceeds the photoacclimation capacity

of the alga and seem to be close to the minimum

light requirements for growth, suggesting that

this factor can play an important role as a me-

chanisms of resistance of P. oceanica habitats to

invasion. However, the methodological approach

used in that study (i.e. non-experimental) preclu-

ded isolating the effect of light availability from

the influence of other environmental factors that

can be related to the development capacity of

the algae and that thus may also be altered by

canopy structure (e.g. water movement, nutrient

availability).

The aim of this study was therefore to experi-

mentally examine the role of light availability in

the colonization of the meadows of P. oceanica

by the alga, testing whether reduced light regi-

mes within this habitat are able per se to explain

the resistance phenomena observed. In order

to evaluate this hypothesis two complementary

experimental approaches were used. We studied

photoacclimation responses (through analysis of

photosynthetic performance and pigment con-

tent) and productive and growth capacity (by

assessment of carbon balance, starch content,

apical elongation and variation in stolon bio-

mass) of C. cylindracea in a mesocosm and a field

experiment in which different light regimes were

experimentally manipulated.

2. Material and methods

2.1. Mesocosm Experiment

Use of a mesocosm system allowed maintaining

controlled environmental conditions (temperatu-

re, light, salinity, pH), enabling us to isolate the

effect of light from the influence of other fac-

tors whose variation could affect the response

of the studied variables. The mesocosm system

consisted of 24 glass independent aquaria of

100 l capacity. Each aquarium had its own light

system (400W halogen lamp, Aqua Medic aquali-

ght-400), water circulation and filtration system,

and contained a plastic tray (22 x 40 cm base

and 10 cm high), filled with previously washed

coarse sediments.

Eight light treatments (L1 to L8) were established

in a range of daily photon flux values (i.e. integra-

ted daily irradiance) comprised between 0 and

13.61 mol quanta m-2 d-1 (Table 1), which inclu-

de light regimes of all natural habitats in which

the alga is found in the study area (unpublished

data). Each light treatment was assigned to 3

randomly selected aquaria. Daily photon flux for

each treatment was determined through daily

integration of instantaneous irradiance values re-

corded in the aquarium on a daily cycle of 12:12

h. These values were obtained using a submersi-

ble light sensor (PAR spherical quantum sensor

MDS MK5, Alec Electronics, Japan) located at

the same depth as the tray, with continuous rea-

dings every ten minutes. The illumination system

is not able to simulate the natural, bell-shaped

light curve (e.g. Fig. 1). Instead, the daily ‘light

curve’ in aquariums had a rectangular shape with

a constant instantaneous irradiance throughout

the illumination period (12 h) that was reached

in a few minutes once the lamps were switched

p. 74 p. 75

CHAPTER 4

TESIS DOCTORAL

on and fell down to total darkness immediately

after the lamps were switched off. As an exam-

ple, the artificial light curve of the L6 treatment

is represented in Figure 1. Unavoidably, the diffe-

rence between the natural and the artificial daily

light curves can have consequences in determi-

nation of photosynthetic parameters (e.g. com-

pensation and saturation light periods, Hc and

Hk, respectively), which should be considered for

the interpretation of the results obtained in the

mesocosm and in the field. In this same context,

differences in light quality between artificial and

natural light sources must be also considered.

High quality natural seawater from a nearby, oli-

gotrophic and unpolluted area was employed in

the mesocosms. Environmental conditions inside

the tanks were similar to those prevailing in the

selected areas for of the stolons of C. cylindracea

(see below) during the time of year in which the

experiment took place. For that purpose, a pe-

riodic analysis of nutrients by colorimetric test

(phosphorus and nitrogen; Merck®), continuous

recording of pH with specific electrodes (Aqua

Medic AT-Control) and a daily monitoring of

the salinity of the water with a conductivimeter

WTW (Model Cond. 197i) were carried out in

each aquarium. Salinity values were maintained

constant (37.5 PSU) by osmosis water addition.

Water temperature during the experiment was

19 ± 0.1 ° C and was controlled by automatic

cooling system (see Marín-Guirao et al. 2011 for

more details).

C. cylindracea stolons were collected by hand in

a nearby population located on the southwest

coast of the Region of Murcia (Isla Grosa, UTM

X: 0701991, Y: 4177942, H 30S) at -11 m deep.

Collection of stolons was done randomly in a

large area (c.a. 1000m2) to capture the natural

variability. The colonized area is an infralittoral

well-illuminated bottom composed by a mosaic

of sand and dead P. oceanica rhizomes (Ruiz et

al. 2011); mean noon irradiance was 195.66 ±

3.99 μmol quanta m-2 s-1 during the experimental

period (i.e. OUT treatment in Fig. 1). Collection

was performed in November 2011, a period of

high vegetative development (Bernardeau-Es-

teller, unpublished results). Immediately after

collection, the cut end of the stolon was sealed

with very small plastic clothes pegs to avoid loss

of internal content and was put in a black plas-

tic bag to prevent overexposure to light. Stolons

selected were transported in refrigerated contai-

ners with seawater to the laboratory. Once here,

they were immediately transplanted into aquaria

for acclimatization for 3 days before the start

of the experiment. Four stolons were planted in

each aquarium, and were of similar characteris-

tics, with an initial length of 25-30 cm, a number

of fronds ranging from 10-15, and a single apical

meristem. During the acclimatization process, ex-

perimental units were subjected to daily photon

fluxes similar to those recorded in the field (ca

4.43 ± 0.05 mol quanta m-2 d-1). After the tran-

sitional period, light conditions were changed in

each aquarium to obtain the experimental light

treatments. Algae were exposed to these treat-

ments for 7 days.

2.2. Field experiment

Simultaneously with the mesocosm experiment,

a field experiment was conducted in the same

area where stolons of C. cylindracea were collec-

ted from for the mesocosms experiment. This

highly colonized area is adjacent to a dense P.

oceanica meadow, but C. cylindracea stolons are

not able to penetrate beyond the seagrass mea-

dow edge (Ruiz et al. 2011, Marín-Guirao et al

2015). Four experimental light treatments (two

inside the seagrass meadow and two outside)

were created: (i) within the meadow (IN), (ii) in

areas within the meadow where the height of the

leaf stratum was experimentally reduced by clip-

ping (CLIPPING), (iii) outside the meadow (OUT),

and (iv) outside the meadow but in areas whe-

re light availability was experimentally reduced

(SHADED) to be similar to those of the IN treat-

ment. The light regime of each experimental

treatment was characterized based on its noon

instantaneous irradiance and the integrated

daily irradiance obtained from daily light cycles

(Fig. 1). To this end, PAR light sensors (spherical

quantum sensors; Alec MK5 MDS) were installed

on the bottom of all experimental plots. Instan-

Table1.

Summary of irradiance measurements in

each light treatment in the mesocosm ex-

periment. Data are presented as means ±

standard error.

Noon Instantaneous Irradiance

μmol quanta m-2 s-1

0.00 ±0.00

6.00 ± 0.20

20.97 ± 0.76

29.95 ± 0.99

43.73 ± 1.09

102.50 ± 3.17

211.77 ± 4.69

314. ± 9.30

Integrated Daily Irradiance

mol quanta m-2 d-1

0.00 ±0.00

0.24 ± 0.01

0.91 ± 0.11

1.29 ± 0.01

1.89 ± 0.04

4.43 ± 0.05

9.15 ± 0.14

13.61 ± 0.52

Treatment

L1

L2

L3

L4

L5

L6

L7

L8

Measurements

Fig. 1.

Daily course of irradiance measured at

the sea floor irradiance and summary of

irradiance measurements obtained for

each field experiment. The dotted line

corresponds to the course of the daily irra-

diance in mesocosm aquaria (only the L6

treatment is represented as an example).

p. 76 p. 77

CHAPTER 4

TESIS DOCTORAL

taneous irradiance measurements were recorded

every 10 minutes during the 7 days of the expe-

riment. The integrated daily irradiance (Etotal,

mol

quanta m-2 d-1) was obtained by the integration

of these instantaneous measurements recorded

in each daily cycle. Throughout the experiment,

water temperature was recorded in situ by HOBO

Pro v2 Water Temperature Data Logger (Onset

Computer, EME Systems, Berkeley, CA, USA). The

average temperature recorded during the experi-

ment was 19.8 ± 0.1 ° C.

Based on the similarities in light regimes obtai-

ned in the IN and the SHADED treatments, we

would expect that algae under these conditions

will present an equivalent photoacclimation res-

ponse, showing a clear limitation in its production

and growth capacity. Moreover, increase in light

availability linked to the experimental manipula-

tion of the canopy (CLIPPING treatment) should

determine an approximation of the responses of

seaweeds under this treatment to those recorded

outside of the meadow (OUT treatment).

Light conditions of the SHADED treatment were

obtained by using floating structures anchored to

the substrate. These structures consisted of a PVC

frame with neutral density filters which determi-

ned that the light regime beneath them was simi-

lar to that recorded within the meadow (IN treat-

ment). A preliminary study showed that this type

of structures do not alter the physico-chemical

conditions of the bottom, minimizing the effect

of any other factor different than light (Bernar-

deau-Esteller, unpublished results).

In the plots of the CLIPPING treatment the

reduction of the leaf layer was carried out ma-

nually to obtain a final leaf length of 15 cm (the

original leaf length of P. oceanica canopy was ca

80 cm). This reduction simulated a high rate of

herbivory (e.g. Tomas et al. 2005) and determines

light conditions intermediate to those obtained

inside and outside the meadow (Fig. 1).

For each experimental treatment, four plots of

2x2 m2 were randomly selected. Five stolons with

the same characteristics as those used in the

mesocosm experiment were transplanted in each

plot. Before the start of the experiments, stolons

were collected and sealed and then transported

in darkness and refrigerated containers to the

laboratory where they were marked and initial

characterization was conducted (see section be-

low referring to the phenological variables used).

Subsequently, stolons were transported back to

the study area where they were transplanted in

the plots. Stolons were attached to the substrate

by a nylon cord anchored by two stainless steel

pegs tied at both ends. After seven days, algae

were collected and transported back to the labo-

ratory to perform all the measurements.

2.3. Alga Analyses

In both experiments (mesocosms and field) the

same response variables were considered.

2.3.1. Photosynthesis vs irradiance curves

(P vs. E curves)

Prior to photosynthetic measurements, C. cylin-

dracea samples taken from the aquarium system

or collection site were held overnight in the dark

under controlled temperature in natural seawa-

ter.

Photosynthesis and dark respiration rates (Rd)

were measured using a polarographic oxygen

electrode and a magnetic stirrer (DW3, Hansa-

tech Instruments Ltd) under controlled tempe-

rature. Incubation was carried out at the same

temperature measured in the mesocosm system

and in the field (19ºC). Three replicated apical

segments of non-epiphytized C. cylindracea

fronds of approximately 2 cm height were em-

ployed for the measurements. Dark respiration

rates were measured by maintaining the fronds

in the dark for 15 min. Net oxygen production

was then determined at 9 different light inten-

sities (from 1 to 700 μmol quanta m-2 s-1) using

a high intensity light source which consists of

an array of 36 red LED’s (LH36/2R, Hansatech

Instruments Ltd). Net photosynthetic rates were

plotted against the light intensities (P vs. E cur-

ves), and the photosynthetic parameters were

calculated as follows: the maximum rate of net

photosynthesis (Pmax

) was determined by avera-

ging the maximum values above the saturating

irradiance (Ek). The photosynthetic efficiency (α,

μmol O2 g FW-1 h-1/μmol quanta m-2 s-1) was calcu-

lated as the slope of the regression line fitted to

the initial linear part of the P vs. E curve, and the

compensation irradiance (Ec) as the intercept on

the X-axis. Ek was calculated as the ratio P

max/α.

Gross photosynthesis (gross-Pmax

) was calculated

as the sum of Pmax

and Rd

2.3.2. Pigment content

Pigment content was determined in the same

apical segments of fronds selected for obtaining

P vs. E curves (n = 3). The analysis was conducted

spectrophotometrically after manual extraction

of a homogenized suspension using 90% ace-

tone (Dennison 1990), with MgCO3 added as a

chlorophyll stabilizer. The acetone extracts (10

ml) were stored at 4 °C in the dark for 24 h and

centrifuged. Chlorophyll a (chl a), Chlorophyll b

(chl b) and carotenoid content was computed

using the equations of Lichtenthaler & Wellburn

(1983).

2.3.3. Estimated daily irradiance regimes and

daily metabolic carbon balances

Mean daily compensation (Hc) and saturation

(Hk) periods were calculated by averaging the

number of hours per day that irradiance values

exceeded the corresponding values of compensa-

tion (Ec) and saturation (E

k) irradiances, respecti-

vely. The values of Ec and E

k used in these calcula-

tions were those obtained from the P vs. E curves.

Daily carbon balance, as a proxy of plant light

limitation (Dennison and Alberte, 1985), was

calculated according to the Michaelis-Men-

ten function (P = [gross-Pmax

E/(E+Ek)] + R

d (Baly

1935)) previously applied to C. cylindracea (Ber-

nardeau-Esteller et al. 2011), where P is the net

production, gross-Pmax

is the maximum gross pho-

tosynthetic rate, E is the irradiance measured in

the field, Ek is the saturation irradiance, and R

d is

the respiration rate. Semicontinuous (i.e. every

10 min) mesocosm and field irradiance measure-

ments were entered into the function to generate

estimates of net production, which were integra-

ted across 24 h periods to yield daily net produc-

tion. If the photosynthetic quotient is assumed

to equal 1, and the ratio g C: g O2 = 0.3 (Matta

and Chapman 1991), then the net production in

oxygen units (μmol O2 g FW-1) can be multiplied

by 0.012 to obtain the equivalent carbon units

(mg C g FW-1). This calculation presumes cons-

tant dark respiration throughout the day and

does not consider other carbon losses (e.g. exu-

dation, grazing) or gains (e.g. light-independent

carbon fixation).

2.3.4. Stolon biomass balance and apical growth rate

To calculate stolon biomass balance and apical

growth rate in a particular stolon, the following

equation was used: Xx = (X

f - X

i) / t, where X

x is the

measurament for a variable (biomass or length)

expressed in units d-1, Xf and X

i are the observed

measurements of the variable at the end and be-

ginning of the experiment, and t, the time dura-

tion of the experiment. The average value of all

stolons from each tray (mesocosm experiment)

or plot (field experiment) constituted each one of

the replicates in both experiments (n=3 and n=4,

respectively).

Total biomass of a stolon was determined using

a precision scale (Metler-Toledo). Growth rate

p. 78 p. 79

CHAPTER 4

TESIS DOCTORAL

based on apical stolon length was determined

according to the methodology described by

Ruitton et al. (2005b). At the beginning of the

experiment the apical region of the stolon was

marked by placing a metal ring on the back of

the last frond before the apical meristem and

manually measured (cm). This mark was used as

a reference for measurement at the end of the

experiment. Length measurements included both

the main axis of the stolon and the stolon ramifi-

cations generated during the experiment.

2.3.5. Starch content

For starch analysis, algae from each tray (meso-

cosm experiment) and plot (field experiment)

were cleaned with distilled water, dried for 48

hours at 50 °C and ground to fine powder. Starch

content was analyzed following the method des-

cribed by Yemm and Willis (1954). Ground ma-

terial (0.150g) was washed with 80% ethanol to

remove all trace of soluble sugars and extracted

with 1 N KOH to solubilize starch. Finally, starch

was determined spectrophotometrically using an

anthrone assay. Starch content was expressed as

percentage of the dry weight of the sample. Each

of these measurements constituted a replicate of

the experiment (n = 3 for the mesocosm experi-

ment; n= 4 for the field experiment).

