Ilustración de la portada por Diana Toledo, Berlín 2011.

TESIS DOCTORAL

Relación entre el tamaño celular, la abundancia y el metabolismo en el fitoplancton marino

Relationship between cell size, abundance and metabolic rate in marine phytoplankton

Memoria presentada por María Huete Ortega para optar al grado de Doctor por la Universidad de Vigo

Emilio Marañón Sainz, profesor titular del Departamento de Ecología y Biología Animal de la Universidad de Vigo,

HACE CONSTAR que la presente memoria, titulada “Relación entre el tamaño celular, la abundancia y el metabolismo en el fitoplancton marino”, presentada por la Licenciada María Huete Ortega para optar al grado de Doctor por la Universidad de Vigo, ha sido realizada bajo mi dirección, cumpliendo con las condiciones exigidas para su presentación, la cual autorizo.

Para que así conste a efectos oportunos firmo la presente en Vigo, a 31 de Enero de 2011.

Fdo. Emilio Marañón Sainz Director de la Tesis Doctoral

Fdo. Mª Jesús Iglesias Briones Directora del Departamento de Ecología y Biología Animal Universidad de Vigo

Agradecimientos

Érase una vez…, no…, así no..., comenzar los agradecimientos de esta tesis como si fuera un cuento no sería apropiado ¿no? Aunque…bien pensado, ¿acaso no lo es al final? Al fin y al cabo es la consumación de un trabajo de cinco años, pero también lo es de una carrera académica que se inició cuando tenía ¿Cuántos? ¿diez? ¿catorce? ¿dieciocho?...y de toda una vida de toma de decisiones, buenas o malas da igual, que al final me han llevado a este punto, a este aquí y ahora, en el que me paro, me bajo de la tierra que da vueltas y miro hacia atrás, uniendo los puntos, uno a uno hasta que llego al mismo instante en que comenzó todo. Y es cuando una se pregunta ¿y dónde comenzó? Sinceramente, ni idea. No sé por qué un buen día se me ocurrió que quería hacer Biología Marina cuando fuese mayor y tampoco es plan aquí de contar la historia de mi vida. Así que, centrándonos un poco, vayamos a uno de esos eventos aislados que tanto definieron lo que soy ahora y dónde estoy.

Sí, imaginémonos entonces un día soleado, de esos de comienzos de primavera en Madrid, cuando la brisa sopla trayendo la frescura de las montañas y el olor de las flores recién nacidas. Imaginemos un césped verde, todavía no excesivamente secado por el sol, y dos chavales, llenos de sueños, cargados de libros y tirados cual lagartijas, mirando hacia el futuro durante unos instantes. Uno de ellos era yo, por supuesto, y el otro era Javi, al que, sin ninguna duda, le debo ese primer comienzo. Por tanto, bicho, para ti va este primer agradecimiento. Porque, sin ti, no habría tenido ni el valor, ni la decisión de liarme la manta a la cabeza, subirme al coche de mi madre y recorrerme 600 kilómetros de carretera hasta una ciudad húmeda, antigua, llena de voces y de historia, y comenzar a realizar el sueño que llevaba siendo mío desde que tenía uso de razón. No fue fácil, eso desde luego, e incluso a veces habré lamentado haber cogido ese camino por diversas razones, pero al final, reflexionando, no me arrepiento. Esta vida va de aprender y mejorar, y ser mejores personas, y todo eso lo hice en Santiago durante esos dos años, y desde luego, sin ti no podría haberlo superado. Y la carrera pasó y, tal y como ocurre ahora, nos enfrentamos al abismo del ¿y ahora qué? Siguiente parada en la carretera de la vida, siguiente punto a unir. Y, una vez más, fuiste tú, Javi el que me ayudaste en la decisión. Una vez más, superando obstáculos y rodeando vayas, apostamos por nuestros sueños y por nuestro futuro juntos. Y, tras mucho mail y meditación, aquí nos instalamos, en esta terra gallega, verde y picuda, en la que ya llevamos viviendo 6 años. Seis años de lecciones, de alegrías y batallas ganadas juntos, como el equipo que somos. Gracias por hacer todo esto posible. Por apoyarme paso a paso, en los comienzos, en las ausencias, durante las estancias, por mail, por teléfono, en casa, por cuestionar, por luchar, por creer en mí y por caminar conmigo paso a paso, de punto en punto con paciencia y decisión. Realmente, sin ti, creo que esta tesis no sería lo que es porque habría terminado volando por la ventana, entre otras cosas.

Por supuesto, también tengo mucho que agradecer a mis padres que, como siempre, me apoyaron en todas mis locuras y me ayudaron a llegar hasta aquí. A ti, mamá, te debo de agradecer el haber aprendido a perseverar, a no rendirme, a ser responsable, a coger el toro por los cuernos y tirar para adelante. Gracias por haberte esforzado tanto y haber estado siempre a mi lado y haberme permitido alcanzar mis sueños. Y a ti papá, te debo ese lado imaginativo de mí que sueña despierto y se divierte creando e inventando. Gracias por nuestras conversaciones sobre ciencia ficción cuando era pequeña, que siempre recordaré como un preciado tesoro. Gracias también a los dos por estar ahí, a pesar de que no llame todo lo que os gustaría y no me veáis muy a menudo, por haberme traído el calor de mi familia hasta estas tierras gallegas y no permitirme olvidar de dónde vengo y a dónde voy. Igualmente, gracias a mis hermanos, que en estos años han pasado de ser sólo eso, mis hermanos, a convertirse en mis mejores amigos. Os quiero, y saber que puedo contar con vosotros siempre es una de las más maravillosas certezas que podría tener en mi vida. A ti, James, bienvenido a la familia.

Gracias también a mi otra familia, la adoptiva. Gracias Jesús y Blanca por apoyarnos a Javi y a mí en nuestra aventura norteña. Gracias por acogerme entre vosotros como si fuera una más. Gracias también a la pequeña duende, y a Mario, por acosarnos a visitas cuando podéis y hacernos más amena nuestra estancia aquí. Os echamos mucho de menos, así que venir siempre que queráis, siempre estaremos con los brazos abiertos, vayamos donde vayamos.

Y retomando ese punto esencial que fue la decisión de comenzar esta tesis, mi más sincero agradecimiento es para Emilio, que a lo largo de estos años no sólo ha sido mi jefe sino también mi mentor. Aún recuerdo el día que, nerviosa, entré en tu despacho para decirte que quería hacer la tesis. Obviamente, no tenía mucha idea de en qué me estaba metiendo y tampoco sabía mucho sobre el tema en sí, por no hablar de que era la primera vez que aparecía por allí. Pero aun así, me escuchaste paciente y me diste una oportunidad. Gracias por confiar en mí entonces y por haberlo hecho desde entonces, ayudándome a formarme profesional y académicamente y ser mi fuente de inspiración. De ti, Emilio, he aprendido muchas cosas, tantas que darían para una larga lista, pero sobre todas ellas destacaría una. Nunca olvidaré el momento en el que, durante esa primera entrevista, me confesaste que realmente, para aquellos a los que realmente les gusta investigar, lo de menos es el tema y lo importante es el hecho en sí de cuestionar, de profundizar, de luchar por encontrar la solución. Ese comentario se me gravó a fuego en la mente y, con el tiempo, ha terminado por confirmarse. Pues aunque durante estos años me ha llegado a apasionar el scaling o la macroecología, si soy sincera creo que investigar cualquier otro tema me parecería igualmente interesante. Y esa certeza creo que me acompañará allá donde vaya a partir de ahora.

Desde el punto de vista profesional también mi más especial agradecimiento es para Pedro, sin el cual habría estado bastante perdida en mis primeros pasos por la vida y milagros del scaling. Gracias por tu paciencia y por contestar a mis más absurdas preguntas con tu característica campechanería zaragozana. Gracias también a Eva Teira por ayudarme en el labo, apoyarme todos estos años y permitirme participar en su proyecto ADDEX. Mi más sincero agradecimiento también a Manuel Varela por ser mi segundo mentor en esos primeros años y ayudarme a desentrañar el lío de la base de datos de la Radial. Gracias también, Manuel, por tus historias y divertidas anécdotas que tantas reuniones, comidas y congresos han amenizado. Gracias a Antonio Bode por sus consejos durante el trabajo de la Radial. Gracias a Ale y Laura, de Gijón, por tener paciencia a la hora de revisar una y otra vez los datos de citometría y soportar mis constantes preguntas. Sin vosotras, realmente, ninguno de los espectros que hecho estos años habría visto el sol. Gracias a Bea, por poner a mi disposición sus conocimientos de oceanografía, a Bernardino por su apoyo estadístico y a Cris por guiarme en la fisiología del fitoplancton. Gracias también a todas aquellas personas que me he ido encontrado a lo largo de estos años durante el desarrollo de mi trabajo y que de alguna forma han ayudado a su consecución. A Jaime Rodríguez, Paco, José María y Andy por recibirme tan cordialmente en el Departamento de Ecología de Málaga y abrirme los ojos al mundo de los espectros. Gracias a Débora Iglesias por darme la oportunidad de iniciarme en el mundo de la ecología molecular en el National Oceanography Centre of Southampton y a John Gittins por guiarme en el camino, poniendo a mi disposición todos sus conocimientos y ayuda. Un especial reconocimiento se merecen también todas aquellas personas que han vivido y trabajado conmigo durante las campañas Carpos, Trynitrop y Aselva. Gracias a todos aquellos marineros, oficiales y científicos del B.O Hespérides y B.O. Sarmiento de Gamboa que han logrado convertir los días de extenuante trabajo en una experiencia increíblemente instructiva y gratificante. Gracias por convertiros durante esos días en mi familia y permitirme reír, llorar, despotricar y hacer ciencia junto a vosotros. Gracias a todos los participantes del proyecto CARPOS, TRYNITROP, ADDEX, ASELVA y PERSEO por vuestro compañerismo y profesionalidad. Y a los IPs y Jefes de Campaña de dichos proyectos por haberme dado la oportunidad de participar.

Y así, desandando los puntos, llego a esa gente que tan importante ha sido para mi estos años y que, de una manera u otra, han alimentado mi espíritu, me han conducido hasta lo que soy y me han ayudado a mantener la cordura. Un especial abrazo y beso para mis niñas, Paulis y Sheila, que a pesar de la distancia seguirán conmigo siempre. Gracias por ayudarme a seguir adelante y hacer de mi casa de Vigo un hogar. Gracias por vuestras conversaciones a la hora de la cena, las fiestas, las pelis y las series, las anécdotas. Gracias por vuestro apoyo. Juntas y siendo tan dispares convertimos cuatro años de convivencia en algo memorable. Un lugar especial y único en mi corazón lo tienen Juan y María que, sin casi conocerme, me abrieron las puertas de su vida desde el primer momento y que, con el tiempo, se han convertido en los pilares de nuestra vida aquí. Gracias por esos findes de rol y conversaciones sesudas, por nuestras locuras y frikadas, por nuestras excursiones y viajes en donde logro desconectar y olvidarme de todo, por ser siempre tan alegres, por vuestra confianza, por vuestro amor y cariño, por estar ahí y ser solo vosotros y sobretodo, por haberos convertido en mi familia y en mi hogar. Me habéis enseñado tanto desde que os conozco que sin duda no sería lo que soy ahora sin vosotros. Un enorme abrazo a mi Daffne, a la que le debo haber recuperado esa parte de mi que había perdido, por haberme abierto nuevos mundos y horizontes y por traer el cariño al labo cada mañana con sus besos. Gracias a Anita, por su ayuda todos estos años, por enseñarme con paciencia, por sus consejos y apoyo, y por haber sido una fantástica compañera de camarote en las campañas, incluso cuando me tiré de la litera. Gracias a Sandris por su apoyo, a Patri P porque aunque te haya visto poco siempre me has hecho reír, a Tamara por ser tan fantástica compañera perseniana, a Meri por su energía contagiosa, su alegría y su buena disposición a echarme una mano cuando la he necesitado, a Leti por nuestras noches bailando salsa y las cervezas después del curro con Paula, a Gon, a Bellas, a Lili, Elena, Fani, las Elsas y la Towers y a las nuevas generaciones del labo que vienen detrás de nosotros, Víctor y Bieito. No hemos coincidido mucho pero espero poder hacerlo en el futuro.

Un especial reconocimiento se merecen mis chicas de siempre, Helen, Didian, Bi y Bea. Las cuales, a pesar de la distancia, me han enseñado que basta con dar al botón de Skype para tenerlas junto a mí y sentir su cariño y alegría. Muchas gracias Didian por esa fantástica portada que has hecho y por tu paciencia durante el diseño, cuando te tirabas de los pelos por la mente cuadriculada de los científicos. De mis estancias, gracias a Miri, Pablo y Maite por ser tan geniales y hacerme pasar tantos buenos momentos. Gracias por enseñarme Málaga y sus paisajes, por el finde en Bolonia, por vuestro cariño y amistad. De Southampton, gracias a mi querido Spanish Team, a Moni, Sonia, Toñito, Marcos, Eva y Romain por haber convertido 4 meses de estancia en una experiencia inolvidable. Gracias por las excursiones, los viernes en el Platform, los bailes en el The Edge, las fiestas hasta el amanecer y las cenas. Gracias Moni por saber escuchar. Gracias a ti y a Sonia por toda vuestra ayuda en el laboratorio. I would like also to thank the English people, Ed, David, Alex, Jennie, Machid and Mike for their support and friendship and to Nicolai for being an awesome tourist guide.

De la gente del SOLAS Summer School, mi más sincero agradecimiento a Sergei, Susan, Cristóbal y David. Gracias por haber convertido un curso de quince días en un genial verano. Gracias por las conversaciones, las risas y los baños en la orilla del Mediterráneo.

Gracias también a la gente de Umbría por haberme ayudado a desconectar durante estos dos últimos años. Por llenar mi vida de aventuras, retos y locuras, por inspirarme y hacerme reir. Gracias a vosotros he sido maga, hada, noble, exploradora espacial, ladrón y docenas de personajes más y me lo he pasado en grande durante el proceso.

Por último, gracias a todos aquellos organismos e instituciones que a lo largo de esta Tesis me han brindado su apoyo. Gracias al Ministerio de Educación y la Xunta de Galicia por las becas predoctorales bajo la cuales se ha desarrollado esta Tesis, a IGBP España por prestarme apoyo económico en el SOLAS Summer School, a la Universidad de Vigo por las ayudas para asistir a los congresos, al IEO de A Coruña por brindarme acceso a su base de datos de la Radial, al IEO de Gijón por prestarme su apoyo en el análisis de las muestras de citometría y la Universidad de Málaga y el National Oceanography Centre of Southampton por acogerme durante mis estancias.

A mi familia y a Javi

Esta Tesis fue realizada durante el disfrute de una beca de Formación de Profesorado Universitario (FPU) del Ministerio de Educación para el período 2006-2010.

Los trabajos que se recogen en esta memoria de Tesis fueron financiados por los proyectos del Ministerio de Ciencia e Innovación

“Trichodesmium spp. y fijación de Nitrógeno (N2) en el Atlántico tropical” y “Patrones macroecológicos en el fitoplancton marino”.

Durante la elaboración de esta Tesis se realizaron dos estancias breves, financiadas por el Ministerio de Educación, en el Departamento de Ecología y Geología de la Universidad de Málaga (España) y en el Nacional Oceanography Centre, Southampton (Reino Unido).

Contents

1. Introduction 3 1.1. Macroecological approaches to the study of phytoplankton 3 1.2. The importance of phytoplankton cell size 3 1.3. Phytoplankton size structure in marine ecosystems 5 1.4. Size-scaling relationships in ecology 7 1.5. Scaling relationship between phytoplankton abundance and cell size 8 1.6. Scaling relationship between phytoplankton metabolic rate and cell size 10 1.7. Hyphoteses and objectives 13 1.7.1. Hyphotheses 13 1.7.2. General objective 14 1.7.3. Specific objectives 15 1.7.4. Thesis outline 15 2. General patterns in the size scaling of phytoplankton abundance in coastal waters during a 10-years time series 19 2.1. Introduction 23 2.2. Methods 25 2.2.1. Sampling 25 2.2.2. Phytoplankton abundance and cell size 26 2.2.3. Size-abundance spectra 27 2.2.4. Time series analysis 29 2.3. Results 30 2.3.1. Hydrography and chlorophyll a 31 2.3.2. Hydrographic periods 33 2.3.3. General size-abundance patterns 35 2.3.4. Size-abundance spectra and hydrographic variability 37 2.3.5. Time series analysis 41 2.4. Discussion 43 2.4.1. General patterns in the size-scaling of phytoplankton abundance 43 2.4.2. Hydrography and phytoplankton size structure 46 2.4.3. Interannual variability in size spectra 49 2.4.4. Conclusions 50 3. Effect of environmental forcing on the biomass, production and growth rate of size-fractionated phytoplankton in the central Atlantic 51 3.1. Introduction 55 3.2. Methods 58 3.2.1. Sampling, hydrography, irradiance and nutrients 58 3.2.2. Size-fractionated chlorophyll a concentration 62 3.2.3. Size-fractionated carbon fixation rate 62 3.2.4. Phytoplankton cell size and abundance 64 3.2.5. Phytoplankton carbon biomass and growth rates 66 3.3. Results 66 3.3.1. Hydrography and chlorophyll a concentration 66 3.3.2. Total phytoplankton biomass, production and growth rates 69 3.3.3. Size-fractionated carbon biomass and production 69 3.3.4. The influence of environmental forcing on size-fractionated carbon biomass, production and growth rates 75 3.4. Discussion 78 3.4.1. Changes in size-fractionated biomass and production in response to environmental forcing 79 3.4.2. Influence of environmental forcing on growth rates 82 4. Metabolic scaling and phytoplankton size structure in the open ocean 87 4.1. Introduction 89 4.2. Methods 93 4.2.1. Sampling, hydrography, irradiance and nutrients 93 4.2.2. Size-fractionated chlorophyll a concentration 95 4.2.3. Size-fractionated carbon fixation rate 95 4.2.4. Phytoplankton cell size and abundance 95 4.2.5. Scaling relationship between abundance and cell size 96 4.2.6. Scaling relationship between cell-specific photosynthesis rate and cell size 97 4.2.7. Methodological considerations 98 4.3. Results 99 4.3.1. General oceanographic conditions 99 4.3.2. Methodological considerations in carbon fixation rate and chl a determinations 100 4.3.3. Size-scaling of carbon fixation rate 103 4.3.4. Size-scaling of phytoplankton total abundance 107 4.3.5. Size-scaling of phytoplankton total energy use 107 4.4. Discussion 108 4.4.1. Size-scaling of phytoplankton metabolic rate 108 4.4.2. Linking the size-scaling relationships of phytoplankton metabolism and abundance 110 4.4.3. Conclusions 113 5. Synthesis 115 5.1. Size-scaling of phytoplankton abundance 118 5.2. Size-differential response of phytoplankton biomass and production to changes in environmental forcing 119 5.3. Methodological considerations 120

5.4. Isometric size-scaling of phytoplankton carbon fixation rate 121 5.5. Higher metabolic rates of large phytoplankton 122 5.6. Size-independence of phytoplankton growth rates 123 5.7. Linking the size-scaling of metabolic rate with phytoplankton size structure 124 5.8. Total energy use along the phytoplankton size spectrum 125 5.9. Perspectives for future research 127 Conclusions 131 References 135 Resumen 159

“Your time is limited. So, don’t waste it living someone else’s life. Don’t be trapped by dogma, which is living with the results of other people’s thinking. Don’t let the noise of other’s opinions drown out your own inner voice. And most important, have the courage to follow your heart and intuition. They somehow already know what you truly want to become.” Steve Jobs, Standford commencement speech, 2005.

Chapter 1 Introduction

Chapter 1. Introduction

1.1 Macroecological approaches to the study of phytoplankton

Macroecology is defined as the study of general patterns in complex ecological systems over extensive spatial and temporal scales (Brown and Maurer 1989, Brown 1995, Gaston and Blackburn 2000). Considered as a research program in ecology by itself (Brown 1995, Brown 1999), macroecology differs from the traditional reductionist approach in its search of those empirical patterns and mechanistic processes by which the emergent structure and dynamics of ecosystems are generated. In this sense, most macroecological studies search for statistical patterns in the abundance, distribution, biomass, diversity and metabolism of organisms across communities and ecosystems (Brown et al. 2002, Blackburn 2004).

Phytoplankton species show a very wide range of spatial and temporal variability in their distribution, abundance and metabolic rates. In addition, phytoplankton communities are so diverse that it is not possible to determine the biological properties of all species that conform them. For these reasons, the macroecological approach is particularly interesting in the study of phytoplankton, as shown by the seminal study of Li (2002), who described general patterns in the relationships between hydrodynamical forcing, phytoplankton cell size and abundance.

1.2. The importance of phytoplankton cell size

Due to the central role of body size as a synthetic tool for analysing the complexity of ecosystems, the scaling relationships between this variable and metabolic rates, abundance and diversity are considered as fundamental macroecological properties of communities and ecosystems

3 Chapter 1. Introduction

Fig. 1.1 A comparison of the size range (maximum linear dimension) of phytoplankton relative to macroscopic objects (from Finkel et al. 2010).

(Peters 1983, Brown et al. 2002, Marquet et al. 2005). The study of body size as an integrative approach to understand the structure and function of phytoplanktonic assemblages is particularly appropriate, if we consider that phytoplankton cell size ranges over more than eight orders of magnitude in volume (Fig. 1.1) and that it influences almost every aspect of their physiology, ecology and evolution (Marañón 2009, Finkel et al. 2010). Phytoplankton stoichiometry, nutrient uptake and metabolic rates are all dependent on cell size (Eppley and Sloan 1966, Banse 1976, Taguchi 1976), and so are loss processes such as susceptibility to predation and sedimentation rates (Kiørboe 1993, Thingstad et al. 2005, Marañón et al. 2009, Finkel et al. 2010). In contrast to other macroecological studies that focus on species-specific analyses (Brown et al. 2002, Marquet et al. 2005), in the case of phytoplankton cell size is commonly used as an ataxonomic aggregation criteria, without disregarding the fact that taxonomic composition changes predictably across the size spectrum (Rodríguez 1994,

4 Chapter 1. Introduction

Cermeño et al. 2005b, Irwin et al. 2006).

1.3 Phytoplankton size structure in marine ecosystems

Phytoplankton size structure is a key factor in the control of the functioning of pelagic ecosystems from both an ecological and a biogeochemical point of view (Chisholm 1992, Kiørboe 1993, Legendre and Rassoulzadegan 1996, Marañón 2009), as it determines the trophic organization of the microbial plankton communities and, hence, the potential for organic carbon export (Legendre and Le Fèvre 1989). A consistent pattern in the ocean is that when total phytoplankton biomass, as estimated from the concentration of cholorophyll a, is low, small phytoplankton (e.g., picophytoplankton, <2 µm in cell diameter) dominate the community (Chisholm 1992, Agawin et al. 2000). In contrast, when total biomass is large, most of the biomass is accounted for by large cells (microphytoplankton, >20 µm in cell diameter) (Fig. 1.2). Thus, oligotrophic regions are characterized by the continuous recycling of organic matter within the microbial food web and, in consequence, a small downward carbon export to the deep ocean (microbial loop, Azam et al. 1993). Conversely, the dominance of large phytoplankton, which typically occurs in highly dynamic and nutrient-rich waters, favours the transfer of the synthesized organic matter towards higher trophic levels through short food-chains, or its downward export out of the euphotic layer by sedimentation (Cushing 1989). This leads to an increase in the potential of these ecosystems for atmospheric CO2 sequestration.

The size distribution of phytoplankton communities is controlled by

5 Chapter 1. Introduction

2 Picophytoplankton Nanophytoplankton 1 Microphytoplankton ) -3

0 (mg m (mg a

-1 total chl total 10 log -2

-3 -1 0 1 log total chl a (mg m-3) 10

Fig. 1.2 Chlorophyll a concentration in picophytoplankton, nanophytoplankton and microphytoplankton versus total chlorophyll a concentration in samples obtained throughout the euphotic layer in coastal and oceanic waters of widely varying productivity (from Marañón 2009).

the physical and chemical characteristics of the environment, since resource uptake and use by phytoplankton depend on cell size (Kiørboe 1993, Raven 1998, Litchman et al. 2007), and water column hydrodynamics determines, to a large extent, nutrient availability within the euphotic layer, as well as affecting the vertical transport of phytoplankton (Parsons and Takahashi 1973, Kiørboe 1993, Malone et al. 1993, Rodríguez et al. 2001). It is well- known that smaller cells dominate those regions with low nutrient concentrations and a higher water column stratification due to their

6 Chapter 1. Introduction

advantage over large phytoplankton in terms of nutrient uptake (Chisholm 1992, Kiørboe 1993, Raven and Kübler 2002). However, small phytoplankton are also present in turbulent and nutrient-rich areas, acting as a background component, whose biomass and primary production keep relatively constant (Raimbault et al. 1988, Thingstad and Sakshaug 1990, Rodríguez et al. 1998). For this reason, it is commonly accepted that most of the geographical and temporal variability observed in phytoplankton biomass and production is the result of the response of large phytoplankton to the environmental forcing, their growth being stimulated by increasing nutrient supply that favours their physiological strategies (Sarthou et al. 2005, Thingstad et al. 2005, Litchman et al. 2007, Verdy et al. 2009). However, recent studies have highlighted that the picophytoplankton are also able to respond, in terms of biomass and primary production, to the occasional enrichment of oligotrophic regions of the ocean, although the magnitude of this response may be lower than that presented by larger cells (Barber and Hiscock 2006, Tarran et al. 2006, Glover et al. 2007).

1.4 Size-scaling relationships in ecology

Size-scaling relationships in ecology are formalised mathematically by a power function in which the biological characteristics of organisms are related to body size following the equation Y = a Vb, where Y is the studied biological property, a is a taxon-related constant, V is the organism body size and b is the size scaling exponent (Brown et al. 2002, Marquet et al. 2005, Finkel et al. 2010). Logarithmic transformation gives the linear model: log Y = log a + b log V, where log a and b are the Y-intercept and the slope values respectively. Thus, the logarithmic data can be fitted to a

7 Chapter 1. Introduction

straight line by a linear regression analysis. Many of these relationships are considered as universal scaling laws that reflect the existence of powerful constraints on the organization of complex systems, as they can be applied consistently to all kinds of organisms (microbes, plants and animals) spanning more than 20 orders of magnitude in body size and living in all types of environments (e.g., terrestrial, marine and freshwater habitats) (Brown 1995, West 1999, Brown et al. 2002, DeLong et al. 2010). The biological properties that can be described by these macroecological, size- scaling relationships include abundance, lifespan, home range size, metabolic rate and resource use (Peters 1983, Brown et al. 2004).

1.5 Scaling relationship between phytoplankton abundance and cell size

The relationship between abundance and body size has been extensively studied in ecology, given that it represents a fundamental link between the traits of species at the individual and population level and the structure and dynamics of ecological communities (Kerr and Dickie 2001, Woodward et al. 2005). Therefore, the importance of the abundance (or density) size-scaling relationships has been widely recognized for both terrestrial and aquatic ecosystems (Brown 1995, Gaston and Blackburn 2000, Kerr and Dickie 2001).

