Tesis Soler-Hurtado.Pdf

Tesis Doctoral

Biología de la conservación de las comunidades de gorgonias tropicales en el Pacífico oriental (Ecuador)

María del Mar Soler Hurtado

Universidad Internacional Menéndez Pelayo

Madrid, 2016

Consejo Superior de Investigaciones Científicas

Museo Nacional de Ciencias Naturales

Biología de la conservación de las comunidades de gorgonias tropicales en el Pacífico oriental (Ecuador)

María del Mar Soler Hurtado

Directores:

Dra. Annie Machordom

Departamento de Biología Evolutiva, MNCN-CSIC

Dr. Pablo José López González

Departamento de Zoología, Universidad de Sevilla

Universidad Internacional Menéndez Pelayo

Madrid 2016

A mi madre

“Un científico en su laboratorio no es solamente un técnico, es también un niño colocado ante fenómenos naturales que le impresionan como un cuento de hadas”

Marie Curie

"El azar afortunado suele ser casi siempre el premio del esfuerzo perseverante"

Santiago Ramón y Cajal

“En el fondo, los científicos somos gente con suerte: podemos jugar a lo que queramos durante toda la vida”

Lee Smolin

"Mi consejo a los estudiantes de ciencia es que si desean ardientemente investigar, deberían hacerlo por todos los medios. Nada debería interponerse al deseo intenso de dedicar la vida a la Ciencia. Si tienes el anhelo de llevar a cabo investigación científica adquiere el aprendizaje preciso y por todos los medios hazlo. Difícilmente alguna otra cosa te dará tanta satisfacción y, sobre todo, tal sentido de logro"

Severo Ochoa

Agradecimientos

Intentar resumir en unas líneas todo lo que quiero expresar es muy complicado, pero me gustaría empezar diciendo que esta tesis, realizada entre Sevilla, Madrid y Ecuador, no es un trabajo único hecho por mi, es un trabajo donde he tenido la suerte de contar con un sinfín de colaboradores tanto en el terreno profesional como en el personal y a los que espero poder agradecer con hechos o con palabras toda su aportación y apoyo.

Las primeras personas a las que quiero agradecer infinitamente su esfuerzo, meticulosidad y dedicación en cada una de las líneas de esta tesis es a mis tutores. Sin ellos nunca hubiera llegado hasta aquí, he tenido la suerte de tener a dos personas “diferentes” en sus campos y por tanto, de los que he podido exprimir mucho. Pablo, me iniciaste en todo este mundo, me diste la oportunidad que buscaba y necesitaba, hiciste que me enamorara de estos bichejos y me enseñaste que hay muchas implicaciones más detrás de todo esto que la puramente científica, hay cosas que nunca se olvidarán. Annie, has sido un apoyo increíble (tanto en lo laboral como en lo personal), siempre has tenido un lugar y un momento para mi, me has enseñado mucho más de lo que crees, gracias por reñirme y a la vez por tener una paciencia infinita y por esos días eternos de trabajo donde no importaba el día o la hora. Gracías por estar siempre ahí, hasta el último minuto, las cosas nunca jamás caen en saco roto.

A lo largo de estos años he tenido la suerte de contar con dos grupos de investigación o más bien dos familias. En Sevilla he pasado la mayor parte de mi tiempo (si excluimos los eternos meses en Ecuador). Gracias a Iri y a Badii, por nuestros cafés, comidas de tape y zumitos de naranja en el momento más oportuno, por dejarlo todo para apoyarnos y desahogarnos, por ser mucho más que unas compañeras, unas amigas y por todas las ganas de reír cuando ya no podíamos más, siempre me tendréis. Espe, mi Antonia, sin tí los “coffees” no son nunca lo mismo, sabes que eres un poco la madre de todos nosotros, te guste o no, y siempre sabes decir las palabras adecuadas aunque no fueran las que queríamos oír, gracias por todo. Cesarius, creo que nunca me han dado mayores ataques de risa con nadie que contigo, gracias por apoyarme, por enseñarme, por

poder contar contigo y por tu paciencia, gracias por ese “espuminglish” que nunca olvidaré y por hacerme ver las cosas desde otro punto de vista.

A mis madrileños, a todas esas personas del Museo que siempre, por mucho tiempo que pasara fuera, me hacían sentir como en casa. Gracias a Viole, Paula e Iván, por ser unos amigos con los que contar siempre, por escucharme y ayudarme siempre en todo lo que hiciera falta, sois especiales y grandes, por dentro y por fuera, nunca lo olvidéis. A Ricardo, por ayudarme tanto y porque nos han faltado churros para desayunar entre geles y PCRs. Gracias a toda esa súper familia, Anna, Miriam, Lourdes, Patricia, “el primo”, Silvia, Virginia, César, Paloma, Jorge, Samu ... da gusto y alegría ir a trabajar y estar rodeado de gente como vosotros. Y a los “súper” como Rafa Araujo, Ignacio (Oso), Carolina, Mario, Pepe Templado, Noemí, David Buckley, Marta ... gracias por vuestro interés en este trabajo y por saber que puedo contar con vosotros.

A mis botánicos favoritos, empezando por ti Jesús, no olvidaré nunca TODO lo que has hecho por mi!. Desde el día que nos conocimos por teléfono cuando me llamaste para entrar en el Máster, hasta el último momento de esta Tesis, señor Director. Siempre quedarán las risas, las listas negras (que ya estamos en unas cuantas) y por creer en mí y demostrármelo siempre. A ti Javi, que más se puede pedir contigo?, en lo personal y lo laboral nunca he disfrutado tanto de colaborar en las mil cosas que nos enredamos (y las que nos quedan), en las horas charlando y reliándonos, y en estar cuando hiciera falta, gracias de verdad. Vladi, mi colombiano, mi amigo y mi apoyo, por muchas más cosas que nos quedan por hacer juntos y porque nos guste o no, nos conocemos demasiado bien.

A mi familia del Máster, donde me vais a permitir que resalte a dos personas. La primera para mi gorda, Ali, nuestra vida cambió un día comiendo en Reina Mercedes y desde ese momento comenzaste a ser algo indispensable en mi vida. He vivido contigo 24h durante 365 días, donde tu apoyo incondicional, tu cariño y todo lo que no puedo expresar por escrito, han estado conmigo hasta el final y sé que seguirán estando siempre, eres mucho más que una amiga. A Mica, mi hermana del otro lado del charco, sin tí esta tesis no hubiera podido ser, jamás, jamás olvidaré todo lo que has hecho por mí, esa sensación de que sólo con mirarnos a 30m de profundidad sabíamos lo que queríamos, esos muestreos sin tí hubieran sido eternos. Hemos aprendido muchísimo la una de la otra,

gracias de todo corazón por tu cariño, compresión y apoyo. A mi "parse" (mi Vale) y a mi morenito (mi Damián), porque somos el súper equipo de las gorgonias con y sin Tsunami, vivir y compartir tanto con vosotros fue increíble y lo seguirá siendo, esto no acaba aquí, ni mucho menos. A mi Almendra, Amadita y Lentalia (mis tres niñas que os adoro), a Gloria, Michael, Octavio, Chío, Javier, Silvia, Melinda, Johana, Juan Carlos, Carlitos, Melinda y esa larga y completa familia que formamos en un año juntos, todo empezó con vosotros.

A los caprelidos sevillanos, que pase el tiempo que pase os habéis interesado por cada punto y coma de este proyecto y de mi futuro. Jose gracias por tu apoyo y por estar cuando sea y donde sea siempre, nunca fallas y sé que nunca fallarás. Pili, Elena, Maca, Juanjo, Carlos... sois unos amigos de los de corazón.

Y porque la familia no se elije pero los amigos son a veces mejor que la familia, sin orden de prioridad. A mis niñas tomareñas, es increíble vuestra incondicionalidad, sea cuando sea, habéis aprendido de las “coliflores” como las que más y siempre me hacéis sentir orgullosa de lo que hago, Lore, Ro, Pelu, Rorru, Ra, Marinilla, Espe y Javi (el niño), que más deciros que no sepáis, os quiero!. Bárbara, mi gordita, porque 25 años juntas no son todavía suficientes y así estés en Barcelona o en el fin del mundo siempre presumo de hermana, siempre has estado, estás y estarás a mi lado y ahora mi ahijada, mi gran regalo. A Cristina, que te digo?, si sólo con una llamada cruzas el mapa para apoyarme y decirme “lo chica que soy y lo grande que soy”, por algo nacimos “casi” el mismo día, muchos años la una pa´la otra y los que quedan. A mi Dory, alias Bárbara, porque nunca he conseguido trabajar tanto y tan efectivamente con alguien ya sea en 10 minutos o en 24 horas, da igual lo que nos veamos o no, porque mi melli es mi melli, nunca lo olvides. A mi Manu, que cuando me mudo a Madrid se me va a tierras inglesas, pero siempre encontramos un hueco para saber que los amigos de verdad existen por muy lejos que estén, su apoyo lo sientes en tu hombro. A mi sevillana, polaca, china Izabella, cada día contigo es una aventura, unas risas y una forma de ver la vida que sólo nosotros entendemos, gracias por todo, nunca que me creo como nos conocimos y como hemos acabado. A todos sabéis que os quiero con todo mi corazón y sin vosotros no lo hubiera conseguido.

A Marco mi fotógrafo favorito, por poner ese punto de vista diferente y por ayudarme con ese ojo tuyo.

A Modesto Villamarín, por darme ese otro punto de vista de objetividad y racionalidad que tanto necesitaba y levantarme más de una vez.

A mi familia, lo más grande que tengo, lo más especial y donde puedo presumir de tener la mejor del mundo, con mis cinco sobrinas preciosas que tratan entender que hace su tita y por qué esta todo el día con la maleta a cuestas. Siempre lucháis por mí. Y en especial a mi madre, mi mejor compañera, a la cual dedico esta tesis porque no es una frase hecha, nunca lo hubiera logrado sin ella, la primera que entendió que mi futuro estaba en un viaje a mitad de la selva de Ecuador, hace ya casi 10 años. A mi tía Isi y mi tío Jose, por hacer de su casa la mía y tratarme como una hija. A toda la familia San Martín, con Juanma y Raquel a la cabeza, porque los amigos también son familia, por echarme esa mano siempre hasta el codo cuando hizo falta y hacerme sentir una más. A Oscar y Sonia, por dejarme invadir siempre esa maravillosa “Casa Pepa”.

A ti Luis, porque llegaste en la recta final y te convertiste en mi fiel compañero, cuando menos lo esperaba, porque te has comido lo peor de esta tesis y a su vez me has apoyado de una forma que nunca podría imaginar, has creído en mi como el que más, y pase lo que pase en el futuro, eso jamás lo olvidaré.

Y finalmente, a todas aquellas personas que en cada uno de mis viajes y estancias han estado a mi lado y han sido una parte fundamental de todo este proyecto. En Ecuador, Michel amigo del alma y al que admiro con todo su equipo de Exploramar Diving. Santiago siempre puedo contar contigo y todo tu apoyo. A la familia de Bienvenido, sin vosotros nunca hubiera sido tan especial esas largas horas de muestreo. En Yale, a More y Marta, mis “crownfounding”, sin conocerme me abristeis la puerta de vuestra casa y más... My special thanks to Gonzalo Giribet and Adam J. Baldinger (MCZ, Harvard University); Aude Andouche (MNHN, París); Andrew Cabrinovic and Miranda Lowe (NHM, Londres); Eric Lazo-Wasem and Lourdes Rojas (gracias amiga) (YPM, Yale), and Stephen Cairns and Herman H. Wirshing (Smithsonian, NMNH), for their support and friendship.

A TODOS y cada uno de vosotros, ¡¡¡GRACIAS!!!.

Lista de abreviaciones

• MCZ: Museum of Comparative Zoology, Harvard University, Cambridge, Boston.

• MNHN: Muséum National d’Histoire Naturelle, Paris.

• NHM: Natural History Museum, London.

• YPM: Yale Peabody Museum of Natural History, New Haven.

• NMNH (USNM): National Museum of Natural History, Smithsonian, Washington.

• MECN: Museo Ecuatoriano de Ciencias Naturales, Quito.

• BEIM: Colección de referencia del grupo de investigación “Biodiversidad y Ecología

de Invertebrados Marinos”, Universidad de Sevilla, Sevilla.

• MNCN: Museo Nacional de Ciencias Naturales (MNCN-CSIC), Madrid.

• UTI: Museo de Zoología de la Universidad Tecnológica Indoamérica, Quito.

• MZB: Museu de Barcelona, Barcelona.

Lista de publicaciones originales

Soler-Hurtado, M.M & López-González, P.J. (2012) Two new gorgonian species (Octocorallia, Gorgoniidae) from Ecuador (Eastern Pacific). Marine Biology Research, 8: 380-387. (Capítulo 4.1).

Soler-Hurtado, M.M, López-González, P.J., Muñoz, J. & Machordom, A. (2016) New records of the genera Leptogorgia, Pacifigorgia and Eugorgia (Octocorallia, Gorgoniidae) in Ecuador, with a description of a new species. Scientia Marina. Aceptado. (Capítulo 4.1).

Soler-Hurtado, M.M., Megina, C., Machordom, A., & López-González P.J. Foxed intra- and interspecific differentiation in Leptogorgia (Octocorallia: Gorgoniidae). A description of a new species through multiple evidences. Enviado. (Capítulo 4.2).

Soler-Hurtado, M.M., Machordom, A. & López-González P.J. Molecular phylogenetic relationships reveal contrasting evolutionary patterns in Gorgoniidae (Octocorallia) in the Eastern Pacific. Enviado. (Capítulo 4.3).

Soler-Hurtado, M.M & López-González, P.J. Sexual reproductive cycle of two representatives of the families Gorgoniidae and Plexauridae (Octocorallia) in Ecuadorian waters. Enviado. (Capítulo 4.4).

Soler-Hurtado, M.M., Megina, C. & López-González P.J. Biodiversity patterns of gorgonians epifaunal community in Ecuador (Eastern Pacific). Enviado. (Capítulo 4.5).

Soler-Hurtado, M.M., Sandoval-Sierra, J.V., Machordom, A. & Diéguez-Uribeondo J.. Aspergillus sydowii and other potencial fungal pathogens in gorgonian octocorals of the Ecuatorial Pacific. Coral Reef. Enviado. (Capítulo 4.6).

Otra publicación relacionada:

Soler-Hurtado, M.M. & Guerra-García, J.M. 2016. The caprellid Aciconula acanthosoma (Crustacea: Amphipoda) associated with gorgonians from Ecuador (Eastern Pacific). Pacific Science 70(1):73-82.

Índice

Summary 23 Resumen 27 1. INTRODUCCIÓN GENERAL 31 1.1. Cnidarios, evolución y clasificación 33 1.2. Biología y Ecología de los Octocorales 38 1.3. Gorgonias 41 1.4. Historia paleo-biogeográfica de las regiones del Pacífico oriental Tropical y sus arrecifes 45 1.5. Rasgos generales acerca de la problemática actual y conservación de los ecosistemas de coral 46 1.6. Área de estudio 48 1.7. Referencias 53

2. OBJETIVOS 77

3. MATERIAL Y MÉTODOS 81 3.1. Puntos de estudio 83 3.2. Análisis morfológicos 86 3.3. Análisis moleculares 92 3.4. Técnicas histológicas 95 3.5. Análisis estadísticos 97 3.6. Referncias 97

4. CAPÍTULOS 105 4.1 – Taxonomic and faunistic studies of the genera Leptogorgia, Pacifigorgia and Eugorgia (Octocorallia, Gorgoniidae) in Ecuador, with a description of a new species. 107 4.1.1 Abstract 107 4.1.2 Introduction 107

4.1.3 Materials and methods 109 4.1.4 Results 113 4.1.5 Discussion 175 4.1.6 References 179

4.2 - Foxed intra- and interspecific differentiation in Leptogorgia (Octocorallia: Gorgoniidae). A description of a new species based on multiple sources of evidence. 189 4.2.1 Abstract 189 4.2.2 Introduction 189 4.2.3 Materials and methods 192 4.2.4 Results 198 4.2.5 Discussion 210 4.2.6 References 214

4.3 - Molecular phylogenetic relationships reveal contrasting evolutionary patterns in Gorgoniidae (Octocorallia) in the Eastern Pacific. 225 4.3.1 Abstract 225 4.3.2 Introduction 225 4.3.3 Materials and methods 228 4.3.4 Results 236 4.3.5 Discussion 243 4.3.6 References 251

4.4 – Sexual reproductive cycle of two representatives of the families Gorgoniidae and Plexauridae (Octocorallia) in Ecuadorian waters. 261 4.4.1 Abstract 261 4.4.2 Introduction 261 4.4.3 Materials and methods 264 4.4.4 Results 266 4.4.5 Discussion 276

4.4.6 References 282

4.5- Biodiversity patterns of gorgonians epifaunal community in Ecuador (Eastern Pacific). 295 4.5.1 Abstract 295 4.5.2 Introduction 296 4.5.3 Materials and methods 298 4.5.4 Results 301 4.5.5 Discussion 311 4.5.6 References 316

4.6 - Aspergillus sydowii and other potential fungal pathogens in gorgonian octocorals of the Ecuadorian Pacific. 329 4.6.1 Abstract 329 4.6.2 Introduction 329 4.6.3 Materials and methods 331 4.6.4 Results 336 4.6.5 Discussion 340 4.6.6 References 344

5. DISCUSIÓN FINAL 351 5.1 Referencias 367

6. CONCLUSIONES 385

7. CONCLUSIONS 387

8. APPENDICES 389

Summary

Within the benthic marine organisms zoological groups, located in the tropical waters of both of the American Continent’s coastlines, one of the groups that has the highest endemism is that of the gorgonians (Cnidaria: Alcyonacea: Octocorallia). These organisms represent an important challenge from both taxonomic and phylogenetic viewpoints. Although geographic areas, such as the Ecuadorian littoral, are famous for their global biodiversity, it is important to emphasise that there is a great lack of information about these areas’ invertebrate species inhabitants. For this reason, it has becomed essential to work towards completing information containing different aspects such as diversity, evolution, reproductive biology, ecology and possible diseases.

With this in mind, the principal goal of the present PhD thesis was to improve the knowledge of the gorgonian communities (Gorgoniidae) through diverse perspectives and multidisciplinary methodologies (morphology, morphometry, histology and molecular systematics) in the unexplored area of continental Ecuador, located within the context of the Eastern Pacific Ocean eco-regions.

According to our results, by means of faunistic and taxonomic studies of the three genera, Eugorgia, Leptogorgia and Pacifigorgia (Gorgoniidae) present in the Ecuadorian littoral, fourteen species have been identified, constituting to seven of them being new records for Ecuador. Furthermore, through the use of classical morphological taxonomy and integrative taxonomy framework with the incorporation of morphometric and molecular characters, in this study have been described four new species, E. ahorcadensis, L. mariarosae, L. manabiensis and P. machalilla. This achievement has verified the usefulness of searching for new characters in the definition and identification of the studied species in question.

Furthermore, the study of phylogenetic relationships of found known organisms, including their closest conspecific species from the oriental Pacific, demonstrated that: 1) The combination of mitochondrial markers (mtMutS and Cox) and nuclear (ITS and 28S) provided the greatest resolution of the evolutionary relationships among these organisms, whereas each molecular marker individually analysed resolved at a different taxonomic

level; 2) Among the studied genera of Gorgoniidae, Pacifigorgia showed a monophyletic profile, unlike in the case of Leptogorgia and Eugorgia, who integrated together int0 the same clade; 3) Different evolutionary processes were proposed for Pacifigorgia species, such as an explosive radiation with a recent diversification, or a possible ancient hybridisation with a subsequent genomic re-homogenisation; 4) And finally, for Leptogorgia species complex, reticulate evolution and/or incomplete lineage sorting processes were proposed to explain the shown patterns.

Within the wide variability of reproductive cycles present in octocorals, the two representative species studied here showed the same reproductive model but with some differential characteristics. It was observed that the species Leptogorgia alba and Muricea plantaginea were gonochoric, broadcast spawners, without a specific annual synchronisation, nor any mechanism of oocytes retention, or even possible larvae (mucus) in the proximity of female colonies. Although the broadcast process was not seen to be directly influenced by the environmental temperature, there was indication that they were possibily influenced by water currents, mainly by the Humboldt Current.

Throughout this Thesis, the effectiveness of gorgonians along the Ecuadorian littoral as benthic heterogeneity bio-constructors has been demonstrated. The greatest abundance of fauna related to these hosts has reached the highest so far recorded values in gorgonians, scleractinians and other “ecosystem engineers” benthic groups. Significant differences were found in: the associated community structure; the abundance; and in the diversity between different studied gorgonian species. These differences appear to be related to the host’s morphology. Moreover, seasonal differences suggested the importance of the Humboldt Current’s influence and Panama Current’s fluctuations in the spatio- temporal modelling of these associated communities. Also, a decrease in the epifauna diversity coupled with an increase of the available gorgonian host space was observed. This finding was contrary to “the heterogeneity of habitat” hypothesis.

Finally, a diverse fungi community, associated with study area’s two species of gorgonians was identified. Some of these organisms have never had been recorded before in these ecosystems. In this fungi community, some pathogenic fungi, such as Aspergillus

sydowii, were detected on healthy individuals. Such circumstances may lead to the development of several diseases, for example the proven lethal Aspergillosis. Thus gorgonians species studied in this thesis could be carriers of these potential pathogens.

Through this thesis, novel information through different studies encompassing the multidisciplinary integrative perspective has been provided. Thus, the present thesis provides a solid basis for future investigations in the areas of biodiversity, conservation and management of these tropical ecosystems.

Resumen

Uno de los grupos zoológicos del bentos marino con mayor endemicidad en aguas tropicales, en ambas costas del continente americano, lo constituyen las gorgonias (Cnidaria: Alcyonacea: Octocorallia). Estos organismos representan un importante reto desde el punto de vista tanto de la taxonomía, así como de sus relaciones filogenéticas. Además, en áreas geográficas como el litoral de Ecuador, reconocido por su amplia biodiversidad global, es importante destacar la gran falta de información existente sobre estas especies. Por eso se convierte en algo esencial completar su conocimiento en varios aspectos, como su diversidad, evolución, biología reproductiva, ecología o posibles enfermedades.

Conscientes de esto, el objetivo principal de la presente Tesis fue mejorar el conocimiento sobre las comunidades de gorgonias (familia Gorgoniidae), a través de diversos enfoques y metodologías multidisciplinares (morfología, morfometría, histología o sistemática molecular), en un área tan inexplorada como es Ecuador continental, en el contexto biogeográfico del Pacífico oriental.

Acorde a nuestros resultados, por medio del estudio faunístico y taxonómico de los tres géneros, Eugorgia, Leptogorgia y Pacifigorgia (Gorgoniidae) presentes en la costa de Ecuador, se identificaron catorce especies, de las cuales siete representaban nuevos registros para Ecuador. Además, a través de una taxonomía morfológica clásica o bien por medio de una taxonomía integradora, con la incorporación de análisis morfométricos y moleculares, durante la realización de esta memoria se describieron cuatro nuevas especies para la ciencia, E. ahorcadensis, L. mariarosae, L. manabiensis y P. machalilla, comprobándose la utilidad de la búsqueda de nuevos caracteres en la delimitación e identificación de las especies bajo estudio.

Por otro lado, el estudio de las relaciones filogenéticas de los organismos encontrados, incluyendo a sus congéneres más cercanos del Pacífico oriental mostró que: 1) Los resultados obtenidos para cada uno de los marcadores moleculares utilizados ayudaban a la resolución filogenética de los diferentes grupos de especies estudiados a diferentes niveles taxonómicos y por ello, la combinación de marcadores mitocondriales

(mtMutS y COX) y nucleares (ITS y 28S) proporcionaba la mayor resolución de la relaciones evolutivas entre estos taxones; 2) Dentro de los géneros de Gorgoniidae estudiados, Pacifigorgia mostró un perfil claramente monofilético, mientras que los géneros Leptogorgia y Eugorgia se integran en un mismo clado; 3) Diferentes patrones evolutivos fueron propuestos para las especies de Pacifigorgia, como un proceso de radiación explosiva con una diversificación muy reciente o una posible antigua hibridación con una posterior re-homogeneización del genoma; 4) Finalmente, para un complejo de especies de Leptogorgia se pudieron observar posibles procesos de evolución reticulada o resolución incompleta de linajes.

Entre el amplio espectro de ciclos reproductivos presentes en octocorales, las dos especies representativas estudiadas, mostraron el mismo modelo de reproducción con ciertas características diferenciales. Se observó que las especies Leptogorgia alba y Muricea plantaginea eran gonocóricas y emitían sus gametos al medio (broadcast spawners) sin una sincronización anual determinada, y no existiendo aparentemente ningún mecanismo de retención de ovocitos o posibles larvas (mucosidad) en las proximidades de las colonias femeninas. Los procesos de emisión no parecieron tener una relación directa con la temperatura ambiental, pero sí con las corrientes predominantes de la zona, como la corriente de Humboldt.

A través de esta Memoria se ha demostrado la efectividad de las gorgonias del litoral de Ecuador como bio-constructoras de la heterogeneidad bentónica. Se observó una hiper- abundancia de fauna asociada a estos hospedadores, alcanzando los mayores valores hasta ahora registrados en gorgonias y escleratinios u otros grupos bentónicos considerados también “ingenieros ecosistémicos”. Además, se encontraron diferencias significativas en la estructura de la comunidad asociada, abundancia y diversidad entre las especies de gorgonias estudiadas, aparentemente relacionadas con la propia morfología del organismo hospedador. Por otro lado, las diferencias estacionales encontradas sugieren la importancia de la influencia de la corriente de Humboldt y la posterior incursión de la corriente de Panamá en el modelado espacio-temporal de estas comunidades asociadas. También se observó que, con el aumento del espacio habitable en la gorgonia, se producía una disminución de la diversidad de la epifauna, lo que parece oponerse a la hipótesis de

heterogeneidad del hábitat.

Finalmente, se identificó una diversa comunidad fúngica asociada a dos especies de gorgonias del área de estudio. Algunos de estos organismos no habían sido registrados anteriormente en estos ecosistemas. Así mismo, se detectaron hongos patógenos tales como Aspergillus sydowii en individuos sanos, lo que podría favorecer el desarrollo de enfermedades como la conocida aspergilosis, que podría estar latente en este área, siendo las especies de gorgonias estudiadas portadoras de estos potenciales patógeneos.

Esta Tesis proporciona una base sólida para futuras investigaciones en el campo de la biodiversidad, conservación y gestión de estos ecosistemas tropicales, proporcionando nuevos datos, a través de los diferentes estudios englobados en un enfoque integrador multidisciplinario.

1. INTRODUCCIÓN GENERAL

Introducción general

El tema fundamental de esta memoria versa sobre el conocimiento de la diversidad de gorgonias y su papel en los ecosistemas litorales de Ecuador. Por ello, en esta introducción se pretende dar una visión general de su contexto taxonómico y biogeográfico, acerca de todos los aspectos relacionados con la identificación, así como del marco evolutivo gracias a los estudios filogenéticos llevados a cabo en las especies de la familia Gorgoniidae, con especial hincapié en la zona de estudio, el Pacífico oriental. A través de los diferentes capítulos de la presente Tesis, se tratarán temas relacionados con la taxonomía, la biología, las relaciones filogenéticas y la ecología de estos organismos.

1.1. Cnidarios, evolución y clasificación

El filo Cnidaria comprende aproximadamente unas 10.000 especies de organismos relativamente simples desde el punto de vista de su estructura corporal, en el que se incluyen corales, hidroides, medusas, anémonas y abanicos de mar (Daly et al. 2007). Estos organismos son frecuentes y abundantes en ambientes marinos, aunque también hay un porcentaje muy reducido que habitan en agua dulce (Gooderham & Tsyrlin 2002). Los primeros registros fósiles de cnidarios están presentes desde el Cámbrico temprano, donde se produce un evento único de intensa diversificación biológica, conocida como “radiación cámbrica” (Cartwright & Collins 2007; Liñán & Gámez-Vintated 1999).

En términos generales, los Cnidarios son metazoos diblásticos, con una epidermis (originada a partir del ectodermo), una gastrodermis que tapiza la cavidad gastrovascular (originada a partir del endodermo), y entre ambas capas se encuentra la mesoglea, la cual, en antozoos, presenta elementos celulares de origen ectodérmico o endodérmico (Harrison 1991), aunque puede ser acelular (e.g. hidrozoos). La gastrodermis delimita una cavidad gastrovascular en forma de saco que se comunica con el exterior a través de una abertura única, que sirve al mismo tiempo de boca y ano y actúa en los procesos de circulación, digestión y distribución de nutrientes (Brusca & Bruca 2005; Harrison 1991). Los cnidaros poseen un sistema nervioso sencillo (difuso y no centralizado) donde, a nivel ultra-

estructural, muchas de sus neuronas pueden ser multifuncionales, organizadas en una red nerviosa con puntos de interacción (Grimmelikhuijzen & Westfall 1995; Satterlie 2011). Poseen una estructura y organización celular consideradas como una de las líneas más primitivas dentro de los metazoos (Harrison 1991; Marshall & Williams 1985).

Además de la conocida diversidad morfológica del grupo, estos relativamente simples metazoos, se caracterizan por la posesión de cnidocistos (Collins 2002). De los tres tipos de cnidocistos (nematocistos, pticocistos y espirocistos) (Mariscal 1974), sólo los nematocistos se encuentran en todo el filo. Los cnidocistos son productos elaborados de secreción intracelular, están formados por una cápsula que contiene un filamento empaquetado, que se dispara al recibir un estímulo químico y/o mecánico apropiado, y cuyas funciones en general pueden ser ofensivas o defensivas (Anderson & Bouchard 2009; Fautin & Mariscal 1991; Mariscal 1974). Otras tres características que han sido consideradas como diagnósticas para el filo Cnidaria son: la simetría radial, una larva plánula y poseer en el desarrollo una etapa pólipo (Ball et al. 2004; Harrison 1991). No obstante, esto no es generalizable (Daly et al. 2007): aunque algunos cnidarios presentan simetría radial (Zapata et al. 2015), algunos sifonóforos (hidrozoos) poseen asimetría direccional (Dunn & Wagner 2006), y muchos otros una organización birradial o bilateral, llevando a algunos autores a concluir que la simetría bilateral es la condición ancestral para el filo (Daly et al. 2007; Dunn & Wagner 2006; Salvini-Plawen 1987). De esta forma se considera que los cnidarios pueden aportar información sobre la evolución del plano corporal de metazoos, tanto por lo que comparten con los bilaterales, como por lo que no (Barfield et al. 2016; Finnerty 2001; Nielsen 1995).

Acorde con los estudios realizados sobre la evolución de los metazoos, los poríferos aparecerían como grupo hermano del clado de los eumetazoos, dentro del cual los cnidarios junto con los ctenóforos (como representantes de los celentéreos), quedarían relacionados filogenéticamente con los bilaterales como su grupo hermano (Fig. 1) (Barfield et al. 2016; Edgecombe et al. 2011; Finnerty 2001; Vargas & Zardoya 2012). Sin embargo, es cierto que todavía existe cierta controversia acerca de estas relaciones filogenéticas basales de los metazoos (Edgecombe et al. 2011; Schierwater et al. 2016).

Figura 1. Imagen ilustrativa sobre las relaciones filogenéticas entre Bilateria y Cnidaria, dentro de los metazoos, sobre la base de estudios filogenéticos moleculares, recogidos en los trabajos de Edgecombe et al. (2011) (izq.) y Philippe et al. (2009) (dcha.).

El filo Cnidaria posee un complejo ciclo de vida, incluyendo un proceso de metagénesis (alternancia de generaciones), donde una larva tipo plánula, producida sexualmente, metamorfosea en una fase pólipo bentónica sésil, que puede reproducirse asexualmente formando colonias (e.g. Anthozoa, Hydrozoa), o en las formas más derivadas del filo (Medusozoos) adquirir además una fase medusoide (Daly et al. 2007). Según las variaciones en la presencia y dominancia de las diferentes fases, polipoide y medusoide en el ciclo de vida (Fig. 2), y cuestiones anatómicas, los cnidarios tradicionalmente han sido divididos en cuatro clases (Anthozoa, Scyphozoa, Hydrozoa y Cubozoa) (Collins et al. 2005; Finnerty 2001).

Figura 2. Imagen tomada del trabajo de Finnerty (2001) donde se puede observar, esquemáticamente, las alternancias de las formas pólipo y medusa, en la evolución de Cnidaria, acorde al grupo taxonómico al que pertenezcan.

Sin embargo, en términos de relaciones evolutivas, el filo Cnidaria se divide en dos grandes clados, Anthozoa y Medusozoa (Scyphozoa, Staurozoa, Hydrozoa y Cubozoa) (Fig. 3) (Collins 2002; Collins et al. 2006; Marques & Collins 2004). La distinción entre Anthozoa y Medusozoa está bien apoyada tanto por su anatomía, como por su ciclo de vida (Daly et al. 2007; Bridge et al. 1995; Salvini-Plawen 1978). Además, ambos clados muestran una notable variación en su ADN mitocondrial (ADNmt), tanto en el contenido de genes, como en la organización de la estructura del genoma: en Anthozoa (hexa- y octocorales) se encuentra organizado en una sola molécula circular, mientras que en Medusozoa, el ADNmt consta de una o varias moléculas lineales (Bridge et al. 1992; Ender & Schierwater 2003; Kayal et al. 2012).