2.4. Statistical analysis

2.4.1. Multivariate analysis

To explore the photoacclimative response of C.

cylindracea to the different treatments in the

mesocosm experiment, Principal Component

Analysis (PCA) was performed based on the co-

rrelation matrix of photoacclimatation variables

(which includes photosynthetic parameters, dark

respiration rate and pigment content). Data were

previously transformed to achieve centralization

and standardization. PCA was performed with

the program CANOCO version 4.5 (Microcompu-

ter Power Ltd).

2.4.2. Univariate analysis

For both the mesocosm and field experiments,

differences among treatments for each varia-

ble were tested using one-way ANOVAs. Prior to

analysis, data were tested for heterogeneity of

variance using Cochran’s C-test and transformed

when necessary. Where variance remained hete-

rogeneous, untransformed data were analysed,

as ANOVA is a robust statistical test and is relati-

ve unaffected by the heterogeneity of variances,

particularly in balanced experiments (Underwood

1997). A probability level of 0.05 was regarded

as significant except when data transformation

was not possible. In such cases the level of signi-

ficance was reduced to P < 0.01 to minimize type

I error. The Student-Newman-Keuls (SNK) test

was used for a posteriori pairwise comparisons

of means. ANOVA analyzes were developed with

the program GMAV® version 5 for Windows (Un-

derwood and Chapman 1998).

In addition, for both experiments, relationships

between photoacclimation variables and light

were explored using simple regressions. These

analyzes were developed with the program Sig-

maplot 10.0 (Systat Software Inc.).

3. Results

3.1. Mesocosm experiment

The first axis of the PCA performed on Caulerpa

cylindracea’s photoacclimative response (Fig

2A) represents most of the variance explained

(67%), which had a strong correlation (r > 0.7)

with all variables except Pmax

. This correlation was

negative in the case of Ek, R and E

c, while it was

positive for pigment content and photosynthetic

efficiency (α). The second ordination axis explai-

ned 23.8% of the variation and was highly corre-

lated (r =0.91) with Pmax

(Fig. 2A).

Distribution of treatments on AXIS 1 can be in-

terpreted in terms of light availability with higher

irradiance treatments on the left side of the PCA

and lower irradiance treatments in the right side.

Indeed, the ordination of treatments along AXIS

1 showed a high and significant correlation with

the Integrated daily Irradiance (R2 = 0.5833, p <

0.0001, n =3 ; Fig. 2B).

P vs. E curves allowed the estimation of several

photosynthetic parameters, most of which (Pmax

,

Rd, E

c and E

k) having a positive and significant li-

near relationship with irradiance (Figure 3A). The

highest values of these parameters were recorded

in the treatment of highest irradiance (L8; 13.61

mol quanta m-2 d-1) and were significantly higher,

except for Ec, to the other treatments (SNK, Fig.

3A, Annex Table 1). Minimum values were obser-

ved in the low irradiance treatment L3 (0.91 mol

quanta m-2 d-1) in the case of Rd and E

c, and dark-

ness treatment (L1) in the case of Pmax

and Ek (Fig.

3A). Values recorded in these treatments repre-

sented a reduction of about 70%, in the case of

Rd and E

c, and close to 50% for P

max and E

k com-

pared to treatment L8. Photosynthetic efficiency

(α) decreased with increasing irradiance (Fig. 3A),

although significant differences were found only

between treatment L3, which yielded the highest

Fig. 2.

Multivariate Analysis of photoacclimation

responses of C. cylindracea in the meso-

cosm experiment: A. Ordination diagram

of the Principal Component Analysis (PCA)

with selected photoacclimative variables for

mesocosm experiment. B Relationship be-

tween X-axis position and Integrated Daily

Irradiance (Table 1).

p. 80 p. 81

CHAPTER 4

TESIS DOCTORAL

value (30% greater than the value recorded in

higher irradiance treatments) and the rest of the

treatments (SNK, Fig. 3A, Annex Table 1).

Chl a and chl b concentrations and the ratio chl-

b/a showed a similar pattern to that observed

for photosynthetic efficiency, characterized by a

negative linear relationship with irradiance (Fig.

3B). Higher values of chlorophyll were generally

observed in the low – intermediate irradiance

treatments, and were highest in treatment L3

(0.91 mol quanta m-2 d-1). No clear response for

carotenoid content was found regarding diffe-

rent light levels. However, the ratio of these ac-

cessory pigments in relation to chlorophyll a and

b content showed a positive linear relationship

with irradiance, registering the highest values

above 9.15 mol quanta m-2 d-1 (L7) (Fig. 3B, An-

nex Table 1).

Daily compensation period (Hc) remained at

maximum values (12h) in all treatments except

for L1 and L2, in which values were significantly

lower (0 and 10.42 h respectively, Fig. 4). Daily

saturation period (Hk) values progressively in-

creased with increasing irradiance, being close

to 0 hours for darkness treatment L1 and the low

light treatment L2 (0.24 mol quanta m-2 d-1) and

with maximum values (12h) in treatments with

more than 5 mol quanta m-2 d-1of daily irradiance

(L7 and L8) (Fig. 4, Annex Table 1). In accordan-

ce with Hc and photoacclimation variables, daily

carbon balance also showed a positive response

with increasing light. All treatments (except L3

and L4) significantly differed from each other,

with carbon balance being negative in treatment

L1 and nearly 0 in L2 (0.24 mol quanta m-2 d-1)

(Fig. 4, Annex Table 1). Stolon biomass balance

showed negative or very close to 0 values for irra

diance levels below 0.3 mol quanta m-2 d-1 (treat-

ments L1 and L2, Fig. 4). A progressive increase

in this variable was identified above these values

of irradiance, reaching maximum values from 4

mol quanta m-2 d-1 (treatment L6 and L7, Fig. 4).

Finally, a significant reduction occurred at maxi-

mum irradiance (treatment L8, 13.61 mol quanta

m-2 d-1, Fig. 4, Annex Table 1). Apical growth rate

had a similar pattern to stolon biomass balance,

although values were always positive even in the

absence of light (L1), with rates ranging from

0.4 (treatment L1) to 3.4 cm d-1 (treatment L7 )

(Fig 4). Starch content ranged from 1.99 ± 0.04

% DW in treatment L1 to 2.57 ± 0.06 % DW in

treatment L2, and while there were significant

differences among treatments, there was no sig-

nificant correlation with irradiance.

Fig. 3.

Photoacclimation response of C. cylindracea for mesocosm experiment: A. Photosynthetic

parameters derived from P vs. E curves. B. Pigment content. Symbols are mean ± standar error.

Solid lines represent the regression line fitted to data and the smoothed dashed line illustrates

the trajectory of the response variable as irradiance increases.

p. 82 p. 83

CHAPTER 4

TESIS DOCTORAL

Fig. 3.

Fig. 4.

Productive and growth capacity of C. cylindracea in the mesocosm experiment: Li-

ght-compensation period (Hc), light-saturation period (H

k), daily carbon balance, stolon

biomass balance, apical growth rates and starch content for the mesocosm experiment.

Data are mean ± standard error. The smoothed dashed line illustrates the trajectory of

the response variable as irradiance increases.

p. 84 p. 85

CHAPTER 4

TESIS DOCTORAL

Fig. 5.

Photoacclimation response of C. cylindracea for the field experiment: A. Photosynthetic

parameters derived from P vs. E curves. B. Pigment content. Data are mean ± standard

error. Solid lines represent the regression line fitted to data and the smoothed dashed line

illustrates the trajectory of the response variable as irradiance increases. Mean values ob-

tained in treatment L2 and L6 of the mesocosm experiment (asterisks) have been included

as a reference.

Fig. 5.

p. 86 p. 87

CHAPTER 4

TESIS DOCTORAL

3.2 Field experiment

Similarly to the mesocosm experiment, parame-

ters derived from P vs. E curves showed a signi-

ficant linear relationship with irradiance. This

relationship was positive in all cases, except for

photosynthetic efficiency (α), for which a signifi-

cant reduction was observed with increasing irra-

diance (Fig. 5A). SNK test detected differences

between treatments for the variables Pmax

, Ek y R

d,

in which the recorded values were significantly

higher in OUT than in the other treatments, while

contrarily α values were significantly lower in the

OUT treatment than in the IN and SHADED ones

(Fig. 5A, Annex Table 2).

With regard to pigment content, no significant

differences were observed amongst treatments.

However, the ratio chl b/a showed a positive li-

near relationship with irradiance, registering sig-

nificant differences between the conditions of

low irradiance (IN-SHADED) and the rest light

treatments. On the contrary, ratios between caro-

Fig. 6.

Productive and growth capacity of C. cylindracea in the field experiment: Light-compensation pe-

riod (Hc), light-saturation period (H

k), daily carbon balance, stolon biomass balance, apical growth rates

and starch content for field experiment. Symbols are mean ± standard error. The smoothed dashed

line illustrates the trajectory of the response variable as irradiance increases. Mean values obtained in

treatment L2 and L6 of the mesocosm experiment (asterisks) have been included as a reference.

tenoids and chlorophylls were significantly higher

at lower irradiances (IN-SHADED; Fig. 5B). While

there were no significant differences in Hc among

treatments (always exceeding 7 hours), we did

detect differences in Hk, which were significantly

higher in the OUT and CLIPPING (>5h) in compa-

rison to SHADED and IN treatments (2h). Maxi-

mum values of carbon balance were observed

outside the meadow (OUT; 0.53 mgC g-1 FW d-1),

with a significant reduction of 42% in CLIPPING

and of about 80% in lower irradiance conditions

(IN and SHADED; Fig. 6, Annex Table 2).

Algae outside the meadow (OUT) exhibited the

highest values of stolon biomass balance, apical

growth rate and starch reserves. Stolon biomass

balance was negative in the rest of treatments,

being significantly lower within the meadow (IN)

than in SHADED and CLIPPING conditions. In

contrast, no significant differences were found

between these low light availability treatments

in terms of apical growth rate and starch content

(Fig. 6, Annex Table 2).

In figures 5 and 6 mean values obtained in L2

and L6 treatments of the mesocosm experiments

are represented in order to allow some compa-

rative, graphical analysis with those obtained in

the field experiment under similar field regimes

i.e. the IN and OUT treatments. It can be seen

how most of the response variables followed si-

milar patterns in both experimental approaches

(except chl b and ratio chl b:a). However, there

were appreciable quantitative differences for

some of the variables, particularly in the high li-

ght treatments (OUT and L6). Major quantitative

differences are appreciated in Rd, α, pigment con-

tent and composition (Fig. 5), Hk, stolon biomass

and growth and starch content (Fig. 6).

4. Discussion

Under the light gradient set in the mesocosm

experiment, C. cylindracea showed a clear pho-

toacclimation response to reduction in light

availability which included (i) a reorganization

of the photosynthetic apparatus illustrated by an

increase in pigment content (and antenna size)

and changes in photosynthetic performance (i.e.

reductions in Pmax

, Ek, and E

c, and increments in α),

as well as (ii) a reduction in respiration rate (Rd).

These physiological mechanisms are considered

common strategies to overcome low light regi-

mes in marine macrophytes (Littler et al. 1986,

Lünning 1990, Kirk 1996, Lobban and Harrison

1997, Falkowski and Raven 2007) and were con-

sistent with results from previous descriptive field

studies that examined photoacclimation capaci-

ty of the alga under natural light gradients in the

Mediterranean Sea (Raniello et al. 2004, 2006,

Bernardeau-Esteller et al. 2011; Marín-Guirao et

al. 2015). While increase in pigment content and

photosynthetic efficiency (α) reflect an improve-

ment in both light harvesting capacity and ener-

gy conversion efficiency (Lobban and Harrison

1997, Hanelt and López-Figueroa 2012), reduc-

tion in Rd reveals a decrease in metabolic demand

in order to maximize carbon gains (Markager and

Sand-Jensen 1994, Peréz-Llorens et al. 1996, Ber-

nardeau et al. 2011). These responses enable

the alga to reduce light requirements for grow-

th (illustrated by the decrease in compensation

and saturation irradiance [Ec and E

k]) and extend

the daily period at which algae photosynthesizes

at saturating irradiance (Hk) in order to main-

tain the photosynthetic and productive capacity

under low light regimes (Denisson and Alberte

1982, 1985, Litter et al. 1986, Gantt 1990, Matta

and Chapman 1996, Gómez et al. 1997).

Even though there were quantitative differences

in some photosynthetic variables (mainly Ek, R

d, α

and Chlorophyll b in high light treatments), the

photoacclimative patterns described above for

the mesocosm experiment were very similar to

p. 88 p. 89

CHAPTER 4

TESIS DOCTORAL

those observed in the field experiment, except

for pigment content (Chlorophyll b) and compo-

sition (Chlorophyll b and carotenoid ratios). Such

differences could be explained by unavoidable

differences in some key factor related with the di-

fferent nature of both experimental approaches,

despite the fact that in the mesocosm system

we tried to simulate field conditions as similar

as possible. For example, carotenoids (relative to

chlorophyll a and b) differences between meso-

cosm and field data are likely explained by di-

fferences in the spectral composition of light (i.e.

change not only in quantity but also in quality of

light) related to depth and light capture within

the seagrass canopy. As it was already mentio-

ned in the methods section, we were not able to

reproduce exactly the natural light regime in the

mesocosm system. In the mesocosm system, li-

ght has a greater component of the red part of

the spectrum, while these wavelength are vir-

tually nonexistent in field conditions due to the

absorption by water column and the P. oceani-

ca leaf canopy, where there is a predominance

of green and blue wavelength. These differences

in light quality would promote the development

of accessory pigments such as some carotenoids

(e.g. siphonaxanthin) that have an enhanced

ability to absorb green light (Kirk 1994, Dalla Via

et al. 1998). In fact, increased siphonaxanthin

concentrations as a response to light reductions

have been previously described in this (Raniello

et al. 2006) and other species of the genus Cau-

lerpa (Riechert and Dawes 1986). The influence

of other key factors such as nutrients or tempera-

ture in explaining divergences between field and

laboratory results is much less probable since the-

se conditions were highly controlled in the meso-

cosm system. Seawater used in the mesocosm

was obtained from the same area where the

field experiment was performed and frequently

renewed to maintain nutrient levels. Therefo-

re, quality of seawater in both experiments can

be considered similar at least in relation to the

studied responses. Such differences in pigment

composition (or in any other photoaclimative va-

riable) were not as evident between the IN and

the SHADED treatment of the field experiment,

revealing that while shading structures success-

fully reproduced the light environment inside the

seagrass leaf canopy, they did not modify other

factors linked to meadow structure (e.g. hydro-

dinamism, nutrient availability, etc.) that also

appear to have influenced the photoacclimatory

responses observed in the field experiment.

In the mesocosm experiment maximum growth

rates and biomass production were observed in

light levels ranging between 4.43 (L6) and 9.15

(L7) mol quanta m-2 d-1. Light reductions below

these optimum light levels lead to photoacclima-

tory responses and reductions in the production

and growth capacity of the alga. When light le-

vels ranged between 1.89 (L5) and 0.91 (L3) mol

quanta m-2 d-1, photoacclimation mechanisms

allowed for an optimization of light capture and

use, maintaining positive Hk values and net car-

bon gains. In fact, while these treatments suffe-

red a reduction in light availability which ranged

between 60% and 80% compared to treatment

L6 (i.e. optimum light conditions), carbon gains

decreased only between 20% and 30% in com-

parison to L6, and allowed the alga to produce

new biomass. In this light range, reduction in

growth capacity is not only a consequence of

the decrease in carbon production but it is also

related to the costs of the process of acclimation.