One of the most common approaches to the study of the scaling relationship between abundance and body size in pelagic ecosystems is the construction of an individual size distribution, known as size-abundance spectrum (White et al. 2007). In this approach, the total abundance (N) or

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Fig. 1.3 Examples of size-abundance spectra in phytoplankton. A1, A2, C1 and C2 referee to different eddy structures found in the Alboran Sea. Continuous line: flow cytometry subrange; broken line: image analysis subrange (from Rodríguez et al. 1998).

biomass (B) of all organisms within each size class is plotted against the nominal cell size of each size class expressed in carbon or volume units (V) (Fig. 1.3). To construct size-abundance spectra, taxonomic composition is not considered, and cell size is used as a single aggregation criteria (Sheldon et al. 1972, Rodríguez and Mullin 1986, Rodríguez et al. 2001). The

9 Chapter 1. Introduction

relationship between total abundance and cell size typically results in a power function, whose size scaling exponent, b, can be regarded as a synthetic descriptor of the community size structure, and varies depending on the productivity of the ecosystem. For phytoplankton size-abundance spectra, the slope values obtained in oligotrophic ecosystems are more negative (-1.3 to -1.1) than those found in more productive waters (-0.8 to - 0.6), reflecting the increased dominance of larger cells in nutrient-rich environments (Cavender-Bares et al. 2001, Reul et al. 2005, Cermeño and Figueiras 2008, Marañón et al. 2007). Although a strong regularity in the linearity of the size-abundance spectra has been found in oligotrophic regions, more productive ecosystems, which are often subjected to a higher degree of hydrodynamical variability, often show irregularities (i.e. nonlinearities) in the phytoplankton abundance-cell size relationship, as a consequence of the accumulation of species from a given size class (Sprules and Munawar 1986, Marquet et al. 2005, Reul et al. 2006). However, it has not been determined yet whether this lack of a linear relationship is a general pattern that can be applied to all ecosystems subjected to a highly variable environmental forcing, or if it is only the result of the relatively scarce number of size-scaling studies carried out in this kind of environments. Finally, the problem of the origin of the actual slope values in the phytoplankton-cell size spectra (e.g. why does the slope take a value between -1.3 and -1 in oligotrophic waters) has not yet been solved.

1.6 Scaling relationship between phytoplankton metabolic rate and cell size

The ¾-power relationship between metabolic rates and body size has

10 Chapter 1. Introduction

been one of the most widely accepted scaling laws in ecology since first formulated by Kleiber in 1923 (Kleiber 1947). If metabolic rate is divided by body mass or volume (V), the resulting mass- (volume-) specific rate (units of time-1) scales as V-1/4, which means that as organisms increase in size their biomass-specific metabolic rate tends to decrease, or, in other words, that large organisms have a slower metabolism than small ones. Given the ecological implications of this metabolic pattern, the universality of Kleiber’s law has been tested both in terrestrial (Enquist et al. 1998, Savage et al. 2004, McNab 2008) and aquatic ecosystems (Banse et al. 1976, Quiñones et al. 1992, Glazier et al. 2006), although a great controversy about this topic still remains (Dodds et al. 2001, Bokma 2004, Glazier et al. 2006, Kolokotrones 2010). Several models have also been developed in order to obtain a general explanation for the origin of quarter- power scaling laws in biology that could be applied to different life-forms and levels of organization (West et al. 1999, Dodds et al. 2001, Banavar et al. 2002, West and Brown 2005).

In the case of phytoplankton, several studies have reported the applicability of the ¾-power relationship (Eppley and Sloan 1966, Taguchi 1976, Blasco et al. 1982, Finkel 1998, López-Urrutia et al.2006). Niklas and Enquist (2001) showed a continuity in the ¾-power size-scaling of biomass production rate in all photoautotrophic organisms, from microscopic, unicellular algae to large trees (Fig. 1.4). However, all these studies were based on data obtained with laboratory cultures. In contrast, recent works that use observations from natural assemblages in the field have highlighted the existence of an isometric relationship (size-scaling exponent, b, close to

11 Chapter 1. Introduction

Fig. 1.4 Size-scaling relationship for biomass production rate (here called growth rate) in photosynthetic organisms including unicellular algae and vascular plants. Solid lines are reduced major axis (Model Type II) regression lines (hair lines indicate 95% confidence intervals); resulting scaling exponent in the upper left (from Niklas and Enquist 2001).

1) between phytoplankton photosynthesis rate and cell size, which also seems to vary depending on the productivity of the ecosystem (Marañón et al. 2007, Marañón 2008). Given that a nearly isometric relationship implies that larger cells may have higher metabolic rates than expected for their size, and even higher growth rates than smaller cells, this result gives new insights on the factors that control phytoplankton structure and the role of large phytoplankton on the functioning of pelagic marine ecosystems, with important ecological and biogeochemical implications (Cermeño et al. 2005a, Marañón 2008, Marañón 2009). For instance, an isometric size- scaling of phytoplankton metabolism implies that the dominance of large

12 Chapter 1. Introduction

cells in nutrient-rich environments may result from a purely physiological mechanism, without invoking top-down trophic effects. However, all these scaling relationships had a number of methodological limitations. In the case of Marañón et al. (2007), they used measurements of metabolic rate in only a small number of size classes, which prevented them from determining the size-scaling of metabolism in particular phytoplankton assemblages at the local scale. In the case of Marañón (2008), the inter- specific relationships reported are based on data collected from the literature, which, due to differences in the methods used by each worker, leads to additional uncertainty. For this reason, it is crucial to determine the size-scaling of phytoplankton metabolism in natural conditions using more accurate methods.

1.7 Hypotheses and objectives

1.7.1 Hypotheses

Cell size plays an important role in many aspects of the biology and ecology of phytoplankton and there is a strong relationship between the taxonomic composition of phytoplankton communities and their size distribution. Moreover, environmental factors, both biotic and abiotic, seem to exert a differential effect among different phytoplankton size classes. Therefore, the underlying basis for the present work is that phytoplankton cell size constitutes a macroecological tool for reducing the complexity of phytoplankton communities to general patterns that help us to understand the functioning of marine ecosystems. Within this framework, the following specific hypotheses are formulated:

13 Chapter 1. Introduction

 The scaling relationship between phytoplankton abundance and cell size shows irregularities in ecosystems that are subjected to highly dynamical environmental forcing.

 The seasonal and interannual variability in resource availability and hydrographic conditions experienced by coastal ecosystems leads to changes in phytoplankton size structure, represented by variations in the slope of the size-abundance scaling relationship.

 Large phytoplankton are more limited by nutrient availability than smaller cells in the oligotrophic regions of the ocean, so their biomass, production and growth rate will increase more than those of smaller cells when nutrient supply is increased.

 Phytoplankton metabolic rate, and in particular photosynthesis rate, scales with cell size following the ¾-power law.

 Phytoplankton size structure in near-steady state ecossytems, such as the oligotrophic open ocean, can be inferred from the size-scaling of phytoplankton metabolic rate.

1.7.2 General objective

The main objective of the present Thesis is to determine the nature and variability of the relationship between cell size, abundance, biomass and metabolic rate in natural phytoplankton communities.

14 Chapter 1. Introduction

1.7.3 Specific objectives

 To test the persistence in time of the power-law scaling relationship between phytoplankton abundance and cell size in a highly dynamic and productive marine ecosystem.

 To ascertain the seasonal and interannual variability of the size- scaling of phytoplankton abundance in a productive marine ecosystem, and its relationship with hydrodynamical conditions.

 To determine the size-differential response of open ocean phytoplankton biomass and production to changes in environmental forcing.

 To determine the size-scaling relationship of phytoplankton photosynthesis rate, in order to elucidate the applicability of Kleiber’s law to phytoplankton metabolic rate.

 To investigate the linkage between the size-scaling of metabolic rate and the size structure of phytoplankton.

1.7.4 Thesis outline

In order to test the hypotheses and to achieve the specific objectives stated previously, two lines of work were carried out. Firstly, to study the temporal variability and regularity of the size-scaling relationship of phytoplankton abundance in a dynamic and productive ecosystem, as well as to test the variability in the size-scaling associated to changes in the environmental forcing, we analysed a large data set from a 10-year time-

15 Chapter 1. Introduction

series in a shelf station off A Coruña. The dataset consisted on monthly collected data of phytoplankton abundance and cell size from which size- abundance spectra at different levels of integration were constructed. The resulting size-scaling slope and Y-intercept values were subsequently analyzed in relation to the different hydrographic periods that characterized that pelagic ecosystem throughout the annual cycle. The seasonal and interannual variability of the phytoplankton size distribution was studied by a time-series analysis. The results of this first line of work are reported in Chapter 2 of the present Thesis and have been recently published in Huete- Ortega et al. (2010).

The second line of work, related to the analysis of the metabolic size-scaling of natural phytoplankton communities and the size-differential response of phytoplankton to changes in the environment, was conducted during an oceanographic cruise in the tropical and subtropical Atlantic Ocean. During this cruise, the in situ size-fractionated photosynthesis rate and chlorophyll a concentration was measured and samples were obtained for determination of phytoplankton cell abundance and biovolume. The resulting data were analysed by two different approaches.

On one hand, an analysis of the variability of size-fractionated phytoplankton primary production, carbon biomass and turnover rates was carried out focusing exclusively on those stations directly influenced by the Atlantic Equatorial upwelling. The main aim of this analysis was to assess the differential response of phytoplankton size groups to changes in environmental forcing, especially nutrient availability and water column stability, and to elucidate if the nutrient limitation of phytoplankton

16 Chapter 1. Introduction

communities in the oligotrophic gyres was restricted to the phytoplankton standing stocks or affected also their physiology. The details and results of this approach are shown in Chapter 3 of the present Thesis and have been submitted for publication (Huete-Ortega et al. in review).

On the other hand, by constructing size-spectra of photosynthesis rate and abundance, it was possible to study the large-scale spatial variability in the size-scaling of phytoplankton metabolic rate and to assess the linkage between metabolic scaling and the size distribution of natural phytoplankton communities. The results of these size-scaling analyses and their ecological implications are presented in Chapter 4 and have been submitted for publication (Huete-Ortega et al. in review).

Finally, Chapter 5 presents a synthesis of the main results of this Thesis and suggests some perspectives for future research.

17 Chapter 1. Introduction

18

Chapter 2 General patterns in the size scaling of phytoplankton abundance in coastal waters during a 10-year time series

Chapter 2. Size-scaling of phytoplankton abundance

Abstract

To ascertain the general patterns in phytoplankton size structure of a temperate, coastal ecosystem, we monthly determined the scaling relationship between total abundance and cell size (size spectrum) for nano- and micro-phytoplankton in a shelf station off NW Iberian Peninsula during the period 1993-2002. The inverse linear relationship between log abundance and log cell size was persistent throughout the water column and across seasonal and interannual timescales. In addition, and despite the high productivity and marked temporal variability in water column structure at our study site, departures from linearity in the size spectra were rare. The slope (-0.96) of the overall size spectrum for the entire time series indicated that roughly equal amounts of biomass were present over different logarithmic size classes in the size range considered. The phytoplankton size spectra had similar average slopes during winter mixing, early upwelling, summer stratification and autumn downwelling, suggesting that, in these oceanographic conditions, both nano- and microphytoplankton respond similarly to environmental variability. In contrast, significantly less negative slopes were observed during upwelling relaxation, indicating an increased importance of larger cells. Our results illustrate the utility of individual size distributions to provide a synthetic description of phytoplankton community structure in dynamic, non steady-state marine ecosystems.

21 Chapter 2. Size-scaling of phytoplankton abundance

22 Chapter 2. Size-scaling of phytoplankton abundance

2.1 Introduction

Phytoplankton size structure is of critical importance for the functioning of pelagic ecosystems both from an ecological and a biogeochemical point of view, as it determines the trophic organization of microbial plankton communities and, hence, their role in the global carbon cycle (Kiørboe 1993, Legendre and Rassoulzadegan 1996, Marañón 2008).

Given that body size affects all aspects of the biology of organisms, including lifespan, home range size, metabolic rates and resources use (Peters 1983, Brown et al. 2004), the relationship between body size and abundance has long been recognised as a fundamental property of communities and ecosystems. One of the most widely used approaches to study this relationship in aquatic ecosystems is to construct a size spectrum (White et al. 2007) where the total abundance or biomass of all individuals within each logarithmic size class, irrespective of species, is plotted against the nominal or mean cell size of each size class (Sheldon et al. 1972, Rodríguez and Mullin 1986, Sprules and Munawar 1986, Rodríguez et al. 2001).

When the size spectrum is constructed using total abundance (N), the resulting distribution usually follows a simple decreasing power function of cell size (V) such that N  Vb, where b is the size scaling exponent. Logarithmic transformation yields log N = a + b log V, where a and b are the intercept and slope, respectively, of the linear relationship. The value of b, which typically ranges between -1.3 and -0.6 depending on the productivity of the ecosystem, can be regarded as a synthetic descriptor of

23 Chapter 2. Size-scaling of phytoplankton abundance

the phytoplankton community size structure (Cavender-Bares et al. 2001, Reul et al. 2005, Marañón et al. 2007). In addition, it has been shown that more productive ecosystems that are also subject to stronger hydrodynamical forcing can show irregularities (i.e. non-linearities) in the size spectrum (Sprules and Munawar 1986, Reul et al. 2006), resulting from the accumulation of a small number of species.

Despite the fact that the relationship between water column structure and phytoplankton size spectra has been studied for decades, most previous analyses either did not consider seasonal variability or were based on a small number of observations conducted during just one year. Thus, the emphasis has been placed mostly on the relationship between particular hydrodynamic events and the resulting changes in phytoplankton size structure (Rodríguez et al. 1987, Reul et al. 2005, 2006) rather than in the search of broader patterns across a range of temporal scales. The present study used a large set of monthly data of phytoplankton composition and abundance collected in a shelf station off A Coruña (NW Iberian Peninsula) during 1993-2002. The region is characterised by the typical seasonal variability of coastal temperate seas and, in addition, is subjected to intermittent upwelling pulses during the period April-September (Fraga 1981, Casas et al. 1997, Teira et al. 2003), thus leading to a particularly dynamic pelagic ecosystem. Our main objectives were i) to verify the validity of the power-law relationship between phytoplankton abundance and cell size to describe phytoplankton size structure in a non steady-state ecosystem; ii) to describe the seasonal and interannual variability in phytoplankton size structure; and iii) to obtain general patterns in the size-

24 Chapter 2. Size-scaling of phytoplankton abundance

scaling of phytoplankton abundance in relation to contrasting hydrographic conditions. The results presented in this chapter have been published by Huete-Ortega et al. (2010a).

2.2 Methods

2.2.1 Sampling

The data used in the present study were obtained within the framework of the time series project RADIALES, conducted by the Instituto Español de Oceanografía in the Ría of A Coruña (NW Spain) (Valdés et al. 2002, 2007). This project involves a monthly sampling of 5 stations along a coast-ocean transect and has been running since 1989. Our analysis is focused on the abundance and cell size of phytoplankton collected at station 2 (depth = 80 m; 43° 25.30’N, 08°26.20’W; Fig. 2.1) during the period January 1993-December 2002. On each sampling visit, the vertical distribution of temperature and salinity was measured with a CTD SBE-25 probe. Water samples were collected with 5-L Niskin bottles or a rosette sampler from 0, 5, 10, 20, 30 and 40 m depth. For each sampling depth, samples were collected for the determination of nutrient concentration, chlorophyll a (chl a) concentration and phytoplankton abundance following the methods previously reported in Casas et al. (1997, 1999) and Teira et al. (2003). In addition, the daily averaged Ekman offshore transport of surface water across a transect parallel to the shoreline was estimated as described by Lavín et al. (1991), using wind speed and direction data measured every 6 hours at the Centro Meteorológico Zonal of A Coruña (Agencia Estatal de Meteorología). The sign and value of this transport is used as indicative of

25 Chapter 2. Size-scaling of phytoplankton abundance

the occurrence and intensity of the upwelling events, respectively, as positive values indicate upwelling-favourable, offshore Ekman transport, whereas negative values indicate downwelling-favourable, onshore Ekman transport. Water-column stability was estimated using the Brunt-Väisalä frequency (BV) calculated from the density difference between 0 and 40m depth:

1/ 2  4g 2   1   BV     2   1 z2  z1  where g is the gravity acceleration value, σ2 and σ1 are the densities at 40 and 0 m, respectively, and z2 and z1 are the sampling depths.

2.2.2 Phytoplankton abundance and cell size

Phytoplankton samples of 50-100 mL in volume were preserved in Lugol’s solution and kept in the dark until analysis. Samples were always counted by the same person using a Nikon Diaphot TMD microscope from 1993 to May 1997 and a Nikon Eclipse TE300 microscope from June 1997 to the end of the time series, following the technique described by Utermöhl (Lund et al. 1958). A magnification of 100 was used for big forms, 250 for intermediate forms and 400 and 1000 for microflagellates. Whenever possible, organisms were classified at the species or genus level. Cell biovolume for each species was estimated in some samples from measurements of cell dimensions under the microscope, assigning a certain geometric shape to each species (Edler 1979), and in the case of colonial species only cell size and cell abundance were considered. An average cell size obtained from these measurements was used in subsequent analysis. For

26 Chapter 2. Size-scaling of phytoplankton abundance

those species or genera for which no cell size measurements were available, cell volume was taken from the literature. The cell size range considered in our analysis was the nano- and micro-phytoplankton [2-20 µm and >20 µm of equivalent spherical diameter (ESD), respectively]. The total number of phytoplankton samples analysed for the present study was 571.

48° Ría de Ferrol study site

44°

IberianIberian St. 2 Ría de Ares 40° Peninsula de

u o 43 o 25' 43 30' 36°

NLatit 8° 4° 0°

43 o 20' Bay of A Coruña

8 o 30' 8 o 25' 8 o 20' 8 o 15' 8 o 10' W Longitude

Fig. 2.1 Map of sampling site off A Coruña (NW Iberian Peninsula).

2.2.3 Size-abundance spectra

Size-abundance spectra were constructed using the phytoplankton abundance and cell size data. Size classes were established on an octave

(log2) scale of biovolume and total cell abundance was calculated for each class by summing the abundance of all species included in it. The maximum number of size classes obtained was 19, ranging from 2 µm to ~200 µm of

27 Chapter 2. Size-scaling of phytoplankton abundance

ESD. Afterwards, the log10 of total abundance was plotted against the log10 of the lower limit of the corresponding octave size class in order to obtain a linear relationship (Blanco et al. 1994, Reul et al. 2005). Given that methodological error was present in both variables, a Model II regression analysis by the reduced major axis method was carried out for each spectrum in order to estimate the regression slope and the Y-intercept (Laws and Archie 1981). When a comparison between spectral slope values was necessary, Student’s-t test following the Clarke method was used (Clarke 1980). Size-abundance spectra with a determination coefficient (r2) lower than 0.5 (less than 15% of all spectra) were eliminated and discarded from further analysis.

In order to analyse the phytoplankton size structure along the whole time series, we obtained size-abundance spectra at different levels of integration. At a first level, we constructed the size-abundance spectra for each month and sampling depth, obtaining approximately 600 spectra. In addition, data from the different sampling depths on each month were pooled together, thus yielding a size-abundance spectrum for the whole water column, hereafter referred to as water column macrospectra. These macrospectra result from plotting together all the individual spectra obtained for a given sampling date. Reduced major axis regression (r.m.a.) was applied to these macrospectra to verify the existence of linearity in the size- abundance relationship. As explained above for the individual spectra, those macrospectra with a determination coefficient below 0.5 were not considered for further analysis. At a third level of integration, annual macrospectra were also obtained, by plotting together all the water column

28 Chapter 2. Size-scaling of phytoplankton abundance

macrospectra for each year. Finally, a single macrospectrum for the entire data set was also constructed by combining all annual macrospectra.

2.2.4 Time series analysis

With the aim to analyse the interannual and seasonal variability in our data set, we carried out a Fourier analysis in order to assess the periodicity of the time series, and later the variables were broken down into different sources of variability such as the seasonality and the interannual trend or the residuals (Nogueira et al. 1997). The variables subjected to time series analysis were temperature, salinity, mixing layer depth, nutrient concentration, the size-abundance spectra slopes at 5 m and 40 m depth, the water column macrospectra slopes, Ekman transport and the monthly values of the North Atlantic Oscillation index. Once variables were broken down, correlation coefficients between the sources identified were calculated and cross-correlation tests between them were carried out to assess the relationship between the abiotic factors (hydrographic variables and nutrient concentration) and phytoplankton community size structure. Although seasonal sources were obtained for some of those variables, our analysis was focused on the interannual variability of the data set. The level of significance of the identified interannual trends was tested by first order regression analysis. Statistical analyses were made using MATLAB software (Component Run Time version 7.7).

29 Chapter 2. Size-scaling of phytoplankton abundance

2.3 Results

2.3.1 Hydrography and chlorophyll a

The sampling station presented a hydrodynamic pattern typical of the NW Iberian peninsula, which is highly influenced by the predominant winds in each period of the year. Thus, in autumn and winter there is a clear dominance of downwelling, whereas in spring and summer upwelling pulses prevail (Fig. 2.2). In spring, the onset of vertical stratification, mainly as a result of surface water warming, favours the development of phytoplankton blooms (Fig. 2.3). In addition, sequences of upwelling-relaxation- downwelling events are frequent in summer, causing the advection of cold and nutrient-rich water into the photic layer and the development of summer blooms during the relaxation period.

In spite of its interannual variability, station 2 showed the characteristic seasonal patterns of temperate coastal ecosystems: low temperatures, high nitrate concentrations and low chl a concentration during late autumn and winter, and warmer temperatures and higher chl a concentrations during the rest of the year (Figs. 2.3 and 2.4). Vertical mixing was evident from January until April, with temperatures around 13ºC throughout the water column (Figs. 2.3A and 2.4B). Warmer temperatures occurred during summer and early autumn, when thermal stratification was present, mainly in summertime (Fig. 2.3A). The highest temperatures, observed in early autumn, coincided with the intensification of downwelling events which cause the accumulation of shelf waters against the coast (Fig. 2.4B). In addition, the intense mixing of the water column in

30 Chapter 2. Size-scaling of phytoplankton abundance

1500

1000

) 500 -1 km

-1 s 0 3 (m

x

-Q -500

-1000

-1500 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 Fig. 2.2 Monthly averaged values of the Ekman transport (m3 s-1 km-1) during the period of study. Positive and negative values of this index indicate upwelling-favourable and downwelling-favourable conditions, respectively.

winter resulted in a vertically homogeneous distribution of nutrients. Conversely, during summer, nutrient concentration increased with depth as a result of active phytoplankton consumption in the upper layers and the remineralisation of sinking organic matter below the photic layer (Figs. 2.3B and 2.4C). Finally, the seasonal pattern was also clearly observable in chl a concentrations, with high values being measured during summer and lower values in winter (Figs. 2.3C and 2.4D). Thus, the monthly averaged values of chl a at 5 m depth ranged between 3.6 mg m-3 in June and 0.72 mg m-3 in January. The maximum values were observed in summer, coinciding

31 Chapter 2. Size-scaling of phytoplankton abundance

0

A 18

-20 17

16 -40 14 Depth (m)

-60 13

11

0

B 12 -20 8

-40 4 Depth (m)

2 -60

0

0 C 10 -20 8 )

m 6 h ( t -40 4

Dep 2

-60 1

0 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002

Fig. 2.3 Temporal and vertical variability in A) temperature (ºC), B) nitrate concentration (µmol L-1) and C) chl a concentration (mg m-3).

with a moderate stratification of the water column, and higher incident irradiances that favour the development of summer blooms after the enrichment of the photic layer by upwelling events (Fig. 2.4D).

32 Chapter 2. Size-scaling of phytoplankton abundance

800 18 AA) B 17 400

) 16 -1 0 km 15 -1 s 3

(m 14 -400 x

-Q 13 Temperature (ºC) Temperature -800 12

-1200 11

12 5 C)C D

10 4 ) -3 )

-1 8 3

6 m (mg 2 mol L a  4 1

2 0 Nitrate (

0 Chlorophyll -1 Nitrate Chlorophyll a -2 -2 Jan Feb Mar AprMayJun Jul Aug Sep Oct NovDec Jan Feb MarAprMayJun Jul AugSep Oct NovDec

Fig. 2.4 Monthly mean values of A) upwelling index (m3 s-1 km-1), B) temperature (ºC), C) nitrate concentration (μmol L-1) and D) chl a concentration (mg m-3). In B-D, filled circles correspond to 5 m depth values and open circles to 40 m depth values. Error bars indicate ± 1 standard deviation.

2.3.2 Hydrographic periods

With the aim of analysing the seasonal variability of phytoplankton community size structure, we identified five hydrographic periods relevant to the phytoplankton sucession during the annual cycle. This classification was based on the temporal variability of the following environmental

33 Chapter 2. Size-scaling of phytoplankton abundance

factors: surface temperature, nitrate and chl a concentrations, averaged Ekman transport over the 6 days prior to sampling date, and water column stability estimated by the Brunt-Väisalä frequency (BV).

The five hydrographic periods identified, whose characteristics, described in Table 2.1, are similar to those previously reported for the study area (Casas et al. 1997, Varela et al. 2001), were: winter-mixing, upwelling type I (initial stage), upwelling type II (final stage), stratification and downwelling. The two types of upwelling periods identified differ mainly in nutrient and chl a concentration. Upwelling I corresponds to the initial

Table 2.1 Characteristics of the different hydrographic periods identified for the 10-year time series.

Brunt- Hydrographic Ekman n Temp. Nitrate Väisälä Chl a period transport freq.

Winter mixing 41 13.8 (0.8) 3.9 (1.8) 0.003 1.0 (0.9) -388

Upwelling I 12 14.0 (1.5) 2.2 (2.4) 0.003 1.8 (3.1) 434

Upwelling II 20 14.2 (1.4) 1.1 (1.2) 0.005 2.4 (1.1) 312

Stratification 23 15.9 (1.0) 0.8 (0.9) 0.013 2.5 (2.6) 50

Downwelling 11 16.4 (0.8) 1.8 (1.2) 0.008 1.8 (2.4) -624

Shown are mean surface values (5 m depth) for temperature (ºC), nitrate concentration (μmol L-1) and chl a concentration (mg m-3). n indicates the number of stations included in each period. Brünt-Väisäla frequency was calculated between 0 and 40 m. Ekman transport (m3 s-1 km-1) was averaged over the 6 days prior to sampling date. Standard deviation for each variable is in parentheses.

34 Chapter 2. Size-scaling of phytoplankton abundance

stages of the upwelling event, when nitrate concentration is higher and chl a concentration is still relatively low. Upwelling type II corresponds to the final stages of upwelling events, when the northern winds relax and a moderate stratification of the water column leads to bloom development. The winter mixing period shows the characteristics of temperate waters in late autumn and winter, with a high nutrient concentration, strong vertical mixing and low phytoplankton biomass. In contrast, the stratification period is characterised by a strong vertical thermal gradient and a high water column stability. Finally, downwelling conditions are frequent in early autumn and involve the advection of warm water from the shelf towards the coast.

2.3.3 General size-abundance patterns

A significant, inverse linear relationship between abundance and cell size was persistent throughout the study and at different levels of integration, including i) individual samples from a given depth, ii) water column spectra and iii) annual spectra (Fig. 2.5). During the entire study period, the slope of the log-log relationship between phytoplankton total abundance and cell size in individual samples ranged from -1.39 to -0.66. In addition, we found that the slope of the size-abundance spectra from different depths on the same sampling date were not significantly different from the slope of the corresponding water column macrospectra (Student’s t-test always P < 0.05). Consequently, we assumed that the water column macrospectra were representative of the phytoplankton community size structure for a given sampling date. Finally, when we pooled together all the observations collected along the time series, the resulting overall

35 Chapter 2. Size-scaling of phytoplankton abundance

macrospectrum had a slope of -0.96 (Fig. 2.6).

