Environmental Factors Affecting the Trophic Ecology of Benthic Suspension Feeders

Sergio Rossi Heras

Environmental factors affecting the trophic ecology of benthic suspension feeders

Environmental Environmental factors affecting the trophic ecology of benthic suspension feeders Sergio Rossi Heras Environmental factors affecting the trophic ecology of benthic suspension feeders

Factores ambientales que afectan la ecología trófica de los suspensívoros bentónicos

Tesis Doctoral

Universitat de Barcelona Facultat de Biologia, Departament d’Ecologia

Programa de doctorado: Ecología. Bienio: 1996-1998

Environmental factors affecting the trophic ecology of benthic suspension feeders

Memoria presentada por Sergio Rossi Heras para optar al título de Doctor en Ciencias Biológicas en el Departamento de Ecología, Facultad de Biología, Universitat de Barcelona, bajo la dirección del Doctor Josep María Gili Sardà

Sergio Rossi Heras Barcelona Diciembre 2001

El Director de Tesis Dr. Josep María Gili Sardà Investigador del CMIMA (CSIC)

A Rosanna

CONTENTS

Agradecimientos ...... 9

Introducción general ...... 15

General introduction ...... 23

Temporal variability of near-bottom seston concentration and composition in a warm temperate sea over an annual cycle ...... 31

Short-time-scale cycles of near bottom seston composition: comparison of early and late spring conditions in a warm temperate sea ...... 49

Biochemical characteristics of sedimenting POM available to north-western Mediterranean benthic communities: a seasonal and geographical comparison ...... 63

Activity rhythms in six temperate species of passive suspension feeders: observations at different time and space scales related to environmental conditions...... 79

Spatial and temporal variability in the diet and prey capture rates of a passive benthic suspension feeder: Leptogorgia sarmentosa , a case study ... 107

The “memory” of the Protein-Carbohydrate-Lipid seasonal balance in two sessile marine invertebrates: A new approach to understand the bentho-pelagic coupling processes ...... 129

Conclusions...... 149

Conclusiones ...... 157

References...... 165

Resumen de la Tesis...... 185

Agradecimientos

La tesis doctoral que aquí se presenta tiene un problema obvio de entrada: ¿cómo es posible que una sola persona haya podido hacer todas esas cosas y no esté en tratamiento médico agudo por agotamiento físico y mental? Hay truco. Ha sido un privilegio (y una enorme ventaja) el poder contar con la ayuda de una serie de personas que de una manera u otra me han ido soportando durante estos cinco años y medio... La primera víctima obvia ha sido mi mujer, Rosanna, a quien dedico esta tesis por el sen- cillo hecho de estar siempre allí, de creer en lo que hacía y de armarse de paciencia cuando he tenido que hacer campañas, estancias fuera o un crucero oceanográfico de dos meses (dejándola con una criatura de un año). A ti Livia, también tengo que agradecerte el haberme dado alegría (excepto a la hora de cenar!!!) y un giro completo a mi vida. A Clara y Carmen, que nos han ayudado a educar a nuestra hija durante ese lapso de tiempo (todo el día...) en el que no veíamos a Livia. Mi padre Fernando y mi madre Plácida, mi hermano Marco mi hermana Silvia y su marido Lincoln, mi suegra Claudia y su familia, observán- dome desde diferentes ángulos, me han apoyado y dado ánimos en todo momento. Mis amigos íntimos también han estado allí: Daido y Eva (os quiero mucho pareja!), Toni (con una novela compartida a punto de acabarse...y otra en marcha), Cristina (siempre nos hemos entendido muy bien y me has ayudado mucho), Enrique (entre todos, esto empieza a parecer una mafia chaval!), Ana (tu entusiasmo y fe mueven auténticas montañas), Ricardo (aunque nos veamos poco, parece como si acabásemos de quedar el día anterior), Marta y Ernest (por esos momentos compartidos) o Gemma y Quim (a ver cuando volve- mos al agua!!). Elisabetta es otra persona que ha tenido que aguantar mis neuras (aparte de trabajar como una loca en algo que espero que se publique antes de que la especie estudiada se extinga); todavía recuerdo el día que llegaste, miraste en el interior del despacho de Josep-María y te fuiste; al rato llegó el habitante usual del despacho y yo le pregunté: “Tenemos estudiantes de BUP ahora?” La experiencia de tener estudiantes de la Universidad, de Erasmus, de programas de colaboración con Universidades extranjeras o de Cursos de Doctorado se lo debo a Joandomènec Ros y a Josep-María Gili. A vosotras/os, estudiantes, aparte el enorme trabajo que habéis realizado (cientos de pólipos, decenas de muestras de bioquímica, imágenes de gorgonias, contajes de zooplancton...) y que quedará plasmado en una serie de trabajos que espero tengan salida algún día, debo agradeceros momentos muy divertidos y charlas muy animadas, consejos prácticos y vuestra enorme capacidad de trabajo. Siempre se me ha tomado el pelo porque la imagen de Sergio caminando como una locomotora y dos estudiantes femeninas detrás con una gradilla llena de probetas era frecuente por los pasillos del antiguo ICM, pero ¿Qué culpa tengo yo de que las mujeres sean mucho más curiosas en cuestiones biológicas que los hombres? Helena Pardell fue la primera estudiante (de FP) que estuvo mirando contenidos estomacales de gorgonia, con paciencia religiosa; después vino “la polaca”, Susanne Wisniowska, que miro zooplancton (la muestra entera, sin hacer ali- cuotas!) y Serena Zaccara (durante el verano del 98, ya habíamos aprendido lo de las ali- cuotas). Dos alemanes que hablaban un perfecto castellano vinieron a hacer cosas de repro- ducción y contenidos estomacales (Anngret Lieb y Alfonso Cugat), y con ellos compartí unas cuantas inmersiones de trabajo y de placer en las Medes y Banyuls. Sonia Solorzano fue la primera estudiante que vino de la Autónoma de Barcelona, pero a ella le siguieron Judith Guixà (fallecida en un accidente de coche poco después de acabar el período de prác- ticas, siempre te recordaré), Ana Gálbez y Anna Espasa, todas ellas con ganas de trabajar,

AGRADECIMIENTOS 11 entender y discutir. Manel Cascante (uno de los pocos estudiantes masculinos!) hizo de todo, obligando a su timidez a dejar paso a las innumerables preguntas que tenía sobre el trabajo desarrollado por el equipo. En esa época también entró Elisabet Reyes, que procesó innumerables muestras de lípidos, proteínas y carbohidratos, con una capacidad de trabajo fuera de lo común y un sentido del humor genial. Dos italianas se aplicaron a fondo en hacer un trabajo riguroso y a disfrutar del buen ambiente del departamento, Laura Cerasi y Marta Micheli, con las que volví a hablar italiano hasta que se cansaron de mi masacre ligüística y prefirieron esforzarse con el castellano. Cristina Roldán también estuvo padeciendo el mirar reproducción de gorgonias con la lupa, así como Ero Jiménez y Ana Romero pade- cieron mirando contenidos estomacales de una gorgonia que no comía nada. Y Áurea Peralba, que llegó en Febrero y en Junio ya había hecho zooplancton, contenidos estomacales, densidad de pólipos y pesos secos de un ciclo Ártico de alimentación (por duplica- do!). Todas vosotras (y vosotros) nos habéis facilitado muchísimo el trabajo, y a mi me habéis dado la oportunidad de soltaros el rollo de qué estaba haciendo y por qué lo estaba haciendo mientras compartíamos laboratorio. Marta Ribes y Rafel Coma han orientado parte de las bases de esta tesis y ha sido de gran ayuda en la toma de una serie de buenas decisiones que luego han revertido en un trabajo bien encauzado (como por ejemplo aconsejarme que no eligiese Eunicella como gorgonia central de mi tesis...); ambos han sido clave, especialmente en la parte inicial del proyecto, donde habían de definirse unos objetivos y darle forma a una serie de campañas. Por otro lado, Imma Llobet se encargó desde el principio de la parte del zooplancton, optimi- zando el método en los momentos en que podía escaparse de su trabajo. Francesc Pagés, con un humor tendente a lo más puramente británico, me ha ayudado en innumerables ocasio- nes a responder preguntas que no lograba enmarcar correctamente (y otras que me servirán en un futuro próximo...eh?). Pep Gasol, aunque no sea muy consciente, ha sido de una gran ayuda en muchos aspectos, especialmente por una serie de charlas y consejos que compar- timos en el Polastern y más allá. Gracias también a Ana Sabatés, por haber creído en algunas de mis ideas y haberlas puesto en práctica, y por haberme enseñado a ser más realista en algunas de las propuestas que hemos discutido. Covadonga Orejas ha sido una compa- ñera maravillosa que me ha enseñado muchas cosas sobre cómo comportarse en campañas en alta mar, y Nuria Teixidó, aparte ayudarme muchas veces en el campo, me sugirió una serie de trucos para facilitarme la existencia en el montaje del crucero con el Polastern. Neus Vert entró a formar parte del equipo hace ya unos cuantos años, mirando gónadas de gorgonia de forma imparable e incansable, y pasando después al mundo bioquímico. A ella tengo que agradecerle no sólo el trabajo realizado, sino unas ganas de aprender y un excelente sentido del humor incluso en situaciones no muy favorables. A Jordi Corbera le debo, aparte de la maquetación de esta tesis, un montón de charlas y ratos muy agradables. Montse y Josep, compañeros de “cubículo” durante cuatro años, gracias por haber compartido momentos buenos y malos. Joandomènec Ros es una persona a la que debo agradecerle, aparte el engranar parte de la estructura burocrática que permite sobrevivir a nuestro equipo, el despertar mi inquietud cuando apenas tenía 22 años en biología marina; recuerdo cómo escuchó con el rostro impa- sible una serie de alocados planes que pretendían describir ecología terrestre y marina a la vez (en Cap de Creus) durante una charla que tuvimos a finales de tercero de carrera. Mikel Zabala también contribuyó sustancialmente a este empeño por parte mía a continuar en

12 AGRADECIMIENTOS investigación, y fue un excelente profesor de Ecología (cuarto de carrera) que aplicó su pragmatismo para hacerme ver que las cosas pasan por un filtro práctico, un filtro de rea- lismo del cual yo carecí a lo largo de toda mi vida (y sigo careciendo). Durante el proyecto de tesis se desarrollaron dos campañas intensivas en las que parti- cipó mucha gente, yendo al agua a horas impensables, con una meteorología a veces mala y un frío que pelaba. Estoy muy agradecido a todos, especialmente a Bernat Hereu y a David Diaz, sin los cuales gran parte del trabajo de campo habría sido sencillamente imposible; he pasado momentos muy intensos e inmersiones inolvidables (como una nocturna en que pudimos apagar las linternas por haber luna llena). Gracias de todo corazón, tengo el privilegio de teneros como compañeros. En Banyuls-sur-Mer he tenido siempre el apoyo incondicional de Esther Jordana que realizó su tesis doctoral allí, así como la experiencia y el soporte logístico dado por Antoine Grémare y su equipo que me acogieron en Noviembre del 96 y me enseñaron parte de la bioquímica que ahora se. La otra parte me la enseñó con paciencia Elisa Berdalet, con la que todavía ahora sigo haciendo cosas con gran ilusión. No me olvido de Jean Claude Roca, el capbuçador pofessional de Banyuls, que durant 16 mesos va recollir les mostres de repro- ducció y balanç P-C-L de Leptogorgia a la zona sovint amb l’Esther J. En el Estartit, está claro que quien más ayuda logística (y humorística) ha aportado es la familia Llenas-Elías. Gracias de todo corazón por tener paciencia con otra generación de biolocos desmadrados, que aportan más quebraderos de cabeza que otra cosa. Thanks Mark Snyder, you believed in me when I came to California few years ago. We shared excellent moments and a scientific objective that I’m completely shure that we will get soon. Gracias a Luisa (por ese café de primera hora de la mañana y tu humor salao) y Severina (por esas charlas que hemos tenido entre pólipo y pólipo). Varias personas del Instituto han hecho mi trabajo posible, dando agilidad a trámites, pedidos y otras cosas que son funda- mentales (y que pasan desapercibidas cuando te las hacen tan bien). Estoy muy agradecido a Conchita Borruel, Justo Martínez, Jordi Estaña, Marta Ezpeleta, Gloria Medina, Eva López y Nuria Angosto por estar allí cuando os necesitaba aguantando prisas, rollos, etc. Tengo que agradecer profundamente la avalancha de bolitas de C/N que Isidre Casal y Pilar Fernández han soportado (y por supuesto los técnicos/becarios que asisten los apara- tos!!), así como a Laia Balart y Pilar Teixidor por proporcionar las herramientas y la experiencia para procesar muestras de lípidos fraccionados y TOC. Creo que es innegable el hecho de que el Departamento de Biología Marina goza de una salud humorística y de buen rollo óptima. Mis agradecimientos van dirigidos a vosotros (Celia, Dolors, Alicia, Rafel, Marta, Magda, Esther, Albert, Vanesa, Lluïsa, Ramón, Mikel, Carles, Victor, Pep, Laura, Andrea, Isabel, Bea, Kees, Jordi, Enrique, Renate, Miquel, Francesc, Enric, Evarist, Xelu), que me aguantasteis estoicamente durante estos años...sien- to decir que me tendréis que aguantar como mínimo un par de años más (se que algunos de vosotros se hacía la ilusión de que me fuese inmediatamente de post-doc, para poder así darse de baja en el psicoanalista)! Y a ti, Josep María (JM): nunca has sido sólo el director de esta tesis, sino un verdade- ro amigo que has tenido la cruz de tener al lado una persona como yo, a la que has dado coba para que se metiese en mil cosas a la vez y que compartiese ideas y proyectos que poco a poco se han ido transformando en realidades. No se por qué, pero siempre has creído en

AGRADECIMIENTOS 13 mi, dándome la oportunidad de desarrollarme como profesional y como persona dentro de mi trabajo. De ti admiro el eclecticismo y el excelente humor que siempre te acompaña, te debo mucho y me queda mucho todavía por deber. Por último, quiero volver a agradecerte a ti Rosanna el amor que me has dado y la oportunidad de compartir contigo una vida.

14 AGRADECIMIENTOS INTRODUCCIÓN GENERAL

Las escalas de variabilidad de las comunidades marinas (tanto planctónicas como ben- tónicas) son todavía poco conocidas. Por lo general, cuando hablamos de factores que afectan a una comunidad los enmarcamos en dos tipos de variabilidad: ambiental (físico-quími- ca de la columna de agua) y biológica (fisiología, genética, etc.). Cualquier masa de agua está afectada por procesos físicos que a su vez estructuran tanto las comunidades pelágicas como las bentónicas. En el Mediterráneo, los procesos físicos siguen una clara pauta estacional que se plasma en unas oscilaciones recurrentes de la producción planctónica (Estrada et al. 1985, Estrada 1996) de la columna de agua. Se produce un reflejo de estas oscilaciones tanto en la alimentación como en la reproducción, crecimiento, respiración, etc. de las comunidades bentónicas (Boero 1984, Ballesteros 1989, Sardà et al. 1999, Coma et al. 2000). Las masas de agua costeras y las más próxima a los fondos marinos puede ver atenuada la oscilación estacional tan evidente cerca de la superficie debido a los procesos de interac- ción con el continente y con las comunidades bentónicas que allí se producen. Por un lado, una mayor complejidad en dirección e intensidad de los vientos debido a la orografía, la influencia de avenidas súbitas de los aportes continentales, y/o las perturbaciones esporádi- cas de las tormentas hacen que la frontera delimitada por la costa y la masa de agua colin- dante añada complejidad a la dinámica del seston. Por otro lado la topografía del fondo, una dinámica diferente de las partículas cerca del mismo, y/o las comunidades de organismos a veces muy complejas hacen que las características de esa masa de agua del lecho marino sean difíciles de interpretar. Se ha podido constatar que a medida que nos acercamos a la costa los ciclos de fitoplancton y zooplancton se tornan menos estacionales aumentando su variabilidad temporal (Colebrook y Robinson 1965). Por otro lado, cerca del fondo se producen una serie de fenómenos físicos y biológicos sobre las concentraciones de partículas que pueden enmascarar también una otrora clara variación temporal (Thomsen 1999). De esas partículas se alimentarán los suspensívoros bentónicos (y otros organismos), por tanto, la comprensión de su dinámica y tratar de comprender las escalas de variabilidad temporal y espacial en las que se observan la concentración y calidad del seston permitirá acercarnos un poco más al acoplamiento plancton-bentos. Los organismos que viven en el bentos están adaptados a dicha dinámica, estando su biología íntimamente ligada a la heterogeneidad del seston. En este estudio se propone hacer un análisis tanto a las causas que pueden afectar a los parámetros físicos como biológicos que influyen la dinámica de las comunidades de sus- pensívoros bentónicos, como a los efectos que estos parámetros puedan ejercer sobre dichas comunidades. Para ello, es necesario abordar los dos bloques (causas y efectos) a diferentes escalas temporales y espaciales. Estudiar la concentración y calidad del seston cerca del fondo a lo largo de ciclos anuales (con una frecuencia elevada de muestreo) ha de ser el primer paso que ayude a comprender qué parámetros físicos están implicados en la dinámica estacional de las distintas partículas que lo componen. Asimismo, estos ciclos permiten dis- criminar qué fracciones (tamaño de partícula) y qué composición (Clorofila a, Carbono Total, Carbono Orgánico, etc.) siguen en esa masa de agua un régimen estacional detectable con muestreo de frecuencia quincenal o semanal. Sin embargo, el estudio anual puede ser insuficiente para detectar cambios estacionales claros si la influencia de los factores relacionados con el efecto costa y el lecho marino es demasiado acusada (provocando una serie de pulsos que aumentarán la variabilidad, enmascarando las tendencias estacionales).

INTRODUCCIÓN 17 Para buscar una variabilidad más detallada ha de considerarse un análisis más intenso de los parámetros físicos y biológicos que gobiernan el seston, teniendo en cuenta lapsos de tiempo más cortos que sean capaces de detectar, cerca del fondo, cambios que puedan ayu- dar a entender la dinámica de alimentación, reproducción, crecimiento, etc. de los suspen- sívoros bentónicos. Estos muestreos a corto plazo (cada 4-6 h) permitirán una comparación entre momentos diferentes dentro de una misma época del año considerada como productiva en el Mediterráneo. Los pulsos pueden ser cruciales para comprender los balances ener- géticos de estos organismos: su más o menos elevada frecuencia nos dará indicios de si, dentro de una misma época productiva (la primavera), un aporte más continuado de energía disponible puede revertir en la reproducción o el crecimiento, junto con otros parámetros como la luz o la temperatura. Para poder detectar a nivel estacional las tendencias de la materia en suspensión disponible para los organismos bentónicos el estudio ha de realizarse con una herramienta que permita integrar la variable temporal. De esa forma, los pulsos, aún no siendo detectados a lo largo del tiempo con una precisión extrema, si que quedan “memorizados” y puede ser mejor observada la estacionalidad tanto de la producción en la columna como de los eventos de resuspensión más evidentes. Para ello se han utilizado trampas de sedimento, que recogen la materia orgánica (e inorgánica) en sedimentación antes de que acabe formando parte bien de la dieta del bentos, bien del propio sedimento. En efecto, las trampas de sedimento han demostrado que por lo general pueden distinguirse dos épocas bien diferencia- das a nivel de cantidad y calidad de materia en sedimentación en el Mediterráneo: la época de otoño-invierno, en la que hay una gran cantidad de materia en suspensión pero de escaso valor nutricional (traducido en Materia Orgánica y balance Proteína-Carbohidrato- Lípido), y otra época primavera-verano, en la que la materia en suspensión se vuelve más escasa pero de mayor valor nutricional (Fichez 1991, Grémare et al. 1997). Precisamente por el hecho de poder detectar estacionalidad en la dinámica de la materia orgánica sedimentada en dichas trampas de sedimento, nos permite el paso siguiente: estudiar la heterogeneidad espacial del seston a través de las tasas de sedimentación y des- criptores bioquímicos (lo que nos permitirá calcular la similitud o diferencia del seston disponible para los suspensívoros bentónicos). Por lo general, pocos son los trabajos que hayan intentado hacer una aproximación a pequeña escala (cientos de metros o unos pocos kiló- metros), especialmente en el Mediterráneo (Pusceddu et al. 1999a). Está claro que en pocos metros podemos encontrar diferencias de microhabitat que influyan en la biomasa, biología, fisiología, etc. de los organismos (Airoldi y Virgilio 1998, Underwood y Chapman 1998, Benedetti-Cecchi 2001), pero a nivel de comunidad hemos de buscar, en global, un espectro más amplio en los inputs que provienen de la columna de agua. La comparación espacial se ha hecho paralela a los ciclos temporales (anuales), lo que en el caso de este estudio se hizo siguiendo dos trampas de sedimento de forma quincenal en Banyuls-sur-Mer y en las Illes Medes. La hipótesis de partida es que dos lugares con un hidrodinamismo potencialmente diferente y con una influencia terrestre (a través de los ríos anexos a la zona de muestreo) también diferenciada, pueden repercutir en la dinámica de la materia en sedi- mentación, materia orgánica que después puede repercutir unas comunidades bentónicas que pueden ser muy distintas en biomasa, diversidad y estructura. Los organismos bentónicos regulan sus ciclos de actividad de acorde con la variabilidad ambiental. Este acoplamiento físico-biológico se interpreta como un ahorro energético

18 INTRODUCCIÓN (Sebens 1987). Los ritmos de actividad de los suspensívoros bentónicos han de verse influi- dos por la variabilidad temporal y espacial de los parámetros ambientales tales como corrientes, y biológicos tales como concentración y calidad de partículas transportadas por las mismas. La aproximación temporal en este sentido también ha de ser a diferentes escalas: estacional y diaria. También es útil “simplificar” la actividad de una comunidad a tra- vés de los organismos más representativos que la compongan. En la comunidad del coralí- geno Mediterráneo, que posee una muy elevada diversidad y por tanto de un elevado núme- ro de respuestas a la variabilidad ambiental potencialmente diferentes por parte de los organismos, los suspensívoros bentónicos pasivos forman el componente más representativo (True1970, Gili y Ros 1985). Mediante el estudio estacional de la actividad de seis especies de cnidarios se intentará comprobar el nivel de acoplamiento físico-biológico y además si la crisis trófica estival (Coma et al. 2000). En general, se trata de observar si hay una respuesta diferenciada a lo largo de las estaciones en consonancia a épocas de mayor o menor productividad que vayan paralelas a un aprovechamiento de energía en forma de reproduc- ción, crecimiento, etc. Por otra parte, los ritmos de actividad a escala diaria nos permiten ver por un lado cuales son los parámetros ambientales específicos (intensidad de corrientes, concentración de partículas y calidad de esas partículas) que activan dichos ritmos en los suspensívoros pasivos. Por otra parte si hay o no una respuesta de actividad sinérgica a nivel de comunidad cuando las condiciones de corriente y partícula convergen en sus niveles más adecuados para los organismos que han de alimentarse del seston. También es importante plantear una comparación espacial, aunque en este caso a nivel de pocos metros de distancia, debido a que la respuesta frente a cambios ambientales en forma de actividad de los organismos es muy rápida. Sí cabe la posibilidad de que subpo- blaciones de una misma especie tengan muy diferente respuesta a corrientes similares debido a su ubicación en el espacio, porque la propia topografía del substrato hace que dirección e intensidad del flujo iniciales se tornen muy distintas. La captura de presas es el siguiente paso necesario cuando tratamos de entender cómo repercute la variabilidad de parámetros ambientales físicos y biológicos en la ecología tró- fica de los suspensívoros bentónicos. Esta variabilidad, tanto espacial como temporal, ten- dría que verse reflejada en la dieta y tasas de captura de un suspensívoro pasivo y reflejan el acoplamiento plancton-bentos. Un ejemplo es el de la gorgónia Leptogorgia sarmentosa , habitante de substratos blandos-detríticos del Mediterráneo. El conocimiento de su alimen- tación ha de ser un paso previo para después poder abordar el análisis temporal y espacial de la variabilidad de dieta y captura. De esta forma se reunirán dos objetivos en uno: cono- cer la dieta y tasas de captura anuales de un suspensívoro pasivo que habita fondos blandos- detríticos (hecho de por sí novedoso debido a la escasísima información que hay de estos organismos en tales fondos) y profundizar más en las consecuencias de una variabilidad espacial y temporal del flujo energético proveniente del seston en un mismo organismo que vive en unas condiciones ambientales distintas. Por ello se eligieron tres poblaciones de la gorgonia Leptogorgia sarmentosa : una población en la que se estudío el ciclo anual de tasas de captura integrando diferentes intervalos de tiempo de muestreo y estudiando sus tiempos de digestión a diferentes temperaturas, y teniendo en cuenta sus ritmos de actividad; con esta población también se harían experimentos para calcular la tasa de ingestión de detritus y organismos unicelulares (diatomeas, dinoflagelados, ciliados, etc.) no cuantificables a tra- vés de los contenidos estomacales. Por otro lado, la aproximación espacial se hizo a través

INTRODUCCIÓN 19 de dos poblaciones diferentes, separadas varios kilómetros de distancia (Banyuls-sur-Mer, Francia; Illes Medes, España; ca 50 km). El estudio de los contenidos estomacales será un buen registro de las entradas aproximadas de carbono, y daría pie a interpretar similitudes o diferencias según las condiciones ambientales en ambas poblaciones de la misma especie, se espera poder demostrar que las diferencias en el seston, en la materia orgánica particulada en sedimentación entre ambas zonas, deben verse reflejadas en las tasas de captura de un suspensívoro pasivo como Leptogorgia sarmentosa que depende totalmente de las partícu- las que son transportadas o resuspendidas por las corrientes. Como herramienta de acercamiento al acoplamiento plancton-bentos, la alimentación es esencial para comprender qué fracción del seston es depredada y cual es el impacto sobre la columna de agua. Sin embargo, debido a la extrema variabilidad del seston cerca del fondo, el estudio del acoplamiento plancton-bentos de forma estacional (esta vez como repercusión en el organismo) ha de hacerse con otros métodos que permitan integrar episodios más lar- gos que los puntuales de la captura. Al igual que para el seston se busca un modo de registrar pulsos a través de las trampas de sedimento, una buena aproximación a cómo las variables ambientales afectan el estado de la población a lo largo de ciclos anuales es el balance Proteína-Carbohidrato-Lípido. Este balance puede aportarnos mucha información acerca de cual es la manera de acumular energía por parte de una especie, cual es la relación de ese acumulo energético con la reproducción de dicha especie, si hay o no coincidencia de este algún elemento de este balance con épocas de crecimiento, si pueden o no haber indivi- duos/colonias más o menos favorecidas dentro de una población (según las condiciones hidrodinámicas potencialemente más o menos favorables), y por último el registro estacional de épocas de carestía durante un ciclo anual. Este balance macromolecular actúa a modo de “memoria” de procesos estacionales (Clarke 1977). Un ejemplo a este respecto es el siguiente: hay indicios claros para una serie de organismos suspensívoros de que en mares templados en verano (debido a la estratificación de las aguas, disminución de las corrientes, empobrecimiento de nutrientes y falta de materia en suspensión) sufren un proceso de esti- vación, es decir una ralentización de su actividad y metabolismo (Coma et al. 2000); esto se traduce en muchos casos en una ingesta menor (Coma et al. 1998) y por tanto habría de tra- ducirse en ritmos de menor actividad (o sea, en el caso de antozoos poblaciones con más colonias con pólipos contraídos) y una depauperación de las reservas energéticas. Pero por otro lado, a finales de otoño inicio de invierno, la columna de agua se empobrece al colap- sar la producción primaria debido a un exceso de turbulencia y turbidez (Sverdrup 1953), proveniente de la disminución de irradiancia, de un exceso de oleaje (y resuspensión de par- tículas) y de un exceso de materia pobre (debido también a las fuertes avenidas de ríos y rie- ras cargadas de material muy refractario). El alimento potencial durante esta época, aunque muy abundante, es muy pobre y muy poco asimilable (Grémare et al. 1997). Estudiando tan sólo la tasa de captura, ritmos de actividad de los organismos o un muestreo puntual del seston pueden darnos una imagen equivocada, debido a la extrema variabilidad de estos pará- metros; sin embargo, el propio registro de la materia orgánica particulada en sedimentación (trampas de sedimento que registrarán adecuadamente las fluctuaciones del seston) y el balance Proteína-Carbohidrato-Lípido de las comunidades bentónicas (balance metabólico que registrará las épocas de carestía alimenticia de las especies) pueden proporcionar una aproximación más completa para interpretar el acoplamiento plancton-bentos a lo largo de ciclos anuales. Es por este motivo que se siguieron ciclos estacionales del balance Proteína-

20 INTRODUCCIÓN Carbohidrato-Lípido en dos especies de suspensívoros pasivos que habitaban zonas diferentes ( Paramuricea clavata , substrato rocoso; Leptogorgia sarmentosa , substrato blando- detrítico, Weinberg 1978) y que, potencialmente, tenían una distribución, estructura de población, morfología, alimentación, crecimiento y reproducción diferentes. Estos balances se relacionaron con dieta, tasas de captura, esfuerzo reproductor y crecimiento, para intentar ver si había o no un efecto de registro de estos factores con dicho balance. En este trabajo se han tratado de abordar todos los puntos mencionados para comprender mejor las relaciones entre la columna de agua y las comunidades bentónicas, así como los factores ambientales que influyen en los flujos de energía de los organismos bentónicos y los procesos de acoplamiento plancton-bentos. Se presentan una serie de capítulos ordenados de la siguiente manera: 1) El primer capítulo presenta relaciones entre la concentración del seston cerca del fondo con diferentes factores ambientales que potencialmente puedan influirlo (temperatura del agua, oleaje, descarga de un río anexo); al mismo tiempo se analiza por fracciones la composición bioquímica y biológica de dicho seston a través de un muestreo quincenal que permita hacer un estudio estacional de estos parámetros. 2) El segundo capítulo presenta dos ciclos intensivos ubicados en una misma época del año (la primavera), en los que se trata de ver la variabilidad de los factores ambientales y cómo pueden afectar a la composición bioquímica del seston; de hecho, se trata de intentar registrar pulsos de producción en la columna de agua cercana al fondo y relacionar su frecuencia con períodos de reproducción y crecimiento en las comunidades bentónicas. 3) El tercer capítulo relaciona condiciones hidrodinámicas, con- centración de Clorofila a en la columna y cantidad y calidad de la materia orgánica particulada en sedimentación de dos lugares a unos 50 km de distancia; la finalidad de esta comparación espacial del seston es intentar hacer una composición ambiental que pueda afectar a las comunidades bentónicas que viven en ambos lugares a mediante la utiliza- ción de las trampas de sedimento que puede integrar pulsos de producción-resuspensión en la columna de agua. 4) El capítulo nº 4 engloba ritmos de actividad de seis suspensí- voros pasivos (los antozoos Paramuricea clavata , Eunicella singularis , Leptogorgia sarmentosa , Corallium rubrum , Alcyonium acaule y Parazoanthus axinellae ) a diferentes escalas temporales (estacional y diaria) para entender en qué forma afectan las variables ambientales (corrientes), y biológicos (concentración y composición del seston) a su diná- mica anual y diaria. Ello permitirá encontrar un nexo entre la variabilidad ambiental y la respuesta de la comunidad bentónica para explotar esa variabilidad de la forma energéti- camente más adecuada. 5) El quinto capítulo hace una descripción exhaustiva de la dieta de un suspensívoro pasivo que habita en fondos blandos-detríticos ( Leptogorgia sarmentosa ) a través de contenidos estomacales de un ciclo anual, experimentos de digestión a diferentes temperaturas (dieta natural), implementación de los ritmos de actividad a las tasas de captura y experimentos puntuales (primavera) de la fracción no cuantificable a través de contenidos estomacales (campanas de incubación); También se incluye un estudio de la variabilidad espacial, en la que dos poblaciones de dicho suspensívoro se comparan en su dieta y tasas de captura teniendo en cuenta factores ambientales diferentes (Capítulo III). 6) El último capítulo se centra en el balance Proteína-Carbohidrato-Lípido como una herramienta que permita detectar cambios estacionales en el acoplamiento plancton-bentos, y relacionar estos cambios con distintos procesos de producción secundaria (reproducción, crecimiento, etc.) en dos especies de suspensívoros pasivos

INTRODUCCIÓN 21 (Paramuricea clavata y Leptogorgia sarmentosa ) que potencialmente difieren en cuanto a sus estrategias ecológicas y tróficas. A pesar de que esta tesis doctoral presenta un objetivo común (intentar comprender un poco mejor cómo afectan los parámetros ambientales y biológicos, y su variabilidad a diferentes escalas en la ecología trófica de los suspensívoros bentónicos), cada uno de los capí- tulos han sido planteados con estructura de artículos para revistas científicas; de esta forma podemos identificar cada una de las preguntas dentro de un contexto que después pueda ser directamente presentado a la comunidad científica por separado y facilitar así su difusión.

22 INTRODUCCIÓN General Introduction

Scales of variability in both planktonic and benthic marine communities are still poorly understood. In general, the factors that influence variability in a community are placed in one of two categories, environmental (physico-chemical factors affecting the water column) and biological (physiological, genetic, etc.). All water masses are affected by physical processes which in turn affect the structure of both pelagic and benthic communities. In the Mediterranean Sea physical processes follow clear seasonal patterns that result in repeated fluctuations in planktonic production in the water column (Estrada et al. 1985, Estrada 1996). These fluctuations are reflected in the feeding, reproduction, growth, respiration, etc. of benthic communities (Boero 1984, Ballesteros 1989, Sardà et al. 1999, Coma et al. 2000). The pattern of seasonal fluctuations that is so distinctly discernible in surface waters may be less perceptible in the water column inshore and next to the sea bed as a result of interactions with the continental land mass and with the benthic communities dwelling on the bottom in those areas. On the one hand, increased complexity in patterns of wind speed and direction driven by terrestrial geographic features, the effect of sudden continental inputs, and/or the sporadic disturbances caused by storms add to the complexity of seston dynamics in the border region between the shore and the neighbouring water masses. On the other hand, bottom topography, differences in near-bottom particle dynamics, and/or at times extremely complex communities of organisms mean that the features of the water mass along the sea floor can be hard to interpret. Both phytoplankton and zooplankton cycles have been reported to be less seasonal in nature and subject to higher levels of temporal variability as the distance from shore decreases (Colebrook and Robinson 1965). Furthermore, a series of physical and biological processes affecting particle concentrations also occur close to the bottom and may act to mask what would otherwise be clear temporal variations (Thomsen 1999). Benthic suspension feeders and other organisms feed on this particulate matter, hence an understanding of the dynamics of this material and an attempt to determine the scales of temporal and spatial variability that affect seston concentration and variability will help improve our understanding of coupling between the plankton and the benthos. Benthic-dwelling organisms have adapted to these dynamic patterns, and their biology is closely linked to sestonic heterogeneity. Accordingly, this study presents an analysis of the factors that affect both the physical and biological factors that influence the dynamics of benthic suspension feeder communities as well as of the effects on such communities exerted by these factors. To this end, it has been necessary to address two different categories of event (causes and effects) that follow different temporal and spatial scales. The first step in elucidating the physical factors involved in the seasonal dynamics regulating the particles making up the seston was to con- duct frequent sampling of sestonic quality and concentrations near the bottom over an entire annual cycle. The annual cycle also provided information on the seasonal patterns in the fractions (particle size) and composition (chlorophyll a, total carbon, organic carbon, etc.) of the seston in the water mass that can be detected by weekly or bi-weekly sampling. Nevertheless, an annual analysis may not suffice to detect clear seasonal fluctuations where the influence of shore and bottom-related factors is most pronounced as a result of a series of pulses that can augment variability and thus mask seasonal trends. To elucidate aspects of variability in greater detail requires a more intensive analysis of the physical and biological factors regulating the seston over shorter time periods, so that

INTRODUCTION 25 changes taking place near the bottom that will help explain the feeding, reproduction, growth, etc. dynamics of benthic suspension feeders will be discernible. Intensive sampling of this kind, over short time periods of 4-6 h, are adequate for comparison of different points in time within a single season, e.g. the spring, the time of year considered most productive in the Mediterranean. Pulses can be critical in elucidating the energy balance of this type of organism, and the high sampling rate should furnish data that will indicate whether more continuous inputs of available energy within a single season with high productivity (spring), together with such other factors as light and temperature, can be invested in reproduction or growth. A tool that enables the variable time to be taken into account is needed to be able to detect trends in suspended matter available to benthic suspension feeders. Even though pulses will not be followed with high precision over time, such a tool will nonetheless “memo- rize” their effects and thereby provide for better observation of seasonality of both production in the water column and the most conspicuous resuspension events. Sediment traps have been used as such a tool here. The traps collect organic and inorganic material settling to the bottom before it becomes either part of the diet of the benthos or part of the bottom sediment itself. On the whole, sediment traps have revealed that there are two clearly dif- ferentiated time periods in the Mediterranean in respect of both the quantity and the quality of the sedimentary matter: autumn-winter, in which there are large amounts of suspended matter but that matter is of poor nutritional value (as indicated by the organic matter and protein-carbohydrate-lipid balance), and spring-summer, in which the amount of suspended matter is smaller but the matter available is of higher nutritional value (Fichez 1991, Grémare et al. 1997). The information on seasonal aspects in the dynamics of sedimentary organic matter provided by the sediment traps is the basis for the next step: examining spatial heterogeneity of the seston by means of the sedimentation rate and biochemical descriptors, thus providing an indication of the similarities or differences in the seston available to benthic suspension feeders. Few studies have undertaken a small-scale approach (on a scale of hundreds of metres or a few kilometres), especially in the Mediterranean (Pusceddu et al. 1999a). It is quite clear that microhabitat differences capable of affecting the biomass, biology, physiology, etc. of organisms can be found in a span of just a few metres (Airoldi and Virgilio 1998, Underwood and Chapman 1998, Benedetti-Cecchi 2001), but at the community level it is necessary to develop a more global picture of a broader spectrum of inputs from the water column. This spatial comparison was carried out in parallel to the study of annual temporal cycles, which in terms of this study meant monitoring two sediment traps, one in Banyuls- sur-Mer and the other in the Medes Islands, on a bi-weekly basis. The initial hypothesis was that two locations with potentially quite dissimilar hydrodynamics as well as differing continental inputs from the rivers adjacent to the sampling sites could have different implications for the sedimentation dynamics of the organic matter, which in turn could produce benthic communities differing in biomass, diversity, and structure. Benthic organisms regulate their activity cycles in response to environmental variability. This physical-biological coupling has been regarded as an energy saving measure (Sebens 1987). Activity rhythms of benthic suspension feeders are affected by temporal and spatial variability in such environmental factors as currents and such biological factors as the quality and concentration of particulate matter carried by the currents. Here too, the tem-

26 INTRODUCTION poral coverage needs to employ different scales, seasonal and daily. It is also helpful to “simplify” community activity by reducing it to the community’s most representative organisms. Passive suspension feeders are the most representative component in the coralligenous community in the Mediterranean, with its high diversity and hence large number of potentially different responses by component organisms to environmental variability (True 1970, Gili and Ros 1985). A seasonal study of the activity of six cnidarian species has been undertaken in an attempt to elucidate the level of physical-biological coupling as well as whether the summer trophic crisis (Coma et al. 2000). The study is intended to observe whether responses differ with the season according to the level of productivity and with energy use for reproduction, growth, and the like. Additionally, daily activity levels should indicate the specific environmental factors (current flow rate, particle concentration, and particle quality) that trigger these rhythms in passive suspension feeders, as well as whether or not there is a synergistic response in the activity of the community as a whole when current flow and particle conditions are both at their optimal levels for organisms that feed on the seston. A spatial comparison is likewise of great interest in this connection, though in this case on a scale of just a few metres, because organisms are extremely fast in regulating their activity in response to environmental change. Subpopulations of the same species may exhibit quite different responses to similar current flows as a result of their spatial placement, in that substratum topography may considerably alter the initial flow rate and direction. The next step in trying to understand the effect of variability in physical and biological environmental factors on the feeding ecology of benthic suspension feeders is the study of prey capture. Both spatial and temporal variability should be reflected in the diet and capture rates of passive suspension feeders, which should in turn reflect coupling between the plankton and the benthos. The gorgonian Leptogorgia sarmentosa , which dwells on soft, detrital substrata in the Mediterranean, is a good example of this. A knowledge of its feeding pattern is a preliminary step for later being able to undertake a spatial and temporal analysis of variability in both diet and prey capture. This also achieves two objectives simultaneously: it furnishes data on the yearly diet and prey capture patterns for a passive suspension feeder dwelling on soft, detrital bottoms (information that is in itself novel in view of the paucity of data available for suspension feeders on this type of bottom) while at the same time providing an in-depth picture of the effects of spatial and temporal variability in energy transfer from the seston in one and the same species under differing environmental conditions. To this end, three different populations of Leptogorgia sarmentosa were selected. One population was chosen for a study both of the annual cycle of prey capture rates using different sampling intervals and considering different temperatures (experiments) of digestion, taking into account the organism’s activity rhythm; additionally, experiments were carried out on this same population to calculate the ingestion rate for detritus and single-celled organisms (diatoms, dinoflagellates, ciliates, etc.) not quantifiable in the stomach contents. Also, a study of spatial aspects was performed using two different populations sep- arated by a distance of around 50 km, one in Banyuls-sur-Mer, France and the other in the Medes Islands, Spain. Stomach content analysis normally provides a good, though some- what rough, record of carbon inputs and a basis for estimating similarities and differences according to environmental conditions in each of two separate populations of the same species. It is expected that differences in the seston, i.e., in the particulate organic matter set-

INTRODUCTION 27 tling to the bottom at the two locations, will be reflected in the prey capture rates of this passive suspension feeder, which is wholly dependent upon the particles carried to it or resuspended by currents. Feeding is an essential tool for describing coupling between the plankton and the benthos and in elucidating the fraction of the seston preyed upon and the impact on the water column. However, because of the extremely high variability in the near-bottom seston, any seasonal study of coupling between the plankton and the benthos (and its effects on L. sarmentosa ) has to employ methods that will enable intervals longer than the very moment of capture of prey items to be taken into account. Just as sediment traps are used as a means of recording pulses in the case of the seston, the protein-carbohydrate-lipid balance is a good means of examining how environmental factors affect a population over annual cycles. The balance can provide large amounts of information as to how a species accumulates energy, how the accumulation of energy relates to reproduction in that species, whether or not there is a correspondence between any of the components of the balance and periods of growth, whether or not some individuals or colonies within a population may be able to benefit more than others (from potentially more or less favourable hydrodynamic conditions), and finally whether there are seasonal periods of scarcity within the annual cycle. This macromolecular balance acts as a sort of record or “memory” of seasonal processes (Clarke 1977). The following example may be illustrative. There are clear indications that a series of suspension feeding organisms inhabiting temperate seas undergo aestivation, i.e., a slowdown in metabolism and activity, in summer because of stratification of the water column, lower current flow, nutrient depletion, and a paucity of suspended matter (Coma et al. 2000). In many instances this takes the form of reduced food intake (Coma et al. 1998), which should in turn result in lower activity rhythms (e.g., in the case of anthozoans, populations in which there are more colonies with contracted polyps) and depleted energy reserves. Furthermore, in late autumn early winter, the water column undergoes impoverishment because of depressed primary production brought about by excessive turbulence and turbidity (Sverdrup 1953) caused by lower light levels, high wave action (and particle resuspension), and excessive amounts of low-quality matter (in turn caused by large outflows of highly refractory material from rivers). Though potential food is extremely abundant at this time of year, the food that is available is of low quality and is hard to assimilate (Grémare et al. 1997). Focusing solely on capture rates, an organism’s activity rhythms, or discrete seston samples could yield an erroneous picture given the high level of variability to which all these factors are subject. Nevertheless, the record of sedimentary particulate organic matter collected by sediment traps, which will adequately profile the fluctuations taking place in the seston, and the protein-carbohydrate-lipid balance of benthic communities, which will reflect the metabolic balance and thus provide a record of periods of trophic crisis suffered by individual species, should provide a more accurate basis for interpreting coupling between the plankton and the benthos over annual cycles. For this reason, the protein-carbohydrate-lipid balance has been monitored over seasonal cycles in two passive suspension feeding species dwelling in different habitats, namely, Paramuricea clavata , which inhabits rocky substrata, and Leptogorgia sarmentosa , which inhabits soft, detrital substrata (Weinberg 1978), on the assumption that the distribution, population structure, morphology, feeding pattern, growth, and reproduction of these species were all potentially different. The data on the protein-carbohydrate-lipid balance for these species were related to the infor-

28 INTRODUCTION mation on the diet, prey capture rate, reproductive effort, and growth, in an attempt to determine whether or not there was any record of these factors in the balance. This study has endeavoured to address all the aforementioned aspects in an effort to better understand the relationships between the water column and benthic communities and to elucidate coupling between the plankton and the benthos and the environmental factors that affect energy flows to and from benthic organisms. The presentation has been divided into the following chapters. Chapter One discusses the relationships between near-bottom seston concentrations and the different environmental factors potentially exerting an influence on the seston, namely, water temperature, wave action, and run-off from a nearby river. It also examines the biochemical and biological composition of the seston by fraction based on bi-weekly samples suitable for a seasonal analysis of the factors considered. Chapter Two describes two intensive sampling cycles carried out within the same season of the year (spring), in an attempt to elucidate environmental variability and how it may affect the biochemical composition of the seston. More specifically, this portion of the study sought to record production pulses in the water column near the bottom and to relate the frequency of such pulses with periods of reproductive activity and growth by benthic communities. Chapter Three compares hydrodynamic conditions, chlorophyll a levels in the water column, and the amount and quality of sedimentary particulate organic matter at two sampling sites located some 50 km apart. The purpose of this spatial comparison of the seston was to try to derive a clear picture of the environmental factors affecting the benthic communities at each of the sampling sites. Sediment traps were used to record production pulses and episodes of resuspension in the water column Chapter Four covers the activity rhythms of six passive suspension feeders, the anthozoans Paramuricea clavata , Eunicella singularis , Leptogorgia sarmentosa , Corallium rubrum , Alcyonium acaule , and Parazoanthus axinellae , on two different time scales (seasonal and daily) to see how environmental factors (current flow) and biological factors (seston concentration and composition) affect the annual and daily activity patterns of these species. This portion of the study was designed to link environmental variability and the response by the benthic community to derive the most energy benefit from the variability. Chapter Five presents a comprehensive analysis of the diet of a passive suspension feeder that inhabits soft, detrital bottoms, Leptogorgia sarmentosa , based on the stomach contents collected over a yearly cycle, digestion experiments carried out at different temperatures (natural diet), and application of the activity rhythms to the non-quantifiable fraction and to the stomach contents. This chapter also includes a consideration of spatial variability in two populations of this suspension feeder, comparing the diet and prey capture rates in the light of the differences in the environmental factors (Chapter Three). Chapter Six, the final chapter, uses the protein-carbohydrate-lipid balance as a tool for observing seasonal changes in coupling between the plankton and the benthos and relating changes to the various secondary production processes (reproduction, growth, etc.) in two species of passive suspension feeders, Paramuricea clavata and Leptogorgia sarmentosa , with potentially different ecological and feeding strategies. Although this doctoral dissertation has a single object, that of increasing our knowledge of the effect of variability in environmental and biological factors, when considered on dif-

INTRODUCTION 29 ferent spatio-temporal scales, on the feeding ecology of benthic suspension feeders, each of the chapters has been structured as a self-contained paper for a scientific journal. This structure is intended to allow each of the issues examined here to be more specifically addressed in a context suitable for direct presentation to the scientific community separately and thus to facilitate dissemination of the content.