Changes in photosynthetic apparatus determine

an increase in maintenance costs of the maco-

phytes (Raven 1984, Copertino et al. 2006), while

reducing respiratory rates imply a lower provision

of internal resources for growth (Kirk 1994, Pe-

réz-Llorens et al. 1996).

Under more severe light reductions (i.e. below L3

light levels), no further photoacclimation took

place, suggesting that photosynthetic plasticity

capacity of the alga was exceeded. This uncou-

pling between acclimation response and light

availability determines an inefficient use of light,

as illustrated by the extremely low values of Hk

and carbon balance (very close to 0), which in

turn determine a limitation in the capacity of

the alga to develop new biomass. According to

these results, it can be inferred that minimum

light requirements for growth under mesocosm

conditions are very close to the L2 light regime

(0.24 mol quanta m-2d-1). In fact, based on light

extinction coefficient mean values (Kd) obtained

by authors in the same experimental area (Ma-

rín-Guirao et al. 2015), we estimated that the

extremely low light levels measured inside the P.

oceanica meadow are within those prevailing at

the maximum depth range of C. cylindracea in

the Western Mediterranean Sea (but considering

that the spectral composition under seagrass ca-

nopies and maximum distributional depths can

differ). Positive growth rates based on apical sto-

lon length described in L2 and L1 (i.e. darkness)

treatments could be explained as a mechanism

of talus expansion resulting from the dilution

of internal biomass in response to light-limiting

conditions (Sand-Jensen 1988, Pérez-Llorens et

al. 1996).

In the field experiment, light availability in treat-

ments that reproduce light regime within a P.

oceanica meadow (IN and SHADED = 0.41-0.47

mol quanta m-2d-1) laid between the L2 and L3

mesocosm treatments, suggesting that in these

treatments the algae were probably close to the

limit of its photoacclimation plasticity and its

minimum light requirements for growth. In fact,

the very low (although still positive) values of Hk

and daily carbon balance registered support the

idea that this light regime overcomes the alga’s

acclimation capacity. Despite the positive values

registered for these productivity variables, stolon

biomass balance was negative in both treat-

ments, indicating that carbon fixation was not

enough to maintain new biomass production and

thus that light availability limits the development

capacity of the alga. In this field experiment, the

growth capacity of the alga was in general con-

siderably lower than that reported in the meso-

cosm experiment under comparable light levels,

suggesting an apparently higher light require-

ment under field conditions due to, for example,

an increase in maintenance costs associated with

the impacts of other environmental factors such

as grazing and mechanical damage (Markager

and Sand-Jensen 1992). However, once again di-

fferences in light regime provided in both experi-

mental approaches could also be involved in such

discrepancies in algal productivity. As explained

in the methods section, the illumination system

of the mesocosm produced rectangular-shaped

daily light ‘curves’ (Fig. 1), which necessarily re-

sulted in daily Hk periods (i.e. the daily period at

which the alga is photosynthesizing at its maxi-

mum rate) much larger that those derived from

typical, natural bell-shaped light curves. Since

Hk is crucial in determining carbon balance and

growth (Dennison and Alberte 1985, Dunton and

Shell 1986, Gómez et al. 1997) it could reasona-

bly account for the higher rates of algal growth

and biomass accumulation in the aquariums, li-

kely based on the consumption of internal resour-

ces (as indicated by the similar starch content

between light treatments). Furthermore, the hi-

gher algal productivity of the mesocosm system

is consistent with other quantitative differences

previously reported for some photosynthetic va-

riables (Ek, R

d and α) between both experimental

approaches.

p. 90 p. 91

CHAPTER 4

TESIS DOCTORAL

Increases in light availability provided by expe-

rimental manipulation of the seagrass leaf ca-

nopy (i.e. the CLIPPING treatment) allowed C.

cylindracea to achieve a carbon balance three

times higher than those recorded in the IN and

the SHADED treatments, as well as Hk daily pe-

riods longer than 5 hours. These results reinforce

the hypothesis supported by the other results ob-

tained in this study that light availability inside

the seagrass canopy limits the photosynthetic

performance of the algae, and are consistent

with recent experimental studies demonstrating

that the removal of P. oceanica leaves promotes

the establishment and spread of the invasive

seaweed (Tamburello et al. 2014). However, des-

pite the efficient use of light demonstrated by

the alga, stolons transplanted to the CLIPPING

plots presented apical growth rates and biomass

losses similar to that recorded in IN and SHADED

treatments. Furthermore, these stolons were de-

pleted in starch content relative to those in the

OUT plots. These results could be explained if car-

bon gains and internal reserves are being used to

cope with some kind of additional stress instead

of for biomass growth and maintenance. In fact,

stolons from the CLIPPING plots showed some

physical damages at the end of the experimental

period; algae stolons from IN plots also displayed

these wounds, but not those from SHADED plots,

which could explain the significant higher bio-

mass losses measured in stolons from IN treat-

ments. These unexpected results suggest that in

addition to light, other stressful factors linked to

the meadow structure can be limiting the grow-

th and development of C. cylindracea inside the

seagrass leaf canopy. Macrophyte canopies may

affect the distribution of plant understory species

through several differing effects other than sha-

ding, such as scouring (Black 1974, Velimirov and

Griffiths 1979), or exudation of chemicals subs-

tances (Fletcher 1974, Dayton et al. 1984). Since

wounds appeared in stolons just after a short,

stormy event that occurred during the experi-

mental period, a scouring effect caused by sea-

grass leaves over the bottom could be proposed

as a candidate factor. In fact, scouring (Gambi et

al. 1989, 1990) and chemical exudation (Cuny et

al. 1995) are mechanisms by which P. oceanica

can influence the understory assemblages within

the meadow. In addition, a shorter seagrass ca-

nopy can decrease protection from fish (Farina et

al. 2009), some of which being avid consumers of

this alga (Tomas et al. 2011).

In summary, our results are consistent with the

presumed high photosynthetic plasticity of C.

cylindracea and its capacity to colonize Medite-

rranean habitats within a broad range of light

regimes (Piazzi et al. 2000, Klein and Verlaque

2008). However, acclimation mechanisms de-

veloped by the alga represent an energy cost

which may affect its ability to grow in low light

environments, as illustrated by the lower abun-

dances shown by the alga at depths greater than

25-30 m (Klein and Verlaque 2008, Katsanevakis

et al. 2010, Bernardeau et al 2011). Despite the

influence of certain experimental factors (mainly

light quality and quantity) on algal productivity,

results obtained from mesocosm and field expe-

rimental approaches consistently showed that

light levels inside the P. oceanica leaf canopy

overcome the phenotypic plasticity capacity of

C. cylindracea, strongly limiting its photosynthe-

tic performance and leading to carbon balances

unable to sustain algal development. A similar

conclusion was achieved in a previous field study

under winter conditions (Marín-Guirao et al.

2015), confirming that the alga is subjected to

light regimes under or very close to its minimum

light requirements for growth over long periods

of its annual life cycle. In fact, the presence of

C. cylindracea stolons growing at the meadow

edge zone can only be explained by net carbon

gains obtained by the alga during summer, when

Variable

Pmax Rd Ec Ek α Chl a Chl b Carotenoids Chl b/a Ratio Carotneoids/chl a Ratio Carotneoids/chl b Hc Hk Carbon balance Stolon biomass balance Apical growth rate Carbohydrate content

Effect

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

df

7

16

7

16

7

16

7

16

7

16

7

16

7

16

7

16

7

16

7

16

7

16

7

16

7

16

7

16

7

16

7

16

5

12

MS

7,41

1,01

0,65

0,04

8,83

0,75

149,49

11,94

0,0035

0,0002

3095,98

311,43

1357,40

79,43

198,09

24,03

0,0059

0,0001

0,0024

0,0001

0,0344

0,0005

52,90

0,0009

81,96

0,42

0,61

0,0002

0,02

0,0003

1,53

0,14

0,11

0,019

SE

0,58

0,12

0,50

2,00

0,01

10,19

51,46

2,83

0,01

0,00

0,00

0,02

0,38

0,01

0,01

0,22

F

7,35

15,48

11,73

12,52

15,25

9,94

14,09

8,24

64,28

28,66

65,01

6094,9

193,08

3300,79

60,38

10,85

5,88

P

***

***

***

***

***

***

***

***

***

***

***

***

***

***

***

***

n.s.

SNK test

L1=L2=L5=L6=L4=L7=L3<L8

L3=L2=L4=L1=L5=L6=L7<L8

L3<L2=L4=L5=L1=L6=L7=L8

L1=L2=L3=L5=L4=L6=L7<L8

L7=L1=L8=L6=L5=L4=L2<L3

L1=L6=L7=L8=L5=L4=L2<L3

L7=L8=L6=L1<L5=L4=L2<L3

L6=L1=L7=L4=L5=L8=L2=L3

L7=L8<L6<L1=L4=L5=L3=L2

L4=L6=L3=L1=L5=L2=L7<L8

L4=L3=L6=L2=L5=L1<7<8

L1<L2<L3=L4=L5=L6=L7=L8

L1=L2<L3<L4<L5<L6=L7=L8

L1<L2<L4=L3<L5<L6<L7<L8

L1=L2<L3=L8=L4=L5<L6=L7

L1=L2<L8=L3=L6=L5=L4=L7

Mesocosm Experiment

Table1.

Summary of the one way ANOVA and SNK tests performed to assess the effect of treatment on all C. cylindracea

variables in the mesocosm experiment: df = degree of freedom, MS = Mean Squares, F = F-statistics, p = P value,

SE= standard error; ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001

light availability allow highly positive carbon ba-

lances (Marín-Guirao et al.2015). Thus, results

obtained in this and previous studies support the

hypothesis that light plays a major role in deter-

mining the resistance of P. oceanica meadows to

C. cylindracea bioinvasion in the Mediterranean

Sea, but also indicates that other factors linked

to the meadow structure could also be involved in

the growth and colonization capacity of the alga.

Therefore, further experimentation would be ne-

cessary in future research to attain a better un-

derstanding of the vulnerability of this seagrass

habitat to C. cylindracea invasions.

5. Annex 1:

p. 92 p. 93

CHAPTER 4

TESIS DOCTORAL

Variable

Pmax Rd Ec Ek α Chl a Chl b Carotenoids Chl b/a Ratio Carotneoids/chl a Ratio Carotneoids/chl b Hc Hk Carbon balance Stolon biomass balance Apical growth rate Carbohydrate content

Effect

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

Treat.

Res.

df

3

8

3

8

3

8

3

8

3

8

3

8

3

8

3

8

3

8

3

8

3

8

3

24

3

24

3

24

3

12

3

12

3

12

MS

2,89

0,41

0,03

0,002

252,85

0,26

0,006

15,28

1,35

0,001

153,40

423,96

65,96

65,70

93,50

58,57

0,01

0,0008

0,0023

0,0002

0,11

0,001

8,23

2,47

55,50

4,41

0,30

0,019

0,0041

0,0003

0,12

0,031

28,60

1,42

SE

0,37

0,02

0,30

22,56

0,01

0,01

0,00

0,01

0,45

0,05

0,01

0,10

1,42

F

7,05

19,02

16,55

10,19

5,16

0,36

1,00

1,6

14,87

12,59

15,23

3,34

12,58

15,83

15,81

3,75

20,09

P

*

***

***

**

*

n.s.

n.s.

n.s.

**

**

**

n.s.

***

***

***

*

***

SNK test

SHADED=IN=CLIPPING<OUT

SHADED=IN=CLIPPING<OUT

SHADED=IN=CLIPPING=OUT

IN=SHADED=CLIPPING<OUT

OUT=CLIPPING=SHADED=IN

SHADED=IN<OUT=CLIPPING

CLIPPING=OUT=SHADED=IN

CLIPPING=OUT<SHADED<IN

IN=SHADED<CLIPPING=OUT

IN=SHADED<CLIPPING=OUT

IN<CLIPPING=SHADED<OUT

IN=CLIPPING=SHADED<OUT

IN=SHADED=CLIPPING<OUT

Mesocosm Experiment

Table 2.

Summary of the one way ANOVA and SNK tests performed to assess the effect of treatment on all C. cylindracea

variables in field experiment: df = degree of freedom, MS = Mean Squares, F = F-statistics, p = P value, SE= stan-

dard error; ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

DISCUSIÓNGENERAL

G E N E R A L D I S C U S S I O N

p. 97

1. Dispersión y dinámica poblacional de C. cylindracea a escala regional

Tras más de 20 años presente en el Mediterrá-

neo, C. cylindracrea puede ser considerada una

especie naturalizada, donde actualmente se en-

cuentra en fase de propagación, según el marco

general definido para la dinámica de especies

exóticas introducidas (Theoarides y Dukes 2007,

Blackburn et al. 2011). La observación de las pri-

meras colonias del alga en la Región de Murcia

en 2005 constata la continuación del proceso de

dispersión a través del Mediterráneo Occidental

en sentido este-oeste, que posteriormente ha

continuado hasta presentarse en la actualidad

en toda la cubeta occidental (Rivera-Ingraham et

al 2010). Los resultados de los trabajos iniciales

de seguimiento de la distribución del alga en esta

región del sureste peninsular, pusieron en eviden-

cia diversos aspectos sobre las pautas de disper-

sión y los mecanismos empleados que ya habían

sido puestos de manifiesto en otras regiones del

Mediterráneo (Klein y Verlaque 2009). Por un

lado, se observó un patrón espacial de dispersión

muy discontinuo, caracterizado por la aparición

de nuevas colonias aisladas y separadas entre si

distancias que oscilan entre centenas de metros

a decenas de kilómetros entre años sucesivos, lo

que indica (i) la importancia de los mecanismos

de reproducción vegetativa en la colonización

de nuevos hábitats y la elevada resistencia de

los fragmentos y propágulos generados y (ii) la

intervención de vectores secundarios de origen

antrópico en su dispersión a escala local y regio-

nal. Recientemente Papini et al. (2013), a través

de técnicas de Perfil Geográfico, han propuesto

un modelo para la dispersión del alga en el Me-

diterráneo en base al cual se establece que los

principales vectores secundarios implicados en

la propagación del alga a escala regional son el

fondeo de embarcaciones y el transporte asocia-

do a las artes de pesca. En efecto, la primera po-

blación detectada en Murcia se corresponde con

una zona frecuentada habitualmente por embar-

caciones de artes menores y muchos de los pun-

tos donde se ha detectado posteriormente son o

bien zonas con un elevado atractivo turístico don-

de es frecuente la presencia de embarcaciones

deportivas o zonas utilizadas habitualmente por

Las investigaciones desarrolladas en la presente tesis doctoral analizan diversos aspectos de la ecología

de C. cylindracea en el Mediterráneo aportando información sobre cuestiones relevantes relacionadas

con el éxito invasor como son (i) la dispersión y capacidad de desarrollo del alga en una nueva región,

(ii) los factores que controlan la introducción y propagación de la nueva especie y (iii) y el impacto sobre

las comunidades nativas.

En el capítulo 1 así como en el Anexo se ha examinado la capacidad de expansión del alga tras su

llegada a una nueva región, los patrones de dispersión a escala regional y la dinámica poblacional a

medio-largo plazo y en los capítulos 2, 3 y 4 se ha evaluado la influencia de factores abióticos del medio

(luz) sobre el potencial invasor del alga y la interacción con los hábitats nativos, más concretamente

sobre las praderas de P. oceanica. Los resultados obtenidos en los diferentes trabajos han sido discuti-

dos en cada capítulo pero se considera necesario presentarlos, en un contexto general, en el presente

apartado.

p. 98 p. 99

DISCUSIÓN GENERAL

TESIS DOCTORAL

la flota pesquera de la región (Ruiz et al. 2014).