Fig. 2.5 Representative abundance-size spectra at different levels of integration: A) abundance-size spectra at different depths on a particular sampling day (November 16, 1994), B) water-column abundance-size macrospectra for January 1995, March 1995 and August 1995 and C) abundance-size macrospectra for 1995, 1998 and 2002. Each spectrum was fitted to a linear model by r. m. a. regression.

36 Chapter 2. Size-scaling of phytoplankton abundance

5

4

3

2

1 abundance (cell/mL)

10 0 log -1

-2 01234567 log cell size (m3) 10

Fig. 2.6 Overall size-abundance macrospectrum for the whole time series. The 2 regression line is: log10 total abundance = 4.12 – 0.97 log10 cell size (r = 0.59; P < 0.0001, n = 5964).

2.3.4 Size-abundance spectra and hydrographic variability

Size-abundance macrospectra were obtained from each hydrographic period identified by plotting together all the water column macrospectra of the different sampling dates belonging to each period (Table 2.2). All hydrographic periods showed similar values of the slope of the size- abundance relationship (Student’s t-test, P > 0.05), with the exception of upwelling II, which had a significantly less negative slope (Table 2.2;

37 Chapter 2. Size-scaling of phytoplankton abundance

Student’s t-test always P < 0.0001), indicating an increased importance of larger species. The intercept of the size-abundance macrospectra had its lowest value in winter, reflecting low levels of phytoplankton abundance, whereas the highest values were recorded during the upwelling type II and stratification periods (Table 2.2).

We analysed the vertical variability in the size-abundance spectra during two contrasting hydrographic periods, winter mixing and stratification, by calculating the averaged slope of size-abundance spectra for each depth within each period (Fig. 2.7). During the winter-mixing period, the slope of the size-abundance spectra throughout the water column

Table 2.2 Statistical parameters for the relationship between log10 total cell abundance and log10 nominal size for the different hydrographic periods analysed during 1993-2000.

Hydrographic n Y-intercept b (slope) r2 period

Winter mixing 2107 3.92 (3.85, 4.00) -0.97 (-0.99, -0.95) 0.65

Upwelling type I 669 4.22 (4.07, 4.38) -0.98 (-1.03, -0.94) 0.58

Upwelling type II 1066 4.05 (4.05, 4.16) -0.85 (-0.88, -0.82) 0.50

Stratification 1188 4.35 (4.24, 4.47) -0.99 (-1.03, -0.96) 0.60

Downwelling 470 4.11 (3.96, 4.27) -1.01 (-1.05, -0.96) 0.68

The Y-intercept and slope (b) were obtained using r.m.a. regression analysis. n indicates the number of observations included into the regression analysis. Confidence limits (95%) for the intercept and slope are given in parentheses.

38 Chapter 2. Size-scaling of phytoplankton abundance

had very similar values, ranging between -0.95 and -1.02, which reflected the vertical homogeneity of phytoplankton community size structure. By contrast, a higher variability in slope values was observed during the stratification period. In this case, more negative values were registered in surface waters and less negative values appeared below the depth of 20 m, indicating an increasing importance of larger phytoplankton in deeper waters.

0 AB

-20

-40 Depth (m)

-60

-80 -1,3 -1,2 -1,1 -1,0 -0,9 -0,8 -0,7 -1,3 -1,2 -1,1 -1,0 -0,9 -0,8 -0,7 Slope Slope

Fig. 2.7 Vertical variability of the mean macrospectrum slopes during A) winter mixing and B) summer stratification.

Finally, we tried to ascertain whether or not there was any relationship between phytoplankton biomass, as represented by chl a concentration, and the parameters of the scaling relationship between abundance and cell size. We classified all the sampling dates into three groups, according to the values of surface chl a concentration: <0.2, 0.2-2

39 Chapter 2. Size-scaling of phytoplankton abundance

and >2 mg chl a m-3. Size-abundance macrospectra were then constructed for each group by plotting together all the water column macrospectra corresponding to each chlorophyll level. The slope of the high chlorophyll macrospectrum (-0.93) was significantly less negative (Student’s t-test, P < 0.001) than that of the low chlorophyll macrospectrum (-0.97), indicating a higher relative importance of larger cells during conditions of high phytoplankton biomass (Table 2.3). In contrast, no significant differences were found between the medium chlorophyll macrospectrum and the other chlorophyll macrospectra (Student’s t-test, P > 0.05). In addition, the intercept of the low chlorophyll macrospectrum had a significantly lower value, reflecting a lower phytoplankton abundance in all size classes when compared to the other two situations. In contrast, the high chlorophyll macrospectrum showed the highest value of the intercept, corresponding to the highest abundance levels.

Table 2.3 Statistical parameters of the macrospectra obtained with samples of different levels of chl a concentration (mg m-3).

Chl a n Y-intercept b (slope) r2

<0.2 mg m-3 2349 3.89 (3.83, 3.86) -0.97 (-0.99, -0.95) 0.70

0.2> mg m-3 <2 1440 4.08 (3.98, 4.18) -0.95 (-0.98, -0.92) 0.59

mg m-3 >2 1936 4.27 (4.18, 4.36) -0.93 (-0.95, -0.90) 0.52

The Y-intercept and slope (b) were obtained using r.m.a. regression analysis. n indicates the number of observations included into the regression analysis. Confidence limits (95%) for the intercept and slope are given in parentheses.

40 Chapter 2. Size-scaling of phytoplankton abundance

2.3.5 Time series analysis

The temporal variation of the slope of the water column macrospectra, together with the slopes of the 5 m and 40 m depth size- abundance spectra for the entire time series, are shown in Fig. 2.8. We observed a significant trend towards less negative slopes in all three variables (trend slope = 0.014, P < 0.05 for water column macrospectra slopes, trend slope = 0.015, P < 0.05 for 5 m depth spectra slopes, and trend slope = 0.015, P < 0.01 for 40 m depth spectra slopes). In order to determine if these interannual trends were due to an increase in the abundance of larger cells or a reduction in the abundance of smaller species, we examined the temporal evolution in the biovolume of the three main phytoplankton groups, namely flagellates (2-9 µm in ESD), dinoflagellates (7-72 µm in ESD) and diatoms (3-230 µm in ESD) (data not shown). Only the flagellates showed a significant change in biovolume during the study, with lower values towards the end of the sampling period (P < 0.001 at 5 m and < 0.05 at 40 m depth). We also found a significant, negative correlation between flagellate biovolume at a depth of 5 m and 40 m and the spectral slopes at these depths (r2 = 0.23, P < 0.05; r2 = 0.19, P < 0.05 respectively). It thus seems that the temporal trend towards less negative slopes in the size- abundance relationship (e.g. an increase in the relative importance of larger cells) would result from a decrease in flagellate biomass rather than an increase in the biomass of diatom or dinoflagellates.

In order to explore the possible causes for the observed interannual trends in the size-abundance slope and the biomass of flagellates, we analysed the temporal variability in sea surface temperature and salinity,

41 Chapter 2. Size-scaling of phytoplankton abundance

-0,4 A -0,6

-0,8

-1,0

Slope -1,2

-1,4

-1,6

-1,8

-0,4 B -0,6

-0,8

-1,0

Slope -1,2

-1,4

-1,6

-1,8 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002

Fig. 2.8 Temporal evolution in the slope of A) water-column macrospectra and B) spectra from 5 m (white circle, dashed line) and 40 m (dark circle, solid line) depths.

42 Chapter 2. Size-scaling of phytoplankton abundance

nutrient concentration at 5 and 40 m depth, mixing layer depth, Ekman transport and North Atlantic Oscillation index, as well as their cross- correlation with the phytoplankton size structure. However, we did not find any significant correlation between the slope of the size-abundance spectrum (calculated both on an annual and a monthly basis) and these hydrographic and climatic variables. On the contrary, when we analysed separately the relationship between deseasonalised flagellate biovolume and deseasonalised hydrographic variables, we found that the biomass of this phytoplankton group was inversely correlated with mixing layer depth. This means that, when mixing depth was shallower (and, therefore, water-column stratification was more intense) flagellates tended to be more abundant.

2.4 Discussion

2.4.1 General patterns in the size scaling of phytoplankton abundance

Although several studies have previously addressed the temporal (Gilabert 2001, Reul et al. 2005) and vertical (Gin et al. 1999, Rodríguez et al. 2001) variability in the size-scaling of phytoplankton abundance in marine ecosystems, to our knowledge this is the first study that includes repeated monthly observations over both seasonal and interannual timescales. The present work thus provides a robust description of general patterns in the phytoplankton size structure of coastal waters, which is based on the analysis of some 600 size-abundance spectra. Our data indicate that the linear inverse relationship between abundance and cell size i) is persistent throughout the water column and across seasonal and interannual timescales, and ii) can be adequately described by a power law.

43 Chapter 2. Size-scaling of phytoplankton abundance

It has been often emphasised that more productive ecosystems subjected to stronger hydrodynamical forcing can show irregularities (i. e., dome-like patterns) in the linear relationship between abundance and cell size (Sprules and Munawar 1986, Dickie et al. 1987), as blooms of one or a few species would cause a significant departure from linearity in the log-log relationship between abundance and cell size (e. g., Reul et al. 2006). In our study, however, even though >10-fold changes in phytoplankton chlorophyll were observed over seasonal time scales, the size-scaling relationship could be always adequately described by a statistically significant regression line and major departures from linearity were seldom observed (Fig. 2.5). Recent modelling work has shown that the power-law relationship between abundance and cell size results from the size scaling of cellular nutrient requirements and growth (Irwin et al. 2006), without the need to invoke other mechanisms such as competition or size-dependent changes in grazing and sedimentation. Although the precise parameters of the functions relating cell size to resource use and growth rate change according to taxonomic affiliation and resource availability, among other factors (Banse 1982, Finkel 2001, Marañón et al. 2007), these relationships tend to conform to a power law. This explains our observation that the scaling of phytoplankton abundance and cell size, even in a productive ecosystem, can also be described by a power relationship, and the persistence of this relationship throughout the study and at different levels of integration suggest the importance of bottom-up control of the phytoplankton size structure in the study site.

The scaling of phytoplankton abundance and cell size depends on

44 Chapter 2. Size-scaling of phytoplankton abundance

resource availability: less productive waters are characterised by steeper (more negative) slopes, indicating a higher relative importance of smaller species (Gin et al. 1999, Cavender-Bares et al. 2001, Marañón et al. 2007). Cermeño and Figueiras (2008) presented a review of size spectra for nano- and micro-phytoplankton obtained in regions of contrasting trophic status (their Table 1). They found that the slope of the size spectrum ranged from values around -0.8 in coastal, eutrophic ecosystems (where mean primary production over the year is usually higher than 2 g C m-2 d-1) to values as low as -1.3 for the ultraoligotrophic waters of the Atlantic subtropical gyres (where typical primary production rates are below 0.3 g C m-2 d-1). The average slope observed in the present study (-0.96) agrees with the fact that our study site is characterised by high productivity (mean primary production over the period 1993-2002 was 1.7 g C m-2 d-1). In the Ría de Vigo, a coastal embayment which is located some 150 km to the south of our study site and has an average primary production of ca. 2.5 g C m-2 d-1 (Marañón et al. 2004, Cermeño et al. 2006), the slope of the phytoplankton size spectra takes values between -0.9 and -0.8 (Marañón et al. 2007, Cermeño and Figueiras 2008). These results illustrate the macroecological pattern whereby increasingly productive regions tend to show an increasing dominance by larger phytoplankton (Chisholm 1992, Marañón et al. 2001).

Our observations of the scaling between cell size and abundance have direct implications for the distribution of phytoplankton biomass along the size spectrum. If phytoplankton total abundance (N) scales with cell size (V) according to N  Vb and phytoplankton carbon biomass per cell (C) scales with V as C  Vc, then total biomass (N × C) will scale as Vb+c.

45 Chapter 2. Size-scaling of phytoplankton abundance

Although the determination of the value of c is subject to several methodological uncertainties, most recent estimates fall in the range 0.9-1 (Montagnes and Berges 1994, Menden-Deuer and Lessard 2000, Finkel 2001), which implies that, for our study system, b + c will take an average value close to zero. This would mean that, within the large nano- to micro- phytoplankton size range, approximately equal amounts of biomass are contained within each logarithmic size class. This result is in accordance with the early observations of Sheldon et al. (1972), although in their case biovolume rather than biomass was considered.

2.4.2 Hydrography and phytoplankton size structure

With the exception of the upwelling relaxation phase, we found that size macrospectra had similar slopes in the different hydrographic conditions identified (Table 2.2). This suggests that, in these oceanographic conditions, both nano- and micro-phytoplankton respond similarly to changes in hydrodynamic forcing and resource availability. Similar observations have been reported for the southern Bay of Biscay, where the slopes of size spectra of seston particle biovolume exhibited low variability despite a large range of Brunt-Väisalä values observed during the seasonal cycle (Bode and Fernández 1992). Although the spectra constructed by Bode and Fernández (1992) were normalized biomass size spectra, the direct comparison with our slope values is possible, as the regression parameters of both scaling approaches are equivalent (Blanco et al. 1994). In addition, in the Galician shelf the biomass of both nanoplankton and microplankton increased similarly (by a factor of 2) from winter to spring bloom conditions (Bode et al. 1994). Lower standing stocks of

46 Chapter 2. Size-scaling of phytoplankton abundance

phytoplankton in winter, which result from excessive mixing and low light availability, are also reflected in our analysis, since the winter size macrospectra had a significantly lower Y-intercept than those of the stratification and upwelling periods (Table 2.2).

Upwelling relaxation (defined as upwelling II in our analysis) brings about the largest increases in phytoplankton biomass and productivity in the Galician Rías and shelf (Bode and Varela 1994, Casas et al. 1999, Tilstone et al. 2000). During the peak of the upwelling event, when upward water velocities are highest, relatively strong turbulence and offshore wash-out of cells, together with the physiological time lag required for communities to adjust to the new conditions, prevent the accumulation of large amounts of phytoplankton. During upwelling relaxation, however, high nutrient concentrations combined with enhanced water-column stability and reduced dispersion allow the onset of phytoplankton blooms, which are typically dominated by chain-forming diatoms. In our study, this process is evidenced in a significantly less negative slope (-0.85) of the size macrospectrum (Table 2.2), which indicates the increasing dominance of larger cells. The common association between increased phytoplankton biomass and productivity and a higher dominance of large phytoplankton, and in particular diatoms, has been often attributed to trophic effects (Kiørboe 1993, Irigoien et al. 2005). In this view, large phytoplankton dominance is not caused by higher intrinsic growth rates, relative to those of smaller phytoplankton, but rather from the higher grazing pressure experienced by the latter. However, there is recent evidence, both from modelling (Irwin et al. 2006) and experimental studies (Cermeño et al. 2005a, Marañón et al.

47 Chapter 2. Size-scaling of phytoplankton abundance

2007), which suggests that large phytoplankton are capable of sustaining higher growth rates than small phytoplankton under conditions of high resource availability.

Our observations also allow us to extract some conclusions regarding the vertical variability in phytoplankton size structure (Fig. 2.7). During the winter mixing period, the slope of the size spectrum was constant with depth, reflecting a relatively homogeneous size structure throughout the euphotic layer. However, during the stratification period the slope became less negative with depth (-0.8 at 30 m compared with -1 at the surface), which indicates that the relative importance of large phytoplankton was greater near the bottom of the euphotic layer. This pattern contrasts with that reported for the stratified waters of the tropical and subtropical open ocean, where the deep chlorophyll maximum (DCM) is characterised by an increased dominance of small phytoplankton, compared with the upper mixed layer (Gin et al. 1999, Pérez et al. 2006, Poulton et al. 2006). These authors have interpreted this pattern as a result of the higher light-use efficiency of smaller cells, which would be advantageous in the low- irradiance conditions of the bottom of the euphotic layer. It has to be noted, however, that the DCM in the low-latitude open ocean is a permanent structure characteristic of a near steady-state ecosystem. In our study site, a coastal location subject to intermittent upwelling during summer, stratification is never a persistent feature but rather appears only between successive upwelling pulses. After each upwelling-caused bloom, intense sedimentation of large, fast-sinking species takes place (Figueiras and Pazos 1991, Varela et al. 1991, Cermeño et al. 2006), which explains the observed

48 Chapter 2. Size-scaling of phytoplankton abundance

trend towards less negative size spectrum slopes in subsurface waters. By contrast, the surface layer, where nutrients are scarcer, is dominated by smaller species (Varela et al. 1991), which are better adapted to use low concentrations of nutrients (Chisholm 1992, Kiørboe 1993).

2.4.3 Interannual variability in size spectra

Although longer time series are needed in order to establish significant, long-term trends in the structure and functioning of marine ecosystems (e.g., Leterme et al. 2005), our data set allowed us to explore the interannual variability in phytoplankton size structure, as represented by the parameters of the size spectra during a 10-year period. We found that community size structure showed a significant interannual trend towards less negative slope values (Fig. 2.8). While there was no significant correlation between the slope of the size spectrum and the interannual variability in the physico-chemical variables, we did find that the observed trend in slope was related to a decrease of flagellate abundance, rather than to any change in the abundance of diatoms or dinoflagellates. Moreover, the biomass of flagellates (<10 µm in ESD) was inversely correlated to the mixing layer depth, which would suggest that on more stratified settings flagellates are favoured relative to other phytoplankton groups. As mentioned above, the association between enhanced stratification, reduced nutrient supply and increased dominance of small cells is a well-established pattern in biological oceanography (Chisholm 1992, Kiørboe 1993). Additional studies are required, however, to ascertain the occurrence and causal mechanisms of interannual trends in the composition and size

49 Chapter 2. Size-scaling of phytoplankton abundance

structure of phytoplankton in our study region.

2.4.4 Conclusions

Even though our study ecosystem is highly productive and characterised by marked temporal changes in hydrodynamics, the inverse linear relationship between phytoplankton abundance and cell size was persistent over seasonal and interannual time scales. In addition, it could be adequately described by a power-law model, which makes it easy to incorporate into size-based mathematical models of plankton dynamics. The slope of the size macrospectrum obtained from the overall time series took a value of -0.96, which indicates that roughly equal amounts of biomass were present over the different logarithmic size classes in the nano- to micro- phytoplankton size range. During the different hydrographic periods that occur over the year, the slope of the size macrospectrum took a similar value, suggesting that both the nanophytoplankton and the microphytoplankton often respond similarly to environmental forcing. An exception was the upwelling relaxation period, when a stronger dominance of large phytoplankton resulted in a significantly less negative (-0.85) slope. The vertical variability in the size spectrum slope during stratification showed a trend towards less negative values with depth, probably as a result of sedimentation of large cells after an upwelling-caused bloom. Our results illustrate the utility of individual size distributions to provide a synthetic description of phytoplankton community structure in dynamic, non steady- state marine ecosystems.

50

Chapter 3 Effect of environmental forcing on the biomass, production and growth rate of size-fractionated phytoplankton in the central Atlantic

Chapter 3. Size-fractionated phytoplankton biomass and production

Abstract

To ascertain the response of the different size classes of phytoplankton to changes in water-column stability and nutrient availability, we determined size-fractionated biomass, carbon fixation rates and growth (production/biomass) rates in surface waters along the central Atlantic Ocean (26ºN-5ºS). As a result of the enhanced input of nutrients into the euphotic layer and the higher water-column stability found at the equatorial upwelling, we observed increases not only in phytoplankton standing stocks and primary production, but also in turnover rates, suggesting nutrient limitation of phytoplankton physiology in the oligotrophic central Atlantic. In addition, the four phytoplankton groups analysed (pico-, small nano-, large nano- and micro-phytoplankton) showed different responses to environmental forcing, associated with the equatorial upwelling, in terms of carbon biomass, primary production and growth rate. Large nano- and micro-phytoplankton consistently showed higher growth rates and carbon fixation to chl a ratios than smaller phytoplankton, and thus contributed to total production more than could be expected on the basis of their share of biomass. We observed a higher stimulating effect of increased nutrient supply on the growth rates of small phytoplankton. This observation can be explained by considering the dynamics of the equatorial upwelling, where the continuous but small nutrient input into the euphotic layer provide a competitive advantage for smaller cells adapted to oligotrophic conditions. The size-fractionated approach to phytoplankton biomass, production and growth reveals important group-specific differences in the response to environmental forcing, which cannot be appreciated in bulk measurements of the whole community.

53 Chapter 3. Size-fractionated phytoplankton biomass and production

54 Chapter 3. Size-fractionated phytoplankton biomass and production

3.1 Introduction

Phytoplankton size structure is a critical property of pelagic ecosystems as it largely determines their food-web organization and biogeochemical functioning. In addition, both chemical and physical characteristics of ecosystems are key factors in controlling the size distribution of phytoplankton community. Among other factors, a higher nutrient supply tends to stimulate in particular the growth of large phytoplankton, and the upwelling water motion increases the time of permanence of large cells inside the photic layer, thus increasing primary production (Parsons and Takahashi 1973, Malone et al. 1993, Kiørboe, 1993).

It is commonly accepted that in pelagic ecosystems picophytoplankton constitute a background and constant component of the phytoplankton community (Raimbault et al. 1988, Thingstad and Sakshaug 1990, Rodríguez et al. 1998). However, recent studies have demonstrated that, in addition to the spatial and temporal variability observed for picophytoplankton abundance in both open-ocean and coastal ecosystems (Partensky et al. 1996, Zubkov et al. 1998, Calvo-Díaz et al. 2006, Li et al., 2006), there is also a response of small phytoplankton biomass and primary production to the enrichment of oligotrophic regions of the ocean (Barber and Hiscock 2006, Tarran et al. 2006, Glover et al. 2007). Given the importance of the nutrient-impoverished regions of the ocean in global biogeochemical cycles (Longhurst et al. 1995), these results open new questions about which factors control the size-differential dynamics of phytoplankton metabolism and, in consequence, the size structure of

55 Chapter 3. Size-fractionated phytoplankton biomass and production

phytoplankton in open-ocean ecosystems.

Few studies have focused on the simultaneous analysis of size- fractionated biomass and productivity of phytoplankton in the open ocean over larger spatial scales. In most cases, biomass has been almost always estimated from chlorophyll a concentrations, rather than from direct estimations of cell carbon content (Marañón et al. 2001, Poulton et al. 2006). Moreover, instead of covering all size classes [the pico- (0.2-2 μm in espherical equivalent diameter, ESD), nano- (2-20 μm in ESD) and micro- phytoplankton (>20 μm in ESD) (Sieburth 1979)], studies generally have focused on the picophytoplanktonic component of the community (Zubkov et al. 1998, Li and Harrison 2001, Worden et al. 2004) or they have only distinguished between picophytoplankton and large phytoplankton (including within this group all cells >2 μm ESD) (i.e. Joint and Pomroy 1986, Jochem and Zeitzschel 1993, Poulton et al. 2006). However, recent studies have highlighted the importance of conducting this kind of analysis for the whole phytoplankton size range, in order to better understand the carbon fluxes through the microbial plankton community (e.g. Latasa et al. 2005). This importance arises from the different dynamics, in both biomass and production, showed by the main phytoplankton taxonomic groups under different environmental conditions, which can be translated into a different contribution of each group to carbon cycling.

In addition, a more informative approach to the study of the functioning of planktonic communities is the analysis of the variability of phytoplankton turnover rates in combination with primary production estimations and carbon biomass measurements for the different

56 Chapter 3. Size-fractionated phytoplankton biomass and production

phytoplankton size groups. In this sense, the simultaneous variation of primary production and turnover rates under changing environmental forcing will mean that phytoplankton are not only limited in their standing stocks but also in their physiological state (Goldman et al. 1979). However, the studies carried out in order to determine the growth rates for different size groups of phytoplankton in the field are very scarce, and most of them have considered only the smaller size classes such as the picophytoplankton (i.e. Liu et al. 1997, Hirose et al. 2008, Zubkov and Tarran 2008).

The latitudinal variability in nutrient concentration and water- column structure of the tropical and subtropical Atlantic Ocean is well documented (e.g. Marañón et al. 2000, Marañón et al. 2001, Poulton et al. 2006). A “Typical Tropical Structure” (TTS; Herbland and Voituriez 1979), whereby a nutrient-depleted upper mixed layer is separated from a light- limited deep layer by a strong density gradient, is found in those regions under the influence of the oligotrophic gyres, while a persistent upwelling takes place in the equatorial region (Herbland et al. 1987, Longhurst 1993, Monger et al. 1997, Pérez et al. 2005a). This equatorial upwelling is the result of the differential effect of the Ekman transport on both sides of the Equator, which causes the entrance into the photic layer of nutrient-rich water from the equatorial undercurrent and the shallowing of the deep chlorophyll a maximum (DCM) that typically develops at or close to the pycnocline depth (Longhurst 1993, Tomczak and Godfrey 1994). Therefore, the study of the variability of biotic and abiotic factors throughout a long latitudinal transect of the Atlantic central ocean crossing the equatorial upwelling constitutes a good opportunity for testing the effect of

57 Chapter 3. Size-fractionated phytoplankton biomass and production

environmental forcing, mainly nutrient availability and water-column stability, on the dynamics of the phytoplankton community. Here we report the latitudinal variability of size-fractionated phytoplankton carbon biomass, primary production and growth rates in a survey conducted from 26ºN to 5ºS along the 29ºW meridian, with two main objectives: i) to determine the response of the different size groups of phytoplankton, in terms of both biomass and production, to changes in water-column stability and nutrient availability; and ii) to determine the latitudinal variability of size- fractionated phytoplankton growth rates in order to assess the physiological response of different size classes to environmental forcing and, in particular, nutrient supply. The results of this chapter are reported in an article which has been sent for publication (Huete-Ortega et al. in review).

3.2 Methods

3.2.1 Sampling, hydrography, irradiance and nutrients

We sampled 10 stations during the TRYNITROP (Trichodesmium and N2 fixation in the tropical Atlantic) cruise, carried out in November- December 2007 in the tropical Atlantic Ocean (Fig. 3.1) on board R/V Hespérides. At each sampling station, vertical profiles (0-300 m) of temperature, salinity and in situ fluorescence were obtained using a Conductivity-Temperature-Depth (CTDSBE911) probe attached to a rosette sampler equipped with Niskin bottles. Using the density computed from the temperature and salinity data binned over 1-m intervals, we calculated the Brunt-Väisäla frequency (BV), averaged over the upper 125 m. Sampling was conducted before dawn and different water subsamples were collected

58 Chapter 3. Size-fractionated phytoplankton biomass and production

Fig. 3.1 Location of the sampling stations superimposed on a map of the climatological mean surface chlorophyll a concentration for the period September-November of the years 2002-2008 in the Atlantic Ocean. Ocean color data are from MODIS Aqua (9 Km). from the Niskin bottles to determine nutrient concentration, phytoplankton biomass and abundance, chlorophyll a concentration and primary production.

59 Chapter 3. Size-fractionated phytoplankton biomass and production

During our cruise and a subsequent cruise conducted along the same track, in April-May 2008, 16 vertical profiles of photosynthetically active irradiance (PAR) were obtained at noon with a Satlantic OCP-100FF radiometer. A highly significant relationship was obtained between the depth of the 1% PAR (Zeu) and the depth of the DCM identified by in situ 2 fluorescence (ZDCM): Zeu = 9.3 + 0.98 × ZDCM (r = 0.93, P < 0.001, n = 16).

This relationship was used to calculate Zeu and the light extinction coefficient (Kd) in all sampled stations.