El clado Anthozoa se considera el miembro basal de filo, ya que el ciclo de vida- pólipo que poseen sus integrantes, la estructura circular del ADN mitrocondrial y la estructura del cilio en los cnidocistos representan las condiciones primitivas para los cnidarios (Marques & Collins 2004; Bridge et al. 1995; Odorico & Miller 1997). Los integrantes de este clado pueden reproducirse tanto sexual como asexualmente (Finnerty 2001) y pueden ser coloniales o solitarios (Collins et al. 2005), sin esqueleto o con un

esqueleto mineral y/o proteico. Además, dentro de la clase Anthozoa (literalmente “animales de flores”), la cual comprende unas 7.500 especies, podemos diferenciar dos linajes monofiléticos, Octocorallia y Hexacorallia (Daly et al. 2007; Zapata et al. 2015).

Figura 3. Ilustración tomada del estudio realizado por Collins et al. (2006), donde se observan las posibles relaciones filogenéticas dentro del filo Cnidaria.

Sin embargo, existe una controversia sobre la consideración del clado como entidad natural. Reconstrucciones filogenéticas recientes, basadas en los genomas mitocondriales completos, han sugerido que Anthozoa es un grupo parafilético, siendo Octocorallia el grupo hermano de Medusozoa y no de Hexacorallia (Kayal & Lavrov 2008; Kayal et al. 2012; Lavrov et al. 2008; Park et al. 2012; Shao et al. 2006). Esta observación no está de acuerdo con la clasificación morfológica actual y con las reconstrucciones filogenéticas basadas en marcadores nucleares, que apoyan la naturaleza de Anthozoa (Octocorallia + Hexacorallia) como un clado monofilético (Berntson et al. 1999; Daly et al. 2007; France et al. 1996; Odorico & Miller 1997; Won et al. 2001). Figueroa & Baco (2015) recogen estas diferentes hipótesis y recomiendan que la reconstrucción filogenética a niveles taxonómicos superiores dentro de Cnidaria, debe estar apoyada por el uso de genes nucleares, incluso cuando los genomas mitocondriales completos estén disponibles. Finalmente, el reciente trabajo basado en análisis filogenéticos con la combinación de genomas y transcriptomas,

publicado por Zapata et al. (2015), confirma la división de los Cnidarios en dos clados monofiléticos, Anthozoa, con Octocorallia y Hexacorallia (monofiléticos a su vez), y el clado Medusozoa que estaría compuesto por Staurozoa, Cubozoa, Hydrozoa y Scyphoza.

1.2. Biología y Ecología de los Octocorales

La subclase Octocorallia está formada por las formas estolonadas, los corales blandos, gorgonias (abanicos y látigos de mar), plumas de mar y los corales azules. Actualmente incluye aproximadamente unas 3.000 especies conocidas, de las cuales la gran mayoría son coloniales (Daly et al. 2007). Estos organismos poseen una amplia distribución geográfica, asociada a zonas de alta productividad y una igualmente amplia tolerancia ambiental (Pérez 1999; Silva & Pérez 2002). Se encuentran desde el Ártico hasta el océano Antártico, en diferentes densidades poblacionales desde zonas someras, incluso intermareales, hasta profundidades abisales (Bayer 1981; Figueroa & Baco 2015; Morris et al. 2012; Sánchez 2007; Stock 2004). Su zonación, abundancia, distribución, riqueza de especies, forma y tamaño, están influenciados por factores como la iluminación, el régimen hidrodinámico, la presencia e inclinación de sustrato de fijación, la profundidad y la sedimentación (Botero 1987; Goldberg 1973; Preston & Preston 1975; Rey-Villiers & Alcolado 2012; Sánchez et al. 1998). Los octocorales son relativamente resistentes al fenómeno del blanqueamiento o la expulsión de sus simbiontes (Drohan et al. 2005; Lasker et al. 2003), uno de los principales problemas que afectan a los arrecifes de hexacorales.

Los octocorales constituyen uno de los grupos bentónicos más abundantes en las comunidades marinas (Brito 1993; Silva & Pérez 2002), donde representan importantes componentes estructurales, ya que aportan a los arrecifes una gran cantidad de carbonato de calcio en forma de espículas calcáreas (Bayer 1961; Opresko 1973) y constituyen refugio para numerosas especies (Kumagai 2008; Pérez 1999; Silva & Pérez 2002). Estos organismos pueden crecer de forma individual, como colonias aisladas, o formando parches o jardines de coral (McFadden et al. 2010). Las colonias poseen un gran número de formas que van desde incrustantes, filiformes y membranosas, hasta complejas y elaboradas arquitecturas ramificadas (Silva & Pérez 2002). Sin embargo, algunas de las características morfológicas

pueden ser modificadas por el ambiente y la interpretación de las morfologías puede ser confusa cuando es un carácter utilizado en algunas identificaciones (Breedy & Guzmán 2007; Sánchez et al. 2004, 2007; Vargas et al. 2010a).

Como su nombre sugiere, los octocorales poseen un carácter invariable dentro del grupo, como es la presencia de elementos (tentáculos, mesenterios) siguendo una simetría octorradiada (mostrada en general a nivel de mesenterios) (Daly et al. 2007; Miyazaki & Reimer 2014). La presencia de pínulas (extensiones laterales) en los tentáculos, también se considera diagnóstico, aunque este carácter puede no estar presente en varios taxones (Alderslade & McFadden 2007; Daly et al. 2007) (Fig. 4). El pólipo es el zooide individual en octocorales, pudiendo ser monomórficos (sólo hay un tipo de pólipo, denominado autozooide), dimórfico (dos tipos, autozooides y siphonozooides), o presentarse situaciones más complejas. Hasta cinco tipos de pólipos han sido descritos en octocorales, y suelen asumir diferentes funciones en la colonia (alimentación, reproducción sexual, reproducción asexual, soporte hidrostático de la colonia, flujo y control de la presión hidrostática en el interior de la colonia, etc.) (Bayer 1973; Breedy et al. 2009; Fabricius & Alderslade 2001; McFadden et al. 2010; Williams et al. 2012). En las formas monomórficas el pólipo (autozooide) asume todas las funciones vitales, la captura de los alimentos, el transporte de nutrientes, la excreción, la defensa y la reproducción (Bayer 1973; Sánchez et al. 2004).

Otro de los puntos esenciales a la hora de hablar de estos organismos, es conocer su biología reproductiva. Aunque en la mayoría de estas especies las estructuras reproductivas se encuentran separadas en colonias masculinas y femeninas, mostrándose como organismos gonocóricos (Kahng et al. 2011), estudios previos muestran la gran diversidad de patrones reproductivos que pueden mostrar los octocorales (Coma et al. 1995; Kahng et al. 2008, 2011; Orejas et al. 2007; Quintanilla et al. 2013; Rossi & Gili 2009). De hecho, algunas especies pueden ser hermafroditas (Achituv & Benayahu 1990), como por ejemplo Heteroxenia coheni Verseveldt, 1974, o incluso el caso de Heteroxenia elizabethae Kölliker, 1874, una especie que se muestra gonocórica en la Gran Barrera de Coral, pero hermafrodita en el Mar Rojo (Goffredo et al. 2010; Hwang & Song 2007). Además, presentan variación en la duración de su ciclo reproductivo, fertilización interna, externa, o

externa permaneciendo en la superficie de la colonia, o diferencias en el tamaño de los productos reproductivos (Kahng et al. 2011).

Figura 4. Imágenes del detalle de los tentáculos en los pólipos de octocorales, con y sin pínulas, tomadas de Alderslade & McFadden (2007), van Ofwegen et al. (2010) y Williams (2013).

Por otro lado, la clasificación taxonómica actual usada para Octocorallia fue propuesta por Bayer (1981), donde se divide al grupo en tres grandes órdenes: Helioporacea (corales azules), Alcyonacea (estolonados, corales blandos y gorgonias) y Pennatulacea (plumas de mar) (Bayer 1981; Daly et al. 2007; Fabricius & Alderslade 2001; McFadden et al. 2010). En este contexto, los caracteres asociados con los elementos esqueléticos y la forma de las colonias, han sido ampliamente utilizados en la identificación y clasificación de los diferentes niveles taxonómicos (Bayer 1956; Fabricius & Alderslade 2001; McFadden et al.

2010). Sin embargo, las descripciones antiguas de las especies dentro del grupo son problemáticas (Bayer 1981), y en ocasiones esto se agrava al carecer de ilustraciones informativas detalladas (Breedy & Guzmán 2011; Morris et al. 2012; Sánchez 2007). Además, las relaciones filogenéticas dentro de Octocorallia son discutibles, sobre todo en los niveles taxonómicos más altos, siendo uno de los grupos menos comprendidos (McFadden et al. 2006, 2010; Vargas et al. 2014).

El orden Alcyonacea Lamouroux, 1812 reúne unas 30 familias de formas estolonadas, masivas, ramificadas y flageliformes (Bayer 1981; Fabricius & Alderslade 2001). Sus especies habitan en aguas de todo el mundo, con una amplia distribución batimétrica, y a las cuales se puede distinguir principalmente por una estructura colonial, la presencia o ausencia de un eje, por su composición axial, y características del escleroma (distribución, tipos, etc.) (Daly et al. 2007; Fabricius & Alderslade 2001).

1.3. Gorgonias

El nombre “gorgonia” carece actualmente de categoría taxonómica, y se utiliza para denominar aquellos octocorales que contienen en general una morfología colonial ramificada, aunque también pueden presentar una forma de “látigos de mar” como, por ejemplo, las especies de los géneros ellisélidos Viminella Gray, 1870, o Junceella Valenciennes, 1855.

Estudios previos, como los realizados por Bayer (1961) y Sánchez et al. (2003, 2007) muestran a las gorgonias como uno de los grupos de organismos marinos bentónicos de mayor endemicidad en las aguas tropicales de ambas costas de América (Atlántico y Pacífico). Son los cnidarios más abundantes en los arrecifes tropicales del Atlántico occidental, al igual que en los arrecifes del Pacífico oriental y en el Caribe (Sánchez & Wirshing 2005), a diferencia de los organismos escleractinios y corales blandos, que dominan los arrecifes del Indo-Pacífico (Fabricius & Alderslade 2001).

La variabilidad morfológica dentro de gorgonias, así como su patrón de ramificación, puede presentar una cierta y reconocida plasticidad fenotípica (Brazeau &

Lasker 1990; Gori et al. 2012; Manrique-Rodriguez et al. 2006; Sánchez et al. 2007).

Factores como el estrés provocado por el oleaje influyen en el crecimiento de estos organismos; sin embargo, otros aspectos como su flexibilidad y su orientación respecto a las corrientes (que pueden incluso modificar durante su crecimiento), les permiten ciertas ventajas en su desarrollo y alimentación (Brazeau & Lasker 1988; Goh et al. 1999; Graus et al. 1977; Ryland & Warner 1986; Wainwright & Dillon 1969). Procesos como la capacidad competitiva (Jackson 1979; Kaufmann 1973), o el balance entre la disponibilidad de alimento y la tasa metabólica (Lasker et al. 2003), han demostrado afectar a la morfología colonial (Brazeau & Lasker 1990; Manrique Rodríguez et al. 2006).

Las gorgonias contribuyen con su presencia a incrementar la biodiversidad general del arrecife. Al ser organismos sésiles de mediano a gran tamaño, colaboran en la complejidad del sistema arrecifal, por lo que son conocidos como “ingenieros ecosistémicos” (Cerrano et al. 2000; Jones et al. 1994), sirviendo de lugar de refugio, alimentación o reproducción, a un sinnúmero de especies (Arntz W 1999; Coma et al. 1995; Kinzie 1973; Tsounis et al. 2006). Además, pueden suponer una fuente importante de alimento (o un lugar donde encontrarlo) para la epifauna que vive en su superficie (Buhl- Mortensen & Mortensen 2004; Plaisance et al. 2011; Wendt et al. 1985).

En los dos últimos siglos, se han realizado una gran cantidad de aportaciones taxonómicas a la fauna de octocorales y concretamente de gorgonias en aguas del Pacífico oriental, incluyendo entre los autores a Valenciennes (1846, 1855), Milne-Edwards & Haime (1850, 1857), Horn (1860), Duchassaing & Michelotti (1864), Verrill (1864, 1866, 1868a,b, 1870), Bayer & Macintyre (2001), Bayer (1951, 1961, 1981), Bielschowsky (1918, 1929), Kükenthal (1919, 1924), Hickson (1928), Stiasny (1941, 1943), Horn (1860), Prahl et al. (1986), Breedy & Guzmán (2002, 2003a; b, 2004, 2005, 2007, 2013), Breedy (2001), Breedy et al. (2009b, 2012), Williams & Breedy (2004), Horvath 2011), etc. Quizás las contribuciones más significativas fueron las realizadas por autores como Verrill o Bayer. Sin embargo, posiblemente debido al mayor desarrollo de técnicas como la microscopía óptica y SEM (Sánchez & Wirshing 2005) y a las facilidades de acceso a las colecciones de referencia depositadas en los principales museos, el número de publicaciones acerca de estas especias

ha aumentado casi exponencialmente en las últimas dos décadas, gracias en gran parte al esfuerzo revisionario de autores como Breedy y Guzmán.

Entre el amplio grupo de gorgonias, encontramos la familia Gorgoniidae, objeto de esta memoria, la cual comprende 19 géneros y unas 300 especies, siendo una de las familias más diversas dentro del grupo de los octocorales (Sánchez 2007) (Fig. 5). En términos generales, los individuos de esta familia se caracterizan por poseer pólipos monomórficos, retráctiles, un eje (axis) de gorgonina, con calcificaciones en los espacios intersticiales (Jeyasuria & Lewis 1987), rodeado por un delgado cenénquima con la solenia y escleritos cuya ornamentación es uniforme y simétrica (Bayer 1953, 1961; Bayer et al. 1983; Daly et al. 2007; Grasshoff & Alderslade 1997).

Figura 5. Algunos ejemplos de la variabilidad morfológica colonial mostrada en representantes de la familia Gorgoniidae, en este de caso, de Ecuador.

En la actualidad, son reconocidos cuatro géneros de la familia Gorgoniidae en el Pacífico oriental: Phycogorgia Milne Edwards y Haime, 1850, actualmente incluyendo una única especie, Phycogorgia fucata (Valenciennes, 1846); Pacifigorgia Bayer 1951, con 36 especies conocidas, todas ellas restringidas a la costa americana del Pacífico, excepto

Pacifigorgia elegans (Milne Edwards & Haime 1857) procedente de Brasil; Leptogorgia Milne Edwards y Haime, 1857, el más abundante y cosmopolita, con aproximadamente 30 especies descritas para el Pacífico oriental; y Eugorgia, Verrill 1868, el cual es considerado exclusivo del Pacífico oriental, con 18 especies conocidas (Breedy & Guzmán 2002, 2003b, 2007, 2013; Breedy et al. 2009, 2013).

1.4. Historia paleo-biogeográfica de las regiones del Pacífico oriental tropical y sus arrecifes Las masas de tierra no son las únicas barreras existentes para la dispersión de los organismos marinos del Pacífico (e.g. estrecho de Panamá). Para las especies sedentarias de aguas poco profundas, existen otras barreras marinas como, por ejemplo, la Barrera del Pacífico oriental que está formada por 5.000 kilómetros de extensión de agua profunda, y ha separado las faunas oceánicas del Pacífico central y oriental, desde el inicio del Cenozoico (Lessios 2012). Al final del Cretácico (o principios del Terciario), la conexión entre el Caribe y las regiones del Pacífico oriental aún persistían y, por tanto, la fauna concerniente a los arrecifes de coral era común (Vermeij 1978). Sin embargo, durante la época del Plioceno un evento tectónico conduce a la separación del Pacífico oriental y las regiones del Atlántico occidental (Cowman & Bellwood 2013). Por medio del levantamiento del istmo centroamericano, un proceso que se desarrolló durante 12 millones de años (Ma), completado hace 2.8 Ma (Coates et al. 2005; Lessios 2008), y que produjo el aislamiento de ambas regiones. Este evento tuvo consecuencias oceanográficas fundamentales, así como cambios en el clima e importantes implicaciones biológicas (Coates et al. 1992). La aparición del istmo centroamericano afectó al flujo de corriente, salinidad, temperatura, y productividad primaria del Pacífico y el Atlántico, y lanzó organismos marinos de los dos océanos en trayectorias evolutivas independientes (Lessios 2008). Además, se crearon las condiciones apropiadas para la generación de eventos como “El Niño”, dando lugar a alteraciones debidas a aumentos bruscos de la temperatura en el Pacífico oriental, como veremos más adelante. Por otro lado, dieron comienzo eventos de surgencia costera “upwelling”, con movimientos surgentes de aguas frías, que se producían cíclicamente

(Cortés 1997). Este aumento de los nutrientes superficiales ha sido mayor en el Pacífico que en el Atlántico en los últimos 5 Ma y aumenta constantemente desde entonces (Cannariato & Ravelo 1997; Lessios 2008). La corriente cálida superficial Circumtropical que fluía hacia el oeste a través de la vía marítima fue bloqueada (Kameo & Sato 2000) y la corriente del Atlántico Norte Ecuatorial fue desviada hacia el norte, intensificándose el flujo de la corriente del Golfo (Berggren & Hollister 1974; Burton et al. 1997; Lessios 2008). Tales regímenes térmicos y cambios del nivel del mar, no eran propicios para el crecimiento y el desarrollo de los corales presentes en los arrecifes (Cortés 1997), lo que dio lugar a una serie de extinciones de estos organismos en el Pacífico oriental (Guzmán & Cortés 1993). En general, la fauna coralina del Pacífico oriental, ha recibido gran atención de los zoogeógrafos. De hecho, varios estudios sugieren que la fauna en este área derivó del Pacífico occidental (Glynn & Wellington 1983) y central (Glynn 2001; López-Pérez 2005) por medio de la colonización larvaria a través de los movimientos de las corrientes oceánicas (Glynn & Ault 2000; Glynn 1997; Grigg & Hey 1992). Por otro lado, se ha observado un conjunto de corales endémicos descendientes de faunas del océano Atlántico, junto con especies que se han originado en el Pacífico de manera independiente (Reyes-Bonilla & Bárranza 2003; Veron 1995). En la actualidad, el proceso de colonización ha continuado, siendo especialmente intenso el transporte de larvas en los años donde se presenta el fenómeno del Niño (Glynn 2001; Reyes-Bonilla & Bárranza 2003). De esta forma, el continente americano presenta hoy día tres principales áreas arrecifales: Brasil, Caribe y Pacífico oriental (Cortés 2003). Los arrecifes del Pacífico oriental se extienden desde el mar de Cortés (Golfo de California) hasta Chile, incluyendo Ecuador, y desde Colombia al oeste de la isla de Clipperton (Cortés 2003; Fabricius 2005).

1.5. Rasgos generales acerca de la problemática actual y conservación de los ecosistemas de coral

Aunque se han realizado importantes avances científicos en las ciencias del mar en

las últimas décadas, los procesos que controlan la distribución, estructura y funcionamiento de la diversidad biológica en el medio marino, así como los efectos de su deterioro deben aún ser estudiados con mayor detalle (ver Bianchi & Morri 2000; Bremner et al. 2003; Cheung et al. 2009; Edgar et al. 2008; Occhipinti-Ambrogi & Savini 2003; Roberts et al. 2002). Como es generalmente conocido, el aumento de la frecuencia y el impacto de las perturbaciones antropogénicas han provocado la pérdida de diversidad y cambios en el funcionamiento de una amplia gama de ecosistemas marinos (Edwards & Richardson 2004; Halpern et al. 2008; Hoegh-Guldberg & Bruno 2010; Obura & Grimsditch 2009).

Uno de los ecosistemas más amenazados es el de los arrecifes coralinos (Roff & Mumby 2012) debido, entre otras razones, a su propia biología: la mayoría de las especies muestra un crecimiento lento, una madurez tardía y una baja fecundidad (Musick 1999), haciendo que éstos sean especialmente vulnerables a las perturbaciones externas (Coma et al. 2004; Garrabou & Harmelin 2002; Linares et al. 2007, 2008). Además, los aumentos en las enfermedades en corales, tanto infecciosas como no, representan una amenaza creciente para estos ecosistemas (Harvell 2002; Jessica R. Ward, Kiho Kim 2007; Ward & Lafferty 2004). De hecho, desde mediados de los años ochenta del pasado siglo, los corales formadores de arrecifes están sufriendo el mayor declive de los últimos 10.000 años (Aronson et al. 2000; Bischof 2010; Kim 2015; O’Brien et al. 2016; Pandolfi et al. 2003). En concreto, factores relacionados con el estrés ambiental, como el cambio climático (elevada temperatura del mar), agentes contaminantes como herbicidas o metales pesados, así como infecciones bacterianas como aquellas provocadas por el género Vibrio (Ben-haim et al. 2003; Marshall & Schuttenberg 2006; Rosenberg & Falkovitz 2004; Weil et al. 2008; Wolff et al. 2015), provocan procesos como el conocido blanqueamiento coralino. Además, variables ambientales, en particular el aumento de la temperatura y/o la disposición de nutrientes, pueden afectar a la tasa y prevalencia de las enfermedades en estos organismos, como es el caso de las enfermedades de la “plaga blanca”, de la “banda amarilla”, de los “lunares oscuros”, así como la aspergilosis (Alker et al. 2001; Kuta & Richardson 2002; Rypien et al. 2008; Ward et al. 2006).

Desde un punto de vista sociológico, y aunque deba profundizarse aún en su

conocimiento, los arrecifes de coral y los jardines de gorgonias, son algunos de los ecosistemas donde se han realizado recientemente más esfuerzos para entender su funcionamiento, debido a su capacidad bioconstructora ya mencionada, así como por constituir una reserva de extraordinaria diversidad, entre otras razones ecológicas, económicas y farmacológicas (Conell 1978; Ibarra-Alvarado et al. 2007; Moberg & Folke 1999; Stella et al. 2011). En general, su presencia es importante para dilucidar características del medio en los que se encuentran, tales como el grado de tensión hidrodinámica, la presencia de contaminantes y la sedimentación (Alcolado 1981; Goatley & Bellwood 2013; Madin & Connolly 2006; Rogers 1990). Además, los arrecifes coralinos son igualmente importantes debido a sus significativas contribuciones en la economía mundial, aportando gran número de recursos (i.e. pesca) y servicios (i.e. turismo, recreación), siendo la principal fuente de ingresos para numerosos países (Brander et al. 2007; Cesar 2002; Cesar et al. 2003; Gomez et al. 1994; White et al. 2000). En concreto, proveen de alimentos y recursos para aproximadamente 500 millones de personas en el mundo (Mooney et al. 2009).

1.6. Área de estudio

1.6.1. Ecuador y su geografía marina

Ecuador ejerce jurisdicción sobre la franja de 200 millas paralela a la línea costera continental y el territorio insular del archipiélago de Galápagos. Este área marina cubre alrededor de 1.095.000 km2, de los cuales al continente corresponden 238.000 km2 y a la Provincia de Galápagos 857.446 km2. La costa continental de Ecuador, formada por una sucesión de bahías y cabos alternados, tiene una longitud aproximada de 1.256 km (Majluf 2002) (Fig. 6). Además, Ecuador cuenta con 79 cuencas de drenaje, 71 de las cuales desembocan en el océano Pacífico (Cucalón 1986, 1996).

El mar ecuatoriano está formado por aguas tropicales cálidas procedentes del norte de la línea ecuatorial y aguas subtropicales provenientes del sur (Thiel et al. 2007). El rasgo sobresaliente en la región submarina, es el margen continental (plataforma continental, talud y emersión continental) que bordea al continente y representa la transición con los

fondos oceánicos. El fondo oceánico se diferencia por la presencia de la dorsal de Carnegie, que es una cordillera submarina cuyas cimas constituyen las islas Galápagos, y por la prolongación de la fosa Perú-Chile que alcanza más de 4.000 m de profundidad (Gutscher et al. 1999).

Figura 6. Imagen satélite de la región de Ecuador continental y las islas Galápagos. ©Google Earth.

1.6.2. Condiciones climáticas marinas de Ecuador

El litoral de Ecuador se caracteriza por una zona compleja de confluencia de corrientes oceánicas, donde se produce una transición entre las aguas frías provenientes del sur a través de la corriente de Humbolt, que con su retirada deja paso a las aguas cálidas de la corriente de Panamá desde el norte (Cucalón 1986, 1996; Félix et al. 2010; Sonnenholzner et al. 2014) (Fig. 7).

De una forma más detallada, entre las corrientes conocidas que afectan a Ecuador, la Corriente superficial Ecuatorial del Sur, perpendicular a la costa, se caracteriza por ser relativamente pobre en nutrientes, con una transición entre aguas tropicales y subtropicales que fluye hacia el oeste (Coloma-Santos 2007).

Por otro lado, entrando desde el sur, encontramos el mencionado sistema de corrientes de Humboldt (SCH), el cual es el sistema de afloramiento más productivo de los

océanos del mundo, en términos de pesca (ver Chavez et al. 2008; Echevin et al. 2012). Se extiende a lo largo de la costa oeste de América del Sur desde el sur de Chile hasta Ecuador y las islas Galápagos. La oceanografía general del SCH se caracteriza por un flujo hacia el norte predominante de las aguas superficiales de origen subantártico y por la fuerte corriente ascendente de aguas frías sub-superficiales ricas en nutrientes de origen ecuatorial (Thiel et al. 2007).

El período de mayor intensidad de esta corriente se produce de junio a septiembre, y desaparece gradualmente durante noviembre (Coloma-Santos 2007; Duxbury et al. 2002; Peixoto & Oort 1992) por la incursión de la corriente cálida y pobre en nutrientes de Panamá (Chavez & Brusca 1991; Coello et al. 2001; Glynn 2003c). Numerosos estudios han examinado los efectos de las condiciones oceanográficas en una variedad de procesos ecológicos en el SCH (Alheit & Niquen 2004; Arntz W 1999; Coloma-Santos 2007; Echevin et al. 2012; Thiel et al. 2007). La surgencia es la principal fuerza impulsora de los procesos ecológicos que promueven la alta producción primaria en las aguas cercanas a la costa, tanto en el plancton como en el bentos (Iriarte & González 2004; Wieters et al. 2003). Además, tiene efectos positivos en la composición de la comunidad de zooplancton, la dinámica poblacional de peces y la dispersión larvaria (Escribano et al. 2004; Halpin et al. 2004; Poulin et al. 2002; Thiel et al. 2007).

Figura 7. Mapa ilustrativo de las principales corrientes oceánicas influyentes para Ecuador (Instituto de Ecología Aplicada, ECOLAP, Universidad San Francisco de Quito).

Por otro lado, hay que hablar de uno de los fenómenos que afecta a los ecosistemas marinos en Ecuador, conocido como El Niño. Se trata de una perturbación climatológica periódica a escala planetaria, que conlleva cambios en la abundancia y la distribución de los recursos pesqueros, así como de otros organismos marinos, provocando diferentes fenómenos de mortandad (Glynn & Wellington 1983; Glynn 2001; López-Ibarra & Palomares-García 2006; López-Pérez et al. 2016; Mendo & Wolff 2003; Rojo-Vázquez et al. 2001). Durante El Niño, las aguas cálidas fluyen hacia el este desde Ecuador, dominando sobre la corriente de Humboldt y produciendo cambios como el aumento de la temperatura del agua de 2 a 3 °C, la elevación del nivel del mar de 40 a 50 cm y una reducción en la disponibilidad de nutrientes en superficie. Hay que destacar que entre estos fenómenos algunos de los más intensos fueron los registrados en los años 1982-83 y, posteriormente, en 1997-98, aunque parece que actualmente estamos ante un posible nuevo período de este proceso (Allen 2016; Li et al. 2015). Finalmente, otro de los fenómenos característicos de la zona es el conocido como La Niña, que suele

contraponerse con el Niño. Durante este evento se produce una bajada de las temperaturas marinas, asociada con una época de disminución de las precipitaciones, relacionada con el incremento de los vientos alisios del este (Allen 2016; Cazes-Boezio & Talento 2016; Maturana et al. 2004; Meyers et al. 2007). Sin embargo, en este trabajo no haremos un mayor hincapié en estos fenómenos ya que no ha sido detectado durante nuestros años de estudio.

1.6.3. Áreas marinas de Ecuador y sus arrecifes

Poco se sabe de las formaciones de gorgonias o corales en general en el Ecuador continental. Sin embargo, parece que poseen una estrecha afinidad taxonómica con la fauna del Pacífico central y el Caribe (ver Breedy & Guzmán 2002, 2007, 2011; Breedy et al. 2002; Glynn & Ault 2000; Guzmán 2003; Hickson 1928; Williams & Breedy 2004).

Generalmente, las comunidades de coral necesitan condiciones determinadas para poder vivir, como aguas cálidas, claras, poco profundas y con de moderada a vigorosa circulación (Glynn 2003a; Glynn et al. 2003). Al parecer, estos factores son un limitante para el desarrollo de arrecifes en las costas de Ecuador, donde muchas veces se encuentran aguas frías como efecto de la corriente de Humboldt, y además con una alta sedimentación como resultado de los aportes de materiales continentales a través de las desembocaduras de los ríos. No obstante, aunque no se encuentren verdaderos arrecifes coralinos, existen parches de coral, cuya dinámica es muy parecida a la de los arrecifes, albergando también a una gran diversidad de especies (Glynn 2003b). Ejemplos de ello son las ya bien conocidas colonias de las islas Galápagos (Breedy & Guzmán 2005; Glynn 2003c; Williams & Breedy 2004) y las algo menos conocidas grandes colonias de coral en las zonas costeras del Parque Nacional Machalilla e isla de la Plata (Bo et al. 2012; Glynn 2003b; c).

Ecuador tiene en la actualidad 25 unidades de conservación como parte del Sistema Nacional de Áreas Protegidas, de las cuales siete incluyen ambientes marinos costeros (ECOLAP & MAE 2007). Una de las principales áreas protegidas marino-costeras de Ecuador es el Parque Nacional de Machalilla, la cual se encuentra localizada al suroeste de la provincia de Manabí, dentro de los cantones de Jipijapa, Puerto López y Montecristi,

abarcando una superficie de 56.184 ha, e incluye la isla de la Plata y 2 millas náuticas alrededor de sus límites (Instituto Ecuatoriano Forestal y de Areas Naturales y Vida Silvestre 1998). En Machalilla las playas son el ecosistema costero mejor representado, aunque incluye también costas acantiladas, costas rocosas y dunas. El Parque Nacional de Machalilla es el único área protegida continental que incluye muestras de ecosistemas marinos como plataforma continental, islas, islotes y bajos (Glynn 2003).

Todo plan de manejo, gestión o conservación debe basarse en el conocimiento del hábitat y de las especies de flora y fauna que allí se desarrollan (ver Kim & Byrne 2006; Mace 2004; Zapata & Vargas-Ángel 2003). Desgraciadamente, estudios de este tipo en las áreas marino-costeros de Ecuador son prácticamente inexistentes (Cruz et al. 2003). Además, la información disponible sobre los organismos marinos se encuentra muchas veces sesgada hacia las conocidas como especies bandera o especies claves (ver Isasi-Catalá 2011), como ballenas, tortugas marinas y otros vertebrados marinos, o aquellas con interés comercial (Cobo & Massay 1969; Cruz et al. 2003; Massay 1983; Seminoff et al. 2008)).

Como consecuencia de lo anteriormente mencionado, los esfuerzos científicos han aumentado, con el fin de discernir la estructura y funcionamiento de estas comunidades marinas, con especial énfasis en los grupos taxonómicos claves que las constituyen (Fabricius 2005). Estos esfuerzos deben abarcar la distribución, composición y estado de las comunidades, así como evaluar adecuadamente la diversidad de especies en los diferentes niveles taxonómicos (Gladstone 2002; Guzmán & Guevara 1998; Guzmán et al. 2004; Hughes et al. 1999). Todos estos enfoques han sido los que hemos tratado de desarrollar a una escala u otra, a lo largo de esta Tesis, y para ello nos planteamos los objetivos que se enumeran a continuación.

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2. OBJETIVOS

Objetivos

El objetivo general de esta Tesis se centra en el estudio de las gorgonias de la familia Gorgoniidae en las costas de Ecuador, con el fin de comprender aspectos fundamentales para su conservación, tales como diversidad, sus relaciones evolutivas, su biología reproductiva o sus asociaciones ecológicas con otros organismos. Para ello se han planteado los siguientes objetivos específicos:

1. Estudio de la diversidad de las gorgonias pertenecientes a la familia Gorgoniidae (Cnidaria, Octocorallia) presentes en las costas ecuatorianas. Para ello, este objetivo será complementado con el estudio faunístico y revisión taxonómica de las especies de Gorgoniidae en el Pacífico oriental ecuatoriano. 2. Caracterización de la variación inter- e intraespecífica en el contexto de una taxonómica integradora, con análisis morfométricos y moleculares. 3. Aproximación a la historia evolutiva de los tres géneros que componen a la familia Gorgoniidae en el litoral de Ecuador. Para ello se inferirán la relaciones filogenéticas de las especies encontradas en sus costas, junto con aquellos materiales tipo albergados en colecciones de diversos museos europeos y americanos. 4. Determinación de patrones de reproducción en gorgonias presentes en aguas ecuatorianas, por medio de la descripción del ciclo gametogénico en dos especies representantes de las familias Gorgoniidae y Plexauridae, respectivamente. 5. Exploración de la estructura de la comunidad asociada a gorgonias, patrones estacionales de diversidad y abundancia de esta fauna asociada en el área de estudio y su relación con diferentes parámetros morfológicos de la gorgonia hospedadora. 6. Identificación de la comunidad fúngica asociada en dos especies del género Leptogorgia (Gorgoniidae) en el litoral de Ecuador. Se pondrá énfasis en la detección de hongos patógenos y sus posibles consecuencias en este ecosistema.