Estos vectores secundarios podrían explicar tam-

bién la propagación del alga desde las colonias

más cercanas conocidas, localizadas entonces en

la provincia de Alicante. A escalas espaciales lo-

cales, factores naturales como la hidrodinámica

local pueden también contribuir a la dispersión

del macrófito debido al transporte de fragmentos

y propágulos entre zonas próximas, al igual que

ya ha sido descrito para C. taxifolia en el Medi-

terráneo (Thibaut 2001). Por último, tampoco se

puede descartar la posibilidad de otros vectores

como el tráfico marítimo pesado vinculado con

las grandes infraestructuras portuarias, que juega

un papel clave en la dispersión del alga a escala

geográfica (Papini et al 2013). De hecho, las pri-

meras colonias observadas en las localidades de

Calblanque y Cabo Tiñoso comprenden una am-

plia área marina caracterizada por una elevada

densidad de este tipo de tráfico a consecuencia

de la presencia del puerto de Cartagena y la refi-

nería de petróleo de la dársena de Escombreras,

uno de los nudos más importantes de conexión

de rutas marítimas del Mediterráneo. Al igual que

en otras zonas del Mediterráneo (ver revisión en

Klein y Verlaque 2009), C. cylindracera en las cos-

tas murcianas se ha desarrollado principalmente

en zonas dominadas por fondos detríticos y de

Mäerl, fondos rocosos con comunidades de algas

fotófilas y fondos con mata muerta de P. oceani-

ca. Se han registrado tasas de colonización muy

elevadas en los primeros tras su aparición (de

hasta 1ha año-1), similares a las reportadas pre-

viamente en otras regiones (Piazzi et al. 1997b,

De Biasi et al 1999) y que confirman la alta ca-

pacidad de expansión que puede llegar a desa-

rrollar el del alga bajo condiciones ambientales

favorables una vez se ha establecido.

Tras esta fase de dispersión inicial, se continuó

el seguimiento de la abundancia del alga en un

número limitado de localidades para conocer en

detalle la tendencia de la dinámica de las pobla-

ciones establecidas, y sus variación intra-anual

(estacional) e interanual. Los resultados recogi-

dos en el Anexo constituyen la primera serie tem-

poral a medio-largo plazo (8 años, 2007-2014)

sobre la abundancia del alga en el Mediterráneo.

La elevada frecuencia de regresiones invernales

observada en el periodo estudiado (Anexo) y los

bajos balances de carbono obtenidos durante el

invierno (Capítulo 3) sugieren un claro patrón

estacional similar al observado en otras zonas

del Mediterráneo (Ruitton et al. 2005b, Lenzi et

al 2007), definido por notables diferencias en la

abundancia del alga entre invierno y verano. Es-

tudios posteriores del ciclo de crecimiento anual

del alga realizados en la zona (Bernardeau-Es-

teller, datos no publicados y no presentados en

esta tesis) han confirmado este patrón estacio-

nal en el que se identifica una época de máximo

crecimiento y abundancia en verano y principio

de otoño, y una época en la que su crecimiento

se muestra severamente ralentizado en invierno

y principio de primavera. Aunque las poblaciones

del alga pueden persistir durante las condiciones

más adversas del invierno (Capítulo 3 y Anexo) es

probable que en esta época desfavorable su resi-

liencia se vea reducida y por tanto se incremente

la vulnerabilidad ante otras fuentes de pertur-

bación mecánica, como por ejemplo el elevado

hidrodinamismo propio de las tormentas inver-

nales. En esta época precisamente se ha podido

comprobar que las poblaciones del alga son muy

vulnerables a los efectos de los grandes tempora-

les, incluso en las zonas más profundas (hasta 26

metros), lo que explicaría las bruscas regresiones

invernales observadas en la mayoría de los años.

Sin tener en cuenta las variaciones estacionales,

durante todo el periodo de estudio C. cylindracea

ha mantenido poblaciones estables en las esta-

ciones monitorizadas, con niveles de abundancia

que oscilan dentro de los rangos ya definidos

para la especie (ver revisión en Klein y Verlaque

2009), aunque con importantes fluctuaciones

interanuales que parecen sugerir dos etapas en

su desarrollo en la región. La primera etapa está

asociada a los primeros años de la aparición del

alga, en la que se observan los valores máximos

de biomasa (hasta 70g/m2), y la segunda etapa

a los siguientes años del periodo de seguimiento,

en los que los valores de abundancia se mantie-

nen dentro de unos niveles más bajos, inferiores a

30g/m2, y más o menos constantes a lo largo del

tiempo. Esta evolución parece reflejar una diná-

mica característica de muchas especies exóticas

en fase de propagación, caracterizada por la apa-

rición de fluctuaciones importantes en la abun-

dancia a lo largo del tiempo como consecuencia

de la interacción más o menos compleja con

factores abióticos y bióticos del medio (i.e. com-

petencia, predación, etc; Boudouresque 1999,

Blackburn et al. 2011). Estas dinámicas impiden

por tanto descartar proliferaciones del alga en el

futuro tal y como se ha descrito en otras macro-

algas exóticas en el Mediterráneo (Boudoures-

que 1999). En cualquier caso parece descartarse

un declive de la especie, como ha sido sugerido

en otras zonas del Mediterráneo (Barbara et al.

2013) o una dinámica regresiva a largo plazo

como la observada para C. taxifolia (Jauber et al.

2003, Iveša et al. 2006) Los datos más recientes

del seguimiento del alga en la región de Murcia

(Ruiz et al. 2014) indican que la especie sigue

presente con abundancias apreciables en todas

las localidades monitorizadas (Capítulo 1).

p. 100 p. 101

DISCUSIÓN GENERAL

TESIS DOCTORAL

Como ya se comentaba en la introducción de la

presente tesis, el éxito en la introducción de una

especie en un nuevo hábitat o ecosistema esta

condicionado, entre otros factores, por (i) las

características y atributos de la propia especie

y (ii) la resistencia de los hábitats y ecosistemas

receptores a la colonización. Atributos como la

elevada capacidad de crecimiento o el desarrollo

de mecanismos de reproducción vegetativa han

sido ya señalados en apartados anteriores como

determinantes para comprender el éxito de esta

especie en el Mediterráneo y han sido reconoci-

dos en general como rasgos característicos de

otras especies sifonales con una alta capacidad

invasora (Willianms y Smith, 2007). Por otro lado,

existen diversas evidencias que sugieren que el

alga presenta una elevada plasticidad fenotípi-

ca a nivel fisiológico y morfológico ante factores

abióticos decisivos capaces de limitar el desarro-

llo y distribución de los macrófitos marinos como

la salinidad, la temperatura o la luz (Lobban

1997). Esta importante capacidad aclimatativa

explicaría, por tanto, la elevada tolerancia am-

biental y el amplio nicho ecológico mostrado por

C. cylindracea en muchas zonas del Mediterráneo

(ver revisión en Klein y Verlaque 2009).

En concordancia con estos estudios, los trabajos

desarrollados en el Capítulo 1 permitieron identi-

ficar poblaciones del alga en aguas de la Región

de Murcia sobre gran variedad de sustratos y con-

diciones ambientales. Sin embargo, y al igual que

ha sido descrito por otros autores (Katsanevakis

et al.2010, Bulleri et al. 2011), se observó que una

de las comunidades más resistentes a la coloniza-

ción son las constituidas por macrófitos de porte

erecto y que forman grandes doseles vegetales

(canopy formers) y más concretamente las pra-

deras de Posidonia oceanica. La susceptibilidad a

la invasión de una determinada comunidad o há-

bitat está, entre otros factores, relacionada con

la disponibilidad de los recursos abióticos y con

la forma en que las interacciones con las comu-

nidades nativas modifican dicha disponibilidad.

Aunque diversas investigaciones habían anali-

zado ya la plasticidad fotoaclimatativa del alga

ante diversos gradientes de luz en el Mediterrá-

neo (Raniello et al. 2004 y 2006), los trabajos de-

sarrollados en los capítulos 2, 3 y 4 han permitido

por primera vez (i) estudiar diversas respuestas

de fotoaclimatación bajo condiciones controla-

das de laboratorio y (ii) valorar la repercusión de

estos mecanismos sobre el crecimiento y capaci-

dad productiva del alga,. Los principales resulta-

dos obtenidos en estos capítulos son analizados

en los dos apartados siguientes.

2. Mecanismos que regulan la introducción de C. cylindracea en el Mediterráneo

a fotoaclimatación es el conjunto de respuestas

desarrollados por los organismos autótrofos ante

cambios en la condiciones lumínicas con el ob-

jetivo de mantener un balance positivo entre la

energía generada a través de la fotosíntesis y la

energía metabólicamente consumida (Kirk 1994,

Raven y Geider 2003). El análisis de los mecanis-

mos de fotoaclimatación en condiciones contro-

ladas de mesocosmos (Capítulo 4) permitió cons-

tatar que las distintas respuestas identificadas

previamente en condiciones naturales (gradiente

de profundidad y gradiente generado por el dosel

vegetal de P. oceanica; capítulos 2, 3 y 4) estaban

relacionadas inequívocamente con las variacio-

nes en los regimenes lumínicos. Estas respuestas

fotoaclimatativas (resumidas en la Tabla 1) fue-

ron fundamentalmente de tres tipos, (i) variacio-

nes a nivel morfológico, (ii) cambios en el aparato

fotosintético y (iii) ajustes en la actividad meta-

bólica. Estas respuestas reflejan la activación de

mecanismos de aclimatación habitúales en ma-

crófitos marinos (Kirk 1994), incluyendo esta y

otras especies congéneres en el Mediterráneo

(Gacia et al. 1996b, Häder et al. 1997, Raniello

et al. 2004, 2006). Curiosamente, los ajustes a

nivel metabólico expresados por la modificación

de las tasas respiratorias y que jugaron un papel

determinante en la aclimatación de la especie,

sólo habían sido descritos hasta la fecha en po-

blaciones tropicales del alga (Riechert y Dawes

1986). Algunas de estas respuestas no sólo refle-

jan cambios cuantitativos de la disponibilidad

de luz, sino también cambios cualitativos del régi-

men lumínico debido a la modificación que expe-

rimenta el espectro de luz con la profundidad o al

atravesar el dosel foliar de P. oceanica. En sínte-

sis, el objetivo último de todos estos mecanismos

es (i) mantener la capacidad absorción de luz y su

uso eficiente a nivel fotosintético y (ii) reducir la

demanda metabólica para preservar el balance

energético del alga y reducir los requerimientos

lumínicos para su crecimiento (Kirk 1994, Raven

y Geider 2003, Falkowski et al. 2007).

2.1. El papel de la luz

2.1.1. Mecanismos de fotoaclimatación de C. cylindracea

p. 102 p. 103

DISCUSIÓN GENERAL

TESIS DOCTORAL

Mecanismo de fotoaclimatación

Cambios morfológicos

Reorganización del aparato

fotosintético

Cambios metabólicos

Tipos de respuesta Indicadores Gradiente lumínico

Cambios contenido

pigmentario

Cambios funciomaniento

fotosintético

Cambios tasa

respiratoria

Longitud Fronde

Chla

Chlb

Carotenoides

Ratio pigmentos

Pmax

α

Ek

Ec

Rd

profundidad

dosel P. oceanica

mesocosmos

profundidad

dosel P. oceanica

mesocosmos

profundidad

dosel P. oceanica

mesocosmos

dosel P. oceanica

mesocosmos

profundidad

dosel P. oceanica

mesocosmos

profundidad

dosel P. oceanica

mesocosmos

profundidad

doselP. oceanica

mesocosmos

profundidad

dosel P. oceanica

mesocosmos

profundidad

dosel P. oceanica

mesocosmos

profundidad

dosel P. oceanica

Las elevadas variaciones registradas en la mayor

parte de las variables analizadas (e.g. reduccio-

nes de hasta el 70% en las tasas respiratorias

e incrementos en el contendido pigmentario de

hasta el 40% bajo condiciones severas de limita-

ción de luz) evidencian la importante plasticidad

de los mecanismos fotoaclimatativos desarrolla-

dos por la especie.

En condiciones naturales, los patrones de acli-

matación expresados mostraron divergencias

entre los distintos experimentos realizados. Se

observaron variaciones tanto cualitativas como

cuantitativas en las respuestas manifestadas

por el alga entre los distintos niveles de un gra-

diente espacial, ante gradientes similares en

épocas distintas del año e incluso ante gradien-

tes similares en la misma época del año pero en

experimentos realizados en años diferentes. Por

ejemplo, en el Capítulo 2 se puso de manifiesto

como el alga es capaz de desarrollar diferentes

estrategias fotoaclimatativas en función de los

cambios del régimen lumínico que se producen

con la profundidad. A profundidades intermedias

del rango batimétrico que ocupa C. cylindracea

en la zona de estudio, los mecanismos desarrolla-

dos por el alga para compensar la reducción en la

disponibilidad de luz y optimizar su utilización, se

basan fundamentalmente en cambios en el apa-

rato fotosintético manifestados por incrementos

en la eficiencia fotosintética (α). A profundida-

des mayores, donde la reducción de la luz es más

severa, dichos mecanismos compensatorios se

basan principalmente en estrategias de reduc-

ción de la demanda metabólica (reducción de

la respiración, Rd). Sin embargo, este patrón de

aclimatación mostraba ciertas variaciones signi-

ficativas cuando se midió al año siguiente sobre

los mismos sitios, y en la misma época (verano),

para la realización de los experimentos descritos

en el Capítulo 4. Por ejemplo, se pudo observar

como en profundidades intermedias como les

mecanismos de respuesta además de cambios en

el aparato fotosintético incorporaban también

una reducción de las tasas respiratorias. Por otro

lado, en cada nivel de profundidad, este patrón

de aclimatación varía también considerablemen-

te según la época del año. Así, por ejemplo, en

invierno se observa un mayor esfuerzo de fotoa-

climatación (reflejado por la mayor reducción de

las tasas fotosintéticas, respiratorias, así como

por los valores de Ec y Ek en relación al verano)

como consecuencia de la mayor reducción en la

disponibilidad de luz característica de esta esta-

ción, especialmente en las zonas mas profundas.

Esta variabilidad espacio-temporal de la respues-

ta se justifica si tenemos en cuenta que la acli-

matación a la luz es un proceso energéticamente

costoso condicionado por multitud de factores,

aparte de las características que definen el régi-

men lumínico, que pueden ser tanto ambientales

(temperatura, disponibilidad de nutrientes, etc.)

como inherentes al propio organismo (contenido

nutricional interno, ciclo de vida, requerimientos

para el crecimiento, reservas de carbono, etc.)

(Lobban y Harrison 1997, Hanelt et al. 2003). Por

tanto, las respuestas fotoaclimatativas expresa-

das por el alga en cada situación determinada

(profundidad, época, etc.) serán las que permitan

una mejor optimización del uso de la luz en base

a los recursos disponibles y posibilidades de la es-

pecie. En este contexto, Flagella et al. (2008) han

sugerido que C. cylindracea presenta un compor-

tamiento tipo “anticipador estacional” (seasonal

anticipator), por lo que las respuestas menciona-

das podrán también estar condicionadas por los

programas de aclimatación estacional internali-

zados por la especie.

Tabla 1.