30 INTRODUCTION Temporal variability of near-bottom seston concentration and composition in a warm temperate sea over an annual cycle

Abstract

Trends in seston composition in the near-bottom water layer and the influence of environmental factors were studied in a temperate climate (NW Mediterranean Sea) over an entire annual cycle. Chlorophyll a (Chl a), total and organic particulate carbon (TPC and POC), particulate organic nitrogen (PON), particulate proteins and carbohydrates, and zooplankton were analysed from samples collected every two weeks at a depth of 20 m (0.5 m above the bottom) from May 1997 to August 1998. Biochemical analyses were performed on the <100 µm and <10 µm size fractions in order to evaluate the role of the microplankton and the nanoplankton in seston composition. As a further estimate of overall seston concentration, in situ horizontal Secchi disk (HSD) and vertical Secchi disk (VSD) measurements were made in the immediate vicinity of the sampling site. The environmental factors water temperature, river runoff, and wave height were recorded from January 1997 to August 1998. The <10 µm Chl a fraction was prevalent over the sampling period, and was the only biochemical parameter to exhibit seasonal trends. Other than during late winter-early spring blooms, the <10 µm fraction predominated over all the rest of the parameters analysed (e.g., accounting for more than 80% of the <100 µm POC and more than 90% of the <100 µm PON). The 10-100 µm TPC fraction represented nearly 32% of the <100 µm TPC fraction. The <10 µm fraction was the principle POC fraction over the sampling period, suggestive of a sizeable proportion of inorganic material in the 10-100 µm fraction. Protein and carbohydrate carbon made up nearly 50% of the POC over the sampling period, suggesting a rich near-bottom seston composition. The >100 µm zooplankton was dominated by copepods and did not display any apparent seasonal trends. Seasonal trends were observed only for meroplankton larval and gelatinous filter feeding zooplanktonic (i.e., Salpae and Appendicularia) pulses, which coincided with winter- spring and summer Chl a peaks. River runoff and wave action explained 57% of the variance in the vertical Secchi disk measurements and 44% of variance in the horizontal Secchi disk measurements, suggesting a direct influence on seston concentration in the study area. Water temperature explained 61% of the variance in the VSD and HSD readings. This chapter presents the first report on seasonality in the near-bottom seston in a temperate littoral area. The results indicate that seston in the near-bottom water layer behaves differently from that in the rest of the water column. Stochastic processes in the boundary system described here may mask seasonal trends, differing from the clear seasonal trends in seston composition observable at a greater distance from the coast or higher in the water column off the bottom. It is suggested that pulses in environmental factors (e.g., wave height or river runoff), benthic community activity, and properties particular to the seston in this near-bottom water layer may mask seasonal trends in seston composition. The near-bottom seston could have more important consequences for the rest of the water column than previously thought.

Introduction

Coastal seston composition may be influenced by several physical and biological factors, such as water temperature, light intensity, river runoff, tidal flows, wind stress, and sea bed morphology. The influence of each of these factors has previously been assessed and found to be a key contributor to seston variability (Margalef 1998). Seston composition is highly variable inshore near the bottom, because coastal hydrography is also variable due to fluctuations in wind stress and continental runoff near the coast (Millot 1979, Nogueira et al. 2000) and to the effect of bottom topography (Riedl 1971). Lower sedimentation rates as

TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON 33 3º 13' E Figure 1. Study area location (Medes Islands). Black dot indicates the sampling area. 0 0.5 1 km

L'ESTARTIT

MEDES ISLANDS

42º 02' N

Ter River

Sea ean Mediterran

compared to the surface portion of the water column, horizontal transport, and higher resi- dence times for particles near the bottom are responsible for differences in seston dynamics near the substratum (Reynolds 1993, Thomsen et al. 1995, Thomsen and Weering 1998). The activity of the benthic community can also influence seston composition and concentration (Cloern 1982, Fabricius and Domisse 2000). In general, near-bottom processes are similar to those in littoral systems: boundaries where inputs-outputs of matter in the water column increase the richness of the seston composition but at the same time also increase variability through the recurrence of unpredictable, sporadic pulses (Margalef 1998). In temperate environments like the Mediterranean Sea, variability in physical factors affects both the production and the physiology of plankton communities (Estrada 1996). For instance, primary production peaks in the water column contribute to seston composition. Inputs of organic detritus contribute seasonally to benthic primary production (macroalgae, etc.) and thus to the composition and abundance of the near-shore seston (Mann 1988). Such

34 TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON environmental and biological factors affect both the seston in the water column and benthic communities (Coma et al. 2000). Activity by benthic communities may also be responsible for a portion of near-bottom seston variability, as the activity of benthic organisms may deplete (predation), add to (reproduction, egesta), and recycle (excretion) large amounts of organic matter (Gili and Coma 1998). Even though the magnitude and importance of near- bottom seston-planktonic communities and benthic-pelagic coupling of food chains has been demonstrated, these aspects have received scant attention by marine scientists (Arntz et al. 1999). Few studies have focused on temporal changes in seston composition in the near-bottom water layer (Fegley et al. 1992, Navarro and Thompson 1995, Ribes et al. 1999). The temporal dynamics of near-shore bottom seston communities differs from that of water-column seston in the open sea. Accordingly, resuspension and associated processes taking place in the benthic boundary layer must be considered key to our understanding of the dynamics of coastal environments (Graf 1992). We have hypothesized that seston concentration should exhibit seasonal trends, but the biological composition of the seston does not necessarily follow the same seasonal pattern. Despite the accepted influence of bottom communities on seston composition and concentration (Gili and Coma 1998), the effect of the activity of benthic organisms on the near-bottom water layer is difficult to assess. Information of this sort has mostly been obtained for cases of monospecific communities or in controlled situations (Cloern 1982, Asmus and Asmus 1991). Quantification of the biological characteristics of benthic organisms that may affect near-bottom seston composition is complicated because of the difficulty inherent in studying the activity and impact of a highly diverse biota. In contrast, physical factors (e.g., temperature, river runoff, wave height) that may affect seston variability in the water column are easier to record (Zingone et al. 1995, Nogueira et al. 2000). In coastal ecosystems seston communities are influenced by the environmental and biological processes taking place in the water column and the near-bottom layer. For an overview of biological production, both these processes must be taken into account, along with the role of benthic communities on water column dynamics. Given the paucity of studies on the near-bottom seston, the present study had the following four objects: 1) to quantify seston composition in the near-bottom water layer in a temperate ecosystem to better understand the dynamics of coastal seston communities; 2) to study which environmental factors may influence overall seston concentration near the bottom; 3) to study the relationship between seston concentration and composition and variations over an annual cycle; and 4) to compare patterns of seston dynamics in the near-bottom water layer with previous known trends observed at higher levels in the water column in similar temperate ecosystems. To fulfill these objectives, various biological parameters were quantified to assess the proportions of inorganic and organic carbon and ascertain the proportion of particulate organic carbon (POC) available to higher trophic levels. Most biochemical parameters were estimated for both the <100 µm and the <10 µm size fractions in order to assess the contribution of micro- and nanoplanktonic organisms to total seston composition, given that the <10 µm fraction has been reported to account for more than 80% of total planktonic biomass in the open sea and coastal waters of the NW Mediterranean (Raimbault et al. 1988, Delgado et al. 1992, Ferrier-Pagès and Rassoulzadegan 1994).

TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON 35 Materials and methods

General characteristics of the study area. The physical and biological parameters of the sea water in the Medes Islands, NW Mediterranean (40º02’55’’N, 3º13’30’’E, Fig. 1) were studied between January 1997 and August 1998. This small archipelago (emerged area 21.92 ha) comprises a group of seven islets and a few rocky reefs. The underwater topography of the Medes Islands is markedly asymmetric, with prominent slopes on the northern side and gentle slopes on the southern side. Spring-summer is characterized by strong, dry winds from the north-northwest. In autumn (September-December) strong storms (wet) arise suddenly off the northeastern coast of Catalonia. Winter usually combines heavy storms from the north and west with long periods of calm without wind (Cebrián et al. 1996). The Ter River is the main source of inputs of continental waters in the area, with a highly seasonal pattern of runoff (see Pascual et al. 1995, Cebrián et al. 1996), consisting of heavy discharges between November and February and low discharges in spring-summer. Water temperature is also highly seasonal (see Pascual et al. 1995, Cebrián et al. 1996), with a highly developed thermocline between April and October at a depth of 30 m (with differences of 7-10ºC). Salinity is usually around 37.5-38‰, with maximum values in winter during mixing events produced by heavy storms caused by the north winds. The influence of Ter River discharges can be high, lowering the salinity values to 34-35‰ exceptionally even to 32‰ (Pasqual and Flos 1984). Seston measurements. Biological sampling of the biochemical characteristics of the seston and zooplankton composition was carried out from 30 May 1997 to 27 August 1998 at 19 m on the northern side of the Medes Islands. For the biochemical parameters, natural seawater (22-24 L) was collected by SCUBA diving every 15 days. The water was sampled using 2-L plastic bags, 0.2 - 0.5 m above the horizontal rocky surface, avoiding any movements that might result in resuspension of the sediment. The plastic bags were rinsed with deionized water after each use. On board the bags were emptied into 5-L bottles stored in ice (6-10ºC) until arrival at the laboratory on shore (maximum elapsed time between collection and arrival: 1 h). Seawater samples were processed at 6-10ºC in the dark. The seawater samples were pre-filtered through a 100 µm mesh to reduce variance. Part of this <100 µm water was filtered again through a 10 µm filter to estimate nanoseston composition. Biochemical characterization of the seston was performed for both the <100 µm and <10 µm size fractions, assessing chlorophyll a (Chl a), total particulate carbon (TPC), particulate organic carbon (POC), and particulate organic nitrogen (PON) concentrations. Total particulate protein and carbohydrate carbon was measured only for the <100 µm fraction. To determine the Chl a concentration in the <100 µm and <10 µm fractions, three 200- mL replicates were filtered through GF/F glass fiber filters. The filters were immediately frozen in liquid nitrogen and stored at – 80ºC until processing. Chl a extraction was performed by grinding the filters in 6 mL of 90% acetone, the homogenate being left in the dark at 4ºC for 30 min. After centrifugation at 2 000 rpm at 4ºC for 10 min, the supernatant was read in a Turner Designs fluorimeter as per Parsons et al. (1985). For the TPC (including both organic and inorganic carbon) and PON readings for the <100 µm and <10 µm fractions, three 800 mL replicates were filtered through precombusted (450ºC, 5 h) GF/F filters and immediately frozen in liquid nitrogen. Subsequently, the filters were dried at 60ºC for 24 h. The samples were analysed using a C:N autoanalyser

36 TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON (Perkin-Elmer 240). To determine the contribution of the POC to the two size fractions, samples were collected in the same way as for TPC. After deep-freezing and drying (60ºC, 24 h), the filters were left in an HCl-saturated atmosphere for 48 h to destroy any inorganic material (Ribes et al. 1999). Another 24 h were necessary to completely dry the filters before analysis. For the total particulate protein concentration three 1 400-mL replicates of seawater were filtered through precombusted GF/F filters that were immediately frozen in liquid nitrogen and stored at –80ºC until processing. Protein concentration was estimated spectrophotometrically by a slightly modified version of the method of Lowry et al. (1951). The filters were extracted by cold grinding in 1N NaOH (2 mL) in a tissue homogenizer. The assay was performed on 0.5 mL of the homogenate. After colorimetric reaction, the mixture was centrifuged at 3 000 rpm to avoid glass fibre interference in the 750-nm spectrophotometric reading. Bovine serum albumin (Sigma) was used as standard. Protein was converted to nitrogen and carbon using the equations µg protein N x 6.25 = µg protein and µg protein carbon = 0.50 x µg protein (Freifelder 1982). For total particulate carbohydrate concentration three 1 400-mL replicates of seawater were filtered through precombusted GF/F filters and again immediately frozen in liquid nitrogen and stored at –80ºC until processing. The methodology used was a slightly modified version of the method of Dubois et al. (1956). The filters were extracted by cold grinding in 2 mL deionized water using a tissue grinder. Once ground, an aliquot of 1 mL was taken for chemical reaction. At the end of the process, the samples were centrifuged at 3 000 rpm to avoid glass fibre interference in the 485-nm spectrophotometric readings. Glucose was used as standard. Carbohydrate was converted to carbon using the formula µg carbohydrate carbon = 0.44 x µg carbohydrate (Freifelder 1982). Zooplankton samples were collected in duplicate using 22 cm diameter nets with a mesh size of 100 µm. The nets were towed for a distance of 40 m by a diver (30 to 50 cm above the bottom-dwelling community). Topography in the sampling zone is complex, with large boulders and steep walls bearing dense populations of gorgonians, mainly Paramuricea clavata and Eunicella singularis (Gili and Ros 1985). Although other zooplankton studies near the benthos have used larger net diameters (see Stanwell-Smith et al. 1999), we decided to use smaller but more manoeuvrable nets. From January 1997 to April 1997 we tested 100 µm and 60 µm-mesh nets (Rossi, unpublished data) and concluded that although the 60 µm-mesh net caught significantly more nauplii and eggs, other groups were underestimated because nets rapidly clogged and the zooplankton exited again before the sampling transect had been fully covered. As a consequence, zooplankton smaller than 100 µm was underestimated, and in any case nauplii and eggs have not been considered in the results. The zooplankton samples were fixed in 6% formaldehyde in seawater and identified to the level of the main taxonomic groups. Zooplankton samples were observed under a binocular microscope after rinsing with DDW to avoid vapours from the fixative. When the zooplankton captured was too dense, making too many individuals present in the formalin-fixed sample, an aliquot was taken according to the following procedure: 1) the sample was rinsed with DDW and left to stand in a 50-mL cylinder for 2-8 h until sedimentation was complete; 2) the supernatant was removed, leaving a total volume of 20-40 mL (depending on sample concentration); 3) the cylinder was covered with plastic film and shaken to homogenise the volume; 4) an aliquot of 2-5

TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON 37 mL (depending on sample concentration) was extracted quickly and carefully spread on a Kolmogorov counter plate. Secchi disk. A vertical Secchi disk (VSD) was observed from a boat 20-30 m distant from the sampling point. A horizontal Secchi disk (HSD) was observed by chaining a disk with a float to a boulder in the study area adjacent to the sampling site and recording visual fading of the disk as the diver was swimming away from it in a straight line. The precision of the measurement was ±0.10 m. Both VSD and HSD were estimated from January 1997 to August 1998, between 9:00 a.m. and 2:00 p.m. to avoid light incidence problems (Preisendorfer 1982). Environmental data collection. Temperature (20 m depth), Ter River runoff, and wave height were sampled in the study area from January 1997 to August 1998. Water temperature and Ter River runoff were measured every 3-6 days and wave height was estimated daily. These physical data were collected according to the methods described by Pascual et al. (1995) and Cebrián et al. (1996). Statistical analysis . Correlation matrices were constructed for the physical and biological parameters collected every two weeks from 30 May 1997 to 27 August 1998. Standard methodology (Pearson’s product-moment correlation) was employed when the variables were normally distributed, and Spearman’s rank-order correlation matrices were constructed when variable distribution was not normal (Zar 1996, Dytham 1999). To analyse the possible effect of seasonality and/or size fraction on the variability of each parameter measured, ANOVA was run using Tuckey’s honest significant differences post-hoc comparison (Dytham 1999). To test the effect of seasonality, the data were grouped by season (Winter: January / February / March; Spring: April / May / June; Summer: July / August/ September; Autumn: October / November / December) and by the year in which they had been collect-

Table 1. Mean ± SD values for the different environmental and biological parameters analysed over the 20 month data collection period, by season (description in Materials and Methods)

Temp Wave height River runoff VSD HSD Chla<100 Chla<10 (ºC) (m) (m 3s-1 ) (m) (m) (µg l -1 ) (µg l -1 )

W1 (1997) 12.3±0.6 0.57±0.18 7.8±2.5 16.7±7.3 10.6±5.5 S1 15.1±2.1 0.65±0.26 5.7±5 18.7±2.5 13.7±4.3 0.530±0.140 0.442±0.151 Sm1 21.8±1.4 0.66±0.29 4.0±2.2 20±4.6 21.2±4 0.478±0.225 0.267±0.088 Au1 19±2.7 0.80±0.43 5.6±4.4 19.8±4.3 17.3±7.2 0.520±0.121 0.463±0.121 W2 (1998) 13.4±0.6 0.45±0.30 6.9±2.5 16.7±4.4 9.3±3 1.170±0.664 0.690±0.166 S2 14.8±1.8 0.61±0.16 2.8±0.8 18.2±3.1 12.2±4.4 0.830±0.459 0.309±0.106 Sm2 21.1±1.7 0.56±0.17 1.6±1.4 24.6±3.2 23.6±6.5 0.246±0.155 0.151±0.062

TPC<100 TPC<10 POC<100 POC<10 PON<100PON<10 Prot<100 Carb<100 Zoop>100 (µg l -1 ) (µg l -1 ) (µg l -1 ) (µg l -1 ) (µg l -1 ) (µg l -1 ) (µg l -1 ) (µg l -1 ) (ind m -3 )

S1 298±51 284±175 287±64 202±108 18±3 16±20 164±34 52±27 1654±1337 Sm1 349±145 154±25 217±46 111±21 25±5 29±3 165±76 80±64 2400±2542 Au1 234±35 164±52 191±20 124±18 17±2 17±3 101±27 70±27 1116±596 W2 (1998) 310±65 157±22 236±57 128±31 21±4 18±4 138±39 107±35 1536±1453 S2 336±41 160±26 287±53 133±32 25±5 22±5 170±43 101±85 1845±1106 Sm2 277±67 161±47 237±47 134±32 19±7 19±5 134±19 143±62 2078±1442

38 TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON a b

c d

Figure 2. (a) Chl a, (b) TPC, (c) POC and (d) PON over the 16 month sampling period. Solid line-solid symbols, <100 µm fraction; dashed line-blank symbols, <10 µm fraction. Mean ± SD (N=3 per sample). ed (1997 and 1998). The sampling seasons will hereinafter be referred to respectively as W1, S1, Sm1, and A1 for 1997 and W2, S2, and Sm2 for 1998.

Results

Biological parameters. The mean ± SD values for the different environmental and biological parameters in the water column in the different seasons are presented in Table 1. Considering the data set as a whole, the Chl a <100 µm (mean 0.648 ± 0.440 µg l -1 , n=112) -1 was significantly higher than the Chl a <10 µm (0.401 ± 0.203 µg l , n=111, F 1,221 =28.51, p<0.00001, Fig. 2a). The two Chl a size fractions exhibited differing trends over the 16 months of sampling. In S1, Sm1, A1 and Sm2, the <10 µm size fraction constituted between 72% and 95% of the <100 µm fraction. However, important phytoplankton blooms (winter- spring blooms) consisting mainly of >10 µm organisms occurred in W2 and S2. During these two seasons the Chl a <10 µm was only 58% and 37%, respectively, of the Chl a <100 µm (F 6,206 =4.47, p<0.0003). Overall, the Chl a values recorded in the period W2-S2 were significantly different from those measured during the rest of the study for both size fractions (F 6,106 =11.90, p<0.0001 for the <100 µm fraction and F 6,103 =27.23, p<0.0001 for the <10 µm fraction, Table 1).

TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON 39 a TPC and POC values for both the <10 µm and the <100 µm size fractions did not undergo seasonal variations over the study period (Fig. 2b,c). Considering the data set as a whole, the <10 µm size fraction was an important contributor to both the <100 µm TPC (62%, F 1,214 =83.94, p<0.00001) and POC (84%, F 2,214 =12.5, p<0.0005). In turn, POC contributed 57% of TPC in the <100 µm fraction b (F 1,224 =176.37, p<0.00001), and up to 77% of TPC in the <10 µm fraction (F 1,204 =14.45, p<0.0002). Considering the data by season (Table 1), TPC in the <100 µm fraction was always significantly higher than the corresponding POC value. In contrast, no significant differences between TPC and POC were found for the <10 µm fraction, except in the case of S1 (F 6, 192 = 0.93, p<0.4732). In general, POC <100 µm was similar to POC <10 µm (Fig. c 2c). Finally, TPC <100 µm was significantly higher than TPC <10 µm (Fig. 2b, except in S1 and A1). PON averaged 21.02 ± 4.12SD µg l -1 . Considering the data set as a whole, PON <100 µm was in no case significantly different from PON <10 µm (F 1,213 =0.23, p<0.6300), because the <10 µm fraction made up 97% of the PON, except in S2, when the small fraction was only 85 % of the <100 µm fraction (see Fig. 2d). No significant differences by season were found within the <100 µm fraction (F 6,95 =2.86, Figure 3. C:N ratio (a) POC:PON <100 µm (solid p<0.0132). The <10 µm fraction behaved line) and <10 µm (dashed line) over the 16 month similarly, with maxima in spring and mini- sampling period, (b) proportion (%) of autotrophic ma in autumn (Table 1). Overall, no clear (dashed line) and heterotrophic (solid line), (c) 10- 100 µm fraction <10 µm fraction. seasonal cycle in PON concentration was observed in either size fraction. The C:N ratio (POC:PON) is depicted for both fractions in Fig. 3a. The annual mean value for the <100 µm fraction (11.7) was higher than for the <10 µm fraction (7.1). Over the year the C:N <100 µm was higher than the C:N <10 µm. No significant differences were found between seasons for either the <100 µm fraction (F 5,23 =2.79, p<0.412) or the <10 µm fraction (F 5,22 =0.91, p<0.4931). Fig. 3b graphically represents the values for the autotrophic POC fraction (autotrophic POC = Chl a x 40, Delgado et al. 1992) and those for the het-

40 TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON erotrophic fraction. Autotrophic biomass peaks are generally related to summer and winter-spring blooms (Fig. 2a). The <10 µm fraction (Fig. 3c) was basically composed of the heterotrophic fraction all year long, with small peaks of autotrophic biomass in the same periods as the 10- 100 µm fraction. Samples of protein concentration in the water column were significantly lower in autumn than in the other seasons (Table 1); no correspondence with phytoplankton in Figure 4. Particulate protein (dashed line-empty late winter-early spring was found symbol) and carbohydrate (solid line-solid symbol) (F 6,107 =6.48, p<0.0001) [Fig. 4]. There <100 µm over the 16 month sampling period. were considerable fluctuations in carbohy- Mean ± SD (N=3 per sample). drate concentration without any clear seasonal pattern (Fig. 4, Table 1). On the other hand, no significant interannual differ- a ences were found for this parameter within each season. Proteins and carbohydrates made up 48% (mean over the 18 month cycle, Fig. 5a) of the <100 µm POC. The <10 µm POC fraction, which accounts for most of the POC (see POC results above), has been depicted in Fig. 5a. PON <100 µm and PON <10 µm have been graphically represented in Fig. 5b, along with the calculated protein nitrogen . b Protein nitrogen accounted for nearly 100 % of the PON (97 % over the year) in both fractions (very similar, see PON results). Tables 2 present the >100 µm zooplankton composition. Because 100 µm does not efficiently collect nauplii and copepod eggs, these zooplankton components have been omitted (see Materials and Methods). Copepods were the dominant group, accounting for 76% of the individuals. Maximum quantities of copepods were Figure 5. (a) Proportion of carbohydrate + protein recorded in late June 1997, minimum carbon (dotted line-”x” symbol) and the two POC quantities in March 1998. Over the 16- fractions (<100 µm, solid line-solid symbol; <10 µm, dashed line-blank symbol), (b) protein month cycle, Appendicularia abundance nitrogen (dotted line-dotted circle) and the two was an order of magnitude lower, con- PON fractions (<100 µm, solid line-solid symbol; tributing 7% of the individuals sampled, <10 µm, dashed line-solid symbol).

TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON 41 Table 2. Changes in density (ind m -3 ) of the main groups of near bottom water layer zooplankton (>100 µm) over 16 month sampling period.

1997 1998 30/5 16/6 28/6 28/7 28/8 8/9 23/9 6/10 16/10 13/11 26/11 12/12 14/1 30/1 13/2

COPEPODS 329 1256 6773 437 622 957 397 760 1467 640 360 179 300 744 251 APPENDICULARIA 29 272 261 41 26 29 75 61 11 21 33 9 25 68 23 LAMELLIBRANCHIA 16 27 5 7 104 19 1 0 16 66 19 3 61 51 52 PROTOZOA 15 16 0 11 7 48 79 83 139 79 19 4 85 117 23 TINTINNIDA 9 3 144 59 43 56 8 3 5 6 1 1 16 73 32 POLICHAETA 15 37 32 4 2 0 0 5 32 98 38 32 15 37 31 ECHINODERMATA 7 77 21 4 2 0 1 16 27 6 1 3 5 81 29 CLADOCERA 0 21 75 7 17 8 1 0 0 0 0 0 0 0 0 SALPIDA 4 21 171 25 10 8 13 5 0 0 1 0 0 2 0 SIPHONOPHORA 68 109 32 0 3 0 8 0 0 0 0 0 5 0 0 OTHER GROUPS 57 96 75 9 38 27 16 19 43 66 14 11 9 55 53

Total organisms 549 1936 7590 604 875 1152 600 952 1739 983 486 242 522 1228 494

27/2 19/3 26/3 9/4 23/4 29/4 15/5 13/6 28/6 15/7 31/7 19/8 27/8 N %

COPEPODS 1621 173 971 505 507 545 1749 1451 240 195 2187 1718 464 27799 76 APPENDICULARIA 147 4 80 57 53 17 21 473 32 33 315 260 32 2511 7 LAMELLIBRANCHIA 224 7 11 9 8 4 59 89 9 24 60 120 77 1146 3 PROTOZOA 43 9 12 31 27 8 0 14 18 37 9 41 40 1012 3 TINTINNIDA 75 0 3 4 4 0 21 0 1 21 26 3 3 619 2 POLICHAETA 51 8 7 13 5 15 16 14 3 5 17 29 11 572 2 ECHINODERMATA 75 0 7 15 0 5 32 21 7 7 3 0 0 452 1 CLADOCERA 5 1 0 0 1 1 0 50 0 39 207 3 3 440 1 SALPIDA 3 0 0 3 0 4 0 28 7 33 43 3 5 391 1 SIPHONOPHORA 48 11 1 8 5 3 5 25 3 1 6 0 0 343 1 OTHER GROUPS 99 36 41 17 12 27 32 82 24 39 62 41 53 1153 3

Total organisms 2390 249 1132 662 623 630 1936 2247 345 435 2932 2217 688 but they sometimes attained relatively high abundance levels (June 1997). Bivalve (lamellibranchia) larvae were also sizeable (3%), especially in summer and winter. No significant differences in zooplankton abundance were found season/year, nor was any a clear seasonal pattern observable (F 5,22 =0.47, p<0.7917). Environmental data . Table 1 gives the mean seasonal values for temperature, river runoff, wave height, VSD, and HSD. The data collected over 20 months revealed seasonal patterns for temperature at 20 meters (Fig. 6a) and for Ter River runoff (Fig. 6c). In contrast, wave height did not exhibit any seasonal trend (Fig. 6b). No significant interannual differences (F 6,115 =18.83, p<0.00001) were found on comparing data for the same seasons in different years. The highest variability in temperature was observed in summer (Fig. 6a). River Ter runoff was significantly higher in winter 1997 than in winter 1998 (F 6,72 =17.04, p<0.0003). The highest variability was recorded in winter-spring, and strong storms (caused mainly by East winds) were more frequent in autumn 1997. In particular, a mean wave height of ca. 5 m was measured at the Estartit Meteorological Station on 29 October and 17 December 1997, i.e., the strongest storms recorded in the region in the last 10 years. However, by season over the sampling period there were no statistical differences in wave height (F 6,107 =1.50, p<0.1855).

42 TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON a

b Figure 7. Seston concentration as measured by vertical (VSD, N=12-16 monthly, dashed line-blank symbols) and horizontal (HSD, N=2-6 monthly, solid line-solid symbols) Secchi disk readings over the 20-month sampling period, January 1997 to August 1998 (mean ± SD).

Secchi disk. The VSD values exhibited high variability over the entire sampling period yet nonetheless followed seasonal trends (Table 1, Fig. 7). There were no sta- c tistical differences in the interannual VSD values by season (F 6,83 =9.48, p<0.00001). Similarly, HSD values did not display any significant differences on comparing the different years by season (F 6,79 =12.3, p<0.00001). The environmental parameters wave height and River Ter runoff explained 57% of the variance in the VSD values and 44% of the variance in the HSD values, the two indicators of seston concentration (Table 3). In addition, significant positive Figure 6. Physical parameters: (a) temperature relationships between HSD and both VSD (N=12-16 monthly), (b) wave height (N=28-31 monthly) and (c) River Ter runoff (N=8-12 monthly) and temperature were found. Both VSD and over the 20 month sampling period, January 1997 to HSD were positively and significantly relat- August 1998 (mean ± SD). ed (see Table 3).

Table 3. Relationships between environmental parameters (wave height, river runoff, and water temperature) and seston concentration (horizontal and vertical Secchi disk); N=20

Regression curve R 2 p

VSD = 27.37 – 11.1wave height – 0.16river runoff 0.568 <0.001 HSD = 29.51 – 18.3wave height – 0.16river runoff 0.438 <0.01 VSD = 2.22 + 0.977temperature 0.611 <0.0001 HSD = -8.93 + 1.499temperature 0.615 <0.0001 HSD = -5.50 + 1.164VSD 0.580 <0.0001

TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON 43 Discussion

The biological and environmental parameter values collected during the 16 months of this study have for the first time furnished a complete characterization of the near-bottom water layer in a temperate littoral area. On average, POC accounted for 43% of the TPC <100 µm (Table 1; Figs. 1b,c) in the study area during that period. In other words, the POC made up 57% of the TPC. In its turn, more than 80% of the POC consisted of the nanoplanktonic size fraction i.e., <10 µm (Table 1; Fig. 1b,c), except in late winter-early spring and late summer, when microphytoplankton (10-100 µm) blooms (Table 1; Fig. 1a,b,c) occurred. Aside from the regular seasonal periodicity of Chl a concentration, none of the other biochemical parameters employed to characterize the nano- and microplanktonic size fractions of the seston displayed any clear seasonal trends. This is not surprising, as Chl a is related to light levels and nutrient availability (Smetacek and Passow 1990, Huismann et al. 1999), and these two factors are also related to light penetration and river discharges, which did follow seasonal trends. The absence of seasonal trends for most of the biological parameters contrasted with the clear seasonal pattern observed for temperature at 20 m depth, River Ter runoff, and both the VSD and HSD measurement values (Fig. 6a,c; Fig. 7). Wave height, on the other hand, did not follow any seasonal pattern. On the whole, significant relationships were found only among the different environmental parameters (Table 3). The water column exhibits clear seasonal trends in primary production in coastal waters and the open sea in the NW Mediterranean, with high primary production in late winter- early spring and a lower but detectable peak in autumn (Margalef and Castellví 1967, Estrada 1996). The phytoplankton blooms recorded in these periods mainly comprise the microplanktonic size fraction (10-100 µm) and occur in response to supplies of nutrient rich waters associated with water column mixing (Margalef 1978). In contrast, nanophytoplank- ton predominate in the periods after the blooms, times of water column stratification and nutrient exhaustion (Delgado et al. 1992, Ferrier-Pagès & Rassoulzadegan 1994). This same pattern was also found in the near-bottom water layer in the study area, as revealed by the size fraction make-up of both the Chl a and POC. The main phytoplankton blooms that occurred in winter-spring are attributable to general mixing of the water column and higher levels of river runoff. Small Chl a peaks were also found near the bottom in late summer (coinciding with a well-developed thermocline). These unexpected increases in phytoplankton biomass were associated with nutrient pulses resulting from sudden short bursts of rainfall or land-driven winds commonly observed in the Mediterranean (Cebrián et al. 1996, Duarte et al. 1999). Our results indicate that river inputs may indeed sometimes mask clear seasonal trends in seston composition in coastal waters in general (Duarte et al. 1999) and in near-bottom waters in particular. The results of autotrophic-heterotrophic contributions to total POC in the near-bottom water layer over the year (Figs. 2b,c) pointed up the prominent role of the 10-100 µm autotrophic fraction in the late winter-early spring diatom bloom. In the remainder of the sampling cycle, the <10 µm fraction predominated in the biomass. No seasonal trends were found for POC, PON, or protein and carbohydrate concentrations. These biochemical parameters are a different type of estimator of the total (i.e., autotrophic and heterotrophic) biomass and include detritus as well. The absence of clear relationships among them and between these parameters and the seasonal environmental parameters (i.e., temperature, river runoff) may be indicative of: 1) other sources of living and detri-

44 TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON tal material coexist with the succession of plankton in the water column, e.g., macroalgal productivity (Ballesteros 1989) or 2) the existence of other sources of variability, e.g., organic matter utilization and recycling by the near-bottom seston (Ritzrau et al. 1997). Heterotrophic flagellates and bacteria are abundant in the bottom layer throughout the year and display productivity peaks, except during the late winter-early spring blooms (Ribes et al. 1999). This heterotrophic biomass may be an important contributor to the total seston biomass and may be increased by the resuspension of detritus (Ritzrau 1996). In this sense it is very interesting to note that protein nitrogen accounted for most of the PON (Fig. 5b) and that when both carbohydrates and protein were expressed in terms of carbon, the protein nitrogen contributed a high proportion (almost 50%) of the POC (Fig. 5a), while other studies have reported values lower than 30% (see Fabiano et al. 1984, Arin et al. 1999). To date it has not been easy to distinguish living from detrital biomass (Rice 1982, Arin et al. 1999). It would not be surprising if a sizeable part of the near-bottom seston was detrital in origin, as has previously been observed in the open sea and coastal waters of the NW Mediterranean (Arin et al. 1999, Ribes et al. 1999), but in any case it is clear that a large proportion of the <100 µm bioseston in this area is available to higher trophic levels. The C:N ratio may also help in assessing the biological composition of the seston (Fig. 3a). The <100 µm fraction had a high C:N value (nearly 12), which is similar to the values observed for benthic macroalgae, seaweed, and phytodetritus of land origin (Poklington and Leonard 1979). In contrast, the <10 µm fraction had a lower C:N ratio (around 7), more typical of unicellular organisms (Poklington and Leonard 1979, Atkinson and Smith 1983). This suggests that a different kind of autotrophic organism or detritus is the source of part of the 10-100 µm POC material and that most of the nanoseston (<10 µm) is composed of live heterotrophic biomass, driven by resuspension and horizontal and vertical nutrient processes near the bottom. In any case, biochemical estimators indicate that the seston in the benthic boundary layer is quite rich and could be a food source both for the planktonic community itself and for the benthic community that may depend on it. Furthermore, a sub- stantial proportion of suspended material could have its origin in benthic metabolic activity. Such a scenario could explain the low seasonal variability of the biochemical composition of the near-bottom seston, thanks to the inputs from both benthic and planktonic activities. At the same time, such random physical parameters as wave height, which did not vary seasonally, could make a more appreciable contribution to the near-bottom seston than to the seston in the water column. Zooplankton may be another source of variability in seston composition, due to grazing activity (Cottingam and Schindler 2000). In general, the zooplankton composition did not show any clear seasonal trends, though there were peaks in accordance with warm temperate column patterns (Mazzocchi and Ribera d’Alcalà 1995, Vives 1966, Calbet et al. 2001). The zooplankton consisted principally of copepods (Table 2). The overall absence of seasonal trends in the near-bottom water layer may, for example, be due to horizontal swarms (Hamner and Carleton 1979). Near-bottom zooplanktonic communities receive inputs of benthic and planktonic components via vertical migration (e.g., Ohlhorst 1982). Meroplanktonic larval pulses, i.e., lamellibranchs, polychaetes, echinoderm larvae (see Table 2), occur in association with Chl a peaks (see also Fig. 2a), as pointed out by Starr et al. (1990). A portion of the gelatinous zooplankton, i.e., Appendicularia and Salpae, also seems to be related to winter-spring Chl a increases in the near-bottom layer. Although sig-

TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON 45 nificant grazing pressure on the microphytoplankton, nanoplankton, and detritus by both meroplanktonic larvae and gelatinous zooplanktonic groups has been demonstrated (Martín et al. 1996, Deibel 1998), the proportion of these groups present over the study period (14%) as compared to copepods (76%) suggests a low impact on the <100 µm seston. Seston biochemical composition and zooplanktonic concentration levels were low in late autumn (November-December) [see Table 2; Figs. 1,3]. It has been suggested that high nutrient levels may collapse primary production in the water column if not accompanied by optimal light levels (Sverdrup 1953, Smetacek and Passow 1990), thus affecting trophic relationships throughout the water column. Several studies have reported such an autumn Chl a collapse (San Feliu and Muñoz 1975, Azov 1989, Mura et al. 1996, among others). Low zooplanktonic abundance, observed in various studies, may also reflect such late autumn low Chl a concentrations (Massutí 1959, Vives 1966, Mazzocchi and Ribera d’Alcalà 1995, Calbet et al. 2001, among others). Our results suggest that this same effect may also occur during a brief period (perhaps 30-45 days) between late November and December in warm seasonal near-bottom environments. Despite the general non-seasonality of the biochemical composition of the seston (except for Chl a), the two indirect estimators of seston concentration, VSD and HSD, were significantly correlated with temperature and river discharges (Table 3). These two environmental parameters did exhibit clear seasonal trends (Fig. 7). It would thus appear that, overall, variations in seston concentration roughly follow seasonal trends, whereas the biological composition of the seston does not. The immediate consequences of this lack of temporal correspondence between the Secchi disk readings and the biochemical composition of the seston is that visibility is influenced by the inorganic seston (TPC minus POC), which could be related to resuspension and river runoff, represented mainly by CaCO 3 (Emerly and Milliam 1978). These inorganic particles are the main components of littoral sediments of terrestrial origin (Carter 1993). In addition, wave height, which does not vary by season, could influence the seston concentration over the year. Indeed, river runoff and wave height explained 57 % and 44 % of the variance in the VSD and the HSD values, respectively (Table 3). River inputs may account for pulses of detritus (Emerly and Milliam 1978) and higher nutrient levels that may affect visibility, either directly by interference by detritus or indirectly by fuelling primary production in the water column or by supplying potential food to the benthos (Danovaro et al. 2000). Sometimes an increase in wave height and high river discharges occurred together, lowering the VSD; in our study this was the case in December 1997 (Figs. 5b-c,6). Other times these two events occurred separately, e.g., in October 1997, when there was high wave action and low river runoff, and in February 1997, when there was low wave action but high river runoff, though the effect on water visibility was similar. Resuspension prevents sedimentation by organic particles, because of the special features of the near-bottom water layer (Graf 1999). While inorganic particles sedimented quickly, organic components remain suspended in the water longer or are transported by near-bottom currents (Thomsen et al. 1995). According to our results, near-bottom seston composition did not exhibit seasonal trends, unlike the water column further offshore. The absence of seasonal trends in near-bottom seston composition could have several explanations: 1) the proximity of the substrate creates a set of special hydrodynamic features which enhance particle suspension time (Graf 1999); 2) seasonal seston variability could be masked by small (daily) yet significant biological and physical disturbances difficult to eval-

46 TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON Table 4. Chlorophyll a comparison of different areas in the Mediterranean sea and Atlantic ocean. All data from different studies in mg Chl a m-3 . The Chl a variation index is the standard deviation/mean (for the sampling periods) x 100

Location Depth of bottom Chl a Number of samples Reference surface (m) Variation Index (%)

Water column Estuary (inside) 9 47 22 Flemer (1970) Estuary (inside) 9 47 19 Bruno et al (1983) Estuary (inside) 10 61 24 Patten et al (1967) Estuary (outside) 12 36 22 Flemer (1970) Shelf coast 15 49 25 Mura et al (1996) Shelf coast 20 60 20 San Feliu and Muñoz (1975) Shelf coast 30 65 22 Azov (1989) Shelf coast 40 38 11 Estrada (1982) Shelf coast 100 34 66 Bustillos-Guzmán et al (1995) Pelagic 200 42 12 Fernandez and Bode (1991) Pelagic 250 52 21 Azov (1989) Pelagic 2000 39 12 Fernandez and Bode (1991)

Near bottom water Shelf coast 25 146 18 Navarro and Thompson (1995) Shelf coast 18 73 29 This study uate (Sournia 1974); and 3) the seston in the benthic boundary layer is strongly affected by benthic organisms, which may alter variability in the water column, thereby generating a more dynamic and heterogeneous system as compared to seston communities higher up in the water column. For example, Colebrook and Robinson (1965) demonstrated that Chl a cycles followed a well-defined seasonal trend in open oceanic waters, while higher variability in near-coastal environments may mask seasonal effects. This has a major effect on variability in the seston in the near-bottom water layer. Using the variability index (variance/mean %) we compared variability in Chl a concentration in different ecosystems (Table 4). Only Chl a concentration was chosen for comparison because it is the easiest parameter that can be compared across different seasonal cycles in different parts of the world. The highest variations were found in coastal shelf environments and the lowest in the open sea. Similar trends are observable on comparing the variability index values for POC, proteins, and carbohydrates with those published in other seasonal studies carried out in similar temperate areas (e.g., Neveux et al. 1975, Fabiano et al. 1984, Fichez 1991), but less information is available on seasonal cycles for these parameters. In fact, seston dynamics in the near-bottom water layer is different from that in the water column in the open ocean. Seasonally independent annual fluctuations in certain biochemical parameters and the high proportion of the heterotrophic nanoseston suggest continuous cycling of nutrients and POC in the near-bottom layers, as pointed out above. Furthermore, topography inshore tends to be complex, making hydrodynamics and flow direction more variable (Riedl 1971), thus contributing to unpredictable pulses that drive the seston community in the near-bottom layer during the year. Less pronounced seasonal trends in the HSD readings as compared to the VSD readings were indicative of a seasonal component affecting the seston concentration (Tables 1,3; Fig. 7), influenced by seasonal (river runoff and temperature) and random (wave height) environmental factors, which also influence inorganic components (Tyson

TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON 47 1995). Despite its obvious importance, the effect of the benthic community on this water layer (Larsen and Riisgård 1996, Gili and Coma 1998) is difficult to assess. Feeding, reproduction, and excretion may affect the composition of the benthic boundary layer (Asmus and Asmus 1991, Riisgård and Larsen 2000). In warm temperate seas indistinct seasonal trends in this water layer suggest the possibility of greater food availability for some suspension feeders than previously thought. Further research will improve our knowledge of the composition of the near-bottom seston, an essential tool in understanding benthic-pelagic coupling and the role of the water column in coastal processes.

48 TEMPORAL VARIABILITY IN NEAR-BOTTOM SESTON Short-time-scale cycles of near bottom seston composition: comparison of early and late spring conditions in a warm temperate sea

Abstract

In this study, various biochemical (Chlorophyll a [Chl a], total particulate carbon [TPC], particulate organic carbon [POC], particulate organic nitrogen [PON)], proteins, and carbohydrates) and environmental (temperature, wave height, river runoff, and currents) factors were analysed in Mediterranean coastal waters using a short-time-scale approach. The experiment was performed in spring, a period of intense production in the Mediterranean, for the purpose of explaining shifts in biomass and within-season variability. The near-bottom water layers were sampled in recognition of the importance of the plankton-benthos relationship. Though the Chl a concentration was higher in early spring (end of March), the POC, PON, and protein concentration levels were significantly higher in late spring (end of June). The index of variability (variance/mean) for most of the biochemical parameters studied was higher in late spring, and this was also true for the environmental factors (i.e. temperature, river runoff and currents, not wave height). Short-time-scale cycles have been shown to be appropriate to an understanding of variability during seasonal periods of high productivity. Our hypothesis is that brief but intense periods of productivity in near bottom seston may be important for the benthic community. It is suggested that higher biomass and variability in biochemical parameters, together with higher temperatures and more frequent pulses in the environmental factors in the near-bottom water layers in late spring may be a key factor in explaining the high production levels of benthic communities at this time of the year.

Introduction

Determination of the time scales underlying patterns of environmental variability has been one of the main research objectives of marine ecologists in recent decades. Environmental changes can take place rapidly in marine coastal systems across a variety of spatial and temporal scales. In contrast to large-time-scale studies, short-time-scale observations have been carried out mainly under laboratory conditions because of logistical constraints bearing on field work (e.g., Innes 1998). As more empirical data from short-time- scale studies become available, the role of environmental variability at this time scale becomes more evident (Marrasé et al. 1992, Taylor and Howes 1994). For instance, in a general model of variability for beaches, Carter (1993) showed that the two peaks of maximum sensitivity in the model corresponded to the diel tidal period and the annual seasonal cycle. Beaches display high variability on both scales, as Carter (1993) was able to explain by using an adequate sampling range. In another example, Taylor and Howes (1994) showed that without adequate sampling frequency production peaks in coastal ecosystems go undetected, and the final interpretation of the seasonal cycle may be flawed. Short-time-scale variability has already been compared with seasonal and interannual variability in estuarine and brackish-water ecosystems (Alongi 1998). In such systems a set of environmental factors (i.e., storms, runoff, tidal currents, etc.) may generate discontinuous disturbances in biological communities by introducing inputs that can be quantitatively equivalent to seasonal changes. Due to variability in seston composition and concentration in these waters, short- time-scale events would appear to afford a suitable tool for explaining seasonal trends. Physical forces are generally accepted as the factors driving variability in most biological processes (Denman and Powel 1984). Nogueira et al. (2000) demonstrated the effect of meteorological and hydrographic factors on species diversity and succession in phyto-

SHORT-TIME CYCLES OF NEAR-BOTTOM SESTON 51 plankton communities in coastal waters. On the whole, physical-biological interactions in the water column are related both to the organisms considered and observational scales employed (Steele 1978). Short time scale observations are needed to assess the influence of environmental factors on biological and biochemical variability in the seston (Reynolds 1994, Tilstone et al. 2000). Warm temperate seas lie the Mediterranean Sea have strong seasonal primary production patterns in the water column (Estrada 1996, Longhurst 1998). In coastal ecosystems this is also reflected in the quality and quantity of sedimented particulate organic matter (Gremare et al. 1997), and in the dynamics of the benthos (Coma et al. 2000), with organisms becoming specially active in late winter-spring, following the maximum production peaks in the water column and increases in the quality of the sedimenting organic matter. Although the near-bottom seston will clearly exhibit a high level of seasonality, only the total concentration of particulate matter and Chlorophyll a have been shown to conform to that pattern (Chapter I). Other biological and biochemical parameters apparently do not follow seasonal trends in near-bottom water layers (Chapter I), but the short-time-scale changes remain largely ignored and could help to explain the events taking place during seasonal periods of high productivity. The principal object of this paper is to consider how short-time-scale variability of environmental and biochemical factors in the near-bottom water layers may explain seasonal trends affecting the seston in the Mediterranean Sea, a body of water representative of temperate seasonal ecosystems. To that end, two weekly cycles of highly intense sampling were carried out in a littoral area. Early spring (end of March) and late spring (end of June) were chosen. The spring has been considered a key period because a major portion of the annual primary and secondary production is concentrated then [though other peaks also occur in early autumn and early winter (Margalef 1985)]. Seston composition (live and detritic) in near-bottom water layers is affected by inputs of particulate and dissolved organic matter from river runoff, resuspension, and benthic community processes. Besides the organic input, the near- bottom water layer is subject to highly variable levels of turbulence due to bottom effects which also influence seston dynamics (Wainright 1990). All these events can be expected to be more variable and more pronounced in late spring than in early spring. This paper also suggests an additional hypothesis: a possible relationship between higher variability in environmental and biochemical features of the near-bottom water column in late spring results in higher seston biomass. Consequently, this enhanced production in the water column could be consistent with the principal production dynamics of benthic communities.