3. MATERIAL Y MÉTODOS

Material y métodos

En este apartado se comentarán características generales de los puntos de muestreo realizados y se abordarán los cuatro tipos de análisis utilizados a lo largo de la presente Tesis: análisis morfológicos, moleculares, técnicas histológicas y análisis estadísticos. Todos estos procedimientos serán desarrollados y descritos con mayor detalle en cada uno de los capítulos de este estudio.

3.1. Puntos de estudio

Para nuestro estudio se seleccionaron 11 puntos de muestreo (Fig. 8), de los cuales, ocho se encontraban dentro de la jurisdicción del Parque Nacional de Machalilla (PNM) y tres se encontraban fuera de la misma. Bajo la jurisdicción del PNM se encuentran isla de la Plata, el Barbullo, Punta Machalilla, los Frailes, Punta Gruesa, Punta Mala y la isla de Salango. Por otro lado, encontramos los Ahorcados, el Cope y Salinas, donde no existe figura de protección medioambiental marina. Todas las descripciones acerca de los puntos de estudio que se detallarán a continuación se basan en los trabajos e informes de Flachier et al. (1997), ECOLAP & MAE (2007) y en observaciones personales a través de los periodos de muestreo (Fig. 9).

• La isla de la Plata (1°16'25.84"S 81° 4'11.70"W) es de origen continental y está formada por roca volcánica. Localizada a unas 25 millas náuticas de Puerto López, su perfil presenta pequeñas bahías, importantes conjuntos de parches rocosos y acantilados altos, algunos muy accidentados. La morfología del arrecife de la isla de la Plata parece estar determinada en gran medida por el tipo de corrientes superficiales y por el impacto de los vientos alisios que dominan la zona de manera estacional e impactan sobre los acantilados. Posee su propio microclima y es una zona con bastante afluencia turística debido a una cierta similitud faunística con Galápagos.

Figura 8. Situación del área de muestreo, así como los puntos concretos donde se han realizado los mismos.

• Los islotes del norte, la Viuda (1°26'5.95"S 80°46'7.30"W) y el Burbullo (1°28'23.59"S 80°51'23.04"W) se encuentran alejados de la línea de costa y se localizan entre las comunas de Salaite y Puerto Cayo. Son islotes rocosos, que llegan a formar un talud, el cual se extiende hasta los 25-30 m de profundidad. • Punta Machalilla (1°28'33.53"S 80°47'38.04"W) se encuentra al norte de Punta los Frailes, a 300 m de la comuna de Machalilla y está rodeada por una plataforma de fondo rocoso paralela a la costa. Su relieve es bastante parecido a los de Punta Mala y Punta Gruesa, siendo su profundidad media de unos 15 m. • Los Frailes (1°30'14"S 80°48'33”W), dentro de los cuales tenemos el islote Horno de Pan, se encuentran en la zona central del Parque Nacional de Machalilla, frente a una playa prístina de 5 km de arena blanca. Posee largas plataformas rocosas llanas y barreras emergidas que dan lugar al islote Horno de Pan. Su profundidad media es de unos 15-20 m. • Punta Mala (1°33'41.37"S 80°50'8.79"W) y Punta Gruesa (1°33'38.15"S 80°50'5.28"W) poseen plataformas rocosas y barreras emergidas, con un relieve bastante regular y una profundidad hasta los 20-25 m aproximadamente. Punta Mala además es el saliente más prominente del perfil costero comprendido entre las comunas de Puerto López y Salango. 84

• La isla Salango (1°35'55.13"S 80°52'0.01"W) es una formación de origen continental, separada por movimientos tectónicos y se encuentra rodeada de rocas sumergidas, con formaciones de paredes verticales, con zonas de grandes llanos a poca profundidad. La isla posee diferentes zonas de buceo gracias a sus áreas de exposición a las corrientes. Entre esa formación rocosa se localizan importantes parches de corales duros y gorgonias, con una profundidad bastante variable entre 5 y 30 m. • El islote de los Ahorcados (1º40'44”S 80º50'08”W) se encuentra en el extremo sur del Parque Nacional de Machalilla a 2,2 millas náuticas al oeste de Ayampe. Se compone de dos formaciones rocosas que surgen de la misma base y un ecosistema submarino que se caracteriza por numerosas crestas rocosas que contienen parches de corales duros y gorgonias extensos, con profundidades muy variables entre los 5 y los 35 m. Es una zona caracterizada por sus fuertes corrientes submarinas y oleaje. • El bajo Cope (1°43'34"S 80°59'85"W) se encuentra a unas 20 millas náuticas en frente de la localidad de Ayampe. Es una extensión plana rocosa, con relieve bastante irregular y de extensión y forma desconocida. De las características más significativas de este bajo, destacan las múltiples emanaciones de gas que burbujean entre los resquicios del fondo rocoso. Su profundidad puede llegar hasta los 40 m. En él se concentra una gran variedad de peces y grandes vertebrados, como mantas gigantes y ballenas. • El relieve submarino de Salinas (2°12'50.01"S 80°56'5.93"W), localizado en la provincia de Santa Elena, sufre un fuerte impacto antrópico debido a que es una zona sin figura de protección marina y con una fuerte afluencia de pescadores y turismo. Por ello se trata de una zona degradada y arenosa, con numerosos barcos hundidos y con parches de coral muy dispersos, difíciles de encontrar y de extensión desconocida.

Figura 9. Imágenes tomadas en algunos de los fondos marinos encontrados en las áreas de estudio.

3.2. Análisis morfológicos

El tratamiento morfológico llevado a cabo en esta Tesis sigue las directrices descriptivas sugeridas en Bayer et al. (1983) y los trabajos revisionarios de Bayer (1951, 1953, 1961, 1973) donde se incluyen aspectos de la diversidad de las especies en la familia Gorgoniidae, así como las revisiones de los géneros aquí estudiados, Eugorgia, Leptogorgia

y Pacifigorgia (Breedy & Guzmán 2002, 2003a, 2007, 2013; Breedy et al. 2009; Williams & Breedy 2004). De esta forma, la descripción aportada de las especies estudiadas, estarán basadas en la combinación de diferentes caracteres asociados con la morfología colonial, las dimensiones y color de diferentes estructuras tanto a nivel de los pólipos como de la colonia, y los escleritos de cada uno de los ejemplares examinados. Además desglosaremos y ampliaremos con más detalle algunos de los caracteres empleados usualmente, para hacer las descripciones lo más completas posibles (Tabla 1).

Tabla 1. Tipos de caracteres y sus diferentes estados, utilizados en la identificación y descripción de las especies de Eugorgia, Leptogorgia y Pacifigorgia (familia Gorgoniidae).

Carácter Código

Anastomosis ausente / presente

Patrón de malla regular / irregular

Nervio central ausente / presente Patrón Densidad de la malla desde 0-2 hasta 18-20 colonial (cm2)

Tipo de red ausente / abierta / fina/ cerrada

blanco / rosa / rojo / morado / naranja / amarillo / Color de la colonia marrón

Organización separada / arborescente / en malla

Tipo malla / dicotómica / irregular / pinnada / laxa

Max nº ramas desde 3 hasta ≤ 9 Ramificación Grosor (mm) desde ≈ 0.7 hasta ≤ 4.9

Ramificación final desde 0-3 hasta ≤27 (mm)

Organización separada / estrecha Pólipo Forma sobre la rama prominente / poco elevada / plana o a ras

Tabla 1. Continuación.

Tipo de esclerito husos / disco doble / cabestrantes / husos en forma dominante de disco

Disco doble ausente / presente

Husos en forma de ausente / presente disco

Tamaño del huso desde ≈ 0.08 hasta ≤ 0.24

Huso curvado ausente / presente Huso redondeado ausente / presente

Tamaño del huso desde ≈ 0.08 hasta ≤ 0.24 redondeado

Cruces ausente / presente

Color del esclerito sin color / rosa / rojo / morado / naranja / amarillo Escleritos Husos > 0.1 mm ausente / presente

Tamaño del desde ≈ 0.05 hasta ≤ 0.12 cabestrante Escleritos ausente / presente bicoloreados Color escleritos del sin color / rosa / rojo / morado / naranja / amarillo antocodio

Forma escleritos del barras/ barras aplanadas / forma galleta antocodio

Tamaño escleritos desde ≤ 0.07 hasta ≥ 0.18 del antocodio

Escleritos del antocodio ausente / presente bicoloreados

Las gorgonias pueden contar con más de una veintena de patrones de ramificación diferentes (Bayer 1953, 1973; Bayer et al. 1983). De hecho, las formas de crecimiento de las gorgonias en el Pacífico oriental, muestran una amplia gama de variación, incluyendo formas dicotómicas, pinnadas, reticuladas (anastomosis), filiformes, arborescentes e irregulares (Guzmán & Breedy 2008). Además, estas y otras caracteristicas del patrón de ramificación puede ser consideradas como caracteres propios de cada una de las especies,

aunque en muchas ocasiones pueden ser comunes a algunas de ellas (Fig. 10)(e.g., Breedy & Guzmán 2003b, 2007; Breedy et al. 2009; Gori et al. 2012).

Figura 10. Algunos de los tipos más comunes de organización colonial y patrones de ramificación presentes en Gorgoniidae. A) Reticulado (anastomosis), B) pinnado, C) dicotómico (Bayer 1953); D) arborescente (Bayer 1973).

Por otro lado, la coloración de las colonias en los octocorales suele depender de los pigmentos incorporados en los escleritos y en los tejidos, así como de algas simbióticas intracelulares alojadas en el endodermo en algunos casos (Bayer 1961). En el caso de los gorgoniidae examinados, la coloración de la colonia usada en la descripción de las especies es debida a los escleritos, y ésta permanece en las muestras conservadas, de una forma muy poco afectada por el alcohol, el secado o la luz (Bayer 1961).

En todo caso, en las descripciones más actuales de Gorgoniidae, se suele incorporar la coloración de la gorgonia tanto en estado vivo, como en seco o conservado, debido a la posible diferencia de coloración que pudiera existir entre ambos estados (ver Breedy y Guzmán 2002, 2007; Breedy et al. 2009). Sin embargo, este carácter a veces está sujeto a cierta subjetividad de los taxónomos, puede ser coincidente entre especies diferentes y además puede darse el caso de determinadas especies como Leptogorgia virgulata (Lamarck, 1815) que puede presentar cierta variabilidad cromática intraespecífica.

Los pólipos pueden también aportar información específica, ya que existen diferentes posibilidades en su disposición, morfología y apertura (Sánchez et al. 2004). Pueden disponerse de forma aleatoria, bilateral a lo largo de los bordes de las ramas o de manera uniforme sobre las mismas, teniendo una forma prominente, poco elevada o a ras del cenénquima de la rama (e.g. Breedy y Guzmán 2003b).

En general, las variaciones en el tamaño, la tipología, la forma y la coloración de los escleritos, así como, en ocasiones, su posición relativa dentro de la colonia, son algunos de los principales y más usados criterios para la caracterización de estas especies (Tabla 1) (Bayer 1953; Bayer et al. 1983; Vargas et al. 2010b). En los octocorales existe una amplia variedad de tipos de escleritos (Bayer et al. 1983); sin embargo, dentro de las especies de la familia Gorgoniidae en el Pacífico oriental, los más comunes son conocidos como husos (spindles) y cabestrantes (capstans) en el cenénquima, y aquellos escleritos pertenecientes al antocodio (Fig. 11). Además, la presencia de ciertos escleritos ornamentados como los conocidos como “cruces” o “mariposa” (Fig. 11) (Bayer et al. 1983), aunque no suelen encontrarse de forma muy abundante dentro de los individuos, puede ayudarnos a la diferenciación interespecífica. En cada uno de estos tipos, se establecen rangos de tamaños característicos para las especies, así como el número de ciclos de tubérculos que presentan, su coloración (simple o bicoloreados), terminación puntiaguda o redondeada, y si tienen una forma curvada o no (Tabla 1) (ver Bayer 1953; Breedy y Guzmán 2002, 2007; Breedy et al. 2009).

Por otro lado, la presencia/ausencia de un tipo u otro de esclerito puede determinar la diferencia no sólo entre especies, sino también a nivel de géneros. Por ejemplo, las especies del género Eugorgia son indistinguibles a nivel de morfología externa colonial de las del género Leptogorgia; sin embargo, las especies del primero (Eugorgia), cuentan con la presencia de unos escleritos característicos denominados como disco doble o “double- disc” (Fig. 11) (Breedy et al. 2009), ausentes en Leptogorgia. Algo similar ocurre con el género Eunicella (Gorgoniidae), el cual es distinguible por la presencia de escleritos “balloon club” o en forma de globo o maza (Bayer et al. 1983).

Figura 11. Variación morfológica de los diferentes tipos de escleritos presentes en la familia Gorgoniidae del Pacífico oriental (Leptogorgia, Eugorgia y Pacifigorgia): A) husos puntiagudos; B) husos redondeados; C) cabestrantes; D) escleritos inmaduros; E) escleritos del antocodio; F) forma de mariposa; G) cruces; H) husos en forma de disco; I) disco doble; J) disco doble completo. H-J sólo están presentes en el género Eugorgia.

La aplicación práctica de estos caracteres en la taxonomía morfológica de las especies de Gorgoniidae identificadas y descritas en Ecuador, se desarrollarán principalmente en los capítulos 1 y 2 de la presente Tesis, con el apoyo del material revisado de las colecciones de los principales museos. Además en todas las especies trabajadas, se muestran las citas originales y las sinonimias más importantes incluidas en la

taxonomía de cada una de ellas.

3.3. Análisis moleculares

La taxonomía tradicional de los corales, basada en su morfología, ha estado lastrada por la plasticidad fenotípica y una serie de caracteres discretos sobre los cuales se han establecido las descripciones sistemáticas (Concepcion et al. 2008). Entre los grupos taxonómicos superiores de cnidarios, el grupo de Octocorallia es posiblemente el menos comprendido en lo que se refiere a sus relaciones filogenéticas a nivel de orden y familia y es por ello que los niveles taxonómicos actuales se consideran inestables (McFadden et al. 2006) y posiblemente artificiales. Para abordar este conflicto, el siguiente paso en el estudio de la taxonomía de estas especies consiste en incluir caracteres moleculares con el fin de abordar las relaciones en base a caracteres neutrales y contrastar las diferentes hipótesis postuladas en base a caracteres morfológicos (e.g., López-González et al. 2014; Vargas et al. 2014).

En este contexto, las herramientas moleculares pueden ser útiles para resolver también relaciones filogenéticas entre las especies (Avise 2012; Godoy 2009; Hebert et al. 2004; Simon et al. 2006), permitiendo dilucidar su historia evolutiva y los posibles factores o eventos históricos que han promovido su diversificación (Sá-Pinto et al. 2005).

Los marcadores utilizados en los análisis moleculares revelan sitios de variación a nivel de la secuencia de ADN. A diferencia de los marcadores asociados a caracteres morfológicos (fenotipos de fácil identificación visual), las variaciones en los marcadores de ADN pueden no mostrarse por sí mismas en el fenotipo, pero sí en diferencias en un solo nucleótido del gen o en una parte del ADN repetitivo (Avise 2012).

El ADN mitocondrial ha sido extensamente utilizado en las últimas dos décadas como marcador genético en análisis de estructuras poblacionales, de flujo génico, fenómenos de hibridación (en este caso combinado con ADN nuclear), patrones biogeográficos o filogenéticos entre poblaciones, especies o taxones superiores (Avise & Ball 1990; Avise 1994; van der Ham et al. 2009; McFadden et al. 2004; Morris et al. 2012; Simons et al. 1994). El ADN mitocondrial, de herencia materna, salvo excepciones (Hoeh et

al. 2002; Zouros et al. 1994), tiene generalmente una tasa de substitución más alta que la del ADN nuclear; de hecho, se estima que los genes mitocondriales en animales evolucionan típicamente de 5 a 10 veces más rápido que el ADN nuclear de copia única (Avise 2012; Wan et al. 2004), lo cual hace más fácil la resolución de diferencias entre individuos cercanamente emparentados y se convierte en una buena herramienta de diagnóstico de los taxones más próximos (Sunnucks 2000). Éstas son algunas de las razones por las que, durante las últimas décadas, el genoma mitocondrial ha sido una de las principales fuentes de caracteres utilizados para estudios de sistemática molecular en animales. Desafortunadamente, los intentos de utilizar genes mitocondriales para análisis similares en octocorales, no han tenido tanto éxito, debido a la pronunciada falta de variación en las secuencias de este grupo (McFadden et al. 2004). Estudios recientes han sugerido que el genoma mitocondrial de las especies de octocorales (Fig. 12) varía a un ritmo de 10 a 20 veces más lento que en otros grupos de animales (Chen & Yu 2000; Shearer et al. 2002). Las posibles explicaciones para las menores divergencias observadas, incluyen una menor tasa de evolución de los genomas mitocondriales en octocorales y la presencia de un gen, mtMutS (Mutator S, msh1), que puede codificar para un sistema de reparación en apareamientos erróneos del ADN mitocondrial, reduciendo así el número de sustituciones (France & Hoover 2002).

La tasa de evolución de los genes mitocondriales en octocorales es, de hecho, más baja que en su grupo hermano compuesto por los hexacorales, que carece del gen mtMutS. Sin embargo, paradójicamente, este gen sí tiene mayores tasas de mutación que otros genes mitocondriales en octocorales (Bilewitch & Degnan 2011).

Debido por tanto, entre otros aspectos, a la presencia de mtMutS, actualmente se sugiere que la mejor manera de abordar los estudios sobre sistemática en octocorales, es la combinación de varios marcadores, en los cuales se incluyan genes del ADN mitocondrial y zonas más variables del ADN nuclear (McFadden et al. 2004; Sánchez et al. 2003b).

Figura 12. Genoma mitocondrial de algunas familias de octocorales, donde se incluye Gorgoniidae. Tomado de Figueroa & Baco (2015).

Sin embargo, en la actualidad, se han encontrado pocos marcadores moleculares que sean realmente útiles en el estudio de los procesos evolutivos en octocorales. La región del ADN ribosómico nuclear que comprenden el 28S y las regiones ITS (espaciadores internos transcritos), junto con los genes mitocondriales mtMutS y citocromo oxidasa, son los que más extensamente se han utilizado en estudio filogenéticos entre especies de octocorales (Aguilar & Sánchez 2007; McFadden & Hutchinson 2004; McFadden et al. 2001, 2014; Sánchez & Dorado 2008; Sánchez et al. 2003b; Taylor & Rogers 2015). No obstante, creemos que es muy necesaria la búsqueda de nuevos marcadores, debido a la falta de resolución, inter- e intraespecífica, de los anteriormente citados.

Para situar la revisión taxonómica en un contexto evolutivo, ésta debe ir acompañada del estudio de las relaciones filogenéticas entre los taxones que lo componen ( Carranza & Amat 2005; Finston & Peck 2004; Gonder et al. 2006; Granzow de la Cerda 1990; Lefébure et al. 2006). La finalidad de los análisis filogenéticos es estimar la genealogía, las relaciones de parentesco, que encuadren la historia evolutiva del grupo taxonómico de estudio (Peña 2011).

Para ello, existen distintos métodos de inferencia, según los principios y los algoritmos que se empleen para confeccionar redes o árboles. Dichos métodos pueden

distinguirse entre los que utilizan un modelo (como la máxima verosimilitud –ML por sus siglas en inglés- o la inferencia bayesiana –IB) y los que no lo hacen (máxima parsimonia - MP). Dichos modelos tienen en cuenta principalmente cuatro parámetros: la frecuencia de las bases nucleotídicas, la sustitución entre los nucleótidos y la distribución de los cambios a lo largo de la secuencia y la existencia de posiciones invariables.

3.4. Técnicas histológicas Las técnicas histológicas son un pilar fundamental para llevar a cabo estudios acerca de la biología reproductiva de estos organismos, con el fin de poder apreciar las estructuras internas, permitiendo una valoración y descripción adecuadas de sus productos reproductivos y ciclos.

Dentro de esta técnica se agrupan una serie de pasos como la fijación del material, generalmente en formaldehído en concentraciones próximas al 4%. Posteriormente, las muestras serán descalcificadas en ácido fórmico al 10% para eliminar toda la materia inorgánica de origen calcáreo y dejar sólo los tejidos. A continuación procederemos a la deshidratación completa de la muestra (Tabla 2), para una posterior inclusión en un medio no hidrosoluble, que en nuestro caso será parafina plastificada (Paraplast Plus, McCormick), en una estufa a 55-60ºC (se realizarán varios cambios de parafina). Seguidamente se llevará a cabo el proceso de cortes seriados (entre 6-10 µm de grosor según permita el material) con un microtomo del tipo Leitz (de cuchilla fija). Los cortes serán posteriormente colocados sobre un portaobjetos y fijados con Poly-L-Lysine 1:10.

Tabla 2. Ciclos de cambio de medio químico y tiempo llevados a cabo a lo largo del proceso de deshidratación del tejido. (TBA: Terciary Butiric Alcohol, 2-Metil-2-Propanol, (CH3)3COH, Panreac).

Alcohol Aceite de Etanol Etanol Butírico H2O Parafina Tiempo 100% 90% Terciario (Hodernal) TBA 1 10 % - 40 % 50 % - 1 h TBA 2 20 % - 50 % 30 % - 1 h TBA 3 35 % - 50 % 15 % - 1 h TBA 4 50 % - 50 % - - 1 h TBA 5 75 % 25 % - - - 1 h TBA puro 100 % - - - - 12 h TBA- 50 % - - - 50 % 1 h Hodernal

Posteriormente procederemos a la tinción del material. Para nuestro estudio, la tinción elegida fue la tricrómica de Ramón y Cajal (Gabe, 1968) y los colorantes usado fueron la Fuchsina de Ziehl (básico) y el Pícrico-indigocarmín (ácido) (Fig. 13).

Figura 13. Esquema ilustrativo de las distintas etapas por donde pasa la muestra durante la tinción, así como sus tiempos correspondientes, todos realizados a temperatura ambiente.

Finalmente las preparaciones histológicas se observarán y estudiarán por medio de un microscopio óptico (Leica DMLB) a diferentes magnificaciones para observar los distintos detalles. Además se tomarán fotografías de las preparaciones histológicas con una cámara digital (ZEISS modelo AXIOCAM- MRc5).

3.5. Análisis estadísticos Para poder tratar los datos procedentes de los estudios de la comunidad asociada a las especies de gorgonias y de los caracteres morfométricos, nos apoyaremos en diferentes análisis estadísticos. Para ello, se utilizará el paquete estadístico PRIMER PERMANOVA v1.0.1 (Clarke & Gorley 2006). A través de este conjunto de programas realizaremos diferentes análisis multivariantes para conocer la estructura de la comunidad, por medio del índice de distancia de Bray-Curtis. Por otro lado, se examinarán, a través de análisis univariantes, las diferencias significativas en abundancia (N) o diversidad (Índice de Shannon-Wiener, H'), mediante pruebas en matrices de PERMANOVA a través de distancias euclídeas, en un enfoque similar al ANOVA paramétrico (Anderson 2001; Schultz et al. 2012; Scyphers et al. 2011). La homogeneidad en la dispersión será comprobada con la prueba PERMDISP (Anderson et al. 2008). Además nos apoyaremos en el programa R (Rstudio) (Team 2015) para obtener las representaciones gráficas.

Todos estos procedimiento serán más ampliamente desarrollados en los correspondientes capítulos de la presente Tesis.

3.6. Referencias

Aguilar, C. & Sánchez, J.A. (2007) Molecular morphometrics: Contribution of ITS2 sequences and predicted RNA secondary structures to octocoral systematics. Bulletin of Marine Science 81, 335–349.

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Anderson, M.J., Gorley, R.N. & Clarke, K.R. (2008) PERMANOVA+ for PRIMER: Guide to Software and Statistical Methods. PRIMER-E, Plymouth, UK.

Avise, J.C. (2012) Molecular markers, natural history, and evolution. 2rd ed. Sinauer, Sunderland, MA.

Avise, J.C. & Ball, R.M.J. (1990) Principles of genealogical concordance in species concepts and biological taxonomy. Oxford Surv. Evol. BioI. 7, 45–67.

Bayer, F.M. (1951) A revision of the nomenclature of the Gorgoniidae (Coelenterata: Octocorallia) with an illustrated key to the genera. Journal of the Washington Academy of Sciences 41, 91–102.

Bayer, F.M. (1953) Zoogeography and evolution in the octocorallian family Gorgoniidae. Bulletin of Marine Science 3, 100–119.

Bayer, F.M. (1961) The shallow-water Octocorallia of the West Indian region. A manual for marine biologists. Studies of the Fauna Curacao and other Caribbean Islands 12, 1–373.

Bayer, F.M. (1973) Colonial organization in octocorals. In Boardman, R. S; Cheetham, A. H. and Oliver, W. A. Jr., (Ed). Animal Colonies. Stroudsburg:Dowden, 69–93.

Bayer, F.M., Grasshoff, M. & Verseveldt, J. (1983) Illustrated trilingual glossary of morphological terms applied to Octocorallia. Brill, E.J., Leiden.

Bilewitch, J.P. & Degnan, S.M. (2011) A unique horizontal gene transfer event has provided the octocoral mitochondrial genome with an active mismatch repair gene that has potential for an unusual self-contained function. BMC Evolutionary Biology 11, 228.

Breedy, O. & Guzmán, H.M. (2002) A revision of the genus Pacifigorgia (Coelenterata: Octocorallia: Gorgoniidae). Proceedings of the Biological Society of Washington 115, 782–839.

Breedy, O. & Guzmán, H.M. (2003)a) A new species of Pacifigorgia (Coelenterata: Octocorallia: Gorgoniidae) from Panama. Zootaxa 10, 1–10.

Breedy, O. & Guzmán, H.M. (2003)b) Octocorals from Costa Rica: The genus Pacifigorgia

(Coelenterata: Octocorallia: Gorgoniidae). Zootaxa 281, 1–60.

Breedy, O. & Guzmán, H.M. (2007) A revision of the genus Leptogorgia Milne-Edwards and Haime, 1857 (Coelenterata: Octocorallia: Gorgoniidae) in the Eastern Pacific. Zootaxa 1419, 1–90.

Breedy, O. & Guzmán, H.M. (2013) A new species of the genus Eugorgia (Cnidaria: Octocorallia: Gorgoniidae) from mesophotic reefs in the eastern Pacific. Bulletin of Marine Science 89, 735–743.

Breedy, O., Guzmán, H.M. & Vargas, S. (2009) A revision of the genus Eugorgia Verrill, 1868 (Coelenterata: Octocorallia: Gorgoniidae). Zootaxa 2151, 1 – 46.

Carranza, S. & Amat, F. (2005) Taxonomy, biogeography and evolution of Euproctus (Amphibia: Salamandridae), with the resurrection of the genus Calotriton and the description of a new endemic species from the Iberian Peninsula. Zoological Journal of the Linnean Society 145, 555–582.

Chen, C. a. & Yu, J.-K. (2000) Universal primers for amplification of mitochondrial small subunit ribosomal RNA-encoding gene in scleractinian corals. Marine Biotechnology 2, 146–153.

Clarke, K.R. & Gorley, R.N. (2006) PRIMER v6: user manual/tutorial. PRIMER-E,. , 192.

Concepcion, G.T., Crepeau, M.W., Wagner, D., Kahng, S.E. & Toonen, R.J. (2008) An alternative to ITS, a hypervariable, single-copy nuclear intron in corals, and its use in detecting cryptic species within the octocoral genus Carijoa. Coral Reefs 27, 323–336.

Finston, T.L. & Peck, S.B. (2004) Speciation in Darwin’s darklings: taxonomy and evolution of Stomion beetles in the Galapagos Islands, Ecuador (Insecta: Coleoptera: Tenebrionidae). Zoological Journal of the Linnean Society 141, 135–152.

France, S.C. & Hoover, L.L. (2002) DNA sequences of the mitochondrial COI gene have low levels of divergence among deep-sea octocorals (Cnidaria: Anthozoa). Hydrobiologia 471, 149–155.

Godoy, J.A. (2009) La genética, los marcadores moleculares y la conservación de especies. Ecosistemas 18, 23–33.

Gonder, M.K., Disotell, T.R. & Oates, J.F. (2006) New genetic evidence on the evolution of chimpanzee populations and implications for taxonomy. International Journal of Primatology 27, 1103–1127.

Granzow de la Cerda, I. (1990) Aplicación del método cladístico a un caso real: análisis filogenético de Anomodon Hook. & Tayl. (Musci). Anales del Jardín Botánico de Madrid 2, 305–325.

Guzmán, H.M. & Breedy, O. (2008) Leptogorgia christiae (Octocorallia: Gorgoniidae) a new shallow water gorgonian from Pacific Panama. Journal of the Marine Biological Association of the UK 88, 719–722. van der Ham, J.L., Brugler, M.R. & France, S.C. (2009) Exploring the utility of an indel-rich, mitochondrial intergenic region as a molecular barcode for bamboo corals (Octocorallia: Isididae). Marine Genomics 2, 183–192.

Hebert, P.D.N., Penton, E.H., Burns, J.M., Janzen, D.H. & Hallwachs, W. (2004) Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences of the United States of America 101, 14812–14817.

Hoeh, W.R., Stewart, D.T. & Guttman, S.I. (2002) High fidelity of mitochondrial genome transmission under the doubly uniparental mode of inheritance in freshwater mussels (Bivalvia: Unionoidea). Society for the Study of Evolution 56, 2252–2261.

Lefébure, T., Douady, C.J., Gouy, M. & Gibert, J. (2006) Relationship between morphological taxonomy and molecular divergence within Crustacea: Proposal of a

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molecular threshold to help species delimitation. Molecular Phylogenetics and Evolution 40, 435–447.

López-González, P.J., Grinyó, J. & Gili, J.-M. (2014) Chironephthya mediterranea n. sp. (Octocorallia, Alcyonacea, Nidaliidae), the first species of the genus discovered in the Mediterranean Sea. Marine Biodiversity, 37–49.

McFadden, C.S., Brown, A.S., Brayton, C., Hunt, C.B. & van Ofwegen, L.P. (2014) Application of DNA barcoding in biodiversity studies of shallow-water octocorals: Molecular proxies agree with morphological estimates of species richness in Palau. Coral Reefs 33, 275–286.

McFadden, C.S., Donahue, R., Hadland, B.K. & Weston, R. (2001) A molecular phylogenetic analysis of reproductive trait evolution in the soft coral genus Alcyonium. Evolution; international journal of organic evolution 55, 54–67.

McFadden, C.S. & Hutchinson, M.B. (2004) Molecular evidence for the hybrid origin of species in the soft coral genus Alcyonium (Cnidaria: Anthozoa: Octocorallia). Molecular Ecology 13, 1495–1505.

McFadden, C.S., Tullis, I.D., Hutchinson, M.B., Winner, K. & Sohm, J. a. (2004) Variation in coding (NADH dehydrogenase subunits 2, 3, and 6) and noncoding intergenic spacer regions of the mitochondrial genome in Octocorallia (Cnidaria: Anthozoa). Marine Biotechnology 6, 516–526.

Morris, K.J., Herrera, S., Gubili, C., Tyler, P. a., Rogers, A. & Hauton, C. (2012) Comprehensive phylogenetic reconstruction of relationships in Octocorallia (Cnidaria: Anthozoa) from the Atlantic ocean using mtMutS and nad2 genes tree reconstructions. Biogeosciences Discussions 9, 16977–16998.

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Peña, C. (2011) Métodos de inferencia filogenética. Revista Peruana de Biología 18, 265–267.

Sá-Pinto, A., Branco, M., Harris, D.J. & Alexandrino, P. (2005) Phylogeny and phylogeography of the genus Patella based on mitochondrial DNA sequence data. Journal of Experimental Marine Biology and Ecology 325, 95–110.

Sánchez, J.A. & Dorado, D. (2008) Intragenomic ITS2 variation in Caribbean seafans. Proceedings of the 11th International Coral Reef Symposium, 1383–1387.

Sánchez, J.A., Lasker, H.R. & Taylor, D.J. (2003)a) Phylogenetic analyses among octocorals (Cnidaria): Mitochondrial and nuclear DNA sequences (lsu-rRNA, 16S and ssu-rRNA, 18S) support two convergent clades of branching gorgonians. Molecular Phylogenetics and Evolution 29, 31–42.