Nanomolar and micromolar nutrient concentrations were determined with a segmented-flow automatic analyser (Technichon/Bran Luebbe), following the method of Raimbault et al. (1990). Given that the concentrations of nitrate, phosphate and silicate concentrations were highly correlated along the latitudinal transect (r2 = 0.97, P < 0.001, n = 109 and r2 = 0.85, P < 0.001, n = 103 for nitrate vs. phosphate and nitrate vs. silicate correlations, respectively), and considering that neither silicate (mean concentration in the upper mixed layer was 1.4 µmol L-1) nor phosphate (the mean nitrate to phosphate ratio in the upper mixed layer was 3.0) were likely to be limiting, our analysis was subsequently focused on the latitudinal variability of nitrate concentration and its limiting role on the phytoplankton metabolism. The nitracline depth was defined as the depth below which nitrate concentration was equal to 0.5 µmol L-1. Vertical nitrate fluxes into the euphotic layer were calculated from the product of the estimated diffusion coefficients (kz) and the gradients of nanomolar nitrate concentration, computed across the depth of the euphotic layer by using linear interpolation. The photic layer depth corresponded to the 1% of PAR

60 Chapter 3. Size-fractionated phytoplankton biomass and production

(photosynthetic active radiation). Vertical diffusivity (kz) was estimated from Osborn (1980) as:

 kz  e N 2

where e is a mixing efficiency (e = 0.2 herein),  is the dissipation rate of turbulent kinetic energy, and N is the Brunt-Väisäla frequency averaged over depth intervals of 10 m length. Assuming that the dissipation of turbulent kinetic energy balances the local production due to the shear induced by the wind stress,  was parameterized from wind data (Terray et al. 1996) as:

3 u *   KZ where k is the von Karman’s constant (0.4) and u* is the friction velocity, related to the surface wind stress by:

2 2  = wu* = a CD W

where w is the water density, a is the air density, CD is a drag coefficient and W is the wind speed, determined with an Aanderaa meteorological station during the cruise. For each station, wind speed was averaged during 24 h before the CTD sampling. The drag coefficient was 1.14  10-3 for 4 < -1 -3 W ≤ 10 m s and computed as CD = (0.49 + 0.065 W)  10 for 10 < W < 26 m s-1 (Large and Pond 1981).

61 Chapter 3. Size-fractionated phytoplankton biomass and production

3.2.2 Size-fractionated chlorophyll a concentration

Two replicates of 500 mL surface (5 m) seawater were sequentially filtered through 40 µm net filters and 20, 10, 5, 3, 2, 0.8 and 0.2 µm polycarbonate filters under low vacuum pressure (<50 mmHg). In order to avoid the undersampling of larger cells, for 40, 20, 10 and 5 µm filters, the whole 500 mL were filtered, whereas for 3, 2, 0.8 and 0.2 µm filters only 250 mL were filtered. After filtration, pigments were extracted in 90% acetone during 24 h in the dark and at 4ºC. Chlorophyll a (chl a) concentration was estimated with the non-acidification technique using a Turner Designs Fluorometer (TD-700), previously calibrated with pure chl a. The detailed analysis of each of the 8 assayed size classes was used to calculate the size-scaling of carbon fixation and abundance, which is reported in Chapter 4. Here, to calculate the chl a concentration in the phytoplankton size classes commonly used in the literature, concentrations measured in different filters were added: 0.2 and 0.8 μm for picophytoplankton (<2 μm in ESD), 2 and 3 μm for small nanophytoplankton (2-5 μm in ESD), 5 and 10 μm for large nanophytoplankton (5-20 μm in ESD), and 20 and 40 μm for microphytoplankton (>20 μm in ESD). Total chl a values were calculated as the sum of all size-fractionated chl a concentrations.

3.2.3 Size-fractionated carbon fixation rate

Photosynthetic carbon fixation rate was measured with the 14C- uptake techique. One-litre polycarbonate bottles (3 transparent and 1 dark bottles) were filled with surface (5 m) seawater from each station, taking

62 Chapter 3. Size-fractionated phytoplankton biomass and production

care to avoid any light shock to the plankton populations. Bottles were 14 inoculated with ~100 µCi NaH CO3 and then incubated during 6-9 hours in on-deck flow-through incubators which were cooled with running surface seawater. The irradiance received by the incubation bottles was attenuated to 80% using a neutral density screen. At the end of the incubation time, water samples were sequentially filtered, under low vacuum pressure, through the same types of filters used for the estimation of the size- fractionated chl a concentration. In order to ensure adequate sampling of larger cells, for 40, 20, 10 and 5 µm filters the whole content of the 1-L bottles was filtered, whereas in the case of the 3, 2, 0.8 and 0.2 µm filters only 500 mL were filtered. The unassimilated dissolved inorganic 14C was removed from the filters by exposing them to concentrated HCl fumes during 10-12 hours. Filters were then placed in 5-mL scintillation vials to which 4 mL of scintillation cocktail were added. The radioactivity of each filter was measured with a Wallac scintillation counter. Disintegrations per minute (DPMs) from the dark bottle filter were subtracted from the DPMs of each light bottle filter and the carbon fixation rate of each size class was calculated taking into account the filtered volume. Carbon fixation rates from each light bottle were subsequently averaged. To obtain daily carbon fixation rates, the hourly rates were multiplied by the duration of the daylight period and it was assumed that 20% of the carbon fixed during the light period is lost through respiration during the night (Geider 1992). Carbon fixation rates for pico-, small nano-, large nano- and micro- phytoplankton were calculated from the rates determined for each individual size class, as explained before for chl a concentration. Total carbon fixation rates for each sample were calculated as the sum of the size-fractionated

63 Chapter 3. Size-fractionated phytoplankton biomass and production

carbon fixation rates.

3.2.4 Phytoplankton cell size and abundance

The abundance of pico- (<2 µm in ESD) and small nano- phytoplankton (2-5 µm in ESD) was determined by flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson) with a laser emitting at 488 nm. Samples (4 mL) were fixed with 1% (v/v) paraformaldehyde and 0.05% glutaraldehyde, frozen in liquid nitrogen and subsequently kept at - 80ºC until analysis at the laboratory. Aliquots of each sample were used for the analysis of the cell size and abundance of pico- and small nano- phytoplankton. As our aim was to determine the total cell abundance in a given size range, taxonomical groups such as Prochlorococcus spp. or Synechococcus spp. were not distinguished. Calibration of the cytometer flow rate was performed daily (Marie et al. 1999) for estimating the abundance of both size ranges. In order to increase the number of counting events, the analyses for large picoeukaryotes and small nanophytoplankton were always conducted during 10 minutes at high flow rate (around 40 µL min-1). An empirical calibration between relative side scatter (SSCrel) and cell diameter (D) following Zubkov et al. (1998) was used to estimate the individual cell biovolume (V) of picophytoplankton cells, D = 1.0049  SSCrel + 0.6297. For the small nanophytoplankton, V was estimated using a calibration curve that relates the light scattering signal to cell biovolume estimated by image analysis, log V = 0.0079 x FSC + 1.0699 (Rodríguez et al. 1998). This latter calibration curve was constructed with measurements of monospecific cultures of Nannochloropsis gaditana (2-5 μm in ESD), Isochrysis galbana (3-5 μm in ESD), Phaeodactylum tricornutum (3-6 μm

64 Chapter 3. Size-fractionated phytoplankton biomass and production

in ESD), Rhodomonas spp. (7 μm in ESD), Heterocapsa spp. (15 μm in ESD) and Alexandrium spp. (20 μm in ESD).

Large nano- (5-20 µm in ESD) and micro- (>20 µm in ESD) phytoplankton were determined by image analysis under an inverted microscope. With the aim of increasing the number of large-sized cells sampled, we used 2 replicates of 2 L of seawater. Samples were filtered through 5 μm polycarbonate filters and then the material collected on the filters was gently resuspended with 0.2 μm-filtered seawater until completing a sample volume of 125 mL. One of these replicate samples was fixed with 2% Lugol’s solution whereas the other one was fixed with 4% formaldehyde. Samples were stored in the dark until their analysis following the method of Utermöhl (Lund et al. 1958). Subsamples of 100 mL in volume were allowed to settle in sedimentation columns during 48 hours and then counted using an Olympus IX50 inverted microscope. A magnification of 100 was used for large forms, 200 for intermediate forms and 400 for smaller forms. In the samples fixed with Lugol’s solution, the cell biovolume of 400 cells was determined using the geometric shapes recommended in Olenina et al. (2006) and the NIS- Elements BR 3.0 image analysis program. Samples fixed with formaldehyde’s solution were only analyzed in order to estimate the cell abundance and biovolume of coccolitophores. In the case of chain-forming species (i.e., Trichodesmium spp., Rhizosolenia spp. or Pseudonitzchia spp.), measurements of the average cell size of the cells that formed each trichome or chain and the size of each trichome and chain were carried out in order to estimate the cell abundance of these species in each sample.

65 Chapter 3. Size-fractionated phytoplankton biomass and production

3.2.5 Phytoplankton carbon biomass and growth rates

Picophytoplankton biovolume (V) was converted to carbon biomass (C) using the conversion factor of 235 fg C μm-3 obtained from averaging the values proposed in Worden et al. (2004) for the different picophytoplankton groups. Small nanophytoplankton carbon was estimated with the conversion equation proposed by Verity et al. (1992), pg C cell-1 = 0.433 x V0.863, and large nano- and micro-phytoplankton carbon was calculated according to Montagnes et al. (1994), pg C cell-1 = 0.109 x V0.991. Phytoplankton carbon biomass (Phyto-C) was estimated by multiplying cell abundance by cellular carbon biomass and the resulting values were added in order to obtain the Phyto-C for pico-, small nano-, large nano- and micro- phytoplankton. Growth (turnover) rates for both total and size-fractionated phytoplankton were calculated by dividing production (carbon fixation rates) by biomass, as advised by Kirchman (2002).

3.3 Results

3.3.1 Hydrography and chlorophyll a concentration

The vertical distribution of temperature shown in Fig. 3.2A indicates strong thermal stratification in the upper mixed layer (UML) throughout the transect. Warmer UML temperatures (~28ºC) occurred south of 17ºN, coinciding with the strongest stratification of the water column. The 16ºC isotherm was relatively shallow (<120 m) between 17ºN and the Equator, indicating the upwelling of cold and nutrient-rich deep waters in this region. Very low nitrate concentrations (<0.05 μM) were measured in the UML along the whole transect and those stations most affected by upwelling

66 Chapter 3. Size-fractionated phytoplankton biomass and production

28 A 26 24 100 22 20 18 Depth 200 16 14 12 10 8

0 30 B 25 20 15 h

t 100 10 5

Dep 2 1 200 0.5 0.3 0.05

0 0.8 C 0.7 50 0.6 0.5 0.4 Depth 100 0.3 0.2 0.1 0 5 10 15 20 25 0 Latitude Fig. 3.2 Latitudinal and vertical variability of A) temperature (ºC), B) nitrate concentration (µmol L-1) and C) chlorophyll a concentration (μg L-1).

presented higher sub-surface concentrations (Fig. 3.2B). Similarly, the highest chlorophyll a concentration (chl a) values (>0.25 μg L-1) in the UML were found in those stations most influenced by the equatorial upwelling, whereas near both ends of the transect UML chl a below 0.2 µg

67 Chapter 3. Size-fractionated phytoplankton biomass and production

L-1 was measured, coinciding with the beginning of the subtropical gyres (Fig. 3.2C). A deep chl a maximum (DCM) was observed throughout the transect at the base of the photic layer. As a consequence of the equatorial upwelling, the DCM was shallower (~60 m) between 15ºN and the Equator.

Coherent patterns in the latitudinal variability of water-column stability (indicated by the Brunt-Väisäla frequency, averaged over the upper

125 m), PAR extinction coefficient (Kd), nitracline depth and nitrate diffusive fluxes computed across the euphotic zone were observed during the transect (Fig. 3.3A and 3.3B). In general, stronger water stability was observed between 15ºN and the Equator, as a result of the upwelling of cold -1 subsurface waters. In the case of Kd, higher values (>0.05 m ) were found between ca. 15ºN and the Equator, reflecting the presence of higher phytoplankton abundances in this region. Northwards and southwards of these latitudes, progressively smaller Kd values were measured, indicating the presence of very clear and oligotrophic waters with small phytoplankton abundances. Nitracline depth and nitrate diffusive flux showed opposite latitudinal patterns. The shallowest nitracline depths (<70 m) and the highest nitrate diffusive fluxes (~2500 µmol N m-2 d-1) were found in those stations under the influence of the equatorial upwelling (between 17ºN and the Equator), while the deepest nitraclines (>100 m) were registered northwards of 20ºN, coinciding with the lowest nitrate diffusive supply through the base of the euphotic layer.

68 Chapter 3. Size-fractionated phytoplankton biomass and production

3.3.2 Total phytoplankton biomass, production and growth rates

The concentration of surface phytoplankton carbon biomass (Phyto- C) showed a marked increase in the equatorial upwelling region, where values >20 µg C L-1 were measured (Fig. 3.3C). Outside this region, Phyto- C took values around 10 µg C L-1. A significant correlation between total Phyto-C and total chl a was found (r2 = 0.87, P < 0.01, n = 10). Surface total primary production varied from 0.5 µg C L-1 d-1 to 6 µg C L-1 d-1 and total turnover rates ranged between 0.05 d-1 and 0.2 d-1. In general, both variables showed a similar latitudinal pattern, with markedly higher values in the region affected by the equatorial upwelling (between 17ºN and the Equator). Phytoplankton turnover rates increased to values of up to 0.2 d-1 in the region most affected by the upwelling, whereas values below 0.08 d-1 were estimated for the stations near both ends of the transect. In addition, a significant correlation between surface total primary production and euphotic layer-integrated total carbon fixation rates was also found (r2 = 0.80, P < 0.01, n = 10; euphotic layer-integrated primary production data were obtained from Moreno-Ostos et al. in press).

3.3.3 Size-fractionated carbon biomass and production

Surface phytoplankton biomass was dominated by the <2 µm size fraction (picophytoplankton), with values between 5-20 μg C L-1 (Fig. 3.4A) and a relative contribution of >40% to total carbon biomass in most stations (Fig. 3.4C). Phytoplankton biomass of the 2-5 and 5-20 μm size fractions (small and large nanophytoplankton, respectively) were much lower (0.3-3.2 μg C L-1) with a relative contribution not higher than ca. 20% to total carbon

69 Chapter 3. Size-fractionated phytoplankton biomass and production

3,5e-4 0,08 A 3,0e-4 0,07

2,5e-4 0,06 ) -1

2,0e-4 0,05 (m d

1,5e-4 0,04 K

1,0e-4 0,03 Brunt-Väisäla frequency

5,0e-5 0,02 ) -1 d

160 3000 -2 140 B 2500 120 2000 mol N molm N  100 1500 80 1000 60 500

Nitracline depth (m) depth Nitracline 40 0 diffusive flux( 3

20 ) NO -1 d ) -1

-1 30 7 0,24

C 0,22 )

6 -1 g C L g C L 25 0,20   5 0,18 20 4 0,16 0,14 15 3 0,12 2 0,10 10 0,08

1 Total turnover rate (d 0,06

Total carbon biomass ( biomass Total carbon 5 0 0,04 -10-50 5 1015202530 Total carbon fixation rate ( fixation rate carbon Total Latitude Fig. 3.3 Latitudinal variability of A) Brunt-Väisäla frequency (circles, solid lines) and Kd (squares, dashed lines), B) nitracline depth (circles, solid lines) and nitrate diffusive flux computed across the euphotic zone (squares, dashed lines), and C) total carbon biomass (circles, solid lines), total carbon fixation rate (squares, dashed lines), and total turnover rate (triangles, dotted lines). Positive and negative latitude values correspond to the North and South hemisphere, respectively.

70 Chapter 3. Size-fractionated phytoplankton biomass and production

25 3,0 -1 d )

A B -1 -1 20 2,5 g C L g C L 

 2,0 15 1,5 10 1,0

5 0,5 Carbon biomass ( biomass Carbon

0 0,0 ( rate fixation Carbon

100 100 C D 80 80

60 60

40 40

20 20

0 0

Contribution to total carbon biomass (%) biomass carbon to total Contribution -10-50 5 1015202530-10-50 5 1015202530 Latitude Latitude (%) rate fixation carbon to total Contribution Fig. 3.4 Latitudinal variability of A) carbon biomass, B) carbon fixation rate, C) relative contribution (%) to total carbon biomass and D) relative contribution (%) to total carbon fixation rate of picophytoplankton (0.2-2 µm in ESD; black circles), small nanophytoplankton (2-5 µm in ESD; white circles), large nanophytoplankton (5-20 µm in ESD; black triangles) and microphytoplankton (>20 µm in ESD; white triangles). Latitude values as in Fig. 3.3

biomass along the transect. In several stations between ca. 11ºN and the Equator, the carbon biomass represented by the size fraction >20 µm (microphytoplankton) was also relatively high, with values varying between 11 and 17 µg C L-1. This size class, with a relative contribution to total

71 Chapter 3. Size-fractionated phytoplankton biomass and production

carbon biomass of ca. >40% in this region, displaced the picophytoplankton in their dominance of the phytoplankton biomass. This increase in the biomass of the microphytoplankton in this region was mainly due to the enhanced abundance of the filamentous cyanobacteria Trichodesmium spp. (Fernández et al. 2010), although an increase in the abundance of other chain-forming species was also observed (data not shown).

A higher degree of latitudinal variability was found in the contribution of the different size classes to carbon fixation rates (Fig. 3.4D). Although picophytoplankton showed, on average, the largest contribution to total production, the partitioning of carbon fixation among size classes (Fig. 3.4B) was more equitable than that of biomass. All the size fractions showed a marked latitudinal pattern with higher production in the upwelling region (between 17ºN and the Equator), although the degree of response to the enhanced nutrient supply varied depending on the size class. While small nano- and large nano-phytoplankton primary production rates were between 0.1-0.5 μg C L-1 d-1, pico- and micro-phytoplankton carbon fixation rates varied by ca. 16-fold and an opposite pattern in their relative contribution to total primary production was observed in the region of the equatorial upwelling.

The turnover rates of the pico- and small nano-phytoplankton typically ranged between 0.05-0.3 d-1, and showed an increase in several stations affected by the upwelling (between 10°N and the Equator) (Figs. 3.5A and 3.5B). The growth rates of the large nanophytoplankton were higher, taking values in the range 0.07-0.6 d-1, without any clear latitudinal pattern of variation (Fig. 3.5C). Similarly unclear latitudinal variability was

72 Chapter 3. Size-fractionated phytoplankton biomass and production

shown by microphytoplankton turnover rates, whose values ranged between 0.05-0.5 d-1 along the transect (Fig. 3.5D).

0,35 0,35 AB0.2-2 m ESD 2-5 m ESD 0,30 0,30 ) -1 0,25 0,25

0,20 0,20

0,15 0,15

0,10 0,10 Turnover rate (d rate Turnover 0,05 0,05

0,00 0,00 0,8 0,6 5-20 m ESD >20 m ESD 0,7 CD 0,5 ) -1 0,6 0,4 0,5 0,4 0,3 0,3 0,2 0,2 Turnover rate (d rate Turnover 0,1 0,1 0,0 0,0 -10-50 5 1015202530 -10-50 5 1015202530

Latitude Latitude

Fig. 3.5 Latitudinal variability of turnover rate of A) picophytoplankton (0.2-2 µm in ESD), B) small nanophytoplankton (2-5 µm in ESD), C) large nanophytoplankton (5-20 µm in ESD) and D) microphytoplankton (>20 µm in ESD). Latitude values as in Fig. 3.3.

73 Chapter 3. Size-fractionated phytoplankton biomass and production

The carbon fixation rate to chl a concentration ratio of all size classes increased in the region most affected by the upwelling (Fig. 3.6). Throughout the transect, lower values were consistently found in the pico- and small nano-phytoplankton (range 1.4 to 14.5 μg C μg chl a-1 d-1) than in the larger size classes (range from 3 to 35.5 μg C μg chl a-1 d-1).

16 10 AB0.2-2 m ESD 2-5 m ESD 14 9

) 8 -1 12 d

7 -1

a 10 6 8 5

g chl 4  6 3 4 g C g C 2  2 1 0 0 ratio(

a 18 40 CD5-20 m ESD >20 m ESD 16 35 30 14 25 12 20 10 15 8 10 Carbon fixation to chl to chl fixation Carbon 6 5 4 0 -10-50 5 1015202530 -10-50 5 1015202530

Latitude Latitude

Fig. 3.6 Latitudinal variability of size-fractionated carbon fixation rate to chl a concentration ratio of A) picophytoplankton (0.2-2 µm in ESD), B) small nanophytoplankton (2-5 µm in ESD), C) large nanophytoplankton (5-20 µm in ESD) and D) microphytoplankton (>20 µm in ESD). Latitude values as in Fig. 3.3.

74 Chapter 3. Size-fractionated phytoplankton biomass and production

3.3.4 The influence of environmental forcing on size-fractionated carbon biomass, production and growth rates

In order to study the differential response of phytoplankton size fractions to changes in water-column stability and nutrient input into the euphotic zone, we carried out a correlation analysis between Brunt-Väisäla frequency (BV), nitracline depth, nitrate diffusive flux, Phyto-C, carbon fixation rate, turnover rate and the carbon fixation to chl a ratio (Tables 3.1 and 3.2). All the variables that did not meet the assumption of normality were log transformed before the statistical analysis. With the exception of microphytoplankton carbon biomass, which correlated with the water- column stability (r2 = 0.77, P < 0.05, n = 10), neither Brunt-Väisäla frequency nor nitracline depth showed any significant correlation with pico-, small nano- and large nano-phytoplankton biomass. Primary production in all size fractions except large nanophytoplankton correlated with BV (r2 = 0.68-0.82, P < 0.05, n = 10), reflecting the association between higher stability and increased productivity in the upwelling region. The primary production, turnover rate and carbon fixation to chl a ratio of pico- and small nano-phytoplankton showed an inverse relationship with nitracline depth, suggesting a stimulating effect of higher nutrient input on both absolute and biomass-specific productivity in the smaller size classes. Although no significant relationships between nitrate diffusive flux and biotic variables were found, there was an association between the increasing in size-fractionated primary production, biomass and turnover rates found under the influence of the equatorial upwelling and the higher nitrate diffusive supply estimated for this region (see Figs. 3.3, 3.4 and 3.5).

75 Table 3.1 Pearson correlation coefficients between phytoplankton biomass and primary production and water column stability, represented by Brunt-Väisäla frequency (BV freq.), nitrate diffusive flux (μmol N m-2 d-1) and nitracline depth (m).

Biomass Primary production

Bt B 0.2 B 2 B 5 B 20 Pt P 0.2 P 2 P 5 P 20

0.86 0.46 0.42 -0.19 0.77 0.79 0.82 0.82 0.58 0.68 BV freq. (0.002) (0.181) (0.223) (0.597) (0.010) (0.006) (0.003) (0.004) (0.079) (0.031)

Nitracline -0.71 -0.66 -0.22 0.22 -0.46 -0.68 -0.79 -0.73 -0.40 -0.49 depth (0.032) (0.051) (0.562) (0.567) (0.211) (0.046) (0.012) (0.026) (0.293) (0.183)

Nitrate 0.55 0.59 0.43 0.12 0.24 0.38 0.45 0.54 0.42 0.48 diffusive (0.124) (0.095) (0.243) (0.755) (0.535) (0.313) (0.219) (0.134) (0.265) (0.193) flux

Bt and Pt are total carbon biomass and total carbon fixation rate respectively. B 0.2, B 2, B 5 and B 20 refer to the carbon biomass of pico-, small nano-, large nano- and micro-phytoplankton. P 0.2, P 2, P 5 and P 20 refer to the carbon fixation rate of pico-, small nano-, large nano- and micro-phytoplankton. Between brackets are the correlation P-values.

Table 3.2 Pearson correlation coefficients between phytoplankton turnover rates, carbon fixation rate to chlorophyll a (chl a) ratio and the water column structure, represented by Brunt-Väisäla frequency (BV freq.), nitrate diffusive flux (μmol N m-2 d-1) and nitracline depth (m).

Turnover rate Carbon fixation to chl a ratio

Tt T 0.2 T 2 T 5 T 20 P/Chl 0.2 P/Chl 2 P/Chl 5 P/Chl 20

0.74 0.82 0.69 0.52 -0.04 0.81 0.79 0.37 0.62 BV freq. (0.014) (0.004) (0.027) (0.121) (0.925) (0.005) (0.007) (0.296) (0.057) Nitracline -0.73 -0.65 -0.67 -0.49 -0.30 -0.75 -0.71 -0.28 -0.46 depth (0.025) (0.058) (0.047) (0.181) (0.471) (0.024) (0.031) (0.464) (0.219)

Nitrate 0.39 0.25 0.31 0.08 -0.10 0.36 0.48 0.29 0.32 diffusive (0.303) (0.511) (0.415) (0.843) (0.807) (0.339) (0.187) (0.444) (0.406) flux

Tt is the total turnover rate. T 0.2, T 02, T 5 and T 20 refer to the turnover rate of pico-, small nano-, large nano- and micro- phytoplankton and P/Chl 0.2, P/Chl 2, P/Chl 5 and P/Chl 20 refer to the carbon fixation to chl a ratio of pico-, small nano-, large nano- and micro-phytoplankton. Between brackets are the correlation P-values.

Chapter 3. Size-fractionated phytoplankton biomass and production

3.4 Discussion

The Atlantic equatorial upwelling constitutes a scenario of environmental forcing in which phytoplankton carbon biomass (Phyto-C) and primary production are stimulated (Marañón et al. 2000, Marañón et al. 2001, Pérez et al. 2005b, Poulton et al. 2006). In our study, both Phyto-C values and primary production rates were similar to those previously reported for the same region (e.g. 6-47 μg C L-1 and 1.2-18 μg C L-1 d-1 respectively: Buck et al. 1996, Marañón et al. 2000), and the latitudinal pattern observed for both variables, characterized by higher values in those stations under the influence of the equatorial upwelling, coincided with previous studies in the Atlantic Ocean (Marañón et al. 2000, Morán et al. 2004, Pérez et al. 2005b). In addition, given the significant correlation found between surface and euphotic layer-integrated primary production (P < 0.01, r2 = 0.63, n = 10) we can consider that our surface measurements of total carbon fixation rate were representative of the primary production of the whole euphotic layer. Two new contributions arising from our study should be highlighted. Firstly, in addition to the determination of phytoplankton primary production and chlorophyll a concentration, we also obtained direct biomass measurements, so phytoplankton turnover rates could be derived. Therefore, it was possible to elucidate whether the primary production enhancement observed under the upwelling influence was due only to the increased phytoplankton standing stocks, or if it was also the result of the favourable effect of nutrient inputs on phytoplankton physiology, translated into higher phytoplankton growth rates. Secondly, phytoplankton biomass, primary production and turnover rates were estimated in four different size classes, so we have been able to determine if there was a differential

78 Chapter 3. Size-fractionated phytoplankton biomass and production

response among different phytoplankton groups to the environmental forcing associated with the equatorial upwelling. The results of this size- fractionated analysis conducted from surface samples can be extrapolated to the rest of the water column, as the geographical variability in phytoplankton size structure along latitudinal transects in the Atlantic is much higher than the variability observed in the vertical profile (Marañón et al. 2001, Moreno-Ostos et al. in press).