Mecanismos de fotoaclimatación de C. cylindracea. Chla y Chlb, contenidos en clorofila a y b; Pmax, fotosíntesis neta máxima; α;

eficiencia fotosintetica; Ek, irradiancia de saturación; Ec, irradiancia de compensación; Rd, dark respiration rate.

p. 104 p. 105

DISCUSIÓN GENERAL

TESIS DOCTORAL

El objetivo final de los procesos de fotoaclimata-

ción es mantener cierta capacidad productiva y

una tasa de crecimiento positiva cuando las con-

diciones de luz son subóptimas (Kirk 1994, Raven

y Geider 2003). Por tanto, la distribución poten-

cial de un organismo en ambientes con diferen-

tes regímenes lumínicos dependerá de la mayor

o menor plasticidad de sus mecanismos de acli-

matación (Kirk 1994, Falkowski et al. 2007). Estos

aspectos son especialmente relevantes en el caso

de la introducción de macrófitos marinos, ya que

el potencial colonizador estará condicionado por

su capacidad productiva y de crecimiento. En el

Capítulo 3, mediante la simulación de la ausen-

cia de mecanismos de fotoaclimatación en las

poblaciones profundas del alga, se puso de ma-

nifiesto cómo las respuestas fotoaclimatativas

pueden haber jugado un papel determinante en

el éxito colonizador de la especie en los amplios

rangos batimétricos en que se ha desarrollado

en el Mediterráneo, ya que se traducen de forma

efectiva en una optimización de las tasas produc-

tivas bajo condiciones de luz limitante.

La plasticidad de la respuesta a las variaciones

en los regimenes de luz puede estar limitada por

diversos factores entre los que evidentemente se

incluye la propia disponibilidad del recurso (Van

Kleunen y Fischer 2005). La respuesta de aclima-

tación se encuentra condicionada por los propios

costes de producción de los elementos implica-

dos en dicha respuesta (p.e. pigmentos, enzimas,

etc.) así como por los asociados al propio mante-

nimiento de la maquinaria de aclimatación (Ra-

ven y Geider 2003). Por tanto, unos niveles de luz

muy reducidos pueden determinar una reducción

de los recursos internos disponibles, reduciendo la

capacidad del alga de invertir en mecanismo de

fotoaclimatacion y determinado por tanto una li-

mitación en su respuesta. La falta de adecuación

biológica derivada de los límites en la capacidad

de aclimatación puede a su vez determinar un

desequilibrio entre la energía generada a través

de la fotosíntesis y los propios costes metabólicos

del organismo, que son en definitiva expresados

como una inhibición de la capacidad productiva

y de crecimiento. Estas condiciones determinan

por tanto los requerimientos mínimos de luz que

permiten el desarrollo de un organismo vegetal.

En condiciones de mesocosmos, los niveles mí-

nimos de luz que permitían el crecimiento del

alga se obtuvieron en valores próximos a 0,24

mol quanta m-2d-1 (tratamiento L2, Capítulo 4).

Bajo estos niveles de irradiancia, C. cylindracea

no fue capaz de continuar desarrollando ningún

tipo de respuesta fotoaclimatativa adicional

respecto a la respuesta observada a niveles su-

periores de irradiancia. Esta incapacidad se tra-

duce en periodos de saturación (Hk) y balances

de carbono próximos a cero y, en consecuencia

una fuerte limitación de su capacidad producti-

va y de crecimiento. En condiciones naturales, se

ha comprobado que estos escenarios limitantes

para el desarrollo del alga tienen lugar dentro del

dosel de la pradera, en las estaciones de invierno

(Capítulo 3) y otoño (Capítulo 4), e independien-

temente de la profundidad, y también fuera del

dosel foliar de la pradera, pero solo en invierno y

en la estación más profunda (26m; Capítulo 3).

En muchos de estos casos, como se observa en

la tabla 2, la irradiancia total diaria presentaba

valores medios algo superiores al establecido en

condiciones de mesocosmos como limitante del

desarrollo algal, es decir, 0,24 mol quanta m-2d-1.

Sistema experimental

MESCOSMOS

CAMPO IN

OUT

Tratamiento/Localidad Irradiancia Total Diaria(mol quanta m-2d-1)

Época muestreo

L2

I. grosa (-11m)

C. Tiñoso (-18m)

Calblanque (-26m)

I. grosa (-11m)

C. Tiñoso (-18m)

Calblanque (-26m)

11.74±0.56

9.47±0.30

7.52±0.24

1.78±0.13

2.30±0.02

1.59±0.26

15.74±0.92

10.50±0.30

4.75±0.34

0.71±0.06

0.20±0.04

0.11±0.13

5.06±0.31

1.61±0.23

0.63±0.10

0.24±0.01

0.41±0.08

4.56±0.72

Verano

2008

Verano

2009

Otoño

2011

Invierno

2009

Estas divergencias entre los resultados obtenidos

en el sistema de mesocosmos y los experimentos

in situ, no son más que un reflejo de las inevita-

bles diferencias entre las condiciones de labora-

torio, más controladas, y las condiciones natura-

les, sujetas a mayor variabilidad y complejidad

(interacción con otros factores). En primer lugar,

en el mesocosmos, los niveles de luz son contro-

lados y mantenidos a niveles constantes, y en la

naturaleza experimentan cierta variabilidad cau-

sada por los numerosos factores que afectan a la

cantidad de radiación solar incidente o a la turbi-

dez de la columna de agua. Por otro lado, existen

otros factores (p.e. herbivoría, hidrodinamismo,

etc) que pueden (i) condicionar o limitar la plas-

ticidad de aclimatación a la luz (Valladares et al.

2007) y/o (ii) incrementar los costes de manteni-

miento del organismo (Markager y Sand-Jensen

1994), lo que en definitiva resulta en un incre-

mento de los requerimientos de luz necesarios

para el desarrollo.

En cualquier caso, los experimentos realizados

ponen de manifiesto que el régimen lumínico

que prevalece bajo el dosel foliar de P. oceanica

es limitante para el desarrollo del alga durante la

mayor parte del año. Además, en la época más

favorable (verano), los niveles de irradiancia, pese

a superar los requerimientos mínimos para el cre-

cimiento, son también considerablemente bajos

en comparación con los registrados fuera de la

pradera. Esta situación es consistente con la ca-

pacidad del dosel foliar de P. oceanica de mante-

ner abundancias del alga muy bajas a lo largo del

tiempo, como se observa en las praderas monito-

readas entre 2007 y 2014 (Anexo). Por tanto, los

resultados obtenidos apoyan la hipótesis inicial

de que la disponibilidad de luz dentro de las pra-

deras de P. oceanica juegan un papel relevante

para explicar la limitada capacidad colonizadora

del alga en estos hábitats. Al igual que sucede en

otras especies de Caulerpa (Terrados y Ros 1992,

Robledo y Freile-Pelegrín 2005) y otros macrófi-

tos marinos (Rosemberg y Ramus 1982, Gagne et

al. 1982, Dunton y Schell 1986, Gomez y Wiencke

1998) sometidos a ambientes lumínicos limitan-

tes de forma estacional, es probable que durante

la época favorable se favorezca la producción de

sustancias de reserva que faciliten la subsisten-

cia de la población en la época desfavorable. Es

2.1.2 Consecuencias de los procesos de fotoaclimatacion en la capacidad de colonización

de C. cylindracea.

Tabla 2.

Resumen de las medidas de irradiancia obtenidas en las poblaciones de C. cylindracea donde se observaron condiciones limi-

tantes para el crecimiento del alga.

p. 106 p. 107

DISCUSIÓN GENERAL

TESIS DOCTORAL

posible que otros mecanismos puedan estar tam-

bién interviniendo en el mantenimiento de las

poblaciones, como por ejemplo, la obtención de

carbono mediante heterotrofía, como ya ha sido

documentado en C. taxifolia (Chisholm y Jaubert

1997), o la translocación de recursos desde fuera

de la pradera gracias a la naturaleza cenocítica

del alga (Collado-Vides y Robledo 1999). La au-

sencia de este mecanismo de resistencia podría

explicar también la mayor susceptibilidad mos-

trada por otras angiospermas en el Mediterrá-

neo, que dado su menor porte determinan cam-

bios menos drásticos en los regimenes lumínicos

(ver revision en Klien y Verlaque 2009).

Por otro lado, y de acuerdo con los resultados ob-

tenidos, la disponibilidad de luz perece jugar un

papel importante no solo bajo el dosel foliar de P.

oceanica, sino también para explicar la variación

de su capacidad colonizadora en los sustratos

fuera de las praderas a lo largo de gradientes na-

turales de irradiancia asociados a la profundidad.

Si no tenemos en cuenta episodios puntuales de

regresión, entre 2007 y 2014 la abundancia de

las poblaciones más someras fuera de la pradera

fue superior al registrado en la localidad más pro-

funda (Anexo). Como se ha demostrado, durante

el invierno en esta localidad más profunda las

condiciones lumínicas son totalmente limitantes

para el desarrollo de C. cylindracea (fuera de la

pradera), lo cual limita a su vez la capacidad de

desarrollo del alga en las épocas más favorables.

Esto es consistente con el mencionado gradien-

te de biomasa del alga y sugiere que, al menos

en la zona estudiada, la capacidad colonizadora

del alga se encuentra limitada a partir de profun-

didades de 25 m. Esta idea no es incompatible

con la observación de poblaciones del alga a

profundidades superiores en ésta y otras zonas

del Mediterráneo español. De forma similar, se

ha obtenido evidencia de la importancia de la

disponibilidad de luz para explicar la distribución

vertical de otras especies invasoras como Wo-

mersleyella setacea (Cebrian y Rodríguez-Prieto,

2012). El patrón de abundancia de C. cylindracea

a lo largo de gradientes de profundidad descri-

to en la zona de estudio, ha sido observado en

otras áreas del Mediterráneo (Capiomont et al.

2005, De Biasi et al. 1999), pero no se ha des-

crito en otras zonas (Cebrian y Ballesteros 2009).

Esto es probablemente debido a la influencia

de otros factores capaces de enmascarar este

patrón de abundancia subyacente. Por ejemplo,

en las estaciones someras contempladas en este

trabajo, en algún año la abundancia del alga se

mantuvo a niveles tan bajos como las estaciones

más profundas debido a la incidencia de fuertes

temporales antes de la realización de los mues-

treos. Por otro lado, en estas zonas someras se

ha descrito que la herbivoría sobre C. cylindracea

es muy intensa y mantiene la abundancia de sus

poblaciones a niveles muy bajos. Ambos factores,

hidrodinamismo y herbivoría, están sujetos a una

elevada variabilidad interanual.

Diversas investigaciones sobre los mecanismos

asociados a la resistencia que ofrecen las comu-

nidades marinas a la introducción de macrófitos

exóticos revelan que, dicha resistencia parece

estar estrechamente ligada a la identidad y di-

versidad de los grupos funcionales de producto-

res primarios que componen la comunidad y a la

forma en que dichos grupos usan y compiten por

los recursos disponibles (Arenas et al. 2006, Brit-

ton-Simmons 2006). Estas ideas se contraponen

a las teorías basadas en los postulados de Elton

(1958) que establecen que la resistencia biótica

a la invasión de una comunidad estaría vincula-

da con su diversidad específica, de manera que

una mayor diversidad determinaría un uso más

completo de los recursos, reduciendo su dispo-

nibilidad para las nuevas especies introducidas

(teoría del uso complementario de los recursos;

Hooper 1998)). Una investigación reciente basa-

da en técnicas de metanálisis ha mostrado que

mientras en las comunidades terrestres la diver-

sidad funcional tiene un papel relevante en la

resistencia a la introducción, su papel en los eco-

sistemas marinos es menos importante ya que

parece ser determinante la presencia de ciertos

grupos funcionales con una elevada capacidad

de competencia por los recursos primarios (Kim-

bro et al. 2013).

Los estudios desarrollados en los capítulos 3 y 4

muestran que el severo control definido por el

dosel vegetal de P. oceanica sobre la disponibili-

dad de la luz es un mecanismo competitivo fun-

damental para explicar la resistencia biótica a la

colonización del alga y aportan por tanto, nue-

vas evidencias sobre el papel del grupo funcional

constituido por “canopy -former species” en los

mecanismos de resistencia de comunidades na-

tivas frente a la introducción de especies exóticas

marinas. La baja capacidad colonizadora mos-

trada por el alga durante el periodo 2007-2014

bajo el dosel foliar de P. oceanica (Anexo) indica

la prevalencia de estos mecanismos de resisten-

cia a largo plazo y refleja que las praderas de P.

oceanica en buen estado de conservación actúan

a modo de barrera ecológica frente a la disper-

sión del alga, algo que ya había sido sugerido por

diversas investigaciones en otras zonas del Medi-

terráneo (Katsanevakis et al. 2010, Bulleri et al.

2010). La eficacia de estos mecanismos de resis-

tencia implica el mantenimiento de la estructura

tridimensional definida por el dosel foliar de la

pradera. En las zonas colonizadas por C. cylindra-

cea, las praderas de P. oceanica han mostrado es-

tabilidad estructural, como evidencia el análisis

de sus tendencias poblacionales realizado en el

Anexo, que son estables o positivas, y similares a

las observadas en praderas de zonas no invadidas

de la misma zona. Estas tendencias se observan

también en praderas invadidas de otras zonas de

la Región de Murcia (Ruiz et al. 2014) así como

en praderas no invadidas de otras regiones medi-

terráneas (Sanchez-Rosas et al. 2009, Álvarez et

al. 2009, Guillén et al. 2013, González-Correa et

al. 2015) Por tanto, de acuerdo con estos resulta-

dos, en las zonas invadidas por C. cylindracea, no

parece existir algún tipo de interacción negativa

entre el alga y la angiosperma que implique un

deterioro de la vitalidad de la pradera de P. ocea-

nica, al menos a nivel estructural y poblacional en

la región estudiada. Aunque estos resultados no

se pueden generalizar y extrapolar a otras áreas

geográficas, no se dispone de evidencia científi-

ca consistente de que dicha interacción negativa

2. 2. Interacción entre C. cylindracea y P. oceanica: Resistencia biótica de las praderas

de P. oceanica

p. 108 p. 109

DISCUSIÓN GENERAL

TESIS DOCTORAL

haya tenido lugar en otras localidades mediterrá-

neas.

Estos resultados contrastan con el potencial atri-

buido a C. cylindracea de alterar el desarrollo

vegetativo de la angiosperma, bien mediante la

acción de substancias alelopáticas (Raniello et

al. 2007, Dumay et al. 2002b) o bien a través de

la anoxificación de los sedimentos que coloniza,

con la consiguiente acumulación de fitotóxicos

frente a los que P. oceanica ha mostrado cierta

vulnerabilidad (Holmer et al. 2009). Sin llegar

a cuestionar el potencial de estos mecanismos

de acción, es necesario matizar que se requiere

nueva y robusta evidencia experimental que de-

muestre de forma efectiva la relación entre di-

chos mecanismos y el deterioro de la estructura

y vitalidad de la pradera de P. oceanica. Por otro

lado, los mecanismos de acción mencionados

estén probablemente vinculados (i) al desarrollo

de grandes biomasas del alga y su persistencia

en el tiempo de manera que se puede dar una

alteración de las condiciones del sedimento y (ii)

a una elevada disponibilidad de recursos internos

que permitan la sisntesis de compuestos secun-

darios que actúen a modo de compuestos ale-

lopaticos. Sin embargo, como se ha descrito en

apartados anteriores, por un lado, dentro de las

praderas el desarrollo del alga se encuentra muy

limitado (capacidad productiva y de crecimiento

reducidas); por otro lado, fuera de las praderas,

C. cylindracea puede colonizar los sustratos adya-

centes a los límites de las mismas, pero su bioma-

sa sigue una dinámica altamente fluctuante en

el tiempo, entre años y estacionalmente dentro

de cada año.