Sánchez, J.A., McFadden, C.S., France, S.C. & Lasker, H.R. (2003)b) Molecular phylogenetic analyses of shallow-water Caribbean octocorals. Marine Biology 142, 975–987.

Schultz, A.L., Malcolm, H.A., Bucher, D.J. & Smith, S.D.A. (2012) Effects of reef proximity on the structure of fish assemblages of unconsolidated substrata. PLoS ONE 7, e49437.

Scyphers, S.B., Powers, S.P., Heck, K.L. & Byron, D. (2011) Oyster reefs as natural breakwaters mitigate shoreline loss and facilitate fisheries. PLoS ONE 6, e22396.

Shearer, T.L., van Oppen, M.J.H., Romano, S.L. & Wörheide, G. (2002) Slow mitochondrial DNA sequence evolution in the Anthozoa (Cnidaria). Molecular Ecology 11, 2475–2487.

Simon, C., Buckley, T.R., Frati, F., Stewart, J.B. & Beckenbach, A.T. (2006) Incorporating molecular evolution into phylogenetic analysis, and a new compilation of conserved polymerase chain reaction primers for animal mitochondrial DNA. Annual Review of Ecology, Evolution, and Systematics 37, 545–579.

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Simons, C., Frati, F., Beckenbach, a, Crespi, B., Liu, H. & Flook, P. (1994) Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Annals of the Entomological Society of America 87, 651–701.

Sunnucks, P. (2000) Efficient genetic markers for population biology. Trends in Ecology and Evolution 15, 199–203.

Taylor, M.L. & Rogers, A.D. (2015) Evolutionary dynamics of a common sub-Antarctic octocoral family. Molecular Phylogenetics and Evolution 84, 185–204.

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Vargas, S., Breedy, O., Siles, F. & Guzmán, H.M. (2010) How many kinds of sclerite? Towards a morphometric classification of gorgoniid microskeletal components. Micron 41, 158–64.

Vargas, S., Guzmán, H.M., Breedy, O. & Wörheide, G. (2014) Molecular phylogeny and DNA barcoding of tropical eastern Pacific shallow-water gorgonian octocorals. Marine Biology 161, 1027–1038.

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Williams, G.C. & Breedy, O. (2004) The Panamic gorgonian genus Pacifigorgia (Octocorallia: Gorgoniidae) in the Galápagos Archipelago, with descriptions of three new species. Proceedings of the California Academy of Sciences 55, 55–88.

Zouros, E., Oberhauser Ball, A., Saavedra, C. & Freeman, K.R. (1994) An unusual type of mitochondrial DNA inheritance in the blue mussel Mytilus. Proceedings of the National Academy of Sciences of the United States of America 91, 7463–7467.

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4. CAPÍTULOS

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4.1 – Taxonomic and faunistic studies of the genera Leptogorgia, Pacifigorgia and Eugorgia (Octocorallia, Gorgoniidae) in Ecuador, with a description of a new species.

4.1.1. Abstract

New records of the genera Leptogorgia, Pacifigorgia and Eugorgia (Octocorallia, Gorgoniidae) on the coast of Ecuador are reported. These new records redefine the current known limit of distribution of these species on the Eastern Pacific coast (from southern California to Chile). Some of these species are reported for the first time since their original descriptions. The newly collected specimens allow for the measurement of the variability of several morphological characters, from colonial to sclerite levels. Additionally, Eugorgia ahorcadensis n. sp., Leptogorgia mariarosacea n. sp., and Pacifigorgia machalilla n. sp., are described and compared with their closest relatives. Morphological differentiation among related species with Pacifigorgia machalilla n. sp., is supported by genetic divergence estimated from an extended barcode of MutS + Igr + COI.

4.1.2. Introduction

The octocorals are found in marine habitats ranging from intertidal to abyssal waters and are distributed from the Arctic to the Antarctic (Bayer 1961). The gorgonian octocorals are one of the best represented taxonomic groups in sublittoral marine ecosystems, are ecologically important and are the dominant macrofaunal group on many tropical reefs (Sánchez et al. 2003a; Williams & Breedy 2004). The study of the Eastern Pacific gorgonian octocorals, specifically in Ecuador, has not matched the intensity and number of publications dedicated to Caribbean species (see Bayer 1981). Despite important contributions by authors such as Breedy and Guzmán (2002, 2007), Williams and Breedy (2004) and Breedy et al. (2009) in the Eastern Pacific, the knowledge on the gorgonian fauna of Ecuador is far from complete (but see Bielschowsky 1929). Four gorgoniid genera (Gorgoniidae) were previously reported in the Eastern Pacific, Phycogorgia Milne-Edwards

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and Haime, 1850, Leptogorgia Milne-Edwards and Haime, 1857, Eugorgia Verrill, 1868, and Pacifigorgia Bayer, 1951. The genus Leptogorgia, with about 60 valid species (27 species in the Eastern Pacific) is one of the most frequent genera in the shallow water communities of the Eastern Pacific (Breedy & Guzmán 2007), with a wide distribution from southern California to Chile. This genus is also present in the Caribbean, Western and Eastern South African coasts, Eastern Atlantic, and the Mediterranean Sea, and one species is known from the Subantarctic (Williams & Lindo 1997). The genus Pacifigorgia is geographically confined to the Pacific coast of tropical America, with the exception of Pacifigorgia elegans (Milne-Edwards and Haime, 1857) from the tropical western Atlantic. Pacifigorgia includes about 36 recognized species distributed throughout the Eastern tropical Pacific region (Breedy & Guzmán 2002, 2003b, 2004; Guzmán & Breedy 2011; Williams & Breedy 2004). The genus Eugorgia, which includes 16 species, is considered exclusive to the Eastern Pacific (from southern California to Peru) (Breedy & Guzmán 2013). Finally, the genus Phycogorgia, with only one species, Phycogorgia fucata (Valenciennes, 1846), is distributed along the west coast of Central and South America (Bayer 1953).

The aim of this report is to present new information derived from material of the genera Eugorgia, Leptogorgia and Pacifigorgia collected in Ecuador. We expand the known distribution of some species in the Eastern Pacific and describe new morphological variability for several taxa. Finally, using morphological and genetic data, we describe three new species of Eugorgia, Leptogorgia and Pacifigorgia, emphasizing the set of morphological characters considered to be of taxonomic importance for this genus (Breedy & Guzmán 2002, 2003b, 2004; Guzmán & Breedy 2011). We include the sequence analysis of the barcode of mtMutS plus COI with a short, adjacent intergenic region (Igr1) proposed by McFadden et al. (2011), with its closest congeners and available sequences in GenBank for Pacifigorgia new species.

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4.1.3. Materials and methods

Study area and sampling methodology

Eleven stations in Ecuadorian waters were sampled for gorgonian octocorals from February 2010 to June 2014 (Fig. 1). Gorgonians were collected by SCUBA diving in a depth range between 5 and 30 metres. Substrata found in these habitats mainly include sand and rocky bottoms. During sampling, we took colour photographs of the living specimens in their environment; specimens were also photographed outside the water, just after collection to ensure the accuracy of our colour descriptions.

Figure 1. Station map in Ecuadorian waters where gorgonians octocorals were sampled from February 2010 to June 2014.

Subsamples were fixed in either 96% ethanol for future molecular analyses, or in buffered 4% formalin (after previous relaxation, performed by adding menthol crystals for a few hours); the rest of the colonies were allowed to air dry. Buffered formalin-fixed subsamples were subsequently transferred to 70% ethanol.

Fragments from different parts of the colonies were prepared for study by scanning electron microscope (SEM), employing the usual methodology previously described by several authors (e.g. Bayer and Stefani 1989; Alderslade 1998), and permanent mounts were made for light microscopy observation (Zapata-Guardiola and López-González 2010). The

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species are described and illustrated according to standard terminology in Bayer et al. (1983), as well as that of the recent descriptive revisions (Breedy & Guzmán 2005, 2007; Breedy et al. 2009).

For either morphological and molecular comparisons, we studied types and additional materials deposited in the Museum of Comparative Zoology, Harvard University (MCZ); Muséum National d’Histoire Naturelle, Paris (MNHN); Natural History Museum, London (NHM); Yale Peabody Museum of Natural History, New Haven (YPM); and National Museum of Natural History, Smithsonian, Washington (NMNH).

Museum specimens were revised for morphological comparative purposes, and in some cases, for molecular analysis. These samples are listed in the text after the newly collected material of each species. Additional museum specimens of species revised but not found in Ecuador, were the following:

− Eugorgia ampla (Verrill, 1864): NHM 69.4.15.53, Baja California (Mexico), no further data. YPM 399, Baja California, 11-15 m depth, 1865. MCZ 65167, Mexico, no depth given, date unknown. − Eugorgia aurantica (Horn, 1860) YPM (4051), Baja California (Mexico), no depth given, 1867-1870. NHM 40.8.22.8, Baja California (Mexico), no depth given, date unknown. MCZ 36185, Baja California (Mexico), no depth given, date unknown. − Eugorgia nobilis Verrill, 1868: YPM 1552a-e, type material, Las Perlas (Panama), 11-15 m depth, 1866. YPM 401, type material, Baja California (Mexico), no further data. MCZ 36316, Baja California (Mexico), no further data. USNM 49371, Gulf of Nicoya (Costa Rica), no depth given, 15 Jan. 1930. USNM 49372, Baja California (Mexico), no depth given, 16 Mar. 1911. − Eugorgia multifida Verrill, 1870: YPM 4605, type material, Mazatlan (Mexico), no depth given, date unknown. USNM 49368, Baja California (Mexico), 11-15 m depth, date unknown. − Pacifigorgia adamsii Verrill, 1868: MCZ 391, type material, Panama, no depth given, 1863. MCZ 36079, type material, Panama, no depth given, 1866-1867. NHM 1930.6.173.9, South Pacific, no depth given, date unknown. YPM 1173, type material,

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Panama, no depth given, 1866-1867. USNM 49688, Las Perlas (Panama), 33 m depth, 5 Mar 1888. USNM 49586, Mexico, no depth given, date unknown. − Pacifigorgia agassizii (Verrill, 1864): MCZ 374, type material, Panama, no depth given, 1863. MCZ 4035, Acapulco (Mexico), no depth given, 1859-1860. MCZ 36266, Baja California (Mexico), no depth given, date unknown. NHM 1885.5.18.2, type material, Mexico, no depth given, 1859-1860. YPM 956, type material, Baja California (Mexico), no depth given, 1860. MNHN-IK 1589, no depth given, date unknown. USNM 49369, Baja California (Mexico), no depth given, date unknown. − Pacifigorgia arenata (Valenciennes, 1846): MNHN-IK 1411, type material, New Zealand, no depth given, 1839. − Pacifigorgia firma Breedy and Guzmán, 2003: MCZ 51918, type material, Culebra Bay (Costa Rica), 20 m depth, 21 Sept. 1997. − Pacifigorgia pulchra var. exilis (Verrill, 1870): YPM 4059, type material, Baja California (Mexico), no depth given, 1867-1870. USNM 75076, Baja California (Mexico), no depth given, date unknown. − Pacifigorgia rutila (Verrill, 1868): MCZ 184, type material, Acapulco (Mexico), no depth given, date unknown. YPM 2266, type material, Acapulco (Mexico), no depth given, 1869-1870.

The newly collected specimens are deposited in the Museo Ecuatoriano de Ciencias Naturales (MECN), the octocoral reference collection of the research group “Biodiversidad y Ecología de Invertebrados Marinos” at the University of Seville (BEIM), Museo Nacional de Ciencias Naturales in Madrid (MNCN-CSIC), Museo de Zoología de la Universidad Tecnológica Indoamérica in Quito (UTI), and Museu de Ciénces Naturals in Barcelona (BCN).

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DNA extraction, PCR amplification, and sequencing

DNA was extracted from 20-30 mg of tissue using the DNeasy extraction kit (Qiagen, Inc.) according to the manufacturer’s protocol. Partial COII-COI (including Igr1 region) and MutS sequences were amplified by PCR using the following primers: COII8068F (McFadden et al. 2004), COIOCTR (France & Hoover 2002), the newly developed COI- Gorg1-R3 (5´-AGAGAAGGTGGTAATAACCAGAAA-3´) and COI-Gorg2-F2 (5´- GATTCGGAAATTGGTTTGTG-3´) for COI + Igr1; ND42599F (France and Hoover 2002) and MUT3458R (Sánchez et al. 2003) for MutS. Amplifications were carried out in a final volume of 50 µl containing 5 µl of 10x buffer (containing 10 x 2 mM MgCl2), 1 µl dNTPs mix (10 mM), 0.8 µl of each primer (10 µM), 0.5 µl of Taq DNA polymerase (5U/µl) (Biotools) and 2 µl of genomic DNA. Thermocycling for the COI fragment included an initial 4 min denaturation step at 94 ºC, followed by 40 cycles of 45 s at 94 ºC, 1 min at 58 ºC and 1 min at 72 ºC. The cycle ended with 10 min of sequence extension at 72 ºC. For MutS, we used an initial 4 min denaturation step at 94 ºC, followed by 35 cycles of 90 s at 94 ºC, 90 s at 58 ºC and 1 min at 72 ºC. The cycle ended with 5 min of sequence extension at 72 ºC. The amplification products were purified by ethanol precipitation. The amplicons were sequenced for both strands using BigDye Terminator in an ABI 3730 genetic analyzer (Applied Biosystems). The sequences obtained were edited using the Sequencher v.4.6 program (Gene Code Corporation, Ann Arbor, MI, USA). The resulting alignments were inspected by eye and manually checked and adjusted with Se-Al v2.0a11 (Rambaut 2002). The distance matrix was obtained using PAUP*v4.0b10 (Swofford 2003). A molecular data matrix was created with the morphologically closest species, together with other published sequences for Gorgoniidae in GenBank (see Table 1).

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Table 1. Pacifigorgia species involved in the molecular comparisons carried out in this paper. Materials in bold are species sequenced for this study. Note that all GenBank sequences are considered here with the names as they appear in GenBank and their original publications (including numbers or letters). For sequences with duplicate complete names, we have include (1), (2), (3), etc., for the purpose of correctly identifying the sequence in Table 5.

Species Catalog. Nos Igr + COI mtMutS

Pacifigorgia bayeri Breedy 2001 voucher HGM77 HG917061 HG917044

Pacifigorgia catedralensis (1) Breedy & Guzmán 2004 voucher HGM109 HG917065 HG917019

Pacifigorgia catedralensis (2) Breedy & Guzmán 2004 voucher HMG112 HG917066 HG917020

Pacifigorgia exilis Verrill 1870 YPM 4059 KX351878 KX351871

Pacifigorgia firma Breedy & Guzmán 2003 MCZ 51918 KX351879 KX351872

Pacifigorgia irene Bayer, 1951 voucher HMG10 HG917070 HG917024

Pacifigorgia irene Bayer, 1951 MNCN 2.04/1174 KX351880 KX351873

Pacifigorgia machalilla n. sp. (1) MECN Ant0058 KX351881 KX351874

Pacifigorgia machalilla n. sp. (2) MECN Ant0059 KX351882 KX351875

Pacifigorgia machalilla n. sp. (3) MECN Ant0061 KX351883 KX351876

Pacifigorgia media Verrill 1864 Parrin et al. 2009 GQ342421 GQ342497

Pacifigorgia sculpta Breedy & Guzmán 2004 MCZ 57053 KX351884 KX351877

Pacifigorgia smithsoniana Breedy & Guzmán 2004 voucher HMG59 HG917076 HG917023

Pacifigorgia stenobrochis Valenciennes 1846 voucher HMG100 HG917078 HG917026

Pacifigrogia rubicunda (1) Breedy & Guzmán 2003 voucher HMG01 HG917073 HG917032

Pacifigrogia rubicunda (2) Breedy & Guzmán 2003 voucher HMG29 HG917074 HG917033

4.1.4. Results

Order Alcyonacea Verrill, 1866

Suborder Holaxonia Studer, 1887

Family Gorgoniidae Lamouroux, 1812

4.1.4.1. Genus Eugorgia Verrill, 1868

Lophogorgia Horn, 1860: 233.

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Gorgonia Verrill, 1864:33, 1866:327.

Leptogorgia Verrill 1864: 32.

Eugorgia Verrill, 1868a: 406–407; Studer, 1887: 64–65; Bielschowsky 1918: 39, 1929: 170; Kükenthal 1924: 343; Stiasny 1951:63; Bayer 1951: 99, 1981: 921; Breedy et al. 2009: 8.

Eugorgia ahorcadensis Soler-Hurtado & López-González, 2012

(Figures 2, 3)

Type material: MECN (Ant0056), holotype, one whole colony with holdfast, Los Ahorcados, Manabi (Ecuador), 1º40’44”S 80º50’08”W, 5 m depth, 27 Feb 2010. BEIM-CRO (072), fragments of the holotype. MECN (Ant0040), paratypes, El Chichó, Manabí (Ecuador), 1°31'15.39"S 80°49'21.37"W, 20 m depth, 24 Nov. 2013, three whole colonies without holdfast.

Description of the holotype: The colonies are up to 190 mm in high by 100 mm in width. The colony is bushy, branching and irregularly dichotomous. Two main branches arise from a short stem, approximately 5 mm in diameter. The unbranched distal twigs can reach up to 20 mm in length, 1.5 mm in diameter, compressed proximally, being more cylindrical and slightly tapered at the ends (Figure 2A,B). There are slightly marked longitudinal grooves near the base and along the thick basal branches. The holdfast circular is 0.9 mm in diameter. The polyps retract within slightly raised polyp-mounds, almost flat, with a rounded aperture; on all sides of branches, in multiple rows; scarce on the stem, absent on the holdfast. The colony is brownish-orange, with red rings on polyp- mounds (Figure 2B). The coenenchymal sclerites are red, yellow or bicoloured (Figure 2C). The spindles and disc-spindles are abundant, up to 0.17 mm in length, 0.10 mm in width, with 4-11 whorls of warty tubercles; they are straight or bent, some with a marked waist (Fig. 2C, 3A). The capstans reach up to 0.09 mm in length, 0.06 mm in width (Fig. 2C, 3B). Reddish sclerites are dominant in deep coenenchymal layers, while yellow ones are mainly present on the surface. The mostly double discs reaching up to 0.06 mm in length, 0.05 mm in width (Fig. 2C, 3C). No anthocodial sclerites were obtained from the colony for

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study.

Figure 2. Eugorgia ahorcadensis n. sp. Holotype (MECN Ant0056). A, colony; B, detail of a branch; C, light micrograph of sclerites.

Bathymetric and geographic distribution: Currently, Eugorgia ahorcadensis n. sp. is known only from the type locality (Los Ahorcados, Manabí, Ecuador); at 5 m depth.

Etymology: The species name was chosen to reflect the type locality where the new taxon was discovered (Los Ahorcados, Manabí, Ecuador).

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Figure 3. Eugorgia ahorcadensis n. sp. Holotype (MECN Ant0056). SEM photographs. A, spindles and disc- spindles; B, captans; C, double-disc.

Comparison with other species of Eugorgia: The taxonomic characteristics of Eugorgia are similar to those used in the taxonomy of the other gorgoniid genera Leptogorgia and Pacifigorgia, a combination of morphological and chromatic features as stated previously, including the detailed structure of the sclerites (see also Grasshoff 1988; Breedy and Guzmán 2007; Breedy et al. 2009) (Table 2).

In examining the recent review of the genus Eugorgia carried out by Breedy et al. (2009), E. ahorcadensis n. sp. appears, from a morphological point of view, most similar to

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Eugorgia ampla (Verrill 1864) and Eugorgia bradleyi Verrill, 1868. They share a similar colony shape (bushy), branching pattern (irregular-dichotomus), and prominence of the polyp-mounds (slightly raised).

However, Eugorgia ahorcadensis n. sp. is differentiated from E. ampla and E. bradleyi by the absence of complete double discs and crosses. Moreover, there are differences in spindle size (up to 0.17 mm in length in E. ahorcadensis n. sp.; only up to 0.15 in the other two species) and spindle ornamentation (E. ahorcadensis n. sp. has 4-11 whorls of warty tubercles, while there are 4-5 in E. ampla, and 4-9 in E. bradleyi) (Table 2). In addition, E. ahorcadensis n. sp. has red-coloured rings on its polyp-mounds (Eugorgia aurantiaca is the only other species with rings (yellow) in the genus; see Breedy et al. 2009: 15, Figure 2C). Eugorgia bradleyi and E. ahorcadensis n. sp. share the presence of disc spindles (a spindle with whorls of tubercles more or less completely fused into discs), bent spindles and bicoloured sclerites, but these sclerite types are absent in E. ampla. Moreover, E. bradleyi lacks capstans, a sclerite type present in the other two species. Anthocodial rods were not found in any of these species. The colour of the colonies of all species used in this comparison is similar (orange, purplish red, brown). The colour of the sclerites is similar in the three species (orange), but in E. ahorcadensis n. sp. the presence of reddish sclerites is dominant, especially in the deeper coenenchymal layers.

Concerning the geographical distribution, E. bradleyi has been reported in Costa Rica and Panama, while E. ampla has a wide distribution in the eastern Pacific, with known records from Mexico, Panama and Peru (see summarized data in Breedy et al. 2009). At present, E. ahorcadensis n. sp. is known only from its type locality, Los Ahoracados (Machalilla National Park, Ecuador).

From a bathymetric point of view, verified records show E. ampla and E. ahorcadensis n. sp. from shallow water (≤ 30 m depth). For E. bradleyi the available information is clearly insufficient, and no bathymetric data have been indicated in the literature (Breedy et al. 2009).

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Table 2. Comparative morphological features of Eugorgia ahorcadensis n. sp. with its closest congeners.

Characters Eugorgia Eugorgia Eugorgia bradleyi ahorcadensis ampla

Color of colony brownish-orange orange purplish red/ orange/ light brown

Number of fans one (or more small) one (or more one (or more small) small)

Polyp mounds sligthly raised or flat sligthly raised sligthly raised or flat

Colour of sclerites red/yellow orange yellow/orange

Spindles max. size (mm) 0,17 legth 0,13-0,15 legth 0,15 legth

0,10 width 0,05 width 0,05 width

Double disc (mm) 0,06 legth 0,06 legth 0,08 legth

0,05 width 0,06 width 0,06 width

Capstan max. size (mm) 0,09 legth 0,09 legth -

0,06 width 0,06 width Anthocodial max. size (mm) - - 0,10 legth

0,03 width

Shape and colour of - - - anthocodial rods

Bicolour sclerites present; coenenchyme absent present; coenenchyme

References This paper Breedy et al. Breedy et al. 2009 2009

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Eugorgia daniana Verrill, 1868

(Figs. 4,5)

Eugorgia daniana Verrill, 1868a: 409–410; Bielschowsky, 1918: 45, 1929: 181; Kükenthal, 1924: 346; Stiasny, 1951: 65; Harden, 1979: 123–125; Prahl et al., 1986: 17; Breedy et al., 2009: 17.

Newly collected examined material: MECN (Ant0033), Salinas, Santa Elena (Ecuador), 2°12’50.01”S 80°56’5.93”W, 15m depth, 9 Dic. 2011, one whole colony. MNCN (2.04/1173), Salinas, Santa Elena (Ecuador), 2°12’50.01”S 80°56’5.93”W, 15m depth, 10 Dic. 2011, one whole colony. MECN (Ant0060), Punta Gruesa, Manabí (Ecuador), 1°33’38.15”S 80°50’5.28”W, 18 m depth, 20 Feb. 2013, one whole colony. MECN (Ant0056), El Chichó, Manabí (Ecuador), 1°31’15.39”S 80°49’21.37”W, 20 m depth, 24 Nov. 2013, one whole colony. MNCN (2.04/1179), El Burbullón, Manabí (Ecuador), 1°28’23.59”S 80°51’23.04”W, 25 m depth, 23 Nov. 2013, one whole colony.

Additional examined materials: Eugorgia daniana MCZ 7080, type material, Gulf of Nicoya (Costa Rica), no depth given, May 1868; MCZ 723, type material, Las Perlas (Panama), no depth given, 1866-1867; NHM 69.4.15.55, type material, Panama, no depth given, data unknown. Eugorgia daniana MCZ 02138, Cali (Colombia), 3 m depth, Dec. 1979; USNM 59084 Gulf of Guayaquil (Ecuador), 20 m depth, Sept. 1966.

Description: The colonies are up to 110 mm in length by 190 mm in width and arise from a circular holdfast, 0.5 mm in diameter. They are profusely and pinnately branched in multiple planes. Main stems are 3-5 mm in diameter, compressed, and very short (up to 10 mm long) or absent (Fig. 4A). The branches arise directly from the base, and are cylindrical, ranging from 0.8 to 1.2 mm in diameter. Some of the branches are pseudoanastomosed (anastomosis to the coenenchyme level, but not including the axes). The unbranched distal twigs can reach up to 15 mm in length and are blunt at the ends (Figs. 4A, B). In some branches, there are numerous and evident yellow, longitudinal

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grooves. The polyps retract within prominent polyp-mounds, leaving a small bilabiate aperture; they occur in multiple rows on all sides of the branches, having sometimes yellow spots. Colonies are dark orange or bright red streaked with bright yellow on the branches or on the polyp-mounds (Fig. 4B). The coenenchymal sclerites are red, yellow or bicoloured (Fig. 4C). They are double discs reaching up to 0.07 mm in length, 0.06 mm in width (Figs. 4C, 5B). The capstans reach up to 0.08 mm in length, 0.05 mm in width (Figs. 4C). The dominant sclerite types are spindles (up to 0.15 mm in length, 0.05 mm in width); disc-spindles (up to 0.14 mm in length, 0.05 mm in width) are also abundant, with 4-6 whorls of warty tubercles; they are straight or bent, some with a marked waist (Fig. 5A, D). The crosses are up to 0.14 mm in length, 0.06 mm in width (Figs. 4C, 5C). No anthocodial sclerites were obtained.

Geographic and bathymetric distribution: This species has a wide distribution along the Eastern Pacific. It has been recorded in USA, Mexico, El Salvador, Nicaragua, Costa Rica, Panama, Colombia, Ecuador (Galapagos and the Gulf of Guayaquil) and Peru (Fig. 23K), at 3-30 m depth (Verrill 1868; Breedy et al. 2009, present record).

The only specimen of Eugorgia daniana previously found in continental Ecuadorian waters was collected in the Gulf of Guayaquil (identified by Bayer in 1966, and published by Breedy et al. 2009). Our record from Manabí and Santa Elena is the second finding of this species in continental Ecuador, providing information about the possible existence of widespreadrich populations of this species in this area. Currently, Eugorgia daniana and E. ahorcadensis Soler-Hurtado and López-González, 2012 are the only species of this genus found in Ecuadorian waters.

Remarks: Differentiation between E. daniana and E. aurantica is difficult at first sight due to a similar branching pattern and colour of their colonies. However, E. aurantica has polyp-mounds with characteristic yellow rings and lacks disc-spindles. Eugorgia daniana, E. aurantica and E. multifida Verrill, 1870 conform the “daniana-group” (Breedy et al. 2009, 2013), which includes species with irregular pinnate branching, prominent polyp-mounds

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and a similar colour of colonies and sclerites.

Figure 4. Eugorgia daniana (MNCN 2.04/1179). A, colony; B, detail of a branch; C, light micrograph of sclerites.

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Figure 5. Eugorgia daniana (MNCN 2.04/1179) SEM photographs. A, coenenchymal sclerites, spindles; B, double disc; C, crosses; D, disc-spindles; E, complete double disc.

Genus Leptogorgia Milne-Edwards and Haime, 1857

Gorgonia Pallas, 1766: 160; Milne-Edwards and Haime, 1857: 157.

Leptogorgia Milne-Edwards and Haime, 1857: 163; Verrill, 1868b: 387, 1869: 420; Kükenthal, 1924: 324; Bielschowsky, 1929: 81; Stiasny, 1943: 87; Bayer, 1961: 214; Grasshoff, 1988: 97, 1992: 54; Williams, 1992: 231; Williams and Lindo, 1997: 500; Breedy and Guzmán, 2005: 2,

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2007: 6.

Lophogorgia Milne-Edwards and Haime, 1857: 167; Kükenthal 1924: 322; Bielschowsky 1929: 73; Bayer 1956: 212, 1961: 194.

Filigorgia Stiasny 1937: 309, 1939: 301; Bayer 1956: 206.

Leptogorgia alba (Duchassaing and Michelotti, 1864)

(Figs. 6, 7)

Lophogorgia alba Duchassaing and Michelotti, 1864: 19; Rossi, 1956: 198; Prahl et al., 1986: 17; Volpi and Benvenuti, 2003: 58.

Gorgonia (Leptogorgia) rigida var. laevis Verrill, 1866: 327.

Gorgonia (Litigorgia) laevis Verrill, 1868a: 415.

Leptogorgia alba Verrill, 1868b: 398-399, 1869: 421; Studer 1894: 68; Bielschowsky 1918: 30, 1929: 107-108; Kükenthal 1919: 771, 1924: 329; Hickson 1928: 400-405; Breedy and Guzmán 2007: 12-19.

Leptogorgia alba var. sulcata Kükenthal, 1919: 771, 1924: 329; Bielschowsky, 1929: 108-110.

Euplexaura lemasti Hickson, 1928: 349-351; Stiasny, 1941: 266-267, 1943: 63.

Leptogorgia fasciculata Bielschowsky, 1918: 29, 1929: 94-96; Kükenthal, 1919: 912, 1924: 326; Stiasny 1951: 60.

Newly collected examined material: MECN (Ant0001), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 15 m depth, 27 Feb. 2010, three whole colonies. MECN (Ant0019), Isla de Salango, Manabí (Ecuador), 1°35’55.13”S 80°52’0.01”W, 7 m depth, 20 Nov. 2011, two whole colonies. MECN (Ant0020), Salinas, Santa Elena (Ecuador), 2°12’50.01”S 80°56’5.93”W, 15 m depth, 18 Dec. 2011, one whole colony. MECN (Ant0027), Los Frailes, Manabí (Ecuador), 1°30’14”S 80°48’33”W, 10 m depth, Dec. 2011, three whole colonies. MECN (Ant0030), Los Frailes, Manabí (Ecuador), 1°30'14”S 80°48'33”W, 15 m depth, 19 March. 2012. BEIM (0071), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 15 m

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depth, 27 Feb. 2010, one whole colony. BEIM (0074), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 5 m depth, 27 Feb. 2010, one whole colony. MNCN (2.04/482), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, Feb. 2010, one whole colony. MNCN (2.04/483), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 15 m depth, 28 Feb. 2010, one whole colony. MZB (2016-2989), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 15 m depth, 27 Feb. 2010, one whole colony. UTI (MZUTI- Inv02), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 15 m depth, 28 Feb. 2010, one whole colony. MECN (Ant0040), Punta Mala, Manabí (Ecuador), 1°33’41.37”S 80°50’8.79”W, 16 m depth, 02 Feb. 2013, one whole colony. MECN (Ant0041), Islote La Viuda, Manabí (Ecuador), 1°26’5.95”S 80°46’7.30”W, 15 m depth, 23 Nov. 2013, one whole colony. MECN (Ant0050) Punta Machalilla, Manabí (Ecuador), 1°28’33.53”S 80°47’38.04”W, 13 m depth, 24 Nov. 2013, one whole colony.

Additional examined materials: Litigorgia laevis, MCZ 5416, type material, Las Perlas (Panama), no depth given, date unknown; MCZ 7008, type material, Golfo de Nicoya, (Costa Rica), no depth given, May 1868. Leptogorgia laevis, YPM IZ 001639, type material, Las Perlas, (Panama), no depth given, 1868. Leptogorgia alba, NHM 1946.1.14.52, Toboga (Panama), no depth given, date unknown; NHM 30.6.17.13, Toboga (Panama), no depth given, date unknown. Leptogorgia alba, USNM 59078, Ecuador, 8-9 m depth, 8 May 1966; USNM 1016223, Ecuador, no depth given, Dec. 1937.

Description: The colonies measure up to 350 mm in length and 105 mm wide, irregularly pinnate; branches slender, mostly in a plane (Fig. 6A). Unbranched distal twigs up to 100 mm in length and 20 mm in diameter, compressed proximally, more cylindrical and slightly tapered distally (Fig. 6A, B). The holdfast circular, up to 5 mm in diameter. Slightly marked longitudinal grooves along the thick basal branches and near the base. The polyps retract within slightly raised polyp-mounds, sparsely distributed all around the branches with oblong apertures (Fig. 6B). The colour of the colony is white. The coenenchymal sclerites hyaline (Fig. 6C). The dominant sclerite type spindles up to 0.14 mm in length and 0.03 mm wide, with 4-6 whorls of tubercles; straight or bent, some with a marked waist

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(Figs. 6C, 7A). The capstans up to 0.07 mm in length and 0.03 mm wide (Fig. 7B). Crosses not found. The anthocodial sclerites hyaline rods up to 0.08 mm in length and 0.02 mm wide, with some marginal projections (Figs. 6C, 7C).

Geographic and bathymetric distribution: Leptogorgia alba has been reported in Mexico, El Salvador, Costa Rica, Panama, Colombia, and the Galápagos Islands (Ecuador) (Bielschowsky 1929; Breedy & Guzmán 2007; Duchassaing & Michelotti 1864), at 3-30m depth (Fig. 29A).