3.4.1 Changes in size-fractionated biomass and production in response to environmental forcing

Relatively few studies have previously analysed the partitioning of phytoplankton carbon biomass and carbon fixation rates among several size classes and over large spatial scales in the Atlantic Ocean (Buck et al. 1996, Marañón et al. 2001, Pérez et al. 2006, Poulton et al. 2006). It is commonly accepted that picophytoplankton biomass and production in the ocean keep relatively constant, independently of changes in the environment (Raimbault et al. 1988, Thingstad and Sakshaug 1990, Rodríguez et al. 1998). However, our results confirm that not only microphytoplankton biomass and productivity respond to the environmental forcing associated with the equatorial upwelling, but also the other phytoplankton size classes identified, even the picophytoplankton, increased their biomass and carbon fixation rates in those stations under the influence of the upwelling. If we examine the latitudinal pattern of size-fractionated Phyto-C and carbon fixation rates (Fig. 3.4) and also the relationships found between these variables and the Brunt-Väisäla frequency and nutrient supply (as reflected in the vertical diffusive flux of nitrate and also on the location of the

79 Chapter 3. Size-fractionated phytoplankton biomass and production

nitracline depth) (Table 3.1), we can see that the response to the equatorial upwelling of all the size fractions was more intense in terms of primary production than in carbon biomass. This result reflects the fact that phytoplankton standing stocks are also influenced by loss processes such as grazing and sedimentation rates. As a consequence of this differential response in terms of biomass and production to the nutrient enrichment and the increased water-column stability, turnover rates were higher in those stations under the upwelling influence.

The effect of environmental forcing, reflected in changes in water- column stability and nutrient supply, varied depending on the size class considered. While primary production of all phytoplankton size fractions correlated with the water-column stability, the response of pico- and small nano-phytoplankton carbon fixation rates to the enhanced supply of nitrate into the euphotic layer seemed to be stronger than that showed by the large sized classes. Although this result contrasts with that previously reported by other authors (Landry et al. 1996, Poulton et al. 2006, McAndrew et al. 2007) and with the commonly accepted preference of smaller cells for the ammonium (Chisholm 1992, Sunda and Hardison 2010), it would confirm the observations by Glover et al. (2007) regarding the physiological response of picophytoplankton to nitrate perturbations. In their addition experiments conducted in the Sargasso Sea, these authors showed an enhancement of picophytoplankton photosynthesis in response to nanomolar nitrate supplements. Similarly, from the results derived from the JGOFS program, Barber and Hiscock (2006) concluded that other phytoplankton taxa, in addition to diatoms, are able to respond by increasing their growth

80 Chapter 3. Size-fractionated phytoplankton biomass and production

rates during the development of diatom-dominated blooms.

The fact that microphytoplankton production does not correlate with the nitracline depth or nitrate diffusive flux may seem paradoxical, as it is well known that large phytoplankton grow mainly when resource availability increases (Chisholm 1992, Kiørboe 1993, Marañón 2009). In our survey, although other chain-forming species enhanced their abundance in those regions under the upwelling influence, the dominant species within microphytoplankton size class was the N2-fixing cyanobacteria Trichodesmium spp., whose contribution to total Phyto-C was 30% from 14ºN to the Equator (data not shown). The presence of Trichodesmium spp. coincided with a higher rate of N2 fixation between ca. 20ºN and the Equator (Fernández et al. 2010). The dominance of microphytoplankton by a diazotroph species may explain the lack of relationship between the enhanced nitrate flux and the productivity of large phytoplankton in the region affected by the Equatorial upwelling.

As it has been found in previous studies carried out in the oligotrophic Atlantic Ocean (Buck et al. 1996, Marañón et al. 2001, Pérez et al. 2005b, Teira et al. 2005), along the whole latitudinal transect Phyto-C was dominated by picophytoplankton (relative contribution always >40%) and the contribution of the small and large nanophytoplankton never exceeded 20%. However, a marked shift in the relative contribution of pico- and micro-phytoplankton to total Phyto-C in those stations under the influence of the upwelling must be highlighted, as a decrease in the picophytoplankton biomass contribution of 40% coincided with an increase of ca. 50% in the contribution of microphytoplankton. Similarly, in these

81 Chapter 3. Size-fractionated phytoplankton biomass and production

stations where the highest production was found, the highest contribution to total primary production was due to microphytoplankton and this peak coincided with a decrease of ca. 20% in the picophytoplankton contribution to total carbon fixation rate. However, as we discuss below (subsection 3.4.2), our data suggest that the pico- and small nano-phytoplankton were the size classes that showed the strongest physiological response to the upwelling. The increased dominance of larger phytoplankton in the upwelling area may have been partly favoured by the retention effect of upward water motion (e.g. Rodríguez et al. 2001).

3.4.2 Influence of environmental forcing on growth rates

The knowledge of phytoplankton growth rates in combination with primary production rate and Phyto-C concentration is useful in order to elucidate whether the phytoplankton community is only limited in its standing stocks or also in its physiological state (Goldman et al. 1979). Moreover, by comparing the growth rates estimated with those expected for the temperatures registered it is possible to assess if phytoplankton are growing at their maximum theoretical rate (Eppley 1972, Marañón 2005). In our study, the turnover rates of the whole phytoplankton assemblage fall within the range of values estimated previously by several authors for the same oceanic regions (e. g. 0.05-0.2 d-1: Goericke and Welschmeyer 1998, Lessard and Murrell 1998, Marañón et al. 2005, among others). These values were clearly smaller than the maximum theoretical growth rates (1.5 d-1) estimated by Eppley (1972) for microalgae growing at the warm temperatures observed in tropical oceans. A nutrient limitation of phytoplankton growth has been suggested as an explanation for the

82 Chapter 3. Size-fractionated phytoplankton biomass and production

suboptimal rates found (Marañón et al. 2005). In addition, a significant inverse correlation between total growth rate and nitracline depth has been reported in basin-scale terms for the Atlantic Ocean (Marañón et al. 2000) and several experiments carried out in the tropical and subtropical Atlantic regions have shown compelling evidence of nutrient limitation of phytoplankton physiology and growth (Graziano et al. 1996, Mills et al. 2004, Moore et al. 2008, Martínez-García et al. 2010). In our case, the association, observed in the upwelling region, between increased nitrate diffusive fluxes, shallower nitracline depths and enhanced total phytoplankton turnover rates strongly suggests a response of phytoplankton growth to nitrate supply. Therefore, not only phytoplankton standing stocks but also their physiological states were limited by nitrate availability.

Most previous studies on phytoplankton growth rates in specific size classes have focused on the <2 μm size fraction (Liu et al. 1997, Hirose et al. 2008, Zubkov and Tarran 2008, among others). To the best of our knowledge only Pérez et al. (2006) and Poulton et al. (2006) reported some growth rates estimations for all phytoplankton in the >2 µm size fraction, so our study is the first assessment of the variability in phytoplankton turnover rates of four different size classes over basin-wide scales. The picophytoplankton turnover rates measured were within the range reported by previous studies for the same region (Pérez et al. 2006, Poulton et al. 2006) and both pico- and small nano-phytoplankton turnover rates were, in general, much lower than those estimated for large nano- and micro- phytoplankton. This observation contrasts with the higher growth rates expected for small-sized phytoplankton, if we consider their advantage over

83 Chapter 3. Size-fractionated phytoplankton biomass and production

larger cells in nutrient-poor environments (Raven and Kübler 2002). In addition, recent studies have reported that phytoplankton taxonomic groups with average large size such as diatoms sustain higher growth rates than other taxonomic groups that belong to the pico- and nano-phytoplankton size range, such as green algae or prymnesiophytes (Latasa et al. 2005). These results highlight the importance of determining the growth rates of different taxonomic and size categories, because a single, overall turnover rate for the whole community often masks the presence of contrasting growth dynamics in different algal groups. As a result, the relative importance of different groups in terms of carbon fluxes can vary considerably (Latasa et al. 2005). Thus, we can conclude that in our study the large nano- and the micro-phytoplankton contributed to primary production more than what could be expected on the basis of their biomass share.

The latitudinal pattern showed by pico- and small nano- phytoplankton turnover rates, with higher rates coinciding with shallower nitracline depths and higher nitrate diffusive fluxes, indicates that these small-celled photoautotrophs were the most responsive to the enhanced nitrate supply in the equatorial region. This result is supported by the significant correlations found between the carbon fixation to chl a ratio estimated for pico- and small nano-phytoplankton and the nitracline depth. The fact that the smaller phytoplankton, rather than the microphytoplankton, responded to the enhanced nutrient supply with an increase in their turnover rates and carbon fixation to chl a ratios must be interpreted taking into account the dynamics of the equatorial upwelling. Coastal upwelling and

84 Chapter 3. Size-fractionated phytoplankton biomass and production

water mixing events occur episodically in pelagic ecosystems, and cause sudden and large increases in the available nutrient concentration. In this scenario, large-sized phytoplankton such as diatoms are strongly favoured by their high maximal uptake rates and nutrient storage capacities (Sarthou et al. 2005, Thingstad et al. 2005, Litchman et al. 2007, Verdy et al. 2009), resulting in their dominance of the phytoplankton community (Margalef 1978, Smayda and Reynolds 2001). In contrast, the equatorial upwelling, although subject to temporal variability (Longhurst 1993, Pérez et al. 2005a), represents a more steady hydrodynamical setting that results in modest and persistent increases in nutrient supply. In our study, although the nutrient supply rate into the euphotic layer was clearly enhanced in the upwelling region, nitrate concentration never exceeded 0.2 µmol L-1 in the upper mixed layer. The more marked growth response of small phytoplankton would thus be a result of the small magnitude of the nutrient input and its relatively continuous nature. These conditions provide a competitive advantage for small cells adapted to oligotrophic conditions (Verdy et al. 2009, Cermeño et al. in press). Our results highlight the importance of the simultaneous analysis of biomass, production and growth in different size classes in order to better understand the response of phytoplankton communities to environmental variability.

85 Chapter 3. Size-fractionated phytoplankton biomass and production

86

Chapter 4 Metabolic scaling and phytoplankton size structure in the open ocean

Chapter 4. Metabolic scaling and phytoplankton size structure

Abstract

To explore the linkage between the size-scaling of metabolic rate and size structure in natural phytoplankton communities, we determined simultaneously phytoplankton carbon fixation rates and abundance across a cell volume range spanning over six orders of magnitude in tropical and subtropical waters of the Atlantic Ocean. An isometric relationship between cell-specific carbon fixation rates and cell size was found (slopes range 1.03-1.32), negating the idea that Kleiber’s law is applicable to unicellular autotrophic protists. The observed isometric size-scaling of carbon fixation is likely to result from physiological traits and ecological strategies evolved by large-sized species to overcome the cellular constraints associated with resource uptake and use. Based on the scaling of individual resource use with cell size, we predicted a reciprocal size-scaling of phytoplankton metabolic rate and abundance. This prediction was confirmed by the observed slopes of the relationship between phytoplankton abundance and cell size, which took values between -0.97 and -1.29. Thus, we conclude that the size structure of phytoplankton communities in the open ocean largely results from the size-scaling of metabolic rate. We also infer that total energy processed by carbon fixation is constant along the phytoplankton size spectrum, which may explain the persistence, over evolutionary time-scales, of large-sized species in oligotrophic, steady-state open ocean ecosystems.

89 Chapter 4. Metabolic scaling and phytoplankton size structure

90 Chapter 4. Metabolic scaling and phytoplankton size structure

4.1 Introduction

Body size is a fundamental property that controls individual-level metabolism, community structure and ecosystem functioning (Peters 1983, Marquet et al. 2005). Metabolic rates such as carbon fixation, nutrient acquisition or heterotrophic respiration are related to body size by a power function in the form, M = c Vd, where M is a metabolic rate, c is a taxon- related constant, V is organism size and d is the allometric exponent, which commonly takes a value of ~¾. If logarithms are taken, the power function yields the linear relationship, log M = log c + d log V, where d is the slope value. Given the pervasiveness of this allometric relationship across taxonomic guilds, trophic levels and biomes, it is generally referred to as Kleiber’s law (Kleiber 1947, Peters 1983).

Though previous studies have reported on the applicability of Kleiber’s law to marine and freshwater protists (Eppley and Sloan 1966, Taguchi 1976, Nielsen and Sand-Jensen 1990), currently the extent to which this law holds for unicellular organisms is controversial. Recent evidence shows that carbon fixation and respiration rates in marine planktonic and terrestrial protists scales to cell size with an exponent ~1 (Marañón et al. 2007, Johnson et al. 2009, DeLong et al. 2010). Normalizing these metabolic rates to cell carbon mass, the resulting mass-specific rate becomes independent of cell size for organisms spanning more than eight orders of magnitude in size; that is, by analogy with vertebrates, elephants in the microbial world grow as fast as mice do. It must be noted, however, that the only existing determinations of the size scaling of phytoplankton metabolic rate in natural conditions (Marañón et al. 2007, Marañón 2008) were

91 Chapter 4. Metabolic scaling and phytoplankton size structure

obtained by combining measurements carried out in numerous sampling sites. Experimental determinations of the scaling relationship between metabolic rate and cell size in phytoplankton at the local scale in the field, e. g., in discrete plankton samples, are still unavailable, and, as a result, the variability in the size scaling exponent is unknown.

Phytoplankton size structure plays a fundamental role in pelagic ecosystems as it determines the trophic organization of plankton communities and, hence, the biogeochemical functioning of the ecosystem (Kiørboe 1993, Legendre and Rassoulzadegan 1996). The relationship between phytoplankton abundance and cell size is well established in aquatic ecosystems and follows a power-law model, N = a Vb, where N is the cell density and a is the Y-intercept of the resulting linear regression. The size-scaling exponent, b, is a synthetic descriptor of community size structure (Marquet et al. 2005) and generally takes values between -1.3 and -0.6. The value of the size-scaling exponent is strongly related to ecosystem’s productivity. Despite this connection between ecosystem’s productivity and size structure, the origin of the actual values of the size- scaling exponent for phytoplankton cell abundance is still unclear.

The size distribution of organisms within a given ecosystem can be explained as a function of the requirements for limiting resources by the individuals (Damuth 1981, Enquist et al. 1998). For instance, Enquist et al. (1998) showed that the relationship between population density and plant mass could be derived from the scaling between individual rate of resource use and body mass such that smaller individuals with lower per-capita resource requirements will attain higher abundances than larger individuals.

92 Chapter 4. Metabolic scaling and phytoplankton size structure

In the present study, we use the carbon fixation rate as a proxy for metabolic rate and resource use in phytoplankton. We hypothesize that in a nutrient- limited ecosystem, the size-scaling of individual resource acquisition, or cell-specific carbon fixation rate, determines the size structure of phytoplankton communities, such that a given amount of resource can be partitioned into many small-sized cells or some few larger organisms. To test this hypothesis, we analyse concurrently the size-scaling of phytoplankton cell-specific carbon fixation rate and abundance in the tropical and subtropical Atlantic Ocean, a nutrient-impoverished and relatively stable ecosystem close to the steady-state. Our main objectives are i) to determine, in discrete samples of natural phytoplankton, the scaling relationship between metabolic rate and cell size and ii) to explore the linkage between the size-scaling of metabolic rate and the size distribution of phytoplankton abundance. The results of this Chapter are reported in an article which has been submitted for publication (Huete-Ortega et al. in review)

4.2 Methods

4.2.1 Sampling, hydrography, irradiance and nutrients

We sampled 17 stations during the TRYNITROP (Trichodesmium and N2 fixation in the tropical Atlantic) cruise, carried out in November- December 2007 in the tropical and subtropical Atlantic Ocean (Fig. 4.1) on board R/V Hespérides. At each sampling station, vertical profiles (0-300 m) of temperature, salinity and in situ fluorescence were obtained using a Conductivity-Temperature-Depth (CTDSBE911) probe attached to a rosette

93 Chapter 4. Metabolic scaling and phytoplankton size structure

Fig. 4.1 Location of the sampling stations superimposed on a map of the climatological mean surface chlorophyll a concentration for the period September-November of the years 2002-2008 in the Atlantic Ocean. Ocean color data are from MODIS Aqua (9 Km).

94 Chapter 4. Metabolic scaling and phytoplankton size structure

sampler equipped with Niskin bottles. Using the density computed from the temperature and salinity data binned over 1-m intervals, we calculated the Brunt-Väisäla frequency (BV), averaged over the upper 125 m. Sampling was conducted before dawn and different water subsamples were collected from the Niskin bottles to determine nutrient concentration, phytoplankton biomass and abundance, chlorophyll a concentration (chl a) and primary production. Nanomolar nitrate concentration was determined with a segmented-flow automatic analyser (Technichon/Bran Luebbe), following the method of Raimbault et al. (1990). The nitracline depth was defined as the first depth at which nitrate concentration was higher than 0.5 µmol L-1.

4.2.2 Size-fractionated chlorophyll a concentration

Sampling and methodology used for determining size-fractionated chlorophyll a concentration was carried out as detailed in section 3.2.2.

4.2.3 Size-fractionated carbon fixation rate

Sampling and the 14C-uptake technique used for determining size- fractionated carbon fixation rates was carried out as detailed in section 3.2.3. The carbon fixation rate of the 8 size classes was determined by processing the corresponding filters following the procedures described by Marañón et al. (2001).

4.2.4 Phytoplankton cell size and abundance

The cell size and abundance of pico- (<2 µm in equivalent spherical diameter, ESD), small nano-phytoplankton (2-5 µm in ESD), large nano- (5- 20 µm in ESD) and micro- (>20 µm in ESD) phytoplankton were

95 Chapter 4. Metabolic scaling and phytoplankton size structure

determined by using both flow cytometry and image analysis as detailed in section 3.2.4.

4.2.5 Scaling relationship between abundance and cell size

To determine the scaling relationship between abundance and cell size, size classes were established on an octave (log2) scale of biovolume and total cell abundance was calculated for each size class by adding the abundance of all cells included in it. Individual cell abundances and biovolume chain-forming species found were used when necessary. Considering that analytical subranges of cell size for flow cytometry and image analysis were 0.5-30 μm in ESD and 9-80 μm in ESD, respectively, phytoplankton cell abundances from both flow cytometry and microscopy image analysis were coupled for each sample in order to obtain a single size-scaling relationship for the abundance of the whole autotrophic plankton community, from small cyanobacteria to large dinoflagellates and diatoms (Rodríguez et al. 1998, 2002). The maximum number of size classes found was 24, ranging from 0.5 to 80 μm in ESD. Afterwards, the log10 of total abundance was plotted against the log10 of the lower limit of the corresponding octave size class (nominal size), obtaining as result a linear relationship (Blanco et al. 1994, Reul et al. 2005). Given that methodological error was present in both variables, the regression slope and the intercept of each size-scaling relationship were calculated using a Model II regression analysis by the reduced major-axis (r.m.a) method (Laws and Archie 1981). Ninety-five percent confidence intervals (CIs) for the regression parameters were calculated by bootstrapping over cases (2000 repetitions) using the RMA software designed by the San Diego University.

96 Chapter 4. Metabolic scaling and phytoplankton size structure

When a comparison between slope values was necessary, Student’s t-test following the Clarke method was used (Clarke 1980).

4.2.6 Scaling relationship between cell-specific photosynthesis rate and cell size

With the aim of determining the size-scaling of cell-specific, photosynthetic carbon fixation rate, total cell abundance was calculated for those size classes for which the size-fractionated carbon fixation rate had been previously obtained (0.2-0.8, 0.8-2, 2-3, 3-5, 5-10, 10-20, 20-41 and >41 μm in ESD). The size-fractionated carbon fixation rate measured on each size class was divided by the total cell abundance in that size class, thus obtaining the cell-specific carbon fixation rate for each size class. Cell abundance of Trichodesmium spp. and other chain-forming species found were included within those size classes corresponding to the total biovolume of their chains. Afterwards, the log10 of the cell-specific carbon fixation rate was plotted against the log10 of the corresponding abundance-weighed mean cell size in each size class in order to get a linear relationship (Marañón et al. 2007). The Model II slope and Y-intercept for the carbon fixation versus cell size relationship were calculated using the r.m.a. method (Laws and Archie 1981), and 95% confidence intervals (CIs) for the regression parameters were calculated by bootstrapping over cases (2000 repetitions) using the RMA software. The comparison between the obtained slope values and the ¾ value of Kleiber’s law was conducted by the Student’s t- test following the Clarke method (Clarke 1980).

97 Chapter 4. Metabolic scaling and phytoplankton size structure

4.2.7 Methodological considerations

Although the size-scaling of phytoplankton carbon fixation and chlorophyll a (chl a) has been determined previously in both culture experiments (Finkel 1998, Finkel 2001) and field studies (Marañón et al. 2007, Marañón 2008), to the best of our knowledge this is the first time that the size-scaling of photosynthesis and chl a is determined in local phytoplankton natural communities with a high level of accuracy in terms of number of size classes and sample volume used. The small volume of 14C incubations (75 to 125 mL) and chl a samples (250 mL) normally used for determining primary production and carbon biomass in the ocean might result in the undersampling of larger cells, particularly in those ecosystems where they are in lower abundances, such as the oligotrophic gyres. As a result, the estimated carbon fixation rates and chl a concentrations for the large size fractions may be underestimated. In the present study, we applied several modifications in sampling design in order to minimise the underestimation of the abundance, chl a concentrations and carbon fixation rates of larger cells in the oligotrophic Atlantic Ocean. Specifically, compared with previous studies (Cermeño et al. 2005b, Marañón et al. 2007), the accuracy of the size-scaling relationship was improved by increasing substantially the number of size fractions for which carbon fixation rates and chl a were estimated (from 3-4 to 8). Also, the 1 L- volume incubations and 500 mL-volume samples used for the estimation of carbon fixation rates and the dermination of chl a, respectively, contrasted with the 75 to 250-mL sample volumes commonly used, thus allowing a better representation of the metabolic rates and carbon biomass of larger cells. In addition, the combination of flow cytometry and image analysis

98 Chapter 4. Metabolic scaling and phytoplankton size structure

enabled us to cover the whole phytoplankton size range, from the smallest cyanobacteria to the largest diatoms. Finally, the potential underestimation of large-sized phytoplankton abundance was also avoided by increasing the sample volume used for image analysis (typically 125 mL) to 2 L.

4.3 Results

4.3.1 General oceanographic conditions

During the TRYNITROP 2007 cruise, high incident irradiance, warm surface temperature, strong thermal stratification and low nutrient concentration in the upper mixed layer (UML) were found throughout the tropical and subtropical Atlantic Ocean (see Table 4.1 and Figs. 3.2 and 3.3). The influence of the equatorial upwelling could be observed between 17ºN and 5ºS, where a shallower nitracline depth was found and nitrate concentrations in the UML increased, although without exceeding 0.2 µmol N L-1 (see Fernández et al. 2010). Nutrient-limited conditions thus prevailed throughout the cruise. Based on these general oceanographic conditions observed throughout the studied region, a situation close to the steady-state can be assumed, in which nutrients enter the euphotic zone at a slow rate and are continuously consumed by the phytoplankton, so that nutrient concentration never increases markedly.

99 Chapter 4. Metabolic scaling and phytoplankton size structure

Table 4.1 Hydrographic characteristics of the sampled stations. Nitracline Station Temperature BV frequency Total Chl a depth 26.00º N 25.73º W 24.5 8.8 x 10-5 130 0.24 26.00º N 29.83º W 24.2 7.6 x 10-5 142 0.18 25.99º N 34.12º W 24.6 6.6 x 10-5 - 0.19 26.00º N 38.27º W 25.2 8.1 x 10-5 133 0.19 23.44º N 34.91º W 25.9 7.7 x 10-5 146 0.21 20.92º N 31.69º W 25.7 1.1 x 10-4 129 0.25 18.41º N 29.09º W 25.0 1.6 x 10-4 82 0.27 14.43º N 28.71º W 25.8 2.3 x 10-4 51 0.31 11.17º N 28.00º W 26.6 3.1 x 10-4 41 0.44 7.40º N 29.01º W 27.8 3.0 x 10-4 56 0.35 3.29º N 29.02º W 27.3 3.3 x 10-4 - 0.29 0.33º S 28.99º W 26.9 2.1 x 10-4 61 0.23 4.67º S 28.99º W 26.6 1.6 x 10-4 93 0.21 8.58º S 29.00º W 26.7 1.5 x 10-4 121 0.18 16.58º S 28.99º W 24.7 2.7 x 10-5 173 0.16 24.92º S 28.99º W 23.6 7.2 x 10-5 179 0.20 33.04º S 28.96º W 19.4 1.1 x 10-4 67 0.16 Temperature (ºC) and total chlorophyll a concentration (µg L-1) are surface (5 m) values; Brunt-Väisäla (BV) frequency and nitracline depth (m) were calculated as explained in section 4.2.1.

4.3.2 Methodological considerations in carbon fixation rate and chl a determinations

Figure 4.2A shows the comparison between total carbon fixation rates estimated from the 1-L incubations carried out in the present study and

100 Chapter 4. Metabolic scaling and phytoplankton size structure

)

-1 0.7 h -1 slope = 1.60 (1.27, 1.90) 0.6 Y-intercept = 0.02 (-0.01, 0.04)

g C L g C 2

 r = 0.92 0.5

0.4

0.3

0.2

0.1 A

0.0

1 L-volume total carbon fixation rate ( carbon fixation rate total 1 L-volume 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 75 mL-volume total carbon fixation rate (g C L-1 h-1)

0.50 slope = 1.35 (1.01, 1.52)

) 0.45 Y-intercept = 0.02 (-0.01, 0.07) -1 r2 = 0.94 g L

 0.40 ( a 0.35

0.30

0.25

0.20

500 mL-volume total chl chl total 500 mL-volume 0.15 B

0.10 0.05 0.10 0.15 0.20 0.25 0.30 0.35 -1 250 mL-volume total chl a (g L )

Fig. 4.2 Relationship between A) total carbon fixation rates estimated in 75 mL- and 1 L-volume incubations and B) total chlorophyll a concentration determined in 250 mL- and 500 mL-volume samples. Total carbon fixation rates and chl a were calculated as the sum of all size-fractionated carbon fixation rates and chlorophyll a concentrations. Regression parameters shown were estimated by r.m.a Model II analysis. Between brackets are the 95% CIs.

101 Chapter 4. Metabolic scaling and phytoplankton size structure

those rates resulting from parallel incubations conducted in 75-mL volume bottles (see Moreno-Ostos et al. 2011). Figure 2B shows the comparison between total chl a concentrations determined from 500 mL-volume samples and those obtained from 250 mL-volume samples taken in parallel (see Moreno-Ostos et al. 2011). The slopes of the Model II regression analyses conducted over each comparison reveal that total carbon fixation rates and chl a concentrations obtained from the large volume incubations and samples were 60% and 35% higher, respectively, than those obtained from the small volume incubations. These results suggest the possible underestimation of carbon fixation rates and chl a concentrations when small volume incubations and samples are used in the tropical and subtropical Atlantic Ocean. ) -1 4 ) 6 -1 cell 3 d = 1.15 5 b = -1.29 -1 c = -2.47 2 4 a = 3.61 r2 = 0.99 r2 = 0.98 1 3 (pg C h C (pg

0 2 -1 1 -2 0 -3 -1 -4 -2 total cell abundance (cell mL (cell abundance cell total

carbon fixation rate fixation carbon 10 -3 -5 A B 10

-6 log -4 log -2-10123456 -2-10123456

3 3 log10 cell size (m ) log10 cell size (m )

Fig. 4.3 Example of the log-log relationship between A) cell-specific carbon fixation rate and cell size and B) total cell abundance and cell size for surface phytoplankton collected at 14.43ºN, 28.71ºW. d and b are the slope values of the Model II regression line and c and a the corresponding Y-intercept values.