La demostrada incapacidad de C. cylindracea

de colonizar los sustratos en el interior del es-

trato foliar de P. oceanica, contrasta también

con su mayor capacidad de invadir el interior de

las praderas de otras especies de angiospermas

mediterráneas, como C. nodosa y Z. noltii (Cec-

cherelli y Campo 2002, Raniello et al. 2004). La

estructura del dosel foliar desarrollado por estas

angiospermas es mucho menos complejo que el

formado por las hojas de P. oceanica, cuyas hojas

presentan además concentraciones de pigmen-

tos fotosintéticos mayores y absortancias de luz

incidente superiores al 85% (Sandoval-Gil et al.

2013Esto se traduce en una mayor transmitan-

cia de la irradiancia incidente (del 50%) a través

del dosel foliar de C. nodosa y Z. noltii (Raniello

et al. 2004 ) y, por tanto, en una mayor dispo-

nibilidad de la luz en el interior de las praderas

que forman que, a su vez, permitiría mayores de-

sarrollos del alga invasora dentro de las mismas.

En este caso, el desarrollo de mayores biomasas

de C. cylindracea en el interior de las praderas de

ambas especies podría hacer efectivos los meca-

nismos de acción antes mencionados y explicar

la interacción documentada experimentalmente

entre el alga y las angiospermas, negativa en el

caso de C. nodosa y positiva en el caso de Z. noltii

(Ceccherelli y Campo 2002).

Esta situación descrita para C. cylindracea es

comparable a la observada con C. taxifolia en el

Mediterráneo, donde muestra una capacidad li-

mitada de colonizar praderas de P. oceanica y de-

sarrollar un impacto negativo a largo plazo sobre

las mismas (Jaubert et al. 1999). Por el contrario,

otros macrófitos invasores como Lophocladia

lallemandi han mostrado una mayor capacidad

de afectar a la vitalidad de la angiosperma, de-

teriorando su estructura, probablemente como

consecuencia de la reducción de la disponibilidad

de luz incidente y la alteración del balance de car-

bono (Ballesteros et al. 2007). La capacidad dife-

rencial observada entre las especies de Caulerpa

y la rodofícea para interaccionar con la angios-

perma radica en su diferente naturaleza y mo-

dos de acción, de acuerdo con el modelo general

desarrollado por Thomsen et al. (2012) sobre los

impactos negativos de macrófitos marinos en

praderas de angiospermas. L. lallemandi se com-

porta en el Mediterráneo como epífita de otros

macrófitos mientras que tanto C. cylindracea

como C. taxifolia presentan un tipo de desarrollo

ligado al sustrato. El crecimiento epífito podría

permitir al alga roja eludir las limitaciones lumí-

nicas impuestas por el dosel vegetal de P. ocea-

nica, lo que favorecería el desarrollo de mayores

biomasas que incrementarían su capacidad com-

petitiva por este recurso y determinarían, a su

vez, alteraciones en las condiciones ambientales

(incrementos en las tasas de sedimentación, en el

grado de anoxificación de los sedimentos y en el

enriquecimiento orgánico de los mismos).

La perturbación de las comunidades nativas, ya

sea debida a causas naturales o antrópicas, pue-

de determinar la reducción en la abundancia de

las especies que componen dichas comunidades

o cambiar las condiciones ambientales, incre-

mentando los recursos disponibles y siendo por

tanto un factor que puede facilitar el desarrollo

de las especies exóticas (Olyarnik et al. (2009).

Los cambios en el ambiente lumínico definidos

por la pradera de P. oceanica están basados en la

estructura tridimensional del dosel foliar, de for-

ma que la perturbación o alteración física del mis-

mo podría reducir la resistencia del hábitat a la

colonización por el alga invasora. De acuerdo con

esta hipótesis, Tamburello et al. (2014) y Cecche-

relli et al. (2014) han observado como la reduc-

ción del estrato foliar simulando fenómenos de

herbivoría severos promueve la proliferación del

alga. En el Capítulo 4 se pudo comprobar como

la manipulación de la estructura de la pradera de

P. oceanica incrementaba la disponibilidad lumí-

nica por encima de los requerimientos mínimos

de crecimiento de C. cylindracea. Sin embargo, se

puso también de manifiesto que probablemente

la interacción con otros factores asociados a las

características estructurales de la pradera (p.e.

la abrasión asociada al movimiento de las hojas

durante condiciones de elevado hidrodinamismo

(fuertes temporales)) podrían también contribuir

a limitar el crecimiento del alga. En este sentido,

las perturbaciones severas que pueden causar

alteraciones significativas de la estructura de la

pradera, como por ejemplo el fondeo de embar-

caciones, han sido relacionados con un aumento

de la expansión del alga (Tamburello et al. 2014).

La propagación del alga invasora mediada por

la supresión de los mecanismos de resistencia

del hábitat apenas han sido evaluados hasta la

fecha (Ceccherelli et al. 2014), pero tal y como

se comentaba anteriormente no es descartable

que la reducción de dichos mecanismos pueda

a su vez de manera directa o indirecta promover

los mecanismos de interacción aumentando el

potencial impacto negativo sobre las praderas.

Bullleri et al. (2011) han observado a su vez que

la capacidad de resiliencia de comunidades na-

tivas de fondos rocosos que incluyen macrófitos

de porte erecto sometidos a fuentes de pertur-

bación antrópica se ve mermada como conse-

cuencia de la invasión del alga. En el caso de C.

taxifolia existen evidencias que indican que el

deterioro de la pradera de P. oceanica puede, por

un lado, reducir la resiliencia del hábitat tras el

impacto de otros factores estresantes múltiples

(Molenaar et al. 2009) y por otro, favorecer y ace-

lerar un deterioro adicional de la pradera (Vilelle

y Verlaque1995).

p. 110 p. 111

DISCUSIÓN GENERAL

TESIS DOCTORAL

Dinámica de C. cylindracea en el Mediterráneo e

impacto sobre las comunidades nativas

Aún dentro de las fases de propagación, las po-

blaciones de los macrófitos invasores pueden

reflejar importantes variaciones en el tiempo, tal

y como muestran los resultados obtenidos en el

Capítulo 2. Estas variaciones son la expresión de

las complejas relaciones que se establecen entre

las especies introducidas y los ecosistemas nati-

vos en las que intervienen factores ecológicos y

evolutivos (Diezt y Edwards 2006) y que pueden

modular los efectos de las especies invasoras a lo

largo del tiempo (Strayer et al. 2011). Los estu-

dios que han evaluado la interacción de C. cylin-

dracea con las comunidades nativas se basan en

experimentos a corto-medio plazo que por tanto

pueden ofrecer una idea errónea de los impac-

tos reales del alga en las comunidades nativas.

Por tanto, una adecuada evaluación de dichos

impactos implica el desarrollo de estudios a más

largo plazo, especialmente en aquellas comuni-

dades biológicas ecológicamente relevantes que

han mostrado una mayor susceptibilidad a la

invasión, como por ejemplo las comunidades de

algas fotófilas, las comunidades de coralígeno y

Mäerl o las comunidades de otras angiospermas

marinas como Cymodocea nodosa.

Interacción entre C. cylindracea y P. oceanica.

A pesar de los resultados y conclusiones obteni-

dos sobre la interacción entre ambas especies a

lo largo de la presente tesis, existen todavía nu-

merosos aspectos de dicha interacción que toda-

vía se desconocen y que podrían tener implica-

ciones sobre el propio estado de conservación de

la angiosperma:

• En el Capítulo 4 se mostraba como, además de

la disponibilidad de luz, es muy probable que

existan otras factores vinculados a la estructu-

ra del dosel foliar de la pradera de P. oceanica

implicados en los mecanismos de resistencia

a la colonización de C. cylindracea. La fricción

asociada al movimiento de las hojas o la pro-

ducción de sustancias químicas alelopáticas

han sido directamente relacionadas con los

factores determinantes en la composición de

las comunidades que habitan debajo del dosel

foliar de esta especie (Gambi et al. 1990, Cuny

et al. 1995)) y de los formados por otros ma-

crófitos marinos (Black 1974, Dayton et al.,

1984). El papel de estos u otros posibles me-

canismos, y las interacciones entre ellos, deben

ser evaluados para obtener un conocimiento

más completo acerca de la vulnerabilidad de

este hábitat a la invasión.

3. Futuras direcciones y perspectivas de investigación

• Como ha sido ya comentado anteriormente,

la alteración física de la pradera, ya sea por

efecto de altas tasas de herbivoría, por tem-

porales o el fondeo de embarcaciones, parece

determinar una inhibición de los mecanismos

de resistencia que favorece en última instancia

la capacidad colonizadora del alga dentro del

hábitat. Sin embargo, el papel que otras presio-

nes antrópicas asociadas tradicionalmente con

el deterioro de la pradera tienen sobre la inte-

racción entre ambas especies es todavía des-

conocido. Un caso particularmente interesante

lo representan los procesos de eutrofización

que son una de las presiones más comunes y

extendidas en las praderas mediterráneas. Los

aportes de nutrientes y materia orgánica pue-

den provocar el deterioro de la pradera como

consecuencia de la reducción en la disponibi-

lidad lumínica, la aparición de desequilibrios

metabólicos asociadas a un incremento de la

concentración de nitrógeno, la anoxificación

del sedimento o incremento de las tasas de

herbivoría (Sanchez-Lizaso et al. en revisión).

En el caso de C. cylindracea, el impacto de es-

tos fenómenos ha sido menos estudiado, pero

se ha observado como el exceso de nutrientes

facilita su dispersión en fondos de coralígeno

a través de un incremento en su capacidad de

crecimiento y mediante la reducción de la re-

sistencia a la colonización de las comunidades

nativas (Gennaro y Piazzi 2014). Por tanto, es

posible los efectos de estos fenómenos en am-

bas especies puedan actuar de forma sinérgica

sobre la colonización del alga en la pradera de

P. oceanica.

• Las pocos estudios existentes hasta el momen-

to parecen indicar una capacidad potencial de

C. cylindracea de afectar a la vitalidad de la

pradera de P. oceanica mediante procesos de

alelopatía y anoxificación del sedimento (Hol-

mer et al. 2009. Sin embargo, como se suge-

ría en el Anexo, la influencia efectiva de estos

mecanismos sobre las praderas a largo plazo

no ha sido demostrada hasta la fecha y pare-

ce estar condicionada por la baja capacidad

productiva y de crecimiento del alga Por tanto,

es necesario obtener nuevas evidencias experi-

mentales que demuestre de forma efectiva la

relación entre dichos mecanismos y el deterio-

ro de la estructura y vitalidad de la pradera de

P. oceanica.

• Otra línea de investigación muy interesante

sería analizar como los futuros escenarios aso-

ciados con el cambio global pueden afectar a

la interacción entre ambas especies. En general

el impacto que la variación en los factores re-

p. 112

DISCUSIÓN GENERAL

TESIS DOCTORAL

lacionados con el cambio climático (por ejem-

plo la temperatura o la concentración de CO2)

tienen sobre las invasiones biológicas son poco

conocidos, aunque existen estudios que sugie-

ren que se pueden ver favorecidas por estos

nuevos escenarios como consecuencia de un

incremento de su capacidad colonizadora y de

la susceptibilidad a la invasión de los ecosiste-

mas nativos (Dukes y Mooney 1999).

El impacto que estos cambios pueden tener so-

bre ambas especies no ha sido apenas inves-

tigado. Los eventos de calentamiento extremo

(olas de calor) han sido relacionados a nivel

regional con incrementos en la mortalidad de

P. oceanica (Díaz-Almela et al. 2007, Marbá y

Duarte 2010) o reducción en la producción de

hojas y el crecimiento de los rizomas (Mayot et

al. 2005), lo que reduciría la resistencia de la

pradera a la invasión del alga. En C. cylindracea

dichos eventos parecen también tener un po-

tencial efecto negativo sobre la abundancia del

alga (Bernardeau-Esteller, obs.pers.), aunque

experimentos en condiciones de mesocosmos

no muestran efectos negativos en el desarro-

llo del alga en situaciones de alta temperatura

(Flagella et al. 2008). En cualquier caso, el im-

pacto que otros factores como el incremento

en las concentraciones de CO2 o el aumento de

perturbaciones extremas como los temporales

históricos no ha sido todavía evaluado.

CONCLUSIONESC O N C L U S I O N S

p. 114 p. 115

CONCLUSIONES

TESIS DOCTORAL

Dispersión y dinámica

poblacional de C. cylindracea

1. C. cylindracea fue detectada por primera vez en aguas de la re-

gión de Murcia en el año 2005, año a partir del cual se produjo una

rápida expansión por todo el litoral de esta región, donde ha mos-

trado tasas de colonización muy elevadas.

2. El alga mostró un patrón espacial de dispersión muy disconti-

nuo, caracterizado por la aparición de nuevas colonias aisladas y

separadas entre si distancias que oscilan entre centenas de metros

a decenas de kilómetros entre años sucesivos, lo que indica (i) la

importancia de los mecanismos de reproducción vegetativa en la

colonización de nuevos hábitats y la elevada resistencia de los frag-

mentos y propágulos generados y (ii) la intervención de vectores

secundarios de origen antrópico en su dispersión a escala local y

regional.

3. Las principales comunidades bentónicas colonizadas por el alga

en las costas murcianas fueron zonas dominadas por fondos detríti-

cos y de mäerl, fondos rocosos con comunidades de algas fotófilas

y fondos con mata muerta de P. oceanica. Por el contrario, las pra-

deras de P. oceanica y los fondos sedimentarios fueron los fondos

menos colonizados

4. La dinámica poblacional de C. cylindracea parece evidenciar

un claro patrón estacional similar al observado en otras zonas del

Mediterráneo (Ruitton et al. 2005b, Lenzi et al 2007), definido por

notables diferencias en la abundancia del alga entre invierno y ve-

rano una época de máximo crecimiento y abundancia en verano y

principio de otoño, y una época en la que su crecimiento se muestra

severamente ralentizado en invierno y principio de primavera.

p. 116 p. 117

CONCLUSIONES

TESIS DOCTORAL

Mecanismos de fotoaclimatación de C. cylindracea ante

gradientes espaciales de luz y relación entre disponibilidad lumínica

y éxito invasor

5. Los mecanismos de fotoaclimatación

desarrollados por C. cylindracea ante los

gradientes espaciales definidos por la pro-

fundidad y el dosel vegetal de P. oceancia in-

cluyeron respuestas que actúan a nivel mor-

fológico (cambios en la longitud del fronde),

a nivel del aparato fotosintético (cambios en

el contenido pigmentario y el funcionamien-

to del aparato fotosintético) y a nivel meta-

bólico (cambios en las tasas respiratoria)

6. Las respuestas observadas mostraron

cambios determinados tanto por las varia-

ciones cuantitativas en el régimen lumínico

como por variaciones cualitativas (cambios

en el espectro de luz)

7. Las respuestas de aclimatación del alga

revelaron una elevada variabilidad espa-

cio-temporal (variación entre profundidades,

variaciones interanuales e intranuales), lo

que refleja la fuerte dependencia de dichas

respuestas ante las condiciones locales im-

perantes. En última instancia, las respuestas

fotoaclimatativas expresadas serán aquellas

que permitan una mejor optimización del

uso de la luz en base a los recursos disponi-

bles.