Although Leptogorgia alba has already been collected in Ecuador (Galápagos Islands) (Breedy & Guzmán 2007), this is the first time that it was found on the continental coast since the paper of Bielschowsky (1929).

Remarks: Our material is in agreement with the original description (Duchassaing and Michelotti 1864:19) and its later re-description (Breedy and Guzmán 2007:12-19). Due to its white colour, it is easily recognizable underwater. Breedy and Guzmán (2007) considered that its morphological variability, especially the type of branching, could be perhaps a response to several environmental factors. In our samples we observe a more pinnate pattern, although there is a tendency toward a dichotomous branching in some specimens. In the type material, the spindles are long, up to 0.17-0.18 mm in length and 0.04-0.06 mm in width, with marked and complex tubercles. However, our material shows smaller spindles (up to 0.14 x 0.03 mm) with a less marked ornamentation. In the same way, the anthocodial rods are considered very consistent in size and shape (long and conspicuous) in this species (up to 0.15 mm in length) (Breedy & Guzmán 2007). Nevertheless, the examined Ecuadorian material shows a smaller maximum length (up to 0.08 mm).

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Figure 6. Leptogorgia alba (MNCN 2.04/482). A, colony; B, detail of a branch; C, ligh micrograph of sclerites.

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Figure 7. Leptogorgia alba (MNCN 2.04/482) SEM photographs. Coenenchymal sclerites, A, spindles; B, captans; C, anthocodial sclerites, rods.

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Leptogorgia diffusa (Verrill, 1868)

(Figs. 8, 9)

Litigorgia diffusa Verrill, 1868a: 397.

Gorgonia (Litigorgia) diffusa Verrill, 1868a: 415.

Leptogorgia diffusa Verrill, 1868b: 397, 1869: 421; Nutting, 1910: 5; Bielschowsky, 1918: 30, 1929: 112; Kükenthal, 1919: 771, 1924: 329-330; Hickson, 1928: 413-414; Stiasny, 1938: 29; Breedy and Guzmán, 2007: 32-37.

Leptogorgia rubra Bielschowsky, 1918: 29, 1929: 92-94; Kükenthal, 1919: 911-912, 1924: 325.

Lophogorgia diffusa Prahl et al., 1986:21.

Newly collected examined material: MECN (Ant0032), Punta Mala, Manabí (Ecuador), 1°33’41.37”S 80°50’8.79”W, 13 m depth, 3 Abr. 2012, one whole colony. MECN (Ant0005), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 30 m depth, 28 Feb. 2010, two whole colonies. BEIM (0080), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, Feb. 2010, one whole colony. BEIM (0078), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 20 m depth, 28 Feb. 2010, one whole colony. MNCN (2.04/484), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 15 m depth, 27 March 2012, one whole colony. MNCN (2.04/485), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 30 m depth, 27 Feb. 2010, one whole colony. MZB (2016-2990), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 30 m depth, 27 Feb. 2010, one whole colony. MZB (2016- 2991), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 25 m depth, 27 Feb. 2010, one whole colony. UTI (MZUTI-Inv09), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 30 m depth, 28 Feb. 2010, one whole colony. UTI (MZUTI-Inv01), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, 27 Feb. 2010, one whole colony. MECN (Ant0042), El Burbullón, Manabí (Ecuador), 1°28’23.59”S 80°51’23.04”W, 25 m depth, 23 Nov. 2013, one whole colony.

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Additional examined materials: Leptogorgia diffusa MCZ 7081, type material, Golfo de Nicoya (Costa Rica), no depth given, May 1868. Litigorgia diffusa YPM 1659A, type material, Las Perlas (Panama), no depth given, 1866-1867. Leptogorgia diffusa MCZ 4972, Mexico, no depth given, date unknown. MNHN-IK 2233, no further data.

Description: The colonies are up to 670 mm in length by 950 mm in width. The branching pattern is irregularly pinnate; branches are flat, lax, pinnate, slender and on a plane (Fig. 8A). The unbranched distal twigs can reach up to 22 mm in length and 17 mm in diameter (Figs. 8A, B). The holdfast is slightly flat, up to 15 mm in diameter. The polyps retract within prominent polyp-mounds, sparsely distributed all around the branches with slit-like apertures (Fig. 8B). The colour of the colony and of the coenenchymal sclerites is red (Figs. 8A-C). The spindles are the dominant sclerite type, up to 0.11 mm in length and 0.03 mm in width, with 4-6 whorls of tubercles, they are straight or bent, some with a marked waist (Figs. 8C, 9A). The capstans reach up to 0.07 mm in length and 0.04 mm in width (Figs. 8C, 9B). Some small, scattered crosses are found (0.04 x 0.05 mm) (Fig. 8C). The anthocodial sclerites are orange flattened rods, up to 0.13 mm in length and 0.02 mm in width, with lobe-like marginal projections (Figs. 8C, 9C).

Geographic and bathymetric distribution: Leptogorgia diffusa has been previously reported in California, El Salvador, Costa Rica, Panama, Colombia, and Chile at 5-30 m depth (see Verrill 1868; Bielschowsky 1929; Prahl et al. 1986; Breedy and Guzmán 2007) (Fig. 29B). This new record fills the gap between the northern and southern records of the species. Leptogorgia diffusa has probably a wider distribution, especially in offshore areas of Mexico and Peru, but it may have been previously overlooked. Although this species is quite frequent in Ecuador, it is usually observed isolated within multispecies assemblages and does not form large gorgonian gardens.

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Figure 8. Leptogorgia diffusa (MNCN 2.04/1172). A, colony; B, detail of a branch; C, light micrograph of sclerites.

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Figure 9. Leptogorgia diffusa (MNCN 2.04/1172) SEM photographs. Coenenchymal sclerites, A, spindles; B, captans; C, anthocodial sclerites, flattened rods.

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Remarks: The morphology of this species is very constant in all examined samples. It is easily differentiable from other Leptogorgia species by the lax, pinnate style of branching, with prominent polyp-mounds. Breedy and Guzmán (2007:34-35) noted anthocodial sclerites that were light orange, dark pink, or both; however, our materials are only orange. The coenenchymal sclerites are a bit smaller (up to 0.11 x 0.03 mm) than previously described (up to 0.14-0.15 x 0.05 mm) (Verrill 1868: 398; Breedy and Guzmán 2007: 34). In addition, the Ecuadorian material has scattered small crosses not reported in this species before.

Leptogorgia flexilis (Verrill, 1868)

(Figs. 10, 11)

Gorgonia (Eugorgia) flexilis Verrill, 1868a: 415.

Litigorgia flexilis Verrill, 1868a: 400.

Leptogorgia flexilis Verrill, 1868b: 400; Nutting, 1910: 5; Bielschowsky, 1918: 29, 1929: 96; Kükenthal, 1919: 771, 1924: 326; Hickson, 1928: 414-416; Stiasny 1943: 82; Breedy and Guzmán 2007: 40-44.

Newly collected examined material: MECN (Ant0034), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, 27 Feb. 2010, one whole colony. BEIM (0085), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, 27 Feb. 2010, one whole colony. MNCN (2.04/1171), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 5 m depth, 27 Feb. 2010, one whole colony.

Additional examined materials: Litigorgia flexilis (Eugorgia flexilis) MCZ 722 (4123), type material, Las Perlas (Panama), 6 to 8 m depth, 1860´s. Litigorgia flexilis NHM 69.4.15.15 (1946.1.14.74), type material, Toboguilla (Panama), 5 m depth, 1915. Leptogorgia flexilis YPM 569, Panama, no depth given, 1867-1868.

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Description: The colonies measure up to 200 mm in length by 40 mm in width. The branching pattern is irregularly pinnate, mostly on one plane; branches are lank and bushy with long, slender, and flexible branches, drooping slightly at the ends (Fig. 10A). The unbranched distal twigs can reach up to 50 mm in length and 18 mm in diameter (Figs. 10A, B). The holdfast is circular, up to 10 mm in diameter. The polyps retract within nearly flat polyp-mounds, closely distributed all around the branches, with oblong apertures (Fig. 10B). The colour of the colony is brown (Figs. 10A, B). The coenenchymal sclerites are red, pink and yellow; some of them are bicoloured (Fig. 10C). The capstans are the dominant sclerite type, up to 0.07 mm in length and 0.03 mm in width (Figs. 10C, 11B). The spindles reach up to 0.1 mm in length and 0.03 mm in width, with 4-6 whorls of tubercles; they are straight or bent, some with a marked waist (Fig. 11A). Some crosses or four-radiates (up to 0.03 x 0.03 mm) (Fig. 11D) and six-radiates (up to 0.03 x 0.02 mm) are also found. The anthocodial sclerites are pink flattened rods up to 0.16 mm in length and 0.08 mm in width, with short lobe-like marginal projections (Fig. 10D).

Geographic and bathymetric distribution: This species has been previously reported in California, El Salvador and Panama at 5-30 m depth (Breedy & Guzmán 2007; Verrill 1868b) (Fig. 29C), and thus our specimens represent a sizeable expansion to the south.

Remarks: Under water, the species is characterized by its decumbent or drooping branching pattern and its brown colonies. However, in small colonies the drooping habit is not as evident, and sometimes the holdfast and basal branches are intense yellow. Crosses or four-radiate sclerites were not mentioned by Breedy and Guzmán (2007) in their revision of different type materials (e.g, deposited at MZC and YPM). However, they are evident in our collections and in the type specimens at NHM. This type of sclerite had been noted by Verrill (1868) as well, and it should be considered diagnostic for the species.

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Figure 10. Leptogorgia flexilis (MNCN 2.04/1171). A, colony; B, detail of a branch; C, light micrograph of coenenchymal sclerites; D, light micrograph of anthocodial sclerites.

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Figure 11. Leptogorgia flexilis (MNCN 2.04/1171) SEM photographs. Coenenchymal sclerites, A, spindles; B, captans; C, six-radiate; D, four-radiate.

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Leptogorgia mariarosae Soler-Hurtado & López-González, 2012 (Figures 12, 13)

Examined material: Holotype, MECN (Ant0102), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 25 m depth, 27 Feb 2010, one whole colony with holdfast. BEIM-CRO (073), fragments of the holotype.

Description of the holotype: The colony measures up to 33 mm in length by 73 mm in width. The branching pattern is irregularly pinnate; branches lax, slender, mostly in one plane (Figs. 12A,C). The unbranched distal twigs up to 7 mm in length, 1.2 mm in diameter. The holdfast was not observed. The polyps retract within prominent polyp-mounds, distributed on all sides of branches, with small slit-like apertures. The colour of the colony is red (Figs. 12A,B). The coenenchymal sclerites are red (Fig. 12B). The dominant sclerite types are spindles (up to 0.17 mm x 0.03 mm), with 4-10 whorls of tubercles; straight or bent, some with a marked waist (Figs. 12C, 13A). The capstans reach up to 0.08 mm in length, 0.03 mm wide (Fig. 13B). The anthocodial sclerites are flattened rods (up to 0.11 mm x 0.03 mm) (Fig. 13C), with smooth or short lobe-like marginal projections, light yellow in colour.

Geographic and bathymetric distribution: Currently, Leptogorgia mariarosae n. sp. is known only from the type locality (Los Ahorcados, Manabí, Ecuador); 25 m depth.

Etymology: The species name is a dedication to Maria Rosa Hurtado García, mother of the first author (MMSH).

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Figure 12. Leptogorgia mariarosae n. sp. Holotype (MECN Ant0102). A, colony; B, detail of a branch; C, light micrograph of sclerites.

Comparison with other Leptogorgia species: Based on the recent review of the central eastern Pacific Leptogorgia species published by Breedy and Guzmán (2007), Leptogorgia mariarosae n. sp. appears, from a morphological point of view, similar to Leptogorgia aequatorialis Bielschowsky, 1929 and Leptogorgia diffusa (Verrill 1868b) (Table 3). All share a similar branching pattern (irregular-pinnate), prominent polyp-mounds and reddish colony colour. However, Leptogorgia mariarosae n. sp. is differentiated from L. aequatorialis and L. diffusa by its spindle size (up to 0.17 mm in length; only up to 0.10 mm

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in L. aequatorialis and 0.15 mm in L. diffusa) and spindle ornamentation (L. mariarosae n. sp. has 4-10 whorls of warty tubercles, while there are 3-4 in L. aequatorialis and 4-8 in L. diffusa).

Figure 13. Leptogorgia mariarosae n. sp. SEM photographs. A, coenenchymal sclerites; B, anthocodial sclerites.

Anthocodial sclerites of L. aequatorialis are small and biscuit-shaped rods up to 0.04 mm in length, 0.03 mm in wide, with smooth or lobed margins; in L. diffusa they are long,

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somewhat flattened rods, up to 0.14 mm in length, smooth with short lobe-like marginal projections. In L. mariarosae n. sp., they are flattened rods, up to 0.11 mm in length, 0.03 mm in width, with smooth or short lobe-like marginal projections. In addition, L. aequatorialis has crosses; these are absent in both L. mariarosae n. sp., and L. diffusa (see Breedy and Guzmán 2007: 8, Figure 2). Leptogorgia aequatorialis and L. mariarosae n. sp. share the presence of bent spindles which are absent in L. diffusa. The colour of the colonies for all species in this comparison is similar (red to deep orange). The colour of the sclerites is also similar in the three species (red, pink, or orange). Anthocodial armature coloring is similar in L. mariarosae n. sp. and L. diffusa (light orange, light yellow); those of L. aequatorialis (reddish-pink) (Table 3).

Concerning the known geographical distribution of these species, L. aequatorialis has been recorded from Ecuador, while L. diffusa has a wide distribution in the eastern Pacific, with verified reports from California, Costa Rica, El Salvador, Panama, Colombia and Chile (see summarized data in Breedy and Guzmán 2007). Presently, L. mariarosae n. sp. is known only from its type locality in Ecuador.

From a bathymetric point of view, all three species are present in shallow water (≤ 30 m depth). According to verified records, L. aequatorialis has been found at 4-5 m depth, L. diffusa at 5-30 m, while L. mariarosae n. sp. was found at 25 m.

A few other Leptogorgia species in the eastern Pacific also show a similar colour (reddish-purple, red-rose or purplish-red), branching pattern and prominence of polyp- mounds. They are Leptogorgia parva Bielschowsky, 1929, Leptogorgia ramulus (Milne- Edwards & Haime 1857) (pink variety) and Leptogorgia tabogillae (Hickson 1928). However, in all these species the larger sclerites are up to 0.12 mm, the dominant coenenchymal sclerites are capstans, and there are no bent sclerites. As mentioned previously, sclerites in L. mariarosae n. sp. can reach up to 0.17 mm and the dominant coenenchymal sclerites are spindles, some of them bent. Moreover, coenenchymal sclerites of the other three species are as follows: red, pink or yellow (L. parva; this species also has bicoloured sclerites), red- rose (L. ramulus for its pink variety), or purplish red (L. tabogillae). Anthocodial sclerites are orange (L. parva and L. taboguillae) or light orange (L. ramulus). However, in L.

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mariarosae n. sp., coenenchymal sclerites are red, and anthocodial sclerites are light yellow.

Table 3. Comparative morphological features of Leptogorgia mariarosae n. sp. with its closest congeners.

Leptogorgia Leptogorgia Leptogorgia diffusa Characters mariarosae aequatorialis

Color of colony red deep orange red

Number of fans one two or more one (or more small)

Polyp mounds prominent prominent prominent

Colour of sclerites red orange red/pink

Spindles max. size 0,17 legth 0,10 legth 0,15 legth (mm) 0,03 width 0,04 width 0,05 width

Capstan max. size 0,08 legth 0,08 legth 0,09 legth (mm) 0,03 width 0,04 width 0,06 width

Anthocodial max. size 0,11 legth 0,04 legth 0,14 legth (mm) 0,03 width 0,03 width 0,04 width

Shape and colour of flattened rods/ biscuit-shaped/ flattened rods/ light anthocodial rods yellow reddish-pink orange

Bicolour sclerites absent absent absent

References This chapter Breedy and Gumán Breedy et al. 2009 2007

Leptogorgia obscura Bielschowsky, 1929

(Figs. 14, 15)

Leptogorgia obscura Bielschowsky, 1918: 31, 1929: 119-120; Kükenthal 1919: 914, 1924: 332; Breedy and Guzmán 2007: 57-60.

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Newly collected examined material: MECN (Ant0011), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, 27 Feb. 2010, three whole colonies. MECN (Ant0028), Los Frailes, Manabí (Ecuador), 1°30’14”S 80°48’33”W, 10 m depth, 17 Feb. 2012, one whole colony. BEIM (CRO-0068), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, 27 Feb. 2010, one whole colony. BEIM (CRO-0069), Isla de Salango, Manabí (Ecuador), 1°35’55.13”S 80°52’0.01”W, 7 m depth, 19 Nov. 2011, one whole colony. MNCN (2.04/486), Isla de Salango, Manabí (Ecuador), 1°35’55.13”S 80°52’0.01”W, 7 m depth, 19 Nov. 2011, one whole colony. MNCN (2.04/487), Los Frailes, Manabí (Ecuador), 1°30’14”S 80°48’33”W, 10 m depth, 17 Feb. 2012, one whole colony. UTI (MZUTI-Inv03), Isla de Salango, Manabí (Ecuador), 1°35’55.13”S 80°52’0.01”W, 7 m depth, 19 Nov. 2011, one whole colony. UTI (MZUTI-Inv05), Isla de Salango, Manabí (Ecuador), 1°35’55.13”S 80°52’0.01”W, 13 m depth, 17 Feb. 2012, one whole colony. MZB (2016-2992), Isla de Salango, Manabí (Ecuador), 1°35’55.13”S 80°52’0.01”W, 13 m depth, 19 Feb. 2012, one whole colony. MZB (2016-2993), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, 27 Feb. 2010, one whole colony. MECN (Ant0043), Los Frailes, Manabí (Ecuador), 1°30'14"S 80°48'33”W, 15 m depth, 23 May. 2013, one whole colony. MECN (Ant0044), Islote La Viuda, Manabí (Ecuador), 1°26’5.95”S 80°46’7.30”W, 15 m depth, 23 Nov. 2013, one whole colony. MECN (Ant0049), Punta Machalilla, Manabí (Ecuador), 1°28’33.53”S 80°47’38.04”W, 13 m depth, 24 Nov. 2013, one whole colony.

Description: The colonies are up to 164 mm in length by 75 mm in width. The branching pattern is irregularly dichotomous; branches are bushy, closely ramified and rigid (Figs. 14A). The unbranched distal twigs can reach up to 5 mm in length and 2.3 mm in diameter, are compressed proximally, being more cylindrical and slightly tapered at the ends (Figs. 14A, B). The holdfast is circular, up to 14 mm in diameter. The polyps retract within prominent polyp-mounds leaving oblong apertures, and are closely distributed all around the branches (Fig. 14B). The colour of the colony is purple (Figs. 14A, B). The coenenchymal sclerites are red (Figs. 14C, D). The spindles are the dominant sclerite type, up to 0.13 mm in length and 0.04 mm in width, with 4-5 whorls of tubercles; they are straight or bent, some with a marked waist (Figs. 14C, 15A). The capstans reach up to 0.08 mm in length and

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0.04 mm in width (Figs. 14C, 15B). Some crosses up to 0.06 by 0.04 mm are found (Fig. 15C). The anthocodial sclerites are orange flattened rods, up to 0.07 mm in length and 0.02 mm in width, with lobe-like marginal projections (Figs. 14D, 15D).

Geographic and bathymetric distribution: Leptogorgia obscura was known previously only from the type locality (Bahía de Caráquez, Ecuador) (Bielschowsky 1929) (Fig. 29D). There is an unpublished record from Baja California (Harden 1979), although Breedy and Guzmán (2007) note that this record should be verified. The only reliable previously known depth range for the species was 4-5 m (Bielschowsky 1929), which we now expand to 15 m.

This species is quite abundant in the study area on rocky bottom and usually grows along with Leptogorgia alba.

Remarks: According to Bielschowsky (1929) and Breedy and Guzmán (2007: 57) capstans are the dominant sclerites in the type material. However, spindles are the most common sclerites in our specimens. Crosses or four radiates occur as well, a type of sclerite not reported in this species before.

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Figure 14. Leptogorgia obscura (MECN Ant0011). A, colony; B, detail of a branch; C, light micrograph of coenenchymal sclerites; D, light micrograph of anthocodial sclerites.

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Figure 15. Leptogorgia obscura (MECN Ant0011) SEM photographs. Coenenchymal sclerites, A, spindles; B, captans; C, four-radiates; D, anthocodial sclerites, flattened rods.

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Table 4. Comparative general features of the species of the genera Leptogorgia and Eugorgia in the coast of Ecuador.

Leptogorgia Leptogorgia Leptogorgia Leptogorgia Eugorgia Characters alba diffusa flexilis obscura daniana Color of colony white red brown purple dark orange

irregularly flat, lax, irregularly Type of branching dichotomous pinnae pinnate pinnate pinnate Polyp mounds slightly raised prominent flat prominent prominent Dominant sclerite spindles spindles capstans spindles double-disc type red, pink and Colour of sclerites colourless red red red, yellow yellow

not not yes not yes Bicolour sclerites

Acute spindles 0.15 x 0.05 0.14 x 0.03 mm 0.11 x 0.03 mm 0.1 x 0.03 mm 0.13 x 0.04 mm max. size mm 0.08 x 0.05 Capstan max. size 0.07 x 0.03 mm 0.07 x 0.04 mm 0.07 x 0.03 mm 0.08 x 0.04 mm mm 0.14 x 0.06 Crosses not 0.04 x 0.05 mm 0.03 x 0.03 mm 0.06 x 0.04 mm mm Size and colour of colourless / orange / 0.13 x pink / 0.16 x orange / 0.07 x anthocodial rods not obtained 0.08 x 0.02 0.02 0.08 0.02 (mm) Complete double - - - - yes disc Double disc max. 0.07 x 0.06 - - - - size mm

Disc-spindles - - - - yes

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4.1.4.3 Genus Pacifigorgia Bayer, 1951

Rhipidigorgia Valenciennes, 1855: 13; Milne-Edwards and Haime, 1857: 173; Horn, 1860: 233; Duchassaing and Michelotti: 20, 1864; Verrill 1864: 32.

Litigorgia Verrill, 1868: 414, 1869: 420, 1870: 548.

Gorgonia Bielschowsky, 1918: 32, 1929: 141; Kükenthal, 1924: 338; Stiasny, 1941: 268, 1943: 74.

Pacifigorgia Bayer, 1951: 94.

Pacifigorgia cribrum (Valenciennes, 1846)

(Figs. 16, 17)

Gorgonia cribrum Bielschowsky, 1918: 38, 1929: 150; Kükenthal, 1919: 773, 1924: 340; Valenciennes, 1846: pl. 13 Figs. 1-3.

Rhipidigorgia cribrum Milne-Edwards & Haime, 1857: 175; Valenciennes, 1855: 13.

Leptogorgia cribrum Verrill, 1869: 421.

Pacifigorgia cribrum Bayer & Macintyre 2001: 318; Breedy & Guzmán 2002: 804-808.

Newly collected examined material: MECN (Ant0035), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 15 m depth, 27 Feb. 2010, one whole colony. MECN (Ant0036), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 20 m depth, 28 Feb 2010, one whole colony. MECN (Ant0037), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 15 m depth, 27 Feb. 2010, one whole colony. BEIM (0088), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 25 m depth, 28 Feb. 2010, one whole colony. BEIM (0089), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 20 m depth, 28 Feb. 2010, one whole colony. MECN (Ant0046), El Burbullón, Manabí (Ecuador), 1°28’23.59”S 80°51’23.04”W, 25 m depth, 23 Nov. 2013, one whole colony.

Additional examined materials: Rhipidigorgia cribrum MNHN-IK 1661, type material, New Zealand, no depth given, 1839. Pacifigorgia cribrum MZC 261 (4014), Cape St. Lucas, Baja

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California (Mexico), no depth given, 1859-1861. MCZ 36264, Marcial Point (Mexico), no depth given, date unknown. Pacifigorgia cribrum USNM 49382 (Costa Rica), no depth given, Mar. 1927, F.M. Bayer (Id.). Gorgonia cribrum NMH 58.5.15.237 (Australia), no depth given, date unknown.

Figure 16. Pacifigorgia cribrum (MNCN 2.04/1170). A, colony; B, detail of a branch; C, light micrograph of sclerites.

Description: The colonies are up to 50 mm in length by 73 mm in width, and are formed by two fans: the first fan is the largest and the second one radiates from the holdfast and 147

extends in parallel together with the first fan, until a certain point where both may fused (Fig.16A). The holdfast is very small, up to 4 mm in diameter. The branches are squarish, ranging from 0.6-0.8 mm in diameter (25 meshes/cm2), and the end-branchlets are short, up to 2 mm long. The network is fine and regular; it is formed mostly by square meshes (2 x 2.8 mm by 2.5 x 3 mm) (Fig.16B). There are no midribs; nevertheless, adult specimens have large, slightly compressed principal branches, which arise from near the base, and diverge through the fan, but often for no more than a quarter of the height. The polyps retract within slightly raised polyp-mounds with asterisk-like apertures, placed in multiple rows all around the branches. The colour of the colony is reddish intermingled with yellow. The coenenchymal sclerites are red, light yellow, and bicoloured (Fig. 16C). They are spindles (0.13 x 0.04 mm) having acute ends and 4-6 whorls of tubercles; blunt spindles (0.08 x 0.03 mm) with 4 whorls of tubercles (Figs. 16C, 17A); and capstans (0.07 x 0.04 mm) with tuberculate ends (Figs. 16C, 17B). The dominant sclerite types are acute, straight or bent spindles, some with a marked waist. The anthocodial sclerites are light yellow flattened rods, up to 0.10 mm in length and 0.02 mm in width, with smooth or slightly lobed borders (Figs. 16C, 17C).

Geographic and bathymetric distribution: Pacifigorgia cribrum has been previously reported in Mexico (Breedy & Guzmán 2002) (Fig. 29E), with additional questionable records from New Zealand (MNHN-IK 1661), Australia (NMH 58.5.15.237), and Costa Rica (e.g. USNM 49382, collected in 1927 and identified by M.F. Bayer). In this study we observed this species at 15-30 m depth. A depth range was lacking for it in the literature.

Remarks: According to Bayer and Macintyre (2001) and Breedy and Guzmán (2002), Pacifigorgia cribrum, P. adamsii, P. agassizii and P. rutila may represent a group sharing a set of morphological features, consisting of fine, regular and closely anastomosed networks.

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Figure 17. Pacifigorgia cribrum (MNCN 2.04/1170). SEM photographs. Coenenchymal sclerites, A, spindles; B, captans; C, anthocodial sclerites, flattened rods.

In addition, Pacifigorgia cribrum is morphologically close to P. arenata, both species having a similar colony colour and sclerome characteristics (red, yellow, bicoloured coenenchymal sclerites, and yellow anthocodial sclerites up to 0.10-0.13 mm in length). However, the two species differ in mesh features (presence of midrib and higher meshes in P. arenata (see Breedy and Guzmán 2002). In fact, one of the principal problems in recording the possible morphological and chromatic variability of P. arenata is that the unique known specimen is the holotype, which is deposited in Paris (MNHN).

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Pacifigorgia flavimaculata Breedy and Guzmán, 2003

(Figs. 18, 19)

Newly collected examined material: MECN (Ant0021), Salinas, Santa Elena (Ecuador), 2°12’50.01”S 80°56’5.93”W, 15 m depth, 10 Dec. 2011, one whole colony. MECN (Ant0022), Salinas, Santa Elena (Ecuador), 2°12’50.01”S 80°56’5.93”W, 18 m depth, 11 Dec. 2011, one whole colony. BEIM (0075), Salinas, Santa Elena (Ecuador), 2°12’50.01”S 80°56’5.93”W, 17 m depth, 10 Dec. 2011, one whole colony. BEIM (0070), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, 27 Feb. 2010, one whole colony. MNCN (2.04/490), Los Frailes, Manabí (Ecuador), 1°30’14”S 80°48’33”W, 10 m depth, 13 March 2012, one whole colony. MNCN (2.04/491), Salinas, Santa Elena (Ecuador), 2°12’50.01”S 80°56’5.93”W, 17 m depth, 10 Dec. 2011, one whole colony. MZB (2016-2994), Los Frailes, Manabí (Ecuador), 1°30’14”S 80°48’33”W, 10 m depth, 20 Feb. 2012, one whole colony. UTI (MZUTI-Inv07), Los Frailes, Manabí (Ecuador), 1°30’14”S 80°48’33”W, 10 m depth, 20 Feb. 2012, one whole colony. UTI (MZUTI-Inv08), Los Frailes, Manabí (Ecuador), 1°30’14”S 80°48’33”W, 15 m depth, 10 March 2012, one whole colony. MECN (Ant0047), El Chichó, Manabí (Ecuador), 1°31’15.39”S 80°49’21.37”W, 20 m depth, 24 Nov. 2013, one whole colony. MECN (Ant0048), Punta Machalilla, Manabí (Ecuador), 1°28’33.53”S 80°47’38.04”W, 13 m depth, 24 Nov. 2013, one whole colony.

Additional examined material: Pacifigorgia flavimaculata MCZ 51922, type material, Punta Salsipuedes (Costa Rica), 3 m depth, 22 Jan 1994.

Description: The colonies reach up to 105 mm in length by 70 mm in width and are formed by two parallel fans. The holdfast was not observed (Fig.12A). The branches are cylindrical, ranging from 1.5-2.2 mm in diameter (1-3 meshes/cm2). The branches arise directly from the base and have incomplete anastomoses that form a loose, open and irregular network. The meshes are rounded-square, oblong or triangular (19 x 5 mm, 9 x 4 mm) (Fig.18B). There are no distinct midribs. There are long end-branchlets (up to 11 mm). The polyps

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retract within prominent polyp-mounds having rounded apertures, placed all around the branches in multiple rows. The colour of the colony is light brown when alive and yellow intermingled with light brown and light purple when dry. The coenenchymal sclerites are pink and light yellow, some of them bicoloured (Fig. 18C). They are spindles (0.14 x 0.04 mm) having acute ends and 4-5 whorls of tubercles; blunt spindles (0.11 x 0.04 mm) with 4 whorls of tubercles (Figs. 18C, 19A, B), and capstans (0.09 x 0.04 mm) with tuberculate ends, warty or smooth (Figs. 18C, 19B. The dominant sclerite types are straight or bent spindles, some of them with a marked waist. The anthocodial sclerites are orange flattened rods, up to 0.13-0.14 mm in length and 0.02 mm in width, with pointed projections, and acute and warty ends (Figs. 18C, 19C).

Geographic and bathymetric distribution: Pacifigorgia flavimaculata has only been reported from the type locality in Costa Rica at 3 m depth (Breedy & Guzmán 2003b) (Fig. 29F). In the present study, it was observed at a 3-20 m depth.

Breedy and Guzmán (2003) pointed out that this species was only observed in the type locality (Punta Salsipuedes, Costa Rica), despite a significant sampling effort in the neighbouring areas. Therefore, our record from Ecuador is important in defining the geographic (and bathymetric) distribution of this species.

Remarks: Colonies of this species mainly form loose and irregular networks, sometimes almost pseudoanastomosed, yellow or brown in colour but with purplish or yellowish spots around a prominent polyp-mound, making the species easily recognizable.

The Ecuadorian specimens have a quite constant set of features in comparison to the type specimens examined (Breedy and Guzmán 2003, pers. observ.), except for the size of the anthocodial sclerites, which are longer (up to 0.13-0.14 mm, instead of up to 0.08- 0.09 mm) (see Breedy and Guzmán 2003: 23, present paper).

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Figure 18. Pacifigorgia flavimaculata (MNCN 2.04/1178). A, colony; B, detail of a branch; C, light micrograph of sclerites.

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Figure 19. Pacifigorgia flavimaculata (MNCN 2.04/1178) SEM photographs. Coenenchymal sclerites, A, acute spindles; B, capstans; C, anthocodial sclerites, flattened rods.

Pacifigorgia irene Bayer, 1951

(Figs. 20, 21)

Leptogorgia adamsii Verrill, 1868b: 391-392, 1869: 421.

Gorgonia adamsii Hickson, 1928: 380-383; Kükenthal, 1924: 339.

Gorgonia media Galtsoff, 1950: 27.

Pacifigorgia irene Bayer, 1951: 94-96; Breedy & Guzmán 2002: 825, 2003: 38-40.

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Newly collected examined material: BEIM (0091), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, 8 Dic. 2011, one whole colony. MNCN (2.04/1174), Punta Mala, Manabí (Ecuador), 1°33’41.37”S 80°50’8.79”W, 12 m depth, 16 Feb. 2012, one whole colony. MZB (2016-2996), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 15 m depth, 15 April 2012, one whole colony. MECN (Ant0062), Los Frailes, Manabí (Ecuador), 1°30’14”S 80°48’33”W, 15 m depth, 10 March 2012, one whole colony. MECN (Ant0054), El Chichó, Manabí (Ecuador), 1°31’15.39”S 80°49’21.37”W, 20 m depth, 24 Nov. 2013, one whole colony. MECN (Ant0055), Islote La Viuda, Manabí (Ecuador), 1°26’5.95”S 80°46’7.30”W, 15 m depth, 23 Nov. 2013, one whole colony.