Materials and methods

The study was carried out in the Medes Islands Marine Reserve (Fig. 8, 40º02’55”N, 3º13’30”E). For further details concerning the sampling area see Gili and Ros (1985), Ribes et al. (1999). Sampling was carried out at a depth of 19 m in front of a steeply slanting, north-facing vertical wall surrounded by wide boulders. Sampling for biochemical characterization of seston composition was carried out from 24-29 June 1997 (late spring) and from 27-30 March 1998 (early spring). Natural seawater (22–24 L) was collected by SCUBA diving every 6 h (late spring), and every 4 h (early

52 SHORT-TIME CYCLES OF NEAR-BOTTOM SESTON 3º 13' E Figure 8 . Study area location (Medes Islands). Black dot indicates the sampling area. 0 0.5 1 km

L'ESTARTIT

MEDES ISLANDS

42º 02' N

Ter River

Sea ean Mediterran

spring). The water was carefully collected using 2-L plastic bags 0.2-0.5 m above the horizontal rocky surface, avoiding any movement that might cause resuspension of the sediment. On board the bags were emptied into 5-L bottles stored in ice coolers (6-10ºC) until arrival at the laboratory on land no more than one hour after collection. The plastic bags were rinsed with deionized water after each use. During processing seawater samples were always kept at 6-10ºC in the dark. At the laboratory the seawater samples were pre-filtered through a 100-µm mesh to reduce the variance. For estimation of the Chlorophyll a concentration, three 200-ml replicates were filtered through GF/F glass fibre filters. The filters were immediately frozen in liquid nitrogen and later stored at –80ºC until processing. Chlorophyll extraction was performed by grinding the filters in 6 ml of 90% acetone and leaving the homogenate in the dark at 4ºC for 30’ (Chapter I). After centrifugation at 2 000 rpm at 4ºC for 10 min, the supernatant was analysed in a Turner Designs fluorimeter according to the procedure described by Parsons et al. (1985).

SHORT-TIME CYCLES OF NEAR-BOTTOM SESTON 53 To estimate the total particulate carbon (TPC), encompassing both the organic and the inorganic carbon, and the particulate organic nitrogen (PON), three 800-ml replicates were filtered through precombusted (450ºC, 5 h) GF/F filters and immediately frozen in liquid nitrogen. The filters were then dried at 60ºC for 24 h and the samples were analysed using a C:N auto-analyser (Perkin-Elmer 240). To assay the contribution of the particulate organic carbon (POC) only, samples were collected as for TPC. After deep-freezing and drying (60ºC, 24 h), the filters were left in a saturated HCl atmosphere for 48 h to destroy all inorganic matter. The filters were dried completely for another 24 h before analysis using the C:N auto-analyser. The autotrophic-heterotrophic fraction estimates were calculated using the proportion 40x[µg l -1 Chlorophyll a] = µgC l -1 (Delgado et al. 1992). The total particulate protein concentration was calculated using three replicate 1400-ml seawater samples filtered through precombusted GF/F filters that were immediately frozen in liquid nitrogen and later maintained at –80ºC until processing. The protein concentration was estimated spectrophotometrically according to a slightly modified version of the method of Lowry et al. (1951). The filters were extracted by cold grinding in a tissue-grinding homogenizer in 1N NaOH (2 ml). The assay was performed on 0.5 ml of the homogenate. After the colorimetric reaction the mixture was centrifuged at 3 000 rpm to remove the glass fibres and avoid interference in the 750-nm spectrophotometer reading. Bovine serum albumin (Sigma) was used as standard. The protein contribution to the POC fraction was calculated using the equation 0.5x[µg l -1 Proteins] = [Protein Carbon]µg l -1 (Freifelder 1982). Three replicate 1400-ml seawater samples were filtered through precombusted GF/F filters to estimate the total particulate carbohydrates. Filters were immediately frozen in liquid nitrogen and stored at –80ºC until processing. The methodology used was a slightly modified version of the procedure of Dubois et al. (1956). The filters were extracted by cold grinding in a tissue grinder in 2 ml of DDW. Samples were centrifuged at 3 000 rpm to remove the glass fibres and avoid interference in the 485-nm spectrophotometer reading. Glucose was used as standard. The contribution of carbohydrates to the POC fraction was calculated using the equation 0.44x[µg l -1 Carbohydrates] = [Carbohydrate Carbon]µg l -1 (Freifelder 1982). Temperature at a depth of 20 m was measured as per Cebrián et al. (1996) and Ter River runoff and wave height were measured according to the methods described by Pascual et al. (1995). Water temperature and Ter River runoff were measured at 2-3 days intervals, and wave height was estimated daily. To compile enough measurements for the index of variability (VI) analysis, 15-d periods were considered (15-30 June 1997 and 15-30 March 1998). Although different years were studied (late spring in June 1997 and early spring in March 1998) both periods can be considered representative of spring extremes in the Mediterranean, inasmuch as the differences in environmental conditions (i.e., temperature, river runoff, and wave height) in the early and late spring periods in the years preceding and following each sampling year, respectively (Chapter I) were non significant. Current flow rate and flow direction data were collected using a permanent buoy adjacent to the seston sampling zone by recording the angle and direction (north-south) of buoy inclination daily over the sampling periods. Fig. ure 5 depicts relative values (positive values = northward direction of current flow; negative values = southward direction of current flow), the correspondence being: 0–1 = 0-5.5 cm s -1 ; 1–2 = 5.5–14 cm s -1 ; 2–3 = 14–25 cm

54 SHORT-TIME CYCLES OF NEAR-BOTTOM SESTON s-1 . Estimation of these relative values was discussed in Pasqual et al. (1995) and Pasqual (1999). The purpose of these data was to provide a semi-quantitative approach to the hydrodynamics of the study area during the sampling periods. Pearson’s product-moment correlation was applied when variable values were normally distributed and Spearman’s rank-order correlation matrices were used when variable values were not normally distributed (Zar 1996, Dytham 1999). ANOVA with Scheffé post-hoc comparison (Zar 1996) was used to analyse the possible effect of seasonality (early and late spring). The formula variance/mean x100 was used as the index of variability (VI) for comparing the sampling periods (Zar 1996).

Results

Table 5 presents the means ±SD and index of variability values for the different seston parameters in the two sampling periods. The Chlorophyll a concentrations in late and early spring were significantly different (Figs. 9a, 10a; Tables 5, 6). Differences in the TPC values for early spring and late spring were non-significant, while the POC values for the two

Table 5. Mean ± Standard Deviation values for the different biochemical parameters studied in the seston in the early spring (March 1998) and late spring (June 1997). Index of variability (VI) in both periods calculated as the standard deviation/mean x 100. * Disregarding the 11:00 a.m. peak on 28 March 1998 yields a mean value of 64 ± 13 with an VI of 20 %. V.I. = Variation Index

Early Spring [V.I.] Late Spring [V.I.]

Chl a µg l -1 0.670±0.134 20% 0.414 ±0.094 23% TPC µg l -1 232 ±49 21% 262 ±80 30% POC µg l -1 157 ±24 15% 205 ±40 20% PON µg l -1 17 ±5 29% 24 ±11 46% Proteins µg l -1 143 ±32 22% 176 ±32 18% Carbohydrates µg l -1 76 ±37* 49% 50 ±5 10%

Table 6. Comparison of mean biochemical parameter values for the seston in early spring (27 March 1998) and late spring (24 June 1997) and the corresponding ANOVA functions. Abbreviations are explained in the text. Legends: n.s. = non-significant; * = p < 0.05; ** = p < 0.001; *** = p < 0.0001

Early spring-late spring differences F

Chl a *** F (1,39) =43.65 TPC n.s. F (1,39) =0.95 POC *** F (1,30) =18.92 POC/TPC * F (1,30) =5.45 PON * F (1,39) =6.97 C/N n.s. F (1,30) =0.35 Autotrophic carbon *** F (1,30) =31.24 Heterotrophic carbon *** F (1,30) =31.24 Protein ** F (1,39) =12.42 Carbohydrate * F (1,30) =5.55 Prot C + Carbh C n.s. F (1,30) =0.64

SHORT-TIME CYCLES OF NEAR-BOTTOM SESTON 55 a b

c d

Figure 9 . Biochemical seston parameters in early spring (March 1998): a) Chlorophyll a; b) Total particulate carbon (TPC, solid line) and particulate organic carbon (POC, dashed line); c) Particulate organic nitrogen (PON); proteins (solid line) and carbohydrates (dashed line). Bars represent ± SE of three replicates.

a b

c d

Figure 10 . Biochemical seston parameters in late spring (June 1997): a) Chlorophyll a; b) Total particulate carbon (TPC, solid line) and particulate organic carbon (POC, dashed line); c) Particulate organic nitrogen (PON); proteins (solid line) and carbohydrates (dashed line). Bars represent ± SE of three replicates.

56 SHORT-TIME CYCLES OF NEAR-BOTTOM SESTON a a

b b

c c

Figure 11 . Biochemical estimations in early spring Figure 12 . Biochemical estimations in late spring (March 1998): a) Organic carbon:nitrogen ratio; b) (March 1997): a) Organic carbon:nitrogen ratio; b) Autotrophic (dashed line) and heterotrophic (solid Autotrophic (dashed line) and heterotrophic (solid line) organic carbon; c) Particulate organic carbon line) organic carbon; c) Particulate organic carbon (POC, solid line) and sum of protein-carbohydrate (POC, solid line) and sum of protein-carbohydrate organic carbon (dashed line). organic carbon (dashed line). sampling periods were significantly different (Figs. 9b, 10b; Tables 5, 6). The POC accounted for 85% of the TPC in late spring as opposed to only 67% of the TPC in early spring. Autotrophic carbon values were higher in early spring than in late spring, so heterotrophic carbon was more abundant in June than in March (Figs. 4b, 4b; Tables 5, 6). The PON concentration was lower in early spring than in late spring. The difference in the C:N ratio between the two periods was non-significant (Figs. 9c, 10c; Tables 5, 6).

SHORT-TIME CYCLES OF NEAR-BOTTOM SESTON 57 Protein concentration was significantly higher in late spring than in early spring (Table 6). In contrast, carbohydrate concentration was significantly higher in early spring than in late spring, even without the 11:00 a.m. peak on 28 March 1998 (Figs. 9d, 10d; Tables 5, 6). The protein and carbohydrate carbon concentrations were not significantly different between the two sampling periods, but in early spring these parameters contributed 68% of the POC as opposed to 55% in late spring (Figs. 11c, 12c). Table I shows that the VI values for chlorophyll, heterotrophic carbon, and protein were similar in both sampling periods considered. The VI for TPC, POC, PON, and heterotrophic carbon was higher in late spring, while the converse was true for carbohydrates and protein + carbohydrate carbon. More seston parameters were significantly related in late spring than in early spring (Table 7). Chlorophyll was slightly though significantly related to carbohydrates in both periods but displayed no relationship with any other parameter. The TPC was significantly related to the POC, PON, and proteins in late spring nut only weakly related to the PON in early spring. The POC was significantly related to the PON, proteins, and carbohydrates in late spring but only weakly related to proteins in early spring, while the PON was slightly though significantly related to the proteins only in late spring. There was a significant relationship between proteins and carbohydrates in late spring only (Table 7). Water temperature was significantly higher in June (19.5 ± 0.6ºC) than in March (14.4 ± 0.1ºC). Also, variability was higher (3.3%) in June as compared to March (0.7%) due to the fluctuations in the thermocline. River runoff was slightly higher in early spring (3.9 ± 0.4 m 3 s-1 ) than in late spring (2.6 ± 3 m 3 s-1 ), but the VI was significantly higher in late spring (115%) than in early spring (10%) because the pulses of river runoff were discontinuous in late spring. Wave height was slightly higher in early spring (0.85 ± 0.37 m) than in late spring (0.61 ± 0.23 m), while the VI was similar (43% in March and 38% in June). Figures 12a and 12b present current flow rates in the sampling periods in late and early spring. Flow rates were lower in early than in late spring. There was an abrupt reversal in direction on 22 June 1997 (from south-north to north-south). The direction of current flow was more variable in early spring, but the changes in flow rate were very small. On the four days of seston parameter sampling in early spring, currents flow was less than 5.5 cm s -1 in

Table 7. Relationships between the different seston parameters studied in early spring (26-30 March 1998, italics) and late spring (24-29 June 1997, bold). N = 22 (Chl a, TPC, and protein in June 1997). N = 13 (POC and carbohydrates in June 1997). N = 21 for all the March 1998 parameters. Abbreviations are explained in the text. Legends: n.s. = non-significant; * = p < 0.05; ** = p < 0.001; *** = p < 0.0001

Chl a TPC POC PON Proteins

TPC n.s. n.s. POC n.s. * R 2=0.569 n.s. n.s. PON n.s. *** R 2=0.717 * R 2=0.409 n.s. * R 2=0.316 n.s. Proteins n.s. * R 2=0.358 ** R 2=0.666 * R 2=0.334 n.s. n.s. * R 2=0.27 n.s. Carbohydrates * R 2=0.45 n.s. * R 2=0.568 n.s. * R 2=0.489 * R 2=0.399 n.s. n.s. n.s. n.s.

58 SHORT-TIME CYCLES OF NEAR-BOTTOM SESTON a southward direction. During seston parameter sampling in late spring, current flow was variable in terms of both speed and direction, and flow rates were generally above 5.5 cm s -1 . The VI for both flow rate and direction of flow was 215% in early spring and 2 880% in late spring.

Discussion

Short-time-cycles in the assessment of seasonal trends in the near-bottom water layer

Higher values of Chl a in early spring (compared with late spring) were not indicative of higher biomass or productivity in the near-bottom water layer. Our results showed that on the whole the POC was significantly higher in late spring than in early spring, a finding supported by higher PON and protein values. Only carbohydrates seemed to be higher in early spring, though that may in part be related to phytoplankton biomass (Cullen 1985, Moore and Villareal 1996). Further evidence of this relationship is presented in Table 7, showing Chl a to be significantly related only to carbohydrates in both sampling periods. Autotrophic carbon was higher in early spring and was related to the winter-spring diatom blooms in the Mediterranean, in which the >10-µm fraction of the phytoplankton tends to be more prevalent (Raimbault et al. 1988, Delgado et al. 1992, Selmer et al. 1993, Ferrier-Pagès and Rassoulzadegan 1994, Chapter I). Autotrophic carbon levels were halved in late spring, when heterotrophic carbon (detritic or live) was higher. Heterotrophic biomass, mainly detritic in this water layer, predominated in both sampling periods (Ribes et al. 1999, Chapter I). The C:N ratio was similar ( ≈ 10) and was representative of detritic material (Atkinson and Smith 1983). Resuspension due to higher wave action and constant river runoff may have a more important role affecting the allochthonous detritus in early spring as compared to late spring. The POC:TPC ratio seemed to be consistent with this sugges- tion: the POC only 65% of the TPC in early spring but more than 85% in late spring. This means that a large portion of the particles analysed were inorganic in early spring, again suggestive of resuspension and inputs of inedible river matter. In late spring there was a second input of detritic material resulting from the massive fall of macroalgae in this zone (Ballesteros 1989). The detritic biomass of seaweeds is quite high, and a broad spectrum of heterotrophic organisms proliferates in this bulk near the seafloor (Graf 1992, Danovaro et al. 2000). High variability in both environmental factors and the biochemical composition of the seston has been shown to take place even within the same season. Such observations provide support for short-time-scale field sampling, which can help to uncover smaller-scale changes in ecosystems that go undetected by monthly or seasonal sampling designs. Hydrography and nutrient concentrations may change significantly near the coast within a space of only a few days, altering diatom species succession (Tilstone et al. 2000). By way of another example, changes in environmental conditions in intertidal near-bottom water layers occurring over a span of just a few hours may double or triple the POC, PON, or Chl a concentrations in the seston (Fegley et al. 1992). In our study, environmental factors (temperature, river runoff, and currents) were more variable in late spring than in early spring.

SHORT-TIME CYCLES OF NEAR-BOTTOM SESTON 59 a b

Figure 13 . Relative current values (see Materials and Methods section for the values) in a) early spring (March 1998) and b) late spring (June 1998). N-S indicates north-south direction of current flow.

Temperature was higher in late spring, but stratification was still not complete down to 20 m in depth, which was reflected in a higher VI. River runoff was slightly higher in early spring and was less variable over time as compared to the data for late spring (when river discharge took the form of pulses due to more discontinuous rainfall). Wave height was also slightly higher in late spring, but the similar VI values made further interpretation impossible. Finally, currents were stronger and much more variable (both flow rate and flow direction) in the study area in late spring (Fig. 13). This environmental factor influenced the variability of most of the biochemical parameters observed (Table 5), higher in late spring than in early spring. Biomass in the seston was higher in late spring, the higher biomass levels being partly attributable to greater variability in the environmental factors. Changes in estuaries and tidal shores may be quite pronounced when not stratified and may take place on small scales on the order of tens of meters in just a few hours. These hydrodynamic processes mostly follow patterns on diel and weekly-time scales and are forces that increase water mixing and nutrient transport and consequently stimulate biological production (Valiela 1995). The similar features in temperate shallow coastal and estuarine environments are evident, with the alternation of mixing and stratification in both environments producing seasonal changes in plankton communities in the water column. Differences during periods of mixing (spring) can also be of great relevance, as our study in the Mediterranean has demonstrated. These temporal patterns have been extensively docu- mented in tidal shores and estuaries (e.g., Alongi 1998). In shallow waters where tidal fluxes are weak, such environmental factors as storms or runoff can cause resuspension and lat- eral advection that perturb biological communities continuously, generating inputs that are quantitatively equivalent to those caused by seasonal changes, with different initial and final conditions regulating the processes during periods of mixing (spring in the Mediterranean). It is generally accepted that coastal environments are among the most variable in the world, but many of the changes follow temporal patterns that vary in scale depending on local events. Most of the important environmental factors act at scales that are difficult to discern over a yearly cycle. Events occurring during very short yet hydrodynamically very active periods (spring in warm temperate seas) are capable of having decisive effects over the rest of the year. One example are storms, which can destroy a beach in the span of a few hours, after which recovery of the sandy substrate can take months (Carter 1993). Another exam-

60 SHORT-TIME CYCLES OF NEAR-BOTTOM SESTON ple is tidal bay and estuarine habitats, where water masses flow in and out at least twice a day, with peak production taking place during climatic periods with the highest variability (Mann 2000). Differentiation and identification of this short-time variability is useful in better understanding associated biological processes.

Near-bottom water layer in late spring and benthic community activity

Carbon cycling in the seston at the benthic boundary layer may depend on the amount of time particles are suspended in the water (Thomsen and van Weering 1998, Thomsen 1999). The physics of particles in near-bottom water masses causes organic matter to tend to remain suspended longer than inorganic matter (Thomsen and van Wering 1998). Turbulence may increase resuspension in near-bottom layers, as has been demonstrated for planktonic communities in the water column, because there is more particle cycling and interaction between particles and microorganisms [aggregation-dissolution, predation, nutrient uptake, etc.] (Estrada and Berdalet 1997, Margalef 1997). When turbulence is mod- erate, energy inputs resulting from hydrographic and weather conditions may be conducive to increased diversity and biomass of microorganisms because nutrients are more easily recycled within the system, avoiding losses due to stratification or excessive dynamism (Nogueira et al. 2000). Our findings suggest that more variable patterns of environmental factors in late spring that increase turbulence near the bottom enrich seston composition and production. Furthermore, the biochemical parameters for the seston were more closely correlated in late spring than in early spring (Table 7). This is a result of more synergistic system period, producing more efficient nutrient recycling and thus greater particle diversity (Margalef 1991), with retention of particles within the near-bottom water layer (by grazing, aggregation, etc.). Huisman and Weissing (1999) have shown that in a system with sufficient chaotic pulses, plankton populations may exhibit high levels of diversification and coexis- tence, even when constrained by competition for resources. The greater variability in the environmental factors and in the biochemical composition of the seston together with higher biomass levels leads to a more diverse and productive scenario in the near-bottom water layers in late spring than in early spring. This layer both directly affects and is affected by benthic communities (Cloern 1982, Asmus and Asmus 1991, Gili and Coma 1998). We hypothesize that benthic communities exhibit major reproductive and secondary production in late spring because of the more favourable conditions of seston availability as potential food in the near-bottom water layers (Grémare et al. 1997). Thus, annual coastal soft bottom communities in the Mediterranean attain higher levels of biomass, secondary production, and reproduction between early April and early July than during the rest of the year (Sardá et al. 1999, Medernach et al. 2000). On hard substrata most hydrozoans display high densities and levels gonadal development between April-July (Boero and Fresi 1986), and the gorgonian Paramuricea clavata spawns at the end of June (Coma et al. 1995). Some ophiurids (e.g., Ophiotrix fragilis ) recruit in late spring-early summer (Turón et al. 2000), and it has been registered blooms of sponge larvae (e.g. Cliona viridis ) in coincidence with this late spring period (Mariani et al. 2000) . Vagile benthic organisms also reproduce preferentially in spring [e.g., peak gonadal development in most benthic fishes takes place in late April-early July (Lloret et al. 2000) ]. Generally speaking, most benthic organisms in warm temperate ecosystems exhibit higher levels of activity,

SHORT-TIME CYCLES OF NEAR-BOTTOM SESTON 61 greater secondary production, and higher reproductive output at that time of year (Coma et al. 2000). In our opinion, gonadal and somatic development is brought about not only by the rise in temperature but also by greater seston abundance and more diversified seston composition, making more potential prey available to the benthic community. Along with pulses in spring environmental factors, both higher biomass concentrations and more variable near-bottom biochemical composition of the seston exerted influences on the benthic community. The seston feeds not only adults but also larval and post-larval stages of most benthic organisms. We suggest that the different seston composition and biomass levels at the end of spring can be related to the onset of benthic production and reproduction by benthic species. Temperature is the other key factor for these processes, before the aestivation period diminishes the activity of most benthic organisms (Coma et al. 2000). The data presented here show that short-time variability in periods of high levels for environmental factors and biological production helps account for aspects of system operation that are difficult to explain just by extrapolating on the basis of seasonal data.

62 SHORT-TIME CYCLES OF NEAR-BOTTOM SESTON Biochemical characteristics of sedimenting POM available to north-western Mediterranean benthic communities: a seasonal and geographical comparison

Abstract

Gross sedimentation rates together with the main biochemical characteristics of sedimenting POM were studied at two NW Mediterranean sites (Banyuls-sur-Mer, France, and Medes Islands, Spain) throughout a year to assess possible differences in POM availability to the benthic community. A seasonal pattern was observed at both sites, being more evident in Banyuls-sur-Mer than in the Medes Islands. Organic contents were higher during the spring- summer than during the autumn-winter period. On the other hand, gross sedimentation rates more abundant in the autumn-winter than in the spring-summer period at both sites. Chlorophyll a concentration in the water column was higher in the Medes Islands than in Banyuls-sur-Mer during most of the period under study. The occurrence of stronger winds and higher waves at Banyuls-sur-Mer may explain the tighter negative relationship between gross sedimentation rates and organic contents observed at this site, which supports the role of resuspension in driving sedimentation as already suggested by previous studies. In the Medes Islands this relationship was looser. This suggests that different environmental characteristics may control gross sedimentation rates and sedimenting POM biochemical characteristics at both sites. Although average gross sedimentation rates were not significantly different at both sites, they were much less variable through the studied period in the Medes Islands than in Banyuls-sur-Mer. Average concentrations in organic carbon were not significantly different in both places but average nitrogen concentration was higher in Banyuls-sur-Mer. Consequently average C/N ratio was significantly higher in the Medes Islands. During the period under study, sedimenting POM carbohydrate concentrations tended to be higher in Banyuls-sur-Mer, whereas lipid concentrations tended to be higher in the Medes Islands. There was no such difference for proteins. Differences in the biochemical characteristics of sedimenting POM in the Medes Islands respect the Banyuls-sur-Mer area, suggest different hydrodynamic features in both zones, that may revert in a higher availability of suspended matter to benthic populations. The sedimenting POM characteristics presented above are proposed as a tool to compare sedimentation processes within different coastal zones in view of assessing possible differences in benthic community composition and biomass.

Introduction

The sources of particulate organic matter in the coastal environments are heterogeneous and submitted to strong seasonal patterns both quantitatively and qualitatively (Tenore 1988, Navarro and Thompson 1995, Grémare et al. 1997, Cripps and Clarke 1998). It is generally accepted that seasonal pulses of available organic matter influence the abundance, the biomass and the activity of bottom communities (Graf et al. 1982, Graf et al. 1983). Biochemical characteristics of both sediments and sedimenting Particulate Organic Matter (POM) have been used in several studies to assess the availability of food for benthic organisms (Tenore 1981, Levinton et al. 1984, Valiela et al. 1984, Grémare 1994, Charles et al. 1995, Grémare et al. 1997). However, it is clear that most of the matter associated to the sedimenting POM is refractory, and cannot be readily absorbed by benthic invertebrates: during the last 20 years both biological and biochemical assays have been improved to assess true nutritional organic availability for benthic organisms (Mayer et al. 1986, Grémare et al. 1988, Cheng et al. 1993, Charles et al. 1995, Mayer et al. 1995, Grémare et al. 1997, Dell’Anno et al. 2000), leading more precise tools to evaluate such nutritional availability. It has been shown that only some fractions of the biochemical assays of sedi-

CHARACTERISTICS OF SEDIMENTING POM 65 menting POM were related to availability and growth of benthic fauna (Grémare et al. 1997, Medernach 2000), and lipids in the sedimenting POM was one of the best-fitting parameters (Grémare et al. 1997). The connection of high nutritional sedimenting POM contents with reproduction and growth has been demonstrated in several benthic species (Marsh et al. 1989, Grémare 1994, Charles et al. 1995, Grémare et al. 1997). Relationship between sedimenting POM or sediments characteristics, and benthic abundance or activity has also been observed through seasonal studies (Danovaro et al. 2000a, Danovaro 2000b, Medernach 2000, Shimanaga and Shiriyama 2000). The activity evaluated in form of ATP, heat or biomass production of benthic organisms is also enhanced in accordance with seasonal inputs of high quality organic matter (Graf et al. 1982, Graf et al. 1983, Meyer-Reil 1983, Pfannkuche 1993). The seasonality of pelagic primary production is especially strong in the Mediterranean (Estrada et al. 1985, Estrada 1996) and reverts on the nutritional value of sedimenting POM (Grémare et al. 1997). Suspension, deposit and detritus feeders may show strong seasonal feeding patterns in warm temperate, cold temperate and polar seas (Barnes and Clarke 1995, Coma et al. 1998, Ribes et al. 1998, Riisgard 1998). An important component of their diet is directly derived from sedimenting POM. Even if the study of temporal and spatial variation of potential food items is a key step in understanding seasonal variations in the dynamic of benthic communities, studies considering the biochemical characteristics of sedimenting POM are yet relatively scarce in warm temperate seas (Fichez 1991, Charles et al. 1995, Grémare 1997, Grémare et al. 1998a, Pusceddu et al. 1999a, Medernach et al. 2001). Furthermore, only very few of them have considered small scale spatial variations. In this context, the aim of our work was to compare both the absolute values and seasonal changes in the main biochemical characteristics of sedimenting POM at two sites located only 50 km apart. To achieve this objective, we monitored at both sites gross sedimentation rates together with organic material, total carbon, organic carbon, nitrogen, and total protein, carbohydrate and lipid contents of sediment trap fresh collected materials. Additionally, differences in environmental factors, mainly local winds and waves, water column productivity and river runoff were also considered in order to better asses heterogeneity between sites in sedimenting POM biochemical features analysed (as potential factors affecting both the composition and the biomass of benthic communities at both sites).

Material and methods

Sediment traps. Two sets of sediment traps were moored in Banyuls-sur-Mer (42º29’ 30’’N, 3º08’70’’E, France), and in the Medes Islands (42º02’55’’N, 3º13’30’’E, Spain) (Fig. 14a,b). These two sites are located ca 50 km apart. In Banyuls-sur-Mer, the sediment traps were moored over 27m deep sandy bottoms. In the Medes Islands, the sediment traps were moored northward over a 30m deep sandy-stone bottom. It should be pointed out that the mooring site was located in a much more open area in Banyuls-sur-Mer than in the Medes Islands. The two sets of sediment traps were strictly identical. They consisted of two polyethyl- ene pipes prolonged by a cone and a collector (Charles 1994). The inner diameter of the pipe

66 CHARACTERISTICS OF SEDIMENTING POM a

3º 13' E b 0 0.5 1 km

L'ESTARTIT

MEDES ISLANDS

42º 02' N

Ter River Figure 14. Map showing the location of the two study sites. Arrows indicate study Sea ean Mediterran areas: (A) Banyuls-sur-Mer (the CROSS indicates sediment trap mooring), (B) Medes Islands (the DOT indicates sediment trap mooring).

CHARACTERISTICS OF SEDIMENTING POM 67 was 40cm with a total height of 190 cm giving an aspect ratio of 4.75. The mouths of the traps were located 3 m above the bottom. Trap collectors were collected by SCUBA divers weekly in Banyuls-sur-Mer, and biweekly in the Medes Islands between July 1997 and August 1998. Wind maximum speed and direction was registered daily, together with wave height (Servei Meteorológic de l’Estartit) in the Medes Islands; wind speed and direction was also registered in Banyuls-sur-Mer (Station MeteoFrance du Cap Béar), together with the state of the sea (a rough index that can be used as proxy for wave height). Water samples were collected at both sites to assess phytoplankton biomass. Those samples were collected at 24m depth in Banyuls-sur-Mer and at 20 m depth in the Medes Islands. Samples were collected weekly in Banyuls-sur-Mer and 2 to 5 times a month in the Medes Islands. Chlorophyll a was assessed after Parsons et (al 1985) on triplicates (0.2 L) previously filtered on Whatman GF/F filters. Biochemical assays. Collectors deployment duration was always less than 10 days (usually 5-7 days) to limit microbial degradation. Samples were centrifuged (4000rpm, 15min), frozen, briefly rinsed with distilled water and freeze-dried. The collected material was sieved on a 200 µm mesh, weighed (both fractions <200µm and >200µm) and stored at - 20ºC. All biochemical characteristics were measured on the <200µm size fraction. Organic contents were assessed by measuring the weight lost after combustion (450ºC for 5h, triplicates). Organic carbon and nitrogen were measured on triplicates using a CHN LECO CC- 100 Elemental Analyser, after acidification with HCl. Proteins were measured on triplicates using the Lowry procedure (Lowry et al. 1951) as modified by Rice (1982) to account for the absorption of phenolic compounds. Carbohydrate were analysed on triplicates using the procedure of Dubois et al. (1956). Lipids were analysed on triplicates using the Barnes & Blackstock (1973) procedure. Statistical analysis. Gross Sedimentation Rates (GSR) were compared between sites using a simple linear regression model. Standard regression was also used to relate GSR and organic contents. In this case, data were first log 10 transformed. The same methodology was used to assess the relationships between nitrogen and protein contents at both sites. To assess between-sites differences in OM, C, N, protein, carbohydrate and lipid concentrations during the period under study, analyses of covariance (ANCOVAs) were used to compare slopes and intersections of the regression lines linking those variables and GSRs. To assess seasonal changes, data were pooled into groups (i.e., spring-summer period versus autumn-winter period) as proposed by Grémare et al. (1997) and Grémare et al. (1998a). The mean values of these groups were compared using one-way ANOVA and Scheffé post- hoc tests.

Results

Maximal wind speed tended to be higher in Banyuls-sur-Mer than in the Medes Islands (Fig. 15). Wind direction also differed due to the orography of both sites. Stronger wind speeds resulted in higher wave height in Banyuls-sur-Mer than in the Medes Islands (Fig. 16). Wave height tended to be higher during the autumn at both sites (October- December 1997).

68 CHARACTERISTICS OF SEDIMENTING POM N

WW E BBanyulsanyuls S

MedasMedas

07/97 08/97 09/97 10/97 11/97 12/97 01/98 02/98 03/98 04/98 05/98 06/98 07/98 08/98 09/98 10/98 Figure 15. Wind maximum speed (m s -1 ) and direction in Banyuls-sur-Mer and Medes Islands Stations. a

b Figure 17. Chlorophyll a concentrations (mg m -3 ) in the water column. Solid line, Banyuls-sur-Mer station; Dashed line, Medes Islands station.

Chlorophyll a concentration in the water column was higher in the Medes Islands than in Banyuls-sur-Mer during almost all of the studied period (Fig. 17). At both sites the winter-spring bloom (occur- Figure 16. Estimated wave height from visual ring during February) corresponded to the observations of the state of the sea in Medes higher Chlorophyll a concentrations. Islands Stations (A), and real wave height in Banyuls-sur-Mer station (B). However, there were slight differences in temporal changes between sites: 1) The autumn bloom was only apparent in Banyuls-sur-Mer (November), 2) There was a secondary peak in concentration in the Medes Islands in August which is consistent with other measurements carried at the same site (Chapter I), because of the next river influence. Gross Sedimentation Rates (GSR) were less variable in the Medes Islands than in Banyuls-sur-Mer (Fig. 18a). There was no significant correlation between GSRs recorded at both sites (R 2 = 0.00025, N=24, p=0.942). In Banyuls-sur-Mer GSRs showed a clear sea-

CHARACTERISTICS OF SEDIMENTING POM 69 a sonal pattern with much higher values during the autumn-winter than during the spring-summer period. In the Medes Islands this pattern was less apparent, although maximum GSRs were also recorded in the autumn-winter period. However due to strong variability within each period, the average GSRs do not differ significantly in that zone when periods are compared (Table 9). GSRs were much higher in Banyuls-sur-Mer than in the b Medes Islands during autumn (Fig. 18a, Table 9). There was no significant difference in the average GSRs recorded at Banyuls-sur-Mer during the two spring- summer periods (i.e., 1997 and 1998), which took place during the period under study. In the Medes Islands GSRs were significantly higher during summer 1998 than during summer 1997 (2-way ANOVA with post-hoc Scheffé test, F 1,40 =2.77, p<0.05). Organic contents were always higher in the Medes Islands than in Banyuls-sur- Figure 18. Temporal changes of gross sedimentation rates (a) and organic contents (b) in the Banyuls-sur- Mer except in February 1998 (when we Mer station (solid lines) and the Medes Islands station recorded a little peak in organic contents, (dashed lines). Shaded area indicates spring-summer in coincidence with maximal Chlorophyll time interval, whited area indicates autumn-winter a concentrations in the water column, time interval. Fig. 18b). Organic contents showed the same pattern of seasonal changes at both sites, with significantly higher values during the spring-summer period than during the autumn-winter period (Table 9). In the Medes Islands, peaks in organic contents were recorded in September 1997 and May 1998 and did not coincide with high concentrations of chlorophyll a in the water column. The relationships linking GSRs and organic contents at both sites are presented in Figure 19 In both cases, there was a negative correla- Figure 19. Relationship between organic matter (OM) and gross sedimentation rates (GSR) for the studied tion between these two parameters. period in the the Banyuls-sur-Mer station (N=24) (solid However, this relationship was tighter in line) and the Medes Islands station (N=24) (dashed Banyuls-sur-Mer (R 2 = 0.843, N=24, line). See Table 8 for slopes and interceptions. p<0.0001) than in the Medes Islands (R 2

70 CHARACTERISTICS OF SEDIMENTING POM Table 8. ANCOVA results for OM, Organic carbon, Nitrogen, Proteins, Carbohydrates and Lipids related to GSR (N=48) (n.s.=non-significant; *=p<0,01; **=p<0,001; ***=p<0,0001). d.f. = Degrees of Freedom.

Intercept Intercept p Slope Medes Slope p Sum Squares d.f. Medes Islands Banyuls-sur-Mer Islands Banyuls-sur-Mer

Organic Matter 1.183 1.086 *** -0.219 -0.257 n.s. 5.327 3 Organic Carbon 0.683 0.770 n.s. -0.122 -0.267 n.s. 8.202 3 Nitrogen -0.338 -0.031 *** -0.249 -0.466 * 10.019 3 C/N 1.017 0.803 *** 0.132 0.200 n.s. 9.126 3 Proteins 1.128 1.360 n.s. -0.102 -0.517 *** 11.403 3 Carbohydrates 1.169 1.579 *** -0.157 -0.377 * 8.678 3 Lipids 1.089 1.214 * -0.285 -0.774 ** 25.207 3

= 0.601, N=24, p<0.0001). Slopes did not significantly differ among sites, but the intercept was significantly higher in the Medes Islands (Table 8). Therefore between-sites differences in the organic contents of the sedimenting material did not simply result from differences in GSRs. Temporal changes in organic carbon contents, in nitrogen contents and in C/N ratios at both sites are shown in Figure 20a, b and c. The relationship linking GSRs and organic contents were not significantly different at both sites, whereas both the intercepts and the slopes of the regression models linking nitrogen contents and GSRs were significantly different at both sites (Table 8). This pattern was mainly due to the occurrence of higher nitrogen concentrations in Banyuls-sur-Mer during the summer of 1997. At both sites there were significant differences in the mean organic carbon contents recorded during the spring-summer and the autumn-winter periods. A similar difference was also found for nitrogen in Banyuls- sur-Mer but not in the Medes Islands (Table 9). Organic carbon (both sites) and nitrogen (Banyuls-sur-Mer) contents were higher during the spring-summer period than during the autumn-winter period, The average C/N ratio was higher in the Medes Islands than in Banyuls-sur-Mer (Table 8). There were seasonal differences in Banyuls-sur-Mer with higher values during the autumn-winter than during the spring-summer period (Table 9). This difference was not significant in the Medes islands (Table 9). In Banyuls-sur-Mer a transi- tory increase in both organic carbon and nitrogen contents was recorded during February 1998.There was no such pattern in the Medes Islands.

Table 9. Mean values of summer/spring time (left value)- autumn/winter time (right value) in the Medes Islands station and the Banyuls-sur-Mer station. Two-way ANOVA analysis with Scheffé post-hoc comparison (n.s.=non-significant; *=p<0,05; **=p<0,01; ***=p<0,001).

Medes Islands p Banyuls-sur-Mer p

Gross Sedimentation Rates g DW m -2 day -1 5.8-10.9 n.s. 5.5-23.4 * Organic Matter % 13.5-8.6 ** 9.3-6.3 * Organic Carbon % 5.0-3.0 * 4.6-2.8 * Nitrogen 0.43-0.23 n.s. 0.59-0.30 ** C/N ratio 12-14 n.s. 8-11 * Carbohydrates mg gDW -1 15.3-8.4 * 27.0-14.5 ** Proteins mg gDW -1 13.5-9.0 n.s. 14.0-7.8 * Lipids mg gDW -1 13.0-4.7 ** 7.5-3.1 ***

CHARACTERISTICS OF SEDIMENTING POM 71 a a

b b

c c

Figure 20. Temporal changes of organic carbon con- Figure 21. Temporal changes of the total protein tents (a), nitrogen contents (b) and corresponding (a), carbohydrates (b) and lipid (c) contents in the C/N ratios (c) in the Banyuls-sur-Mer station (solid Banyuls-sur-Mer station (solid lines) and the Medes lines) and the Medes Islands station (dashed lines). Islands station (dashed lines). Shadow zone indi- Shadow zone indicates spring-summer time interval, cates spring-summer time interval, white zone indi- white zone indicates autumn-winter time interval. cates autumn-winter time interval.

Total protein concentrations tended to be higher in Banyuls-sur-Mer than in the Medes Islands except during the period (December 1997) characterized by very high GSRs in Banyuls-sur-Mer and on May 18, 1998 when the protein contents of the material collected in the Medes islands was exceptionally high (Fig. 21a). In Banyuls-sur-Mer there was a clear seasonal pattern with higher concentrations during the spring-summer than during the autumn-winter period. Such difference was not significant in the Medes islands (Table 9).

72 CHARACTERISTICS OF SEDIMENTING POM The February peak of concentrations (see above) was especially pronounced in Banyuls-sur- Mer but did not exist in the Medes Islands. The intercepts of the regression lines linking protein concentrations and GSR were not significantly different at both sites (Table 8), whereas the slope was significantly steeper in Banyuls-sur-Mer (Table 8). This may result from the occurrence in Banyuls-sur-Mer of: (1) high protein concentrations during the summer of 1997 when GSRs were low, and (2) very low protein concentration in December 1997 when GSRs were especially high. The correlation between nitrogen and protein contents was tighter in Banyuls-sur-Mer (R 2 = 0.882, N=24, p<0.00001) than in the Medes Islands (R 2 = 0.542, N=24, p<0.006). Carbohydrate concentration was almost always higher (by a factor usually close to 2) in Banyuls-sur-Mer than in the Medes islands (Fig. 8b). This difference was not linked to GSRs since both the intercepts and the slopes of the regression lines linking carbohydrate concentrations and GSRs were different at both sites (Table 8). There was a strong seasonal pattern in Banyuls-sur-Mer with higher average concentrations during the spring-summer than during the autumn-winter period (Table 9). These differences were also significant although less pronounced in the Medes Islands. There was a peak of carbohydrates in Banyuls-sur-Mer in February 1998, non-existent in the Medes Islands. In the Medes Islands, the carbohydrate concentration recorded on May 18 was not especially high as observed for proteins and lipids (see below). Temporal changes in lipid concentrations at both sites are presented in Figure 21c. In both cases, there was a strong seasonal pattern with higher concentration during the spring- summer than during the autumn-winter period (Table 9). Both the slopes and the intercepts lines linking GSRs and lipid concentrations significantly differed among sites (Table 8). In Banyuls-sur-Mer lipid concentrations tended to be lower during the spring-summer period of 1998 than during the spring-summer period of 1997 (2-way ANOVA with Scheffépost- hoc test, F 1,40 =1.04, p<0.05). In the Medes islands, lipid concentration was especially high on May 18, 1998 (and very low GSR) as already noted for proteins, probably due to labile material. In February 1998, lipid concentration rises in Banyuls-sur-Mer, probably also a reflex of productivity in the water column.