8. Los resultados obtenidos el en capitulo 2

evidenciaron que los mecanismos de fotoa-

climatación desarrollados por C. cylindracea

son un mecanismos eficaz para optimizar la

capacidad productiva del alga a lo largo de

gradientes de profundidad. Sin embargo, los

propios costes asociados a estos mecanis-

mos parecen limitar la capacidad coloniza-

dora del alga a profundidades elevadas (en

la zona de estudio esto parece observarse al

menos a partir de los 25m de profundidad).

9. Ante niveles de luz reducidos se produce

un desacople entre la capacidad de aclima-

tación del alga y la luz disponible, que deriva

en un uso ineficiente de este recurso, refleja-

do por la reducción en la capacidad produc-

tiva del alga

10. Dentro de la pradera de P. oceanica y en

distintas épocas del año se registraron regi-

menes de luz próximos a los requerimientos

mínimos de luz necesarios para el crecimien-

to de C. cylindracea. Estos resultados eviden-

cian que la disponibilidad de luz dentro de

este hábitat parece ser un factor determi-

nante en la resistencia a la colonización.

11. Además de la disponibilidad de luz, los

experimentos realizados indican que existen

otros factores relacionados con la estructura

de la pradera implicados en la resistencia a

la invasión.

p. 118 p. 119

CONCLUSIONES

TESIS DOCTORAL

Interacción entre C. cylindracea y

P. oceanica: resistencia biótica de las praderas

de P. oceanica

12. La baja capacidad colonizadora mostrada por el

alga durante el periodo 2007-2014 bajo el dosel foliar

de P. oceanica indica la prevalencia de los mecanismos

de resistencia a largo plazo y refleja que las praderas

de P. oceanica en buen estado de conservación actúan

a modo de barrera ecológica frente a la dispersión del

alga.

13. La interacción entre C. cylindracea y P. oceanica no

determino un impacto negativo en la abundancia de la

angiosperma marina, al menos en las praderas estudia-

das y durante los ocho años de estudio

ANEXOA N N E X

Assessment of long-term interaction between the endemic

seagrass Posidonia oceanica and Caulerpa cylindracea in the

Mediterranean Sea

p. 123

Assessment of long-term interaction between the ende-mic seagrass Posidonia oceanica and Caulerpa cylindra-cea in the Mediterranean Sea.

Abstract

C. cylindracea has shown a reduced capacity to

colonize healthy P. oceanica meadows throu-

ghout the Mediterranean coast, although it is

suggested that the invasive alga is able to affect

the seagrass vitality through different mechanis-

ms (e.g. allelopathic, sediment anoxia) and hen-

ce alter its population dynamics and meadow

structure in the long-term. To assess the existence

of long-term negative interactions between both

macrophytes, the abundance of both species in

invaded and non-invaded locations of was moni-

tored over an 8-years period (2007-2014). Results

indicate that in all of the invaded locations C.

cylindracea biomass present inside the seagrass

leaf canopy was about 10 to 50 –fold lower than

that measured just outside the leaf canopy. Also,

no differences were highlighted between invaded

and non-invaded meadows and all the monitored

meadows showed stable or progressive trends. In

summary, our results do not support the existence

of a long-term competitive interaction between

the invasive alga and the native seagrass, at least

in the studied meadows and at the meadow level.

C. cylindracea forms huge biomass gradients as-

sociated to the seagrass meadow edges that are

stable with time, which suggests the existence of

highly limiting conditions for algal growth and

survival under the P. oceanica leaf canopy.

1. Introduction

Exotic macrolgae can generate negative impacts

and outcompete native seagrass habitats

(Garbary et al 2004, Eklöft et al. 2006), is being

recognized as a potential threat to these ecolo-

gicaly relevant habitats in coastal areas worldwi-

de (Williams 2007). Seagrass habitats play a key

role in the functioning of Mediterranean coastal

ecosystems (Larkum et al. 2006), therefore, the

knowledge of the interactions between seagras-

ses and highly invasive species such as those of

the genus Caulerpa is an issue of major concern

among scientific and coastal managers (e.g. De

Villele and Verlaque 1995, Meines et al. 1993,

Meinesz et al. 2001, Boudouresque and Verlaque

2002, Ceccherelli et al. 2002, Dumay et al. 2002a,

Belsher et al. 2003).

The green alga Caulerpa cylindracea Sonder. (he-

reinafter C. cylindracea) has rapidly spread throu-

ghout the western Mediterranean during the last

20 years, where it has established in different

habitats including seagrass meadows (Piazzi et

al. 2005b, Klein and Verlaque 2009). The little

available evidence suggests that the capability

of the alga to invade seagrass habitats and in-

teract with its abundance and vitality largely

depends on the type of Mediterranean seagrass

species, its size, growth rate and the complexity

of the tridimensional structures that they form.

In general, the alga seems to be able to pene-

trate in seagrass canopies formed by species of

medium-small size such as Cymodocea nodosa

and Zostera noltei (Raniello et al. 2004), but not

in the more complex leaf canopies of the largest

species, Posidonia oceanica (Marín-Guirao et

al.2015). Enviado para su publicación a Marine Biology.

p. 124 p. 125

ANEXO

TESIS DOCTORAL

Fig. 1.

Location of study area and

monitored stations in the

Mediterranean sea.

(Ruitton et al 2005b,Mezgui et al 2007, Cebrian

and Ballesteros 2009. Enguix et al. 2014).

2.2. Sampling procedures

Seagrass descriptors

In each location four stainless steel pegs were

set up along the meadow edge every 10 meters.

These markers were used as a spatial reference

for all seagrass measurements in order to avoid

confounding effects by the high small-scale spa-

tial heterogeneity of the meadow structure on

interannual variations of the selected seagrass

descriptors. The percentage of meadow cover

was estimated in 10 m transects deployed in

each marker following a fixed compass bearing

(Ruiz et al. 2010a). Within each transect, a visual

estimation of the percentage of the bottom co-

vered by seagrass patches was performed inside

1,600 cm2 quadrats subdivided into four 20x20

cm squares. The values obtained were averaged

for the ten quadrats representing the percenta-

ge cover of the whole transect, which is the true

replicate (n = 4). Shoot density was estimated by

counting the number of shoots inside 400cm2

square frames randomly placed inside living sea-

grass patches. , every five meters in the same

transects used for cover measurements. The ave-

rage of the three measurements obtained along

each transect was used as an independent repli-

cate (n = 4 replicates). In addition, six permanent

plots of 1,600 cm2 were randomly set in October

2007 in the sampled meadow areaand the exact

number of shoots was counted in each one (ni).

These shoot censuses were repeated each year

in the same season for estimating the annual

net population growth (NPGy), which is the re-

lative change in shoot numbers experienced by

the meadow in a per year basis (%·year-1). This

variable was estimated following the equation:

NPGy (% year-1) = [(nf –ni) x 100)/ ni x (365 /

P)], where ni and nf are, respectively, the mean

value obtained at the beginning of the time se-

ries and at the end of each annual period, and

being P the length of that period in days. For a

given permanent quadrat, the sum of all NPGy

values obtained in the whole monitoring period

By means of a short-term manipulative experi-

ment, Cecherelli and Campo (2002) studied the

impact of C. cylindracea on mixed meadows of

the seagrasses C. nodosa and Z. noltei. These

authors showed a reduction in the shoot densi-

ty of C. nodosa in invaded experimental plots.

whereas Z. noltei showed the opposite response,

increasing its shoot density in the invaded plots.

As mentioned above, C. cylindracea has shown a

reduced capacity to colonize healthy P. oceanica

meadows throughout the Mediterranean coast

(Katsanevakis et al. 2010, Bulleri et al. 2011, Ruiz

et al. 2011; Ceccherelli et al. 2014), which repre-

sent one of the main and more extended habi-

tats of the Mediterranean infralittoral bottoms.

However, and in contrast with this apparent re-

sistance, et al. (2002a) reported significant al-

terations in the vegetative development of the

seagrass in invaded areas that were interpreted

as stress symptoms caused by allelopathic effects

of secondary metabolites produced by the alga.

From these results it could be hypothesized that

in the long term such stressful effects would in-

terfere with plant growth (Dumay et al. 2002a)

and perhaps would compromise seagrass survival

and its resilience against the invasion, but the

long-term effects of C. cylindracea on the vitality,

structure and functions of P. oceanica meadows

has yet to be evaluated.. This study represents a

first assessment of the existence of such long-

term negative interactions between the invasive

alga and the native Mediterranean seagrass. To

this end we monitored the abundance of both

macrophytes in invaded and non-invaded locali-

ties of the Southeastern coast of Spain (Murcia)

over an 8-year period. If a negative interaction

between both macrophytes exists over time, then

i) the seagrass meadow structure would decline

and ii) the abundance of the alga into the sea-

grass leaf canopy would increase

2. Material and Methods

2.1. Study area and sampling design

The present study was conducted in the Medi-

terranean coast of Murcia (SE of Spain), where

the exotic seaweed C. cylindracea was observed

for the first time in 2005 (Ruiz et al. 2011). The

study was initiated two years after (2007) in the

three most invaded localities with well-developed

P. oceanica meadows (Fig. 1; Ruiz et al. 2011):Isla

Grosa (I1, -11m), Cabo Tiñoso (I2, -18 m), and

Calblanque (I3, -25 m). For comparison, another

three non-invaded locations with well-developed

meadows were selected encompassing the depth

range of the invaded locations: Calabardina (N1,

-14m), Las Palomas (N2, -17m) and La Azohia

(N3, -20m), where the alga was completely ab-

sent. All the selected locations presented well-de-

veloped and healthy P. oceanica meadows, not

influenced by anthropogenic disturbances and of

similar oceanographic conditions (substrate type,

water quality, etc.) (Ruiz et al.2014).

In each locality, seagrass meadow descriptors

(shoot density, percentage of meadow cover and

net shoot growth in permanents plots) were me-

asured each autumn between 2007 and 2014.

All of these descriptors have demonstrated to

be effective indicators of meadow structure and

vitality and population dynamics and are widely

used in long-term monitoring programs of this

species (Krause-Jensen et al.2004, Marbá et al.

2005, Pergent-Martini et al. 2004). During the

study period, the abundance of C. cylindracea

(standing biomass,g DW m-2) was measured in

invaded localities, both within the P. oceanica

meadow and in substrates outside, adjacent to

the meadow edge; algal biomass was measured

twice a year (Autumn and Winter) over the study

period in order to assess the high seasonal and

interannual variability reported for C. cylindracea

p. 126 p. 127

ANEXO

TESIS DOCTORAL

Algal biomass

A sample collection was performed for the deter-

mination of the algal biomass in two contrasting

times of the annual growth cycle of the seaweed

in this area: October ( Autumn), when growth

rates and biomass are still at its maximum and

January (Winter), when the growth and abun-

dance of the alga is usually lower, at least in the

study area (Bernardeau-Esteller, unpublished

data). Fronds, stolons and rhizoids of C. cylin-

dracea were carefully collected by hand within

400cm2 square frames and introduced in labeled

plastic bags. In each location ten samples were

randomly collected inside the seagrass meadow

(IN) and another ten samples were obtained

in the area outside just in front of the meadow

edge (OUT) (Ruiz et al. 2011). Samples were

transported to the laboratory with seawater in

chilled containers. Sediment, debris and frag-

ments of other algal species were gently removed

from each sample before drying the alga at 60 °C

until constant weight to calculate total standing

biomass (g DW·m-2).

2.3. Statistical analysis

In order to explore the potential influence of C.

cylindracea on seagrass descriptors with time,

a three way repeated measures ANOVA was per-

formed with Condition (two levels: invaded and

non-invaded) as fixed factor, Location (three le-

vels: I1,I2 and I3 for invaded locations and N1,

N2 and N3 for non-invaded ones) as a random

factor nested within Condition. Time was inclu-

ded as the repeated measured factor (eight levels

corresponding to the eight successive annual pe-

riods). Depth was introduced as a covariate due

to the high negative relationships between dep-

th and meadow structural parameters (i.e. shoot

density and meadow cover) (J.M. Ruiz, unpubli-

shed data; Ruiz et al. 2014). Differences in NPG

between invaded and non-invaded mea dows

with Condition ( two levels, invaded, non-inva-

ded) as a fixed factor and Location (three levels)

as a random factor nested within Condition were

examined with a two-way ANOVA. Analyses was

computed using Greenhouse-Geisser adjusted

degrees of freedom when data did not meet the

assumption of sphericity (Mauchly’s test, α =

0.05). Spatio-temporal variation of C. cylindracea

biomass was analysed by a two-way ANOVA with

Position (two levels: IN and OUT) as a fixed fac-

tor and Time as a random factor. Since samples

of winter 2008, 2013 and 2014 were not carried

out, ANOVA was applied to each of these seaso-

nal periods separately.

Prior to carrying out the ANOVAs, the data was

tested for normality (Kolmogorov–Smirnov) and

equal variances (Levene test) and transformed

where necessary. Where variance remained hete-

rogeneous, untransformed data was analysed, as

ANOVA is a robust statistical test and is relatively

unaffected by the heterogeneity of variances,

particularly in balance experiments (Underwood

1997). When appropriate, a posteriori pairwise

and multiple comparisons of means was per-

formed using respectively LSD test for repeated

measures ANOVA and SNK for two way ANOVA.

A probability level of 0.05 was regarded as signi-

ficant except when data transformation was not

possible. In such cases the level of significance

was reduced to P<0.01 to minimize type I error

and special care was taken in the interpretation

of results. Furthermore, shoot density and cover

data were tested in order to assess trends of time

series by using (i) linear regression model and (ii)

non-parametric Kendall’s coefficient of rank co-

rrelation (τ) Relationships between algal biomass

values inside the meadow (autumn values) and

meadow descriptors were explored using simple

regressions. Analysis were performed using the

(2007-2014) correspond to the total net popula-

tion growth (NPGT). This variable represents the

net balance between recruitments and mortality

of the shoot population, taking positive values

when shoot recruitment is higher than mortality

(population growth) and negative values when

mortality overcome recruitment overcomes re-

cruitment (population decline).

Table 1.

Summary of three-way ANOVAs performed to assess the effect of Position, Location and Time on C. cylindra-cea biomass

SourcePositionLocationTimePositionxLocationPositionxTimeLocationxTimePositionxLocationxTimeResidual

Autumn Winter

df

1

2

7

2

7

14

14

432

df

1

2

3

2

3

6

6

216

MS

53259,27

2255,46

3385,49

1724,01

2073,25

1350,03

1464,73

107,88

MS

6375,29

2943,88

11255,51

1816,08

6532,94

23616,08

19175,27

5270,52

F

36,36

1,67

2,51

15,98

1,42

0,92

13,58

F

3,51

0,13

0,48

0,35

0,34

1,23

3,64

p

***

ns

ns

ns

ns

***

***

p

ns

ns

ns

ns

ns

ns

**

Fig. 2.

Temporal variation of biomass (g DWm-2) of C. cylin-dracea stands growing within (IN, full dots) and out-

side (OUT, empty dots) of the three studied invaded

meadows (A=I1, B=I2 and C=I3). Data are presented

as means and standard errors. Asterisks indicate signifi-

cant differences provided by the pair-wise comparison

for the “Position” Factor (two levels, IN and OUT) for

each sampling time obtained in SNK test performed

after ANOVA. *p<0.05, **p<0.01. ND= no data.

p. 128 p. 129

ANEXO

TESIS DOCTORAL

Fig. 5.

Annual net population growth (NPGy) of invaded (A) and non-invaded (B) meadows from 2007 to

2014. Data areis presented as means ± standard error.

Table 3.