102 Chapter 4. Metabolic scaling and phytoplankton size structure

4.3.3 Size-scaling of carbon fixation rate

Figure 4.3A shows an example of the scaling relationship between cell-specific carbon fixation rate and cell size for a particular community of phytoplankton in the subtropical Atlantic Ocean. For the whole data set, the size-scaling parameters for cell-specific carbon fixation rate exhibited little variability (Fig. 4.4A), with slopes values consistently close to, or higher than, 1 (range from 1.03 to 1.32; see Table 4.2). These slopes were found to be significantly higher than 0.75 but, with few exceptions, statistically indistinguishable from 1 (Student’s t-test P-values always < 0.01, see Table 4.2). When the size-scaling slopes from all experiments were averaged, the resulting mean slope was 1.16, indicating a slightly superlinear relationship between metabolic rate and cell size. This overall, mean slope value implies that a 4-fold increase in cell size would be associated with a 5-fold increase in cell-specific carbon fixation rate. This finding was supported by the parallel observation of a nearly isometric relationship between intracellular chlorophyll a content and cell size (Fig. 4.5). It is expected that slopes values would be even higher than those reported here if cell size was expressed as carbon units rather than in cell volume, because the power relationship between cell carbon biomass and cell volume often shows an exponent smaller than 1 (see review in Menden-Deuer and Lessard 2000).

We assessed the validity of the obtained scaling relationships between carbon fixation rate and cell size by comparing the cell-specific rates predicted for different size classes with species-specific rates reported in the literature and rates estimated in previous size-scaling analysis of

103 Chapter 4. Metabolic scaling and phytoplankton size structure

) -1 4 Fig. 4.4 Best fit to data for cell 3 Mean slope = 1.16 (±0.09) the log-log relationship -1 between A) cell-specific 2 carbon fixation rate and cell 1 size, B) total cell abundance 0 and cell size and C) total -1 carbon fixation rate and cell -2 size in all samples obtained -3 during the cruise. Regression -4 parameters for each size- carbon fixation rate (pg C h (pg C rate fixation carbon

-5 A scaling relationship are 10 -6 detailed in Table 4.2. Slope log

values in panels A and B ) 6 -1 Mean slope = -1.15 (±0.09) refer to the mean (± SD) 5 slope for each type of size- 4 scaling relationship, which 3 was obtained by averaging 2 the size-scaling slopes from 1 all experiments. 0 -1 -2 total cell abundance (cell mL (cell abundance cell total

10 -3 B

log -4 )

-1 6 h

-1 5

4

3

2

1 carbon fixation rate (pg C L (pg C rate fixation carbon C 10 0 log -2-10123456 log cell size (m3) 10

104 Table 4.2 Statistical parameters of the scaling relationships between phytoplankton cell size and total cell abundance and carbon fixation rate obtained in the tropical and subtropical Atlantic Ocean. Size-scaling of carbon fixation rate Size-scaling of total abundance Station d c r2 n* P-value b a r2 n 26.00º N 25.73º W 1.20 (0.98, 1.28) -2.56 (-2.78, -2.11) 0.99 7 < 0.0001 -1.23 (-1.32, -1.10) 3.14 (2.79, 3.41) 0.97 20 26.00º N 29.83º W 1.06 (0.79, 1.30) -2.63 (-2.97, -1.92) 0.95 7 0.003 -0.97 (-1.09, -0.83) 2.89 (2.47, 3.24) 0.94 22 26.00º N 34.12º W 1.24 (1.05, 1.38) -3.15 (-3.56, -2.63) 0.97 8 0.0001 -1.16 (-1.37, -0.92) 3.22 (2.79, 3.69) 0.91 20 26.00º N 38.27º W 1.20 (0.79, 1.50) -3.01 (-3.97, -2.37) 0.94 8 0.0003 -1.12 (-1.26, -0.97) 3.22 (2.97, 3.48) 0.95 20 23.44º N 34.91º W 1.16 (0.95, 1.48) -3.19 (-4.17, -2.54) 0.94 8 0.0005 -0.97 (-1.07, -0.84) 3.01 (2.65, 3.34) 0.96 21 20.92º N 31.69º W 1.32 (1.06, 1.60) -2.85 (-3.77, -2.40) 0.96 8 0.0001 -1.22 (-1.35, -1.04) 3.22 (2.80, 3.67) 0.92 21 18.41º N 29.09º W 1.24 (1.09, 1.44) -2.71 (-3.30, -2.37) 0.97 8 < 0.0001 -1.15 (-1.26, -1.02) 3.19 (2.81, 3.56) 0.95 22 14.43º N 28.71º W 1.17 (0.99, 1.32) -2.33 (-2.79, -2.00) 0.98 8 < 0.0001 -1.29 (-1.36, -1.21) 3.61 (3.36, 3.86) 0.98 23 11.17º N 28.00º W 1.03 (0.74, 1.21) -2.17 (-2.57, -1.35) 0.93 8 0.008 -1.13 (-1.25, -0.97) 3.31 (2.94, 3.65) 0.95 21 7.40º N 29.01º W 1.03 (0.73, 1.15) -1.91 (-2.18, -1.16) 0.95 8 0.0028 -1.17 (-1.29, -0.99) 3.28 (2.90, 3.64) 0.94 22 3.29º N 29.02º W 1.07 (0.88, 1.20) -2.19 (-2.58, -1.81) 0.98 8 < 0.0001 -1.11 (-1.21, -0.98) 3.20 (2.82, 3.57) 0.95 23 0.33º S 28.99º W 1.18 (1.04, 1.39) -2.44 (-2.82, -2.19) 0.96 8 < 0.0001 -1.26 (-1.38, -1.10) 3.10 (2.73, 3.45) 0.96 20 4.67º S 28.99º W 1.29 (1.00, 1.38) -2.88 (-3.17, -2.27) 0.98 7 < 0.0001 -1.20 (-1.31, -1.04) 3.04 (2.63, 3.40) 0.95 21 8.58º S 29.00º W 1.03 (0.74, 1.25) -2.19 (-2.68, -1.19) 0.95 8 0.0028 -1.07 (-1.19, -0.92) 2.64 (2.20, 3.02) 0.95 24 16.58º S 28.99º W 1.17 (0.93, 1.26) -2.48 (-2.70, -2.05) 0.98 7 < 0.0001 -1.09 (-1.18, -0.96) 2.72 (2.48, 2.94) 0.96 21 24.92º S 28.99º W 1.15 (1.00, 1.22) -2.47 (-2.67, -2.12) 0.99 7 0.0001 -1.18 (-1.33, -1.00) 3.05 (2.74, 3.32) 0.95 19 33.04º S 28.96º W 1.19 (0.85, 1.40) -2.52 (-2.95, -1.76) 0.97 6 0.0002 -1.16 (-1.28, -1.02) 3.05 (2.71, 3.37) 0.95 18 For each type of relationship, a and c are the Model II intercepts, b and d refer to the Model II slopes, n and n* indicate the number of data points, and r2 is the determination coefficient. 95% CIs for the intercepts and slopes are given between brackets. P-value refers to the comparison between the size-scaling slope of metabolic rate with the expected value of ¾.

Chapter 4. Metabolic scaling and phytoplankton size structure

phytoplankton photosynthesis. The mean carbon fixation rates determined from our size-scaling relationships for small species such as Prochlorococcus spp. and Synechococcus spp. (~4  10-4 pg C cell-1 h-1 and ~1.3  10 -3 pg C cell-1 h-1, respectively), as well as those obtained for 2-µm ESD picoeukaryotes (~2  10-2 pg C cell-1 h-1) and 20-µm ESD microphytoplankton (~58 pg C cell-1 h-1), were all within the range of previous measurements reported for both single species and certain cell )

-1 6

cell Slope = 1.06 (1.01, 1.09) a Y-intercept = -2.07 (-2.16, -1.96) 4 r2 = 0.94 n = 107

2

0 concentration (pg chl a

-2 chlorophyll 10 -4 log -2-10123456 log cell size (m3) 10 Fig. 4.5 Overall log-log relationship between cell-specific chlorophyll a (chl a) concentration and cell size. Similarly to the size-scaling of carbon fixation rate, the size-fractionated chl a determined for each size-class was divided by the respective total abundance obtaining the cell-specific chl a concentration. Slope, Y-intercept, r2 and n are the parameters of the r.m.a. regression analysis carried out from combining all the single size-scaling relationships found for each station. Between brackets are the 95% CIs.

106 Chapter 4. Metabolic scaling and phytoplankton size structure

sizes in natural phytoplankton assemblages (Rivkin and Seliger 1981; Li 1994; Marañón et al. 2007; Jardillier et al. 2010).

4.3.4 Size-scaling of phytoplankton total abundance

We observed a consistent, highly significant, inverse linear relationship between phytoplankton total abundance and cell size (see Fig. 4.3B for an example of a typical cell size-abundance relationship). Throughout the tropical and subtropical Atlantic Ocean, a high amount of the variability in total phytoplankton abundance was explained by cell size, as r2 values were always larger than 0.91. The slope of the cell size- abundance relationships ranged between -0.97 and -1.29 (see Fig. 4.4B and Table 4.2 for regression parameters), with a resulting mean slope of -1.15. These steep slope values coincide with those previously reported for open- ocean, oligotrophic ecosystems (Cavender-Bares et al. 2001, Marañón et al. 2007, Cermeño and Figueiras 2008).

4.3.5 Size-scaling of phytoplankton total energy use

Fig. 4.4C shows the scaling relationship between total carbon fixation per unit volume and cell size for each phytoplankton community analysed. This relationship can be regarded as a proxy for the flow of metabolic energy along the phytoplankton size spectrum. We found that in most cases the regression was not statistically significant, so no size-scaling slopes could be estimated. This result indicates that total energy use by phytoplankton is largely independent of cell size in the oligotrophic Atlantic Ocean.

107 Chapter 4. Metabolic scaling and phytoplankton size structure

4.4 Discussion

4.4.1 Size-scaling of phytoplankton metabolic rate

Metabolic rate has long been assumed to follow a ¾-power relationship with body size in all organisms (Kleiber 1947, Peters 1983, West et al. 1997). Studies using literature data of metabolic rates had confirmed the applicability of the ¾-power rule for photosynthetic organisms from the smallest unicellular algae to the largest trees (Niklas 1994, Niklas and Enquist 2001). Yet, several experimental studies, focused on unicellular organisms, have recently reported an isometric size-scaling relationship not only for heat production and respiratory rates of protists grown in the laboratory (Johnson et al. 2009, DeLong et al. 2010), but also for photosynthetic carbon fixation in natural phytoplankton assemblages (Marañón et al. 2007, Marañón 2008). In the present study, all the slopes were significantly higher than the expected value of ¾, thus constituting another report negating the universal applicability of Kleiber’s law (Dodds et al. 2001, Bokma 2004, Isaac and Carbone 2010). The observed high size- scaling slopes also imply that the carbon fixation rates of large phytoplankton not only are higher than expected for their cell size but can also be even higher than those of smaller species.

The isometry in the metabolic size-scaling of unicellular protists implies that, in addition to the physical and geometrical constraints associated with resource uptake and use (Banavar et al. 1999, Raven 1998, West et al. 1999), other physiological processes, acting over evolutionary timescales, must be involved. Thus, the isometric size-scaling for

108 Chapter 4. Metabolic scaling and phytoplankton size structure

respiratory rates observed in heterotrophic protists has been interpreted as a result of the linear increase with cell size in the total number of mitochondria (DeLong et al. 2010). Although a similar reasoning could be applied to the packing of chloroplasts in eukaryotic photoautotrophs, photosynthetic carbon fixation is ultimately constrained by light absorption and nutrient diffusion into the cell, which are both negatively influenced by increasing cell size through the package effect and changes in the surface to volume ratio (Chisholm 1992, Finkel 2001, Finkel et al. 2004). Therefore, we hypothesize that additional evolutionary mechanisms to those proposed for respiratory rates in unicellular protists should be operating in the case of photosynthetic carbon fixation in phytoplankton.

Microalgal species must maximize their resource acquisition and assimilation rates while minimizing loss rates in order to survive in pelagic ecosystems (Sunda and Hardison 2010). Thus, allometric relationships in phytoplankton might have evolved as a consequence of competition and adaptation processes that involved the acquisition of taxon-specific physiological strategies by different cell sizes (Verdy et al. 2009). A number of structural and physiological properties of large phytoplankton may counterbalance the geometric constraints on resources acquisition and use imposed by cell size, which favour smaller cells when resources are limiting (Thingstad and Sakshaug 1990, Raven 1998). These traits include the possession of intracellular vacuoles and a higher nutrient storage capacity (Stolte and Riegman 1995, Verdy et al. 2009), changes in cell shape (Chisholm 1992, Naselli-Flores et al. 2007), the use of non-limiting substrates to avoid grazing pressure by increasing cell size without reducing

109 Chapter 4. Metabolic scaling and phytoplankton size structure

nutrient uptake (Thingstad et al. 2005), and the ability to migrate vertically in the water column (Villareal et al. 1999).

4.4.2 Linking the size-scaling relationships of phytoplankton metabolism and abundance

Knowledge of the factors that control the size distribution of phytoplankton abundance is a central question in marine ecology (Kiørboe 1993, Legendre and Rassoulzadegan 1996, Marañón 2009). Recently, it has been hypothesized that phytoplankton community size structure can be explained as a function of the use of limiting resources by individuals (Irwin et al. 2006). Given that photosynthetic carbon fixation in surface phytoplankton of low latitudes, where incident irradiance is high, is largely dependent on nutrient availability, we can use the analysis of its scaling along the size spectrum to assess the size-scaling of resource use (Q) by phytoplankton. Thus, we take d, the slope of the scaling relationship between cell-specific carbon fixation rate and cell size, as the size-scaling exponent for the individual rate of resource use, so that Q α Vd. Smaller cells have lower cell-specific nutrient requirements, which means that, for a given limiting resource input, there will be more smaller cells than large-sized ones. Therefore, considering that the maximal number of individuals (Nmax) that can be sustained by an ecosystem in steady state depends on the supply rate of the limiting resource per unit area or volume (R) and the rate of resource use per individual (Q), so that Nmax = R/Q, we can in principle predict the size distribution of a phytoplankton community from the size- scaling of individual metabolic rates.

110 Chapter 4. Metabolic scaling and phytoplankton size structure

In the case of the oceanic ecosystem studied here, if we assume that nutrient supply was the main limiting factor for population growth and that nutrients were equally available along the phytoplankton cell size spectrum 0 0 d -d (R α V ), then Nmax α V / V = V , where d is the slope of the relationship between carbon fixation and cell size. This implies that the scaling exponent in the relationship between abundance and cell size, b, is predicted to be equal to –d, a situation which can be referred to as the reciprocal size- scaling of metabolic rate and abundance. Considering the mean slope value obtained from averaging the slopes of all the relationships between carbon fixation rate and cell size, 1.16 (Fig. 4.4A), the size-scaling relationship of phytoplankton total abundance is predicted to have a slope of -1.16. This expected result is confirmed by the mean slope value of -1.15 obtained from averaging the slopes of all determinations of the scaling relationship between abundance and cell size (Fig. 4.4B). The value of -1.15 was not statistically different from the hypothesized value of -1.16 (see standard deviation of the mean slope, Fig. 4.4B). In addition, this result also holds when each size-scaling relationship for cell-specific carbon fixation rates is analysed individually (see Table 4.2). We thus conclude that the size-scaling of phytoplankton abundance in open-ocean ecosystems, with slopes typically ranging between -1 and -1.3, can be explained as a direct consequence of the size-scaling of phytoplankton metabolic rate.

The simultaneous knowledge of the size-scaling of phytoplankton metabolism and abundance allows us to study the flow of energy along the size spectrum (Marañón et al. 2007). According to Damuth (1981), the total energy used by a given population per unit area (EU) can be assessed by multiplying the average energy use per individual (or resource use, Q)

111 Chapter 4. Metabolic scaling and phytoplankton size structure

and the abundance of the individuals (N). Even though in the present study there was no differentiation of species composition, in practice each size class was an assemblage of species. So, if we consider the size-scaling relationships found for phytoplankton carbon fixation rate and total abundance (mean slope values in Figs. 4.4A and 4.4B, respectively), Q  V1.16 and N  V-1.15. Given that EU = N  Q, EU  V1.16-1.15 = V 0.01. The exponent of this equation (0.01) is not different from 0. Accordingly, we can see in figure 4.4C that phytoplankton total carbon fixation rate was invariant with respect to cell size in the oligotrophic Atlantic, as no significant regression was found between these variables. This result suggests that in an open ocean, steady-state ecosystem, total energy processed by phytoplankton photosynthesis is the same along the size spectrum. This conclusion contrasts with that of Li et al. (2004), who hypothesized a dominance of total energy use by small phytoplankton in oligotrophic oceanic regions (Li et al. 2004). However, this study assumed that phytoplankton metabolic rate follows a ¾-power relationship with cell size, without conducting any in situ measurements of size-fractionated phytoplankton metabolic rates.

The conceptual linkage between the size-scaling of metabolic rate and the size structure of phytoplankton communities developed here is valid only in those marine ecosystems where metabolic rates are resource limited (Enquist et al. 1998). Furthermore, here we assume that loss processes were independent of cell size, a plausible possibility if the higher grazing pressure suffered by small phytoplankton is compensated by the higher sedimentation rates experienced by larger cells (Kiørboe 1993, Thingstad

112 Chapter 4. Metabolic scaling and phytoplankton size structure

1998, Sunda and Hardison 2010). In addition, it must be highlighted that the condition of an uniformly distributed access to the limiting resource along the phytoplankton size spectrum can only be fulfilled in steady-state, open ocean ecosystems, where the limiting nutrients enter the euphotic layer through small and relatively continuous diffusive fluxes from below the thermocline. In these settings, as a result of their taxon-specific competitive strategies, all individuals with a given size class will have equal access to the limiting resources and will grow until reaching the cell abundance that corresponds to their cell. By contrast, highly productive ecosystems, characterized by episodic pulses of strong nutrient injections and non-steady state conditions, typically show a marked dominance by large-sized diatoms, in terms of biomass and resource use, thanks to their higher maximum nutrient uptake and growth rates (Cermeño et al. 2005a, Falkowski and Oliver 2007, Verdy et al. 2009).

4.4.3 Conclusions

We have confirmed, using in situ measurements of carbon fixation in local, natural assemblages, that the ¾-power law is not applicable to phytoplankton metabolism, thus negating the universality of Kleiber’s rule. The isometric size-scaling of phytoplankton carbon fixation is likely to result from taxon-specific physiological strategies of larger species, which allow them to overcome the size-related constraints on resource uptake and use. The inverse power-law relationship between phytoplankton abundance and cell size, with exponents typically between -1 and -1.3, is a well- established property of steady-state open ocean ecosystems, yet its origin has remained elusive. The concurrent analysis of the relationship between

113 Chapter 4. Metabolic scaling and phytoplankton size structure

cell size and phytoplankton abundance and carbon fixation rate suggests that the observed size structure in these ecosystems arises as a direct result of the size-scaling of metabolic rate. As a consequence of the size-scaling of metabolic rate and abundance, total energy use by phytoplankton is invariant along the size spectrum. Over evolutionary timescales, this invariance may help explain the persistence of large-sized species in the oligotrophic, steady-state ecosystems of the open ocean.

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Chapter 5 Synthesis

Chapter 5. Synthesis

Because most aspects of the biology of phytoplankton are a function of cell size, the influence of this variable on phytoplankton physiology, ecology and evolution can be scaled up through individual, population and community levels, constituting a macroecological approach to study the role of phytoplankton in aquatic ecosystems.

Although both phytoplankton metabolism and abundance and their scaling relationships with cell size have been previously addressed from a macroecological point of view, some aspects remain still unresolved. For instance, most determinations of the size-scaling of phytoplankton abundance have been carried out in relatively steady-state open-ocean ecosystems, and those focused on more dynamic and productive aquatic ecosystems have been limited to short temporal scales. In addition, studies of the size-scaling of phytoplankton metabolic rate have been largely based on laboratory work with cultures, whose growth conditions often are not representative of those present in natural conditions. The few determinations of the relationship between phytoplankton metabolism and cell size carried in natural assemblages had several limitations regarding size class resolution and spatial coverage. Finally, as far as we know, no studies have previously attempted to link the size-scaling of individual metabolic rate, or resource use rate, with the size structure of phytoplankton assemblages, in order to find an explanation for the origin of widespread scaling patterns between abundance and cell size.

In the present Thesis we have studied the size-scaling of photosynthesis rate and abundance in natural phytoplankton communities of two contrasting marine ecosystems: the oligotrophic regions of the tropical

117 Chapter 5. Synthesis

and subtropical Atlantic Ocean and the more dynamic and productive coastal ecosystem off NW Iberian Peninsula. Our main goal was to determine the nature and variability under different environmental conditions of the relationship between phytoplankton cell size, abundance, biomass and metabolic rate.

5.1 Size-scaling of phytoplankton abundance

The scaling relationships between total abundance and cell size obtained in the coastal shelf off NW Iberian Peninsula (Chapter 2) and in the oligotrophic central Atlantic Ocean (Chapter 4) highlighted the utility of this approach as a descriptor of the size structure of phytoplankton communities and their response to changes in environmental conditions. The steeper slopes found in the subtropical and tropical Atlantic Ocean, compared with those observed in the coastal system, reflected the increased numerical importance of smaller cells in the oligotrophic waters, whereas the higher Y-intercept values found in the Equatorial upwelling indicated higher phytoplankton standing stocks resulting from enhanced nutrient supply rates (Chapters 3 and 4).

A persistent linearity in the size-abundance spectra was found for the coastal ecosystem off NW Iberian Peninsula over seasonal and interannual time scales. The analysis of the variability in the slope values also showed that, with respect to the nano- and micro-phytoplankton size range, all phytoplankton size classes responded similarly to changes in the environmental forcing associated with different hydrographic settings (Chapter 2). In fact, only in those hydrodynamic conditions characterised by

118 Chapter 5. Synthesis

upwelling relaxation and the beginning of water-column stratification significant departures towards less negative slope values were observed, indicating higher abundance of large phytoplankton. In addition, the interannual trend towards less negative slope values seemed to be related to a decrease of flagellate abundance recorded along the 10-year time-series. The size-scaling approach and its description by a power-law model can be an adequate method for describing plankton dynamics not only in near steady-state ecosystems, but also in highly hydrodynamic and productive regions. Considering the recent interest to include phytoplankton size structure in remote sensing-based estimates of marine primary production (Silió-Calzada et al. 2008, Kostadinov et al. 2009, Brewin et al. 2010), the parameters derived from size-abundance spectra such as those described here may be incorporated into current and future productivity models (Kostadinov et al. 2010, Barnes et al. 2011).

5.2 Size-differential response of phytoplankton biomass and production to changes in environmental forcing

From an analysis of the variability in phytoplankton biomass, primary production and turnover rates found in the central Atlantic Ocean (Chapter 3), we concluded that the enhanced primary production observed in the region under the influence of the Equatorial upwelling was the result of the stimulating effect of increased nutrient supply on phytoplankton physiology. Moreover, the size-differential response of phytoplankton observed in the moderately nutrient-enriched surface waters of the Equatorial Atlantic, in terms of carbon biomass, primary production and turnover rates, highlighted the responsiveness of some phytoplankton size

119 Chapter 5. Synthesis

groups. In this sense, a higher response of small phytoplankton turnover rates to the enrichment of the euphotic layer along the Equatorial upwelling was observed. This result contrasted with the absence of the expected response for large phytoplankton and stressed the importance of conducting size-fractionated analysis of phytoplankton standing stocks and metabolism when the aim is to obtain a more comprehensive picture of the dynamics of phytoplankton communities and their role in the functioning of pelagic ecosystems. Given the contrasting role of large and small phytoplankton size classes in terms of trophic interactions and potential carbon export in pelagic ecosystems, this kind of studies will be complementary to those analyses traditionally focused on the response of the phytoplankton community as a whole to changes in environmental forcing.

5.3 Methodological considerations

We expected to find particularly low abundances of large cells in the oligotrophic Atlantic Ocean (Chapters 3 and 4). Therefore, some methodological improvements were introduced in order to minimise the undersampling of these larger species and to avoid the underestimation of their carbon fixation rates and chlorophyll a concentrations (chl a). Firstly, with the aim of minimising the loss of larger cells, the sample volume used for phytoplankton abundance and biovolume determinations by image analysis was 2 L instead of the traditional volume of 125 mL. Secondly, primary production incubations by the 14C method were carried out in 1-L bottles instead of the traditional small volume of 70-125 mL. And, finally, chl a concentrations were determined in 500 mL-volumes samples instead of the commonly used volume of 250 mL. The resulting total carbon

120 Chapter 5. Synthesis

fixation rates obtained with these larger volumes were 60% higher than those measured in parallel incubations carried out in 75-mL bottles (Slope = 1.60, r2 = 0.92, n = 16), while the obtained chl a concentrations were 35% higher than those determined in 250 mL samples taken in parallel. These are striking results that put into question the validity of most primary production estimates and biomass determinations carried out so far in the oligotrophic open ocean using small volumes. If confirmed, this underestimation of open ocean primary productivity can have important implications for global carbon budgets, including the balance between photosynthesis and mesopelagic respiration (del Giorgio and Duarte 2002). In addition, the underestimation of chl a concentrations can be relevant for those studies that estimate marine primary production from remote sensing of surface chl a concentrations and that need to be calibrated by in situ determinations of this variable.

5.4 Isometric size-scaling of phytoplankton carbon fixation rate

The isometric scaling relationship between phytoplankton carbon fixation rate and cell size observed in the present Thesis (Chapter 4) agreed with recent reports on the size-scaling of respiration rates in protists (Johnson et al. 2009, DeLong et al. 2010). However, important differences exist regarding the underlying evolutionary mechanisms by which this isometric scaling may have been generated. The isometric size-scaling for heterotrophic protists has been suggested to result from the organization of respiratory complexes within mitochondria, which allows eukaryotic cells to increase the number of respiratory complexes linearly as cell volume increases. Conversely, we suggest that in the case of photoautotrophic

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organisms the evolutionary incorporation of chloroplasts within the cell may have led to the appearance of other problems related to light acquisition, such as the package effect, which increases exponentially with cell volume. In addition, photosynthetic carbon fixation is strongly nutrient dependent, and therefore the decrease in the surface to volume ratio as cell volume increase poses further limitations to larger cells. Thus, we hypothesize that several additional, taxon-specific strategies may compensate the geometric constraints on resource uptake and use imposed by large cell size, yielding the observed pattern of isometric metabolic scaling.

5.5 Higher metabolic rates of large phytoplankton

The high slope values obtained for the size-scaling of phytoplankton carbon fixation rate means that not only carbon fixation rates by large phytoplankton would be higher than expected for their size, but also that they can be even higher than those shown by smaller size classes (Chapter 4). In fact, higher photosynthetic efficiencies and turnover rates for large phytoplankton have been previously reported for both coastal and open- ocean ecosystems (Tamigneaux et al. 1999, Marañón et al. 2001, Hashimoto and Shiomoto 2002, Cermeño et al. 2005a). Based on these observations, the dominance of phytoplankton biomass and primary production by larger forms in more productive marine ecosystems has been suggested to be a consequence of their own physiological characteristics, rather than being the result of the action of trophic and hydrodynamic mechanisms (i.e. lower grazing pressure over larger cells due to the decoupling of generation times between preys and consumers, or the higher time of permanence of large phytoplankton in the euphotic layer under

122 Chapter 5. Synthesis

increased upward water motion).

The difference between the realized growth rate of a species as measured in the field and the maximal growth rate that this species can achieve in optimal growth conditions is indicative of the degree of its growth limitation. Given that maximum nutrient uptake and growth rates are higher in large phytoplankton, particularly in diatoms, we suggest that the smaller cells were less limited by the low nutrient concentrations found in the tropical and subtropical Atlantic Ocean.