Discussion

At both sites, GSRs are within the range of those already measured in the Mediterranean Sea (Table 10). This was also the case for most of the considered biochemical parameters. The organic matter in Banyuls-sur-Mer (Table 10) was slightly lower than the observed in the Têt river delta (3.7-29%, Monaco et al. 1990), but the Medes Islands showed similar values. In other studies, the difference of Banyuls-sur-Mer respect this Têt river observations was lower (Medernach 2000). In the Medes Islands C/N ratio seems to be related with min- eralisation processes (12-26, Table 10, Parsons et al. 1984), and can be compared to sedimenting POM in Posidonia oceanica beds (in Calvi Bay, Corsica, Dauby et al. 1995; but see Danovaro 1996). In Banyuls-sur-Mer the C/N ratio range is wider, but also similar to other literature data (Table 10). It seems that the lower mean C/N value could be related to more fresh material collected in Banyuls-sur-Mer than in the Medes Islands. In Banyuls-sur-Mer, the coincidence of high C/N ratio values seems to be more related to resuspension events

CHARACTERISTICS OF SEDIMENTING POM 73 (i.e. easterly storms and heavy autumn rains) than in the Medes Islands. Proteins, carbohydrates and lipids are within the range found in previous studies (see Grémare et al. 1998a, Medernach 2000), but higher than other systems (Fabiano et al. 1995, Danovaro 1996, Pusceddu et al. 1999a, Table 10). This could be due to great differences between the studied areas. For example, Posidonia oceanica beds, seems to be a high recycling system, where labile material is immediately captured by the dwelling organisms (Danovaro 1996). On the

; other extreme, we find Banyuls-sur-Mer and -1 )

-1 Medes Islands values lower than Najdek et al.

g l (1994) work in the North Adriatic (12.5-275 µg µ

g mgDW mg DW -1 ), probably because of the presence in µ

;(* that study of lipid droplets (result of a deficient -1

) ** phytoplankton digestion) in the collected faecal -1

g l pellets. µ A seasonal pattern in GSRs and in the main biochemical components of sedimenting POM has been observed at both sites. The general trend is to higher gross sedimentation rates and lower concentrations of sedimenting POM in ; (%) (*µg mgDW

-1 the autumn-winter period than during the day

) ** spring-summer period. This trend is in accor- -2 -1 dance with previous works carried at a shallower station in the Bay of Banyuls-sur-Mer **mg l (Grémare et al. 1997, 1998a). However, these (*g DW (*g m DW seasonal patterns were much more pronounced in Banyuls-sur-Mer than in the Medes islands. bedsbeds 0-13* - 5-25 7-14 - 2-6 - 0.5-8* 0.8-6* - 1996 Danovaro Dauby et 1995 Maximal GSRs were much higher at Banyuls- sur-Mer than in the Medes islands. These GSRs were measured during the autumn-winter period and were associated to sediment resuspension, which is consistent with the results of

Posidonia oceanica Posidonia oceanica Posidonia Charles (1994) and then of Grémare et al. (1997 and 1998a). The absence of such very high GSRs during wintertime in the Medes islands suggests that resuspension may be less important at this site. This is supported by the occurrence of higher wave height in Banyuls- Comparison of gross sedimentation rates, C/N index, protein Organic and matter, lipid concentrations from different areas of the Mediterranean sur-Mer (even if caution should be taken in comparing the data sets corresponding to the two sites). Another factor which may con- Têt Delta delta River 8-2379* 4.6-15 - - - Buscail et al 1990 Marconi Gulf (NW Italy)Calvi Bay (Corsica) Shelf Bay - 6-13 9-29 0.015-0.07* 0.025-0.214* Fabiano et al 1995 Prelo Bay (NW Italy) Banyuls (S France) Shelf Bay 0.5-160* 8-18 5-14 3-35* 1.3-20* Grèmare et al 1998 Table 10. Table Area EcosystemMedes Islands (NE Spain) Shelf Coast Gross rates C/N Index Organic Matter Proteins 0.5-23* Lipids 12-26 Reference 6-22 7-29* 1.2-22* This study sea. Maximum and minimum values are shown. Marseille (South France) Submarine (Entrance)Cave 0.2-4.2** 8-11 - 12-75** 10-110** Fichez 1991 Marsala (SE Italy) StationI Banyuls (S France) Coastal Lagoon Shelf Bay - 0.6-96* - 7-27 - 3-14 3-8* 1-26* 0.3-4* 0.2-19* This study Pusceddu et al 1999a

74 CHARACTERISTICS OF SEDIMENTING POM tribute to differences in GSRs between both sites is sediment granulometry. In Banyuls-sur- Mer, sediment traps were moored over a sandy mud (Grémare et al. 1998c) inhabited by benthic soft bottom communities (Guille 1971a). In the Medes Islands, mud and sandy mud are rare and most of the bottoms is composed of rocks and gravels (Gili and Ros 1985), which are less sensitive to resuspension. Furthermore, although both sampling areas showed the same general orientation pattern, sediment traps were located closer to the coast in the Medes islands than in Banyuls-sur-Mer. Resuspension may thus be more important in the fine-medium particulate substrates of Banyuls-sur-Mer exposed to open sea conditions than in the medium-large particulate substrates of the Medes Islands which are located within a more sheltered area. Nevertheless and because of the negative relationship between GSRs and concentrations, a lower range in GSR variations in the Medes islands may thus account for a weaker seasonal pattern in seasonal changes in the biochemical characteristics of sedimentary organics at this site. For all the biochemical parameters considered during the present study (except organic carbon), the regression models linking GSRs and concentrations differed among sites. Medernach (2000) compared the degree of POM lability associated with various biochemical parameters based on the slopes of the regression lines linking those parameters with GSRs at a shallower site in the Bay of Banyuls-sur-Mer. Among the parameters, considered during the present study, both organic matter and carbohydrates were associated to the most refractory fraction, whereas both proteins and lipids were associated to the most labile one. Given, the occurrence of stronger resuspension in Banyuls-sur-Mer it is thus not surprising that : (1) carbohydrate concentrations are higher in Banyuls-sur-Mer during the autumn- winter period, and (2) lipid concentrations tended to be higher in the Medes Islands. However, it should be stressed that: (1) between sites differences in carbohydrate concentrations tended to be larger when GSRs were low, and (2) sedimenting POM concentrations in proteins were not significantly lower in Banyuls-sur-Mer than in the Medes islands. Together with the occurrences of significant between-sites differences in regression models linking GSRs and concentrations, both these observations suggest that GSRs are not the only factor driving sedimenting POM biochemical characteristics at both sites. Phytoplankton biomass tended to be higher in the Medes islands than in Banyuls-sur- Mer during most of the period under study. However patterns of seasonal changes in Chlorophyll a concentrations at both sites tended to be similar with a clear peak corresponding to the winter-spring bloom during February. Interestingly, the consequence of this peak was much clearer in Banyuls-sur-Mer than in the Medes islands in spite of the possible con- founding effects of resuspension due to strong hydrodynamics during that period of the year (see above). This is in agreement with the results of Graf et al. (1982 and 1983) who showed that spring pulses in primary production had a significant effect on benthic community activity (quantified as ATP, biomass and heat production). The absence of the February peak in the Medes islands may be connected to the occurrence of high GSRs during February. However, it is very unlikely that those GSRs are due to sediment resuspension since wave height was relatively low at that time of the year. This is probably indicative of the existence of another source of (mostly) refractory POM at this site: the next river influence (Chapter I). This hypothesis is further supported by the occurrence of higher C/N ratios in the Medes Islands. Along the same lines, the peak of concentrations recorded on May 1998 in the Medes islands was not associated to any maximum in chlorophyll a concentration but rather

CHARACTERISTICS OF SEDIMENTING POM 75 to low GSRs as well. This may explain why this peak was much more pronounced for biochemical associated to labile fractions of sedimening POM (i.e., proteins and lipids) than for carbohydrates or to lesser extent organic contents. Based on the results of other studies (Chapter I), we believe that the River Ter runoff may affect both seston quantity and composition in the Medes islands as already shown by Danovaro et al. (2000a) in an other area. Detrital output by the Ter River together with the extensive algal and phanerogam coverages (Gili and Ros 1985, Ballesteros 1992) may thus influence sedimenting POM biochemical characteristics in the Medes islands. Moreover, River runoff could also account for the occurrence of a tighter relationship between nitrogen and protein contents in Banyuls-sur-Mer than in the Medes islands. Almost all the nitrogen probably corresponds to proteins in Banyuls-sur-Mer, whereas in the Medes Islands part of the nitrogen could be derived from the decomposition of humic material transported by the river as already observed by Bushaum et al. (1991). Our conclusion, is thus that between sites differences in both absolute values and in the modalities of the seasonal pattern reflect the existence of different pathways of POM transfer. In Banyuls-sur-Mer, resuspension is clearly dominant in driving GSRs and sedimenting POM concentrations. This results in a strong seasonality because this process is mostly related to winter storms. Resuspension is less important in the Medes islands. Interestingly the correlation between water column chlorophyll a concentrations and sedimenting POM biochemical characteristics is also looser at this site. Moreover, the concentrations of both lipids and proteins are not higher in the Medes islands than in Banyuls-sur-Mer. This suggests the existence of another source of (refractory) POM in the Medes islands. This source may be the Ter River runoff, which may result in a weaker seasonality than in Banyuls-sur-Mer. Responses of benthic assemblages to variations in the characteristics of seston or sedimenting POM may vary with changes over spatial scale and may be dependent on the con- comitant action of other physical and biological factors (Airoldi and Virgilio 1998, Gardner 2000). An important result from the present study is that both GSRs and the biochemical characteristics of sedimenting POM may differ between sites, which are only 50km apart. This certainly complicates the generalisation of surveys assessing changes in sedimenting POM biochemical characteristics since most of those surveys have been conducted at a single site (Charles et al. 1994, Danovaro 1996, Grémare et al. 1997, 1998a , Medernach et al. 2001, among others; but see Airoldi and Virgilio 1998, Pusceddu et al. 1999a). Table 10 shows GSRs and sedimenting POM recorded during at different sites in the Mediterranean. For each site and each parameter, the absolute range of variation is high, suggesting that feeding constraints on benthic organisms vary during the year. A similar pattern has been found in polar zones, where the strong summer phytoplankton blooms increase POM availability to benthic organisms (Cripps et al. 1998, Pusceddu et al. 1999b). Therefore, sedimenting POM biochemical characteristics and thus nutritional value in coastal shelf waters may be occasionally high, but is seasonally constrained, and results from different factors such as resuspension, river runoff and primary production. Regarding this last factor, it should be stressed that sedimenting POM characteristics integers discontinuous water column processes on a larger time-scale (Boero 1994). In the Mediterranean, algal blooms are sometimes difficult to detect because of their short duration. These blooms sink to the sediments when the turbulence slows down (Margalef 1997) resulting in an increase in the nutritional value of sedimenting and sedimented POM. Thus punctual analy-

76 CHARACTERISTICS OF SEDIMENTING POM ses carried out on water column samples may less reflect POM availability to the benthic community than measurements carried out on sedimenting or sedimented POM. During the present study, this was especially the case in the Medes islands, where there was no clear relationship between Chlorophyll a concentrations and sedimenting POM concentrations in various biochemical compounds. Therefore, small scale spatial comparisons of sedimenting POM may constitute a useful tool to understand how different hydrographical features affect sedimentation regimes and then benthic processes. Changes in food supply is one of the most important factor influencing the production and the biomass of coastal benthic communities. One clear expression of the enrichment processes in coastal area is the sediment type (Alongi 1998). Organic matter in sediments is a mixture of particulate and dissolved material derived from various autochthonous and allochthonous sources (Tyson 1995). Proteins, carbohydrates and lipids, which are essential in sustaining benthic communities are derived from these sources. However, the availability of these organic components for benthic organisms is largely depending on local hydrodynamics (Shimeta and Jumars 1991), with a range of flow and frequency that determines animal diversity, biomass, structure and distribution. Recruitment of benthic organisms may also be drastically affected by POM availability. According to the settlement timing hypothesis, the larval release in the water column is mainly cued by Chlorophyll a concentrations (Starr et al. 1990). Moreover, in the Mediterranean polychaetes tend to settle during the Spring when sedimenting POM nutritional value is at its maximum (Sardà et al. 1999). Therefore, the availability of organic matter could also be one of the key factor controlling the survivorship of new recruits, and we have showed that both studied zones differ in biochemical characterisation of sedimenting POM. Results from previous studies carried out at both studied sites, showed that benthic community is more dense and with a higher biomass per unit of surface area in the Medes Islands than in Banyuls-sur-Mer (Guille 1971a, Guille 1971b, Gili and Ros 1985, Grémare et al. 1998b). Moreover, those communities are dominated by suspension-feeders in the Medes islands and by deposit-feeders in Banyuls-sur-Mer. Medernach (2000) has compared the ability of several biochemical parameters to describe changes in sedimentary organic nutritive value. Based on the correlations between sediment concentrations and benthic fauna standing stocks in the Gulf of Lions, she concluded that lipids was one of the best biochemical descriptors of POM nutritive value. Interestingly, this is also the only biochemical parameter whose concentrations tended to be higher in the Medes islands than in Banyuls- sur-Mer. Differences in suspending POM nutritional value may thus contribute to explain differences in benthic standing stocks at both sites. It would now be interesting to carefully study, the interaction of this factor with local hydrodynamics, which is probably much more important in the Medes islands as indicated by the occurrence of much coarser sediments (Gili and Ros 1985, Grémare et al. 1998). We suggest that this hydrodynamic (i.e. higher wave heights in Banyuls-sur-Mer than in the Medes Islands) and substrate (i.e. more fine sediments in Banyuls-sur-Mer than in the Medes Islands) differences make the Banyuls-sur- Mer area more sensitive to resuspension events than the Medes Islands area (reflected in the GSRs). Moreover, river inputs are significant for the seston dynamics in the Medes Islands all over the year (Rossi et al. Submitted), while only in the hard rain autumn conditions is evident the influence in Banyuls-sur-Mer (Charles et al. 1995, Grémare unpublished data). We concluded that differences in those factors may influence structure and biomass of ben-

CHARACTERISTICS OF SEDIMENTING POM 77 thic communities in both zones, and such temporal and spatial scale approaches are needed to understand ecological processes and bionomic features of different areas.

78 CHARACTERISTICS OF SEDIMENTING POM Activity rhythms in six temperate species of passive suspension feeders: observations at different time and space scales related to environmental conditions

Abstract

The activity rhythms of six passive suspension feeders of a warm-temperate sea (Mediterranean) were registered in a subtidal zone (11-19 m depth) of the coralligen, considered a diverse and complex community. From September 1996 to August 1998, the seasonal trends of the anthozoans Paramuricea clavata , Eunicella singularis , Leptogorgia sarmentosa , Corallium rubrum , Alcyonium acaule and Parazoanthus axinellae were observed as expanded/contracted colonies 1-14 times a month. Furthermore, some environmental parameters were tested within short time periods concurrently with activity rhythms to link such factors with polyp expansion (periods tested: early spring [late March]; late spring [late June]). The environmental parameters tested were currents, chlorophyll a, seston proteins/carbohydrates concentrations and zooplankton density. In addition, diel and spatial activity rhythm variability was tested in June in the six cnidarian species. There is a seasonal tendency for all but C. rubrum species, in which colonies show a clear low activity in July-September, suggesting a response to water stratification (i.e. lower hydrodynamism and low seston concentration). The studied anthozoans show different response to natural environmental factors. Although variability is high, P. clavata activity rhythms seems to be solely related to currents, while E. singularis , L. sarmentosa and A. Acaule were also related to seston parameters (i.e. proteins and zooplankton concentrations). C. rubrum and P. axinellae were only related to seston parameters when the activity rhythms were tested in the two spring situations tested (i.e. March and June). Even when results show high heterogeneity among species, there is a clear synergistic effect on the activity rhythms at the level of community when both, currents and seston concentration, show concurrently its higher values. It is suggested a different temporal behaviour of the community in front of different environmental fluctuations in early (late March) and late spring (late June), being such environmental features more pulse-like in the late than in the early spring. Spatial heterogeneity (considering cnidarians patches 20-50 m apart) gives striking results: one specie’s patch may have a clear active behaviour while the other is concurrently almost non-active at all. Activity rhythms proof to be a useful and easy to observe tool to test for all these combined effects. Activity rhythms may help us to understand synergy levels in front to environmental energetic fluctuations: 1) seasonally, because these organisms will show less activity in the less favourable times of the year (i.e. summer in warm temperate seas); 2) Daily, because the tendency will be to take advantage of the more favourable pulses (i.e. when currents and seston are in its optimal ranges). Our conclusion is even daily activity rhythms are regulated by external environmental factors, seasonal rhythms must be in accordance with endogenous control acquired by the learning from repeated exposure and response to short periods that permit the adjust of internal clocks, as it has been suggested for marine mobile invertebrates (e.g. crabs, copepods, etc.).

Introduction

Establish the role of environmental control in regulating the frequency of community activity is one of the key factors to understand energy fluxes in marine ecosystems. Environmental variability, particularly as regards hydrodynamics, is conducive to enhance local benthic production, and community development and stability depends on constant hydrodynamic conditions and bursts of pelagic production in the area (Fréchette and Lafaivre, 1990; Josefson et al. , 1993). In general, organisms have a tendency to optimise

ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS 81 prey capture depending, among others, on disposable resources which depends on surrounding environmental factors (Coma et al. 2001). Responses of populations to environmental changes are generally assumed to be behaviour related (Sutherland 1996). The more significant change to be solved by one species is food availability, which is driven by external forces in suspension feeders. The daily search of food generates a set of ecological strategies designed to find prey at the lower energetic cost (Hughes 1980). To do that, marine organisms have an activity behaviour synchronized with the environmental variability at different temporal and biological scales. This behaviour is highly diversified but could be summarized in two general tendencies: a daily activity set out to cover energy demands of individual and population maintenance, and seasonal activity to guarantee population growth and reproduction. In both, daily and seasonal scales, mobile organisms have been developed specific activity rhythm at both daily and seasonal time scale. The marine species rhythmicity is controlled for internal or physiological and external clock, phenomena extensively studied during the last 50 years for benthic (Naylor 1976) and plankton (Ohman 1990) organisms. The literature is full of examples related with, for example, vertical migration of zooplankton or synchronized tidal displacements of crabs. Guided by endogenous or exogenous rhythms, these organisms show quantifiable activity, more or less regular, as a response to environmental and biological factors. In this way, very little information is available for sessile organisms. A common concept is that such organisms have spontaneous rhythms, as for example anemone individual expansion, valve opening or cirral beat in barnacles (Rodriguez and Naylor 1972). Other works suggested that behaviour response of sessile invertebrates could be more synchronic than has been though before (Sebens and DeReimer 1977). Much of these organisms have daily feeding rhythms that have been correlated with the major prey abundance (Sebens 1987) but, also exhibit seasonal patterns that recently have been correlated with feeding activity (Coma et al. 2001). Sessile organisms have evolved mechanisms at both scales, daily and seasonal, to allow animals capture particles with high frequency, and many species probably developed rhythmic behaviour to facilitate low feeding costs. An open question is if the feeding behaviour patterns of sessile organisms could be com- parable with mobile ones and, if it is true, what is the ecological significance and adaptive value of rhythmic behaviour. Much of sessile species are benthic suspension feeders, a group that dominate most of sublittoral communities in all oceans (Gili et al. 2001) and that plays a paramount role in littoral marine ecosystems (Gili and Coma 1998). Certain species of benthic suspension feeders present patterns of expansion and contraction. Following the activity of several species within a diverse community, seasonal patterns have been demonstrated in warm temperate (Coma et al. 1994, Garrabou 1999) and polar (Barnes and Clarke 1995) ecosystems. Periods of maximum expansion (e.g open polyps, opened oscula, active lophophores) in a given colony are closely related to periods of maximum prey capture (Sebens and DeRiemer 1977; Robbins and Shick 1980, Barnes and Clarke 1995, Riisgard and Goldson 1997). Conversely, when polyps are contracted the metabolism slows; it appears to be an adaptation to restrict energy expenditure when prey availability is low (Lasker 1981; Zamer 1986; Sebens 1987). In fact, most of the suspension feeders observed in warm temperate, cold temperate and polar ecosystems show low activity when trophic environmental conditions become unfavourable (i.e. low food availability, low flow speeds, etc; Sebens 1983, Zamer 1986, Coma et al. 1994, Barnes and Clarke 1995, Garrabou 1999).

82 ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS One of the main objectives of this paper is understand rhythmic patters of sessile organisms which will be coupled with physical and biological environmental variability, considering the detailed behaviour study of six species of passive suspension feeder species. To better understand which environmental and biological factors are correlated with community behaviour activity, and how they may influence energy flow between plankton and benthos, temporal and spatial heterogeneity has to be considered. Levels of population heterogeneity span a continuum running from monospecific patches of colonial or individual suspension feeders to complex three-dimensional formations comprising communities with high species and functional diversity. The more variable the water column environmental factors, the more diversified mechanisms to enhance prey capture and feeding optimisation, as organisms adopt different strategies to take advantage of every potential source of food (Gili and Coma 1998, Coma et al. 2001). Small and medium-scale variations in the density and quality of the seston have been reported to determine the density of suspension- feeder populations (Fegley et al. , 1992; Smaal, 1994). For the seston to be continuously available to suspension feeders, the water layer surrounding the suspension feeders must be continuously renewed (Eckman and Duggins, 1993). The problem becomes more difficult when the community as a whole is considered, since components of a single species population present varying patterns of capture and capture efficiencies depending on their spatial location, size and age (Sebens and Johnson, 1991). Thus, is essential to apply short time and space scales of observations in the activity of benthic sessile organisms to understand such rhythmic complex response to environmental heterogeneity. The coupling of environmental factors with activity rhythms of organisms representative of a benthic community is presented here to understand how energetic constraints could be controlled by physical and/or biological parameters. Three main goals are developed in this study: 1) Observe seasonal variations of activity rhythms in warm temperate suspension feeders related to environmental factors (i.e. different food availability periods); 2) Study which environmental factors are involved in such activity; and 3) Understand the role of high-resolution temporal and spatial variability in the relationship between activity of species of organisms owing diverse life histories within the same community. To reach these objectives, the activity rhythms (i.e. polyp expansion-contraction) of six passive suspension feeders (the soft corals Paramuricea clavata , Eunicella singularis , and Leptogorgia sarmentosa , the hard coral Corallium rubrum , the alcionarian Alcyonium acaule, and the zoantharian Parazoanthus axinellae ) have been observed at different temporal and spatial scales in the coralligenous community of the North Western Mediterranean Sea. Passive suspension feeders where chosen for its paramount role in the studied community (Gili and Coma 1998), but also because activity rhythms are easy to observe and quantify in such organisms (Coma et al. 1994, Barnes and Clarke 1995, Garrabou 1999). Concurrently, in two high frequency sampling periods (early spring, end of March, and late spring, end of June), several environmental parameters (flow speed as a physical factor, chlorophyll a concentration, particulate protein and carbohydrate concentrations, and zooplankton concentration as biological factors) were tested on the field to understand their influence on polyp colony expansion. This is the first study which analyses on the field the activity of seasonal and short-time/space-scale changes at the community level of benthic passive suspension-feeders, influenced by warm temperate environmental conditions. The last objective of this paper is compare the activity rhythms patterns observed in sessile organisms with the better known activity rhythms of the mobile ones. The question pro-

ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS 83 3º 13' E a b 0 0.5 1 km

L'ESTARTIT

MEDES ISLANDS

42º 02' N

Ter River Figure 22. Study area location (Medes Islands). Black dot indicates the sampling

Sea area. Detailed map: (A) Wall zone and (B) ean Mediterran Boulders zone for the observation of the activity rhythms.

posed is: are such activity rhythm patterns different, or all marine organisms follow similar trends irrespective of their biological constraints (i.e. sessile vs. mobile)?

Material and methods

Study area. The study area is located in the Medes Islands, NW Mediterranean (40º02’55’’N, 3º13’30’’E) (Fig. 22). Sampling and observations were carried out at 11-19 m depth in front of a north-oriented vertical wall (in further explanations called Wall, Fig. 22a) and a channel with wide boulders (in further explanations called Boulders, Fig. 22b). Suspension feeder patches of both zones were 20 to 50 m apart. The Boulders zone (channel) has North to South and South to North alternate currents that may reach high speeds (Pasqual et al. 1995); these currents are supposed to complicate its direction in the Wall zone do the topography of the area. Attending to zonation patterns established for this area, we focused on the coralligen community considered one of the most diversified in the Mediterranean benthos and in which passive suspension feeders have a paramount role (Gili and Ros 1985).

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Figure 23. Colonies considered with expanded polyps. (A) Paramuricea clavata ; (B) Eunicella singularis ; (C) Leptogorgia sarmentosa ; (D) Corallium rubrum ; (E) Alcyonium acaule ; (F) Parazoanthus axinellae .

ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS 85 Seasonal observations. Activity rhythms were observed in six species in the Wall zone from September 1996 to August 1998. The passive suspension feeder species studied are: the soft corals Paramuricea clavata , Eunicella singularis , and Leptogorgia sarmentosa (this gorgonian dwells only the Boulders zone), the hard coral Corallium rubrum , the alcyonarian Alcyonium acaule, and the zoantharian Parazoanthus axinellae . Colonies with expanded polyps (Fig. 23a-d), semi-contracted polyps (i.e. polyps were tur- gent but polyp crown was retracted), and contracted polyps were registered 1-14 times a month as follows: Ten groups of ten Figure 24. Alcyonium acaule dormant colony colonies were observed each time for each (Garrabou 1999), which has contracted polyps and specie, except Parazoanthus axinellae which a thin film covering the surface. were counted only ten groups of ten polyps. Colonies wich were halve expanded-halve contracted were not registered. The alcionarian Alcyonium acaule has also dormancy colonies (i.e. colonies which had contracted polyps and a thin film covering the surface, Fig. 24, Garrabou 1999) that were also registered. Environmental factors. To test which factor/s were involved in the polyp expansion-contraction behaviour of the six cnidarian species, several environmental parameters were sampled the 24-29/6/97 and the 27-30/3/97 in the Boulders zone concurrently with the activity rhythms of the six cnidarians. Hydrodynamic data set was registered with the aid of an ADP-doppler effect current meter. Unfortunately, in March 1998 the ADP-doppler data were lost, and no current regis- tration is available. Concurrently to the rhythms of activity of the gorgonians, zooplankton samples were collected in duplicate using 22 cm diameter nets of 100 µm mesh size. The nets were towed over a distance of 40 m by a diver (30 to 50 cm from the benthic surface). The zooplankton samples were fixed in 6% formaldehyde in seawater and identified to the level of main taxonomic groups (in the June 1997 cycle, each 6h; the March 1998 zooplankton data are not available). Further details of zooplankton observations are available in Chapter I. The sampling for seston Chlorophyll a, Proteins and Carbohydrates (only March 1998) was carried concurrently with the zooplankton fishing. Natural seawater (22-24 L) was collected by SCUBA diving. Once in the lab, the seawater samples were pre-filtered through a 100 µm mesh ( sensu Ribes et al. 1999a). Further details of sample treatment, filtered water volumes, and laboratory biochemical processes are available in Chapter I. Activity rhythms high-resolution time comparisons. Due to the extreme variability in the activity rhythms of the cnidarians and with the aim to clarify possible diel changes in the different species, two different temporal situations were chosen to observe the behaviour of the species with a high frequency sampling: a) Early Mediterranean spring, where there is a phytoplankton bloom and water column has a uniform nutrient, salinity and temperature

86 ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS distribution, and b) Late Mediterranean spring, where production in the water column is still very high, but environmental conditions are more unstable due to the sudden wind and water current changes, the thermocline formation and a less uniform river landing (Estrada et al. 1985, Hopkins 1985, García et al. 1995, Chapter I and II). In the Boulders zone, from 24- 29/6/97 (In further explanations Late spring) activity rhythms of the six species were registered each 6 h as above mentioned. In the same zone, from 27-30/3/98 (In further explanations Early spring) the activity rhythms of the same species were registered each 4 h. Furthermore, in the Wall zone, two activity rhythms samplings were made the 18-19/6/97 and the 20-21/6/97 each 3 h to compare diel cycles in this zone with all species (and Leptogorgia sarmentosa that was only present in the Boulders zone). Space comparisons. Concurrently to the Boulders zone sampling in June 1997 (from 24- 29/6/97 each 6 h), observations were made in all species in the Wall zone (except Leptogorgia sarmentosa , that was present only in the Boulders zone). The distance between patches between zones covered 20-50m (see Figure 22a,b). Statistics. One-way ANOVA with Scheffé post-hoc comparison was used to test for differences between seasons in the activity rhythms of the six cnidarians. The field observations were gathered seasonally taking into account astronomical dates to avoid subjective interpretations. Standard multiple regression analysis is used to test which parameter (and in which proportion) affects the polyp expansion of the colonies. In this way, Flow speed- Chlorophyll a-Proteins-Plankton has been tested for the six species in June 1997; Chlorophyll a-Proteins-Carbohydrates have been tested in March 1998 for the six cnidarians (Zar 1996). To test temporal similarities/differences (i.e. to compare early with late spring situations) in the activity rhythms of the six passive suspension feeders two statistics have been applied: 1) Compare Variation Index of each species (Variance/Mean) in both periods; 2) Correlate colony expansion between species in the same monitored time. The final aim of these two statistical proofs is test if environmental conditions (apparently more stable in Early spring than in Late spring, see Chapter II) are reflected in the synergy of the community (i.e. more stable conditions will lead lower VI and higher correlation between expanded colonies observations). To test spatial variability, VI is also applied in Corallium rubrum and Parazoanthus axinellae , and standard correlation is used to test colony expansion between patches of the Wall and Boulders zones (Dytam 1999).

Results

Seasonal trends . All species show high variability in the activity rhythms (shown by the high Standard Deviation bars, Fig. 25a-f) when observed few times a month. Paramuricea clavata showed no between seasons significant differences (ANOVA F 3,129 = 0.74, p = 0.5325). Eunicella singularis showed differences between winter and summer colony activity (68 ±38SD% expanded colonies in winter; 28 ±35SD % expanded colonies in summer), but no other significant differences (ANOVA F 3,118 = 5.52, p < 0.002). Leptogorgia sarmentosa and Corallium rubrum showed no differences between seasons in the activity rhythms observed (ANOVA F 3,47 = 2.03, p = 0.1231 , and ANOVA F 3,100 = 0.52, p = 0.6675

ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS 87 a b

c d

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Figure 25. Seasonal trends in the six studied anthozoans. Alcyonium acaule shows expanded colonies + dormant colonies. Mean % expanded colonies (or polyps in the Parazoanthus axinellae case) ± Standard Deviation.

88 ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS Figure 26. Effect of the flow speed (cm s -1 , left) in the activity rhythms (% expanded colonies, right) of Paramuricea clavata in the late spring period (24-29/6/97). respectively), nor the Alcyonium acaule colonies (ANOVA F 3,102 = 1.98, p = 0.1218). The last tested anthozoan, Parazoanthus axinellae , showed lower summer activity, and summer polyps were significant shrinked compared to all other seasons (ANOVA F 3,101 = 7.63, p < 0.0001). All the tested organisms have a tendency: 1) A regular period of expansion from January to June (more than 50% of the colonies full expanded, except Alcyonium acaule Fig. 25e), 2) A low expansion period (July-August); this is more evident in Alcyonium

Table 11. Multiple regression analysis to estimate the variance in the variable proportion of expanded polyps per colony from Paramurica clavata, Eunicella singularis, Parazoanthus axinallae, Corallium rubrum, Leptogorgia sarmentosa and Alcyonium acaule explained by flow speed (cm s -1 ), Chorophyll a (mg l -1 ), proteins (mg l -1 ), and plankton (ind m -3 ). Data collected in situ between 6h intervals between June 24 and June 29, 1997. N: 19. (a) Slopes (± SE) for the different variables, intercept (A), adjusted squared r (Adj R 2). (b) Standarized partial regression coefficients.

P. clavata E. singularis P. axinellae C. rubrum L. sarmentosa A. acaule

(a) Flow speed 3.15±0.52 2.09±0.61 n.s. n.s. 1.51±0.73 1.46±0.39 Chl a n.s. n.s. -83.42±64.99 n.s. n.s. n.s. Proteins n.s. n.s. n.s. n.s. n.s. 0.19±0.11 Plankton n.s. 0.007±0.002 0.006±0.003 n.s. 0.01±0.003 0.003±0 A n.s. —— n.s. n.s. Adj R 2 0.66*** 0.55*** 0.27* —— 0.48* 0.61**

(b) Flow speed 0.82 0.55 n.s. n.s. 0.35 0.57 Chl a n.s. n.s. -0.27 n.s. n.s. n.s. Proteins n.s. n.s. n.s. n.s. n.s. 0.28 Plankton n.s. 0.50 0.45 n.s. 0.60 0.28

Non-significant (n.s.), *** P<0.0001, ** P<0.001, * P<0.05.

ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS 89 Table 12. Multiple regression analysis to estimate the variance in the variable proportion of expanded polyps per colony from Paramurica clavata, Eunicella singularis, Parazoanthus axinallae, Corallium rubrum, Leptogorgia sarmentosa and Alcyonium acaule explained by Chorophyll a (mg l -1 ), Proteins (mg l -1 ) and Carbohydrates (mg l -1 ). Data collected in situ between 4h intervals between March27 and March 30, 1998. N: 21. (a) Slopes (± SE) for the different variables, intercept (A), adjusted squared r (Adj R 2). (b) Standarized partial regression coefficients.

P. clavata E. singularis P. axinellae C. rubrum L. sarmentosa A. acaule

(a) Chl a n.s. n.s. 88.3±37.2 n.s. n.s. n.s. Proteins n.s. 0.58±0.24 0.35±0.16 0.72±0.3 n.s. 0.88±0.71 Carbohydr. n.s. n.s. n.s. n.s. n.s. n.s. A Adj R 2 —— 0.2* 0.19* 0.27* —— 0.39**

(b) Chl a n.s. n.s. 0.48 n.s. n.s. n.s. Proteins n.s. 0.49 0.44 0.57 n.s. 0.72 Carbohydr. n.s. n.s. n.s. n.s. n.s. n.s.

Not significant (n.s.), *** P<0.0001, ** P<0.001, * P<0.05.

acaule where, from June to September, the dormant colonies grow in number, reach their maximum values (July-August), and decrease (Fig. 25e), 3) A third irregular period between September and December. In November all six species have expanded colonies that reach maximum values. Environmental factors . Paramuricea a clavata is influenced by the flow speed when tested the expanded colonies in front of the currents the 24-29/6/97 (Fig. 26 and Table 11). The colony expansion in this period is highly significant related to current speed but with no other tested factor. The 27-30/3/98 period, in Paramuricea clavata there was no relationship of tested factors with colony expansion (Table 12). Eunicella singularis is significant affected by flow speed and zooplankton b concentration during the 24-29/6/97 week Figure 27. (a) Effect of near bottom zooplankton density (Table 11), and protein concentration is (ind m -3 , left) in the activity rhythms (% expanded weekly although significant related the 27- colonies, right) of L. sarmentosa in the late spring period 30/3/98 (Table 12). (24-29/6/97). (b) Effect of near bottom seston protein concentration (µg Protein l -1 , left) in the activity rhythms In the 24-29/6/97 period, flow speed (% expanded colonies, right) of Alcyonium acaule in the but specially zooplankton concentration early spring period (27-30/3/98) (Fig. 27a) are significant related with

90 ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS a b

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Figure 28. Rhythms of activity of the six observed anthozoans during the early spring period in the boulders zone (27-30/3/98). Mean % expanded colonies (or polyps in the Parazoanthus axinellae case) ± Standard Deviation.

ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS 91 polyp expansion in Leptogorgia sarmentosa colonies (Table 11). In March (27-30/3/98), this gorgonian is not significant affected by any tested factor (Table 12). Corallium rubrum colony expansion is significant affected by protein concentration in March, but no in June. No other factor is significant related with this hard coral in the multiple regression analysis that has been applied in both periods. Alcyonium acaule shows high sensibility for many factors when tested the colony expansion in the Boulders zone. In June, is significant related with flow speed, protein and zooplankton concentration (Table 11), and in March is again related with water column protein concentration (Fig. 27b, Table 12). The behaviour of Parazoanthus axinellae in June and March is opposite when tested the effect of Chlorophyll a concentration on polyp expansion. In fact, in the 24-29/6/97 period is negatively correlated, while in 27-30/3/98 is positively correlated with similar (but opposite sign) slopes (Table 11 and 12). Zooplankton is significant correlated with polyp expansion in 24-29/6/97 cycle (Table 11). High-resolution temporal variability cycles. Early spring cycle (27-30/3/98) has a temperature of 13.7ºC (0.5 m depth), 13,2ºC (5 m depth), and 13.1ºC (20 m depth) in the water column (Josep Pasqual, Estartit Meteorological Station). All six cnidarians are concurrently expanded from 15:00 (29/3/98) to 11:00 (30/3/98) (Fig. 28a-f). All the rest of the cycle, each patch shows different behaviours in front of the environmental factors. In the above mentioned elapsed time, standard deviation of all the species gets its lowest values (Fig. 28a-f). This period is in coincidence with the protein concentration rise (see Fig. 27b). When protein concentration gets low values again (19:00 of the 30/3/98), all suspension feeder studied patches contract their polyps (except Parazoanthus axinellae , see Fig. 28f). In late spring (24-29/6/97) period, water column temperature was 20.6ºC (0.5 m depth), 20.4ºC (5 m depth), and 20.1ºC (20 m depth). There is also a punctual co-ordinated colony expansion behaviour at 3:00 of the 29/6/97 (Fig. 29a-f), the moment when water currents, protein concentration (see Chapter II) and zooplankton concentration gets concurrently high values (Fig. 26, 27a). After this brief period, all colonies contract their polyps. More irregular, this cycle seems to have two period, one from 15:00 of the 24/6/97 to 9:00 of the 27/6/97, in coincidence with minimum zooplankton and protein concentrations (except Paramuricea clavata , Fig. 29a-f), and a second period from 15:00 of the 27/6/97 to 3:00 of the 29/6/97 where zooplankton and proteins are significant more abundant than the first period (see Fig. 27a and Chapter II). Paramuricea clavata follow similar recurrent patterns of colony expansion and contraction in both studied cycles: an alternate of expanded and contracted colonies during both periods (see Fig. 28a, 29a). In June 1997 this seems to be related with a regular flow pattern in the Boulders zone (Fig. 26). No other species follow such pattern, but no other species is so tightly related with flow speed. When zooplankton and/or protein concentration coincide with high flow speed, the gorgonian leave the polyps opened. Eunicella singularis do not follow such pattern (Fig. 28b, 29b), nor Leptogorgia sarmentosa (Fig. 28c, 29c) or Alcyonium acaule (Fig. 28e, 29e), that seems to be more weakly related with the Boulders zone flow patterns. The species which dwell the crevices in the Boulders ( Corallium rubrum and Parazoanthus axinellae , see Fig. 28d, 29d and 28f, 29f) show a more chaotic polyp expansion behaviour, but coincide with other species in the above mentioned propitious moments. Variability in the expansion behaviour of Parazoanthus axinellae is very high, but it has to be considered that expand-

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Figure 29. Rhythms of activity of the six observed anthozoans during the late spring period in the boulders zone (24-29/6/97). Mean % expanded colonies (or polyps in the Parazoanthus axinellae case) ± Standard Deviation.

ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS 93 Table 13. Variation Index (mean of the VI, Standard Deviation/Mean) of the different cnidarians tested in early spring (3/98) and late spring (6/97) periods.

Variation Index Early Spring (March) Variation Index Late Spring(June)

Paramuricea clavata 48% 80% Eunicella singularis 70% 86% Leptogorgia sarmentosa 31% 62% Corallium rubrum 92% 30% Alcyonium acaule 102% 103% Parazoanthus axinellae 54% 55%

Table 14. Relationships of colony expansion (%) between different studied species during early spring (27- 30/3/98, ITALIC , N=21) and late spring (24-29/6/97, BOLD , N=19) periods. Legend: n.s.=non significant; *=p<0.05; **=p<0.02; ***=p<0.001; ****=p<0.0001.

P. clavata E. singularis L. sarmentosa C. rubrum A. acaule

E. singularis **R 2=0.37 **R 2=0.29 L. sarmentosa *R=0.24 * *R 2=0.68 ***R 2=0.54 **R 2=0.37 C. rubrum **R 2=0.28 **** R 2=0.33 ** R 2=0.33 **R 2=0.25 **** R 2=0.68 **R 2=0.35 A. acaule **R 2=0.28 ****R 2=0.79 ** R 2=0.47 n.s. n.s. **R 2=0.41 ** R 2=0.27 ****R 2=0.69 P. axinellae n.s. **R 2=0.43 ** R 2=0.47 **R 2=0.27 * R 2=0.27 * R 2=0.23 ****R 2=0.58 n.s. ***R 2=0.44 **R 2=0.41 ed polyps were counted individually, no colonies as a whole as in the other cnidarians. In this Boulders zone, Alcyonium acaule has no dormancy colonies in March, but more than 6% over the studied period are in such stage in June (see Fig. 29e). In early spring, Paramuricea clavata , Eunicella singularis and Leptogorgia sarmentosa had lower Variation Index (Variance/Mean) than in late spring (Table 13). Corallium rubrum Variation Index is higher in March period, and Alcyonium acaule and Parazoanthus axinellae had very similar Variation Index in both studied cycles. On the other hand, in Table 14 are shown different relationships among the passive suspension feeders studied. The coordination degree among cnidarians is variable, and seems to be a slightly more correlative pattern in early spring (March cycle) than in late spring (June cycle). In the diel cycles studied in June 1997 on the Wall zone, no circadian activity rhythm was found (Fig. 30 and 31). Both cycles show different trends in all six studied species. The 18-19/6/97 all species have expanded colonies from 10:00 until 19:00, when expansion decrease (except in Leptogorgia sarmentosa , which dwell the Boulders zone, Fig. 30c). The 20-21/6/97 cycle is more irregular, although synergy responses to environmental factors are evident (e.g. 13:00 of the 20/6, Fig. 31; exception again Leptogorgia sarmentosa in the Boulders zone, Fig. 31c). All the six studied organisms have polyp contraction (including Leptogorgia ) the 21/6 at 4:00. The 18-19/6/97, Alcyonium acaule has 8% of the colonies in dormant stage, while there is a slight increase the 20-21/6/97 with 11% of the colonies in dormant stage.

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Figure 30. Rhythms of activity of five anthozoans during the first 24h diel cycle in the wall zone (18- 19/6/97, except Leptogorgia sarmentosa , not present in this zone). Mean % expanded colonies (or polyps in the Parazoanthus axinellae case) ± Standard Deviation.

ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS 95 a b

c d

e f

Figure 31. Rhythms of activity of five anthozoans during the second 24h diel cycle in the wall zone (20- 21/6/97, except Leptogorgia sarmentosa , not present in this zone). Mean % expanded colonies (or polyps in the Parazoanthus axinellae case) ± Standard Deviation.

96 ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS a b

c d

e Figure 32. Rhythms of activity of five anthozoans during the late spring period in the wall zone concurrently to the boulder patch observations (see Fig. 29) (24-29/6/97, except Leptogorgia sarmentosa , not present in this zone). Mean % expanded colonies (or polyps in the Parazoanthus axinellae case) ± Standard Deviation.

Spatial variability . All the species studied in the Wall zone (i.e. Paramuricea clavata , Eunicella singularis , Corallium rubrum , Alcyonium acaule and Parazoanthus axinellae ) show more contracted polyps than the concurrently studied patches in the Boulders zone (Fig. 32). The flow related pattern shown in the Boulders zone is non-existent in the Wall

ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS 97 zone in Paramuricea clavata . P. clavata and E. singularis coincide with the others species at the beginning of the cycle with a maximum colony expansion. After this brief period (from 9:00 24/6/97 to 3:00 25/6/97), only P. clavata , E. singularis and A. acaule concurrently contract their polyps, while C. rubrum and P. axinellae variably expand and contract polyps. At the end of the period, all species expand again the polyps. Alcyonium acaule shows maximum dormancy stage colonies, with 14% of the colonies with the thin layer covering the external surface (Fig. 32d). Parazoanthus axinellae shows greater variability than in the boulders zone in the Wall zone (Fig. 32e, with a Variation Index of 78% in the Wall in front of the 55% of the Boulders), and reach 0% polyps expanded, a value never reached in the studied period on the Boulders zone; in the same way, Corallium rubrum has higher variability in the Wall zone than in the Boulders zone (Fig. 32c, with a Variation Index of 80% in the Wall in front of the 30% of the Boulders). There is no significant relationship between species when compared the colony expansion between zones (e.g P. clavata Wall versus P. clavata Boulders). In fact, colony expansion is high at the beginning of the cycle in the Wall, while is minimum in the Boulders (compare Fig. 29 and 32).

Discussion

Is there a seasonal activity rhythms pattern?

As in any other ecosystems, physical factors are the main responsible of community energy fluxes and structure in the Mediterranean sea. In such warm temperate seas, temperature influence water mass dynamics with the introduction of energy and structures (Flos 1985) that affect directly plankton community productivity (Estrada et al. 1985). In summer, such communities are affected by thermocline formation, which constitutes surface low flow rates, nutrient depletion and high temperature levels of the water column. Summer (July-August) sharp impoverishment of the plankton community reverts in part of the benthic community: energetic constraints are reflected in lower organisms activity and production (Boero and Fresi 1985, Sardà et al. 1999, Coma et al. 2000). In the study area, summer is characterised by a decrease in sea storms and associated water motion (Pasqual and Flos 1984), and a decrease in zoo and phytoplankton biomass (Coma et al. 1994, Ribes et al. 1999a, but see Chapter I). Subtidal zones as the coralligen (20-50 m depth) are influenced by physical but also by biological forces that structure the community (i.e. intra and interspecific competition, complex bi-dimensional and three-dimensional morphologies to enhance diversified nutrient or food acquisition, low growth rates) that make more difficult seasonal interpretations, because temporal changes are less detectable at the community level (Ballesteros 1991, Garrabou et al. 1998). Aestivation processes in warm temperate benthic communities have been described as an energetic constraint period for many benthic sessile organisms (Coma et al. 2000). The activity rhythms reported here (Fig. 25) are indicative of such aestivation process. All six analysed species show a tendency to have retracted polyps during the summer. The high variability expressed as large standard deviation bars indicate a complex behaviour, and a particular response to microhabitat conditions (Garrabou 1999). It is well known that polyp contraction reflects (among other things) an energy economy trend

98 ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS (Pearse 1974, Sebens and De Reimer 1977). At least one case of the observed organisms (Paramuricea clavata ) has been deeply studied (Coma et al. 1998). The lower ingesta registered in summer periods (Coma et al. 1994, Ribes et al. 1999b) is reflected in a lower respiration rates (Coma et al. in Press). This could be the reason of a general response among the six studied cnidarians, which show high proportion of the colonies contracted during the July-August period. To reduce respiration rates, low activity (i.e. polyp contraction) could be understood as a community response to energetically unfavourable conditions. In Paramuricea clavata such respiration rates are lower in summer than in winter time, even when the temperature shift is about 10ºC (Coma et al. in press). The other two gorgonians (i.e. Eunicella singularis , Fig. 25b; Leptogorgia sarmentosa , Fig. 25c) may follow a similar behaviour, although the presence of zooxhantellae by E. singularis (Chapman and Theodor 1969), and a different diet and capture rates of L. sarmentosa (Chapter V) may complicate this interpretation. In fact, although a common community response can be suggested in front of an energetic constraint period (Coma et al. 2000), the particular behaviour of each species reflects the diversity and complexity of behaviour responses in complex communities. One extreme is represented by Alcyonium acaule , which present dormant colonies increasing proportion when energetic unfavourable situation is more conspicuous (Fig. 25e). In this and other species of Alcyonacea dormancy is a common feature (e.g. A. digitatum , Hartnoll 1975; A. siderium , Sebens 1983; A. acaule , Garrabou 1999) and has been related to decrease in food availability and the storage of ripe gametes. Dormancy is interpreted as the extreme response to unfavourable feeding input periods, lowering the metabolic demand though the isolation of the organisms from the environmental constraints (Ricklefs 1984). Also Parazoanthus axinellae seems to show clear response to the summer environment. P. axinellae has been decribed as a dynamic species (i.e. expansion-contraction of the polyps, growth and shrinkage of the colonies, Garrabou 1999), and in our study the tendency of an aestivation response exists (Fig. 25f) as previously observed (Garrabou 1999). Polyp expansion seems to be collapsed in summer (specially in July-August), perhaps because of the feeding strategy. Polyps are large compared with the other studied cnidarians, and it can be hypothesised large size prey capture and then extensive periods of expansion to compensate such energy investment (Sebens 1987). But the lack of studies on feeding in P.axinellae prevent any definitive conclusion. Corallium rubrum seems to have the more variable response to seasonal environmental changes (Fig. 25d). This hard coral dwells on crevices in this depth range (15-40 m, True 1970), near to the benthic substrate. Other processes linked to both hydrodynamic and microhabitat variability such resuspension could increase prey availability and therefore condition colony expansion response. Growth and module size in modular animals has been described as a response to environmental energetic constraints (O’Dea and Okamura 1999). Is difficult to see seasonal energetic constraints at community level, because a diverse community as the coralligen gather organisms with very different feeding strategies with different life histories. Rhythms of activity proved to be very useful to interpret such physical and biological fluctuations in a strong seasonal environment with an also diverse community as the Antarctic benthos (Barnes and Clarke 1995). Barnes and Clarke showed a short but significant pause in the activity of several organisms when fast ice covered completely the studied areas, but also stressed the importance of each species strategy, and suggested different feeding adaptations. Caution has to be taken in the extrapolation of our seasonal activity results. In at least

ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS 99 one study of another Mediterranean suspension feeding species ( Ditrupa arietina ), lower activity has been suggested not because food constraints but related to reproductive period and ageing (Jordana et al. 2001). This soft bottom polychaete feeds on microphytoplankton, nanoplankton, and picoplankton in different proportions during the year (Jordana et al. 2000), and energetic needs to maintain the activity in summer may be compensated by the relative constancy nanoplankton and picoplankton food in this season (Ferrier-Pagés and Rassoulzadegan 1994, Lantoine 1995, Chapter I). Another example of full feeding activity in summer comes from one warm temperate active suspension feeder, the ascidian Halocyntia papillosa (Ribes et al. 1998). This is not surprising, as the diet of this tunicate is composed mainly by detritus food (Ribes et al. 1998), and in summer, although more scarce, supplied food as sedimenting particulate organic matter have a very high nutritional value (i.e. rich in lipids, Grémare et al. 1997, Medernach et al. 2001). With these conditions in mind, energy invested in pumping activity in ascidians is low (Riisgard 1988), and again can be compensated by retention of diluted but highly nutritive food. In passive suspension feeders the whole colony open polyp energy costs in summer may exceed the environmental food compensations (Sebens 1987), and the contraction strategy most of the time may be an adequate response. Other communities, as the intertidal, show clear activity rhythms related with physical and associated biological factors (i.e. daily energy flow and low tidal physical stress, Kuipers et al. 1981). There can be assessed clear energetic seasonal trends in such intertidal community together with diel fluctuations (Newell 1979), being the low and high tide fluctuations, and a set of factors related, responsible of such registered activity. In warm temperate subtidal as the studied here, the physical and biological stimulus that bring to the benthic fauna to be active or not have been hypothesised (Coma et al. 1994, Garrabou 1999), but, until now, no direct cause-effect have been observed.

Which environmental factors may affect warm temperate passive suspension-feeders activity rhythms?