Summary of the two-way Repeated Measured ANOVA performed to assess the effects of Condition,

Location and Time on Annual Net Population Growth (NPGy). ns=not significant, ***P<0.001.

SourceBetween-Subjects EffectsConditionLocation (Condition)ResidualWithin-Subjects EffectsTimeTimexConditionTimexLocation(Condition)Residual

df

1

4

30

4,53

4,53

18,12

158,55

MS

38,3088629

319,388108

520,059365

6617,36154

1343,36163

818,203926

1159,25942

F

0,11994455

0,61413779

8,08766779

1,64184207

0,70579882

p

ns

ns

***

ns

ns

Table 4.

Mean and standard error of Total Population Growth (NPGt) measurements at each location and

results of two-way ANOVA and SNK test performed to assess the effects of Condition and Location on

this variable. ns=not significant, *P<0.05.

NPGt

ANOVASourceConditionLocation(Condition)ErrorSNK: I3<N1=N3=N2=I2=I1

Variable Location

I1

27,61

± 9,28

df

1

4

30

I2

23,47

± 11,84

MS

785,94

3461,00

1176,36

I3

-24,91

± 8,90

F

0,21

2,94

N1

-18,66

± 19,65

p

ns

*

N2

15,83

± 15,39

N3

1,87

± 8,05

Fig. 3.

Shoot density of Posidonia oceanica of invaded (A) and non-invaded (B) meadows from 2007 to 2014. Data are

presented as means ± standard error.

Fig. 4.

Percentage of cover of invaded (A) and non-invaded (B) meadows from 2007 to 2014.

Data are presented as means ± standard error.

Table 2.

Summary of the two-way Repeated Measured ANOVA performed to assess the effects of Condition, Location

and Time on Shoot Density and Meadow Cover. Depth factor was include as covariate. ns=not significant,

***P<0,001.

SourceBetween-Subjects EffectsDepth [covariate]ConditionLocation (Condition)ResidualWithin-Subjects EffectsTimeTimexDepthTimexConditionTimexLocation(Condition)Residual

Shoot Density Variable Meadow Cover

df

1

1

4

18

7

7

7

28

144

df

1

1

4

18

7

7

7

28

144

MS

1.89

8,84

357,56

28,04

35,84

35,58

32,37

354,11

976,18

MS

10,07

28,24

65,62

41,90

81,86

81,53

86,28

42,59

16,79

F

0.067

0.02

12,75

0,40

0,40

0,37

1,87

0,40

F

0,240

0,43

1,57

1,92

1,91

2,03

2,54

p

ns

ns

***

ns

ns

ns

***

p

ns

ns

ns

ns

ns

ns

***

p. 130 p. 131

ANEXO

TESIS DOCTORAL

tion of this growth during the winter (Ruitton et

al. 2005b, Lenzi et al. 2007). However, this does

not seem to be a general pattern as for some

years the algal abundance did not only decline in

winter, but it had maintained close to mean va-

lues recorded in previous and following autumns

(locations I1 and I3 in 2009 and location I2 in

2011). This interannual variability could explain

the fact that other authors did not find a seaso-

nal pattern of C. cylindracea during shorter time

periods (≤ 2 years; South Italy, Giaccone and Di

Martino, 1995; Balearic Islands, Cebrian and Ba-

llesteros 2009). This algal species has demonstra-

ted the ability to balance its carbon budget in the

winter although with reduced growth rate, which

suggests that the algal biomass is able to persist

under conditions of light and temperature typical

of winter periods (Flagela et al. 2008. Marin-Gui-

rao et al. 2015, Bernardeau-Esteller et al. Unpu-

blished data). Nonetheless, in such conditions the

algae could have a reduced resilience and hence

a higher vulnerability to additional physical dis-

turbance such as, the action of hydrodynamic

forces associated to winter storms, which inten-

sity and frequency can be subjected to great in-

terannual variability. Strong hydrodinamism has

been identified as a possible factor involved in C.

cylindracea winter regressions (Klein and Verla-

que 2008), however this hypothesis must be ad-

dressed with specific experimental work.

The populations of C. cylindracea showed very

low biomass within the seagrass leaf canopy,

which highlight the low capacity of the alga to

colonize this habitat. These results are in line

with other works showing that P. oceanica mea-

dows with a good conservation state can act as

ecological barriers against the spread of the in-

troduced seaweed (Katsanevakis et al. 2010, Bu-

lleri et al. 2010). Although the factors underlying

this incapacity of of the alga to penetrate into

P. oceanica meadows are still poorly understood,

Marín-Guirao et al. (2015) and Bernardeau-Este-

ller et al. (2015) have shown that physical factors

linked to the leaf canopy structure such as light

limitation can play a relevant role, regardless the

depth and meadow structure properties asso-

ciated to that depth. Similarly, the inability of C.

cylindracea to colonize the P. oceanica meadow

was evident even in the deepest meadow of the

I3 location, probably as consequence of the clo-

se coupling between meadow structure and light

availability described for P. oceanica across dep-

th gradients (Pergent et al 1995, Ruiz et al. 2014).

Descriptors of seagrass meadow structure indica-

te that most of the monitored meadows followed

a stable or progressive trend throughout the stu-

died 8-years period (Table 5), without differences

between invaded and non-invaded meadows. In

location I3 a negative total population growth

(NPGt) was obtained in the permanent quadrats,

which contrast with the positive and stable trends

Table 5.

Summary trends.

I1I2I3N1N2N3

Shoot density Meadow Cover NPGt

++-===

Balance 2007-2014

+===++

Balance 2007-2014

=++===

Lineal Regression

==+===

Lineal Regression

=+====

Tau Kendall

==+===

Tau Kendall

===+==

Locationstatistical package SPSS version 17.0 (SPSS Inc.

Chicago, Ill) and Sigmaplot 10.0 (Systac Softwa-

re Inc.).

3. Results

C. cylindracea biomass outside of the meadow in

the autumn season were in overall significantly

higher (between 10 and 50 fold) than inside (Fig.

1, Table 1, SNK, α=0.05). Abundance outside of

the meadow ranged from 70.3 to 9.4 in I1, 54.4

to 15,4 in I2 and 23.5 to13.9 in I3. In years in

which these differences were not observed (2012

and 2014 for I1 and I2; 2010 and 2012 for I3; Fig.

4) algal biomass was low (<6 g/m2), registering in

some cases a complete regression of the popula-

tion (p.e. location I1 in autumn 2013) (Fig. 1). For

the winter season biomass ranged from 0.0 to 3.2

g/m2 in all locations and in both position (Fig. 1)

except in 2011 for I1(69.9 g/m2) and in 2009 for

I2 (53.4 g/m2) and I3 (14.5 g/m2) (Fig. 1). Only in

these years biomass outside of the meadow was

significantly higher than that recorded inside (14

to 70 fold higher; Table 1, SNK, α=0.05).

Invaded and non- invaded P. oceanica meadows

did not show differences in their structural para-

meters (shoot density and meadow cover) and

population dynamics (NPGy, NPGt) throughout

the study period (Table 2, Table 3, Table 4) and

no correlations with algal biomass within the

meadow canopy were detected in those invaded.

Both invaded and non-invaded meadows showed

significant interannual changes (LSD test,

α<0.05) in meadow descriptors. Variations up

to 50% for shoot density (location N1 between

2007 and 2008, Fig 3) and 100% for meadow

cover (location N3 between 2010 and 2011; Fig

. 4) were detected. Time also had a statistically

detectable effect on NPGy, with maximum net

gains and losses close to 40% (locations N1 and

N2 in 2007) and -20% (location N3 in 2013)(Fig.

4, Table 3). For the whole time series meadow

descriptors showed stable or progressive trend

in most locations (Table 5). In I1, N2 and N3

a significant increase in shoot density between

the first and the last year of the time series was

observed, while for meadow cover this increase

was significant in I2 and I3. In the rest of the

locations no changes for both variables were de-

tected at the end of the study period (LSD test,

α=0.05). Both meadow descriptors showed posi-

tive lineal relationships and significant positive

correlations based on Kau coefficient with time

in I3 (r = 0.42, p = 0.048; 0.88, p = 0.0004 and τ =

0.86, p = 0.001). These significant positive trends

were also observed for meadow cover in I2 (r

= 0.51, 0.028) and N1 (τ = 0.50, p = 0.042). The

rest of the locations had not shown significant

trends. NPGt showed values greater than zero in

all locations except in N1 and I3, although SNK

test only found significant differences between

I3 and other locations (Table 4).

4. Discussion

Over the study period (2007-2014), populations

of C. cylindracea outside of the meadow showed

biomass values within the ranges reported for

this species in other invaded localities at a similar

depth range (Capiomont et al. 2005, Mezgui et

al. 2007, Cebrian and Ballesteros 2009, Enguix

et al. 2014 ). Results obtained in this study sug-

gest the existence of marked fluctuations of algal

biomass in all studied locations with maximum

values in the autumn sampling time and a dras-

tic decline in the winter time, when the alga even

disappear in most years of the study period. This

seasonal pattern of algal biomass, including win-

ter regressions, has been reported in populations

of several areas of the Western Mediterranean

Sea (Buia et al. 2001, Ruitton et al. 2005b, Len-

zi et al. 2007, Enguix et al. 2014) and is consis-

tent with the greater growth rates measured in

C. cylindracea stolons in summer and the inhibi-

p. 132 p. 133

ANEXO

TESIS DOCTORAL

netheless the scientific knowledge at this level is

very low and more robust experimental evidence

is necessary to conclude something about the

operation of such competitive mechanisms in

Mediterranean seagrass habitats.

In summary, our results indicate that C. cylindra-

cea populations are well established in inffralito-

ral bottoms of the studied area, although with a

high temporal variability mainly accounted for

by seasonal forces but also by local oceanogra-

phic and climatic conditions. Our results reinforce

the idea of P. oceanica meadows as ecological

barriers against the spread of C. cylindracea, as

indicated by the observation of stable, persistent

gradients of algal biomass at both sides of the

meadow edge. The resistance mechanisms of

P. oceanica meadows to bioinvasions seems to

be linked to its highly complex canopy structure

(Ceccherelli et al. 2014, Marín-Guirao et al. 2015,

Bernardeau-Esteller et al. 2015). Interactions be-

tween C. cylindracea and Mediterranean seagras-

ses have only been demonstrated in the much less

complex leaf canopies of C. nodosa and Z. noltei

(Cecchererelli and Campo 2002) within which the

alga is able to grow and develop high biomasses

(Raniello et al. 2004).. Our results show that the

presence of C. cylindracea in invaded areas did

not affect the structure of the P. oceanica mea-

dow during the studyperiod and hence resistance

mechanisms of the seagrass to the invasion also

remained intact. Therefore, this study does not

support the existence of a long-term competiti-

ve interaction between the invasive alga and the

native seagrass, at least in the studied meadows

and at the meadow level, although more in depth

research is necessary to address the inconsisten-

cy of these results in other geographical regions

and the involved mechanisms of competitive in-

teractions.

showed by shoot density and meadow cover me-

asured in the same meadow. This apparently

contradictory result could probably reflect the di-

fferent scales at which each descriptor is measu-

red and their differential sensitivity to changes in

meadow structure. Permanent quadrats were ins-

talled very close to the meadow edges, adjacent

to the densely colonized sediments, which could

suggest a possible specific negative effect of the

algal population on seagrass shoots of the mea-

dow limit. However, this possibility is not suppor-

ted by the observation of very similar trends in

the non-invaded locality N1 (Table 4), although

it was not statistically significant in this case due

to the high variability between replicates. Moreo-

ver, when we analyse the shoot population grow-

th per annual basis (i.e. annual net population

growth, NPGy; Fig. 4) a general trend towards

negative values was observed both for most in-

vaded and non-invaded locations since 2011. In

addition, local factors could also account for the

particular declining behaviour of the shoot popu-

lation in I3 permanent quadrats. Meadow edges

are vulnerable to physical disturbances caused

by sediment dynamics and hydrodynamic forces

(Fonseca and Bell 1998, Infantes et al. 2009, Ben-

Brahin et al 2014). This could explain the particu-

larly negative NPGt values observed in I3 since

in this location was frequent the observation of

deeply buried P. oceanica shoots caused by the

migration of large sand waves. Persistent burial

of P. oceanica shoots above 3 cm have been de-

monstrated to be a cause of shoot mortality in

this seagrass species, with one of the lowest rates

of vertical growth among seagrass species (Bou-

douresque et al. 1984; Manzanera et al 2014).

Consistently, the P. oceanica meadow at the I3

location presented a fragmented seascape for-

med by patches of tens of meters spread over

the sandy bottom revealing the existence of a

particular regime of physical disturbance in this

locality (Hemminga and Duarte 2000).

Therefore, our results do not support the existen-

ce of a negative interaction between C. cylindra-

cea and P. oceanica meadows, at least at the po-

pulation/meadow level and in the studied region.

Two possible mechanisms have been suggested

as being able to cause potential effects of C.

cylindracea on P. oceanica meadows in the long

term: (i) allelopathic interaction by which the re-

lease of a phytotoxic compound would affect and

damage the competitor physiology (Dumay et al.

2002a), and (ii) modifications of sediments con-

ditions to turn these adverse to support seagrass

growth (Holmer et al. 2009). From this study the

operation of such mechanisms cannot be addres-

sed in the invaded locations, but some additio-

nal information allows us to speculate that they

could be of low relevance in this case. First of all,

concentrations of phytotoxic compounds in C.

cylindracea tissues are of 1 order of magnitude

lower than those measured in congeneric species

C. prolifera and C. taxifolia (Dumay et al. 2002b.

Box et al. 2010). This fact, together with the high

fluctuations of algal abundance reported in inva-

ded locations, considerably reduces the probabili-

ty of allelopathic, negative effects of C. cylindra-

cea populations on the seagrass meadow since it

would probably require a very high and persistent

algal biomass over time. The formation of sedi-

ment anoxia, and the consequent accumulation

of phytotoxic compounds in sediments, is a plau-

sible hypothesis, but it also probably depends on

the persistence of high algal biomasses. Consis-

tently, no sediment anoxia was observed during

sampling surveys in all monitored invaded areas

throughout the 8-years periods. Furthermore,

since the development of the alga is limited wi-

thin the P. oceanica meadow, the probability of

sediment anoxia within the leaf canopy (where

the plant is rooted) should be also very low. No-

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p. 158

CODA

Seguro que fueron más, muchas más, pero estas sonaron una y otra vez du-rante los últimos compases de elaboración de esta tesis, quedando grabadas a modo de banda sonora perenne de las sensaciones y estados vividos.

The Caulerpa Remix: the role of life

1. Fjögur píanó. Sigur Ros2. Bye Bye. Destroyer3. They Harder they Come. Jimmy Cliff4. Snowflakes Falling on the Sea.The Windy Hills5. Heroes and Villains. The Beach Boys6. Via Lattea. Franco Battiato7. Is That Enough. Yo la Tengo8. Change Your Mind. Neil Young and Crazy Horse9. Le onde. Ludovico Einaudi10. Remember Our Heart. Alexander11. Stranizza d’amuri. Franco Battiato12. Ya Hey. Vampire Weekend13. Fire Scene. S Carey14. How Can You Really. Foxygen15. Sketch for a Summer. The Duruti Column16. Slash Your Tires. Luna17. Kaputt. Destroyer18. Did i Tell You. Yo la Tengo19. This Old Heart of Mine. The Isley Brothers20. Helpless. Neil Young 21. Wia. Wim Mertens22. Apple Tree. The Windy Hills

https://open.spotify.com/user/jaimito78/playlist/5Bpm7yXKhe5pGCIcE6JlTv

OCT · 2015


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