Additional examined material: Pacifigorgia irene USNM 49379, type material, Nicoya (Costa Rica), no depth given, Mar. 1927; USNM 33611, type material, Costa Rica, no depth given, date unknown. Pacifigorgia irene MZC 36232, Gulf of Fonseca (Costa Rica), no depth given, date unknown.

Description: The colonies reach up to 600 mm in length by 500 mm in width, and are formed by one to several fans (Fig. 20A). The holdfast was not observed. The branches are slender, ranging from 0.3 to 0.7 mm in diameter (about 35 meshes/cm2). The branches form a fine and regular network. The network consists of small and squarish meshes (usually up to 1.2 by 0.9 mm) (Fig. 20B). The fans present several stout, rounded midribs. There are short end-branchlets (<1 mm long). The polyps retract within slightly raised and crowded polyp-mounds, placed all around the branches. The colour of the colony is reddish intermingled with yellow both when alive and dry, with a slight discoloration at the edges of the colony. The coenenchymal sclerites are red, lemon yellow, and orange, some of them bicoloured (Fig. 20C). They consist of long spindles (0.17 x 0.05 mm) having acute ends and 5-6 whorls of tubercles (Figs. 20A, 21A), and capstans (0.08 x 0.04 mm) with tuberculated ends (Fig. 21B). In our specimens the dominant sclerite type are acute straight or bent spindles, some with a marked waist. Some crosses up to 0.06 by 0.05 mm occur as well (Fig. 21C). The anthocodial sclerites are light yellow and light pink flattened rods, up to 0.11 mm in length and 0.02 mm in width, with smooth or slightly lobed borders (Figs. 20, 21C).

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Figure 20. Pacifigorgia irene (MNCN 2.04/1174). A, colony; B, detail of a branch; C, light micrograph of sclerites.

Geographic and bathymetric distribution: Pacifigorgia irene has been previously reported in Panama and Costa Rica at 12-33 m depth (Bayer 1951; Breedy & Guzmán 2002, 2003b) (Fig. 29G). Our finding represents a considerable southerly extension of its known distribution.

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Figure 21. Pacifigorgia irene (MNCN 2.04/1174) SEM photographs. Coenenchymal sclerites, A, spindles; B, captans; C, four-radiates.

Remarks: This species is easily recognized by its characteristic morphology showing a wide fan or fans, marked and thick midribs and small meshes. Some coenenchymal sclerites have been considered here as elongated capstans instead of blunt spindles, as commonly described. The morphology of some sclerites are not always easy to define in this taxon, and they can also be considered transitional forms of blunt spindles, depending on the

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development of the two opposite distal processes on the longitudinal axis with respect to the two central whorls with alternate tubercles (Vargas et al. 2010b). In any case, if these sclerites are considered blunt spindles, they would have equivalent forms with respect to the original description of this species. In situ specimens seen during this study show a more intense reddish and yellow colour than Costa Rican specimens. Bayer (1951) described the colour of the colony as dark purple with greenish borders, but this is not the case in the Ecuadorian material, where colour only slightly fades at the edges of the fans.

Pacifigorgia machalilla Soler-Hurtado, Machordom, Muñoz & López-González, 2016

(Figs. 22-23)

Examined material: Holotype: MECN (Ant0061), Cope, Manabí (Ecuador), 1°43'34"S 80°59'85"W, 23 m depth, 8 Dic. 2012, one whole colony without holdfast. Paratypes: MBZ (2016-2196), Los Ahorcados, Manabí (Ecuador), 1º40’44”N 80º50’08”W, 10 m depth, 1 Dic. 2011, one whole colony. MNCN (2.04/1181), Cope, Manabí (Ecuador), 1°43'34"S 80°59'85"W, 23 m depth, 8 Dic. 2012, a fragment of the colony. Other material: UTI (Inv262), Cope, Manabí (Ecuador), 1°43'34"S 80°59'85"W, 23 m depth, 8 Dic. 2012, one whole colony. BEIM (0095), Isla de la Plata, Manabí (Ecuador), 1°16'25.84"S 81° 4'11.70"W, 22 m depth, 22 Feb. 2012, two whole colonies. MECN (Ant0058), Los Ahorcados, Manabí (Ecuador), 1º40’44”N 80º50’08”W, 10 m depth, 27 Feb. 2010, one whole colony. MECN (Ant0059), Los Ahorcados, Manabí (Ecuador), 1º40’44”N 80º50’08”W, 10 m depth, 1 Dic. 2011, one whole colony.

Description of the holotype: The colony is formed by a single fan 250 mm in length by 290 mm in width (Fig. 22A). The holdfast is circular, up to 15 mm in diameter. The branches are cylindrical, ranging from 1.5-2 mm in diameter (7-9 meshes/cm2) and the end-branchlets reach up to 9 mm in length, and have blunt tips. The network is regular and complete, and is formed of mostly square and rectangular meshes (4 x 5 mm, 10 x 3 mm); in some cases, meshes are oblong (Fig. 22B). There are five or six prominent, long and strong midribs, which divide into others that progressively fuse among the anastomosed structure of the

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mesh (Fig. 22A). The polyps retract within slightly raised or flat polyp-mounds with slit- like apertures, placed in multiple rows all around the branches. The colour of the colony is intense red-brown when dry and bluish-gray when alive. The coenenchymal sclerites are pink, light yellow, and orange, some of them bicoloured (Fig. 22C). They are spindles (up to 0.20 x 0.04 mm) having acute ends and 5-6 whorls of tubercles (Figs. 22A, 23A); blunt spindles are absent, and there are capstans (up to 0.7 x 0.03 mm) with tuberculate ends (Figs. 22C, 23B). The dominant sclerites are spindles, straight or bent, some of them with a distinct waist. There are irregular crosses with different branch lengths (up to 0.11 x 0.05 mm) (Fig. 23C). The anthocodial sclerites are pink, light orange, and light yellow in colour, most of them bicoloured; flattened rods (0.11 x 0.02 mm) with short pointed or lobe-like marginal projections, and acute or rounded ends, occur as well. There are also platelets (0.06 x 0.02 mm) (Figs. 22C, 23D).

Variability: This new species is fairly constant with regards to colonial and sclerome characters. However, in some specimens there are small secondary fans growing parallel to the primary fan. In addition, in some small colonies (about 100 mm in length) the midrib is not very obvious or even absent, or is only formed by two short basal branches.

Geographic and bathymetric distribution: Pacifigorgia machalilla n. sp. is only known from the type locality in Cope, Los Ahorcados, and Isla de la Plata (continental coast of Ecuador), living on rocky bottoms in shallow waters at a depth of 10-23 m.

Etymology: The new species is dedicated to the National Park of Machalilla (Ecuador) and its staff for their constant support and help during field work. Name considered as a noun in apposition.

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Figure 22. Pacifigorgia machalilla n. sp. Holotype MECN (Ant0053). A, colony; B, detail of anastomosed branches; C, light micrograph of sclerites; D, light micrograph of anthocodial sclerites.

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Figure 23. Pacifigorgia machalilla n. sp.. Holotype MECN (Ant0053). SEM photographs. Coenenchymal sclerites, A, acute spindles; B, captans; C, irregular crosses or four radiates and butterfly; D, anthocodial sclerites, flattened rods.

Comparison with other Pacifigorgia species: Pacifigorgia machalilla n. sp. is morphologically close to P. exilis and P. firma, having similar networks with the presence of a midrib, and similar mesh size (9-11.5 meshes/cm2) and anastomosis. The network in P. machalilla n. sp. is formed by almost square or rectangular meshes, 10 x 3 mm, 4 x 5 mm, and 12 x 3 mm, 6 x 2 mm in P. firma (Table 5). Pacifigorgia exilis also has a similar type of network (open or closed and regular); however, it has smaller square or oblong meshes (0.5 x 3 mm). Additionally, P. machalilla n. sp. has blunt spindles while they are absent in both P. exilis and P. firma, and the acute spindles are larger in P. machalilla n. sp. (up to 0.20 x 0.04 mm)

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than in P. exilis (up to 0.11 x 0.05 mm) and P. firma (up to 0.11 x 0.05 mm). Finally, P. machalilla n. sp. has crosses or radiate forms, which are lacking in the other two species (Table 5).

Table 5. Comparative morphological features of Pacifigorgia machalilla n. sp. with its closest congeners.

Pacifigorgia machalilla Pacifigorgia exilis Pacifigorgia firma Characters n. sp.

Light brown/ dark Color of colony Intense red-brown Red-orange purple

Number of fans One or two small several One (or two)

Number of meshes/cm2 7 - 9 7 - 9 11.5

Mesh shape and max. mesh Square -rectangular/ reg/ Square – oblong / rectangular / reg/ size (mm) 10 x 3 – 4 x 5 irreg/ 3 x 0.5 12 x 3 - 6 x 2

Presence of Midrib present present present

Polyp mounds Sligthly raised or flat Sligthly raised Sligthly raised

red/pale Colour of sclerites Pink /yellow/orange Red / yellow yellow/orange

Acute spindles max. size 0,20 legth 0,04 width 0,11 legth 0,05 width 0,11 legth 0,05 width (mm)

Blunt spindles max. size absent 0,09 legth 0,04 width absent (mm)

0,07 legth 0,07 legth 0,09 legth Capstan max. size (mm) 0,03 width 0,04 width 0,05 width

Crosses or radiates present absent absent

Anthocodial max. size (mm) 0,11 legth 0,02 width 0,12 legth 0,02 width 0,10 legth 0,03 width

Shape and colour of flattened rods; flattened rods; flattened rods; pink/ anthocodial rods orange / light yellow yellow ligth orange

present; coenenchyme present; present; and anthocodial Bicolour sclerites coenenchyme coenenchyme

Breedy and Guzmán Breedy and Guzmán References This chapter 2002 2003a

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The molecular distance matrix (COII + Igr + COI + MutS) shows that, within the genus Pacifigorgia, the genetic divergence among species was close to 1% or lower in most cases. The new species showed no intraspecific variation (Table 5). Despite the fact that P. machalilla n. sp. is closely related morphologically to P. firma and P. exilis, as stated previously, the three species are clearly separated molecularly. The maximum genetic divergence is 0.3% between P. machalilla n. sp. and P. firma, and is 1% between P. machalilla n. sp. and P. exilis (Table 6). However, P. machalilla n. sp. is closely related molecularly to P. irene and P. smithsoniana (Breedy & Guzmán 2004). The maximum genetic divergence between P. machalilla n. sp. and P. smithsoniana or P. irene is 0.06 %. However, in a thorough analysis of the sequences for these three species, we observed the following differences: 1) there is a silent mutation in COI for P. machalilla n. sp. and P. smithsoniana; and 2) there is a mutation in the first base of one triplet which changes Lys to Val, in COII for P. machalilla n. sp. and P. irene. It may be interesting to extend the sequencing based on COII in the search of additional variable segments in the mtDNA or nuclear DNA of this group of species.

Figure 24. Comparison of the anastomosed branches in Pacifigorgia machalilla n. sp., holotype (A) and Pacifigorgia irene (B).

Despite its molecular similarities, there are clear morphological differences among these three species. Pacifigorgia machalilla n. sp. differs from P. smithsoniana in having a

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midrib, and larger acute spindles (0.20 x 0.04 vs 0.14 x 0.05 mm). Pacifigorgia machalilla n. sp. differs from P. irene in that its anastomosis forms a network of almost square or rectangular meshes 10 x 3 mm, 4 x 5 mm (only 2 x 0.9 mm in P. irene), its branches are larger, ranging from 1.5-2 mm in diameter (7-9 meshes/cm2), while the branches in P. irene are 0.5-0.7 mm in diameter (35 meshes/cm2), with longer end-branchlets (up to 9 mm long), while these are very short in P. irene, less than 1 mm (Fig. 24). Finally, bicolour anthocodial sclerites are a unique feature for P. machalilla n. sp..

Table 6. Genetic divergence matrix for the species examined based on the COII + Igr + COI and MutS-1 genes sequence.

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Pacifigorgia rubicunda Breedy and Guzmán, 2003

(Figs. 25, 26)

Newly collected examined material: MECN (Ant0023), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, 27 Feb. 2010, two whole colonies. MECN (Ant0025), Punta Mala, Manabí (Ecuador), 1°33'41.37"S 80°50'8.79"W, 15 m depth, 15 Feb. 2012, three whole colonies. BEIM (0083), Isla de la Plata, Manabí (Ecuador), 1°16'25.84"S 81° 4'11.70"W, 22 m depth, 19 Feb. 2012, one whole colony. BEIM (0082), Los Frailes, Manabí (Ecuador), 1°30'14"S 80°48'33”W, 10 m depth, 10 March 2012, one whole colony. BEIM (0081), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 5 m depth, 27 Feb. 2010, one whole colony. MNCN (2.04/488), Isla de la Plata, Manabí (Ecuador), 1°16'25.84"S 81° 4'11.70"W, 20 m depth, 19 Feb. 2012, one whole colony. MNCN (2.04/489), Punta Mala, Manabí (Ecuador), 1°33'41.37"S 80°50'8.79"W, 12 m depth, 27 Feb. 2012, one whole colony. UTI (MZUTI-Inv04) Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, 27 Feb. 2010, one whole colony. MZB (2016-2997), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 5 m depth, 27 Feb. 2010, one whole colony. MZB (2016-2998) Isla de la Plata, Manabí (Ecuador), 1°16'25.84"S 81° 4'11.70"W, 20 m depth, 19 Feb. 2012, one whole colony.

Additional examined material: Pacifigorgia rubicunda MCZ 51917, type material, Gulf of Chiriquí (Panama), 14 m, Dec. 2001.

Description: The colonies reach up to 40 mm in length by 160 mm in width, and are formed by several fans. The secondary fans sprout from the main fan at a right angle and spread perpendicularly to form new fans. Several fans may reunite, adhering together and producing square arrangements like beehives. A short stem divides close to the holdfast (Fig. 25A), which is up to 12 mm in diameter. The branches are cylindrical, ranging from 0.8-1 mm in diameter (9-11 meshes/cm2). The end-branchlets are short. The network is closed and regular; it is formed of mostly square or oblong meshes (10 x 3 mm, 5.5 x 3 mm)

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(Fig. 25B). The polyps retract within slightly raised polyp-mounds with slit-like apertures, placed in multiple rows all around branches. The colour of the colony is brownish-orange when alive or preserved and a conspicuous burnt sienna colour speckled with yellow when dry. The coenenchymal sclerites are pink, orange, and lemon yellow, bicoloured and multicoloured (Fig. 25C). They are spindles (0.1 x 0.03 mm) having acute ends and 10-12 whorls of tubercles (Figs. 25C, 26A); blunt spindles (0.1 x 0.04 mm) with 4-5 whorls of tubercles (Figs. 25C, 26B); and capstans (0.07 x 0.03 mm) with tuberculate ends (Figs. 25C, 26C). The dominant sclerite types are blunt spindles straight or bent, some with a marked waist. Crosses are lacking. The anthocodial sclerites are yellow flattened rods up to 0.12 mm in length and 0.02 mm in width, with short lobe-like marginal projections, and acute and warty or smooth ends (Figs. 25C, 26C).

Geographic and bathymetric distribution: Pacifigorgia rubicunda was originally described by Breedy and Guzmán (2003) from Costa Rica (Fig. 29I). The geographic range of the species is here extended to Ecuador (Manabí). Its bathymetric distribution ranges from 5 to 30 m depth (Breedy & Guzmán 2003b).

Remarks: There are some morphological differences between the specimens of P. rubicunda found in Ecuador and the original description of this species by Breedy and Guzmán (2003). The colour of sclerites is very similar (orange and yellow); however, in Ecuadorian material most of the sclerites are pink (bicoloured or multicoloured) and colourless sclerites are lacking. If compared with the type material (6-9 meshes/cm2, 5-1.2 x 2.5-0.9 mm) (Breedy & Guzmán 2003b), in our colonies the network is less compact, usually more elongate and larger, and there are no crosses.

Finally, the size of sclerites and anthocodial rods seems to be somewhat larger in the type material (spindles 0.09-0.12 x 0.03-0.04 mm, capstans 0.04-0.09 x 0.02-0.04 mm; anthocodial rods 0.14 x 0.05 mm) than in the Ecuadorian material (spindles 0.1 x 0.03 mm, capstans 0.07 x 0.03 mm; anthocodial rods 0.12 x 0.02 mm). In most cases, P. rubicunda was found forming colonies similar to beehives, irregularly stretching across the rocks.

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The midrib extends from the holdfast in parallel to the substrate, but the unique point of union is the holdfast, which is diagnostic for this species (Breedy and Guzmán 2003: 43).

Figure 25. Pacifigorgia rubicunda (MZB 2016-2997). A, colony; B, detail of a branch; C, light micrograph of sclerites.

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Figure 26. Pacifigorgia rubicunda SEM photographs. Coenenchymal sclerites, A, spindles; B, captans; C, anthocodial sclerites, flattened rods.

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Newly collected material of P. rubicunda from Ecuador permits the description of variability of some characters. The species is reported herein for the first time since its original description in Costa Rica.

Pacifigorgia stenobrochis (Valenciennes, 1846)

(Figs. 27, 28)

Gorgonia stenobrochis Bielschowsky, 1918: 39, 1929: 155; Galtsoff, 1950: 27; Hickson, 1928: 387-390; Kükenthal, 1919: 773, 1924: 341-342; Stiasny 1951: 32; Valenciennes 1846: 12.

Rhipidigorgia stenobrochis Milne-Edwards & Haime, 1857: 176; Valenciennes, 1855: 13; Verrill, 1864: 32.

Gorgonia (Rhipidigorgia) stenobrochis Verrill, 1866: 327.

Gorgonia stenobrochis Verrill, 1868b: 414.

Leptogorgia stenobrochis Verrill, 1868a: 393-394, 1869: 421.

Pacifigorgia stenobrochis Bayer, 1951: 94; Breedy & Guzmán, 2002: 833-835, 2003: 50-52.

Newly collected examined material: BEIM (0090), Salinas, Santa Elena (Ecuador), 2°12’50.01”S 80°56’5.93”W, 15m depth, 9 Dic. 2011, a fragment of colony. MNCN (2.04/1176), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, 12 Dic. 2011, a fragment of colony. MNCN (2.04/1177), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 13 m depth, 7 Dic. 2012, a fragment of colony. MZB (2016-2999), Punta Mala, Manabí (Ecuador), 1°33’41.37”S 80°50’8.79”W, 15 m depth, 12 Dic. 2012, a fragment of colony. MNCN (2.04/1175), Salinas, Santa Elena (Ecuador), 2°12’50.01”S 80°56’5.93”W, 15m depth, 11 Dic. 2011, a fragment of colony. MECN (Ant0065), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, 15 Mar. 2012, a fragment of colony. MECN (Ant0064), Punta Mala, Manabí (Ecuador), 1°33’41.37”S 80°50’8.79”W, 15 m depth, 15 Dic. 2011, a fragment of colony. MECN (Ant0063), Punta Mala, Manabí (Ecuador), 1°33’41.37”S 80°50’8.79”W, 13 m depth, 6 April 2012, a fragment of colony. MECN (Ant0051), El Chichó, Manabí (Ecuador), 1°31’15.39”S 80°49’21.37”W, 20 m depth, 24 Nov. 2013, a fragment of

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colony. MECN (Ant0052), El Burbullón, Manabí (Ecuador), 1°28’23.59”S 80°51’23.04”W, 25 m depth, 23 Nov. 2013, a fragment of colony. MECN (Ant0053), Islote La Viuda, Manabí (Ecuador), 1°26’5.95”S 80°46’7.30”W, 15 m depth, 23 Nov. 2013, a fragment of colony.

Additional examined materials: Rhipidigorgia stenobrochis MNHN-IK 1719, type material, New Zealand, no depth given, 1839. Pacifigorgia stenobrochis var. engelmanni (Horn 1860) MCZ 4042, Acapulco (Mexico), no depth given, 1856-1860; Pacifigorgia stenobrochis MCZ 28753, Mexico, no depth given, date unknown; Gorgonia stenobrochis NHM 1930.6.17.12, St. George (Mexico), no depth given, date unknown. Pacifigorgia stenobrochis USNM 49378, Gulf of Nicoya (Costa Rica), no depth given, March 1927. Pacifigorgia stenobrochis USNM 8847, Baja California (Mexico), no depth given, 1911.

Description: The colonies are up to 280 mm in length by 270 mm in width and are formed by a single fan that may divide into smaller fans. The holdfast was not observed (Fig. 27A). The branches are rounded, ranging from 1.1-2.3 mm in diameter (2 meshes/cm2). The branches arise directly from the base and have an open and irregular network. The meshes are rectangular or oblong (55 x 6 mm, 10 x 5 mm) (Fig. 27B). There are no distinct midribs. There are long end-branchlets (up to 26 mm). The polyps retract within flat polyp- mounds, placed throughout the branches, on both sides. The colour of the colony is light brown or dark yellow when alive and yellow intermingled with brown. The coenenchymal sclerites are light yellow (Fig. 27C). There are long acute spindles (0.16 x 0.04 mm), straight or bent, some with a marked waist, having 8 whorls of tubercles, and they are the dominant sclerite types. In addition, there are blunt spindles (0.12 x 0.04 mm) with 6 whorls of tubercles (Figs. 27C, 28A), and capstans (0.09 x 0.04 mm) with tuberculate ends (Figs. 27A, 28B). Some crosses up to 0.08 by 0.06 mm are also found (Fig. 27C). The anthocodial sclerites are light orange in colour, with flattened rods up to 0.09 mm in length and 0.02 mm in width, with smooth or slightly lobed borders (Figs. 27A, 28D).

Geographic and bathymetric distribution: Pacifigorgia stenobrochis has been reported in Mexico, El Salvador, Nicaragua, Costa Rica, Panama, Peru (Breedy & Guzmán 2003b; Verrill

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1868b) and Ecuador (current study) at 3-30 m depth. In the original description, New Zealand is indicated as the type locality, which appears to be an error (see Valenciennes 1846; Breedy and Guzmán 2002, 2003) (Fig. 29J).

Generally, colonies of this species occur both solitary and mixed with other species in the same environment. The Ecuador records nicely connect the previously known localities from Mexico and Peru.

Remarks: The chromatic variability of the colonies and sclerites of this species has been known for a long time (Breedy & Guzmán 2002, 2003b; Hickson 1928). In the Ecuadorian material studied here, all the coenenchymal sclerites are yellow. Pink and grey sclerites noted by other authors are lacking. The colour of Ecuadorian colonies varies between yellow and orange hues, while it is said to be reddish purple and brown in previous descriptions.

The presence of thick and robust branches, shape of the mesh and absence of the midrib are diagnostic for P. stenobrochis, and readily separate it from similar species, like P. firma.

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Figure 27. Pacifigorgia stenobrochis (MNCN 2.04/1175). A, colony; B, detail of a branch; C, light micrograph of sclerites.

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Figure 28. Pacifigorgia stenobrochis (MNCN 2.04/1175). SEM photographs. Coenenchymal sclerites, A, spindles; B, captans; C, crosses; D, anthocodial sclerites, flattened rods.

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Table 7. Comparative general features of the species of the genus Pacifigorgia in the coast of Ecuador.

P. P. P. P. Characters P. irene sculpta P. rubicunda cribrum flavimaculata stenobrochis red, reddish reddish Color of orange brownish- yellow with light brown or (with with colony and orange brown yellow yellow) yellow yellow

two or one or Number of several two two One or more three more fans Number of 2-4 6-9 25 1-3 2 35 meshes/cm2

Mesh shape oblong / square or square / 2 rounded-square, and maximun oblong/ 55 x 6, squarish/ 2 13 x 3, oblong/ x 2.8, 2.5 oblong mesh size 0 x 5 x 0.9 mm 4 x 3 10 x 3, 5.5 x 3 x 3 19 x 5, 9 x 4 (mm)

slightly slightly slightly Polyp mounds slightly raised prominent flat raised raised raised

reddish pink, orange red and red, lemon Colour of pink and light and pale and lemon light light yellow yellow and sclerites yellow yellow yellow yellow orange

Bicolour yes yes yes yes not yes sclerites

Acute spindles 0.24 x 0.13 x 0.04 0.16 x 0.04 0.17 x 0.05 0.1 x 0.03 mm - max. size 0.03 mm mm mm mm

Blunt spindles 0.14 x 0.08 x 0.1 x 0.04 mm 0.14 x 0.04 mm 0.12 x 0.04 mm - max. size 0.04 mm 0.03 mm

Capstan max. 0.06 x 0.07 x 0.03 0.07 x 0.09 x 0.04 0.08 x 0.04 0.09 x 0.04 mm size 0.02 mm mm 0.04 mm mm mm

0.08 by 0.06 0.06 x 0.05 Crosses not not not not mm mm

pale light Size and pale yellow/ pink and yellow/ yellow/ orange/ 0.14 x orange/ 0.09 x colour of 0.12 x 0.02 yellow/ 0.11 0.16 X 0.10 x 0.02 0.02 mm 0.02 mm anthocodial mm x 0.02 mm 0.02 mm mm

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Figure 29. Geographical distribution of the Leptogorgia, Pacifigorgia and Eugorgia new records species mentioned in the paper. The solid circle shows the relative position of the species in the countries where they have been recorded. The empty circles indicate the first record for the species in Ecuador.

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4.1.5. Discussion

Eight of the species identified are new records for the octocoral fauna of Ecuador (Table 1, 2): Leptogorgia diffusa, L. flexilis, Pacifigorgia cribrum, P. flavimaculata, P. irene, P. rubicunda and P. stenobrochis. Another three species, Leptogorgia alba, L. obscura and Eugorgia daniana, had already been reported in Ecuador by other authors (Bielschowsky 1929; Breedy & Guzmán 2007). The new species described, Eugorgia ahorcadensis, Leptogorgia mariarosae, and Pacifigorgia machalilla n. sp., requires additionally records to better establish its geographical and bathymetric distribution.

These species, together with L. aequatorialis (Bielschowsky, 1929), constitute the current list of gorgoniid species from the continental coast of Ecuador. In addition, to complete the species list for Ecuadorian waters, those species described above for the Galapagos Islands like Pacifigorgia darwinii (Hickson, 1928), P. dampieri Williams and Breedy, 2004, P. symbiotica Williams and Breedy, 2004 and P. rubripunctata Williams and Breedy, 2004 should be added.

According to Bayer (1961), gorgonian corals systematics relies on a combination of characters such as axial skeleton (if any), colonial form, polyp arrangement, and sclerites morphology. However, the often unwieldy older literature with no or neglected illustrations and lost or badly preserved type material have played a major role in the taxonomic confusion around octocoral systematics (Sanchez 2004). Currently, variation in sclerite size, sculpture and colouration and the relative proportion of the different sclerite types constitute the main criteria for species delimitation in Gorgoniidae (Breedy 2001; Sánchez et al. 2007; Vargas et al. 2010b; Williams & Lindo 1997). These characters vary widely and the ranges of variation have not been established for all gorgoniid taxa, hindering a robust taxonomy for the group.

The description of sclerite type can sometimes be difficult, because of continuous variation in size and ornamentation within and between species (Sánchez et al. 2003a; Vargas et al. 2010b; Williams & Lindo 1997). This continuum represents a major obstacle to the assignment of size intervals or ranges that could be used to define and separate possible species. Vargas et al. (2010a) proposed for Pacifigorgia, the combined use of continuous and discrete morphological characters, in order to define the relationships

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between species at different resolution level, in an integrative approach with molecular data.

Despite the rebirth of invertebrate systematics due to the ever-increasing availability of DNA sequence characters (Dueñas & Sánchez 2009; Mallet & Willmott 2003; Sanchez 2004), evaluation of the effectiveness of molecular barcodes in octocorals is largely hindered by lack of knowledge regarding species boundaries in these organisms. Moreover, the rate of octocoral mitochondrial gene evolution is very slow. It is estimated to be 10-100 times slower than other metazoans (Brockman & McFadden 2012; Chen et al. 2009), resulting in insufficient resolution to discriminate species within many genera (Sánchez et al. 2003; Lepard 2003 in Cairns and Bayer 2005). Specimens identified as different morphospecies can share the same barcode, which should motivate additional taxonomic work to test species boundaries and quantify intraspecific morphological and molecular variation (McFadden et al. 2010). For instance, Sánchez et al. (2003) exploring mitochondrial genes ND2, ND6 and MutS, observed that in Eunicea spp. and Plexaura spp. the number of substitutions supporting nodes was low (less than 0.005 substitutions/site), and the clade formed by these two genera could be considered unresolved from a molecular point of view. In addition, McFadden et al. (2011) showed that congeners of the genera Isidella, Keratoisis, and Lepidisis, were identical at COI and Igr1 sequences, and only vary 0-2% for MutS.

In this study, we found that pairs of morphologically different species, such as Pacifigorgia exilis and P. media (Verrill, 1864) or P. bayeri Breedy, 2001, and P. catedralensis Breedy and Guzmán 2004, showed no interspecific variation (0%, in COII+Igr+COI+MutS, Table 6); while others, like P. firma and P. catedralensis, P. rubicunda and P. exilis, or P. rubicunda and P. media showed a very low genetic divergence (0.006%). In fact, the maximum genetic divergence observed in this study is 1.3%, between P. media and P. firma, and between P. exilis and P. firma, confirming the reduced variability in the genetic regions examined.

The analysis combining MutS and Igr1-COI for DNA barcoding indicates that these markers are not always suitable and conclusive for species-level identification of Eastern Pacific octocorals (Vargas et al. 2014). However, we can say that these regions can at least

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define different species groups. This may serve as a supporting tool for morphological findings among species within and between groups.

According to our results, based on strong morphological data and molecular tests with sufficient resolution for this set of octocoral species, we propose Pacifigorgia machalilla n. sp. as a new species.

In conclusion, we recommend the use of molecular tools as a necessary complement to morphological identification for future descriptions of new species. Although mitochondrial markers are known to evolve at much lower rates than in other zoological groups, they still provide information that should not be neglected. These mitochondrial markers can be complemented with nuclear regions (e.g. 28S, ITS, and SRP54, among others) that have been demonstrated to be especially useful for some octocoral families and genera (McFadden et al. 2014; Sánchez et al. 2007; Wirshing & Baker 2015).

Although much more work is needed to fully understand the morphological diversification of octocorals, a combination of molecular and morphological data is a very promising approach to disentangling phylogenetic relationships among species (Breedy & Guzmán 2013; López-González et al. 2014; McFadden & Van Ofwegen 2013) and intraspecific population ecology (Calderón et al. 2006; Prada & Hellberg 2013; Prada et al. 2008). Molecular and morphological analytic tools will be essential to quantify the variety of evolutionary pathways within these groups (Concepcion et al. 2010; McFadden et al. 2006; Sánchez et al. 2003a).

However, even when combined, the two approaches do not seem to fully solve the taxonomic problems in this group, and more informative characters in both disciplines must be identified to distinguish closely related morphological species (e.g. McFadden and Van Ofwegen 2013 on stoloniferous octocorals), as well as to obtain more natural classifications, reducing the number of taxonomic synonyms (e.g. Grajales and Rodríguez 2016, on sea anemones). This will help to clarify the evolution of a zoological group that is very important in the structural functioning of benthic marine communities (Conell 1978; Fabricius & Alderslade 2001; Fabricius 2005).

Finally, the presence and abundance of these and other species of octocorals in

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shallow waters could be used as an environmental indicator, suggesting potential areas for protection. Intensive sampling on the coast of Ecuador in biotopes such as submarine ridges and coralligenous bottoms, rich in gorgonian species, could reveal unrecorded gorgonian species in this area. Therefore, much work remains to elucidate Gorgoniidae taxonomy on the coast of Ecuador. The octocorals are an important structural component of the rocky reef fauna of these waters. The present survey contributes towards resolving issues around the distribution and taxonomy of this fauna in this region.

Acknowledgements

This research was funded by the grant “Biología de la conservación de las comunidades de gorgonias tropicales en el Pacífico oriental (Ecuador)” of the Universidad Tecnológica Indoamérica to JM. This research received support from the SYNTHESYS Project http://www.synthesys.info/ financed by the European Community Research Infrastructure Action under the FP7 Capacities Program; and Grant “Ernst Mayr Travel Grants In Animal Systematics” for visiting MCZ at Harvard University. This research was partially supported by a grant from the Spanish Ministry of Economy and Competitiveness (CTM2014-57949- R). Our special thanks to Gonzalo Giribet and Adam J. Baldinger (MCZ) and Aude Andouche (MNHN); Andrew Cabrinovic and Miranda Lowe (NHM); Eric Lazo-Wasem and Lourdes Rojas (YPM), and Stephen Cairns and Herman H. Wirshing (Smithsonian, MNH) for helping in our museum work.We thank Santiago Villamarín (MECN), Machalilla National Park and Ministerio del Ambiente of Ecuador (Manabí) for collection permits; Michel Guerrero and his team (Exploramar Diving) for his special interest and collaboration since we began our research. Special thanks to Micaela Peña for her unconditional help; Alicia Maraver, Valeria García and Damián Ramírez for help on the collection expeditions, for their friendship and hard work. Also thanks to Eva Andrés Marruedo and Mónica Flores for their help during the subsequent fieldwork. Finally, we thank one anonymous referee for his/her suggestions for improving our manuscript.