Our results suggest a high variable response in the passive suspension feeders studied in front of different environmental stimulus. Paramuricea clavata seems to be the species which activity was more tightly related with flow speed (Fig. 26 and Table 11, 12). Chlorophyll a, particulate proteins and carbohydrates concentrations, and zooplankton density seems to have no direct effect on this gorgonian polyp expansion. In fact, it seems to be a periodic flow pattern in the Boulders zone followed by Paramuricea clavata colony expansion both in March and June observations (Fig. 26, 28a, 29a). In a different way, and not so pronounced, this flow pattern seems to affect also Eunicella singularis , Leptogorgia sarmentosa and Alcyonium acaule colony expansion in both periods (March and June, see Fig. 28b,c,e and Fig. 29b,c,e). In these species, flow speed also correlates positively with colony expansion (Table 11). Flow speed has been described as an essential factor to stimulate polyp expansion (Sebens 1987). The range of currents that stimulate polyp colony expansion in anthozoans is wide and depends on colony morphology and polyp size (Sebens and Johnson 1991, Dai and Lin 1993). For example, an excessive flow speed deforms the polyps and reduce drag, so particle retention is non-efficient (Best 1988, Sponaugle 1991). The diverse colony morphologies and polyp sizes in the passive suspension feeders of our

100 ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS study suggest an optimisation, as a whole community, of all potential flow speed ranges, as stated by Dai and Lin 1993. In the Mediterranean subtidal community studied, all organisms are exposed to regular currents (Riedl 1971), but feeding will be enhanced depending on different diet constraints. Passive organisms will thus respond to the same flow event in different ways. It has been suggested that food alone without current does not cause polyp expansion; current may do so, but current and food together have the greatest effect (Sebens 1987). In our observations, Paramuricea clavata respond to flow speed and no (apparently) other stimulus. Other species respond to flow speed, but seems to have a more wide range of environmental responses (Table 11 and 12). For example, P. clavata response to flow speed appears to be gradual, in relation to current velocity, while other species respond immediately to such flow speed increase, and have the tendency to contract their polyps if no preys are available. The species that seems to be more sensible to such combined changes in flow and particle concentration is Alcyonium acaule (Table 11, 12, Fig. 27b). In March and in June this species respond to particulate protein concentration, although different slopes in the multiple regression analysis have been found; the March slope is more pronounced (0.88) than the June slope (0.19) suggesting a different response to protein increase in the early spring (Table 11 and 12). This slope (related to seston protein concentration) is also observed in Eunicella singularis , Parazoanthus axinellae and Corallium rubrum , and perhaps is a response to a short but intense period of water column productivity (response that may be frequent in such environment, Chapter I). E. singularis , P. axinellae , Leptogorgia sarmentosa and A. acaule have a significant correlation with zooplankton concentration in June, which suggest a combined effect of plankton concentration and flow speed in these species (Table 11). The effect of Chlorophyll a in P. axinellae is opposite in both studied periods, with a surprisingly similar slope (see Table 11 and 12). It has been suggested a negative and positive effect on activity depending on the cell concentration and current speed simultaneously (Leonard 1989). From our observations, it is clear that both flow speed and particle concentration gives maximum functional response (full expanded colonies). Previous works agree with our observations, such Leonard 1989 proofs with the passive suspension feeder Antedon mediterranea ; in this case, a combination of microphytoplankton and current speed enhanced feeding activity. Sebens et al. 1996, suggested that not only flow speed-particle concentration, but also the kind of available prey surrounding the passive predator reflect a minor or major efficiency. Also active suspension feeders have a flow speed- prey concentration optimal range. The bryozoans Electra pilosa and Electra crustulenta are influenced by concentration of algal cells in the surrounding water, needing a minimum phytoplancton concentration and current speed to switch on the lophophore activity (Riisgard and Goldson 1997). Also the bivalves respond to such prey concentration-flow speed patterns, having lower activity when tidal flow and seston concentration reach different combined values (Wong and Cheung 2001) or seston quality vary though the time (Iglesias et al. 1992).

Temporal and spatial heterogeneity: common trends in activity patterns?

The comparison of early spring (late March observations, Fig. 28) and late spring (late June observations, Fig. 29) on the Boulders zone, show less inter colony variation through

ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS 101 a lower Variation Index (i.e. Variance/Mean, Table 13), and slightly more correlation in the expansion behaviour between species (Table VI). This could be related with the consequence of a more random meteorological changes (i.e. winds and wave height, river runoff, etc.) that may revert in more sudden water masses changes, which in turn may have more influence in the seston nutrient and particle renewal near the bottom (Chapter II). We suggest this to be one of the causes of an apparently more variable activity of the six studied cnidarians in the Boulders zone in late spring. Even if the variability is high in both studied periods, when environmental factors favourably coincide (for example, high flow speed, plankton and protein concentrations, 3:00 29/6/97, Fig. 26, 27a and Chapter II; high protein concentration, 3:00 30/3/98, Fig. 27b), the whole community seems to respond to such optimal energetic situation with a activity rhythms synergy (i.e. full expanded colonies). In early spring, there is a clear 12 hours synchrony period of colony expansion with the maximum seston biomass in the environment (Fig. 28), followed by an also sharp decrease of activity when seston becomes more diluted (see Chapter II for all variables analysed in this cycle). All the studied passive suspension feeders seems to profit this energetically favourable condition. This is also true in late spring, although a more brief synergy period. Here is rein- forced the idea of more random energy inputs in late spring, due to a more pulse-like situations in this period which are responded with more brief activity of the organisms. Community synergy in colony expansion suggest a clear energy optimisation in front of environmental changes, due to an adequate response of the suspension feeders. For example, at 15:00 of the 26/6/97, all colonies expand their polyps apparently to flow speed (Fig. 26 and 29), but immediately close again when is not detected enough environmental particle concentration (i.e. zooplankton, see Fig. 27a). In optimal foraging theory, Hughes 1980 shows the need, not only as individual, but as a population, to profit favourable feeding pulses. In a complex community as the coralligen (Ros et al. 1985), there will be a broad spectrum of energetic constraints shown by the diverse activity behaviour registered in our studied periods. The tendency to “switch on” concurrently is also reflected in diel cycles (Fig. 30 and 31). Many authors stated maximum prey capture with maximum peaks of environment plankton (Barangé and Gili 1988, Coma et al. 1994, Gili et al. 1996a, Sebens et al. 1996 among others). Those trends show opportunistic behaviour, and have no diel cycle in Mediterranean hard bottom communities (Barangé and Gili 1988, Coma et al. 1994). In intertidal systems, there is a clear daily relationship of energy invested in benthic suspension feeding activity or capture (which have been suggested to be correlated, Sebens 1987) due to the high and low tidal cycles. The hydrozoans Tubularia larynx and Silicularia rosea have maximum feeding rates in coincidence with maximum prey availability, with a suggested relationship on tidal fluxes (Gili et al. 1996a, Gili et al. 1996b). In another study, Wong and Cheung 2001 showed a significant increase of enzyme activity when spring high tide was compared with neap high tide, even higher when seston concentration and richness was maximum. In general, two main activity trends have been identified in the six species studied. First, a daily spontaneous rhythm with short peaks showing a large number of colonies with expanded polyps. Second, a seasonal phasing of behavioural rhythmicity as consequence of maintain a more general pattern by repeated exposure to more constant environmental factors. In both cases, the periods with a great number of polyps expanded coincides with

102 ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS episodes of major flow speed and particle concentration in bottom water layers. At daily scale, the expanding-shrinking polyps in sessile organisms follow a spontaneous behaviour as consequence of a response to the stochastic physical and biological processes occurring in near littoral ecosystems. Near bottom, currents are extremely variable because are driven by the interaction with the substrate (Hiscock 1983). Hydrodynamic processes near the sea floor also affect biological processes, and mechanisms such as resuspension supply food from different sources (water column, benthos, etc.) at the near bottom water layers (Thomsen 1999). Within this stochastic framework, the expected strategy for benthic sessile organisms is a rapid response to food availability. These trends suggest that the degree of endogen activity of a biological rhythm should be weak in these sessile organisms such has been pointed out by Naylor (1976). These organisms are structurally well adapted and spa- tially well located in continuos flow regime, that withstand a wide range of environmental variables (Rodriguez and Naylor 1972). At the same, the fact that these organisms have non- selective diets (Coma et al. 2001) appear to be the most suitable strategy for understand a spontaneous rhythm, probably with no endogenous control. This conclusion could be also corroborate with experimental design such made with barnacles that not follow the same response in the field that in constant controlled conditions (Sommer 1972). The spontaneous rhythms observed in sessile organisms seemsto be different compared with the major patterns described in mobile ones (e.g. zooplankton and other benthic invertebrates). In general, it is accepted that zooplankton undertake vertical migrations regulated by a combined effects of a set of biological and physical factors (Forward 1988). Zooplankton moves throughout the water column to locate food patches (such is coming up) and reducing metabolic costs (coming down) (Gabriel and Thomas 1988). Both processes, at daily scale, are similar to the observed in mobile invertebrates such intertidal animals but in both cases, in comparison with the sessile species, the activity rhythms are governed for endogenous and exogenous clocks (Mauchline et al. 1998, Palmer 1995). The more random changing environmental conditions in near bottom environments in the water column could explain the differences in the behaviour patterns mentioned above. A coincidence aspect is related with the general assumption that, species that expend low levels of energy in foraging are highly successful ecologically such is postulated in the framework of optimal foraging theories (Stephens and Krebs 1986). For zooplankton, the migration to low water layers, with less temperature, is assumed as and physiological adaptation to reduce metabolic costs (Enright 1977). But, also it is true in sessile organisms in which colonies with contracted polyps reduce the 50 % of the metabolic cost (Sebens 1989). Another coincidence is that, even when zooplankton (specially copepods) are considered to feed in a characteristic day-nigh cycle, usually coupled with vertical migration rhythms, recent observations suggest that these organisms also can develop spontaneous rhythms when optimal food is available (Calbet et al . 1999). Temporal behaviour of marine animals is adaptively related to the seasonal rhythmicity of the environment. For example, for planktonic species it has been suggested that patterns of abundance and distribution, including blooms, are probably in accordance with the presence of patch of prey species (Denman 1994). In this way, species must adapt their life cycles in relation with seasonal food availability and the raise of new recruits must be “planed”. This kind of planning require an endogenous rhythm acquired through an evolutive pathway that ensure the population maintenance in a fluctuating environment. For

ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS 103 example, many benthic species have been foresee planktotrophic larvae releasing coinciding the peaks abundance of food in the water column (Starr 1990). A common pattern by zooplankton and benthic invertebrates is that activity periods are discontinuous, with periods of migration, feeding, reproduction etc., alternating with quiescent or aestivation periods (Naylor 1976). For sessile species, it has been demonstrated that the periods of reproduction (including larval release) are programmed to happen in favourable feeding periods and before of unfavourable environmental periods (Coma et al. 1995). On the other hand, mobile organisms, benthic and pelagic, invest much of their year cycles in migrations or displacements to find the habitat for better feeding or spawning (Sutherland 1996). The cost of this moving activity is the critical point in their seasonal rhythms and during them they increase the feeding to guarantee enough energy for invest in reproduction. This pattern means that a repeated response to environmental variability by means of day rhythms permit to adjust internal clocks for seasonal rhythms. For example, the daily benefit of vertical migration in plankton organisms permit the development of ontogenic migrations and adequate the life cycles to environmental variability (Ohman 1990). For benthic sessile organisms studied, the probability of inputs of suspended food is closely associated with the dynamics (intensity and periodicity) of water flow (Wildish and Kristmanson 1997). At the seasonal scale, the inputs are closely related with their critical biological periods because the major feeding periods preceded the reproduction. Such seasonal rhythms required a foresee and should be regulated by internal or endogenous clock. Our conclusion is even daily activity rhythms are regulated by external environmental factors, seasonal rhythms must be in accordance with endogenous control acquired by the learning from repeated exposure and response to short periods that permit the adjust of internal clocks. These behaviour trends described for sessile organisms are not much different that defined for mobile species and represents an evolutive convergence behaviour way in marine animals.

Conclusions

Our results show a great variability of responses in front of a highly variable environmental constraints. Such variability in the expansion of colonies is not surprising due to the different flow speed ranges in which species live such have been observed in other communities studied (see Dai and Lin 1993). Although the wide variability among species, and among temporal and space comparisons, some tendencies can be suggested: 1) There is an aestivation process, in which colonies or polyps show more contraction due to energetic constraints (i.e. low flow speed and low food availability, which makes expansion costly); 2) Flow speed, zooplancton and protein concentrations may be considered as the environmental factors which, within a wide range of responses linked to species microhabitat, diet or behaviour, show higher correlation with colony expansion; 3) It is suggested a different temporal behaviour of the community in front of different environmental fluctuations in early and late spring, being such environmental features more pulse-like in late than in early spring; 4) There is a synergy of the whole community in front to optimal flow speed and particle concentration combination, where all the cnidarians studied expand their polyps to profit such energy pulse; and 5) Spatial scale seems to be very important when considering

104 ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS activity of patches, and may be very different within few meters distance. Activity rhythms proof to be a useful and easy to observe tool to test for all these combined effects. Activity will be related every moment to favourable or unfavourable environmental conditions, but activity rhythms may help us understand synergy levels in front to environmental energetic fluctuations.

ACTIVITY RHYTHMS IN PASSIVE SUSPENSION FEEDERS 105

Spatial and temporal variability in the diet and prey capture rates of a passive benthic suspension feeder: Leptogorgia sarmentosa , a case study

Abstract

Leptogorgia sarmentosa Esper 1791 (Cnidaria: Octocorallia) is a characteristic passive suspension feeder of the gravel-soft bottom benthos in the Mediterranean sea. Several spatial, temporal and methodological approaches were used to asses natural diet and feeding rates. The natural diet (stomach contents) was studied biweekly over an annual cycle, and complemented with a set of spring experiments using continuous flow incubation chambers. The gorgonian feeds mainly on invertebrate eggs, bivalve larvae, copepod eggs and nauplii (stomach contents), all considered low mobility preys, with sizes comprised between 20-800 µm. On the other hand, in situ clearance rates in the experimental chambers showed nanoflagellates, phytoplankton and ciliates significant captures in the spring experiments, representing 24% of the ingested carbon in these spring observations. Detrital POC represents 76% of the ingested carbon in such flow chamber experiments. This represents a high grazing impact in such seston fraction, and an important part of the energy input of this cnidarian. It is the first time that natural diet and feeding rates of a gravel-soft bottom passive suspension feeder over an annual cycle are presented. Furthermore another two populations of Leptogorgia sarmentosa (one in Banyuls-sur-Mer-sur- Mer, France; the other in the Medes Islands, Spain [ca 50 km apart]) were monitored over a period of 16 months to asses possible differences in the natural diet and feeding rates (stomach contents) at both sites. We observe differences in the diet at both sites, and the prey capture was also significant different between studied areas (0.047 ±0.090SD µgC polyp -1 in Banyuls-sur- Mer-sur-Mer and 0.073 ±0.134SD µgC polyp -1 in the Medes Islands). Such differences may revert in the trophic dynamics of this species at both sites, suggesting a lower energy input in the Banyuls-sur-Mer-sur-Mer population compared with the Medes Islands one. All these measures were complemented with the activity rhythms (polyp expansion-contraction) and natural digestion observations (at 13ºC and 21ºC) to make a more realistic approach of the energy input in this gravel-soft bottom dwelling passive suspension feeder. Differences were found when such approaches are considered: we estimated 14371 ±11893 SD prey m -2 day -1 when such approaches are not applied, and 27190 ±23201 SD prey m -2 day -1 when applied. It is concluded that accurate spatial, temporal and methodological approaches are essential to asses realistic bentho-pelagic trophic relationships in passive suspension feeders.

Introduction

Passive suspension feeders play an important role in the plankton-benthos coupling processes. Their importance has been stressed because the abundance in the benthos and the high capture rates (Gili et al. 1998), and increasing evidence of its role as mero and holo- plankton regulators in shallow coastal areas has been recently invoked (Gili and Coma 1998, Gili et al. 2001). It has been accepted that active filter feeders (e.g. bivalves, tunicates) may have a deep impact in the water column plankton communities (Riisgard and Larsen 2000), reducing significantly the plankton production in shallow areas (Cloern 1982, Officer et al. 1982). The lack of more knowledge about passive suspension feeders diet and feeding rates could be related to a great research effort more focused in the active suspension feeder group. In the last two decades, a wide spectra of natural preys and feeding rates of passive suspension feeders is presented on literature (Coffroth 1984, Sebens and Koehl 1984, Zamer 1986, Lewis 1992, Coma et al. 1994, Coma et al. 1995, Fabricius et al. 1995, Sebens et al. 1996, Ribes et al. 1998a, Ribes et al. 1999a, Orejas et al. 2001, among others). Two main conclusions arose from these studies: 1) Almost all benthic passive suspension feeders have

DIET AND PREY CAPTURE RATES OF L. SARMENTOSA 109 mixed diets, and 2) Capture rates are coupled with potential prey availability (Coma et al. 2001). Field studies on diet and prey captures supply essential information which helps to understand their ecological role in marine ecosystems (Coma et al. 1998a). Although the possibility to complement accurate methodologies to estimate annual feeding rates in passive suspension feeders (and the correspondent Carbon input), several ques- tions remain unanswered. For example, up to now the studies have been made in a single local population on a certain geographic area. Differences in habitat features and seston quality may be crucial to understand differences in population feeding performance and demographic parameters (abundance, fitness, etc) (Gardner 2000). Some studies on suspension feeders stress the importance of space comparison (through horizontal or depth gradi- ent considerations) to understand demographic, metabolic or behavioural population differences (e.g. Bayne and Widdows 1978, Walsh and Somero 1981, Sebens 1984, Harland et al. 1992). The importance of space in these studies has been stressed, and other observations in passive suspension feeders demonstrated that the diet show high temporal variability (annual-diel cycles) (Coma et al. 1994, Coma et al. 1995, Ribes et al. 1999a), and temporal and spatial approaches have never been considered concurrently in these organisms. For accurate energy input assessment, different approaches are required: 1) Digestion time is important to calculate capture rates and food intakes. Laboratory digestion time approaches for benthic passive suspension feeders have been made (e.g. Paffenhöfer 1968, Sebens and Kohel 1984), but field approaches are very scarce (Coffroth 1984, Coma et al. 1994, Coma et al. 1995, Gili et al. 1996). Despite its importance, to our knowledge, no one study has been made with natural diet at different temperatures. 2) It is well known that polyps are able to capture preys when are expanded. Periods of polyp contraction are common in benthic colonies or individuals, because contraction of polyps (not feeding) reduce energy costs when preys are not available (Sebens 1987). Expanded polyps is related to external signals (currents and prey concentration, Patterson 1991, Dai and Lin 1993, Sebens et al. 1996) that stimulate the colony to capture, and it seems reasonable to think that expanded polyps will have more probabilities to be full. Very few attempts have been made to calculate the energy input considering the activity rhythms (expanded-contracted polyps) at the level of population of passive suspension feeders (Sebens and Kohel 1984, Coma et al. 1994, Ribes et al. 1999a). This factor is essential to have a realistic approach of population level capture calculations, as the activity rhythms may significant differ depending upon the considered year season (Coma et al. 1994). To our knowledge, never has been quantified this behaviour concurrently to feeding sampling. 3) Testing seasonal trends, annual sampling has been suggested to be done more than the habitual monthly monitoring (at least biweekly, Taylor and Howes 1994, Nogueira et al. 2000). Annual feeding cycles of passive suspension feeders lack this approach, that may be important when seasonal comparisons have to be made. 4) Because the importance of mixed diets in passive suspension feeder energy budgets, it is a flaw to consider ingesta without calculations on all the potential food, including the pico, nano and detrital seston (Ribes et al. 1988, 1999a, Orejas et al. 2001). These are some examples that can be considered important to have a realistic approach to the energy input in passive suspension feeders. However, an annual study that involves simultaneously all these approaches needs a significant experimental effort. The objectives of this study deals with 1) make an annual estimation of the diet and prey capture rates of a passive suspension feeder of a soft bottom-gravel zone (the information

110 DIET AND PREY CAPTURE RATES OF L. SARMENTOSA about these organisms is very scarce), and 2) Compare diet and feeding rates of two natural populations in different zones over an annual cycle (zones potentially different in its environmental features). To reach these objectives, three populations of the detritic ( sensu Gili and Ros 1985) and soft bottom passive suspension feeder Leptogorgia sarmentosa in two different places (Medes Islands, North Catalonia and Banyuls-sur-Mer-sur-mer, South France) were monitored. The high heterogeneity in the feeding processes of benthic organisms let extrapolations from other studies risky, because the prey capture rates of a detritic- soft bottom passive suspension feeder species may not follow patterns previously observed in hard bottom similar species.

Material and methods

Natural diet and prey capture: A total of 32 colonies settled in small stones 20m depth in the Medes Islands (Figure 33b) were gathered in lines to carry on the sampling (final density, 2.3 colonies m -2 ). To study annual variations in the diet, Leptogorgia sarmentosa colonies were sampled biweekly from 28/7/97 to 27/8/98. Each sample consisted of 1 apical fragment collected from 5 randomly selected colonies of the population and immediately fixed in 10% formaldehyde in sea water. Polyps were always expanded in the fragments selected. To study the activity rhythms of the gorgonian, the expansion state of the polyps of 82 colonies was registered concurrently to the sampling ( sensu Coma et al. 1994). Ten polyps of each fragment were dissected under a binocular microscope and the stomach contents examined as described above. To describe possible relationships with zooplankton concentration, data of Chapter I are considered (a seston cycle collected in the same zone and on the same moment as the Leptogorgia sarmentosa branches). To estimate prey digestion time, two experiments were carried out at two different temperatures (26/2/00, 13ºC; 13/8/99, 21ºC). 70-80 branches of the 22 colonies were collected (7:30-8:00 a.m. in the two experiments), and 5 fragments immediately fixed in 10% formaldehyde (time 0h). Fragments were kept in 0.4 µm filtered sea water at constant temperature in the laboratory, and each hour 5 of these fragments were fixed in the same way (8 hours for the 21ºC experiments, 11h for the 13ºC experiment). The stomach contents of 10 polyps on each fragment were examined. To estimate biometric parameters as number of polyps mm -1 , OM% , and mg AFDW mm -1 , three colonies of Leptogorgia sarmentosa were sampled (29/7/99), measured (all >40 cm tall), rinsed with distilled water to remove salts and associated macrofauna, the number of branches counted, and cut in pieces following branch order (Coma et al. 1994). Polyp density and diameter were measured in 2 cm pieces in branch type 1, 2, 3, 4, and 5. The same fragments were peeled, dried (48h at 60ºC), weighed and burned (5h, 500ºC) to know the DW and AFDW of axis and of the tissue without axis. To make final calculations of polyps colony -1 and mgAFDW colony -1 for colonies 20-30 cm tall, three colonies of 24, 29 and 32 cm height with similar height/width ratio (1.2) where photographed and the slides treated as Coma et al. 1998a to have the exact sum of branch types length. Five transects of 100 m length *4m width were made counting and measuring the colonies (height and width) to have an estimate of the natural population density in the

DIET AND PREY CAPTURE RATES OF L. SARMENTOSA 111 a

3º 13' E b 0 0.5 1 km

L'ESTARTIT

MEDES ISLANDS

42º 02' N

Ter River

Sea ean Figure 33. Study areas. Banyuls-sur-Mer Mediterran (a), 10-15m depth (Black Point); Medes Islands (b), 30m depth (White Point) and 20m depth (Triangle).

112 DIET AND PREY CAPTURE RATES OF L. SARMENTOSA Medes Islands (30m depth). The height and width of gorgonians was measured with a ruler (precision ±1mm). After density measures, stones where gorgonians have settled were gathered in straight lines in the same zone to make easier the sampling (see below) (density of gorgonians gathered, 1.55 colonies m -2 ). Flow chamber experiments. To evaluate if the gorgonian actively feeds on the fine fraction ( sensu Ribes 1999b), six feeding experiments were conducted is situ using continuous flow incubation chambers (Ribes et al. 2000) at the Cova del Duï (coastal zone in front of the Medes Islands) from the 14 May to the 28 June 1999. A control of the expanded polyps of the colonies was made each 15 minutes. Protocols of the different treatments of each fraction studied (Heterotrophic Bacteria, Synecococcus sp. , nanoflagellates, dinoflagellates, diatoms, ciliates, DOC and POC) are available in Ribes et al. 1999a (and references there- in). After the experiments, the polyps of each colony were counted, and the gorgonians were rinsed to remove salts and associate macrofauna, dried (48 h at 80ºC) and burned (5h at 500ºC) to assess dry weight and ash free dry weight. Diet and prey capture with mesoscale spatial differences. To evaluate natural diet and prey capture over an annual cycle in two Leptogorgia sarmentosa populations, 20 colonies in the Medes Islands (30m depth, 42º02’55’’N, 3º13’30’’E, Spain, Figure 33a) and 20 colonies in Banyuls-sur-Mer-sur-mer (10 m depth, 42º29’30’’N, 3º08’70’’E, France, Figure 33b) were monitored monthly (from May 1998 to August 1999). One apical fragment of each colony was cut and fixed in 10% formaldehyde. Five polyps of each fragment were dissected under a binocular microscope and the stomach contents examined (minimum sample size as in Barangé and Gili 1988, Coma et al. 1994). Prey items were identified to the level of the major taxonomic groups (see table 15 and 16). The length or maximum diameter of all prey items were measured and prey biomass was calculated following Coma et al. 1994. Sixteen squares 1m x 1m (9-13 m depth) were made randomly in the 10m depth study area (Banyuls-sur-Mer-sur-Mer) to calculate the density of colonies of Leptogorgia sarmentosa in this area. Height and width of the gorgonians was measured with a ruler (precision ±1mm). Prey and Carbon calculations. To evaluate the prey capture polyp -1 day -1 , the following formula has to be applied (Coma et al. 1994):

−  D  1 = −  t  C N ∑1   t =0 D  where N = prey items polyp -1 , D = digestion time (h), t = time (h). For the biomass, the formula is (Coma et al. 1994) −  D  1 =∑ −  t  Cb B  1   t =0 D  -1 -1 where C b = Biomass captured polyp , and B = Total biomass of prey items polyp . Statistics. The standard methodology (Pearson´s product-moment correlation) was followed to relate environmental zooplankton (Chapter I), prey polyp -1 , and full polyps. To test seasonality and differences between populations (Banyuls-sur-Mer/Medes) or methodological approaches (Medes 20 m and Medes 30 m), ANOVA test with Scheffé post hoc test were

DIET AND PREY CAPTURE RATES OF L. SARMENTOSA 113 Table 15. Leptogorgia sarmentosa . Number and type of prey items captured over the annual sampling period (1997- 1998) in the Medes Islands (20 m depth), and total number of stomach contents observed in 1250 polyps. Abbreviation: POM, Particulate Organic Matter.

28/07 11/08 29/08 08/09 23/09 06/10 12/11 26/11 12/12 31/12 14/01 30/01 27/02 11:00 14:00 12:30 12:30 11:30 9:30 9:30 11:00 11:30 17:30 15:45 11:00 11:00

Invertebrate eggs 3 3 7 10 4 2 6 6 5 10 6 9 7 Bivalve larvae 1 0 10 11 1 0 2 5 3 31 9 9 13 POM 2 7 1 6 2 5 1 6 6 6 4 0 1 Phytoplankton 10 1 1 4 8 2 0 3 9 9 3 3 1 Copepod eggs 0 0 1 1 0 1 1 4 1 1 5 5 0 Nauplii 5 1 0 0 0 0 3 2 0 3 0 0 6 Crustacean fragm. 3 1 4 1 1 2 0 2 1 5 1 0 2 Invertebrate larvae 4 1 3 0 1 1 1 5 0 1 0 1 1 Protozoa 0 0 1 0 1 2 3 0 2 3 0 0 1 Others 1 1 1 0 0 0 0 1 5 10 1 0 0 Copepods 0 0 1 2 0 0 0 1 0 2 1 0 0 Unidentified 0 0 5 1 1 0 0 2 0 2 1 0 1

Total prey 29 15 35 36 19 15 17 37 32 83 31 27 33

Full polyp number 20 9 24 20 15 14 13 27 19 43 19 17 25 % 40% 18% 48% 40% 30% 28% 26% 54% 38% 86% 38% 34% 50% Nºprey/Polyp Mean 0,64 0,3 0,72 0,72 0,38 0,32 0,34 0,74 0,66 1,74 0,62 0,58 0,7 SD 1,1 0,93 0,93 1,18 0,67 0,55 0,63 0,83 1,08 1,41 0,9 0,95 0,81 Nº Polyps observed 50 50 50 50 50 50 50 50 50 50 50 50 50

26/03 09/04 23/04 29/04 15/05 07/06 13/06 28/06 15/07 31/07 19/08 27/08 N % 12:00 10:30 18:00 13:00 12:30 12:00 7:45 12:00 10:30 9:15 13:00 12:00

Invertebrate eggs 8 2 6 1 5 37 9 3 5 1 1 4 160 19 Bivalve larvae 12 7 4 1 0 13 15 1 1 0 0 3 152 18 POM 7 6 2 7 6 2 10 3 3 2 1 8 104 12 Phytoplankton 10 12 3 1 7 1 3 2 4 0 2 1 100 12 Copepod eggs 1 5 6 42 4 1 1 0 0 1 0 0 81 10 Nauplii 1 7 3 14 4 0 3 0 0 1 0 5 58 7 Crustacean fragm. 9 2 2 1 0 0 2 1 2 1 2 2 47 6 Invertebrate larvae 1 2 0 1 1 3 0 0 1 0 0 5 33 4 Protozoa 3 6 1 0 0 0 1 1 1 0 0 4 30 4 Others 0 1 1 1 2 2 1 0 0 0 0 1 29 3 Copepods 0 3 3 3 0 0 1 0 1 0 1 3 22 3 Unidentified 0 0 0 0 0 1 0 0 1 0 0 4 19 2

Total prey 52 53 31 72 29 60 46 11 19 6 7 40 835

Full polyp number 26 27 22 22 20 30 26 10 16 6 7 29 % 52% 54% 44% 44% 40% 60% 52% 20% 32% 12% 14% 58% Nºprey/Polyp Mean 1 1,02 0,6 1,4 0,58 1,2 0,92 0,22 0,4 0,12 0,16 0,8 0,7 SD 1,21 1,22 0,81 4,82 0,86 1,48 1,32 0,46 0,64 0,33 0,42 0,86 0,4 Nº Polyps observed 50 50 50 50 50 50 50 50 50 50 50 50

114 DIET AND PREY CAPTURE RATES OF L. SARMENTOSA employed. When made the ANOVA test for the Banyuls-sur-Mer (10m depth) and Medes Islands (30 m depth) populations, because the colonies were tagged (and so were always the same colonies), repeated measures were considered inside the test.

Results

Natural diet and prey capture: The mean size of preys in the 20 m depth colonies is 140 ±106 µm (20-800 µm range), Figure 34. Leptogorgia sarmentosa Medes Islands and the mean of polyps with prey is 40%. population rhythms of activity. Seasonal mean±SE. The more abundant preys are the invertebrate eggs and bivalve larvae with 19 and 18% abundance in gut contents respective- a ly, followed by POM (12%), phytoplankton (12%) and copepod eggs (10%) (Table 15). Copepods (mobile prey) are low when compared with other kind of preys (3%). Activity rhythms of Leptogorgia sarmentosa are shown in Figure 34. There is a tendency in which winter (January-March) and spring (April-June) times the gorgonian polyps seems to be more regularly expanded (expanded colonies mean above 50%), while in summer (July-September) and fall (October-December) times the polyps seems to be more contracted b (expanded colonies mean below 50%). From digestion results, two regression lines were calculated:

Temperature 13ºC Prey polyp -1 = 1.8 – 0.14 Hours R2=0.88,p<0.00001; N=12

Temperature 21ºC Prey polyp -1 = 2.1 – 0.29 Hours R2=0.95, p<0.00001; N=9 Figure 35. (a) Number of prey captured (stomach The slope of the 21ºC experiment was contents, 24 hour integration) per polyp, with digestion and rhythms of activity estimations. (b) twice the slope of the 13ºC one. With these Carbon input per polyp, values of preys calculated results the time of total extinction of preys from Coma et al. 1994 plankton data.

DIET AND PREY CAPTURE RATES OF L. SARMENTOSA 115 Table 16. Biometric parameters of Leptogorgia sarmentosa (following the methodology of Coma 1994). N= number of observed branches (2 cm length).

Polyps mm -1 ∅ (mm) OM % mgAFDW mm -1 OM % mgAFDW mm -1 BRANCH TYPE (Axis) (Axis) (Tissue) (Tissue)

1 (N=36) 2.2 ±0.21 0.84 ±0.16 29.6 ±18.5 0.15 ±0.11 27 ±8.7 0.13 ±0.06 2 (N=36) 2.2 ±0.5 1.07 ±0.39 30.8 ±5.7 0.2 ±0.09 29 ±3.8 0.22 ±0.08 3 (N=36) 2.7 ±0.34 1.57 ±0.36 31.1 ±4 0.6 ±0.24 31.6 ±6.1 0.77 ±0.52 4 (N=25) 3.3 ±0.8 2.5 ±0.59 37.1 ±9.9 1.96 ±0.77 39.4 ±1.9 3.36 ±0.67 5 (N=10) 3.7 ±0.84 3.8 ±0.76 44.4 ±8.2 5.1 ±1.7 41.8 ±1.7 4.25 ±0.78 in the polyp can be calculated: 13h for the 13ºC experiment and 7 h for the 21ºC. Following Paffenhöfer 1968, we extrapolate 10 h of complete digestion for a 17ºC temperature situation. These times of digestion will be implemented in our capture rates formulas. Bivalve larvae were very abundant at the beginning of the 21ºC experiment (51 in 50 polyps), and all were closed (and full). This let us estimate the digestion time of this prey, that seems to be important in the diet of this passive suspension feeder (see Table 15): 6 hours after the point 0h, only 56% of the bivalve larvae counted (9/16 in 50 polyps) were closed (and full), and 7 hours from the beginning of the experiment only 40% (2/5 in 50 polyps) were closed (and full). There are no clear seasonal trends in the gut contents nor in the carbon input analysed through the annual cycle at 20m depth every 15 days (Fig. 35a,b). When considered digestion times, there are only differences between spring 1998 and Autumn 1997 & Summer -1 1998, but no with Winter 1998 & Summer 1998 in prey polyp 24 h (F 4,120 = 4.00, p<0.01, -1 Fig. 35a). No differences have been found when µgC polyp 24 h are considered (F 4,120 = 2.82, p<0.05, Fig. 35b). In the overall cycle, the estimation of polyp mean capture prey is 0.67 ±0.39 SD prey polyp -1 . This is means a mean ingestion of 0.101 ±0.099 SD µg polyp -1 . Relationship with zooplankton abundance in the water column could be tested over this annual cycle (20m depth area), as the plankton was fished concurrently (same day, same moment) with the branch sampling (see Chapter I). The relationship of the total concentration of plankters-concentration of preys in 50 polyps is non-existent trough the annual cycle (R 2=0,06, p<0,282, N=22). Pulses of some passive preys become relevant in the overall number of preys polyp -1 (e.g. bivalve larvae of 31/12/97, 27/2/98, 7/6/98 or 13/6/98, Table 15). This pulses sometimes are in accordance with peaks of abundance in the water column of this preys (see Table 2a,b in Chapter I) (e.g. the relationship bivalve larvae concentration in the water column-bivalve larvae abundance in 50 dissected polyps relationship is weak although significant, R 2=0,37, p<0,01, N=22). The biometrics are shown in Table 16. There is a polyp mm -1 density increase from the first to the fifth branch type (almost double), concurrently to an increase of the branch diameter (4.5 times wider the fifth branches than in the first type ones). The mg AFDW mm -1 show a wide difference from the first to the fifth branch type: there is 34 more biomass in the axis per mm in the fifth branches than in the first ones. Natural density of L. sarmentosa in the Medes Islands (30m depth) was 2.4 ±1.4 colonies 100 m -2 , with 24 ±14 SD cm height. Flow chamber experiments. Feeding on the fine fraction by Leptogorgia sarmentosa is significant (Fig. 36, sensu Ribes et al. 1999a). No significant decrease in prey items smaller than

116 DIET AND PREY CAPTURE RATES OF L. SARMENTOSA 18000 Detritus (incubations)

16000 Gut content

14000 Live C (incubations) -1 d

-1 12000

10000

8000

µg C g AFDW µg C g 6000

4000

2000

0 Jan Feb Mar Abr May Jun Jul Aug Sep Oct Nov Dec

Figure 36. Net growth rates of prey (mean±SE) in Figure 37. Predatory impact estimations of the whole the gorgonian chamber (shadow bars) and in the set of seston fractions over an annual cycle. Data control chamber (empty bars) for each plankton from detritus (incubations) and live carbon (incuba- group. Mean maximum lenght (µm) of each group tions) from Ribes et al. 1999a annual cycle. (mean±SE) is given at the bottom of the Figure. Het B = Heterotrophic Bacteria; Syn = Synecococcus sp . ; Pic = Autotrophic Picoeucaryotes; Nan = nanoeukaryotes (3.8 ±1.4 SD µm) was observed. Autotrophic Nanoeucaryotes; Rhiz = Rhizosolenia From the clearance rates calculated (Fig. 36), sp.; Cha = Chaetoceros sp.; Din = Autotrophic this gorgonian capture 263 ±111 SD nanoflagel- Dinoflagellates; Cil = Ciliates; POC = Particulate lates polyp h -1 , 9 ±3 SD phytoplankton cells Organic Carbon (Detritus). polyp h -1 (diatoms and autotrophic dynoflagel- lates) and 0.3 ±0.1 SD ciliates polyp h -1 . It also feeds significantly on the detritic POM (0.07 ±0.04 µgC polyp -1 day -1 , Fig. 36). When considered the seston composition in the near bottom water layer over an annual cycle (phytoplancton, ciliates, heterotrophyc nanoflagellates, detritus, from Ribes et al. 1999a), 53 ±21% of the ingested carbon of this fraction is represented by detritic POM, 8±5% by phytoplankton, ciliates, and nanoflagellates and 39 ±21 by the gut contents (Fig. 37). There is a tendency in which winter and spring period the carbon input seems to be more regular than summer and autumn period. Energy input and predatory impact. Final calculations on a 2.3 colonies m -2 density population (considering only open polyp) of 20-30 cm height gorgonians (11808 polyps colony, but only 9775 efficient capturing polyps, Coma et al. 1994) lead 65580 ±30320 SD prey m-2 day -1 (considering 3 digestion temperatures) or 9.79 ±5.18 SD mgC prey m -2 day -1 . If activity rhythms are also considered, those calculations fall to 32215 ±21511 SD prey m -2 day -1 or 4.74 ±3.47 SD mgC prey m -2 day -1 . The zooplankton diet represents 128 ±87 SD µgC g AFDW -1 h-1 , or 3082 ±1973 SD µg g AFDW -1 d-1 . Fine fraction represents 260 ±198 SD µgC g AFDW -1 h-1 , or 3548 ±2908 SD µg g AFDW -1 d-1 . Spatial diet and prey capture differences. The density in Banyuls-sur-Mer observed was 2.7 ±1.9 colonies m -2 , with colonies 23 ±14 SD cm height. The density of gathered colonies at 30m depth (1.55 colonies m -2 ) was similar to the natural density of Banyuls-sur-Mer. The mean dimension of the preys was 148 ±115 SD µm in Banyuls-sur-Mer (20-1400 µm range) and 139 ±96 SD µm in Medes (20-800 µm range). There are differences in the diet between the Medes Islands (30 m depth) and Banyuls-sur-Mer (10 m depth) (Table 17 and 18). In both places bivalve larvae represent an important part of the diet (14% in Banyuls- sur-Mer and 15% in Medes). In general, low motility preys are predominant in both annual

DIET AND PREY CAPTURE RATES OF L. SARMENTOSA 117 Table 17. Leptogorgia sarmentosa . Number and type of prey items captured over the annual sampling period (1998-1999) in the Medes Islands (30 m depth), and total number of stomach contents observed in 1665 polyps. Abbreviation: POM, Particulate Organic Matter.

may-10 jun-13 jul-15 aug-19 sep-29 oct-28 30-nov dec-30 feb-02 feb-27

Nauplii 13 5 4 1 4 9 7 9 21 8 Copepod eggs 20 8 20 3 2 1 3 2 37 7 Bivalve larvae 4 2 1 3 1 3 7 6 74 4 Invertebrate eggs 0 0 2 1 9 2 2 2 42 3 Phytoplankton 5 14 5 0 15 6 7 9 4 0 Protozoa 1 6 9 2 5 1 3 2 9 3 POM 0 5 6 4 8 3 1 0 3 3 Copepods 4 1 0 0 2 3 8 8 4 2 Crustacean fragments 2 2 3 2 3 2 5 7 1 0 Invertebrate larvae 2 0 0 1 0 0 1 0 1 3 Unidentified 0 1 4 1 1 1 3 1 0 0 Others 0 1 0 0 3 0 0 0 0 0

Total prey 51 45 54 18 53 31 47 46 196 33

Full polyp number 26 34 35 15 31 24 31 36 78 23 % 26% 32% 32% 14% 30% 22% 29% 34% 74% 22% Nº prey/Polyp Mean 0,51 0,44 0,49 0,17 0,51 0,29 0,45 0,43 1,77 0,31 SD 1,96 0,77 0,87 0,45 1,09 0,61 0,85 0,69 2,82 0,7 Nº Polyps Observed 100 105 110 110 105 110 105 105 105 105

mar-30 apr-30 may-27 jun-26 jul-31 aug-31 N %

Nauplii 99 9 14 8 0 1 212 24 Copepod eggs 8 22 11 18 2 3 167 19 Bivalve larvae 15 0 12 0 5 0 137 15 Invertebrate eggs 22 14 10 2 0 0 111 12 Phytoplankton 1 0 3 3 0 2 74 8 Protozoa 1 3 2 2 0 0 49 5 POM 0 0 0 4 3 2 42 5 Copepods 6 0 2 0 0 0 40 4 Crustacean fragments 3 0 2 0 0 0 32 4 Invertebrate larvae 2 0 2 1 1 0 14 2 Unidentified 0 0 1 0 0 0 13 1 Others 1 0 0 0 1 0 6 1

Total prey 158 48 59 38 12 8 897

Full polyp number 70 29 44 28 9 4 % 67% 29% 44% 28% 9% 4% Mean=31% Nº prey/Polyp Mean 1,48 0,47 0,59 0,38 0,12 0,08 Mean=0,53 SD 1,6 0,86 0,8 0,73 0,41 0,42 SD=0,45

Nº Polyps Observed 105 100 100 100 100 100

118 DIET AND PREY CAPTURE RATES OF L. SARMENTOSA Table 18. Leptogorgia sarmentosa . Number and type of prey items captured over the annual sampling period (1998-1999) in the Banyuls-sur-Mer (10 m depth), and total number of stomach contents observed in 2025 polyps. Abbreviation: POM, Particulate Organic Matter.

may-14 jun-17 jul-17 aug-12 sep-29 oct-23 nov-25 dec-15 jan-22 feb-16 mar-26

Copepod eggs 69 18 0 7 4 0 1 2 4 4 19 Bivalve larvae 3 0 1 0 7 3 2 1 10 7 5 Phytoplankton 4 10 8 13 14 7 7 10 3 0 2 Nauplii 9 6 2 5 0 3 3 3 1 0 6 POM 4 5 5 6 10 8 10 6 0 0 0 Invertebrate larvae 3 2 0 0 4 2 6 0 1 2 5 Invertebrate eggs 8 2 1 2 3 2 8 1 0 1 0 Protozoa 7 4 1 6 2 2 2 0 1 0 0 Unidentified 1 2 1 1 1 2 10 1 0 0 0 Crustacean fragments 0 2 1 1 0 2 2 3 0 0 1 Copepods 1 0 0 2 0 0 1 1 0 1 2 Others 0 0 0 0 0 0 0 0 2 1 1

Total prey 109 51 20 43 45 31 52 28 22 16 41

Full polyp number 42 32 10 30 32 30 36 21 18 14 34 % 38% 29% 9% 21% 29% 27% 33% 19% 16% 13% 31% Nº prey/Polyp Mean 1 0,45 0,16 0,29 0,41 0,31 0,48 0,25 0,2 0,14 0,37 SD 2,88 1,02 0,6 0,73 0,72 0,55 1,04 0,57 0,5 0,42 0,6

Nº Polyps Observed 110 110 110 145 110 110 110 110 110 110 110

apr-26 may-2 jun-30 jul-15 jul-29 aug-19 aug-30 sep-9 N %

Copepod eggs 25 0 0 0 0 0 4 0 157 25 Bivalve larvae 2 0 0 0 2 2 33 7 85 14 Phytoplankton 0 0 2 1 3 1 4 0 89 14 Nauplii 5 3 3 11 6 0 1 0 67 11 POM 0 0 0 3 4 2 0 0 63 10 Invertebrate larvae 9 5 4 0 4 0 1 1 49 8 Invertebrate eggs 2 0 0 0 0 0 0 0 30 5 Protozoa 0 1 0 0 2 0 0 0 28 4 Unidentified 1 0 0 0 0 0 0 0 20 3 Crustacean fragments 1 0 2 2 0 0 3 0 20 3 Copepods 0 0 1 1 2 0 0 0 12 2 Others 1 0 0 0 1 1 0 0 7 1

Total prey 46 9 12 18 24 6 46 8 627

Full polyp number 35 7 9 16 17 6 32 8 % 32% 7% 9% 17% 18% 6% 34% 8% Mean=21% Nº prey/Polyp Mean 0,39 0,1 0,12 0,21 0,25 0,06 0,46 0,08 Mean=0,3 SD 0,64 0,36 0,44 0,52 0,65 0,24 0,74 0,28 SD=0,22 Nº Polyps Observed 110 95 100 95 95 95 95 95

DIET AND PREY CAPTURE RATES OF L. SARMENTOSA 119 a b

Figure 38. (a) Prey per polyp seasonal average (mean±SE) in Banyuls-sur-Mer (10 m depth) and the Medes Islands (30m depth) populations. (b) Carbon estimated average input per polyp (mean±SE, from Coma et al. 1994 plankton data) in Banyuls-sur-Mer (10m depth) and the Medes Islands (30m depth) populations. cycles. In Banyuls-sur-Mer POM and phytoplankton is almost double than in Medes Islands, and zooplankton origin preys seems to be more abundant in the Medes Islands than in Banyuls-sur-Mer (Tables 17 and 18). No significant differences were found in Banyuls-sur-Mer stations through the seasons -1 in the prey polyp (Spring 1998 different from all other seasons, F 5, 125 = 7.84, p<0.0001) -1 nor in the µgC polyp (only Spring 1999 is different from Summer 1998, F 5,125 = 3.81,p< 0.005) estimations (Fig. 38a,b). In the Medes Islands station (30m depth), there was a seasonal tendency in the number of preys per polyp (Winter 1999 and Spring 1999 different from all other seasons, F 5,121 = 9.96, p<0.0001), and when preys are transformed in carbon this tendency is still present (F 5,121 = 5.84, p<0.0001). There were significant differences between both places in Winter (F 1,41 = 46.64, p<0.0001), Spring (F 1,41 = 19.77, p<0.0001) and Summer 1999 (F 1,39 = 6.42, p<0.002), but no between the other seasons sampled when considered the number of preys polyp -1 (Fig. 38a). On the other hand, There were differences between places in Summer 1998 (F 1,42 = 2.54, p<0.05), Winter (F 1,41 = 16.18, p<0.0002) and Spring 1999 (F 1,41 = 2.71, p<0.01), but no between the other seasons sampled when considered the number of µgC polyp -1 (Fig. 38b). Comparison of Prey and Carbon input in the three areas studied (10-20-30 m depth). With the digestion experiments, the biometric measures, and the density approaches, we calculated the feeding rates of Leptogorgia sarmentosa in the three areas considered (Banyuls- sur-Mer, 10 m depth; Medes Islands, 20m depth; Medes Islands, 30 m depth). In Table 19, results of the capture rate are shown (number of preys m -2 day -1 and mgC m -2 day -1 ). For simplicity in the estimates, the same density (2.3 colonies m -2 of 20-30cm height) is considered in the calculations. There are always differences in the capture rates when 10 and 30m areas are compared (compare raws “a”-“b”, “c”-“d”, “e”-“f” and “g”-“h” in Table 19). There are also differences when several parameters are implemented; for example, the same cycle at 30 m shows 17% less prey capture (and 17% less mgC) if the efficiency of the branch type is accounted (i.e. compare raw “a” and “c” of Table 19). When the digestion time is accounted, differences are wider: with an approach of digestion at 17ºC, the capture

120 DIET AND PREY CAPTURE RATES OF L. SARMENTOSA Table 19. Different approaches to annual mean prey capture and predatory impact of a Leptogorgia sarmentosa natural population (2.3 colonies m -2 , 30 cm height with 11808 polyps). A, B rows: 10 m and 30 m populations from raw data (monthly sampling), no calculations. C, D rows: Same as previous rows but taking branch polyp capture efficiency (Coma et al. 1994), from 11808 real polyps to 9775 capturing polyps. E, F: Same as previous rows but applying the Coma et al . 1994 prey capture and carbon ingestion formula with one digestion temperature (17ºC). G, H: Same as previous rows, but with three digestion times (13-17- 21ºC). I: Same as previous rows, but 20m population (taking once a month data), sampling only open polyp branches. J: Same as previous row, but taking rhythms of activity calculations (strictly coincident). K: Same as previous row, but biweekly sampling. Mean ± Standard Deviation.