5.6 Size-independence of phytoplankton growth rates

Knowing the relationship between cell carbon content and cell biovolume, the cell-specific carbon fixation rates estimated in Chapter 4 can be normalized to cell carbon biomass, resulting in a scaling relationship between cell size and biomass-specific production or growth rate. Although subject to a large degree of uncertainty, several studies have shown that carbon biomass scales isometrically with cell biovolume (Montagnes and Berges 1994, Menden-Deuer and Lessard, 2000). Given the obtained slopes of the relationship between cell size and carbon fixation (Chapter 4), this would imply that phytoplankton growth rate is roughly independent of cell size. A similar conclusion was obtained by Marañón et al. (2009) after reviewing the interspecific scaling between phytoplankton production and cell size in natural conditions. While this absence of size-dependence of growth rates had been suggested previously for natural phytoplankton communities (Sommer 1989, Joint 1991, Marañón et al. 2007), studies with laboratory cultures have pointed out the existence of an allometric size-

123 Chapter 5. Synthesis

scaling relationship, with growth rate decreasing with increasing cell size (Blasco et al. 1982, Geider et al. 1986, López-Urrutia et al. 2006), or even a unimodal quadratic distribution (Chen and Liu 2010). The influence of environmental factors, such as nutrient availability, in the relationship between growth rate and cell size, has been also reported (Banse et al. 1976, Kagami and Urabe 2001). In this connection, a possible explanation for the difference in the size-scaling of phytoplankton growth rate observed in phytoplankton cultures versus natural assemblages may be related to the rate of nutrient supply. Constant nutrient supply conditions, which are typical of laboratory cultures, tend to favour small cells, whereas under pulsed nutrient supply regimes, common in natural ecosystems, larger cells have competitive advantage over their smaller counterparts (Stolte et al. 1994, Cermeño et al. in press).

5.7 Linking the size scaling of metabolic rate with phytoplankton size structure

In principle, phytoplankton size structure could be inferred from the rate of resource uptake and use by the individuals that make up the assemblage. The size distribution of phytoplankton is described by the size- scaling of phytoplankton total abundance (Chapters 2 and 4), and cell- specific carbon fixation rate can be considered as a proxy for the rate of individual resource uptake and use by phytoplankton (Chapter 4). Our measurements confirmed the linkage between the size scaling of metabolic rate and phytoplankton size structure, which was reflected in the reciprocity observed between the slope values of the size-abundance and size-carbon fixation rate relationships. This mechanism, whereby the size distribution of

124 Chapter 5. Synthesis

phytoplankton abundance arises from the way in which the different individuals along the size spectrum take up and use resources, implies an essentially bottom-up control of phytoplankton size structure, at least in near steady-state ecosystems such as the oligotrophic central Atlantic.

The linkage between metabolic size scaling and phytoplankton size structure is not expected to apply to coastal, productive environments, which are characterized by non-steady state conditions and where nutrients enter the euphotic zone through pulses. During these events of nutrient enrichment, large phytoplankton, and diatoms in particular, will take up most of the available nutrients, thanks to their higher maximum nutrient uptake rates, their ability to store nutrients intracellularly and their higher efficiency in the conversion of nutrients into biomass (Sarthou et al. 2005, Thingstad et al. 2005, Litchman et al. 2007, Verdy et al. 2009). As a result, access to nutrients will not be uniform across the size spectrum, therefore preventing the linkage between individual resource use and the size scaling of abundance.

5.8 Total energy use along the phytoplankton size spectrum

The total energy processed in a given community can be inferred from the concurrent measurement of the abundance of the organisms that shape the community together with their individual metabolic rates. This approach, which so far had been used only in a few terrestrial ecology studies, is used to scale-up individual energetics to population levels within a community (Damuth 1981, Enquist et al. 1998, Belgrano et al. 2002). In the present Thesis, we used ataxonomic relationships between abundance,

125 Chapter 5. Synthesis

carbon fixation rate and cell size to estimate the total flow of energy across the entire phytoplankton size spectrum. Thus, in Chapter 4 we showed that total energy processed through photosynthetic carbon fixation is the same irrespective of cell size in a steady-state, oligotrophic ecosystem such as central Atlantic Ocean. This pattern resulted from the reciprocal size scaling of abundance and metabolic rate, and coincides with the results of Enquist et al. (1998) who, through a species-specific approach, concluded that all plant populations, no matter their body size, sustain similar rates of resource use in terrestrial ecosystems. In the case of phytoplankton, the constancy of total energy use along the size spectrum applies to near steady-state ecosystems such as the central Atlantic Ocean, but not to more variable and productive ecosystems. For instance, the size-scaling exponents for phytoplankton abundance and carbon fixation rate in the Ría de Vigo during the upwelling season take values around -0.8 and 1.1, respectively (Marañón et al. 2007), which means that total energy use increases with cell size, in agreement with the well-known strong dominance of total biomass and productivity by large phytoplankton in highly productive ecosystems (Chisholm 1992).

The size-independence of total energy use observed in the central Atlantic Ocean is akin to Damuth’s concept of energetic equivalence (Damuth 1981), and could help explain the persistence, over evolutionary time scales, of large-sized species in stable, oligotrophic environments. A recent macroevolutionary model suggests that species using relatively low amounts of energy at any body size have an elevated extinction risk (Damuth 2007). The selective extinction of such species would drive the size-scaling of abundance towards approximate energetic equivalence and

126 Chapter 5. Synthesis

maintain it there. It must be noted, however, that our study deals with total abundance and metabolic rate in ataxonomic size classes, whereas the concept of energy equivalence, strictly speaking, applies to the species level. Additional work is needed to determine the interspecific scaling of population abundance and species-specific metabolic rate in natural phytoplankton assemblages.

5.9 Perspectives for future research

In the present Thesis, we have shown that size-scaling relationships provide a valuable macroecological approach to study the structure and function of phytoplankton communities from an integrative point of view, and to analyze the mechanisms underlying their emergent properties. However, several issues, which have not been addressed here, remain still unresolved. Below are reported some of the challenges that, from our point of view, should be addressed in future research.

Future research should be focused on determining the size-scaling relationship of photosynthesis rate in natural phytoplankton communities of nutrient-rich pelagic ecosystems, in a similar way to that conducted in the present Thesis for the oligotrophic ocean. Through this analysis, it would be possible to study the effect of resource availability on the anabolic size- scaling of phytoplankton, besides elucidating the linkage of the metabolic rate and the phytoplankton size structure and analyzing the total energy use along the phytoplankton size spectrum in these ecosystems. In addition, this study might be completed by extending the scaling analysis to other critical metabolic processes (both anabolic and catabolic) such as respiration rate,

127 Chapter 5. Synthesis

exudation rate or nutrient uptake rate, for which most of the studies conducted so far have been based on laboratory observations or modeling works (Banse 1976, Kriest and Oschlies 2007, Sunda and Hardison 2010).

Considering the suggested role of taxon-specific strategies in determining the isometric size-scaling of phytoplankton photosynthesis, further insight is needed on how functional traits in resource uptake and use of different phytoplankton taxa affect or modulate the size-scaling of metabolic rates. Although previous studies have addressed this topic, most of them have been based on analyzing a dataset from a literature review (Litchman et al. 2007), or they have been focused on analyzing just a few species of phytoplankton (Thingstad et al. 2005, Sunda and Hardison 2010). Then, one step further might be to achieve this goal by conducting laboratory experiments with phytoplankton cultures covering a wide range of cell sizes, wherein size-scaling macroecological patterns for size- and taxon-dependent metabolic rates would be analyzed.

Recent studies have reported shifts in the phytoplankton size structure towards smaller sizes as consequence of the warming of pelagic ecosystems (Daufresne et al. 2009, Yvon-Durocher et al. 2010), this shift being regarded as the third universal ecological response to global warming. This trend can be explained by extending the metabolic theory of ecology (Brown et al. 2004) to phytoplankton metabolism, as there is a close relationship between phytoplankton cell size, metabolic rates and temperature (Agawin et al. 2000, López-Urrutia et al. 2006). However, it is still unclear whether the observed changes in phytoplankton size structure and metabolic rates are exclusively an effect of increasing temperature by

128 Chapter 5. Synthesis

itself (Morán et al. 2009) or, conversely, if it is a nutrient-mediated effect (Li et al. 2009, Tadonléké et al. 2010). For instance, in the context of global warming, the increase of sea temperature is closely associated with an enhancement of water column stratification and a decrease of surface nutrient concentrations, environmental conditions that favor the growth of small phytoplankton (Kiørboe 1993). Then, within the global change framework, further research focused on discriminating the differential effect of nutrient availability and temperature on phytoplankton size and metabolism is needed, and the size-scaling approach might be an adequate way of addressing this issue.

Another line of research might be focused on incorporating phytoplankton size into remote sensing-based estimates of marine primary production (Silió-Calzada et al. 2008, Kostadinov et al. 2009, Brewin et al. 2010, Uitz et al. 2010). Given the reported trend towards a reduced cell size in phytoplankton as response to the global warming, and the role of phytoplankton size structure in the organization of marine trophic food webs and the global carbon cycle, this inclusion is essential in order to model the effect of global change on marine primary production. Thus, size-scaling relationships of phytoplankton abundance determined for different marine ecosystems may be applied to the development of these ocean productivity models. This kind of analysis might be also favored for the development of new approaches to infer phytoplankton size composition from variables that are measured by remote sensing. In this sense, some attempts to estimating the regression parameters of size-scaling relationships from ocean color remote sensing have been carried out recently, showing the possible use of

129 Chapter 5. Synthesis

particulate backscattering coefficient spectrum, chlorophyll a concentration and sea surface temperature as a proxies of these parameters (Kostadinov et al. 2010, Barnes et al. 2011).

As a corollary, macroecology represents a very valid approach for reducing the structural and dynamical complexity of natural phytoplankton communities to general patterns that explain the functioning of marine pelagic ecosystems. By exploring these patterns we are also able to go beyond the boundaries of the traditionally used disciplines and to infer new ecological laws (Brown 1995). However, as we have shown in the present Thesis, and depending on the nature of the patterns identified, the macroecological approach can provide new insight through two different, equally valid avenues. When macroecological patterns are truly accomplished across different systems, communities, trophic levels or life forms, a general model (universal law) is obtained with large predictive power that can be applied to different ecological systems and, therefore, has a high value from a conceptual and a practical point of view. Conversely, in those cases wherein the expected general pattern is not fulfilled or changes across the different systems, trophic levels or life forms considered, the macroecological approach is still very useful. In the process of explaining the departures from universal rules, we learn about the properties of organisms and communities, their differences, and their similarities.

130

Conclusions

Conclusions

1. The inverse power relationship between phytoplankton total abundance and cell size is persistent throughout the water column and across seasonal and interannual time scales in a highly dynamic and productive ecosystem such as the shelf off NW Iberian Peninsula. The slope of the overall size-abundance spectrum (-0.96) suggests that, in this ecosystem, roughly equal amounts of biomass are present over different logarithmic size classes of nano- and micro-phytoplankton.

2. During the different hydrographic periods that occur in the shelf off the NW Iberian Peninsula, the slopes of the phytoplankton size-abundance spectra are similar, suggesting that both nano- and micro-phytoplankton respond similarly to changes in environmental conditions. Only during upwelling relaxation and the onset of upper water-column stratification, significantly less steep values are found, indicative of the increased importance of larger cells.

3. The size-scaling of phytoplankton abundance in the tropical and subtropical Atlantic Ocean is characterized by steeper and less variable slopes (values ranging from -0.97 to -1.29), reflecting the dominance of smaller cells in comparison with coastal waters.

4. Due to the enhanced nutrient supply in the equatorial upwelling region, an increase was detected in phytoplankton total turnover rates, suggesting nutrient limitation of phytoplankton physiology in the central Atlantic Ocean.

133 Conclusions

5. The phytoplankton response to the equatorial upwelling varied among the different size classes: a higher stimulating effect of increased nutrient supply was found on the growth of pico- and small nano- phytoplankton.

6. The use of small sample volumes can lead to significant underestimations of the biomass and production of large phytoplankton in the oligotrophic ocean, with may have implications for global biogeochemical budgets and remote sensing-based studies.

7. The size-scaling of phytoplankton carbon fixation is isometric, negating the applicability of Kleiber’s law to natural phytoplankton communities. The size-scaling exponents obtained for the cell-specific carbon fixation rate (range 1.03-1.29) imply that the growth rates of large phytoplankton are similar to, or even larger than, those of smaller species.

8. Phytoplankton size structure in steady-state ecosystems such as the oligotrophic Atlantic Ocean can be explained as a result of the size-scaling of metabolic rate.

9. Given the observed size-scaling of abundance and metabolic rate, the total use of energy by phytoplankton is constant across the size spectrum in the central Atlantic Ocean, which suggests an explanation for the persistence of large species in ultraoligotrophic environments.

134

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158

Resumen

Resumen

Introducción

Aproximación macroecológica al estudio de los ecosistemas

La macroecología se define como el estudio de aquellos patrones generales que describen los sistemas ecológicos complejos en escalas espaciales y temporales extensas (Brown y Maurer 1989, Brown 1995, Gaston y Blackburn 2000). Siendo considerada en sí misma como un programa de investigación en ecología (Brown 1995, Brown 1999), la macroecología difiere de las aproximaciones tradicionalmente reduccionistas en su búsqueda de aquellos patrones empíricos y procesos mecanísticos por los cuales son generadas la estructura emergente y la dinámica de los ecosistemas. En este sentido, la mayoría de los estudios macroecológicos buscan aquellos patrones estadísticos que definen la abundancia, distribución, biomasa, diversidad y metabolismo de los organismos a través de las comunidades y ecosistemas (Brown et al. 2002, Blackburn 2004). Las especies de fitoplancton varían ampliamente tanto espacial como temporalmente en su distribución, abundancia y tasas metabólicas. Además, las comunidades fitoplanctónicas son tan diversas que no es posible determinar las propiedades biológicas de todas las especies que forman parte de ellas. Debido a estas razones, la aproximación macroecológica resulta particularmente interesante en los estudios del fitoplancton, tal y como muestra uno de los primeros estudios realizados al respecto (Li 2002) en el cual fueron descritos los patrones generales de la relación entre el forzamiento ambiental, el tamaño celular y la abundancia del fitoplancton.

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La importancia del tamaño celular del fitoplancton

Debido al papel fundamental del tamaño corporal como una herramienta sintética para el análisis de la complejidad de los ecosistemas, las relaciones entre esta variable y las tasas metabólicas, la abundancia y la diversidad de los organismos son consideradas como propiedades macroecológicas fundamentales de las comunidades y ecosistemas (Peters 1983, Brown et al. 2002 Marquet et al. 2005). El estudio del tamaño corporal como una forma de aproximación integrativa al conocimiento de la estructura y función de las comunidades de fitoplancton resulta particularmente apropiado si tenemos en cuenta que el tamaño celular del fitoplancton abarca más de ocho órdenes de magnitud y que influye en casi todos los aspectos de su fisiología, ecología y evolución (Marañón 2009, Finkel et al. 2010). La estequiometría del fitoplancton, su capacidad para captar nutrientes y sus tasas metabólicas son todas características que dependen del tamaño celular (Eppley y Sloan 1966; Banse 1976; Taguchi 1976), así como también dependen los procesos de pérdida tales como la susceptibilidad a la depredación o las tasas de sedimentación (Kiorboe 1993, Thingstad et al. 2005, Marañón et al. 2009, Finkel et al. 2010). En contraste con otros estudios macroecológicos que se han centrado en análisis especie- específicos (Brown et al. 2002, Marquet et al. 2005), en el caso del fitoplancton el tamaño celular es usado como un criterio no taxonómico de agregación, aunque sin olvidar el hecho de que la composición taxonómica cambia de manera predecible a lo largo del espectro de tamaños (Rodríguez 1994, Cermeño et al. 2005b, Irwin et al. 2006).

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La estructura de tamaños del fitoplancton en los ecosistemas marinos

La estructura de tamaños del fitoplancton controla el funcionamiento de los ecosistemas pelágicos desde un punto de vista tanto ecológico como biogeoquímico (Kiorbøe 1993, Legendre y Rassoulzadegan 199, Marañón 2009), ya que determina la organización trófica de las comunidades microbianas planctónicas y, por tanto, su potencial en la exportación de carbono orgánico (Legendre y Le Fèvre 1989). Uno de los patrones más consistentes en el océano es aquel en el que la biomasa total de fitoplancton, siendo ésta estimada a partir de la concentración de clorofila a, es baja y el fitoplancton de pequeño tamaño (por ejemplo el picofitoplancton, <2 µm en diámetro celular) domina la comunidad (Chisholm 1992, Agawin et al. 2000). En contraste, cuando la biomasa total es grande, gran parte de ésta se debe a las células de mayor tamaño (microfitoplanton, >20 µm en diámetro celular). Así, las regiones oligotróficas del océano están caracterizadas por el reciclaje continuo de la materia orgánica dentro de la red microbiana y, en consecuencia, por un menor flujo de exportación de carbono hacia las profundidades del océano (bucle microbiano, Azam et al. 1993). Por el contrario, el dominio de la comunidad por parte del fitoplancton de mayor tamaño, que ocurre típicamente en las aguas altamente dinámicas y ricas en nutrientes, determina la transferencia de la materia orgánica sintetizada bien hacia niveles tróficos superiores a través de cadenas tróficas cortas, o bien fuera de la capa eufótica hacia mayores profundidades del océano mediante la sedimentación (Cushing 1989); aumentando como resultado el potencial de dichos ecosistemas para el secuestro de CO2 atmosférico.

Dado que la tasa de captación y uso de los recursos por el

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fitoplancton depende del tamaño celular (Kiørboe 1993, Raven 1998, Litchman et al. 2007) y que el hidrodinamismo de la columna de agua determina en gran medida la disponibilidad de nutrientes dentro de la capa eufótica, así como también afecta al transporte vertical del fitoplancton (Parsons y Takahashi 1973, Kiørboe 1993, Malone et al. 1993, Rodríguez et al. 2001), la distribución de tamaños de las comunidades fitoplanctónicas también está controlada por las características físicas y químicas del ambiente. Así, es bien conocido que, como resultado de su ventaja sobre el fitoplancton grande en términos de captación de nutrientes, las células fitoplanctónicas de pequeño tamaño dominan las regiones del océano con menores concentraciones de nutrientes y una mayor estratificación de la columna de agua (Chisholm 1992, Kiørboe 1993, Raven y Kübler 2002). Sin embargo, el fitoplancton de pequeño tamaño también está presente en áreas del océano más turbulentas y ricas en nutrientes, actuando dichos organismos como un componente de fondo de la comunidad cuya biomasa y producción primaria permanecen relativamente constantes (Raimbault et al. 1988, Thingstad y Sakshaug 1990, Rodríguez et al. 1998). Por esta razón, es comúnmente aceptado que la mayoría de la variabilidad geográfica y temporal observada en la biomasa y producción primaria del fitoplancton es el resultado de la respuesta del fitoplancton de mayor tamaño al forzamiento ambiental, siendo estimulado el crecimiento de dicha clase de tamaño por el aumento de la entrada de nutrientes que favorece el desarrollo de sus estrategias fisiológicas (Sarthou et al. 2005, Thingstad et al. 2005, Litchman et al. 2007, Verdy et al. 2009). Sin embargo, en estudios recientes se ha destacado que el picofitoplancton también es capaz de responder en términos de biomasa y producción primaria al enriquecimiento ocasional de

164 Resumen

las regiones oligotróficas del océano, aunque la magnitud de dicha repuesta podría ser menor a la presentada por las células de mayor tamaño (Barber y Hiscock 2006, Tarran et al. 2006, Glover et al. 2007).

El estudio de las relaciones con el tamaño en ecología

Las relaciones con el tamaño en ecología son expresadas de forma matemática por una función de tipo potencial en la que las características biológicas de los organismos están relacionadas con el tamaño corporal mediante una ecuación tipo Y = a Vb, donde Y es la propiedad biológica estudiada, a es una constante relacionada con la taxonomía, V es el tamaño corporal del organismo en cuestión y b es el exponente de la relación potencial (Brown et al. 2002, Marquet 2005, Finkel et al. 2010). La transformación logarítmica de dicha función da lugar a un modelo lineal: log Y = log a + b log V, donde log a y b son los valores del Y-intercepto y la pendiente de la recta respectivamente. Así, los datos logarítmicos pueden ser ajustados a una línea recta mediante un análisis de regresión lineal. Muchas de estas relaciones son consideradas leyes universales que reflejan la existencia de poderosas limitaciones en la organización de los sistemas complejos, ya que dichas leyes pueden ser aplicadas de forma consistente a todo tipo de organismos que se expanden a lo largo de veinte órdenes de magnitud en tamaño celular (microorganismos, animales y plantas) y que viven en todo tipo de ambientes (por ejemplo, terrestres, hábitats marinos y de agua dulce) (Brown 1995, West 1999, Brown et al. 2002, Marquet et al. 2005, DeLong et al. 2010). Las propiedades biológicas que pueden ser descritas mediante estas relaciones macroecológicas con el tamaño incluyen la abundancia, la tasa de vida media, el tamaño del hábitat, la tasa

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metabólica y el uso de los recursos (Peters 1983, Brown et al. 2004).

Relaciones entre la abundancia del fitoplancton y el tamaño celular

La relación existente entre la abundancia y el tamaño corporal de los organismos ha sido ampliamente estudiada en ecología, dado que dicha relación representa un nexo de unión fundamental entre los rasgos de las especies a niveles individuales y poblacionales y la estructura y dinámica de las comunidades ecológicas (Kerr y Dickie 2001, Woodward et al. 2005). Por lo tanto, la importancia de las relaciones entre la abundancia (o densidad) y el tamaño ha sido ampliamente reconocida tanto en ecosistemas terrestres como acuáticos (Brown 1995, Gaston y Blackburn 2000, Kerr y Dickie 2001).

Uno de las aproximaciones más comunes al estudio de la relación continua entre la abundancia y el tamaño corporal en los ecosistemas pelágicos es la construcción de una distribución individual de tamaños conocida como espectro de tamaño-abundancia (White et al. 2007). En dicha aproximación, la abundancia total (N) o biomasa (B) de todos los organismos incluidos dentro de una clase de tamaño dada es representada frente al tamaño celular nominal de cada clase de tamaño expresado en unidades de carbono o volumen (V). Para construir los espectros de tamaño- abundancia, la composición taxonómica no es considerada y el tamaño celular es usado como criterio único de agregación (Sheldon et al. 1972, Rodríguez y Mullin 1986, Rodríguez et al. 2001). La relación entre la abundancia total y el tamaño celular típicamente resulta en una función potencial cuyo exponente, b, puede ser interpretado como un descriptor

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sintético de la estructura de tamaños de la comunidad que varía dependiendo de la productividad del ecosistema. En el caso de los espectros de tamaño- abundancia del fitoplancton, los valores de pendiente obtenidos en los ecosistemas oligotróficos son más negativos (entre -1.3 y -1.1) que aquellos encontrados para las aguas más productivas (entre -0.8 y -0.6), reflejando un aumento de la dominancia de las células de mayor tamaño en los ambientes ricos en nutrientes (Cavender-Bares et al. 2001, Reul et al. 2005, Cermeño y Figueiras 2008, Marañón et al. 2007). Aunque una fuerte regularidad en la linealidad de los espectros de tamaño-abundancia ha sido encontrada en las regiones oligotróficas, los ecosistemas pelágicos más productivos, que con frecuencia están sujetos a un mayor grado de variabilidad hidrodinámica, suelen mostrar irregularidades (por ejemplo no linealidades) en la relación existente entre la abundancia y el tamaño como consecuencia de la acumulación en la comunidad de especies de una determinada clase de tamaño (Sprules y Munawar 1986, Marquet et al. 2005, Reul et al. 2006). Sin embargo, no ha sido determinado todavía si esta ausencia de relación lineal es un patrón general que puede ser aplicado a todos los ecosistemas sujetos a un altamente variable forzamiento ambiental, o si, por el contrario, es solo el resultado del relativo escaso número de estudios sobre las relaciones continuas con el tamaño celular llevados a cabo en dicho tipo de ecosistemas. Finalmente, la cuestión de cuál es el origen de los valores actuales de la pendiente de los espectros de tamaño-abundancia del fitoplancton (por ejemplo por qué las pendientes toman un valor entre -1.3 y -1 en las aguas oligotróficas) no ha sido aún resuelto.

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Relaciones entre la tasa metabólica del fitoplancton y el tamaño celular

La relación potencial de ¾ entre las tasas metabólicas y el tamaño corporal de los organismos ha sido una de las leyes más ampliamente aceptadas en ecología desde que fuera formulada por primera vez por Kleiber en 1923 (Kleiber 1947). Si la tasa metabólica es dividida entre la masa corporal o volumen (V), la tasa específica por unidad de masa (o volumen) resultante se relaciona con el tamaño de la forma V-1/4, lo que significa que conforme los organismos aumentan en tamaño, su tasa metabólica específica por unidad de biomasa tiende a disminuir o, en otras palabras, que los organismos de mayor tamaño tienen un metabolismo más lento que los de menor tamaño. Dadas las implicaciones ecológicas de este patrón metabólico, la universalidad de la ley de Kleiber ha sido comprobada tanto en los ecosistemas terrestres (Enquist et al. 1998, Savage et al. 2004, McNab 2008) como en los acuáticos (Banse et al. 1976, Quiñones et al. 1992, Glazier et al. 2006), aunque aún existe una gran controversia respecto a este tema (Dodds et al. 2001, Bokma 2004, Glazier et al. 2006, Kolokotrones 2010). Numerosos modelos han sido además desarrollados con el objetivo de obtener una explicación general para el origen de estas relaciones escalares con el tamaño que pueda ser aplicada a diferentes formas de vida y niveles de organización (West et al. 1999, Dodds et al. 2001, Banavar et al. 2002, West y Brown 2005).

En el caso del fitoplancton, diversos estudios han señalado la aplicabilidad de la relación potencial de ¾ sobre el metabolismo de dicho grupo (Eppley y Sloan 1966, Taguchi 1976, Blasco et al. 1982, Finkel 1998, López-Urrutia 2006). En 2001 Niklas y Enquist señalaron la continuidad de

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la relación potencial de ¾ entre la tasa de producción de biomasa y el tamaño corporal en todos los organismos fotoautótrofos, desde los microscópicos y las algunas unicelulares hasta los árboles más grandes. Sin embargo, todos estos estudios han sido basados en datos obtenidos de cultivos en el laboratorio. En contraste, trabajos recientes que se han centrado en el análisis de observaciones procedentes de comunidades naturales de fitoplancton han destacado la existencia de una relación isométrica (exponente de la relación potencial, b, próximo a 1) entre la tasa de fotosíntesis y el tamaño celular, que además parecía variar con la productividad del ecosistema (Marañón et al. 2007, Marañón 2008). Teniendo en cuenta que una relación aproximadamente isométrica implica que las células grandes tienen mayores tasas metabólicas de lo que se esperaría para su tamaño, e incluso mayores tasas de crecimiento que las células de menor tamaño, este resultado plantea nuevos puntos de vista acerca de los factores que controlan la estructura de tamaños del fitoplancton y el papel que el fitoplancton de mayor tamaño ejerce sobre el funcionamiento de los ecosistemas pelágicos marinos, teniendo importantes implicaciones ecológicas y biogeoquímicas (Cermeño et al. 2005a, Marañón 2008, Marañón 2009). De hecho, una relación isométrica entre el metabolismo del fitoplancton y el tamaño implica que la dominancia de las células de mayor tamaño en los ambientes más ricos en nutrientes podría ser el resultado de la manifestación de mecanismos puramente fisiológicos, sin la necesidad de argumentar el efecto de mecanismos de control de tipo trófico. Sin embargo, todas estas relaciones escalares se caracterizaban por tener un gran número de limitaciones metodológicas. En el caso de Marañón et al. (2007), los autores usaron medidas de la tasa metabólica realizadas en

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un pequeño número de clases de tamaño, lo que les impidió además determinar las relaciones existentes entre el metabolismo y el tamaño en comunidades particulares de fitoplancton a escalas espaciales locales. En el caso de Marañón (2008), las relaciones interespecíficas reflejadas son basadas en datos extraídos de la literatura, lo que, debido a las diferencias en la metodología usada por cada autor, podría dar lugar a una incertidumbre adicional. Por esta razón, resulta crucial el determinar las relaciones entre el metabolismo del fitoplancton y el tamaño corporal en condiciones naturales mediante el uso de una metodología con mayor resolución.