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4.2 – Foxed intra- and interspecific differentiation in Leptogorgia (Octocorallia: Gorgoniidae). A description of a new species based on multiple sources of evidence.

4.2.1. Abstract

The challenges of delimiting and identifying marine invertebrates impedes estimations of biodiversity. This is particularly true in studying the biodiversity of gorgonians using only classical morphological characters, which can be homoplastic and continuous. The framework of integrative taxonomy can be a helpful approach in this regard. We explored the utility of classical morphological and morphometric characters, together with four molecular markers (mMutS, Cox, ITS and 28S), also used to infer phylogenetic relationships. We examined two forms initially considered eco-typical variants of Leptogorgia alba (Duchassaing and Michelotti, 1864), which live sympatrically in the littoral area of Ecuador. Based on our results, two different species should be considered, L. alba and L. manabiensis n. sp. They showed distinguishing morphological features that cannot be attributed to phenotypic plasticity. Both species also presented significant differences in morphometric, non-correlated characters. The relationship between the species was polyphyletic, suggesting ancestral polymorphism and incomplete lineage sorting. In conclusion, pooling these techniques and considering different evidence provides the best support for the identification and delimitation of challenging species.

4.2.2. Introduction

In the last few decades, a multitude of evidences has indicated the difficulty of identifying marine species, especially invertebrates, with the exclusive use of morphological characters (see Calvo et al. 2009, Ladner and Palumbi 2012, López-González et al. 2014, Alfaya et al. 2015). Too often, this is compounded by the lack of information about their dynamic life-history and evolutionary processes, giving rise to an underestimation of the true biodiversity. This can lead to inappropriate management of,

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for instance, endangered species or those with restricted distribution areas (see Rocha- Olivares et al. 2001, Hebert et al. 2004, Dincă et al. 2011, Eberle et al. 2016). In addition, it is important to take into account that some traits, which can be used to identify species, are practical tools but not necessarily useful regarding evolutionary relatedness, and vice versa (Carlo et al. 2011). However, recent studies have demonstrated that the use of adequate morphological characters at different levels allow some species to be identified as complexes of species and morpho-species may be subdivided into distinct and valid species (Lattig et al. 2007). Moreover, the improvement of microscopic techniques (optical and SEM) and systematic methods has contributed to the morphological advancement of knowledge of many species of marine invertebrates (Rocha-Olivares et al. 2001, Sánchez and Wirshing 2005).

In this context, understanding species boundaries in corals remains a challenge in different areas of study (taxonomy, evolutionary biology, life-history and ecology) (Vermeij et al. 2007, Stefani et al. 2008, Gori et al. 2012). In fact, the delimitation of species in most studies is still debated (Stefani et al. 2008).

Traditionally, the identification of octocorals has relied on morphological and meristic features (from the seminal studies to Bayer et al. 1983, Breedy and Guzmán 2003, Vargas et al. 2010), but these methods are often problematic due to the use of homoplastic and continuous characters (Sánchez and Wirshing 2005, Sánchez et al. 2007, Gori et al. 2012, Soler-Hurtado and López-González 2012). To detect problems in the characters used, different evidence must be taken into account (i.e., morphological versus ecological, life history or molecular information) (e.g. Breedy et al. 2009).

The confusing range of morphological variation observed in corals is widely attributed to an apparent phenotypic plasticity due to environmental conditions (Weinbauer and Branko 1995, Flot et al. 2008, Sánchez 2009), and evolutionary processes such as widespread interspecific hybridization, explosive radiation, and incomplete lineage sorting (Hatta et al. 1999, Vollmer and Palumbi 2004, Forsman et al. 2010, Ardila et al. 2012, Torres-Suárez 2015). In fact, previous studies suggest that the presence of gene flow among coral species may be more common than previously thought (Calderón et al. 2006, Sánchez

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and Dorado 2008, McFadden et al. 2010).

Thus, in the last decade, studies using molecular analysis have increased (e.g., Sánchez et al. 2003, McFadden et al. 2010, Vargas et al. 2014), but they are not always effective, either in the resolution of species, or even at the genus level. Based on the idea that ‘‘more is better’’ (Winkler et al. 2015), many authors have supported the use and concatenation of nuclear and mitochondrial genes, such as ITS (internal transcribed spacers), 28S (28S rRNA), mtMutS and Cox (partial Cox-II and I including Igr1 region) (McFadden et al. 2006, Aguilar and Sánchez 2007, Sánchez et al. 2007, McFadden et al. 2011, McFadden et al. 2014). This strategy is also advisable because 1) mitochondrial markers evolve unusually slowly in Anthozoa in general, and in octocoral in particular, with their evolution estimated to be 10 to 100 times slower than other metazoans (France and Hoover 2002, Shearer et al. 2002, McFadden et al. 2004, Hellberg 2006), and 2) the support of nuclear genes is essential to detect possible evolutionary events such as hybridization.

Nowadays, and with respect to morphological analyses, other techniques such as morphometric statistics are applied to obtain additional characteristics regarding the shape and structure of organisms. These may better delimit difficult cases of species identification, reducing possible taxonomic subjectivity (Mutanen and Pretorius 2007) and molecular conflicts. These analyses have been shown to be at least as accurate as classical morphological and phylogenetic analyses, in the delimitation of closely related species (see Lumley and Sperling 2010, Gori et al. 2012, Schwarzfeld and Sperling 2014). Indeed, the pool of morphological, molecular and morphometric evidence provides three additional semi- independent datasets with which to better support the evaluation of species identification (Schwarzfeld and Sperling 2014).

Among these challenging organisms, we addressed the study of the genus Leptogorgia Milne-Edwards and Haime, 1857, considered one of the most cosmopolitan genera of the family Gorgoniidae (Grasshoff 1988, Breedy and Guzmán 2007, Soler-Hurtado and López-González 2012). The genus Leptogorgia has a widespread occurrence, with over 100 valid species, in the Mediterranean and Caribbean seas, and Atlantic, Indian and

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Pacific oceans (Bayer 1961, Grasshoff 1988, Williams and Lindo 1997, Breedy and Guzmán 2007). An additional species is found in the sub-Antarctic region (Williams and Lindo 1997). In the Eastern Pacific, it is one of the most frequently encountered genera (in terms of species richness and abundance), with 27 described species (Breedy and Guzmán 2007, Horvath 2011). Its bathymetric distribution has been cited to a depth of 1900 m at the East Pacific Rise (Bayer 2000), although it is present mostly in shallow waters. The genus Leptogorgia is distinguished by combination of a few classical characters including the colour of the colony, branching patterns (filiform, dichotomous or pinnate), in most cases the absence of anastomosis, the polyp mound, and coenenchymal sclerites (spindles and captans), which were described in detail by Bayer et al. (1983). Among those characters are the branching pattern, the morphology and colouration of the colony, the colour of the sclerites, and their sculptural details, size and shape (Grasshoff 1988, Breedy and Guzmán 2007).

Among the taxa encountered in Ecuador, there are two forms initially considered as eco-typical variants of Leptogorgia alba (Duchassaing and Michelotti, 1864). These require a deep analysis, as they may actually constitute two different entities, coexisting in the same habitat. Thus, we studied these two variants within the framework of “integrative taxonomy” (Dayrat 2005, Padial et al. 2010, Schlick-Steiner et al. 2010), using different approaches for species identification, including molecular analyses and classical morphology, with support from morphometric techniques. This analysis contributes towards understanding the relationship between genetic and morphological variation in this case study.

4.2.3. Materials and methods

Samplings

Gorgonian colonies were collected by SCUBA diving from rocky bottoms located in the Machalilla National Park (Manabí, Ecuador). The Machalilla National Park is one of the most important marine-terrestrial reserves in the country. It is located in the central-

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western region of the Ecuadorian coast in the province of Manabí, and it consists of 750 km2 of protected lands and coastlines. The area is known to host a high biodiversity of terrestrial fauna and provides habitat to important marine species (Glynn 2001, Cruz et al. 2003, Bo et al. 2012, Soler-Hurtado and López-González 2012).

Sampling was undertaken from February 2010 to June 2014. We collected 40 colonies from two apparently close variants of Leptogorgia: Leptogorgia sp. and Leptogorgia alba. The two morphs are among the most common gorgonian species in the littoral zone of Ecuador and constitute important gorgonian gardens between 3-30 meters in depth (pers. obs.). These forms are of conservation concerns given the need to clarify their species status. Upon collection, we conserved the attachment of the base of the holdfast such that the colony would regenerate.

The colonies were collected according to four size classes, considering the distance between the holdfast and the most distant branch tip, to take account the high range of variability (class 1: ≤ 70 mm; class 2: between 71 and 140 mm; class 3: between 141 and 210 mm; and class 4: > 211 mm).

During sampling, we took colour photographs of the living specimens in their environment (Fig. 1) and outside of the water, just after collection, to ensure the accuracy of our colour descriptions. Subsamples were fixed in either absolute ethanol for further molecular analyses, or in 4% buffered formalin (after previous relaxation with menthol crystals) for morphological study. The rest of the colonies were allowed to air dry. Buffered formalin-fixed subsamples were subsequently transferred to 70% ethanol (Soler-Hurtado and López-González 2012).

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Figure 1. Image of the rocky bottom gorgonian gardens in Los Frailes, Machalilla National Park (Ecuador), where sympatric location (side by side) of L. alba (white, left) and Leptogorgia sp. (pink, right) was observed.

The newly collected specimens were deposited in the Museo Ecuatoriano de Ciencias Naturales (MECN), the octocoral reference collection of the research group “Biodiversidad y Ecología de Invertebrados Marinos” at the University of Seville (BEIM), in the Museo Nacional de Ciencias Naturales in Madrid (MNCN-CSIC) and Museu de Ciénces Naturals in Barcelona (MZB).

External morphology and SEM study

Fragments from different parts of the colony were prepared for study by SEM according to standard methods (Bayer and Stefani 1989, Alderslade 1998). Additionally, permanent mounts were prepared for light microscopy observation. The colonies were described and illustrated according to standard terminology (Bayer et al. 1983, Breedy and Guzmán 2007).

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Morphometry of the colonies

From each colony, the total area and total area without gaps were calculated (Fig. 2) using the ImageJ software (Java version of NIH image) (Abràmoff et al. 2004), calibrated using a ruler as a reference.

Figure 2. Image showing the differences between total area of the colony (A) and area without gaps (B).

The following features were measured from each gorgonian: the maximum height, maximum width, the average of the length of the primary branches and the maximum length of the primary branches. The number of ramifications and the number of branches of each order were counted.

The following colony parameters were measured and calculated: mean width [mean of three measurements (w1, w2 and w3) done at equidistant positions at a right angle to the height)] (Gori et al. 2012); height to width ratio; height to mean width ratio; ramification density [number of ramifications per surface area (total area and without gaps)] (Weinbauer and Branko 1995); order of the colony [developed by Horton (1945) and later modified by Strahler (1954)]; mean length of the primary branches; maximum length of the primary branches to maximum height ratio; tributary to source ratio of primary and secondary order branches (T/S) ["tributaries" (T) are branches that join a branch of higher order (no change in order), and "source" (S) is any branch that joins another branch of equal order] (Brazeau and Lasker 1988); bifurcation ratio (the ratio of the number of branches of a given order to the number of branches of the next higher order) (Horton 1945, Strahler 1954, Brazeau and Lasker 1988); main thickness of the primary, secondary 195

and tertiary order branches; angle formed between primary and secondary order branches; angle formed between secondary and tertiary order branches; and angle formed by basal branches.

Morphometric statistical analyses

Since two highly collinear variables contain the same information and would be redundant for the purpose of this analysis (Anderson et al. 2008), we checked for correlations among the morphometric variables measured, before proceeding with modelling, leaving only those variables with absolute inter-correlation lower than 0.75.

The data were organized into a morphometric variable/sample matrix, and an Euclidean distance similarity matrix was calculated based on the normalized data (Anderson 2001). The differences in the multivariate structure were analysed in a distance- based permutational multivariate analysis of variance (PERMANOVA) (Anderson 2001, McArdle and Anderson 2001). The experimental design included two crossed fixed factors: species (with 2 levels, Leptogorgia alba and Leptogorgia sp.), and size classes (with 4 levels). The sum of squares (SS) used was Type III, where every term in the model is fitted only after taking into account all other terms in the full model (Anderson et al. 2008). The permutation method used was the permutation of residuals under a reduced model with 9,999 permutations. Homogeneity of dispersions were also tested with PERMDISP routine, which performs a Levene’s-type test using the group means, but obtaining the p-values by permutations (Anderson 2006). Multivariate analyses were performed using the software PRIMER v6.1.11 and the PERMANOVA v1.0.1 statistical package (Clarke and Gorley 2006). Boxplots were made in Rstudio (Team 2015).

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from 20-30 mg of tissue using the DNeasy extraction Kit (Qiagen, Inc.) according to the manufacturer’s protocol. Partial gorgonian Cox,

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mtMutS, ITS and 28S sequences were amplified by PCR using the following primers: COII8068F (McFadden et al. 2004), COIOCTR (France and Hoover 2002), the newly developed COI-Gorg1-R3 (5´ AGAGAAGGTGGTAATAACCAGAAA-3´) and COI-Gorg2-F2 (5´-GATTCGGAAATTGGTTTGTG-3´) for Cox; ND42599F (France and Hoover 2002) and MUT3458R (Sánchez et al. 2003) for mtMutS; ITS 2.1 and ITS 2.2 for ITS (Hugall et al. 1999); 28S-Far and 28S-Rar (McFadden and van Ofwegen 2013), and the newly developed 28S-R3 (ACTGCATRTATGAACTCCA) for 28S.

Amplifications were carried out in 50 µl final volume reactions containing 5 µl of 10x buffer (containing 10x 2 mM MgCl2), 1 µl dNTPs mix (10 mM), 0.8 µl of each primer (10 µM), 0.5 µl of Taq DNA polymerase (5U/µl) (Biotools) and 2 µl of genomic DNA. Thermocycling for the Cox fragment included an initial 4 min denaturation step at 94 ºC, followed by 40 cycles of 45 s at 94 ºC, 1 min at 58 ºC and 1 min at 72 ºC. The cycle ended with 10 min of sequence extension at 72 ºC. For mtMutS, we used an initial 4 min denaturation step at 94 ºC, followed by 35 cycles of 90 s at 94 ºC, 90 s at 58 ºC and 1 min at 72 ºC. The cycle ended with 5 min of sequence extension at 72 ºC. For ITS, we used an initial 4 min denaturation step at 94 ºC, followed by 35 cycles of 60 s at 94 ºC, 90 s at 56 ºC and 1 min at 72 ºC. The cycle ended with 7 min of sequence extension at 72 ºC. Finally, for 28S, we used an initial 4 min denaturation step at 94 ºC, followed by 30 cycles of 90 s at 94 ºC, 90 s at 50 ºC and 1 min at 72 ºC. The cycle ended with 5 min of sequence extension at 72 ºC. The amplification products were purified with 0.5 U Gelase (Epicentre Biotechnologies, Madison, WI). The amplicons were sequenced for both strands using BigDye Terminator in an ABI 3730 genetic analyzer (Applied Biosystems). The sequences obtained were edited using the Sequencher v.4.6 program (Gene Code Corporation, Ann Arbor, MI, USA).

The molecular data matrix was completed with other published sequences for Gorgoniidae in GenBank: Antillogorgia bipinnata (Pseudopterogorgia bipinnata) GQ342499 (mtMutS), GQ342423 (Cox), EU043125 (ITS), and JX203712 (28S); and other newly sequenced species of Gorgoniidae: E. ahorcadensis Soler-Hurtado and López-González, 2012, E. daniana (1 y 2) Verrill, 1868, L. diffusa Verrill, 1868, L. obscura Bielschowsky, 1929, L. mariarosae Soler-Hurtado and López-González, 2012 and P. stenobrochis Valenciennes,

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1846 (Table 2).

The model that best fit for nucleotide evolution for each final alignment was determined by jModelTest (Posada 2008). Phylogenetic analyses were performed using PhyML v3.0 for Maximum Likelihood (ML) (Guindon and Gascuel 2003). Maximum Parsimony (MP) was calculated in PAUP 4b10 (Swofford 2003). MP parameters included a heuristic search with tree bisection-reconnection (TBR) branch swapping. The ML and MP supports were performed through 1000 bootstrap replicates (bootstrap values = bv). Bayesian inference of phylogenetic relationships was performed with MrBayes 3.1. (Huelsenbeck and Ronquist 2001), employing two parallel runs of 5 million generations, with one cold and three heated Markov Chains Monte Carlo (MCMC) for each run, sampling one every 1000 replicates. Ten percent of sampled trees were discarded as burn- in, and support was evaluated based on the posterior probabilities (pp).

4.2.4. Results

Morphological characterization

Family Gorgoniidae Lamouroux 1812

Genus Leptogorgia Milne Edwards and Haime 1857

Leptogorgia alba (Duchassaing and Michelotti 1864)

(Figs. 3-4)

Lophogorgia alba Duchassaing and Michelotti, 1864: 19; Rossi, 1956: 198; Prahl et al., 1986: 17; Volpi and Benvenuti, 2003: 58.

Gorgonia (Leptogorgia) rigida var. laevis Verrill, 1866: 327.

Gorgonia (Litigorgia) laevis Verrill, 1868a: 415.

Leptogorgia alba Verrill, 1868b: 398-399, 1869: 421; Studer 1894: 68; Bielschowsky 1918: 30, 1929: 107-108; Kükenthal 1919: 771, 1924: 329; Hickson 1928: 400-405; Breedy and Guzmán

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2007: 12-19.

Leptogorgia alba var. sulcata Kükenthal, 1919: 771, 1924: 329; Bielschowsky, 1929: 108-110.

Euplexaura lemasti Hickson, 1928: 349-351; Stiasny, 1941: 266-267, 1943: 63.

Leptogorgia fasciculata Bielschowsky, 1918: 29, 1929: 94-96; Kükenthal, 1919: 912, 1924: 326; Stiasny 1951: 60.

Collected examined material: Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 5 m depth, 27 Feb. 2010, two whole colonies. Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, 27 Feb. 2010, seven whole colonies. Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 15 m depth, 27 Feb. 2010, twelve whole colonies. Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 25 m depth, 27 Feb. 2010, two whole colonies. Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 30 m depth, 27 Feb. 2010, one whole colony. Los Frailes, Manabí (Ecuador), 1°30’14”S 80°48’33”W, 10 m depth, Feb. 2012, two whole colonies. Los Frailes, Manabí (Ecuador), 1°30’14”S 80°48’33”W, 15 m depth, March 2012, one whole colony. Punta Gruesa, Manabí (Ecuador), 1°33’38.15”S 80°50’5.28”W, 14 m depth, 12 Sept. 2012, one whole colony. Punta Mala, Manabí (Ecuador), 1°33’41.37”S 80°50’8.79”W, 15 m depth, 20 Feb. 2013, one whole colonies. Punta Mala, Manabí (Ecuador), 1°33’41.37”S 80°50’8.79”W, 15 m depth, 20 Feb. 2013, eleven whole colonies. Punta Mala, Manabí (Ecuador), 1°33’41.37”S 80°50’8.79”W, 16 m depth, 02 Feb. 2013, two whole colonies. Punta Mala, Manabí (Ecuador), 1°33’41.37”S 80°50’8.79”W, 16 m depth, 02 Feb. 2013, nine whole colonies. Salinas, Santa Elena (Ecuador), 2°12’50.01”S 80°56’5.93”W, 15 m depth, 18 Dec. 2011, one whole colony.

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Figure 3. Leptogorgia alba (BEIM-0071). A, colony; B, detail of a branch; C, light micrograph of sclerites.

Description: White colonies up to 274 mm in length and 243 mm wide, irregularly pinnate; branches slender, mostly in a plane (Fig. 3A). Unbranched distal twigs up to 153 mm in length and 27.2 mm in diameter, compressed proximally, more cylindrical and slightly tapered distally (Fig. 3A, B). Slightly marked longitudinal grooves along the thick basal branches and near the base. Polyps retract within slightly raised polyp-mounds, sparsely distributed all around the branches with oblong apertures (Fig. 2B). Coenenchymal sclerites hyaline or colourless (Fig. 3C). Dominant sclerite type spindles, straight or bent, some with a marked waist, measuring between 0.08-0.16 mm in length and 0.03-0.06 mm wide, with 4–6 whorls of tubercles (Figs. 3C, 4A). Capstans measure between 0.03-0.11 mm in length and 0.01-0.07 mm wide (Fig. 4B). Crosses not found. Anthocodial sclerites hyaline or colourless, rods measure from 0.05-0.14 mm in length and 0.01-0.04 mm wide, with some marginal projections (Figs. 3C, 4C).

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Figure 4. Leptogorgia alba (BEIM-0071) SEM photographs. Coenenchymal sclerites: A, spindles; B, captans; Anthocodial sclerites: C, rods.

Geographic and bathymetric distribution: Mexico, El Salvador, Costa Rica, Panama, Colombia, and the Galápagos Islands and continental coast of Ecuador (Duchassaing and Michelotti 1864, Bielschowsky 1929, Breedy and Guzmán 2007), at 3-30m depth.

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Leptogorgia manabiensis Soler-Hurtado, Megina, Machordom & López-González,

2016

(Figs. 5-6)

Collected examined material: Holotype: MECN (Ant0001), Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 15 m depth, 27 Feb. 2010, six whole colonies. Paratypes:, Isla de Salanago, Manabí (Ecuador), 1°35’55.13”S 80°52’0.01”W, 7 m depth, 20 Nov. 2011, one whole colony. Punta Mala, Manabí (Ecuador), 1°35’55.13”S 80°52’0.01”W, 10 m depth, 20 Nov. 2011, one colony. Other material: Los Frailes, Manabí (Ecuador), 1°30’14”S 80°48’33”W, 10 m depth, 1 Feb. 2012, one whole colony. Isla de la Plata, Manabí (Ecuador), 1°16'25.84"S 81° 4'11.70"W, 15 m depth, 22 Feb. 2012, two whole colonies. Isla de la Plata, Manabí (Ecuador), 1°16'25.84"S 81° 4'11.70"W, 22 m depth, 22 Feb. 2012, two whole colonies. Isla de Salango, Manabí (Ecuador), 1°35’55.13”S 80°52’0.01”W, 7 m depth, 20 Nov. 2011, four whole colonies. Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 5 m depth, 27 Feb. 2010, three whole colonies. Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, 27 Feb. 2010, eight whole colonies. Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 15 m depth, 27 Feb. 2010, five whole colonies. Los Ahorcados, Manabí (Ecuador), 1º40’44”S 80º50’08”W, 10 m depth, 27 Feb. 2010, four whole colonies. Los Frailes, Manabí (Ecuador), 1°30’14”S 80°48’33”W, 15 m depth, March 2012, one whole colony. Punta Gruesa, Manabí (Ecuador), 1°33’38.15”S 80°50’5.28”W, 14 m depth, 12 Sept. 2012, one whole colony. Punta Gruesa, Manabí (Ecuador), 1°33’38.15”S 80°50’5.28”W, 14 m depth, 3 Feb. 2013, four whole colonies. Punta Mala, Manabí (Ecuador), 1°33’41.37”S 80°50’8.79”W, 15 m depth, Nov. 2011, three whole colonies. Punta Mala, Manabí (Ecuador), 1°33’41. 37”S 80°50’8.79”W, 16 m depth, 3 Feb. 2012, five whole colonies.

Description of the holotype: Pink colony measures 155 mm in length and 120 mm wide, irregularly pinnate; branches slender, mostly in a plane (Fig. 5A). Unbranched distal twigs up to 50 mm in length and 19 mm in diameter, compressed proximally, more cylindrical and slightly tapered distally (Fig. 5A, B). Slightly marked longitudinal grooves along the

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thick basal branches and near the base. Polyps retract within slightly raised polyp-mounds, sparsely distributed all around the branches with oblong apertures (Fig. 5B). Coenenchymal sclerites hyaline or colourless (Fig. 5C). Dominant sclerite type spindles, straight or bent, some with a marked waist, measuring up to 0.14 mm in length and 0.04 mm wide, with 4–6 whorls of tubercles (Figs. 5C, 6A). Capstans measure up to 0.08 mm in length and 0.03 mm wide (Fig. 6B). Crosses not found. Anthocodial sclerites hyaline or colourless, rods measure up to 0.10 mm in length and 0.02 mm wide, with some marginal projections (Figs. 5C, 6C).

Figure 5. Leptogorgia manabiensis n. sp. (Ant0001), holotype. A, colony; B, detail of a branch; C, light micrograph of sclerites.

Variability: Colonies up to 316 mm in length and 236 mm cm wide. Unbranched distal twigs up to 139 mm in length, and 19 mm in diameter. Spindles measure from 0.06-0.17 mm in length and 0.03-0.06 mm wide. Capstans measure from 0.03-0.10 mm in length and 0.02-

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0.07 mm wide. Anthocodial sclerites rods measure from 0.05-0.14 mm in length and 0.01- 0.06 mm wide.

Figure 6. Leptogorgia manabiensis n. sp. (Ant0001), holotype SEM photographs. Coenenchymal sclerites, A, spindles; B, captans; Anthocodial sclerites, C, rods.

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Geographic and bathymetric distribution: Leptogorgia manabiensis n. sp. is known from the type locality in Los Ahorcados, Cope, Frailes, Punta Gruesa, Punta Mala, Isla de Salango and Isla de la Plata (continental coast of Ecuador), living on rocky bottoms in shallow waters at a depth of 5-23 m.

Etymology: The specific epithet refers to the Province of Manabí (Ecuador), the type locality where the new taxon was discovered. Name considered as a noun in apposition.

Final remarks: According to classical morphological characters, L. alba and L. manabiensis n. sp. species show two main differences. The first is the colony colour, by which they are visually separate and clearly distinct (Fig. 1): while L. alba is white (alive and dry), L. manabiensis n. sp. is pink. The second difference is the shape of the unbranched distal length: L. alba has twigs up to 15.3 cm in length and 27.2 mm in diameter, while L. manabiensis n. sp. has twigs up to 13.9 cm in length and 19 mm in diameter. Other differences found between the two studied species, will be discussed in the morphometric analysis section.

Morphometric Analyses

Nine morphological characteristics were selected: maximum height, maximum length of the primary branches to maximum height ratio, order of colony, bifurcation ratio, tributary to source ratio of secondary order branches, main thickness of the primary order branches, angle formed between primary and secondary order branches, angle formed between secondary and tertiary order branches, and angle formed by basal branches. There were significant differences in the multivariate structure between the species, L. alba and L. manabiensis n. sp., and among size class (Table 1). Interactions between species and size class of the colony were not detected.

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Table 1. PERMANOVA table of results. Two crossed fixed factor: species and size class.

df SS MS Pseudo-F P(perm)

Specie 1 39.80 39.80 5.67 0.00

Size 3 129.5 43.16 6.15 0.00

Specie x Size 3 29.79 9.93 1.41 0.10

Residual 69 484.22 7.01

Total 76 684

Figure 7. Graphical representation of selected morphometric colony features for L. alba (La) and for L. manabiensis (Lm) (n = 80); black line indicates mean value, and dotted vertical line indicates the dispersion range values.

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The multivariate dispersion did not show any significant difference, either between the levels of the factor “species” (PERMDISP F1,75: 2.25; P(perm): 0.14), or among the levels of the factor “size class” (PERMDISP F1,73: 1.22; P(perm): 0.36). In addition, the graphic representations showed differences between the two gorgonian species, according to the selected morphological characteristics (Fig. 7). Colonies of L. alba were characterized by higher maximum length of the primary branches to maximum height ratio, angle formed between primary and secondary order branches, angle formed between secondary and tertiary order branches, and angle formed by basal branches (Fig. 7). In contrast, colonies of L. manabiensis n. sp. had a higher order of colony, bifurcation ratio, and tributary to source ratio of secondary order branches (Fig. 7). In summary, the two forms cannot be considered as ontogenetic stages of a single species.

Molecular Analyses

One matrix was prepared with the concatenated data from the four marker alignments: mtMutS+Cox+ITS+28S (16 specimens, 3353 characters). New sequences were deposited in GenBank (Table 2). The model of nucleotide substitution that best fit our data, obtained using the jModelTest program, was HKY + I. In the concatenated alignment, 152 sites were parsimony informative, 7 between L. manabiensis n. sp. and L. alba. The three approaches used (BI, MP and ML) gave similar topologies.

The reconstruction based on the data obtained from the four concatenated genes (Fig. 8), showed a well-supported (pp = 1; bv = 100) clade, representing a Leptogorgia alba- manabiensis clade (Clade I). Secondarily, the species L. alba and L. manabiensis n. sp. were subdivided into different subclades, but without taxonomic congruence. The sister species was not a Leptogorgia species, but Eugorgia daniana Verrill, 1868, and the addition of E. ahorcadensis Soler-Hurtado and P.J. López-González, 2012 completed this cluster (pp = 1; bv ≥ 85), which constituted a polyphyletic assemblage (Clade II). Finally, L. diffusa and L. obscura (sister species) plus L. mariarosae (Clade III) was the sister group of Clade II (pp =

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1; bv ≥ 97). Pacifigorgia stenobrochis and Antilogorgia bipinnata (designed as an outgroup) were in the basal position of the studied specimens, but without defining their relationships to the rest of the grouped taxa.

The topology of the paired L. alba-manabiensis n.sp. in the presented concatenated tree mainly matched that obtained using the nuclear data (ITS + 28S) (not shown), since the mitochondrial matrix nearly lacked informative characters for their representatives.

Figure 8. Phylogenetic relationship between taxa from Gorgoniidae family. Tree topology was inferred by Bayesian analysis, based on combined mitochondrial (coxII+Igr+coxI and mtMutS) and nuclear genes (ITS and 28S). The numbers of stars indicate the support of clades (pp≥95; bootstrap =70, for MP and ML). For sequences with duplicate complete names, we have included (1), (2), (3), etc., for the purpose of correctly identifying each individual specimen.

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Table 2. Gorgoniidae species involved in the molecular comparisons carried out in this study. Materials in bold are species sequenced for this study. Note that all GenBank sequences are considered here with the names as they appear in GenBank and their original publications (including numbers or letters). For sequences with duplicate complete names, we have included (1), (2), (3), etc., for the purpose of correctly identifying the sequence in Figure 8.

Species Igr + COI mtMutS ITS 28S

Antillogorgia bipinnata Verrill, 1864 GQ342423 GQ342499 EU043125 JX203712

Soler-Hurtado & López-González Eugorgia ahorcadensis 2012

Eugorgia daniana (1) Verrill 1868

Eugorgia daniana (2) Verrill 1868

Leptogorgia alba (1) Duchassaing & Michelotti 1864

Leptogorgia alba (2) Duchassaing & Michelotti 1864

Leptogorgia alba (4) Duchassaing & Michelotti 1864

Leptogorgia alba (5) Duchassaing & Michelotti 1864

Leptogorgia alba (6) Duchassaing & Michelotti 1864

Leptogorgia manabiensis n. sp. (1)

Leptogorgia manabiensis n. sp. (2)

Leptogorgia manabiensis n. sp. (4)

Leptogorgia manabiensis n. sp. (5)

Leptogorgia manabiensis n. sp. (6)

Leptogorgia diffusa Verrill 1868

Soler-Hurtado & López-González Leptogorgia mariarosae 2012

Leptogorgia obscura Bielschowsky 1918

Pacifigorgia stenobrochis Valenciennes 1846

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Finally, L. diffusa and L. obscura (sister species) plus L. mariarosae (Clade III) was the sister group of Clade II (pp = 1; bv ≥ 97). Pacifigorgia stenobrochis and Antilogorgia bipinnata (designed as an outgroup) were in the basal position of the studied specimens, but without defining their relationships to the rest of the grouped taxa.

4.2.5. Discussion

Based on our results, we support the existence of two distinct species, L. alba and L. manabiensis n. sp. in Machalilla National Park (Ecuador), with their own features in terms of colony shape and branching, found coexisting in the same population. This paves the way for better understanding the true diversity within this species group. We have shown that by using an integrative taxonomy of morphology, molecular and morphometric techniques, the delimitation and identification of these challenging species is possible.

Previous morphological studies based on a limited set of characters considered that, in species such as L. alba (Breedy and Guzmán 2007), the variability of colour and shape of some individuals could actually be intraspecific morphological variation, correlated with differences in environmental factors principally due to local currents and depth (Lewis and Wallis 1991, Carlon and Budd 2002, Gori et al. 2012). However, the two species analysed here are living side by side in our area of study; thus, the differences between L. alba and L. manabiensis n. sp. in colony morphology cannot be attributed to phenotypic plasticity due to dissimilar conditions in different ecological niches.

However, most of the morphometric characters that were considered in the delimitation of the two species in the present study had not been noted in previous descriptions and revisions. In addition, our study shows that the differences between the two gorgonian species were significant in all size classes, and, thus, were not restricted to only older individuals or specimens in the first stage of development, for example. This

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integrative approach resulted in an understanding of the role that morphometric and morphological characters play in the systematics of these organisms (Vargas et al. 2010, Gori et al. 2012). This may greatly enhance the delimitation and discrimination power of species, as well as provide certain advantageous features for their biology or ecology. For example, Gori et al. (2012) showed that particular characters, such as branching patterns and colony shape, could represent morphological adaptations in the gorgonian species to different hydrodynamic events, or even feeding.