Prey m -2 day -1 ± SD min max mgC m -2 day -1 ± SD min max

A) 30 m: annual cycle raw data 14371 ± 11893 2173 45922 1.96 ± 2.01 0.17 7.37 (monthly sampling) B) 10 m: annual cycle raw data 9132 ± 5898 2716 27158 1.44 ± 0.79 0.30 2.77 (monthly sampling) C) 30 m: with branch order 11872 ± 9825 1795 37938 1.62 ± 1.66 0.14 6.09 polyp efficiency D) 10 m: with branch order 7544 ± 4872 2244 22437 1.19 ± 0.65 0.25 2.29 polyp efficiency E) 30 m: Digestion at 17ºC 51805 ± 42872 7832 165548 7.08 ± 7.24 0.62 28.58

F) 10 m: Digestion at 17ºC 32920 ± 21261 9790 97905 5.20 ± 2.85 1.08 9.99

G) 30 m: Digestion at 13-17-21ºC 49330 ± 29977 10770 121402 6.61 ± 4.98 0.85 19.49

H) 10 m: Digestion at 13-17-21ºC 35620 ± 23335 9911 99106 5.50 ± 3.43 1.48 13.73

I) 20 m: sampling only open 65580 ± 30320 21539 118927 9.79 ± 5.18 2.58 17.94 polyp colonies J) 20 m: with rhythms of activity 32215 ± 21511 6677 80282 4.74 ± 3.47 0.95 12.93

K) 20 m: sampling every 15 days 27190 ± 23201 2068 114137 5.50 ± 4.53 0.17 19.63 rates (at 30 m) are 77% higher prey capture (and 77% in mgC) than the same data with no digestion approach (i.e. compare raw “c” and “e” of Table 19). On the other hand, the different approach depending on the water temperature with different digestion time seems to be close, with a prey capture of 5% (and 9% in mgC) less when considered the 13-17-21ºC digestion formulas (i.e. compare raw “e” and “g” in Table 19). Now is compared the cycle at 30 m with the cycle at 20 m (Medes Islands, close one another), taking into account sampling once a month in both cases. In the 20m cycle open polyps branches where always taken. The difference is almost 25% less preys (and 34% in mgC) considered in the 30 m cycle (where no open-close polyps are considered in the branch sampling)(i.e. compare raw “g” and “i” of Table 19). Rhythms of activity add more information, and almost halve the estimate of zooplankton capture rates: there is an overestimation of almost the 51% of the prey capture (and 51% in mgC) if not considered the open-closed polyps in the Leptogorgia sarmentosa population (i.e. compare raw “i” and “j” of Table 19). Finally, not a wide difference is found if the 20m cycle is considered with a higher frequency; the difference is an overestimate of the 15% of the prey capture (and 14% in mgC) in the case of a once a month sampling (i.e. compare raw “j” and “k” of Table 19).

DIET AND PREY CAPTURE RATES OF L. SARMENTOSA 121 Discussion

Diet of a detritic-soft bottom passive suspension feeder

It is the first time that is reported the annual diet, capture rates and predatory impact of a detritic-soft bottom passive suspension feeder. The zooplankton diet of Leptogorgia sarmentosa is composed mainly by passive or low motility prey items (see Table 15, 17 and 18), and mean prey size (140 µm) fits very well with polyp size (5mm open crown) and morphology. Other passive suspension feeders also feed on non-motile zooplankton preys of similar size (Sebens and Koehl 1984, Barangé and Gili 1988, Coma et al. 1994, Coma et al. 1995, Orejas et al. 2000). However, environmental zooplankton-prey 50 polyp -1 relationship is almost non-existent in Leptogorgia sarmentosa (except for bivalve larvae, see below). This reflects different behaviour when compared with other passive suspension feeders that have a good correlation between prey capture and environmental zooplankton abundance in annual or diel cycles (Barangé and Gili 1988, Coma et al. 1994, Gili et al. 1996). Orejas et al. 2000 found a similar weak relationship in the hydrozoan Obelia geniculata , suggesting a tight coupling with resuspended waters from upwelling events. Leptogorgia sarmentosa seems not be dependent only on random movements of copepod swarms of near bottom water layers and its associated fauna, but also on resuspension events nearby. Polyp expansion do not follow a strong seasonal pattern (Fig. 34) as in other suspension feeders of rocky bottom substrates due to the summer feeding constraints (Coma et al. 1994, Garrabou 1999), and expansion is significant related to currents but also to fine seston concentration (Chapter IV), suggesting a more continuous exploitation of the fine fraction present in the environment. It has been demonstrated that near bottom fine fraction availability has weak seasonality in warm temperate seas (Medernach et al. 2001, Chapter I), and may be a discontinuous but always present food source for suspension feeders. Seston 4 µm < x < 100 µm (detrital POM, diatoms, dinoflagellates, ciliates and heterotrophic nanoheucariotes) is an important part of the diet in L. sarmentosa , reinforcing the idea of passive prey items specialisation coming mostly from vertical and resuspension fluxes (see Figs. 36 and 37 and Tables 15, 17, and 18). Other gorgonian more extended in vertical hard bottom walls as Paramuricea clavata (Weinberg 1978), had a mixed opportunistic diet, but fine fraction seems to be less important that in Leptogorgia sarmentosa and greatly composed by detritus of low energy value, being the zooplankton the main energy source (Ribes et al. 1999a). It has been suggested that polyp size and predation method are probably the reason why some coelenterates (e.g. L. sarmentosa ) continuously feeds on the fine fraction with high efficiency (Sebens 1987). The bivalve larvae feeding by Leptogorgia sarmentosa is one evidence that particle resuspension is one of the most common hydrodynamic mechanisms used by this species to capture preys. In the three annual cycles studied, bivalve larvae are conspicuous (14-18% of the total prey items captured). Such preys are captured and digested (see results). This is important, as it has been demonstrated that ingestion of invertebrate larvae in general (Mileikovsky 1974), and of bivalve in particular (Purcell et al. 1991) do not involve digestion (and, consequently, absorption). Also the relationship of bivalve larvae abundance- bivalve preyed is significant (R 2=0.37, p<0.01), which means that this passive suspension feeder prey on bivalve larvae peaks coming from adult spawning or substrate resuspension.

122 DIET AND PREY CAPTURE RATES OF L. SARMENTOSA This events could be a source of depletion of such potential bivalve recruits. We suggest that L. sarmentosa could be an important limiting factor in bivalve recruitment in the studied areas. In the 21ºC digestion time experiment, 0.96 ±1.37SD bivalve larvae polyp -1 were captured; in a 30cm height L. sarmentosa colony an estimate of 9000 capturing polyps is acceptable, and this means an impact on the bivalve recruit population of 8600 bivalve larvae individuals colony -1 .Very little is known about the role of predation in such early stages of bivalves (Ólaffson et al. 1994, Beukema et al. 2001), and passive suspension feeders may (together with active suspension feeders, see Davenport et al. 2000) play an active role in detrital-soft bottom substrata bivalve recruitment. Natural habitat of Leptogogia sarmentosa are detrital-soft bottom areas with medium- high turbidity due mainly to resuspension (Weinberg 1978). This gorgonian has a complex branch architecture, with dense ramification and high density of small polyps (Mistri and Ceccherelli 1993, Weinbauer and Velimirov 1998). It has been suggested that optimal branch spacing in hard or soft corals in any given environment is unlikely to result from a single selective pressure (Helmuth et al. 1997). Among others, light (symbiotic Octocorallia), water flow, sedimentation rate, hydromechanical stress and competition for space, acting in concert, may be the key to understand branch spacing in corals and gorgonians (Theodor and Denizot 1965, Velimirov 1976, Helmuth et al. 1997). The trophic strategy observed in L. sarmentosa show a specialisation on low motility preys trapped in low-medium flow speed environments. This feeding strategy suggest a maximum colony feeding effectiveness with a wide range of low-medium flows, observed in other species (Acanthogorgia vegae , 2-16cm s -1 , Dai and Lin 1993). Furthermore, Leptogogia sarmentosa has approximately one third of the area perpendicular to the flow occupied by branches (Weinbauer and Velimirov 1998), and its high polyp density, together with its arrangement on the branch suggest high efficiency in resuspended-low motility preys, exerting drag forces that may retain longer time particles within the branch spaces (Lin and Dai 1996). In near bottom water layers, inorganic fine seston sinks rapidly compared with organic (Thomsen et al. 1995), that remains suspended and recycled by the microbial community (Ritzrau and Thomsen 1997). Is possible that soft bottom active and passive suspension feeders are well adapted to this environmental features, specially erect organisms as Leptogorgia sarmentosa that live mainly in soft bottom substrata normal to the horizontal substrata (Weinberg 1978). In soft Mediterranean bottoms sedimented organic material, although quantitatively less abundant in summer, has a higher quality (Grémare et al. 1997). Even in summer (i.e. high stratified waters), resuspension and water column primary productivity shifts may be frequent, which lets seston available to suspension feeders (Medernach et al. 2001). While soft bottom communities will be adapted to a more or less continuous detritus and fine fraction availability, mainly by vertical settling (McLusky and Elliot 1981), hard bottom substrate passive suspension feeder organisms will be more depend on hydrodynamic features, zooplankton behaviour and epibenthic fauna movements, with a more discontinuous capture prey rates. In general, soft bottom environments in shelf areas are dominated by sediment and detritus feeders benthic organisms (Gray 1981). In hydrodynamic active zones, physical-biological-sedimentary coupling processes leads to the existence of suspension feeder communities in shelf and shelf-slope transition areas. Communities dwelling in such locations, where considerable water masses movement is generated by horizontal current flow patterns, are

DIET AND PREY CAPTURE RATES OF L. SARMENTOSA 123 reminiscent of the highly structured organization and high biomass levels attained by communities dwelling seamounts (Kaufmann et al. 1989) or littoral systems (Gili and Coma 1998). These environmental conditions enhance the development of dense populations of passive suspension feeders (Rogers 1990; Rice et al. 1992). The pennatulacean Pennatula aculeata in the Gulf of Maine represents a major component of benthic macrofauna with patches of 1-8 col. m -2 (Langton et al. 1990), and the ophiuroid Ophiothrix fragilis domi- nates the sessile epifaunal pebble community in the English Channel with patches of more than 200 ind. m -2 (Davoult et al. 1990). The Banks of the coral Lophelia pertusa are frequent throughout the continental shelf and slope in the north eastern Atlantic (Wilson 1979). All these are examples of the dominance and wide distribution of benthic suspension feeders throughout the continental shelf. Currents are also disrupted modifying their velocity by substrate heterogeneity and biogenic structures which enhance benthic suspension feeders particle capture (Witte et al. 1997) in front of other feeding strategies (i.e. sediment and detritus feeders). The increase of these currents could promote the replacement of first sediment feeding fauna by organisms feeding in resuspended particles (Grémare et al. 1998).

Feeding strategy of Leptogorgia sarmentosa : comparison with other suspension feeders.

Other soft bottom suspension feeders have mixed diet typical of high resuspension environments. For example, crinoids feed mostly on protozoa (tintinids), phytoplancton and mollusc larvae (gasteropod and bivalve) (Rutman and Fishelson 1969), and brittle stars on detritus, protozoans and phytoplancton (occasionally on larger preys) (Ölscher and Fedra 1977). In these environments, is usual to take advantage of fine fraction and bacteria associated to the refractory detritic material (McLusky and Elliot 1981), but attention has been usually centred on active suspension feeders. As we have see, an important part of the energy input in Leptogorgia sarmentosa is represented by the fine fraction. If compared with another Mediterranean soft bottom passive suspension feeder (the polychaete Ditrupa arietina ) this energy input is higher ( Ditrupa annual average 70 µgC gAFDW -1 , Jordana et al. 2001). The fine fraction ingested by Leptogorgia is closer to other suspension feeder, the sponge Dysidea avara, 180 µgC gAFDW -1 , Ribes et al. 1999c). Live carbon is more easily assimilated by suspension feeders than detrital carbon (Levinton et al. 1984). The sponge do not show seasonal trends in the carbon ingestion (all from the fine fraction, Ribes et al. 1999c), and it can be expected a similar weak seasonal behaviour in L sarmentosa (suggested specially from slow digestion rates, lack of clear seasonal activity rhythms and expanded polyps stimulation by environmental seston concentration). Our results suggest a strategy where mixed diet (microzooplankton-live fine fraction-detrital fine fraction) permits high growth rates (Mistri and Ceccherelli 1993), and high respiration rates in this soft bottom gorgonian (deduced from his high S/V branch ratios, see Sebens 1987). The strategy seems to be comprised between carnivore hydrozoans, that have high capture rates traduced in high mgC Input mgC Tissue -1 day -1 (due to a ephemeral life cycles and high growth rates-reproductive efforts, see Gili et al. 1998), and, on the other extreme, some Antarctic polar hydrozoans (Gili et al. 1996b, Orejas et al. 2001), anthozoans (Orejas et al. 2001), and zooxhantellae-symbiotic tropical gorgonians (Ribes et al. 1998a), that have lower calculated mgC Input mgC Tissue -1 day -1 ,

124 DIET AND PREY CAPTURE RATES OF L. SARMENTOSA due to feeding constraints, lower carbon demand or a different carbon fuelling (from symbiotic algae in the case of Ribes et al. 1998a). Other communities of benthic suspension feeders in the shelf-slope region, which are related with similar hydrodynamic phenomena, are the high concentrations of hexactinellid sponges in the North Atlantic (Rice et al. 1990), of brittle stars and crinoids associated to corals banks in Florida (Neumann et al. 1977), the anthipatarians at the top of vertical walls of seamounts (Kaufmann et al. 1989) or the diverse and dense benthic suspension feeder communities found along the eastern Weddell Sea shelf and slope (Gutt and Starmans 1998). Differently from the Antarctic communities, the suspension feeding banks are composed by a reduced number of species, mainly by only one such the communities of Leptogorgia sarmentosa studied in the Mediterranean. These communities contrast with the extremely diverse and complex growing on hard bottoms (Gili and Coma 1998). But, despite the importance of these benthic organims, prey capture have never evaluated as far as we know.

Diet and prey capture rates between different populations

While preys are similar in size (139-148µm) and motility (passive-low), its specific proportions are sometimes different over the annual cycles in Banyuls-sur-Mer and Medes Populations (see Table 17 and 18). Different quality and quantity of preys reverts in a lower energy input in the Banyuls-sur-Mer population (Table 19). It cannot be argued colony density and height in such carbon entry spatial differences (trophic shadow, Kim and Lasker 1997), as these two parameters were very similar in both studied populations (and within previous studies observations, Weinberg 1979, Mistri 1995). Depth per se will neither be the source of difference, as prey capture depends on currents and available seston (Sebens 1987), and the 10-14 m depth will be higher than 30m depth hydrodynamism (both populations are northward faced). Furthermore, hydrodynamism (and so the potential interception of passive-low motile preys) is higher in Bayuls-sur-mer area than in the Medes Islands (Chapter III). In fact the seasonality is more marked in the 30 m depth population, which make sense with the continuous food pulses and more continuous hydrodynamism in a shal- lowerzone. Lasker et al. 1983 found higher feeding rates in shallow populations of two tropical gorgonians than in deeper ones, and Harland et al. 1992 found more lipid concentration (used as a trophic parameter) in shallow corals than in deep ones, suggesting more available zooplankton in shallow zones. The studied annual cycles show opposite trends, with a shallow population with lower capture rates than the deeper one. Perhaps in this case are not exclusively the hydrodynamic features of both zones but also the quality of available food. It has been demonstrated that benthic suspension feeder population demography and fitness may be affected by available seston quality (Gardner 2000). The quality of such seston has been pointed out as an essential factor to understand population density, reproductive effort efficiency and growth response in benthic suspension feeders (Beukema and Cadeé 1991, Grémare 1994, Gardner 2000), and evidence is increasing about the importance of particle- type and not only on current speeds in some benthic suspension feeders efficiency (Ribes et al. in press). Banyuls-sur-Mer and the Medes Islands differ also in near bottom water column Chl a concentration (higher in the Medes Islands) and sedimented Particulate Organic Matter

DIET AND PREY CAPTURE RATES OF L. SARMENTOSA 125 (POM) quality (Chapter III). Organic Matter in general and concentration of lipids in particular (an adequate parameter to test for sedimented POM nutritional value, Grémare 1997) has lower values in Banyuls-sur-Mer than in the Medes Islands zone. The diet of L. sarmentosa (passive-low motility preys, partly potentially resuspended and with 140µm mean size) allow us to suggest a relationship of this lower sedimented POM quality in Banyuls- sur-Mer with the lower µgC polyp -1 captured in this area. It has been pointed out that change in community structure and lower biomass values in soft bottom communities in the last 20 years Banyuls-sur-Mer may be due to a change of food availability (Grémare et al. 1998). These authors indicate a change in the sedimented POM characteristics as a consequence of greater intensity and frequency of easterly winds. Possibly such changes are also affecting the Banyuls-sur-Mer Leptogorgia sarmentosa population.

Temporal and methodological considerations.

Time of digestion for Leptogogia sarmentosa goes from 7-13h, depending upon water temperature, and is in accordance with other studied Octocorals (Sebens 1987). The importance to do not extrapolate from other studies is evident. For example, other Mediterranean gorgonian ( Paramuricea clavata ) has a calculated time of digestion of only 4h at 14ºC (Coma et al. 1994). If the digestion time of Paramuricea clavata was accounted for our study, the calculations of zooplankton diet will be double or even triple in formula application (see Methods). Slow digestions can be accompanied by high assimilation rates in organisms feeding on vegetal cells, tissue or debris (e.g. Lawrence 1976, Van Praët 1980, Taghon 1981, Tenore 1983, Frantzis and Grémare 1992). The energy input importance of detritus, phytoplankton, and other groups of the fine fraction in this passive suspension feeder suggest a different assimilation processes compared other passive suspension feeders on hard bottoms (Coma et al. 1994, Coma et al. 1995, Gili et al. 1996). Different digestion rates will be a reflex of similar organisms adapted to this kind of diet (Hawkins et al. 1986, Charles 1993). Low digestion can be also an adaptation to bivalve larvae assimilation. It has been demonstrated that other invertebrates needs until 40 hours to digest efficiently such preys (Purcell et al. 1991). In our case, it seems reasonable to think that both feeding processes (detritic POM-vegetal cells and bivalve larvae) may have adequate assimilation processes in L. sarmentosa . On the other hand, phytoplankton could be a relevant source of energy in soft corals (Fabricius et al. 1995), and its assimilation may trigger several hours (Widding and Schlichter 2001). Another useful tool to calculate real feeding and prey capture rates are the activity rhythms. In warm temperate seas aestivation processes are common in passive suspension feeders (Coma et al. 2000), and activity rhythms are one of the more conspicuous characteristics (Coma et al. 1994, Garrabou 1999). Sampling only expanded colonies and considering rhythms of activity add considerable information and avoid underestimation (expanded polyps, compare rows “g” and “i”, Table 19) or overestimation (activity rhythm, compare rows “i” and “j”, Table 19) at population level prey capture rates and carbon input. All final impact estimations are affected with the activity rhythms considerations (see Final energy input results), suggesting an overestimation of the prey capture and carbon input. It is clear that further studies are needed to have a final evaluation of these two parameters, but is also evident that both have to be considered in future research if the pelagic-benthic coupling of

126 DIET AND PREY CAPTURE RATES OF L. SARMENTOSA passive suspension feeders wants to be adequately evaluated. In fact, field activity rhythms suggest a complex seasonal and diel behaviour of passive suspension feeders depending on water currents and water seston concentration (Chapter IV), varying among species.

Conclusions

Leptogorgia sarmentosa is a detrital-soft bottom passive suspension feeder which feeds primarily on passive-low motile prey items, with a high proportion coming probably from resuspension. This gorgonian may be an important bottle-neck for bivalve population recruits in both studied zones, due to the importance of bivalve larvae feeding rates. The fine fraction covers more than 61% of the ingesta in spring. Our results indicate an important but not preponderant role of the zooplankton diet Leptogorgia sarmentosa . Spatial comparison (two different populations 50 km apart) leads to different annual zooplankton feeding, resulting in a significant different carbon input. Environmental parameters in both zones seems to be also very different, being the environment of Banyuls-sur- Mer (lower carbon input population) poorer than the Medes Islands environment (higher carbon input population). It can also be concluded that temporal and methodological approaches are essential to well understand the benthic-pelagic coupling of a suspension feeder. Methodological approaches as digestion times, open polyp collection, activity rhythms and fine fraction experiments indeed change impact calculations of Leptogorgia sarmentosa . Finally, a mixed diet in which microzooplankton-live fine fraction and detrital carbon seems to be a succesful strategy for a benthic suspension feeder dwelling detritic-soft bottom substrates. All those potential preys may come from vertical, horizontal and resuspension events, available all year long with no clear seasonal trends in the near bottom water layer. In comparison with other hard bottom species more studied (e.g. the gorgonian Paramuricea clavata Coma et al. 1994, Ribes et al. 1999a), the diet of Leptogorgia sarmentosa seems less dependent on zooplankton preys, less seasonally constrained and its physiology more adapted to digest detritus and resuspended material.

DIET AND PREY CAPTURE RATES OF L. SARMENTOSA 127

The “memory” of the Protein-Carbohydrate- Lipid seasonal balance in two sessile marine invertebrates: A new approach to understand the bentho-pelagic coupling processes

Abstract

Seasonal trends in Protein-Carbohydrate-Lipid balance of two passive suspension feeders have been studied. One species ( Paramuricea clavata ) is a hard bottom dwelling species, while the other ( Leptogorgia sarmentosa ) inhabits gravel-soft bottom substrates. Both warm temperate gorgonians (Mediterranean sea) have different feeding, reproductive and growing patterns. One population of P. clavata (colonies >40 cm, considered sexually mature) was monitored at 18 m depth from February 1997 to January 2000 (three years), and analysis of proteins, carbohydrates and lipids were performed. Colonies were tagged and identified (10 males and 10 females). From June 1998 to July 1999 14-20 colonies (<10cm tall, considered sexually immature) were randomly collected, and tissue lipids and carbohydrates were analysed. The reproductive output of the >40 cm height colonies was followed over the three years (samples collected in May 1997, 1998, 1999) to relate with the biochemical balance. On the other hand, one population 30 m depth of Leptogorgia sarmentosa was monitored (19 colonies from May 1998 to August 1999), and the biochemical balance analysed. Colonies were tagged and identified (8 males, 11 females), and reproductive output followed in August 1998 and August 1999 to fit with the Protein-Carbohydrate-Lipid balance. Female and male Paramuricea clavata colonies showed differences in lipid contents only in spring (lipid: 326 ±119 SD µg mg AFDW -1 in females and 248 ±85 SD µg mg AFDW -1 in males). Immature colonies (<10 cm height) showed also significant differences in lipid concentration in spring when compared with mature females (lipid concentration), but no with males. There is a clear seasonal trend in lipid and carbohydrate contents in P. clavata (mature and immature colonies) showing maximum values in winter-spring (e.g. immature colonies lipid concentration: 250 ±49 SD µg mg AFDW -1 in March and 208 ±32 SD µg mg AFDW -1 in May), in coincidence with maximum food concentrations, and minimum values in summer-autumn (e.g. immature colonies lipid concentration: 96 ±10 SD µg mg AFDW -1 in September and 118 ±3219 SD µg mg AFDW -1 in November). The reproductive output-lipid/carbohydrate/protein concentration relationship was non-significant. Protein concentration peaks coincide with previously registered growing times in this gorgonian. In Leptogorgia sarmentosa this protein-growth relationship is also observed, but seasonal trends of lipid and carbohydrate tissue concentrations are different. For instance, there are no male and female biochemical balance differences at any time, and reproductive output is closely related to lipid concentration in both sexes. Lipid storage is different when compared with P. clavata , being higher in L. sarmentosa . In this for- mer gorgonian there is interannual variability, and seasonal drop in lipid and carbohydrate concentration is registered only in autumn (e.g. lipid concentration in spring 1998, 338 ±92 SD µg mg AFDW -1 , and in Autumn 1998, 261 ±44 SD µg mg AFDW -1 ). Interestingly, it seems that both species have hydrodynamically less favoured colonies (i.e. colonies which may be in zones where food is less available) within the patch, as shown by a constant lower lipid values respect to the population mean over the sampling periods studied in these colonies. Both species show drops in lipid and carbohydrate concentrations in fall times, when quality (i.e. nutritional availability) of seston gets also very low values in warm temperate seas. The seasonal biochemical balance (i.e. Protein-Carbohydrate-Lipid concentration on tissues) of marine sessile invertebrates allows this interpretation, as the physiology of these animals fits with environmental conditions, showing trophic crisis in summer and in late fall-early winter. Such balance may be an excellent tool to understand bentho-pelagic coupling processes when parameters as natural diet, feeding rates, reproduction and growth are well understood.

PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE 131 Introduction

Relationships between water column production processes and benthic activity and production are difficult to quantify (Graf 1992). High microphytoplankton production events after fall down to the sea floor in the water column may revert in high oxygen consumption, heat production or bacterial activity in the benthic community (Graf et al. 1982, Graf et al. 1983, Meyer-Reil 1983), and sometimes in a quick lipid accumulation in the benthic organism tissues (Hill et al. 1992, Vanderploeg et al. 1992, Cavaletto et al. 1996). In these examples, water column productivity sharply increases (normally as diatom blooms), giving a large bulk of material ready to be processed by the benthic community. Such mesotrophic-eutrophic environments show production peaks detected in the near bottom water layer (Navarro and Thompson 1995) that directly affects the benthic activity (Thompson and MacDonald 1990). In warm temperate oligotrophic environments such the Mediterranean Sea, water column winter-spring and early autumn blooms are the more conspicuous productivity peaks (Estrada 1996). In such generally considered oligotrophic environments, the water column peaks of production are important for benthic communities because organisms do not receive a continuous amount of food throughout the year (Coma et al. 2000). For this reason, the coupling between water column production events and the benthic coupling response should be key processes to understand ecological cycles in benthic organisms. Detect the coupling levels where sessile organisms interact with seston processes, and in which periods are those bentho-pelagic coupling more conspicuous is not easy, because: 1) Prey capture do not involve immediately translocation of food as energy storage; 2) Environmental factors as temperature or hydrodynamism could be relevant as well as prey availability to the physiology of the organisms (hydrodynamism is closely related to prey availability). 3) Populations could invest reserves independently of the different plankton-benthos trophic episodes, following reproduction or growth events with a shift between high productivity in the water column and such events, due to a certain autoregulation capability of the organisms in front of surrounding environmental factors (Valiela 1995, Margalef 1998). Recent works analyse different parameters such abundance of ephemeral benthic organisms (e.g. hydrozoans, as described in Boero and Fresi 1986, Llobet et al. 1991, Coma et al. 1992), and trophic ecology (e.g. anthozoans as described in Coma et al. 1994, Garrabou 1999) as a response to water column summer food constraints in warm temperate seas (Coma et al. 2000). Benthic suspension feeders may have very different diet constraints along seasonal cycles in warm temperate seas (Coma et al. 1994, Coma et al. 1995c, Ribes et al. 1998, Ribes et al. 1999a, Ribes et al. 1999b, Chapter V), and seston concentration and quality may reflect different feeding trends in accordance to the feeding strategy. In general, organisms depend on energy storage to face seasonal feeding constraints (Lehninger 1982, Prosser 1986, Willmer et al. 2000). It has been suggested that the protein- carbohydrate-lipid balance may reflect trophic constraints in aquatic benthic organisms (Beukema and de Bruin 1977, Zandee et al. 1980, Galap et al. 1997, Cavaletto and Gardner 1998). One of the paradigmatic ecosystems studied from this point of view are marine Polar ecosystems. Polar environments have been widely studied, because it has been detected in several studied organisms a clear accumulation of macromolecules after water column primary production peaks (Clarke 1977, Clarke and Holmes 1986, Hopkins et al. 1993, Hagen

132 PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE et al. 1996, Ahn et al. 2000, Falk-Petersen et al. 2001 among others). Is a direct cause-effect relationship that can be assessed because of the short but very high productivity patterns in the water column primary productivity of polar environments, followed by long periods with lower pelagic and benthic activities (Kopczynska 1992, Barnes and Clarke 1995, Clarke and Leakey 1996). In warm temperate oligotrophic seas, only few works deal with this interpretation (Danovaro et al. 1999, Mayzaud et al. 1999), perhaps because starvation processes, related with the feeding constraints, have not been considered as important as in the above mentioned extreme ecosystems until now (Coma et al. 2000). Several problems arise when this protein-carbohydrate-lipid balance wants to be applied to understand environmental-species relationships and hence the benthic-pelagic coupling processes: 1) Natural diet composition and feeding rates are one of the key factors to understand why the energy storage may or may not have marked seasonal trends (Gardner et al. 1985). In a recent study it has been demonstrated that in warm temperate seas, for example, phylogenetical close passive suspension feeders may have very different diets and prey capture rates, so different environmental constraints through the annual cycle could be reflected in its energy storage (Chapter V). In similar habitat conditions, suspension feeders (in relation with life-history traits) may follow different diet and prey capture patterns (Coma et al. 1998a). Even if trophic ecology is a first step to determine an adequate role in the bentho-pelagic coupling processes, other general biology specie’s features (i.e. distribution, abundance, activity, growth, reproduction, respiration, etc.) is also needed. 2) Which macromolecule is responsible of the energy storage? Different protein-carbohydrate-lipid balances suggest different strategies in this way, and lipids cannot be considered universal storage (see Beukema and de Bruin 1977, Zandee et al. 1980, Peck et al. 1986, Peck et al. 1987, Galap et al. 1997, Costello 1998 among others). 3) It is well known that reproductive effort may mask which part of the energy storage is invested in reproduction and which is invested to face scarce food availability (Clarke 1977, Zandee et al. 1980, Galap et al. 1997). A clear picture of the possible relationship of protein-carbohydrate-lipid with the reproductive investment is essential to know which part is stored to face seasonal low quantity and/or quality of seston. The objective of this work is study the protein-carbohydrate-lipid balance in two benthic invertebrates in a warm seasonal environment such the Mediterranean sea. At the same time, the aim of this work is demonstrate that this balance may be an excellent tool to follow benthic-pelagic coupling. This balance may act as a “memory” of benthic community processes in front of fluctuating water column productivity only if the above mentioned points are considered (i.e. biology of the studied species, complete protein-carbohydrate- lipid balance over an extensive period to know which is/are the macromolecules implied in the storage processes, and relate them with the reproductive output). To reach this objective, two previously well known species were chosen: the gorgonians Paramuricea clavata and Leptogorgia sarmentosa . Both anthozoans have different biological features and are representative of the studied area: natural diet and prey capture, reproductive and growing patterns in accordance to their different morphology, distribution and habitat features (Weinberg 1979, Mistri and Ceccherelli 1993, Mistri 1995, Coma et al. 1998a, Chapter V). Due to the deep knowledge gathered in the last years about this passive suspension feeders, we show an interpretation of the protein-carbohydrate-lipid balance to understand in which way seston seasonal fluctuations are affecting these benthic organisms, and how this balance

PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE 133 may be a useful tool to differentiate the role of environmental or trophic constraints along annual cycles.

Material and methods

Paramuricea clavata . A three years study (February 1997-January 2000) was carried out in the Medes Islands (40º02’55’’N, 3º13’30’’E, North Western Mediterranean, Fig. 39). The population of the gorgonian Paramuricea clavata was 18m depth (Fig. 39). 20 colonies >40 cm height (mature colonies, as stated by Coma et al. 1995a) were tagged in February 1997, and the sex identified on May 1997 by the Coma et al. 1995a procedures. A total of 10 males and 10 females were monitored. Apical branches were cut, preserved in plastic bags and transported to the coast in a cooler full of ice (10ºC). No more than 30 minutes passed until

3º 13' E

0 0.5 1 km

L'ESTARTIT

MEDES ISLANDS A B

42º 02' N

Ter River

Sea Figure 39. Study area location ean Mediterran (Medes Islands). A and B indicates the sampling area of Paramuricea clavata and Leptogorgia sarmentosa respectively.

134 PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE samples were frozen in liquid nitrogen and transported to the main laboratory. Branches were stored at –80ºC, lyophilized 12h at –110ºC and 100 mbar pressure, and kept in a –20ºC cooler until biochemical analysis. All analysis were made using spectrophotometric (colorimetric) procedures. Proteins were quantified using the Lowry et al. 1951 method. A piece of 15-20 mg of each branch (N=20 branches each month) of dry tissue (15-30 polyps per branch + connective ti ssue, peeled branch, no axis) was weighed in a microweighter ( ±0.01mg precision). The tissue was homogenised with 6 ml NaOH 1N. Carbohydrates were quantified using the Dubois et al. 1956 method for the 20 colonies each month. A piece of 15-20 mg of dry tissue was homogenised with 6 ml DDW. Lipids were quantified using the Barnes and Blastock 1973 methodology. A piece of 10-15 mg of dry tissue was homogenised with 4 ml Chloroform- Methanol (2:1 V:V). Results are yield in µg Protein/Carbohydrate/Lipid mg AFDW -1 . AFDW of primary branches transformations were calculated from Coma 1994 (Paramuricea clavata ) . To test Protein/Carbohydrate/Lipid differences within a branch, 4 primary branches of the same colony were cut the 27/2/98. Four pieces of the same branch were subsampled (N=4 x 4 Pieces). To test Protein/Carbohydrate/Lipid differences between branches of the same colony, four different primary branches of the same colony were cut the same day, and three subsamples of each branch were processed. Finally, to test differences between different branch orders (1,2,3,4,5, as Coma et al. 1994) two colonies were picked up (February 1998) and nine branches of each order were analysed following the above mentioned methodologies (AFDW coenenchima differences were considered, following Coma 1994). To evaluate seasonality of the lipid and carbohydrate concentration in immature colonies of Paramuricea clavata (with no gonadal production), 14-20 exemplars <10 cm height (immature, as stated in Coma et al. 1995a) were randomly sampled monthly next to the >40 cm height tagged population (June 1998-July 1999). Apical branches were treated with the above mentioned methodologies for lipids and carbohydrates. Leptogorgia sarmentosa . A population of the aposymbiotic gorgonian Leptogorgia sarmetosa was monitored next to the Paramuricea clavata population (30m depth, 100m distance, Fig. 39b). A total of 8 males and 11 females >30 cm height (mature colonies, Rossi unpublished) were tagged and tip branches cut monthly from May 1998-August 1999. The sex identified on July (maximum gonadal growth, Rossi unpublished) 1998 by the Coma et al. 1995a procedures. Branches were preserved and treated as the Paramuricea clavata ones. In the gorgonian Leptogorgia sarmentosa , only lipid concentration within primary branches (N=4 x 4 Pieces) and differences between branches of the same colony (N=3 x 4 Pieces) were tested the 18/2/99, because of the positive results in Paramuricea clavata . Reproductive output . The reproductive effort was made as follows: one apical branch of each tagged colony was cut and fixed in 6% formaldehyde (May 1997,1998 and 1999 for Paramuricea clavata , July 1998 and 1999 for Leptogorgia sarmentosa ), concurrently to the biochemical sampling. 10 polyps of each branch were opened and the gonads counted and measured as Coma et al. 1995b. Carbon transformation for male and female gonads is made with the Coma et al. 1995b estimations. Statistics . Relationships between sexual reproductive effort-protein/carbohydrate/lipid in AFDW tissue concentrations was calculated with standard regression curve (Pearson´s product-moment correlation). For the statistical comparison within branches, between

PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE 135 branches and between different order branches of the same colony, one-way and two-way ANOVA with Scheffé post-hoc test were used. Male-female and seasonal-annual variability differences were assessed with three-way ANOVA with Scheffé post-hoc test with repeated measures (as colonies sampled were always the same, Zar 1996). Finally, to test differences between colonies, between immature-mature colonies, and between species one way ANOVA analysis with the same post-hoc test were used (Zar 1996).

Results

Biochemical balance in Paramuricea clavata. No differences within primary branches were found in protein/carbohydrate/lipid concentration in Paramuricea clavata , nor in lipid concentration in Leptogorgia sarmentosa (Table 20). Test for differences between primary branches of the same colony were also non-significant for both gorgonians in all the parameters analysed (Table 20). In Paramuricea clavata , branch type 5 had different protein/carbohydrate concentration from all other branch types (Table 21), but non significant differences with branch type 4 were found in lipid concentration. Branch type 1 was also different of branch type 3 and 4 when lipid concentration was tested (Table 21). Lipids followed a decrease from primary to fifth branch type, but this steep process was not shown in protein and carbohydrate concentration.

Table 20. ANOVA with post-hoc Scheffé tests: 1) Comparison within the same primary branch of proteins, carbohydrates and lipids ( Paramuricea clavata ) and of lipids ( Leptogorgia sarmentosa ); 2) Comparison between primary branches of the same colony of proteins, carbohydrates and lipids ( Paramuricea clavata ) and of lipids ( Leptogorgia sarmentosa ). All were non significant (p>0.05).

Protein Carbohydrate Lipid

P. clavata Within Primary Branches F 3,16 =0.00 [p=1.000] F 3,16 =3.73 [p=0.06] F 3,12 =1.10 [p=0.338]

Between Primary Branches F 3,8 =0.39 [p=0.7616] F 3,8 =0.39 [p=0.7607] F 3,8 =0.44 [p=0.5626]

L. sarmentosa Within Primary Branches - - F 3,12 =1.69 [p=0.2224]

Between Primary Branches - - F 3,8 =3.19 [p=0.084]

Table 21. ANOVA with Scheffé post-hoc comparison between different order branches in Paramuricea clavata (sensu Coma et al 1994). 1,2,3,4,5, branch order. Difference column shows which branches were different in the analysis. Values are mean concentration (P-C-L µgP mgAFDW -1 ) ± Standard Deviation.

1 2 3 4 5 Difference

-1 ≠ Protein mgP mgAFDW 660 ±52 640 ±48 642 ±48 627 ±46 583 ±39 5 1,2,3,4 F 4,40 =26.89, p<0.0001 -1 ≠ Carbohy mgC mgAFDW 56 ±5 54 ±5 54 ±5 53 ±5 38 ±3 5 1,2,3,4 F 4,40 =3.36 p<0.02 Lipid mgL mgAFDW -1 357 ±52 339 ±48 242 ±48 176 ±45 134 ±39 5 ≠1,2,3 and 1 ≠3,4 F4,40 =40.14 p<0.0001

136 PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE Table 22. Seasonal Protein-Carbohydrate-Lipid balance, from Winter 1997 to Winter 2000 in Paramuricea clavata . Values are µg Protein-Carbohydrate-Lipid mg AFDW -1 (Mean ± Standard Deviation). Statistics shown in the text.

Protein Carbohydrate Lipid

Winter ’97 693 ±90 81 ±13 269 ±100 Spring ’97 692 ±118 72 ±15 356 ±131 & 228 ±62 ( Summer ’97 631 ±107 61 ±13 176 ±42 Fall ’97 693 ±125 61 ±13 166 ±43 Winter ’98 707 ±120 93 ±13 257 ±83 Spring ’98 770 ±130 74 ±19 300 ±115 & 194 ±74 ( Summer ’98 665 ±109 51 ±10 156 ±63 Fall ’98 799 ±163 71 ±14 190 ±63 Winter ’99 764 ±118 80 ±17 228 ±76 Spring ’99 698 ±82 80 ±16 356 ±103 & 296 ±124 ( Summer ’99 676 ±92 59 ±8 122 ±50 Fall ’99 737 ±85 63 ±22 196 ±84 Winter ‘00 728 ±16 83 ±26 257 ±68

Protein concentration of Paramuricea clavata through the three-years cycle is shown in Figure 40. There was no male- female protein concentration difference (F 3,714 =2.4, p=0.0664). Differences among seasons were found (Table 22) in protein concentration: summer had lower values if compared with fall-winter-spring (F 3,718 = 18.39, p<0.0001), but differences between Figure 40. Three year observations of male (10 fall-winter-spring were non significant. No colonies) and female (10 colonies) of the gorgonian interannual (winter 1997-winter 1998, Paramuricea clavata proteins (February 1997-January etc.) differences were found, with the 2000). Mean±SE. exception of fall 1997 that was different from fall 1998 (see Table 22, F 9,709 =8.71, p<0.0001). In November 1997 and 1998, protein concentration was different from all other months except October, March and April (F 11,710 =11.14, p<0.0001). The carbohydrate triannual cycle is shown in Figure 41. There was no sex difference in carbohydrate concentration (F 3,477 =1.49, p=0.2167). Differences among seasons were found (Table 22), and carbohydrate Figure 41. Three year observations of male (10 colonies) and female (10 colonies) of the gorgonian concentration was significant lower in Paramuricea clavata carbohydrates (February 1997- summer and fall seasons (F 3,481 =44.18, January 2000). Mean±SE. p<0.0001). Non-significant interannual

PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE 137 a b

Figure 42. Three year observations of male (10 Figure 43. Lipid and carbohydrate concentrations colonies) and female (10 colonies) of the gorgonian over a 14 month period in immature colonies Paramuricea clavata lipids (February 1997-January (<10cm height) of the gorgonian Paramuricea 2000). Mean±SE. clavata (June 1998-July 1999). Mean±SE. differences were found over the three year sampling (F 9,472 =11.63, p<0.0001). Lipid concentration in Paramuricea clavata is shown in Figure 42. Female-male lipid concentration was different in spring (see Table 22, F 3,704 =16.08, p<0.0001), but no in other seasons. We found differences between winter (250 ±84SD µg mg AFDW -1 ) and summer-fall (irrespective to sex),and also differences between summer (152 ±57SD µg mg AFDW -1 ) and fall ± -1 (184 66SDµg mg AFDW ) lipid concentrations (F 3,708 =94.67, p<0.0001). No interannual differences among seasons (irrespective to sex, winter-summer-fall) are found in lipid concentration (F 9,699 =35.50, p>0.05). In spring, female colonies were also non-significant different in the different years of the cycle studied, and the males followed the same pattern ± (F 9,686 =6.47, p>0.05). Females had always higher lipid concentration in spring (338 119SD µg mg AFDW -1 ) than in other seasons, and male lipid concentration in spring (240 ±100SD µg mg AFDW -1 ) is different to summer (148 ±57SD µg mg AFDW -1 ) and fall (191 ±75SD µg -1 ± -1 mg AFDW ), but not different to winter (236 71SD µg mg AFDW )(F 9,686 =6.47, p<0.0001). A sharp decrease is observed between June-July/August in the three studied years, and minimum values are also found in late fall (November/December), after an early fall lipid concentration peak (September/October) (Fig. 42). A weak relationship is found between carbohydrate-lipid concentration among the three year sampling (R 2=0.16, p<0.001, N=72). No relationship is found in Paramuricea clavata between protein-carbohydrate nor protein-lipid concentrations. Colonies <10cm height followed a clear seasonal cycle over the studied period (Fig. 43). Carbohydrates were significant more concentrated in winter (83 ± 10SD µg mg AFDW -1 ) than in summer (67 ± 10SD µg mg AFDW -1 ) or fall (70 ± 12SD µg mg AFDW -1 , but were ± -1 similar to spring values (78 12SD µg mg AFDW )(F 5,144 =9.27, p<0.0001). Spring was non-significant different from summer nor fall. Interannual variability was non-significant. Within the sampled period (June 1998-July 1999), no seasonal differences were found between mature-immature colonies in carbohydrate concentration (test: immature-male- female, F 8,244 =17.90, p>0.05). Lipid concentration in such <10cm colonies (Fig. 43) followed also seasonal trends. Winter lipid concentration (216 ±64SD µg mg AFDW -1 ) was significant higher than spring (180 ±52SD µg mg AFDW -1 ), summer (120 ±27SD µg mg -1 ± -1 AFDW ), and fall (124 32SD µg mg AFDW )( F 5,260 =37.44, p<0.0001). Fall and summer were different also from spring time, but very similar between them. Interannual variability

138 PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE was non-significant. Within the sampled a period (June 1998-July 1999), seasonal differences were found between female mature colonies and immature colonies in lipid concentration in spring, but no in other seasons nor with the male mature colonies in spring (test: immature-male- female, F 8,443 =36.65, p<0.0001). A more tight relationship was found between carbohydrate-lipid concentration over the annual cycle in colonies <10cm (R 2=0.51, p<0.01, N=13). b Biochemical balance in Leptogorgia sarmentosa. Less clear seasonal trends are observed in the May 1998-August 1999 cycle of Leptogorgia sarmentosa . There were no differences between male and female seasonal protein concentration (Fig. 44a)(F 5,282 =0.17, p=0.9733). The proteins in winter 1999 and spring 1999 (Table 23) were significant more concentrated than spring, summer and fall 1998 (F =17.98, p<0.0001). Summer 1999 5,288 c was different from winter and spring 1999, but similar to the other analysed seasons. September 1998 (see Fig. 44a) was higher than August and October; there was a significant increase in December through March (different from November and April), and a sudden increase in May 1999 (different from April and June 1999)( F11,278 =413.23, p<0.0001). Carbohydrates (Fig. 44b) had no male-female seasonal differences (F =0.89, p=0.4877). In this 5,232 Figure 44. Protein (a), carbohydrate (b), and lipid (c) case, spring and summer 1999 had signifi- concentrations in male and female of the gorgonian cant higher carbohydrate concentration Leptogorgia sarmentosa over a 16 months period. than all the other analysed seasons (Table Mean±SE. 23)( F 5,238 =14.94, p<0.0001). Also the lipid concentration (Fig. 44c) was, in the Leptogorgia sarmentosa case, non-significant different in the male-female comparison (F 5,305 =0.87, p=0.5033). When seasons are compared (Table 23), spring 1998 was different from fall 1998, but no to summer 1998, winter, spring and summer 1999 (F 5,311 =14.38, p<0.0001). Summer 1998 and 1999 were different from winter and spring 1999. Our results indicate interannual variability in the protein-carbohydrate-lipid balance in Leptogorgia sarmentosa . No significant relationship was found between variables (protein-carbohydrate-lipid) in any case.

PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE 139 Table 23. Seasonal Protein-Carbohydrate-Lipid balance, from Spring 1998 to Summer 1999 in Leptogorgia sarmentosa . Values are µg Protein-Carbohydrate-Lipid mg AFDW -1 (Mean ± Standard Deviation). Statistics shown in the text.

Protein Carbohydrate Lipid

Spring ’98 568 ±111 50 ±9 338 ±92 Summer ’98 554 ±172 48 ±12 304 ±102 Fall ’98 575 ±202 49 ±17 261 ±44 Winter ’99 775 ±135 49 ±15 379 ±71 Spring ’99 724 ±131 73 ±28 362 ±88 Summer ’99 628 ±152 68 ±21 293 ±137

Reproductive output . Once analysed the reproductive effort in both species (May 1997,1998 and 1999 for Paramuricea clavata , July 1998 and 1999 for Leptogorgia sarmentosa ), this parameter was correlated (µgC polyp) with macronutrient concentration (Protein-Carbohydrate-Lipid µg mg AFDW -1 ). In Paramuricea clavata female colonies, no significant relationship was found in any case (N=28) between the above mentioned parameters. In males, only protein concentration was weakly related with reproductive effort (R 2=0.21, p<0.05, N=23). On the other hand, in Leptogorgia sarmentosa there was significant relationship of lipid concentration with reproductive effort in females (R 2=0.69, p<0.0001, N=23) and in males (R 2=0.341, p<0.05, N=15). No relationship was found with protein nor with carbohydrates and reproductive effort. Comparison of biochemical balance in both species . Over the May 1998-August 1999 period, Paramuricea clavata and Leptogorgia sarmentosa protein-carbohydrate-lipid concentrations can be compared. Protein concentration was significant different in spring and fall 1998 when both species are compared (F 5,600 =15.50, p<0.0001), but in no other season (compare Table 22 and 23). Only in fall 1998 and winter 1999 there was a difference in carbohydrate concentration, being higher in Paramuricea than in Leptogorgia (see Table 22 and 23, F 5,391 =9.78, p<0.0001). In the other seasons we do not found any significant difference. The behaviour of lipids was completely different among species. All seasons were different, being more concentrated in Leptogorgia than in Paramuricea (except for spring females 1998 of Paramuricea clavata , and spring 1999 in general, see Table 22 and 23)( F5,623 =10.83, p<0.0001). a b

Figure 45. Mean values of lipid concentration in the tissue of the gorgonian Paramuricea clavata respect to values in single colonies over the three year period of observations: (a) Male colonies 308 (above the mean) and 317 (below the mean); (b) Female colonies 319 (above the mean) and 307 (below the mean).

140 PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE Figure 45a,b shows two different male and female colonies of Paramuricea clavata respect to the mean lipid concentration over the three year cycle. We only represent two for each sex for a major comprehension, but these trends can be applied to other colonies of the population. In Figure 45a, two males (colony 308 and 317) have contrasted behaviours. The colony 317 seems to have less capacity to accumulate lipids over the studied period. As can be observed in the picture (Fig. 46 317 ), this colony dwells on a crevice. The colony 308 seems to be always above the mean lipid concentration, and is on a hydrodynamic favoured zone of the patch (Fig. 46 308 ). Due to the lack of differences between primary branches in lipid concentration (see Table 20), it seems reasonable to think that a synergistic behaviour of the whole colony (similar concentration in primary branches) is followed during the three year period studied. In Figure 46b the same plot is shown, but with two female colonies (Colonies 307 and 319). From the results, it seems that colony 307 has less lipid concentration over the period studied than 319. In Figure 47 two colonies of Leptogorgia sarmentosa (310 Female, 319 Male) are compared respect to the mean lipid concentration. It seems that

308 317

Figure 46. Location in the boulder sampled of the respective male (308, 317) colonies followed during the three year sampled period. Look at the position within the patch: less favoured colony are next to the sand substrate on a crevice, isolated from the rest of the patch (317), while the more favoured are within the patch away from the sandy bottom (308).

PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE 141 319 has less capability to accumulate lipids than 310. In this case, no evident hydrodynamic constraints were observed (personal observation, pictures not shown).

Discussion

In general, energy storage may be related in benthic organisms to several favourable-unfavourable environmental Figure 47. Mean values of lipid concentration in the conditions related to depth, light, substrate tissue of the gorgonian Leptogorgia sarmentosa or water movement (Fitt and Pardy 1981, respect to values in single colonies over the three Jarzebski et al. 1986, Harland et al. 1992, year period of observations in a female colony (310, Cavaletto et al. 1996, Haroon et al. 2000). above the mean), and a male colony (319, below the mean). No significant differences were found Glycogen or/and proteins have been between male and female lipid concentration over described as macromolecules responsible the studied period (see results). for the storage that will be used to face trophic constraints in some organisms (Beukema and de Bruin 1977, Zandee et al. 1980, Peck et al. 1987, Epp et al. 1988, Galap et al. 1997 among others). In the studied anthozoans, lipids seems to be the storage macromolecules. Protein and carbohydrate concentration in our study are in accordance with previous studies made on cnidarians (Hill-Manning and Blanquet 1979, Kellog and Patton 1983, Stimson 1987, Ben-David-Zaslow and Benayahu 1999). Lipids are considered the main stored macromolecules in such studies, and our results indicate same trends in the two monitored gorgonians. Although it seems clear that lipids are the main starviation molecule in the studied cnidarians, different interpretations have been made respect the storage capability. Harland et al. (1992) predicted a larger capacity to store energy depending on the food availability, so shallow coral colonies have more lipids in comparison of deeper ones. On the other hand, Fitt and Pardy (1981) showed a major capability to store lipids in Anthopleura elegantissima in starved conditions, so anemones under feeding stress first catabolise carbohydrates than lipid storage. In that case the author’s emphasise the possession or not of zooxhantellae within the anemone tissue. In fact, Ben-David-Zaslow and Benayahu (1999) also predict an influence over the annual protein-carbohydrate-lipid balance of zooxhantellae physiology. This is not the case for Paramuricea clavata nor for Leptogorgia sarmentosa , both octocorals being aposymbiotic.

Biochemical balance and reproductive output

It has been largely discussed the importance to discern between seasonal energy storage invested in reproduction and in starvation periods (Clarke 1977, Epp et al. 1988, Lucas 1994, Alonzo et al. 2000). Although in Paramuricea there is a clear difference between male and female lipid concentrations in the previous to spawning periods (Coma et al. 1995a, see Fig. 42 and Table 22), non significant relationship of lipids with reproductive output (i.e.

142 PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE male and female gonadal biomass) were observed in three year of study (nor with carbohydrates/protein concentration either). Part of the energy which represent the lipid storage could be invested in reproduction, but we suggest that a large amount of the energy stored during the favourable season (winter-spring), is directly invested to survive during the summer trophic crisis. Paramuricea clavata patches are compact, with up to 32 colonies m -2 (Weinberg 1979, Coma et al. 1994), perhaps surface brooded eggs (Coma et al. 1995a) do not contain large amounts of stored lipids because of a short larval displacement. Dense patch populations and spawning synchronisation may enhance reproductive success among gorgonians (Coma and Lasker 1997), and it has been suggested that marine invertebrates which develop dense populations may have short displacement larvae with low energetic reservoirs (Harrison and Wallace 1998, Uriz et al. 1998, Tioho et al. 2001). Paramuricea clavata store lipids for reproduction (and to front the trophic crisis), but those lipids may contribute to the egg developement, and not necessary have to be stored directly in the eggs (female). Furthermore, colonies <10 cm (immature, Coma et al. 1995a) follow the same male and female seasonal pattern, suggesting lipid accumulation before summer aestivation. In these immature colonies the lipid-carbohydrate relationship among the year cycle is high significant, suggesting resource mobilisation such has been observed in other species (Willmer et al. 2000). In fertile colonies (>40 cm height) this relationship is non-existent. Male and immature colonies have no differences in the winter-spring lipid accumulation, while female and immature colonies have significant lipid concentration differences (more abundant in females). One possible explanation of these differences is that energy invested in gonadal maturation in male colonies is low (spermatic sacks full of proteins) and all lipids stored in such mature colonies are mobilised in food scarcity periods. On the other hand, lipid increase in females (compared to mature males and immature colonies) may be invested in gonadal production. It is clear that the role of lipids in the reproductive output exists in Paramuricea , but not directly related with the number/diameter of eggs per polyp. In Leptogorgia sarmentosa , although there was no significant lipid concentration difference in any season between male and female colonies, a significant relationship between reproductive output and lipids was found (specially in females). Colony density in Leptogorgia is lower than in Paramuricea (up to 2-6 colonies m -2 , Weinberg 1979), and although fecundating processes or larval release have not been observed yet, our unpublished data suggest a punctual reproductive summer effort with a low number of female eggs (3 ±2 SD gonads polyp -1 in primary branches in Leptogorgia sarmentosa , Rossi unpublished data, compared 26 ±4 SD gonads polyp -1 of similar diameter in primary branches of Paramuricea clavata , Coma et al. 1995a) that may concentrate larger amounts of energy to move on wider distances (Harrison and Wallace 1998).

Seasonal trends in Protein-Carbohydrate-Lipid balance.

Warm seasonal marine species, as in other ecosystems, couple their ecological cycles to the variability of environmental factors. Because warm temperate ecosystems have highly variable environmental features, most species have developed mechanisms to avoid strong dependence of such stochastic environment (Margalef 1991). Recently, these ecological factors regulated by endogenous mechanisms, has increased the interest of ecologist, because help to better understand population life histories (Margalef 1998). One of the best known

PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE 143 “isolation” mechanisms is the energy storage (lipid, proteins, glycogen), used in growth or reproductive periods after prey capture peaks (Hill et al. 1992, Vanderploeg et al. 1992). It seems that such is the case shown by the two cnidarian species studied here. In the warm temperate seas, most of the benthic community follows aestivation processes (i.e. low food availability, Sardá et al. 1999, Coma et al. 2000). Lipid and carbohydrate concentration over the three years studied in Paramuricea clavata reflects these aestivation periods, where minimum values are reached in the summer season (Fig. 41, 42 and Table 22). This seems not the case for Leptogorgia sarmentosa , which has variable interannual summer carbohydrate concentration and high lipid contents in this season (Fig. 44, and Table 23). We suggest that this different seasonal biochemical composition is a reflex of the different diet and feeding behaviour of both species (Coma et al. 1994, Ribes et al. 1999a, Chapter V). P. clavata follows a more seasonal feeding pattern, depending on the microphytoplancton and the zooplancton pulse concentrations, while L. sarmentosa depends more on the small seston fraction and resuspended material concentration, more available throughout the year (except in late autumn-early winter, see below). It has been suggested that macrophagous benthic invertebrates that depend on more random food availability (e.g. zooplankton concentration, Paramuricea clavata ) have a tendency to store large amounts of energy to face the poor feeding seasons, while benthic invertebrates that rely on more continuously available food (e.g. microphytoplancton, nanoplankton, detritus, Leptogorgia sarmentosa ) develop less seasonal trends in energy storage (Slobodkin and Richman 1961, Norrbin Bamstedt 1984, Gardner et al. 1985). A more continuous lipid accumulation in Leptogorgia sarmentosa in front of Paramuricea clavata perhaps is a consequence of its ability to continuously feed on the fine seston fraction and on the resuspended material (Chapter V). The high lipid concentration in Leptogorgia tissue in summer may be due to slightly more scarce but very rich content of particulate organic matter in the North-Western Mediterranean shallow areas during summer and to the pulses of the fine fraction (Grémare et al. 1997, Medernach et al. 2001, Chapter III). In the Mediterranean sea it has been detected (although not discussed) the summer drop of lipid contents in some benthic organisms (Vives and Suau 1962, Herrera and Muñoz 1963, Bodoy 1980, Fernández 1997). Other studies show a different trend, specially those related with active suspension feeders (Fraga 1956, Álvarez-Seoane 1960, Establier 1966, Establier 1969). In such studies, lipid values are high or very high, in accordance with reproductive cycles and, we suggest, with sedimenting POM high nutritive values (as Leptogorgia sarmentosa ). In fact, in that case active suspension feeders may take more advantage from resuspension events because of the active feeding capability: they don’t have to depend so strictly on favourable hydrodynamic environmental features as passive suspension feeders (see Chapter IV). Danovaro et al. (1999) suggested a relationship of calorimetric (in general) and lipid (in particular) increase in nematode tissue following sedimenting POM quantity and quality in deep sea Mediterranean areas. This coupling has been shown tight in benthic amphipods and copepods in seasonal lakes (Hill et al. 1992, Vanderploeg et al. 1992), where spring bloom peaks seems to be essential to the overall life cycle of these organisms. In our case, it seems that the higher (and more continuous) late winter-spring productivity of the Mediterranean sea (Estrada 1996) is reflected in the lipid and carbohydrate storage of both gorgonians. In this sense, perhaps because of high contrast between high and low productivity events, polar sea regions have been focused as paradig-

144 PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE matic in the energy storage over annual cycles (Clarke 1977, Clarke et al. 1985, Hopkins et al. 1993, Hagen et al. 1996, Ward et al. 1996, among others). Although it has been demonstrated benthic suspension feeder activity almost all year long (Barnes and Clarke 1995), and an almost continuous presence of pico- and nanoplankton in polar waters (Kang et al. 1997) that is suggested to be a feeding source for some benthic suspension feeders all year long (Orejas et al. 2001, Orejas et al. in Submitted), lipid storage has been invoked as primary energy source during polar winter (Clarke and Holmes 1986, Hagen et al. 1996, Ward et al. 1996, Evanson et al. 2000, among others). In these cases, the trophic coupling (i.e. the energy storage response in front of high productivity events) is clear, although the reproduction output cannot be disregarded. Proteins show a seasonal pattern in both species (Fig. 40, Fig. 44a, and tables 22 and 23), and in both species are related to growth periods. In fact, the maximum somatic growth of primary branches is reached in September-November and February-March in Paramuricea (Coma et al. 1998b); Leptogorgia has more variable somatic growth through the year (Mistri and Ceccherelli 1993, Mistri 1995), but growth peaks are detected in the studied population in September and throughout late winter-spring (Rossi unpublished data). This may be the response to both early autumn and late winter-early spring phytoplankton (and associated zooplankton) blooms in the Mediterranean sea (Estrada 1996). In early autumn there are also small peaks of lipid in Paramuricea tissue (October, see Fig. 42), perhaps because this short fall blooms in the same region (Zingone et al. 1995). Leptogorgia has sharp lipid decrease after August, perhaps because all energy in September is invested in growth. We suggest protein concentration as a useful tool (in the protein-carbohydrate-lipid balance) to detect growing periods in both studied gorgonians. This tool, together with the pothograph monitoring (Coma et al. 1998b, Garrabou 1998) may help to understand energy investments in somatic production in a wide community perspective. This biochemical balance show that, at least in Paramuricea clavata and Leptogorgia sarmentosa , some colonies may be in advantage respect others (Fig. 45a,b, 47) when considered its position in the patch or population (Fig. 46). In fact, the lipid accumulation may be an excellent tool to test the fitness of colonies inside the population, and consider if they are trophic-shadowed (Okamura 1988, Zabala and Ballesteros 1989). Along this study seems that, through the three years period of analysis in Paramuricea and the one year period of analysis in Leptogorgia , some colonies have been stressed in a different way, and the potentially less favoured gorgonian (i.e. worse placed to feed) are always under the mean values of lipid concentration. Lipid in benthic organisms (or the macromolecule that represent energy storage) may be used as index for modular organisms fitness in a monitored population, and may help to understand if the colonies are or not submitted to food stress conditions due to the neighbours influence or as a result of substrate position respect to the main currents.

Late autumn-early winter trophic crisis in the Mediterranean sea.

One of the striking and unexpected results of this study are the very low values of lipids, carbohydrates and even proteins in late autumn-early winter in both species (late November- early January, see Figures 40, 41, 42, 43, 44). This period correspond with a brief but significant low seston availability period for benthic organisms in the Northwestern

PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE 145 Mediterranean as we want to demonstrate. After the two water column production peaks (late winter-spring and early autumn), only the summer crisis aestivation time have been considered as potential stressful for most benthic organisms (Coma et al. 2000). During this period stratification causes nutrient depletion and still waters reduce movement around organisms near the sea floor. In general, because seston concentration and nutrients may be high or very high in autumn (San Feliu and Muñoz 1975, Ballesteros 1989), the repercus- sion of this short fall crisis period on benthic organisms has been neglected (but see Ballesteros 1989 for algal canopy). Sverdrup model (Sverdrup 1953) demonstrates that in the water column, an excess of turbulence and turbidity may low minimum compensation point for phytoplankton or phytobenthos: as a result, even if high nutrient concentration is available, algal productivity get low values (see also Smetacek and Passown 1990, Huisman et al. 1999). In the Mediterranean, after the early fall water column production peak, two seasonal phenomena increase water turbidity (low light penetration) in late autumn: 1) Heavy autumn rains and subsequent river runoff, and 2) Easterly storms (Cebrian et al. 1996, Duarte et al. 1999, Chapter I). As previously mentioned, nutrient concentration may be high, but phytoplankton abundance and productivity get very low values in all the studied annual cycles (Margalef 1957, Neveux et al. 1975, San Feliu and Muñoz 1975, Fabiano et al. 1984, Chapter I among others). The associated microbial loop may also collapse (as a result of poor phytoplankton concentrations) as suggested by the low abundance values of heterotrophic bacteria, heterotrophic nanoeukaryothes, flagellates and ciliates shown in most annual cycles (Selmer et al. 1993, Ferrier-Pagès and Rassoulzadegan 1994, Vaqué et al. 1997). Also the zooplankton during this period in the water column get, in accordance to the food scarcity, very low density values (e.g. crustacean plankton: Vives 1966, Gaudy 1972, Razouls 1975, Benon 1976, Bodiou et al. 1990, Calbet et al. 2001, Chapter I; gelatinous plankton: Massutí 1959, Vives 1966, Berhaut 1969, Chapter I; meroplanktonic larvae: Razouls and Thiriot 1968, Fusté 1982, Chapter I; ictioplankton: Palomera and Olivar 1996). Also the microphytobenthos (Lucchini 1971, Mouneimné 1972, Delille et al. 1990) and macrophytobenthos (Ott 1980, Ballesteros 1989, Benedetti-Cecchi and Cinelli 1993) reach very low biomass and chlorophyll a m-2 values. All these phenomena revert on the sedimented particulate organic matter. Although the seston concentration may reach higher values respect the rest of the annual cycle (Grémare et al. 1997), the availability of such matter reach its lowest nutritive values (i.e. gets low lipid concentration levels as stated by Grémare et al. 1997, see also Fichez 1991, Fabiano and Danovaro 1994, Fabiano et al. 1995, Grémare et al. 1998, Pusceddu et al. 1998, Chapter III). Low phytoplankton, microbial community, zooplankton and scarce quality detritus has to have an influence on the benthic communities. Populations of short lived animals in soft and hard bottom communities may have significant low biomass and density in late autumn- early winter (Duchêne 1980, Boero and Fresi 1986, Bodiou and Albert 1991, Llobet et al. 1991, Sardá et al. 1999 among others, but see exceptions in Boero et al. 1986, Coma et al. 1992). Most of these benthic organisms have also low gonadal indexes and do not release larvae in this period (Suau and Vives 1957, Régis 1979, Hendler and Coma et al. 1995a, Corriero et al. 1996, among others, but see exceptions in Becerro and Turón 1992, Coma et al. 1996). In many cases, growth is stopped or decrease in some species in this late autumn- early winter period (Turon and Becerro 1992, Peirano et al. 1999, Garrabou and Ballesteros

146 PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE 2000, Garrabou and Zabala 2001). Such patterns in population density and dynamics have to be related with the arrival of food quality and concentration to the benthos. For example, in warm seasonal benthic communities on trophic ecology of suspension feeders, suggest that these organisms have low capture rates or low quality food ingested in this period (Coma et al. 1994, Coma et al. 1995c, Ribes et al. 1998, Ribes et al. 1999a, Ribes et al. 1999b, Chapter V). Furthermore, energy storage (protein/carbohydrate/lipid, depending on the species) also have demonstrated to be very low in this period in the Mediterranean sea (Fraga 1956, Vives and Suau 1962, Herrera and Muñoz 1963, Establier 1966, Moueza 1972, Fernández 1997, Mayzaud et al. 1999, among others). The short crisis period described before has not been considered as a period of food constraints for the benthic community. We suggest that in warm temperate seas (at least in the Mediterranean sea), there is one period of large food availability (late winter to late spring, i.e. late January to late June) followed by another period (early July-early January) which is more stressful for benthic organisms for two different reasons: water column strong stratification (summer, Sardá et al. 1999, Coma et al. 2000) and water column scarce food availability (late autumn-early winter). In early autumn all the community take advantage of the thermocline break-up, that fuel phytoplankton blooms with nutrients and still transparent waters, following the aestivation processes. But those blooms are very short in time (one- one and a half month), and the benthic community still have to face a second trophic crisis in the late autumn-early winter period. This assumption leads also to hypothesise that is not only temperature, but also food availability that in warm temperate seas give optimal reproductive conditions for organisms. This could be the reason why most benthic organisms release its sexual products between late winter and late spring (or even early summer), and only scarcely reproduce between late summer and early winter (Boero and Fresi 1986, Sardá et al. 1995, Lloret et al. 2000 as reviews of some groups).

Protein-carbohydrate-lipid tissue balance in benthic communities: a useful tool to register benthic-pelagic couplings.

In this study, it is clear that protein-carbohydrate-lipid balance may be an appropriate tool to register a set of episodes of the ecological cycles of the studied species related with trophic relationships, and hence for bentho-pelagic coupling processes in warm temperate communities. Fortunately, seston data are available concurrently with part of this tissue physiological balance (seston biochemical composition and concentration, chlorophyll a concentrations, zooplancton abundance and sedimented particulate organic matter, see Chapter I and Chapter III), and these data fit very well with our suggested point of view (i.e. in late autumn early winter the seston is very poor) . We consider that, if accompanied by a complete ecological knowledge of the species, the protein-carbohydrate-lipid balance indeed could be a good indicator of the events happened in the water column at the level of population. In summary, the biochemical balance could be a “memory” of the water column fluctuations and their influence in the benthos. Variability of seston concentration and quality, that may change in hours in the water column can be detected only if an intensive seston sampling (both in space and time) is made in the water column (Sournia 1974). Specially near the bottom, seston has special characteristics (Ritzrau et al. 1997, Thomsen and van Weering 1998), and this characteris-

PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE 147 tics make very difficult to asses clear seasonal trends in the biological composition. The “memory” of such water column seston processes has been also invoked for sedimented particulate organic matter (Boero 1994): Sedimented particulate organic matter collected in sediment traps may register periods of 4-10 days of water column biochemical changes, as an integer of such variable processes. But sedimented organic matter cannot reflect physiological behaviour and fitness of the benthic community. At least can r elate seston richness and availability to such community (Grémare et al. 1997). Such seston high or low availability, at the level of community processes, is reflected through this protein-carbohydrate- lipid balance. This balance let us interpret the late autumn-early winter trophic crisis, otherwise neglected because the apparent seston abundance that masks its low quality as a food.

148 PROTEIN-CARBOHYDRATE-LIPID SEASONAL BALANCE Conclusions

Temporal variability in near-bottom seston concentration and composition

• The near-bottom seston concentration (as calculated by means of a Secchi disc) is marked by pronounced seasonal fluctuations, with lower concentrations in late spring and summer and higher concentrations in autumn and winter and early spring. • The factors that best account for the variations in near-bottom seston concentration are intensity of river flow volumes and wave action. • The biochemical composition of the near-bottom seston (i.e., total carbon, organic carbon, organic nitrogen, proteins, and carbohydrat es) did not display any well-defined seasonal trend, except for chlorophyll a, which exhibited a peak coinciding with the phytoplankton peak in late winter/early spring. • The <10 µm fraction was the predominant fraction for all the biochemical factors considered over the entire study period (16 months), though the importance of the 10-100 µm fraction increased substantially during the winter-spring peak. • Inorganic carbon accounted for approximately 30% of the total carbon over the year, and organic nitrogen and proteins were closely related. • Organic carbon concentration in the form of proteins and carbohydrates was high (mean values higher than 50%) throughout the year, indicating that the benthic communities are assured a supply of available food. • There were no clear seasonal fluctuations in the near-bottom zooplankton. No distinct abundance peaks were observed in winter or spring. However, certain organisms (e.g., salps and bivalve larvae) were abundant at the time of the chlorophyll a peaks, especially in late winter, spring, and late summer. • The lack of a seasonal component in the biological factors of the near-bottom seston considered here, together with the pulses occurring over the study period, are suggestive of typical “border” system dynamics influenced by the proximity of the shore (e.g., river runoff, wave action, terrestrial geography, etc.) and by the proximity of the bottom (e.g., topography of the sea floor, the organisms present, near-bottom organic/inorganic particle dynamics, etc.). The findings indicate that unlike what happens in the surface portion of the water column, there is high variability in the processes regulating the near-bottom water layer.

Near-bottom seston composition in an intensive sampling cycle

• Differences in the values of the various biochemical factors considered were found in the two cycles carried out, in early spring (end of March) and late spring (end of June). Chlorophyll a values were higher in early spring and organic carbon concentrations (particularly in the form of proteins) were higher in late spring. • Chlorophyll a correlated well only with the sestonic carbohydrate concentration, probably due to an increase in the cellulose content brought about by higher abundance or a bloom of pigmented phytoplankton (algae). The rest of the biochemical factors were not correlated with chlorophyll a because the values of these factors may be influenced by detrital matter, ciliates, heterotrophic flagellates, heterotrophic bacteria, etc. • Correlations between the biochemical factors were higher in late spring than in early spring. This would seem to suggest that the biological dynamics of the seston in late spring arise from greater interaction among the different factors, probably because pulses and vari-

CONCLUSIONS 151 ability in the physical factors (river run-off, wave action, and temperature) are also more frequent, resulting in continuous resupply of inputs to the system. • Higher concentrations of matter available to suspension feeders in late spring and more continuous replenishment and diversification of that matter in the water column near the bottom may, together with higher light and temperature levels, be key to explaining higher growth and reproductive activity by most benthic organisms at that time of year as opposed to other times of year.

Seasonal and geographic comparison of sedimentary particulate organic matter

• Seasonal patterns in the quantity and quality of particulate organic matter (POM) sedimenting over the annual cycle, measured in parallel at the two sites, were observed in the Banyuls-sur-Mer and Medes Island regions. High seston concentrations with low organic matter contents were recorded in autumn-winter, contrasting with lower seston concentrations with high organic matter contents in spring-summer. This pattern was more readily discernible at Banyuls-sur-Mer than in the Medes Islands. • Hydrodynamic conditions at the two locations differed, with higher wind speed and wave height in Banyuls-sur-Mer than in the Medes Islands. Furthermore, the chlorophyll a concentration in the Medes Islands was higher than at Banyuls-sur-Mer all year round. • The differing hydrodynamic conditions could explain the difference in the concentration of organic matter at the two sites (higher in the Medes Islands than at Banyuls-sur-Mer), translating above all into a higher lipid content values for the sedimentary POM. • Sedimentary POM was more closely linked to primary production in the water column at Banyuls-sur-Mer than in the Medes Islands. In association with other factors, this suggests that river inputs played a greater role in seston dynamics in the Medes Islands, while resuspension appeared to be the factor that most clearly regulates seston dynamics at Banyuls-sur-Mer. • Measurement of sedimentary POM would appear to be an ideal method of recording changes in seston concentration and composition on a medium time scale (“memorizing” pulses that may occur on a shorter time scale), and this method could prove to be extremely important in drawing spatial comparisons over the course of seasonal cycles.

Activity rhythms in six species of passive suspension feeders

• The populations of five of the passive suspension feeding species studied (Paramuricea clavata , Eunicella singularis , Leptogorgia sarmentosa , Alcyonium acaule , and Parazoanthus axinellae ) all followed a seasonal pattern of activity in which colony/polyp expansion was lower in summer, particularly July and August. Corallium rubrum was the exception. This process was most marked in A. acaule , in which the number of dormant colonies rose concurrently with the deficit in available food that occurred at that time of year. • The responses of the activity rhythms of the colonies of the different organisms to the environmental factors considered differed. P. clavata colonies expanded their polyps in response to current flow, whereas E. singularis , L. sarmentosa , and A. acaule responded both to current flow and particle concentration levels (zooplankton and particulate protein).

152 CONCLUSIONS C. rubrum and P. axinellae seemed to respond only to particle concentration levels in the habitat (chlorophyll a, zooplankton, and proteins). • There was a clear synergistic effect among all the patches of the different species when current flow and particles (particularly zooplankton) were at optimum levels of flow rate and concentration during the intensive sampling cycles in early spring (end of March) and late spring (end of June). In those conditions all colonies were fully opened to the maximum extent. • Comparing the two cycles of intensive sampling (early and late spring), behaviour seemed differ, with the species considered responding to more sporadic pulses in late spring than in early spring. This translated into higher variability index values for colony expansion in the various species in late spring and greater coordination among expanded colonies of the different species in early spring. This may be attributable to higher levels of environmental variability in late spring. • Pronounced spatial heterogeneity was observed in the study populations. Patches of the same species only 20-40 m apart had quite different responses. Comparing a sampling site among rocky boulders with recurring daily flow patterns with a sampling site along a rock wall where daily flow patterns seemed to be absent, colonies of the same species might be completely expanded or completely contracted. Furthermore, there were twice the number of “dormant” A. acaule colonies on the rock wall than on the rocky boulders, two sampling locations with different current flow patterns. • There did not appear to be any endogenous factors regulating the daily activity patterns of the passive suspension feeders studied, as there are in other marine organisms (i.e., zooplankton, crabs, sessile intertidal organisms). Nevertheless, short-term responses to repeated environmental factors in winter-spring (i.e., more currents and higher current flow rates and higher seston pulse concentrations than in summer) do provide a basis for main- taining that there is an endogenous pattern to derive the maximum benefit from each and every event contributing to the food supply over the course of the annual cycle, with reserves being stored for subsequent use in reproduction and growth.

Diet and prey capture rates for the gorgonian Leptogorgia sarmentosa

• Leptogorgia sarmentosa , which dwells on soft, detrital substrata, feed principally on invertebrate eggs, bivalve larvae, copepod eggs and nauplii (stomach contents), as well as detrital organic matter, phytoplankton, ciliates, and heterotrophic nanoflagellates (experimental incubation chambers). • The size range of prey observed over the annual cycle was 4 to 800 µm, with most prey items having low mobility. The mean prey capture rate per polyp was 0.67 ± 0.39 SD, that is, around 0.101 ± 0.099 SD µgC·polyp -1 . • A considerable impact on bivalve larvae was observed during the course of the year, probably attributable to exploitation of the seston resulting from resuspension events. A colony measuring about 30 cm in height may contain some 9 000 bivalve larvae in differing stages of digestion. • Digestion in L. sarmentosa was slow, around 13 h at 13ºC and around 7 h at 21ºC. This could be related to highly efficient digestion and subsequent uptake of a variety of such hard to digest foods as phytoplankton, detritus, and bivalve larvae.

CONCLUSIONS 153 • A certain seasonal component was observed in the feeding pattern. L. sarmentosa ingested little carbon in the summer months (July and August) and in late autumn-early winter. • The study of two natural populations at two different localities, Banyuls-sur-Mer and the Medes Islands, about 50 km apart, over an annual cycle yielded significant differences in diet, prey items·polyp -1 , prey-containing polyps, and, in particular, µgC·polyp -1 . Carbon inputs were lower in the Banyuls-sur-Mer population (0.047 ± 0.09 SD µgC·polyp -1 ) than in the Medes Island population (0.073 ± 0.134 SD µgC·polyp -1 ) over the annual cycle considered. • The use of different sampling scales in the study has resulted in more accurate estimates of real capture rates than those obtained in previous studies.

Protein-carbohydrate-lipid balance in two benthic suspension feeders

• Both Paramuricea clavata and Leptogorgia sarmentosa use lipids as the principal organic macromolecule for energy storage (ranging from 120-360 µg lipid·mg AFDW -1 in P. clavata to 250-390 µg lipid·mg AFDW -1 in L. sarmentosa ). • The protein-carbohydrate-lipid balance in these two species was highly seasonal, but specific responses to periods of trophic crisis and food scarcity appeared to differ. Seasonal minima in P. clavata occurred in summer and late autumn-early winter (e.g., lipid values of 176 ± 42 SD µg lipid·mg AFDW -1 in summer and 166 ± 43 SD µg lipid·mg AFDW -1 in late autumn-early winter). In contrast, in L. sarmentosa the minimum values were recorded between late autumn and early winter (e.g., lipid values of 261 ± 44 SD µg lipid·mg AFDW - 1). The seasonal pattern for carbohydrates was quite similar to that of lipids in both species. Furthermore, there were differences in lipid concentrations in male and female P. clavata colonies in spring, while there were no differences in lipid concentrations in L. sarmentosa colonies at any time during the year. • Peak protein values were recorded during the growth periods for both species. This suggests that this biological parameter could be suitable for use as a growth indicator at the populational level. • There was no relationship between lipids and reproductive effort (measured as µg gonadal C polyp -1 ) in P. clavata in either males or females. On the other hand, immature colonies (<10 cm in height) displayed a pronounced seasonal cycle, which suggests the use of lipids primarily as a means of coping with trophic crises like those that occur in summer and late autumn. Lipid storage did appear to be related to the spawning period, but accumulation of lipids did not seem to bear directly on the number and/or diameter of reproductive polyps in this species. Conversely, lipid levels were closely related to reproductive effort in L. sarmentosa , suggesting that these two species use different reproductive strategies. • In both species differing hydrodynamic conditions and/or a more or less favourable position within the population and hence different prey capture rates yielded colonies with lipid reserves that were above average (higher capture rates) or below average (lower capture rates and thus less well-fed colonies). Therefore, the protein-carbohydrate-lipid balance would appear to be a suitable tool for determining whether a colony’s location is more or less favourable vis-à-vis environmental conditions and availability of the seston for capture.

154 CONCLUSIONS • Unexpectedly, the protein-carbohydrate-lipid balance indicated that the condition of the species studied was not good in late autumn-early winter. Primary production undergoes a sharp decline at that time of year because of high turbulence and water turbidity and hence lower light levels, resulting in minimum concentrations in the microbiological food web (ciliates, flagellates, etc.), in the zooplankton, and also in the sedimentary particulate organic matter (of very low nutritional value). All these factors combine to affect the populations of benthic suspension feeders and contribute to a second food deficit following the trophic crisis in the summer, at which time these organisms have minimal digestible carbon inputs and cease reproductive activity. This is clearly reflected in the decrease in the lipid and carbohydrate contents, and to a lesser extent in the protein content, in both species considered. • The protein-carbohydrate-lipid balance may represent an excellent mesoscale tool for recording coupling between the plankton and the benthos by “memorizing” environmental conditions in the habitats of benthic suspension feeders. Environmental pulses are in this way incorporated into the macromolecular balance of organisms. Nevertheless, this tool is unable to convey sufficient information in the absence of a thorough knowledge of a species’ biology and ecology (i.e., feeding, reproduction, growth, distribution, etc.).

CONCLUSIONS 155

Conclusiones

Variabilidad temporal de la concentración y composición del seston cerca del fondo.

• La concentración de seston cerca del fondo (calculada mediante el Disco de Secchi) tiene una marcada estacionalidad, siendo menor su concentración entre finales de primavera y verano, y mayor en otoño, invierno y principios de primavera. • Los factores que mejor explican la variación en la concentración del seston cerca del fondo son el caudal del río y la intensidad del oleaje. • La composición bioquímica del seston cerca del fondo (i.e. concentración de Carbono Total, Carbono Orgánico, Nitrógeno Orgánico, Proteínas y Carbohidratos) carece de una pauta estacional definida, excepto la Clorofila a que tiene un máximo coincidiendo con el máximo de fitoplancton de finales de Invierno y principios de Primavera. • Durante todo el período estudiado (16 meses), predomina en todas las variables bio- químicas estudiadas del seston la fracción <10 µm, excepto en los máximos de Invierno- Primavera donde la fracción entre 10 µm y 100 µm adquiere relevancia. • El Carbono Inorgánico representa a lo largo del año aproximadamente un 30% del Carbono Total y hay una estrecha relación entre Nitrógeno Orgánico y Proteínas. • La concentración de Carbono Orgánico en forma de Proteínas y Carbohidratos es alta durante todo el año (más de un 50% de media), lo que garantiza el alimento disponible para las comunidades bentónicas. • El zooplancton no tiene una variación estacional clara cerca del fondo. No se han observado claros picos de abundancia en invierno o primavera; sin embargo, hay organismos que abundan coincidiendo con los picos de Clorofila a (e.g. salpas y larvas de bivalvo), especialmente a finales de invierno, en primavera y a finales de verano. • La falta de estacionalidad encontrada en los componentes biológicos del seston analizados en la capa de agua cercana al fondo, acompañada de pulsos a lo largo del período estudiado hace pensar en una dinámica típica del sistema de “frontera”, en el que la influencia de la costa (e.g. ríos, oleaje, orografía, etc.) y del fondo (e.g. topografía del bentos, organismos, dinámica de la partícula orgánica /inorgánica cerca del fondo, etc.) son muy rele- vantes y añaden estocasticidad a los procesos de la columna de agua. Con ello podemos afir- mar que hay una gran variabilidad en los procesos que gobiernan esa capa de agua a diferencia de lo que pasa en la capa de agua de superficie.

Composición del seston durante ciclos intensivos cerca del fondo.

• En los dos ciclos estudiados (principio de Primavera [finales de Marzo] y finales Primavera [finales de Junio]) hallamos diferencias en la concentración de diferentes pará- metros bioquímicos analizados; la concentración de Clorofila a es mayor a principios de Primavera y la concentración de Carbono Orgánico (especialmente en forma de Proteínas) es mayor a finales de Primavera. • La Clorofila a sólo se relaciona bien con la concentración de carbohidratos del seston, seguramente por el aumento de celulosa debido a proliferación o mayor abundancia de pigmentos fitoplanctónicos (algas); el resto de los parámetros bioquímicos no están relacionados con la Clorofila a, debido a que pueden pertenecer a material detrítico, ciliados, flagelados heterotróficos, bacterias heterotróficas, etc.

CONCLUSIONES 159 • Hay una mayor correlación entre parámetros bioquímicos a finales de Primavera que a principios de Primavera: esto parece indicar que la dinámica biológica del seston a finales de Primavera es fruto de una mayor interacción entre los elementos, probablemente debido a que los pulsos de variabilidad también son más frecuentes en parámetros físicos (influencia del río, del oleaje y de la temperatura), lo que provoca una realimentación con- tinua del sistema. • Una concentración mayor de materia disponible a finales de Primavera para los sus- pensívoros bentónicos y una renovación y diversificación más continuada de esta materia en la columna cercana al fondo puede ser una de las claves que expliquen (junto con el aumento de luz y temperatura) una mayor actividad en los procesos de crecimiento y repro- ducción de gran parte de los organismos bentónicos durante este período respecto a otras épocas del año.

Comparación estacional y geográfica de la Materia Orgánica Particulada en sedimentación.

• Se ha observado un patrón estacional en la zona de Banyuls-sur-Mer y de las Illes Medes en cuanto a la cantidad y calidad de la Materia Orgánica Particulada (MOP) en sedi- mentación a lo largo de un ciclo anual paralelo. En Otoño-Invierno hay una gran concen- tración de seston con bajo contenido orgánico, mientras que en Primavera-Verano hay una menor concentración de seston pero con un elevado contenido orgánico: esta pauta es más clara en Banyuls-sur-Mer que en las Illes Medes. • Las condiciones hidrodinámicas de ambos lugares son diferentes, habiendo una intensidad de los vientos mayor en Banyuls-sur-Mer que en las Illes Medes. Además, la concen- tración de Clorofila a en las Illes Medes es mayor a lo largo del ciclo anual respecto a Banyuls-sur-Mer. • Las condiciones hidrodinámicas diferentes podrían explicar la diferencia en concen- tración de Materia Orgánica entre ambos sitios (mayor en las Illes Medes que en Banyuls- sur-Mer), lo que se refleja sobre todo en un más alto contenido de lípidos en la MOP en sedi- mentación. • Hay un vínculo más estrecho de la MOP en sedimentación con la producción primaria de la columna de agua en Banyuls-sur-Mer que en las Illes Medes: Esto sugiere, junto con otros factores, una influencia más acusada de los aportes fluviales en la dinámica del seston en las Illes Medes, mientras que la resuspensión parece el factor que gobierna más claramente la dinámica del seston en Banyuls-sur-Mer. • La MOP en sedimentación parece un método ideal para registrar a medio plazo los cambios en la concentración y composición del seston (“memoria” de los pulsos que puedan ocurrir a corto plazo), y puede ser de gran importancia a la hora de hacer comparacio- nes espaciales a lo largo de ciclos estacionales.

Ritmos de actividad en seis especies de suspensívoros pasivos

• Las poblaciones de las especies de suspensívoros pasivos estudiados ( Paramuricea clavata, Eunicella singularis, Leptogorgia sarmentosa, Alcyonium acaule, Parazoanthus axinellae ) siguen una pauta estacional de actividad (excepto Corallium rubrum ), en la que

160 CONCLUSIONES las colonias/pólipos se encuentran menos expandidos en verano (especialmente Julio y Agosto); Este fenómeno es más acusado en A. acaule , especie que incrementa su número de colonias en estado “letárgico” en plena coincidencia con la escasez de alimento disponible en esta época del año. • Las colonias de los diversos organismos responden de forma diferente en cuanto a su actividad respecto a los parámetros ambientales testados; así, las colonias de P. clavata expanden sus pólipos en respuesta a las corrientes, mientras que E. singularis , L. sarmentosa y A. acaule responden tanto a corrientes como a concentración de partículas (Zooplankton y Proteínas del seston); las especies de C. rubrum y P. axinellae parecen responder sólo a la concentración de partícula en el ambiente (Clorofila a, Proteína y Zooplancton) en el marco de las observaciones efectuadas. • En períodos intensivos testados (principio de Primavera [finales de Marzo] y finales Primavera [finales de Junio]), hay una clara sinergia de todas las poblaciones de las distintas especies cuando corriente y partículas (especialmente Zooplancton) se hallan en su velocidad/concentración óptimas; en ese momento todas las colonias se abren al 100%. • Parece haber un comportamiento diferente cuando comparamos ambos períodos intensivos de muestreo estudiados (i.e. principios y finales de Primavera), respondiendo las especies testadas a pulsos más esporádicos a finales de Primavera que a principios de Primavera; esto se ve reflejado en un mayor índice de variabilidad en la expansión de las colonias a finales de Primavera y a una mayor coordinación entre colonias expandidas de las diferentes especies a principios de Primavera; esto podría ser fruto de una mayor variabilidad ambiental en el período de finales de Primavera. • Se ha observado una heterogeneidad espacial muy marcada en las poblaciones estudiadas. Diferentes poblaciones agregadas (patch) sólo a 20-40 m de distancia de la misma especie responden de forma muy diferente: Así, cuando comparamos una zona de bloques de piedra donde hay patrones recurrentes diarios de corriente con una zona de pared donde parece que no hay patrón alguno de corrientes diario, colonias de la misma especie pueden hallarse totalmente expandidas o totalmente contraídas. Además, en A. acaule encontramos el doble de colonias “letárgicas” en la zona de la pared que en la zona de los bloques que presentan un régimen de corrientes distinto. • No parecen haber factores endógenos que rijan los ritmos de actividad diarios de los suspensívoros pasivos estudiados como en otros organismos marinos (i.e. zooplancton, cangrejos, organismos sésiles intermareales). Sin embargo, la respuesta puntual a factores ambientales más repetitivos en Invierno-Primavera (i.e. mayor frecuencia e intensidad de corrientes y mayor concentración de pulsos de seston que en Verano) hacen que a lo largo del ciclo anual sí pueda hablarse de una pauta endógena para aprovechar al máximo todos y cada uno de los eventos que aporten alimento, que se alma- cena para después ser aprovechados para la reproducción y el crecimiento.

Dieta y tasas de captura de la gorgonia Leptogorgia sarmentosa .

• Leptogorgia sarmentosa , que habita substratos detríticos y blandos, se alimenta principalmente de huevos de invertebrado, larvas de bivalvo, huevos de copépodo y nauplios (contenidos estomacales), así como de materia orgánica detrítica, fitoplancton, ciliados y nanoflagelados heterotróficos (cámaras experimentales).

CONCLUSIONES 161 • El rango de tamaño de presas observado a lo largo de un ciclo anual ha sido de 4 a 800 µm, siendo la mayoría de las presas de baja movilidad. La captura anual media por póli- po es de 0.67 ± 0.39 SD, lo que se traduce en unos 0.101 ± 0.099 SD µgC polipo -1 . • Se ha observado un impacto sobre larvas de bivalvo considerable a lo largo del año, probablemente debido a la explotación del seston proveniente de resuspensión. En una colonia de unos 30 cm de altura pueden hallarse unas 9000 larvas de este tipo en distintos estados de digestión. • La digestión de L.sarmentosa es lenta: unas 13 horas a 13ºC y unas 7 horas a 21ºC. Esto podría estar relacionado con una gran eficiencia en la digestión y posterior asimilación de fuentes diversas de alimento poco digerible como el fitoplancton, detritus o larvas de bivalvo. • Se ha demostrado una cierta tendencia estacional en los ritmos de actividad y en la alimentación. L.sarmentosa ingiere poco carbono en los meses estivales (Julio y Agosto) y a finales de Otoño- principios de Invierno. • Al estudiar dos poblaciones naturales durante un ciclo anual paralelo en dos localidades diferentes (Banyuls-sur-Mer y las Illes Medes, a unos 50 km de distancia), se han encon- trado diferencias significativas en la dieta, presas pólipo -1 , pólipos con presa/s y sobre todo µgC pólipo -1 . La población de Banyuls-sur-Mer tiene menos entradas de carbono (0.047 ±0.09 SD µgC polyp -1 ) comparada con la de les Illes Medes (0.073 ±0.134 SD µg polyp -1 ) a lo largo del ciclo anual estudiado. • El considerar para el mismo estudio diferentes escalas de muestreo ha permitido aco- tar mejor las tasas de capturas reales, comparado con estudios anteriores.