Hipótesis y objetivos

Hipótesis

El tamaño celular juega un importante papel en numerosos aspectos de la biología y ecología del fitoplancton y hay una fuerte relación entre la composición taxonómica de las comunidades de fitoplancton y su distribución de tamaños. Además, los factores ambientales, tanto bióticos como abióticos, parecen ejercer un efecto diferente entre las diferentes clases de tamaño del fitoplancton. Por lo tanto, la base subyacente a la realización del presente trabajo de investigación es que el tamaño celular del fitoplancton constituye una herramienta macroecológica adecuada para reducir la complejidad de las comunidades de fitoplancton a patrones generales que nos ayuden a comprender el funcionamiento de los ecosistemas marinos. Dentro de este marco general, las siguientes hipótesis específicas son formuladas:

 La relación entre la abundancia del fitoplancton y el tamaño celular

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puede mostrar irregularidades en aquellos ecosistemas sujetos a un forzamiento ambiental altamente dinámico.

 La variabilidad estacional e interanual en la disponibilidad de los recursos y las condiciones hidrográficas experimentada por los ecosistemas costeros puede dar lugar a cambios en la estructura de tamaños del fitoplancton que serán representados por variaciones en las pendientes de la relación entre la abundancia del fitoplancton y el tamaño celular.

 El fitoplancton de mayor tamaño está más limitado por la disponibilidad de nutrientes que las células de pequeño tamaño en las regiones oligotróficas del océano, por lo que su biomasa, producción y tasa de crecimiento aumentarán más que las de las células de pequeño tamaño ante el incremento en el suministro de nutrientes.

 La tasa metabólica del fitoplancton, y en particular su tasa de fijación fotosintética, se relaciona con el tamaño celular siguiendo una ley potencial con un exponente de ¾.

 La distribución de tamaños del fitoplancton en ecosistemas próximos al estado estacionario, como el océano abierto oligotrófico, puede ser deducida a partir de la relación entre la tasa metabólica del fitoplancton y el tamaño.

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Objetivo general

El objetivo principal de la presente Tesis es determinar la naturaleza y variabilidad de las relaciones entre el tamaño celular, la abundancia, la biomasa y la tasa metabólica en comunidades naturales de fitoplancton.

Objetivos específicos

 Testar la persistencia en el tiempo de la relación potencial continua entre la abundancia del fitoplancton y el tamaño celular en un ecosistema altamente dinámico y productivo.

 Determinar la variabilidad estacional e interanual de la relación continua entre la abundancia del fitoplancton y el tamaño celular en un ecosistema marino productivo, así como su relación con las condiciones hidrodinámicas.

 Determinar la respuesta diferencial entre los distintos tamaños del fitoplancton del océano abierto en términos de biomasa y producción a los cambios en el forzamiento ambiental.

 Determinar la relación continua entre la tasa de fotosíntesis del fitoplancton y el tamaño celular con el objetivo de elucidar la aplicabilidad de la ley de Kleiber a las tasas metabólocas del fitoplancton.

 Investigar la conexión entre la relación de la tasa metabólica con el tamaño y la estructura de tamaños de las comunidades de

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fitoplancton.

Esquema de la tesis

Con el objetivo de testar las hipótesis enunciadas y alcanzar los objetivos especificados anteriormente dos líneas de trabajo fueron llevadas a cabo durante el desarrollo de la presente tesis. En primer lugar, para estudiar la variabilidad temporal y la regularidad de la relación entre la abundancia del fitoplancton y el tamaño celular en un ecosistema dinámico y productivo, así como para determinar la variabilidad de dicha relación ante cambios en el forzamiento ambiental, analizamos una amplia base de datos procedente de un serie temporal de diez años de duración situada en una estación de la plataforma de la ría de A Coruña. Dicha serie temporal consistió en una serie de datos mensuales sobre la abundancia y el tamaño celular del fitoplancton a partir de los cuales los espectros de tamaño- abundancia fueron construidos a diferentes niveles de integración. La pendiente y el Y-intercepto resultantes de las relaciones obtenidas fueron con posterioridad analizados en relación con los diferentes períodos hidrográficos que caracterizan a dicho ecosistema pelágico durante el ciclo anual. La variabilidad estacional e interanual de la distribución de tamaños del fitoplancton fue estudiada mediante un análisis de serie temporal. Los resultados de esta primera línea de trabajo han sido recogidos en el Capítulo 2 de la presente Tesis y han sido recientemente publicados en Huete-Ortega et al. (2010).

La segunda línea de trabajo está relacionada con el análisis de la relación entre la tasa metabólica y el tamaño de las comunidades naturales

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de fitoplancton y la respuesta diferencial entre los distintos tamaños del fitoplancton ante cambios en el ambiente. Dicha línea fue llevada a cabo durante una campaña oceanográfica desarrollada en el océano Atlántico tropical y subtropical. Durante dicha campaña, la tasa de fijación fotosintética y la concentración de clorofila a fraccionadas por tamaños fueron determinadas in situ y fueron tomadas muestras para estimar, con posterioridad, la abundancia y el biovolumen del fitoplancton presente en las distintas estaciones muestreadas. Los datos resultantes fueron analizados mediante dos aproximaciones.

Por un lado, un estudio discreto fraccionado por tamaños de la variabilidad de la biomasa, la producción primaria y la tasa de recambio del fitoplancton fue llevado a cabo centrándonos en aquellas estaciones directamente influidas por el afloramiento ecuatorial del océano Atlántico. El principal objetivo de este análisis fue determinar la respuesta diferencial de los distintos grupos de tamaño del fitoplancton al forzamiento ambiental, especialmente la disponibilidad de nutrientes y la estabilidad de la columna de agua, y elucidar si la limitación por concentración de nutrientes de las comunidades de fitoplancton que ha sido señalada por otros autores para las regiones oligotróficas del océano estaba solamente restringida a la biomasa del fitoplancton o también afectaba a su fisiología. Los detalles y resultados de dicha aproximación son mostrados en el Capítulo 3 de la presente Tesis y han sido enviados para su publicación científica (Huete-Ortega et al. en revisión).

Por otro lado, mediante la construcción de espectros de tamaño-tasa fotosintética y tamaño-abundancia fue posible estudiar la variabilidad de

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largar escala en la relación entre la tasa metabólica del fitoplancton y el tamaño celular y analizar la conexión entre dicha relación escalar y la distribución de tamaños de las comunidades naturales de fitoplancton. Los resultados de estos análisis y sus implicaciones ecológicas son presentados en el Capítulo 4 de la presente Tesis y en un artículo que ha sido enviado para su publicación en un revista científica (Huete-Ortega et al. en revisión).

Capítulo 2. Patrones generales en la relación entre la abundancia del fitoplancton y el tamaño en aguas costeras durante 10 años de serie temporal

Con el objetivo de estudiar los patrones generales de la estructura de tamaños del fitoplancton en un ecosistema costero templado, la relación entre la abundancia total de fitoplancton y el tamaño celular (espectro de tamaños) fue determinada mensualmente para el nano- y micro-fitoplancton en una estación de la plataforma del noroeste de la Península Ibérica durante el período de 1993-2002. La relación lineal inversamente proporcional encontrada entre el logaritmo de la abundancia del fitoplancton y el logaritmo del tamaño celular fue persistente a través de la totalidad de la columna de agua y a lo largo de escalas de tiempo estacionales e interanuales (Fig. 1). Asimismo, a pesar de la alta productividad y la marcada variabilidad temporal observada en la estructura de la columna de agua del sitio de estudio, las desviaciones respecto a la linealidad en los espectros de tamaño resultaron ser escasas. La pendiente (-0.96) del espectro de tamaño-abundancia general para la totalidad de la serie temporal indicó que aproximadamente cantidades iguales de biomasa estuvieron presentes a lo largo de diferentes clases de tamaño logarítmicas en el rango de tamaños

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considerado (Fig. 2). Los espectros de tamaño-abundancia del fitoplancton mostraron pendientes promedio similares durante los períodos hidrográficos de mezcla invernal, comienzo del afloramiento, estratificación estival y hundimiento otoñal, sugiriendo que, en dichas condiciones oceanográficas, ambos grupos de tamaño, el nano- y micro-fitoplancton, respondieron de manera similar a la variabilidad ambiental (Tabla 1). En contraste, pendientes significativamente menos negativas fueron observadas durante el período de relajación del afloramiento, indicando de esta forma un aumento de la importancia de las células de fitoplancton de mayor tamaño en dicha época hidrográfica. Nuestros resultados ilustran la utilidad de las distribuciones individuales del tamaño celular a la hora de proveer de una descripción sintética de la estructura de la comunidad de fitoplancton en ecosistemas marinos dinámicos que están es una situación no estacionaria.

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Fig. 1 Espectros de tamaño-abundancia representativos a diferentes niveles de integración: A) espectros de tamaño-abundancia de diferentes profundidades de un día determinado (16 Noviembre 1994), B) espectros de tamaño-abundancia para la columna de agua de Enero 1995, Marzo 1995 y Agosto 1995 y C) macroespectro de tamaño-abundancia para 1995, 1998 y 2002. Cada espectro fue ajustado a un modelo lineal mediante una regresión r.m.a.

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5

4

3

2

1

abundancia (celúla/mL) 0 10 log -1

-2 01234567 log tamaño celular (m3) 10 Fig. 2 Macroespectro general de tamaño-abundancia para la totalidad de la serie temporal. La línea de regresión es: log10 abundancia total = 4.12 – 0.97 log10 tamaño celular (r2 = 0.59; P <0.0001, n = 5964).

Tabla 1. Parámetros estadísticos para la relación entre log10 abundancia total celular y

log10 tamaño nominal para los diferentes períodos hidrográficos analizados durante 1993-2000. Período hidrográfico n Y-intercepto b (pendiente) r2 Mezcla invernal 2107 3.92 (3.85, 4.00) -0.97 (-0.99, -0.95) 0.65

Afloramiento tipo I 669 4.22 (4.07, 4.38) -0.98 (-1.03, -0.94) 0.58

Afloramiento tipo II 1066 4.05 (4.05, 4.16) -0.85 (-0.88, -0.82) 0.50

Estratificación 1188 4.35 (4.24, 4.47) -0.99 (-1.03, -0.96) 0.60

Hundimiento 470 4.11 (3.96, 4.27) -1.01 (-1.05, -0.96) 0.68 El Y-intercepto y la pendiente (b) fueron obtenidos usando el análisis de regresión r.m.a. n indica el número de observaciones incluidas dentro del análisis de regresión. Los límites de confianza (95%) para el intercepto y la pendiente están entre paréntesis.

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Capítulo 3. Efecto del forzamiento ambiental en la biomasa, producción y tasa de crecimiento del fitoplancton fraccionado por tamaños en el Atlántico central

Con el objetivo de estudiar la respuesta de las diferentes clases de tamaño del fitoplancton ante cambios en la estabilidad de la columna de agua y la disponibilidad de nutrientes, la biomasa, la tasa de fijación de carbono y la tasa de crecimiento (producción/biomasa) fraccionadas por tamaños del fitoplancton fueron determinadas en aguas superficiales del océano Atlántico central (26ºN-5ºS). Como resultado del incremento de la entrada de nutrientes en la capa eufótica y la mayor estabilidad de la columna de agua encontradas en la zona bajo la influencia del afloramiento ecuatorial, observamos no solo un aumento en la biomasa y producción primaria total del fitoplancton, sino también en las tasas de recambio, lo que sugirió la posible limitación por nutrientes de la fisiología del fitoplancton del océano Atlántico oligotrófico (Fig. 3). Asimismo, los cuatro grupos de tamaño de fitoplancton analizados (pico-, nano- pequeño, nano- grande y micro-fitoplancton) mostraron diferentes respuestas ante el forzamiento ambiental asociadas con el afloramiento ecuatorial, tanto en términos de biomasa como de producción primaria y tasas de crecimiento (Fig. 4). Así, el nano- grande y el micro-fitoplancton mostraron consistentemente mayores tasas de crecimiento y ratios de fijación de carbono por unidad de clorofila que el fitoplancton de menor tamaño, contribuyendo de esta forma a la producción primaria total más de lo que podría esperarse si se tuviese en cuenta su contribución a la biomasa. Por otro lado, fue observado un mayor efecto estimulante del incremento en el suministro de nutrientes sobre las

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3,5e-4 0,08 A 3,0e-4 0,07

2,5e-4 0,06 ) -1

2,0e-4 0,05 (m d

1,5e-4 0,04 K

1,0e-4 0,03 Frecuencia Brunt-Väisäla Frecuencia

5,0e-5 0,02 ) -1 d

160 3000 -2 140 B 2500

120 2000 m mol N  (

100 1500 3 80 1000 60 500 40 0 Profundidad nitraclina (m) nitraclina Profundidad 20 Flujo difusivo NO difusivo Flujo ) -1 30 7 0,24

C 0,22 ) g C L 6 -1  25 0,20 5 0,18 20 4 0,16 0,14 15 3 0,12 2 0,10 10 0,08 1 Tasa total recambio (d recambio total Tasa

Tasa fijación carbono total carbono Tasa fijación 0,06 5 0 0,04

Biomasa de carbono total ( -10-50 5 1015202530

Latitud Fig. 3 Variabilidad latitudinal de A) frecuencia Brunt-Väisäla (círculos, línea sólida) y Kd (cuadrados, línea discontinua), B) profundidad de la nitraclina (círculos, línea sólida) y flujo difusivo del nitrato a través de la zona eufótica (cuadrados, línea discontinua), y C) biomasa de carbono total (círculos, línea sólida), tasa de fijación de carbono total (cuadrado, línea discontinua), y tasa total de recambio (triángulos, línea punteada). Latitud positiva indica Norte, latitud negativa indica Sur.

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25 3,0 -1 d ) A B -1 -1 20 2,5 g C L g C g C L g C 

 2,0 15 1,5 10 1,0

5 0,5 Biomasa carbono ( carbono Biomasa

0 0,0 ( carbono fijación Tasa

100 100 C D 80 80

60 60

40 40

20 20

Contribución a biomasa total (%) total biomasa a Contribución 0 0 -10-50 5 1015202530-10-50 5 1015202530

Latitud Latitud Contribucion a tasa fijación carbono total (%) Fig. 4 Variabilidad latitudinal de A) biomasa en carbono, B) tasa de fijación de carbono, C) contribución relativa (%) a la biomasa total y D) contribución relativa (%) a la tasa de fijación de carbono total del pico- (0.2-2 µm en DEE; círculos negros), nano- pequeño (2-5 µm en DEE; círculos blancos), nano- grande (5-20 µm en DEE; triángulos negros) y micro-fitoplancton (>20 µm en DEE; triángulos blancos). Valores de latitud como en Fig. 3.

tasas de crecimiento del picofitoplancton (Fig. 5). Dicha observación puede ser explicada si se considera la dinámica del afloramiento ecuatorial del océano Atlántico, donde la continua pero en pequeña cantidad entrada de nutrientes en la capa eufótica pone de manifiesto la ventaja competitiva de las células de pequeño tamaño adaptadas a las condiciones oligotróficas. La aproximación fraccionada por tamaños al análisis de la biomasa, la

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producción primaria y la tasa de crecimiento llevado a cabo en este estudio revela, por tanto, importantes diferencias específicas para los distintos grupos de tamaño del fitoplancton en respuesta al forzamiento ambiental, que no pueden ser apreciadas cuando se hacen medidas que engloban a la comunidad en su conjunto.

0,8 40 AB

) 0,6

-1 30

0,4 20 0,2 10 Tasa recambio (d recambio Tasa 0,0

0 -10-50 5 1015202530-10-50 5 1015202530 Fijación unidad de carbono de clorofilapor Latitud Latitud

Fig. 5 Variabilidad latitudinal de A) tasa de recambio y B) ratio entre la tasa de fijación de carbono fraccionada por tamaños y la concentración clorofila a (µg C µg Chl a-1 d-1) del pico- (0.2-2 µm en DEE; círculos negros), nano- pequeño (2-5 µm en DEE; círculos blancos), nano- grande (5-20 µm en DEE; triángulos negros) y micro-fitoplancton (>20 µm en DEE; triángulos blancos). Valores de latitud como en Fig. 3.

Capítulo 4. Relación entre la tasa metabólica y el tamaño y la estructura de tamaños del fitoplancton en el océano abierto

Con el objetivo de determinar la relación entre la tasa de fijación de carbono y el tamaño en comunidades naturales de fitoplancton y explorar la conexión entre dicha relación y la estructura de tamaños del fitoplancton,

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determinamos de manera simultánea las tasas de fijación de carbono y la abundancia del fitoplancton a lo largo de un rango de tamaño que abarcaba entorno a seis órdenes de magnitud en el océano Atlántico tropical y subtropical. Fue encontrada una relación isométrica entre la tasa de fijación de carbono específica por unidad de célula y el tamaño celular (rango del valor de las pendientes 1.03-1.32), negando, por tanto, la idea de la aplicabilidad de la ley de Kleiber a los protistas autótrofos unicelulares (Fig. 6A y Tabla 2). La relación isométrica observada entre la tasa de fijación de carbono y el tamaño celular es probable que sea el resultado de estrategias fisiológicas específicas propias de distintos taxones del fitoplancton de mayor tamaño que les permiten superar las limitaciones celulares asociadas a la captación y uso de los recursos. Basándonos en la relación entre el uso individual de los recursos y el tamaño celular, predecimos una relación recíproca entre las relaciones con el tamaño de la tasa metabólica del fitoplancton y la abundancia celular. Dicha predicción fue confirmada por las pendientes observadas para la relación entre la abundancia celular total y el tamaño, que tomaron valores entre -0.97 y -1.29 (Fig. 6B y Tabla 2). Así, concluimos que la estructura de tamaños del fitoplancton en el océano abierto es el resultado de la relación entre la tasa metabólica y el tamaño. Además, deducimos que la energía total procesada debida a la fijación de carbono es constante a lo largo del espectro de tamaños del fitoplancton (Fig. 6C), lo que podría explicar la persistencia, a lo largo de escalas de tiempo evolutivas, de las especies de mayor tamaño en los ecosistemas oligotróficos y en estado estacionario del océano abierto.

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) Fig. 6 Ajuste de la regresión -1 4 cell

Pendiente media = 1.16 (±0.09) lineal sobre la relación log- -1 3 log entre A) la tasa de 2 fijación de carbono 1 específica por unidad de 0 célula y el tamaño celular, -1 B) la abundancia celular -2 -3 total y el tamaño celular y C) -4 la tasa de fijación de carbono -5 tasa fijación carbono (pg C h (pg C carbono fijación tasa A

total y el tamaño celular en 10 -6

todas las muestras tomadas log durante el desarrollo de la ) 6 Pendiente media= -1.15 (±0.09) campaña oceanográfica. Los 5 parámetros de la regresión 4 para cada tipo de relación 3 son detallados en la Tabla 2. 2 Los valores de las pendientes 1 en los paneles A y B se 0 refieren a la pendiente media -1 -2 (± SD) para cada tipo de abundancia total celular (cell/mL celular total abundancia -3 B 10 relación, que fue obtenida -4 calculando el promedio de log )

-1 6 h

todas las pendientes obtenidas para todos los -1 5 experimentos. 4

3

2

1 tasa fijación carbono (pg L C carbono fijación tasa C 10 0 log -2-10123456 log tamaño celular (m3) 10

184 Table 2 Parámetros estadísticos de la relación entre el tamaño celular del fitoplancton y la abundancia celular total y la tasa de fijación de carbono obtenidas en el océano Atlántico tropical y subtropical. Tasa fijación carbono vs. Tamaño celular Abundancia celular total vs. tamaño celular Estación d c r2 n* P-value b a r2 n 26.00º N 25.73º W 1.20 (0.98, 1.28) -2.56 (-2.78, -2.11) 0.99 7 < 0.0001 -1.23 (-1.32, -1.10) 3.14 (2.79, 3.41) 0.97 20 26.00º N 29.83º W 1.06 (0.79, 1.30) -2.63 (-2.97, -1.92) 0.95 7 0.003 -0.97 (-1.09, -0.83) 2.89 (2.47, 3.24) 0.94 22 26.00º N 34.12º W 1.24 (1.05, 1.38) -3.15 (-3.56, -2.63) 0.97 8 0.0001 -1.16 (-1.37, -0.92) 3.22 (2.79, 3.69) 0.91 20 26.00º N 38.27º W 1.20 (0.79, 1.50) -3.01 (-3.97, -2.37) 0.94 8 0.0003 -1.12 (-1.26, -0.97) 3.22 (2.97, 3.48) 0.95 20 23.44º N 34.91º W 1.16 (0.95, 1.48) -3.19 (-4.17, -2.54) 0.94 8 0.0005 -0.97 (-1.07, -0.84) 3.01 (2.65, 3.34) 0.96 21 20.92º N 31.69º W 1.32 (1.06, 1.60) -2.85 (-3.77, -2.40) 0.96 8 0.0001 -1.22 (-1.35, -1.04) 3.22 (2.80, 3.67) 0.92 21 18.41º N 29.09º W 1.24 (1.09, 1.44) -2.71 (-3.30, -2.37) 0.97 8 < 0.0001 -1.15 (-1.26, -1.02) 3.19 (2.81, 3.56) 0.95 22 14.43º N 28.71º W 1.17 (0.99, 1.32) -2.33 (-2.79, -2.00) 0.98 8 < 0.0001 -1.29 (-1.36, -1.21) 3.61 (3.36, 3.86) 0.98 23 11.17º N 28.00º W 1.03 (0.74, 1.21) -2.17 (-2.57, -1.35) 0.93 8 0.008 -1.13 (-1.25, -0.97) 3.31 (2.94, 3.65) 0.95 21 7.40º N 29.01º W 1.03 (0.73, 1.15) -1.91 (-2.18, -1.16) 0.95 8 0.0028 -1.17 (-1.29, -0.99) 3.28 (2.90, 3.64) 0.94 22 3.29º N 29.02º W 1.07 (0.88, 1.20) -2.19 (-2.58, -1.81) 0.98 8 < 0.0001 -1.11 (-1.21, -0.98) 3.20 (2.82, 3.57) 0.95 23 0.33º S 28.99º W 1.18 (1.04, 1.39) -2.44 (-2.82, -2.19) 0.96 8 < 0.0001 -1.26 (-1.38, -1.10) 3.10 (2.73, 3.45) 0.96 20 4.67º S 28.99º W 1.29 (1.00, 1.38) -2.88 (-3.17, -2.27) 0.98 7 < 0.0001 -1.20 (-1.31, -1.04) 3.04 (2.63, 3.40) 0.95 21 8.58º S 29.00º W 1.03 (0.74, 1.25) -2.19 (-2.68, -1.19) 0.95 8 0.0028 -1.07 (-1.19, -0.92) 2.64 (2.20, 3.02) 0.95 24 16.58º S 28.99º W 1.17 (0.93, 1.26) -2.48 (-2.70, -2.05) 0.98 7 < 0.0001 -1.09 (-1.18, -0.96) 2.72 (2.48, 2.94) 0.96 21 24.92º S 28.99º W 1.15 (1.00, 1.22) -2.47 (-2.67, -2.12) 0.99 7 0.0001 -1.18 (-1.33, -1.00) 3.05 (2.74, 3.32) 0.95 19 33.04º S 28.96º W 1.19 (0.85, 1.40) -2.52 (-2.95, -1.76) 0.97 6 0.0002 -1.16 (-1.28, -1.02) 3.05 (2.71, 3.37) 0.95 18 Para cada tipo de relación, a y c son los interceptos del Modelo II, b y d se refieren a las pendientes del Modelo II, n y n* el número de datos incluidos en la regresión, y r2 es el coeficiente de determinación. 95% ICs para los interceptos y pendientes son dados entre paréntesis. P-value se refiere a la comparación de la pendiente de la relación entre la tasa metabólica y el tamaño con el valor esperado de ¾.

Resumen

Conclusiones

1. La relación potencial inversa entre la abundancia total y el tamaño celular del fitoplancton es persistente a través de la columna de agua y a lo largo de escalas de tiempo estacionales e interanuales en un ecosistema altamente dinámico y productivo como la plataforma del Noroeste de la Península Ibérica. La pendiente del espectro general de tamaño-abundancia (-0.96) sugiere que, en dicho ecosistema, aproximadamente iguales cantidades de biomasa están presentes a lo largo de diferentes clases de tamaño logarítmicas del nano- y micro-fitoplancton.

2. Durante los diferentes períodos oceanográficos que ocurren a lo largo del año en la plataforma del Noroeste de la Península Ibérica, la pendiente de los espectros de tamaño-abundancia es similar, sugiriendo que tanto el nano- como el micro-fitoplancton responden de manera semejante a los cambios en las condiciones ambientales. Sólo durante la relajación del afloramiento y el establecimiento de la estratificación en las capas superiores de la columna de agua, se encuentran valores de pendientes significativamente menos negativos, indicando el aumento de la importancia de las células de mayor tamaño.

3. La relación entre la abundancia del fitoplancton y su tamaño celular en el océano Atlántico tropical y subtropical estuvo caracterizada por valores de pendientes más negativos y menos variables (los valores variaron entre -0.97 y -1.29), reflejando la dominancia de las células de menor tamaño en comparación con las aguas costeras.

4. Como resultado del incremento en el suministro de nutrientes en la

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región del afloramiento ecuatorial, fue detectado un aumento en las tasas totales de renovación del fitoplancton, lo que sugiere la limitación por nutrientes de la fisiología del fitoplancton del océano Atlántico central.

5. La respuesta del fitoplancton al afloramiento ecuatorial varió entre las diferentes clases de tamaño: mientras el nano- grande y el micro- fitoplancton mostraron mayores tasas de crecimiento, el mayor efecto estimulante del incremento en el suministro de nutrientes fue encontrado para el crecimiento del pico- y el pequeño nano-fitoplancton.

6. El uso de pequeños volúmenes de muestra puede dar lugar a subestimaciones significativas de la biomasa y producción del fitoplancton de mayor tamaño en el océano abierto oligotrófico, lo que podría tener implicaciones en los balances biogeoquímicos y en los estudios basados en estimaciones por satélite.

7. La relación entre la tasa de fijación de carbono del fitoplancton y el tamaño celular es isométrica, lo que niega la aplicación de la ley de Kleiber en el metabolismo de las comunidades naturales de fitoplancton. Los exponentes obtenidos para dicha relación (1.03-1.29) implican que las tasas de crecimiento del fitoplancton de mayor tamaño son similares a, o incluso mayores que, aquellas tasas presentadas por las células pequeñas.

8. La estructura de tamaños del fitoplancton en ecosistemas en estado estacionario como el océano Atlántico oligotrófico puede ser explicada como resultado de la relación entre la tasa metabólica y el tamaño celular.

9. Como resultado de la relación entre la tasa metabólica y el tamaño

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celular, el uso total de la energía por parte del fitoplancton es constante a través de todo el espectro de tamaños en el océano Atlántico central, sugiriendo una explicación para la persistencia de las especies de mayor tamaño en los ambientes ultraoligotróficos.

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