Balance Proteína-Carbohidrato-Lípido en dos suspensívoros bentónicos

• Las dos especies Paramuricea clavata y Leptogorgia sarmentosa utilizan los lípidos como macromolécula orgánica principal de almacenamiento de energía (oscilando entre 120-360 µg Lípido mg AFDW -1 en P.Clavata y 250-390 µg Lípido mg AFDW -1 en L.sarmentosa ). • Ambas especies tienen un balance Proteína-Carbohidrato-Lípido marcadamente estacional, aunque ambas parecen responder de forma diferente a épocas de crisis trófica. Así, P. clavata posee mínimos estacionales en Verano y finales de Otoño-principios de Invierno (e.g.: lípidos 176 ± 42 SD y 166 ± 43 SD µg Lípido mg AFDW -1 respectivamente), mientras que los mínimos registrados en L.sarmentosa se dan entre finales de Otoño-principios de Invierno (e.g.: lípidos 261 ±44 SD µg Lípido mg AFDW -1 ). Los carbohidratos siguen una pauta estacional muy similar (en ambas especies) a los lípidos. Por otro lado, en P.clavata hay diferencias entre colonias masculinas y femeninas en la concentración de lípidos, aunque sólo en primavera, mientras que en L. sarmentosa dicha diferencia es inexistente a lo largo de todo el año. • Las proteínas tienen máximos coincidentes con las épocas de crecimiento de ambas especies. Por ello se sugiere que la utilización de este parámetro bioquímico puede ser idó- neo para detectar épocas de crecimiento a nivel de población. • La relación entre lípidos y esfuerzo reproductor (medido en µgC gonadal pólipo -1 ) es nula en P. clavata tanto en machos como en hembras. Además, las colonias inmaduras (<10 cm de altura) poseen también un marcado ciclo estacional, lo que sugiere una utilización de

162 CONCLUSIONES los lípidos principalmente para afrontar crisis tróficas como la de verano o finales de otoño. Aparentemente hay una relación entre época reproductora y almacenamiento de lípidos, pero dicho acopio no parece reflejarse directamente en el número y/o diámetro gonadal de la especie. En L. sarmentosa sí existe una estrecha relación entre lípidos y esfuerzo reproductor, lo que sugiere una diferente estrategia reproductora en am bas especies. • Condiciones hidrodinámicas diferentes y/o posición más o menos aventajada dentro la población (capacidad distinta en la captura de presas) dan como resultado en ambas especies colonias cuya capacidad de almacenamiento de lípidos está por encima (mayores capturas) o por debajo (menos capturas o peor alimentadas) de la media; se sugiere el balance P-C-L como una buena herramienta para demostrar una situación más o menos favorable de una colonia respecto a las condiciones ambientales y a la capacidad de capturar seston. • El balance Proteína-Carbohidrato-Lípido ha demostrado una situación desfavorable e inesperada para las especies estudiadas entre finales de Otoño y principios de Invierno. En esta época del año, debido a un exceso de turbulencia y de turbidez de las aguas, hay un colapso de la producción primaria (por falta de luz) que se traduce en mínimas concentraciones en la red trófica microbiológica (ciliados, flagelados, etc.), en el zooplancton y finalmente en la materia orgánica particulada en sedimentación (de muy escaso valor nutritivo); estos factores revierten en las poblaciones de suspensívoros bentónicos, teniendo entradas mínimas de carbono digerible, no reproduciéndose y sufriendo una segunda crisis trófica (después de la estival). Esto se ve claramente reflejado en la disminución de lípidos y carbohidratos en ambas especies de suspensívoros estudiados, y en menor medida en las pro- teínas. • El balance P-C-L puede ser una excelente herramienta para registrar a medio plazo el acoplamiento plancton-bentos, al “memorizar” las condiciones ambientales que rodean los suspensívoros bentónicos. De este modo, los pulsos ambientales quedan integrados en el balance macromolecular de los organismos. Sin embargo, sin una comprensión profunda de la biología y ecología de las especies (i.e. alimentación, reproducción, crecimiento, distri- bución, etc.) la herramienta no transmite suficiente información.

CONCLUSIONES 163

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Factores ambientales que afectan la ecología trófica de los suspensívoros bentónicos

Introducción

El acoplamiento plancton-bentos y por tanto los factores ambientales que influyen la ecología de los organismos bentónicos han sido temas ampliamente debatidos durante los últimos años. La columna de agua y las comunidades planctónicas, por lo general consideradas mucho más dinámicas y variables que el bentos, al interaccionar con el substrato inter- cambian información y energía que posteriormente proporcionan estructura a las comunidades tanto en substratos duros como blandos sea por el hidrodinamismo dominante, el aporte de nutrientes y alimento o por ser la que aporta a la comunidad las perturbaciones ambientales y los nuevos reclutas en forma de larva, propágulo, etc. Por tanto, estudiar cuales son los factores de la columna de agua (especialmente las capas cerca del fondo) y en qué modo esos factores afectan los flujos de energía hacia el bentos (y desde el bentos) es un paso importante para comprender la ecología de los organismos que se hallan fijos en el substrato. En este trabajo se ha abordado el estudio de los factores ambientales que afectan a las comunidades bentónicas intentando integrar escalas de espacio y tiempo diferentes. Se han estudiado los factores ambientales y biológicos de la columna de agua (causa) considerando la biología-ecología de los supensívoros bentónicos (efecto). Las escalas espacio-temporales con las que ha de abordarse tal estudio son importantes, debido a que éstas serán diferentes según el objetivo final de nuestro análisis. Un muestreo anual (mensual, bimen- sual, semanal) es fundamental para registrar las tendencias estacionales de un ecosistema (si las hay). Pero se ha demostrado (en diferentes situaciones) que un muestreo de alta resolu- ción (escala diaria) puede ser clave a la hora de demostrar pulsos ambientales que expliquen a su vez pulsos de producción, aportes de alimento o cambios en la actividad de los organismos. Sin embargo, tanto la dinámica del seston como la actividad de los organismos poseen una variabilidad a veces demasiado elevada para poder hacer un balance global causa-efecto del acoplamiento plancton-bentos. Por eso se han buscado herramientas como, entre otras, las trampas de sedimento (para integrar fluctuaciones y pulsos del seston) y balances bioquímicos (que registren el estado fisiológico y actúen como memoria de los procesos que han ocurrido en la columna de agua y que han quedado registrados en los organismos bentónicos) capaces de explicar de una forma sólida la variabilidad temporal de un sistema. Se han abordado los aspectos anteriormente mencionados para comprender mejor las relaciones entre la columna de agua y las comunidades bentónicas a lo largo de este trabajo, así como los factores ambientales que influyen en los flujos de energía de los organismos bentónicos. Se presentan una serie de capítulos con los siguientes objetivos:

1)En el primer capítulo se presentan las relaciones entre la concentración del seston cerca del fondo con los diferentes factores ambientales que potencialmente puedan determinar su variabilidad (temperatura del agua, oleaje, descarga de un río anexo); al mismo tiempo se analiza por fracciones la composición bioquímica y biológica

RESUMEN 187 de dicho seston a través de un muestreo quincenal que permita hacer una interpre- tación estacional (si la hay) de estos parámetros. 2)En el segundo capítulo se presentan dos ciclos intensivos de muestreo ubicados en una misma época del año (la primavera), en los que se trata de estudiar la variabilidad de los factores ambientales y cómo éstos pueden afectar a la composición bioquí- mica del seston; de hecho, se trata de intentar registrar pulsos de producción en las capas de agua cercanas al fondo y relacionar su frecuencia con períodos de repro- ducción y crecimiento en las comunidades bentónicas. 3)En el tercer capítulo se comparan condiciones hidrodinámicas, concentración de fitoplancton en la columna y cantidad y calidad de la materia orgánica particulada en sedimentación de dos lugares a unos 50 km de distancia; la finalidad de esta com- paración espacial del seston es intentar determinar si las diferencias ambientales (si hay) se corresponden con diferencias en las comunidades bentónicas de ambos lugares a través de una herramienta (las trampas de sedimento) que puede integrar pulsos de producción-resuspensión en la columna de agua. 4)En el capítulo nº 4 se estudian los ritmos de actividad de seis suspensívoros pasivos (los antozoos Paramuricea clavata , Eunicella singularis , Leptogorgia sarmentosa , Corallium rubrum , Alcyonium acaule y Parazoanthus axinellae ) a diferentes escalas temporales (de forma estacional y a corto plazo) para comprender en qué forma afectan las variables ambientales (corrientes, concentración y composición del seston) a su dinámica diaria y anual. Esto permitirá encontrar un nexo entre la variabilidad ambiental y la respuesta de la comunidad bentónica para explotar esa variabilidad siguiendo la estrategia ecológica más adecuada. 5)En el quinto capítulo se hace una descripción exhaustiva de la dieta de un suspensí- voro pasivo que habita en fondos blandos-detríticos ( Leptogorgia sarmentosa ) a tra- vés de contenidos estomacales de un ciclo anual, experimentos de digestión a diferentes temperaturas (dieta natural), implementación de los ritmos de actividad a las tasas de captura y experimentos puntuales (primavera) de la fracción no cuantificable a través de contenidos estomacales (campanas de incubación). Todo ello se collementa con una segunda aproximación, la espacial, en la que dos poblaciones de dicho suspensívoro se comparan en su dieta y tasas de captura teniendo en cuenta factores ambientales potencialmente diferentes (Capítulo III). 6)El último capítulo se centra en el balance Proteína-Carbohidrato-Lípido como una herramienta que permita detectar cambios estacionales en el acoplamiento plancton- bentos, y relacionar estos cambios con distintos procesos de producción secundaria (reproducción, crecimiento, etc.) en dos especies de suspensívoros pasivos (Paramuricea clavata y Leptogorgia sarmentosa ) que potencialmente difieren en cuanto a sus estrategias tróficas y hábitat.

188 RESUMEN Material y métodos

Ciclos anual e intensivos del seston cerca del fondo

Ambos estudios (anual e intensivos) se efectuaron en la cara norte dels Tascons Grossos de las Illes Medes (42º02’55’’N, 3º13’30’’E, Noroeste del Mediterráneo), a unos 20 m de profundidad. Los parámetros físicos tales como altura de las olas, caudal del río anexo al área de muestreo (Ter) y temperatura del agua (a 20 m) fueron recolectados por la Estació Meteorológica de l’Estartit, así como el Secchi Vertical (transparencia del agua). Las corrientes (dirección e intensidad) fueron registradas también por la Estació Meteorológica de l’Estartit, a través de las boyas fijas de la Reserva Natural de las Illes Medes (Pasqual 1999). Los valores del Secchi Horizontal se registraron con ayuda de la escafandra autónoma, observando la extinción del disco a medida que un escafandrista nadaba alejándose del punto fijo en el que el disco se hallaba anclado. Para la obtención de las características bioquímicas y biológicas del seston las muestras se filtraron en tres fracciones: < 10 µm (nanoplancton), entre 10 µm y 100 µm (microplancton), y > 100 µm (microplancton-mesoplancton). En la fracción <10 µm se incluyó el aná- lisis de Clorofila a (fluorescencia), Carbono Total y Orgánico (análisis elemental) y Nitrógeno Orgánico (análisis elemental). En la fracción 10 < x < 100 µm se incluyó el aná- lisis de Clorofila a, Carbono Total y Orgánico, Nitrógeno Orgánico, Proteínas (espectrofo- tometría) y Carbohidratos (espectrofotometría). La fracción >100 µm consideró básicamen- te el zooplancton (sin contar huevos ni nauplios <100 µm), recolectado mediante pescas con redes arrastradas por escafandristas justo por encima de la comunidad bentónica (a 30-40 cm de los organismos) y observando las muestras a través de una lupa binocular. La periodicidad en el muestreo anual (Mayo 1997-Agosto 1998) fue de unos diez-quince días. En los muestreos intensivos, la periodicidad del primer muestreo (24-29 Junio 1997) fue de 6 horas, mientras que del segundo (27-30 Marzo 1998) fue de 4 horas.

Comparación de ciclos anuales de materia orgánica particulada en sedimentación

Dos trampas de sedimentación fueron depositadas a treinta metros de profundidad, una en Banyuls-sur-Mer (42º29’30’’N, 3º08’70’’E, Francia) y la otra en las Illes Medes (42º02’55’’N, 3º13’30’’E, España), a unos 50 km de distancia (Fig. 14a,b). Dichas trampas (idénticas en su forma y estructura) tenían un cilindro terminal que era recolectado sema- nalmente de forma alterna (una semana sí y otra no), con lo que se efectuó un muestreo quincenal en el período comprendido entre Julio 1997 y Agosto 1998. La intensidad y dirección del viento fue registrada diariamente, así como la altura de las olas (Estación Meteorológica del Estartit y Estación Meteorológica de Cap Bear). La con- centración de Clorofila a fue muestreada cada 10-15 días en ambas zonas a unos 20 m de profundidad durante el período de muestreo, el agua fue filtrada y el filtro procesado fluo- rométricamente. El sedimento recolectado en las trampas era trasladado al laboratorio donde se centrifu- gaba, congelaba, liofilizaba y pesaba para posteriores análisis bioquímicos. De cada muestra se analizó materia orgánica (calcinación), carbono y nitrógeno orgánicos (analizador elemental), así como proteínas, carbohidratos y lípidos (espectrofotometría).

RESUMEN 189 Ritmos de actividad de seis cnidarios bentónicos

El estudio de la actividad de seis cnidarios antozoos del Mediterráneo occidental (Paramuricea clavata , Eunicella singularis , Leptogorgia sarmentosa , Corallium rubrum , Alcyonium acaule y Parazoanthus axinellae ) fueron realizados en els Tascons Grossos, Illes Medes, en dos áreas diferentes: una pared (Fig. 22a) y un canal con grandes bloques (Fig. 22b), en los que todas las especies estaban representadas (excepto la pared, donde Leptogorgia sarmentosa no se hallaba presente). En todos los casos se registró el estado de expansión/contracción de los pólipos de las colonias (o sencillamente los pólipos en el caso de Parazoanthus axinellae ): totalmente abiertos /turgentes/totalmente cerrados. En el caso de Alcyonium acaule también se tuvieron en cuenta las colonias en letargo, reconocibles por el fino velo que las envuelve (Fig. 24). Se efectuó un seguimiento estacional de 1 a 14 veces cada mes de las seis especies desde Septiembre 1996 a Agosto 1998. Para testar los factores ambientales que podían influir en los ritmos de actividad de las especies estudiadas se hicieron dos campañas intensivas (24-29 Junio 1997, observaciones y muestros cada 6 horas, y 27-30 Marzo 1998, observaciones y muestros cada 4 horas) en las que se registraba la actividad de los organismos y diversos parámetros: Corrientes (con un correntímetro ADP-doppler), Zooplancton ambiente (pescas mediante escafandristas), Clorofila a (fluorometría), carbohidratos y proteínas (espectrofotometría). Por otro lado, se hizo una comparación espacial, en la que se registraron al mismo tiempo los ritmos de actividad de cinco especies ( Paramuricea clavata , Eunicella singularis , Corallium rubrum , Alcyonium acaule y Parazoanthus axinellae ) en dos lugares separados a escasos 20-40 m de distancia (pared-bloques, Fig. 22a,b ). En la zona de la pared, también se realizaron dos ciclos diarios (18 y 20 de Junio de 1997), cada 3 horas, para registrar posibles pautas diarias a corta escala de tiempo.

Dieta y tasas de captura del suspensívoro bentónico pasivo Leptogorgia sarmentosa

La dieta del antozoo Leptogorgia sarmentosa (habita en fondos blandos-detríticos) se siguió desde Julio 1997 a Agosto de 1998 cada quince días en 32 colonias (pólipos siempre expandidos), a unos 20 m de profundidad en els Tascons Grossos (Illes Medes). Los contenidos estomacales (pólipos) fueron identificados y cuantificados en la lupa binocular. Se hicieron dos experimentos de digestión con dieta natural a temperaturas consideradas extremas en el ciclo anual de la especie (13ºC y 21ºC), recolectando ramas de la población al azar que fueron mantenidas a una temperatura constante y fijadas en intervalos regulares (1 hora) para observar la extinción de presas en los pólipos. Asimismo, se realizó un estudio biométrico exhaustivo (nº de ramas 1ª, 2ª, etc y longitud total, pólipos mm -1 , materia seca y materia orgánica libre de cenizas) para poder hacer extrapolaciones de la captura de presas a nivel de colonia. Para hacer las extrapolaciones a nivel de población se hicieron transectos que calculaban la densidad de colonias (y tallas). Para completar el espectro de presas y la tasa de captura en presas no cuantificables a través de los contenidos estomacales, se realizaron seis experimentos con campanas de incubación de flujo continuo en las que se determinaba la depredación por parte de la gorgonia de fitoplancton, ciliados, bacterioplancton, nanoflagelados, materia orgánica disuelta y materia detrítica.

190 RESUMEN Para evaluar posibles diferencias en la dieta y captura de presas a lo largo de un ciclo anual en dos lugares en los que las condiciones ambientales potencialmente podían ser diferentes (Banyuls-sur-Mer, 10 m de profundidad, 42º29’30’’N, 3º08’70’’E, Francia; Illes Medes, 30 m de profundidad, 42º02’55’’N, 3º13’30’’E, España), se procedió a seguir colonias de ambos lugares (20 colonias en cada lugar) de la misma forma que la descrita para la población de 20 m (durante el período Mayo 1998-Agosto 1999).

Balance Proteína-Carbohidrato-Lípido en dos suspensívoros pasivos a lo largo de ciclos estacionales

Durante un período de tres años (Febrero 1997-Enero 2000) se hizo un seguimiento de una población marcada de la gorgonia Paramuricea clavata a 20 m de profundidad en els Tascons Grossos, Illes Medes (Fig. 39a, 10 colonias machos y 10 colonias hembras mayores de 40 cm, consideradas sexualmente maduras). En dicho muestreo se recolectaron mensualmente dos ramas de cada colonia para proceder a analizar el contenido de Proteínas, Carbohidratos y Lípidos a través de técnicas espectrofotométricas. De esas mismas colonias se procedió a evaluar su esfuerzo reproductor para relacionarlo con dicho balance, recolectando ramas de las mismas colonias en los meses de Mayo de 1997, 1998 y 1999 para cuan- tificar el carbono que representaban las gónadas masculinas y femeninas (este mes puede considerarse como el momento previo a la liberación de productos sexuales). Como trabajo previo al seguimiento anual y para calibrar las variaciones posteriores, se evaluó la diferencia de Proteínas-Carbohidratos-Lípidos en una misma rama de tipo prima- rio (apical), entre ramas primarias, y entre ramas primarias, secundarias, terciarias, etc. Para evaluar si sólo el esfuerzo reproductor es el responsable del acúmulo de reservas energéticas o puede considerarse también un acúmulo destinado a afrontar una posible crisis alimenticia, de 14-20 colonias de a misma gorgonia < 10 cm (consideradas inmaduras) fueron recolectadas mensualmente (desde Julio 1998 a Julio 1999), analizándose espectro- fotométricamente Carbohidratos y Lípidos. Por otro lado, desde Mayo de 1998 a Agosto de 1999, 20 colonias de la gorgonia Leptogorgia sarmentosa (8 machos y 12 hembras, > 30 cm, consideradas sexualmente maduras) fueron recolectadas mensualmente a 30 m de profundidad (Fig. 39b) para compa- rar su balance Proteína-Carbohidrato-Lípido con el de la gorgonia Paramuricea clavata . En este caso también se hicieron test para evaluar diferencias dentro de una misma rama apical (el tipo de rama muestreada) y entre ramas apicales de la misma colonia.

Resultados

1. En el ciclo estacional que se siguió durante los años 1997 y 1998 se ha visto una rela- ción entre el oleaje-descarga del río (Ter) y el Secchi vertical y horizontal (concentración total de seston). Por otro lado, la temperatura también tenía una relación inversa significativa con la concentración total del seston (transparencia). La transparencia del agua sigue un marcado ciclo estacional, con valores mínimos en invierno (Secchi Vertical : 16.7 m, Secchi Horizontal: 9.3 m) y máximos en verano (Secchi Vertical : 24.6 m, Secchi Horizontal : 23.6 m). Hay una relación significativa entre la transparencia tomada verticalmente y horizontalmente.

RESUMEN 191 La fracción < 10 µm domina en la masa de agua cercana al fondo estudiada en todos los parámetros. La Clorofila a < 10 µm representa entre un 72 y un 95 % de la Clorofila a < 100 µm, excepto en los blooms de invierno-primavera, donde oscila entre el 37% y el 58%. La Clorofila a es el único parámetro que muestra una clara pauta estacional. En el caso del Carbono Particulado Total, la fracción < 10 µm representa un 62% de la < 100 µm, pero cuando tenemos en cuenta el Carbono Particulado Orgánico, esa fracción alcanza una media anual del 84%; ninguno de estos dos parámetros muestra una pauta estacional clara a través del período estudiado. El Nitrógeno Particulado Orgánico está compuesto en un 97% por la fracción < 10 µm y tampoco muestra estacionalidad. El índice C/N es mayor cuando se considera la fracción entre 10-100 µm (CN: 12) que cuando se considera la < 10 µm (CN: 7). Proteínas y Carbohidratos no parecen tener una tendencia estacional clara, aunque las pro- teínas adquieren sus mínimos valores entre finales de otoño y principios de invierno. Proteínas y Nitrógeno Particulado Orgánico poseen una estrecha correlación. El zooplancton cercano al fondo no posee, en general, una pauta estacional. Los organismos más abundantes son los copépodos seguidos a distancia por apendicularias y larvas de bivalvos. Larvas de bivalvos y de equinodermos, así como doliolidos tienen una relación significativa con los picos de Clorofila a que se producen a lo largo del período estudiado.

2. Durante los ciclos intensivos estudiados (principios de primavera, Marzo 1998; finales de primavera, Junio 1997), la concentración de Clorofila a fue significativamente mayor a principios (0.670 ± 0.134 µg Chl a l-1 ) que a finales de primavera (0.414 ± 0.094 µg Chl a l-1 ). Por otro lado, los valores de Carbono Particulado Total y Orgánico, así como el Nitrógeno Particulado Orgánico fueron más elevados a finales de primavera que a inicios de la misma; también los índices de variación (varianza/media) fueron mayores a finales de Junio respecto a los de finales de Marzo. La concentración de Proteínas fue superior a finales de primavera que a principios, pero no la concentración de Carbohidratos. Haciendo la correlación de los diferentes parámetros bioquímicos estudiados encontramos que los Carbohidratos sólo correlacionan significativamente con la Clorofila a, y que hay una correlación más estrecha entre los distintos parámetros a finales de primavera (Junio) que a principios de primavera (Marzo). Por otro lado, los índices de variación de los parámetros físicos estudiados (temperatura del agua a 20 m, caudal del río, corrientes) son mayores en Junio que en Marzo (excepto para el oleaje, que son iguales).

3. Los resultados de la comparación entre la zona de Banyuls-sur-Mer y las Illes Medes da como resultado una mayor intensidad de vientos y oleaje en Banyuls-su-Mer que en las Medes; la concentración de Clorofila a a lo largo del año es mayor en las Medes que en Banyuls. No hubo una diferencia significativa entre ambos lugares en cuanto a las tasas de sedi- mentación, pero la variabilidad (y estacionalidad) son más marcadas en Banyuls-sur-Mer que en las Illes Medes. La concentración de materia orgánica era mayor a lo largo del ciclo estudiado en Medes que en Banyuls. La concentración de Carbono Orgánico en la materia en sedimentación no es diferente entre ambos lugares, mientras que la de Nitrógeno sí. La relación C/N sí que muestra diferencias, siendo mayor en las Medes que en Banyuls. La concentración de Proteínas y Carbohidratos de la materia en sedimentación son

192 RESUMEN menos variables en las Medes que en Banyuls; las Proteínas no difieren en su concentración de forma significativamente entre lugares, mientras que los Carbohidratos son más abundantes en Banyuls que en las Medes. La concentración de Lípidos en la materia en sedi- mentación es mayor en las Medes que en Banyuls.

4. El estudio de ritmos de actividad y los parámetros ambientales que están relacionados con la actividad de los seis antozoos observados dan como resultado una tendencia estacional a estar menos activos en el período estival, excepto Corallium rubrum . De Enero a Junio (inclusive) parece haber una cierta regularidad en las poblaciones estudiadas, estando siempre más de un 50% expandidas (excepto Alcyonium acaule ), mientras que de Julio a Diciembre parece que las poblaciones entran en una dinámica de expansión más irregular. Alcyonium acaule empieza a mostrar colonias en fase inactiva en Junio, con máximos en Julio y Agosto y otra vez una disminución en Septiembre. En los ciclos intensivos que se hicieron para comprobar qué factores ambientales influyen en la abertura de los diferentes organismos pudo observarse una tendencia por parte de Paramuricea clavata a responder al ritmo de las corrientes, mientras que los ciclos de actividad (expansión) de Eunicella singularis y Leptogorgia sarmentosa parecen estar relacionados con la concentración de partícula y las corrientes de forma similar; Alcyonium acaule parece sensible a las corrientes, zooplancton y proteínas del ambiente. Corallium rubrum parece estar relacionado sólo por la concentración de proteínas (y sólo en las observaciones de Marzo), mientras que Parazoanthus axinellae responde al zooplancton y a la concentra- ción de clorofila a, aunque con signo opuesto en su correlación en los dos períodos estudiados. Parece haber un patrón más irregular (más variable) en los ritmos de expansión/con- tracción de los pólipos a finales de primavera que a principios. La correlación entre expan- sión/contracción de las especies es mayor en Marzo que en Junio. En ambos períodos, la gorgonia Paramuricea clavata es la única que parece responder a un patrón regular de expansión-contracción de los pólipos según las corrientes dominantes en el canal (bloques). Todos los organismos (en ambos períodos) parecen responder sinérgicamente a un máximo simultáneo de concentración de partícula y de velocidad de corrientes. A corta distancia (entre la zona de la pared y la de los bloques) la expansión de las colonias estudiadas en junio es muy diferente. Sólo a finales del período observado (cuando tenemos el máximo de partículas/corriente antes mentado), todas las colonias de ambos lugares muestran su máxima expansión. No hay un patrón diario en la expansión de los pólipos de los organismos observados (ritmo endógeno) en los dos períodos intensivos de muestreo (18 y 20 de Junio), respondiendo aparentemente a factores externos sin pauta circadial.

5. El estudio de la dieta y tasas de captura del suspensívoro pasivo Leptogorgia sarmentosa muestra un rango de presas no menor a 3.8 µm (nanoflagelados heterotróficos) y no mayor de 800 µm (larva de invertebrado), con una media de 140 ± 106 SD µm. Las presas (huevos de invertebrado, larvas de bivalvo, materia orgánica detrítica, fitoplancton y huevos de copépodo como las más abundantes) son consideradas de escasa movilidad. La captura de presas por pólipo es de 0.67 ± 0.39 SD, mientras que el carbono ingerido por pólipo es del orden de 0.101 ± 0.099 SD µgC. Aparentemente no hay una fuerte estaciona-

RESUMEN 193 lidad durante el período muetreado, aunque hay una tendencia a capturar menos presas durante el verano que en otras épocas del año. La digestión, de unas 13 horas a 13ºC y 7 horas a 21ºC, no parece influeir en las tasas de captura (carbono ingeridi/asimilado) en el período en el que se realizó el estudio. La depredación sobre larvas de bivalvo puede ser especialmente intensa, sobre todo cuando coincide aparentemente con pulsos de liberación/resuspensión de estas presas. Los ritmos de actividad siguen una tendencia estacional, aunque no una pauta clara de contrac- ción de los pólipos en período estival. Por otro lado, esta gorgonia depreda sobre la fracción detrítica de forma activa. No captura presas de tamaño inferior a 4 µm, y se ha calculado en primavera una tasa de captura de 263 ±111 SD nanoflagelados heterotróficos pólipo -1 h-1 , 9±3 SD células de fitoplancton pólipo -1 h-1 (diatomeas y dinoflagelados autotróficos) y 0.3 ±0.1 SD ciliados pólipo -1 h-1 . También se alimenta de forma significativa de MOP detrí- tica (0.07 ± 0.04 µgC pólipo -1 día -1 ). La comparación espacial (Banyuls-sur-Mer y Medes) ha dado como resultado una diferencia significativa entre ambos lugares tanto en el número de pólipos llenos (un 31% a lo largo de al año en las Medes y un 21% en Banyuls), el número de presas(contenidos estomacales) por pólipo (0.53 ± 0.45 SD en Medes y 0.3 ± 0.22 Sd en Banyuls) y el carbono que esas presas significa para cada pólipo (0.073 ± 0.134 SD en Medes y 0.047 ± 0.09 SD en Banyuls) durante el período de dieciséis meses testado.

6. Cuando se analizaron los resultados del balance estacional Proteína-Carbohidrato- Lípido de la gorgonia Paramuricea clavata se pudo ver que no habían diferencias entre machos y hembras excepto para los lípidos en primavera. Las proteínas tienen mínimos en el periodo estival, y picos máximos en otoño y en invierno primavera. Los carbohidratos también siguen una pauta estacional, con máximos en invierno primavera y mínimos en verano-otoño. Esta misma pauta es observada también en los lípidos. Las colonias inmaduras (<10 cm) tam- bién siguen esta pauta estacional de carbohidratos y lípidos en el período estudiado. Leptogorgia sarmentosa tiene una tendencia estacional menos marcada en su balance Proteína-Carbohidrato-Lípido. No hay diferencia alguna entre machos y hembras en la con- centración de ninguna de las macromoléculas estudiadas a lo largo del período estudiado. Las proteínas no parecen seguir una pauta estacional clara, mientras que carbohidratos y lípidos tenen mínimos entre finales de otoño y principios de invierno. Hay una variación interanual mucho más marcada en Leptogorgia sarmentosa que en Paramuricea clavata . Por otro lado, la concentración de lípidos es significativamente más elevada a lo largo del ciclo estudiado en Leptogorgia sarmentosa que en Paramuricea clavata . La relación con el esfuerzo reproductor (expresado en µgC pólipo -1 ) con el balance bio- químico es nula en las hembras de Paramuricea clavata , y sólo posee una cierta relación la concentración de proteínas con el esfuerzo reproductor en machos. En Leptogorgia sarmentosa , la concentración de lípidos está relacionada con el esfuerzo reproductor tanto de hembras como de machos, aunque la relación en hembras es más clarae. Ningún otro pará- metro de los estudiados parece relacionarse con el esfuerzo reproductor. Ambas especies parecen tener colonias más o menos capaces de acumular reservas a lo largo de los ciclos anuales estudiados, colonias que aparentemente están más o menos favorecidas potencialmente por una situación diferente dentro de la población en la que viven. Esto puede estar relacionado con una mayor captura de presas.

194 RESUMEN Discusión y Conclusiones

Ciclo anual del seston cerca del fondo

Por lo que hemos podido observar, hay una buena relación entre dos variables ambientales independientes entre sí (descarga del río anexo a la zona de muestreo, el Ter, y altura del oleaje) y la concentración de seston en la capa de agua cerca del fondo (disco de Secchi). La primera variable ambiental sigue una pauta estacional, mientras que la segunda no, sugi- riendo una fuente de variabilidad considerable por resuspensión que podría provocar en parte la falta de variabilidad en los componentes bioquímicos del seston. Por otro lado, buena parte de la varianza de la concentración del seston (expresada como Secchi Horizontal o Vertical) viene dada por las fluctuaciones de temperatura, siendo la relación inversa e interpretándose como relación directa entre estratificación/mezcla según la época del año. La fracción < 10 µm es predominante durante todo el período estudiado, excepto en invierno primavera, momento en que la Clorofila a entre 10-100 µm adquiere mucha relevancia. No así el Carbono Particulado Total y Orgánico, que, junto con otras variables como el Nitrógeno Particulado Orgánico, las Proteínas o Carbohidratos que tampoco son marcadamente estacionales y muestran una dominación casi absoluta de la fracción < 10 µm en la zona muestreada. El hecho de encontrar una fracción < 10 µm dominante en un mar gene- ralmente oligotrófico como el Mediterráneo era algo común en ciclos realizados en mar abierto, pero menos esperable cerca de la costa. Los procesos estocásticos que se producen en la capa de agua cerca del fondo, relacionados con la proximidad con la costa y con el fondo, enmascararán la estacionalidad que en cambio encontramos lejos de la costa y cerca de la superficie. El efecto costero, la orogra- fía compleja (que puede cambiar intensidad y dirección de los vientos) y la influencia de aportes fluviales pueden ser factores claves a la hora de describir la dinámica del seston en esta capa de agua. Por otro lado, por parte del bentos, la topografía del fondo (que influirá en las corrientes y en la formación de distintas capas de mezcla del agua), la propia diná- mica de la partícula cerca del fondo (descrita como muy compleja a medida que nos acercamos al sustrato) y la existencia de una comunidad bentónica que conlleva procesos de retroalimentación muy acusados cerca del fondo (alimentación, reproducción, formación de detritus, excreción, etc.) hacen que se produzcan pulsos continuos que generan una falta de estacionaidad a escala anual en los procesos físicos y biológicos que ocurren cerca del fondo.

Ciclos intensivos del seston cerca del fondo en primavera

La aproximación intensiva de la dinámica bioquímica del seston se hizo en primavera, pero en dos épocas contrastadas. La primavera es una época de intensa producción y la idea era explicar cambios en la biomasa según si teníamos más o menos variabilidad ambiental. En este sentido, parece que a finales de primavera (Junio) la situación ambiental funciona más a pulsos en cuanto a los aportes del río, movimientos de la termoclina (inexistente en Marzo) y corrientes de la zona (intensidad y dirección). Uno de las observaciones más des- tacables es que a pesar de haber más concentración de Clorofila a (relacionadas con aumen-

RESUMEN 195 tos o disminuciones de carbohidratos), la biomasa (expresada como CPO o como Proteínas) es más abundante a finales de primavera. Por tanto, durante la primavera encontramos dos situaciones contrastadas, con distinta correlación de los parámetros bioquímicos analizados siendo a finales más relacionada que no a principios, quizás por una mayor retroalimentación y variabilidad del sistema. Estos ciclos intensivos han demostrado ser apropiados para entender la variabilidad en períodos de alta productividad, como la primavera. Nuestras observaciones permiten corro- borar la hipótesis es que breves pero intensos períodos de productividad en el agua cercana al fondo pueden ser importantes para la comunidad bentónica. Se sugiere que una más elevada biomasa y una más elevada variabilidad en los parámetros bioquímicos estudiados, junto con temperaturas (e irradiancia) más altas y pulsos de alimento asociados al seston a finales de primavera pueden explicar muy bien los niveles y ritmos de producción (crecimiento y reproducción) de los organismos bentónicos en esta época del año.

Comparación de ciclos anuales de materia orgánica particulada en sedimentación

Los ciclos de materia orgánica particulada en sedimentación efectuados en Banyuls-sur- Mer y en las Illes Medes dieron como resultado una dinámica del seston muy diferente. Las diferentes condiciones atmosféricas e hidrodinámicas, reflejadas en parte por una diferencia de la intensidad de los vientos y el oleaje (mayores en Banyuls-sur-Mer) y en parte por una influencia fluvial menos importante en Banyuls-sur-Mer, hacen pensar que el seston en sedimentación está influido más por factores de resuspensión en Banyuls que en las Medes. Por otro lado, una mayor concentración de fitoplancton (estimado en forma de Clorofila a) en las Medes da una idea de mayor productividad o al menos de mayor biomasa disponible en este lugar. Estas diferencias ambientales y biológicas sugieren que la materia orgánica de ambos lugares (mayor en las Medes) y de las características bioquímicas de la MOP son muy distintas incluso a poca distancia, y que las generalizaciones a partir de un estudio en un solo lugar pueden ser arriesgadas. Aunque la media de las tasas de sedimentación fueron similares en ambos lugares (no hubieron diferencias estadísticas significativas), la variabilidad de dichas tasas fue mucho más marcada (y claramente estacional) en Banyuls respecto a las Medes. Este funciona- miento se da en otros parámetros, donde la señal estacional se ve más atenuada en las Medes que en Banyuls. Las concentraciones de Carbono Orgánico no fueron tampoco diferentes, pero sí las de Nitrógeno Orgánico (mayores en Banyuls), por lo que los índices C/N eran mayores en la zona de Medes que en la de Banyuls. Esto sugiere otra vez que un aporte del río, así como una clara influencia de material detrítico por parte de algas macrófitas y de las praderas de fanerógamas anexas en las Illes Medes tienen una gran importancia para explicar las características del seston observadas. Una mayor concentración de lípidos en las Medes a lo largo del período anual analiza- do (material considerado como buen indicador de la disponibilidad alimenticia de la materia orgánica en sedimentación) sugiere una mayor calidad de la materia orgánica en sus- pensión que podría revertir en la biomasa y producción de la comunidad bentónica. Las características de la MOP presentadas aquí pueden ser muy útiles a la hora de contrastar dos áreas distintas, ya que la materia en sedimentación integra la dinámica y los pulsos del seston cerca del fondo, más difíciles de detectar y enmarcar en un ámbito estacional a través

196 RESUMEN de muestros directos en la columna de agua. Se considera que este procedimiento compara- tivo y la implementación de datos ambientales (como vientos,oleaje, temperatura, descarga fluvial) son un método muy útil para explicar diferencias en composición y biomasa bentó- nica.

Ritmos de actividad de seis cnidarios bentónicos

La tendencia estacional registrada en cinco de los seis organismos estudiados (excepto Corallium rubrum ) en la que los pólipos están contraídos durante el período estival (especialmente Julio y Agosto), parece reforzar la idea de una crisis trófica durante el período de máxima estratificación en verano. Alcyonium acaule presenta un aspecto más evidente si cabe, y es la presencia a partir de Junio de colonias inactivas (recubiertas por un fino velo que aísla del exterior), aumentando en número durante Julio y Agosto y decreciendo súbi- tamente en Septiembre. La falta de eventos de resuspensión frecuentes, la fuerte estratifica- ción y disminución de la velocidad de las corrientes, así como un coste elevado energética- mente manteniendo los pólipos abiertos, son factores que sugieren un inconveniente trófico para algunos organismos bentónicos (como los suspensívoros pasivos). Por otro lado, Corallium rubrum parece ser muy fluctuante en su respuesta sin una tendencia estacional clara, debido probablemente al aprovechamiento de eventos de resuspensión y variabilidad de microhábitat. La respuesta diferencial de los organismos de cara a los factores ambientales testados (corrientes), y biológicas (concentración de diferentes elementos del seston) es quizás sín- toma de una diversidad en las estrategias tróficas y de su propia biología. Así, Paramuricea clavata parece estar estimulada sólo por la intensidad de la corriente, quizás por su estructura de población tendente a formar densas poblaciones que podrían convertirse en trampas para el seston. Eunicella singularis , Leptogorgia sarmentosa y Alcyonium acaule parecen tener una respuesta mixta entre corriente y partícula, estando las colonias más dispersas y quizás siendo capaces de aprovechar más eventos de resuspensión. Corallium rubrum y Parazoanthus axinellae son los únicos que no responden aparentemente a las corrientes en las observaciones llevadas a cabo, siendo posiblemente la concentración de algún paráme- tro bioquímico lo que los activa de forma significativa. Toda esta complejidad en la respuesta de los organismos se ajusta bien a una marcada heterogeneidad propia de la comunidad del coralígeno. Nuestros resultados parecen indicar una diferencia entre los ciclos intensivos, siendo más variable la respuesta a finales de primavera que a principios, quizás debido a una mayor variabilidad de los factores ambientales antes descrita. Sin embargo, un proceso que puede observarse en ambos momentos muestreados es una sinergia de toda la comunidad cuando concentración de partícula (zooplancton, proteína) y la corriente alcanzan su punto máximo de forma paralela durante el período observado. Es como si todos los organismos aprovecharan el pulso de comida y condiciones hidrodinámicas favorables. La diferencia espacial encontrada en la actividad de los organismos testados es importante: mientras una población estaba completamente abierta, 20 m más allá otra población estaba con los pólipos contraídos. La diferencia espacial en cuanto a las condiciones ambientales puede por tanto ser muy grande, lo que sugiere poblaciones que pueden estar tróficamente más o menos favorecidas.

RESUMEN 197 Los ritmos de actividad nos ayudan a entender los niveles de sinergia de los suspensí- voros pasivos frente a fluctuaciones ambientales que revierten en su ecología trófica: 1) Estacionalmente, porque estos organismos mostrarán menos actividad en los tiempos menos favorables del año (verano en los marestemplado-cálidos); 2) Diariamente, porque la tendencia será aprovechar los pulsos más favorables (o sea cuando seston y corriente tengan sus condiciones más adecuadas para la captura). Nuestra conclusión es que mientras los ritmos diarios de estos suspensívoros pasivos son gobernados por los factores exógenos, los ritmos estacionales pueden estar relacionados con factores más endógenos, debido al ajuste del reloj interno a factores repetitivos de más o menos frecuencia en los pulsos de producción de la columna de agua y en concordancia con el período reproductor de cada especie.

Dieta y tasas de captura del suspensívoro bentónico pasivo Leptogorgia sarmentosa

La gorgonia Leptogorgia sarmentosa , que habita substratos detríticos y blandos, se alimenta principalmente de huevos de invertebrado, larvas de bivalvo, huevos de copépo- do y nauplios (contenidos estomacales), así como de materia orgánica detrítica, fitoplancton, ciliados y nanoflagelados heterotróficos (cámaras experimentales). El amplio espectro de presas (que pueden considerarse de baja movilidad o pasivas) es típico de los suspensívoros pasivos que por lo general basan su estrategia trófica en dietas generalistas/ oportunistas. Hay un impacto sobre larvas de bivalvo considerable a lo largo del año, probablemente debido su abundancia en el seston o proveniente de resuspensión; en una colonia de unos 30 cm de altura pueden hallarse unas 9000 larvas de este tipo en distintos estados de digestión. Por otro lado, la digestión de esta gorgonia es lenta: unas 13 horas a 13ºC y unas 7 horas a 21ºC; esto sugiere una gran eficiencia en la digestión y posterior asimilación de fuentes diversas de alimento poco digerible como detritus o larvas de bivalvo. Se encuentra una cierta tendencia estacional en la actividad e ingestión de Carbono en los meses estivales (Julio y Agosto) y a finales de Otoño- principios de Invierno. Al estudiar dos poblaciones naturales durante un ciclo anual paralelo en dos localidades diferentes (Banyuls-sur-Mer y las Illes Medes, a unos 50 km de distancia), se han encon- trado diferencias significativas en la dieta, presas pólipo -1 , pólipos con presa/s y sobre todo µgC pólipo -1 . La población de Banyuls-sur-Mer tiene menos entradas de carbono (0.047 ±0.09 SD µgC polyp -1 ) comparada con la de les Illes Medes (0.073 ±0.134 SD µg polyp -1 ) a lo largo del ciclo anual estudiado. Esto podría ser reflejo de unas condiciones ambientales y biológicas diferentes (como se ha señalado en el apartado de trampas de sedimento), lo que nos lleva a deducir que una aproximación espacial, considerando más de una población, es necesaria para hacer una correcta evaluación de los flujos de energía en poblaciones naturales de suspensívoros en una zona determinada. En nuestro caso, sospecha de que una población a 10 m de profundidad tendría que potencialmente estar más favorecida energéticamente que una población a 30 m (por la tasa de renovación del agua, mayor aporte de detritus, etc.) no se cumple. En general creemos que es fundamental considerar una serie de aproximaciones meto- dológicas, temporales y espaciales para poder evaluar correctamente el posible impacto de los suspensívoros bentónicos en la columna de agua.

198 RESUMEN Balance Proteína-Carbohidrato-Lípido en dos suspensívoros pasivos a lo largo de ciclos estacionales

Las gorgonias Paramuricea clavata y Leptogorgia sarmentosa utilizan los lípidos como macromolécula orgánica principal de almacenamiento de energía (oscilando entre 120-360 µg Lípido mg AFDW -1 en P.clavata y 250-390 µg Lípido mg AFDW -1 en L.sarmentosa ). Ambas especies tienen un balance Proteína-Carbohidrato-Lípido marcadamente estacional, aunque ambas parecen responder de forma diferente a épocas de crisis trófica; así, P. clavata posee mínimos estacionales en Verano y finales de Otoño-principios de Invierno, mientras que los mínimos registrados en L. sarmentosa se dan entre finales de Otoño-principios de Invierno. Los carbohidratos siguen una pauta estacional muy similar (en ambas especies) a los lípidos; por otro lado, en P. clavata hay diferencias entre colonias masculinas y femeninas en la concentración de lípidos, aunque sólo en primavera, mientras que en L. sarmentosa dicha diferencia es inexistente a lo largo de todo el año. Las proteínas tienen máximos coincidentes con las épocas de crecimiento de ambas especies; se sugiere la utilización de este parámetro bioquímico para detectar épocas de crecimiento a nivel de población. La relación entre lípidos y esfuerzo reproductor (medido en µgC gonadal pólipo -1 ) es nula en P. clavata tanto en machos como en hembras. Además, las colonias inmaduras (<10 cm de altura) poseen también un marcado ciclo estacional, lo que sugiere una utilización de los lípidos principalmente para afrontar crisis tróficas como la de verano o finales de otoño. Está claro que ha de haber una relación entre época reproductora y almacenamiento de lípi- dos, pero dicho acopio no parece reflejarse directamente en el número y/o diámetro gonadal de la especie. En L. sarmentosa sí existe una estrecha relación entre lípidos y esfuerzo reproductor, lo que sugiere una diferente estrategia reproductora en ambas especies. Ha podido observarse que condiciones hidrodinámicas diferentes y/o posición más o menos aventajada dentro de la población pueden dar como resultado en ambas especies colonias cuya capacidad de almacenamiento de lípidos está por encima (más favorecidas hidrodinámicamente, capturan más presas) o por debajo (menos favorecidas hidrodinámi- camente, capturan menos presas) de la media. Esta captura puede estar relacionada con la estrategia de cada especie respecto a su fuente de alimento. Se sugiere el balance P-C-L como herramienta óptima para demostrar una situación más o menos favorable de una colonia respecto a las condiciones ambientales y a la capacidad de capturar seston. El balance Proteína-Carbohidrato-Lípido ha demostrado una situación desfavorable inesperada entre finales de Otoño y principios de Invierno; en esta época del año, debido a un exceso de turbulencia y de turbidez de las aguas, hay un colapso de la producción primaria (por falta de luz) que se traduce en mínimas concentraciones en la red trófica micro- biana (ciliados, flagelados, etc.), en el zooplancton y finalmente en la materia orgánica particulada en sedimentación (de muy escaso valor nutritivo). Estos factores revierten en las poblaciones de suspensívoros bentónicos, teniendo entradas mínimas de Carbono, no repro- duciéndose y sufriendo una segunda crisis trófica (después de la estival). Esta situación se ve claramente reflejado en la disminución de lípidos y carbohidratos en ambas especies de suspensívoros estudiados, y en menor medida en las proteínas. El balance P-C-L puede ser una excelente herramienta para registrar a medio plazo el acoplamiento plancton-bentos, al “memorizar” las condiciones ambientales que rodean los

RESUMEN 199 suspensívoros bentónicos. De este modo, los pulsos ambientales quedan integrados en la fisiología de los organismos. Sin embargo, sin una comprensión profunda de la biología y ecología de las especies (i.e. alimentación, reproducción, crecimiento, distribución, etc.) la herramienta no transmite suficiente información sobre la ecología de la especie y su rela- ción con la variabilidad ambiental y biológica del hábitat.

200 RESUMEN