In addition to the importance of understanding the morphological variation present in these organisms, the mechanisms and heritability involved in this variation should be considered (Vermeij et al. 2007). Indeed, these variations could be due to a genetic polymorphism, where the morphology of the species is determined by genotypes, independent of the ecological niche occupied (Carlon and Budd 2002, Vermeij et al. 2007).

In this case, the significant differences observed in the morphometric and morphological analyses were not consistent with our molecular results, where the relationship between L. alba and L. manabiensis n. sp. resulted in a polyphyletic assemblage. The discrepancies among different pieces of evidence suggest that processes of hybridization or incomplete lineage sorting may have occurred. In this scenario, these species showed a genetic polymorphism, not shared equally between two groups. In a speciation framework, some morphological characteristics remain stable for each species (e.g., the colour of the colony, some morphometric branching measures), while others are shared between the two species.

In corals, different examples of molecular phylogenies that show polyphyly or paraphyly have been regarded as hybridization processes (Hatta et al. 1999, Diekmann et al. 2001, McFadden and Hutchinson 2004), but possible ancestral polymorphisms linked to a delayed process of evolution, such as a source of shared haplotypes between species, should also be considered (van Oppen et al. 2001, Vollmer and Palumbi 2004, Torres- Suárez 2015). An incomplete lineage sorting can be a particularly important factor for rapidly and recently diverged species (Hoelzer and Melnick 1994, Funk and Omland 2003, Henning and Meyer 2014). This process could be a problem in the correct identification of

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closely related species where incomplete lineage sorting is likely to produce conflicting or even misleading results (van Velzen et al. 2012, Nater et al. 2015). For example, the study by Eberle et al. (2016) included incomplete lineage sorting in an investigation by integrative approach methods of the scarab beetle genus Pleophylla, with eight putative morphospecies. This may explain the phenotypic variation between the Leptogorgia species studied due to a recent speciation or diversification event that is not yet apparent in the genetic markers examined.

There are a growing number of different cases in the literature regarding incomplete lineage sorting in invertebrates. For example, the study by Pollard et al. (2006) concluded that the phylogenetic incongruence found in the Drosophila species complex could be due to incomplete lineage sorting. Wiemers and Fiedler (2007) presented intra- and interspecific variation in the butterfly family Lycaenidae (blue butterflies), where paraphyly or polyphyly events were likely caused by this process. Carstens and Knowles (2007) exemplified a similar case in Melanoplus grasshopper species, as did Meyer and Paulay (2005) in Cypraeidae marine gastropods.

There are numerous examples of incomplete lineage sorting in corals and octocorals. In the genus Seriatopora, Flot et al. (2008a) detected four clusters where different interrelated species were within the same clade, showing little concordance with species recognized on a morphological basis. In the study of Forsman et al. (2010), closely related species were identified within the complexes of the genus Montipora, where an event of incomplete lineage sorting was suspected, mostly due to their morphological results. Van Oppen et al. (2001) hypothesized that conflicting morphologic and molecular results of Acropora, with incongruence between the nuclear and mitochondrial trees, were because lineage sorting was incomplete, especially if the species were of recent origin. In addition, Vollmer and Palumbi (2004) examined species of Acropora from the Caribbean and noted the challenges in evaluating whether the shared intragenomic variation is the result of a variety of processes, such as incomplete lineage sorting and slow concerted evolution, as well as hybridization. Moreover, McFadden et al. (2010) showed that cases of mito-nuclear discordance and intragenomic variants shared among octocoral species, could also be explained by the permanence of ancestral polymorphisms via an incomplete

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lineage sorting. Sánchez and Dorado (2008) supported the idea of polyphyly of Caribbean sea fans in mitochondrial DNA, where phylogenetic analyses showed intragenomic variation in Gorgonia mariae grouped partially with Pseudopterogorgia spp. and other species, suggesting a hybridization origination of species, or the remote possibility of the preservation of ancestral polymorphisms.

Today, in addition to the utility of molecular analyses, the use of morphological characters (classical or morphometric characters) is essential to species diagnosis (Schwarzfeld and Sperling 2014), due to the fact that incomplete lineage sorting poses a daunting challenge to our efforts to reconstruct the phylogenetic relationships of recently diverged species (Maddison and Knowles 2006).

In addition, it is important to recognize that there may not always be a direct positive relationship between evolutionary status and relatedness of species and the traits that can be used to identify them (Carlon and Budd 2002). However, this is not to say that diagnostic species criteria are not mutually exclusive, since alternate criteria could indicate different aspects of biological information, providing more robust decisions about the identification of species boundaries (Rocha-Olivares et al. 2001).

Finally, the differentiation among the species described in this study, L. alba and L. manabiensis n. sp., could clarify the previously puzzling discrepancies and reduce unexplained variability in this challenging gorgonian group. Moreover, it provides other tools and frameworks for comparative studies in the delimitation of species. This study may be the first step toward future research, where biological, ecological and behavioural characters of these organisms should also be considered. We believe this is essential to understanding the evolution and biodiversity of gorgonian gardens, as well as for implementing effective conservation strategies.

Acknowledgements

This research was partially supported by a grant from the Spanish Ministry of Economy and Competitiveness (CTM2014-57949-R). Our appreciation goes to Santiago Villamarín

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(Museo Ecuatoriano de Ciencias Naturales), Machalilla National Park and Ministerio del Ambiente of Ecuador (Manabí) for permits to collect and their support. We thank Michel Guerrero and his team (Exploramar Diving) for his special interest and collaboration since we began our research. Special thanks to Micaela Peña for her strong support.

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4.3 – Molecular phylogenetic relationships reveal contrasting evolutionary patterns in Gorgoniidae (Octocorallia) in the Eastern Pacific.

4.3.1. Abstract

The description and delimitation of species in an evolutionary framework is essential in the understanding of biodiversity and distribution, and in the assessment of effective conservation strategies of natural resources. This study seeks to clarify the evolutionary history and genetic variation within and between closely related octocoral species within an ecosystem keystone. In this context, we focused on the Gorgoniidae family located in the Eastern Pacific with particular attention to one of the lesser know marine ecosystems, the littoral of Ecuador. According to our results, the monophyly of Pacifigorgia is supported, but it is not for the genera Leptogorgia and Eugorgia integrated a single group. In addition, we found suggestions of “unusual” speciation patterns: morphological differentiation with no apparent genetic differentiation (in Pacifigorgia); and inconsistent results between mitochondrial and nuclear genes, pointing to a hybridisation phenomena (in Leptogorgia). Recent radiation, ancient hybridisation, sympatric speciation on the one side, and reticulate evolution on the other may have contributed to the evolutionary history of the studied taxa. The incongruences between morphological and molecular evidences in octocorals, and in corals in general, suggests that these previous thoughts of as rare incongruences may actually be considered to be common.

4.3.2. Introduction

Species are widely used as fundamental units in taxonomy, biogeography, palaeontology and ecology (Bagley et al. 2015; Barraclough & Nee 2001), and provide the essential information needed to guide research programs in many disciplines (Ardila et al. 2012; Fontdevila, Serra 2013). However, even though this unit is 225

considered crucial in the understanding of biodiversity, where the morphological characters are limited, cryptic or reduced because of miniaturisation, the species delimitation is a debated question in many groups (Delicado et al. 2013). With the combination of morphological and molecular studies, an integrative taxonomy framework (Padial et al. 2010) has given the description and discovery of new species a more solid framework in which to distinguish between basic units. However, the study of discriminant characters alone is not enough to understand the patterns shown by these species or to deduce which processes may have led the changes that gave these different units. Thus, in the last decades, evolutionary studies have tackled the challenges of not only resolving the species identification and ranges of genetic variation within and between closely related species (Moritz & Cicero 2004; Wang et al. 2011), but also in clarifying the speciation processes.

These challenges included the resolution of some phylogenetic questions that presently remain problematic despite significant sampling of characters and taxa (Rokas et al. 2003; Wiegmann et al. 2011). Within these challenges, the interpretation of certain evolutionary patterns, such those resulting from rapid origin of lineages (explosive radiation), events of hybridisation, adaptive introgression (Hatta et al. 1999; Henning & Meyer 2014; Torres-Suárez 2015), and in some cases the search for adequate molecular markers (Winkler et al. 2015) can be especially complex. With consideration to these challenges, currently it is widely recognised that the best approach for establishing phylogenetic relationships among single or a group of organisms is to combine as many different types of data as possible (molecular, morphological, intraspecific population ecology and any other evolutionarily relevant information) in order to offer alternative hypotheses for different observed patterns (Eernisse & Kluge 1993; Guil et al. 2013; López-González et al. 2014; McKinnon et al. 2004; Prada & Hellberg 2013).

In this context, based on the inconsistency between morphological and molecular approaches, the anthozoans (in general) and the octocorals (in particular) represent an example of groups of organisms that are badly understood in regards to their phylogenetic relationships, spanning from family to species level (McFadden et

226

al. 2006). The study of these relationships has been hampered both by the extent of which the environment governs morphological plasticity, and the use of a series of discrete and homoplastic characters on which the morphological descriptions of species have been established (Concepcion et al. 2008; Wirshing & Baker 2015). In fact, previous studies have shown how the traditional taxonomy (classifying based on morphology alone) of these organisms sometimes differs from the results concluded by their molecular phylogenetic analysis (Ardila et al. 2012; Hatta et al. 1999; Sánchez et al. 2003a; Torres-Suárez 2015). Some of these homoplastic and/or overlapping characters can be: colour of the colony and sclerites, types and sizes of sclerites, and possible anastomosis branching patterns. It is also true that in the absence of detailed information, taxonomically defined species can in general reflect the possible evolutionary groups (Barraclough & Nee 2001). However, the use of mitochondrial genes to tackle such phylogenetic reconstructions in molecular analyses is usually preferential (McFadden & van Ofwegen 2013b; Sánchez et al. 2003a; Vargas et al. 2014). Octocorals mitochondrial DNA exhibits low substitution rates, indeed their evolution is estimated to be 10 to 100 times slower than that of other metazoans (Brockman & McFadden 2012; Chen et al. 2009; France & Hoover 2002; Hellberg 2006; Huang et al. 2008; McFadden et al. 2004; Shearer et al. 2002). One of causes for the low differences observed, seems to include the presence of the gene mtMutS, a gene which can encode a system of mismatch repair in mitochondrial DNA, hence reducing the number of substitutions (France & Hoover 2002).

In some octocorals studies, using the octocoral-specific mitochondrial gene mtMutS combined with the CoxII-Igr-CoxI (Cox) extended mitochondrial barcode, have shown that only ~70 % of species could be discriminated (McFadden et al. 2011; McFadden et al. 2014). Furthermore, if species delimitation estimates were based solely on mtDNA, then it was shown that these estimates could fail in detecting boundaries between closely related octocoral to the extent that, at times, closely related species were identified to be identical (Vargas et al. 2014; Yasuda et al. 2015). Currently, in respect to these failures, the utilisation of the nuclear markers such as 28SrRNA (28S) or Internal Transcribed Spacers (ITS) is considered necessary (Aguilar & Sánchez 2007; McFadden & Hutchinson 2004; McFadden et al. 2006, 2014; Sánchez et al. 2007). In the 227

case of ITS, the sequences have been very useful in comparing octocorals species, as well as indicating species hybridisation events (Aguilar & Sánchez 2007; McFadden & Hutchinson 2004).

The octocoral family Gorgoniidae is considered to be one of the most diverse family and includes some of the most abundant, worldwide, shallow-water genera. From Baja California to Peru, members of this family are commonly and widely distributed along the Eastern Pacific and its oceanic islands (Breedy et al. 2009). This described distribution area includes the littoral of Ecuador, a location that, despite harbouring a high level of biodiversity, lacks available and in depth studies of present marine invertebrate organisms, specially (Cruz et al. 2003; Soler-Hurtado & López- González 2012). For example, little research has been conducted to clarify the present marine organisms’ phylogenetic relationships (Vargas et al. 2014).

Hence, the aim of this study is to reveal the genetic diversity and relationships that reside for the majority of recognised species in the Eastern Pacific within the family Gorgoniidae. Ecuador will be the central area of this study. It is believed that this evolutionary analysis will permit the examination of whether boundaries of closely related species are detectable by molecular analyses, and the improvement of the knowledge of sequence of speciation events that gave rise the current gorgonian diversity.

4.2.3 Material and Methods

Samples collection, species and study area

Between February 2010 and June 2014 fresh gorgonian octocoral material was sampled from eleven stations located in Ecuador. Gorgonians located between the depths of 5 to 30 metres were collected by means of SCUBA diving. The samples were fixed in absolute ethanol for molecular analyses.

Additionally, to support this work, we examined samples of type specimens and additional materials deposited at the Comparative Zoology Museum (MCZ), Harvard 228

University; Muséum National d'Histoire Naturelle (MNHN), Paris; Natural History Museum (NHM), London; Yale Peabody Museum of Natural History, New Haven; and National Museum of Natural History, Smithsonian (NMNH), Washington (Table 1).

According to current taxonomy, each sample was identified through its morphological characters (Bayer et al. 1983; Breedy & Guzmán 2002, 2007; Breedy et al. 2009). In the case of Pacifigorgia, some samples were not definitely identified due to lack of clear morphological distinguishable characters. Within this study these particular specimens have been labelled: Pacifigorgia sp1 to sp7.

DNA extraction, PCR amplification, and sequencing

DNA was extracted from 20-30 mg of tissue using the DNeasy extraction Kit (Qiagen, Inc.) according to the manufacturer’s protocol. Partial gorgonians CoxII-CoxI including Igr region (CoxII+Igr+CoxI), hence referred to as Cox; mtMutS; ITS (ITS1 + 5,8S + ITS2) and 28S sequences were amplified by PCR using the following primers: COII8068F (McFadden et al. 2004), COIOCTR (France & Hoover 2002), the developed COI-Gorg1-R3 (5´ AGAGAAGGTGGTAATAACCAGAAA-3´) and COI-Gorg2-F2 (5´- GATTCGGAAATTGGTTTGTG-3´) for Cox; ND42599F (France & Hoover 2002) and MUT3458R (Sánchez et al. 2003b) for mtMutS; ITS 2.1 and ITS 2.2 for ITS (Hugall et al. 1999); 28S-Far and 28S-Rar (McFadden & van Ofwegen 2013a), and the developed 28S- R3 (ACTGCATRTATGAACTCCA) for 28S.

Amplifications were carried out in a 50 µl of final volume reactions containing 5

µl of 10x buffer (containing 10x 2 mM MgCl2), 1 µl dNTPs mix (10 mM), 0.8 µl of each primer (10 µM), 0.5 µl of Taq DNA polymerase (5U/µl) (Biotools) and 2 µl of genomic DNA. Thermocycling for the Cox fragment included an initial 4 min denaturation step at 94 ºC, followed by 40 cycles of 45 s at 94 ºC, 1 min at 58 ºC and 1 min at 72 ºC. The cycle ended with 10 min of sequence extension at 72 ºC. For mtMutS, we used an initial 4 min denaturation step at 94 ºC, followed by 35 cycles of 90 s at 94 ºC, 90 s at 58 ºC and 1 min at 72 ºC. The cycle ended with 5 min of sequence extension at 72 ºC. For ITS, we used an initial 4 min denaturation step at 94 ºC, followed by 35 cycles of 60 s at 94 229

ºC, 90 s at 56 ºC and 1 min at 72 ºC. The cycle ended with 7 min of sequence extension at 72 ºC. For 28S, we used an initial 4 min denaturation step at 94 ºC, followed by 30 cycles of 90 s at 94 ºC, 90 s at 50 ºC and 1 min at 72 ºC. The cycle ended with 5 min of sequence extension at 72 ºC. Due to the intra-individual variations of ITS region, being a multi-copy marker (Álvarez & Wendel 2003; Concepcion et al. 2008), some amplicons, with polymorphic positions were cloned. In these cases, we used competent TOPO® bacterial cells (Invitrogen) with the pGEM-T Vector System (Promega). After plasmid isolation using the Wizard Minipreps DNA Purification System (Promega), up to 10 positive clones were sequenced for both strands with M13 universal primers. The amplification products were purified by ethanol precipitation and with 0.5 U Gelase (Epicentre Biotechnologies, Madison, WI).

The amplicons were sequenced for both strands using BigDye Terminator in an ABI 3730 genetic analyzer (Applied Biosystems). The obtained sequences were edited using the Sequencher v.4.6 program (Gene Code Corporation, Ann Arbor, MI, USA). In the case of the cloned ITS, only the consensus sequence was used. The molecular data matrix was completed through using other published sequences for Gorgoniidae in GenBank (Table 1).

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Table 1. Gorgoniidae species included in the molecular analyses. Materials in bold are species sequenced for this study. Note that all GenBank sequences are considered here with the names as they appear in GenBank and their original publications (including numbers or letters). Numbers after species names indicate different specimens analysed. Type material is marked with an asterisk (*). Leptogorgia and Pacifigorgia specimens from Atlantic Ocean, are indicated with a (A).

Species Species author Igr + COI mtMutS ITS 28S

Gorgoniidae

Adelogorgia phyllosclera Bayer 1958 JN866558

Soler-Hurtado & López-González Eugorgia ahorcadensis (*) 2012

Eugorgia ampla (*) Verrill 1864

Eugorgia daniana Verrill 1868 HG917080

Eugorgia daniana (1) Verrill 1868

Eugorgia daniana (2) Verrill 1868

Eugorgia multifida Verrill 1970 GQ342494 JX203706

Eugorgia nobilis (*) Verrill 1868

Eugorgia pumila Verrill 1868

Eugorgia querciformis Bielschowsky 1918

Eugorgia rubens Verrill 1868 KF874216 JN866557

Eugorgia rubens Verrill 1868

Gorgonia flabellum Linnaeus 1758 GQ342418 GQ342495 JX203708

Leptogorgia alba Duchassaing & Michelotti 1864 HG917081 HG917034

Leptogorgia alba Duchassaing & Michelotti 1864 HG917083 HG917035

Leptogorgia alba (1) Duchassaing & Michelotti 1864

Leptogorgia alba (2) Duchassaing & Michelotti 1864

Leptogorgia alba (4) Duchassaing & Michelotti 1864

Leptogorgia alba (5) Duchassaing & Michelotti 1864

Leptogorgia alba (6) Duchassaing & Michelotti 1864

Leptogorgia alba (7) Duchassaing & Michelotti 1864

Leptogorgia capverdensis (A) (Grasshoff 1986) FJ642931

Leptogorgia chilensis Verrill 1868 KF874214 AY268460

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Table 1. Continuation.

Leptogorgia cofrini Breedy & Guzmán 2005 HG917084 HG917037

Leptogorgia cofrini Breedy & Guzmán 2005 HG917086 HG917040

Leptogorgia cofrini Breedy & Guzmán 2005 HG917085

Leptogorgia cofrini Breedy & Guzmán 2005 HG917087

Leptogorgia cofrini (1) Breedy & Guzmán 2005

Leptogorgia cuspidata Verrill 1865 AY268450

Leptogorgia cuspidata (1) Verrill 1865

Leptogorgia diffusa (1) Verrill 1868

Leptogorgia diffusa (2) Verrill 1868

Leptogorgia flexilis (1) Verrill 1868

Leptogorgia flexilis (2) Verrill 1868

Leptogorgia flexilis (3) Verrill 1868

Leptogorgia flexilis (4) Verrill 1868

Leptogorgia flexilis (5) Verrill 1868

Leptogorgia gracilis AY268454

Leptogorgia hebes (A) Verrill 1869 AY268459

Leptogorgia labiata Verrill 1970 AY268447

Soler-Hurtado & López-González Leptogorgia mariarosae (*) 2012

Leptogorgia obscura (1) Bielschowsky 1918

Leptogorgia obscura (2) Bielschowsky 1918

Leptogorgia obscura (3) Bielschowsky 1918

Leptogorgia ramulus Valenciennes 1855

Leptogorgia ramulus Valenciennes 1855 AY268451

Leptogorgia rigida Verrill 1864 GQ342496

Leptogorgia rubra (A) Bielschowsky 1918

Leptogorgia styx Bayer 2000 AY268453

Leptogorgia virgulata (A) Lamarck 1815 AY268458 AY268463

Pacifigorgia adamsi (*) Verrill 1868

Pacifigorgia agassizii (*) Verrill 1864

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Table 1. Continuation.

Pacifigorgia bayeri Breedy 2001 HG917061 HG917044

Pacifigorgia cairnsi (*) Breedy & Guzmán 2003

Pacifigorgia cairnsi Breedy & Guzmán 2003 HG917062

Pacifigorgia catedralensis Breedy & Guzmán 2004 HG917065 HG917019

Pacifigorgia catedralensis Breedy & Guzmán 2004 HG917066 HG917020

Pacifigorgia catedralensis(1)(*) Breedy & Guzmán 2004

Pacifigorgia cf. cairnsi Breedy & Guzmán 2003 HG917063 HG917046

Pacifigorgia cf. cairnsi Breedy & Guzmán 2003 HG917064 HG917021

Pacifigorgia cribrum (1) Valenciennes 1856

Pacifigorgia cribrum (2) Valenciennes 1856

Pacifigorgia curta Breedy & Guzmán 2003

Pacifigorgia elegans (A) Milne Edwars & Haime 1857 AY126419

Pacifigorgia exilis (*) Verrill 1870 KX351878 KX351871

Pacifigorgia ferruginea (*) Breedy & Guzmán 2004

Pacifigorgia firma Breedy & Guzmán 2003 HG917068

Pacifigorgia firma Breedy & Guzmán 2003 HG917069 HG917028

Pacifigorgia firma Breedy & Guzmán 2003 HG917067

Pacifigorgia firma (1) (*) Breedy & Guzmán 2003 KX351879 KX351872

Pacifigorgia flavimaculata (1) Breedy & Guzmán 2003

Pacifigorgia flavimaculata (2) Breedy & Guzmán 2003

Pacifigorgia irene Bayer, 1951 HG917071 HG917024

Pacifigorgia irene Bayer, 1951 HG917072 HG917022

Pacifigorgia irene (1) Bayer, 1951 KX351880 KX351873

Pacifigorgia irene (2) Bayer, 1951

Pacifigorgia machalilla n. sp. (1) (*) Soler-Hurtado et al. 2016 KX351881 KX351874

Pacifigorgia machalilla n. sp. (2) (*) Soler-Hurtado et al. 2016 KX351882 KX351875

Pacifigorgia machalilla n. sp. (3) (*) Soler-Hurtado et al. 2016 KX351883 KX351876

Pacifigorgia media Verrill 1864 GQ342421 GQ342497

Pacifigorgia media (*) Verrill 1864

Pacifigorgia media (1) (*) Verrill 1864

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Table 1. Continuation.

Pacifigorgia media (2) Verrill 1864

Pacifigorgia rubicunda Breedy & Guzmán 2003 HG917074 HG917033

Pacifigorgia rubicunda Breedy & Guzmán 2003 HG917073 HG917032

Pacifigorgia rubicunda (1) Breedy & Guzmán 2003

Pacifigorgia rubicunda (2) Breedy & Guzmán 2003

Pacifigorgia rubicunda (3) (*) Breedy & Guzmán 2003

Pacifigorgia rubinoffi (1) (*) Breedy & Guzmán 2003

Pacifigorgia rubinoffi (2) (*) Breedy & Guzmán 2003

Pacifigorgia rubripunctata (1) Williams & Breedy 2004

Pacifigorgia rubripunctata (2) Williams & Breedy 2004

Pacifigorgia samarensis (*) Breedy & Guzmán 2003

Pacifigorgia sculpta (1) (*) Breedy & Guzmán 2004 KX351884 KX351877

Pacifigorgia sculpta (2) Breedy & Guzmán 2004

Pacifigorgia sculpta (3) Breedy & Guzmán 2004

Pacifigorgia smithsoniana Breedy & Guzmán 2004 HG917076 HG917023

Pacifigorgia sp1

Pacifigorgia sp2 (1)

Pacifigorgia sp2 (2)

Pacifigorgia sp2 (3)

Pacifigorgia sp3 (1)

Pacifigorgia sp3 (2)

Pacifigorgia sp4 (1)

Pacifigorgia sp4 (2)

Pacifigorgia sp4 (3)

Pacifigorgia sp4 (4)

Pacifigorgia sp4 (5)

Pacifigorgia sp5 (1)

Pacifigorgia sp5 (2)

Pacifigorgia sp6

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Table 1. Continuation.

Pacifigorgia sp7 (1)

Pacifigorgia sp7 (2)

Pacifigorgia sp7 (3)

Pacifigorgia sp8

Pacifigorgia stenobrochis Valenciennes 1846 HG917078 AY126420

Pacifigorgia stenobrochis Valenciennes 1846 HG917079 HG917018

Pacifigorgia stenobrochis (1) Valenciennes 1846

Pacifigorgia stenobrochis (2) Valenciennes 1846

Pacifigrogia rubicunda (1) Breedy & Guzmán 2003

Phyllogorgia dilatata (Esper 1806) AY126428

Pseudopterogorgia bipinnata (Verrill 1864) EU043122 JX203712

Pseudopterogorgia elisabethae (Bayer 1961) EU017561

Pseudopterogorgia rubrotincta Thomson & Henderson 1905 JX152768

Pterogorgia anceps (Pallas 1766) GQ342424 AY126403 JX203714

Plexauridae

Anthomuricea sp. DQ302855

Eunicea tourneforti Milne Edwards & Haime 1857 GQ342445 AY126406

Eunicella tricoronata Velimirov 1971 JX203707

Heterogorgia verrucosa Verrill 1868 HG917052

Paramuricea sp. FJ264913

Muricea laxa Verrill 1864 AY683064

Muricea purpurea Verrill 1864 GQ293304

Muricea sp. HG917060 HG917015

Muriceopsis flavida (Lamarck 1815) GQ342449 AY126416 JX203744

Plexaura flexuosa Lamouroux 1821 AY126411

Plexaurella nutans (Duchassaing & Michelotti 1860) GQ342451 AY126415 JX203745

Scleracis guadalupensis (Duchassaing & Michelotti 1860) KC984626 KC984590

Swiftia pacifica (Nutting 1912) KF874210 KF856083

Swiftia pallida Madsen 1970 JQ241247 KC788259

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Table 1. Continuation.

Alcyoniidae

Alcyonium haddoni Wright & Studer 1889 JX203642

Alcyonium verseveldti (Benayahu 1982) GU355978

Sarcophyton glaucum (Quoy & Gaimard 1833) AF064823 AB759499

jModelTest was used to determine the model that fitted the best in respect with nucleotide evolution for each final alignment (Posada 2008). Phylogenetic analyses were performed using PhyML v3.0 for Maximum Likelihood (ML) (Guindon & Gascuel 2003). Maximum Parsimony (MP) was calculated in PAUP 4b10 (Swofford 2003). MP parameters included a heuristic search with tree bisection-reconnection (TBR) branch swapping. The ML and MP supports were performed through 1000 bootstrap replicates (bootstrap values = bv). Bayesian inference of phylogenetic relationships was performed with MrBayes v3.1. (Huelsenbeck & Ronquist 2001), where two parallel runs of 5 million generations, with each run containing one cold and three heated Markov Chains Monte Carlo (MCMC), with sampling occurring every 1000 generations. The initial 10% of sampled trees were discarded as the burn-in and the support evaluated through examination of the posterior probabilities (pp).

4.3.4. Results

Four matrices were obtained for the four analysed genes: (1) mtMutS (130 specimens, 832 characters); (2) Cox (117 specimens, 990 characters); (3) ITS (64 specimens, 697 characters); and (4) 28S (58 specimens, 886 characters). An additional matrix was prepared, containing the concatenated alignment of specimens with data for the four markers: mtMutS+Cox+ITS+28S (46 specimens, 3371 characters). The new sequences have been deposited in GenBank (Table 1).

The models of nucleotide substitution most appropriate for our data, obtained using the jModelTest program were: HKY+G for mtMutS, HKY+I+G for Cox, K80+I+G

236

for ITS, and 010023+I+G for 28S. In addition, for the concatenated data the selected model was HKY+I+G.

Figure 1. Phylogenetic relationship between taxa from Gorgoniidae family. Tree topology was inferred by Bayesian analysis, based on combined mitochondrial (coxII+Igr+coxI and mtMutS), and nuclear (ITS and 28S) genes. The numbers of stars above the branches indicate the number of phylogenetic methods that supported each clade (pp≥0.95; bootstrap ≥70). Numbers after species names indicate different specimens analysed, and an asterisk (*) type material.

The results showed that the combinations of the four genes gave the most supported and resolved nodes (Fig. 1). Within the concatenated alignment, 265 sites were considered parsimony informative. The inter-species relationships in some

237

groups produced different results for nuclear and mitochondrial genes, although for each gene the three approaches used (BI, MP and ML) gave the same topologies. In addition, the signals providing these phylogenetic reconstructions were unequally distributed among genes, with the ITS region contributing with the highest number of parsimony informative characters (93), followed by Cox (64), mtMutS (60) and 28S (52).

The reconstruction based on the data obtained from the concatenated four genes (Fig. 1) showed two well supported (pp ≥ 0.95; bv ≥ 70) clades: the first representing the monophyletic genus Pacifigorgia (Clade I) (pp = 1; bv = 100); and the second including both Eugorgia and Leptogorgia representatives (Clade II) (pp = 1; bv ≥ 98). The taxa belonging to Eugorgia comprised of two lineages, one conformed by E. ahorcadensis and E. nobilis (pp = 1; bv ≥ 99), and the other by E. daniana (pp = 1; bv = 100), a sister species of a group of Leptogorgia taxa. This last group was integrated by L. alba (in different subclades), L. cofrini, L. flexilis (in two different branches) and L. cuspidata (Clade II.A), but without high support. Finally, L. diffusa, L. obscura (sister species) and L. mariarosae (Clade II.B) completed this big cluster (pp = 1; bv ≥ 99)

The Pacifigorgia representatives were recovered in three different monophyletic groups (Fig. 1). Within Clade I.A clustered together P. irene, P. machalilla n. sp., P. rubripunctata, P. rubinoffi and four of the unidentified species (sp1 - 4) (pp = 1; bv ≥ 99). In one of its fully supported subclades, which included P. irene, P. machalilla n. sp. and Pacifigorgia sp1, Pacifigorgia sp2 and Pacifigorgia sp3, differentiation among the different putative species was not found. Clade I.A was considered to be the sister to Clade I.B, a clade comprising of P. cribrum, P. media, P. rubicunda and two other unidentified species (sp5 and 6) (pp = 1; bv ≥ 87). Clade I.C contained P. flavimaculata, P. stenobrochis, P. sculpta and the final unidentified species (sp7) (pp = 1; bv ≥ 93),

In the analysis of the concatenated information, only complete specimen data sets were used. Additional results from the separate matrices, where more samples and species were represented, provided other clues about the evolutionary history of these taxa.

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Figure 2. Phylogenetic relationships based on separate mitochondrial (coxII+Igr+coxI and mtMutS) and nuclear (ITS and 28S) genes. The numbers of stars above the branches indicate the number of phylogenetic methods that supported each clade (pp≥0.95; bootstrap ≥70). Note that all GenBank sequences are considered here with the names as they appear in GenBank and their original publications (including numbers or letters). Numbers after species names indicate different specimens analysed, and an asterisk (*) type material. Leptogorgia and Pacifigorgia specimens from Atlantic Ocean, are indicated with a (A).

In Clade II.A, L. chilensis, L. styx, L. rigida, L. ramulus (in mtMutS tree) (Figs. 2, 3), and L. rigida (in 28S tree) (Fig. 2, 6) were also included with moderate support. However, it should be mentioned that the two samples belonging to L. ramulus (no type material) were split into two different clades for mtMutS, one in the above mentioned group, and the second was paired with L. labiata in the sister clade of E. daniana and E. multifida. Furthermore, within the Clade II.B, L. rubra was also included with high support by the information supplied by its only successfully obtained marker (ITS) (Figs. 2, 5).

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The Clade II.A of the four genes tree (Fig. 1) could be divided in two subclades. Subclade II.A.1, grouped L. alba (1), L. alba (2), L. cofrini, L. alba (3) and L. alba (4), sister group of L. flexilis (1) (pp ≥ 0.81; bv ≥ 100); and subclade II.A.2 grouped individuals of L. cuspidata, L. flexilis (2) and L. alba (5) (pp ≥ 1; bv ≥ 100).

Figure 3. Phylogenetic relationship based on mitochondrial mtMutS. For symbols explanation see legend of Figure 2.

Within the clade formed by Eugorgia appeared E. ampla (28S not available) and E. querciformis (in mtMutS, individual tree, Fig. 3). Furthermore, the mitochondrial trees (mtMutS and Cox, Figs. 2, 3, 4) observed a third high supported clade, with the species E. pumila and E. rubens.

In the case of individual gene trees in Pacifigorgia, the Clade I.A was completed with P. adamsii, P. agassizii, P. cairnsi, P. elegans and P. smithsoniana for mtMutS gene

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(where P. rubripunctata represented another lineage), with a moderate support (Fig. 3). The clade formed by P. rubinoffi, Pacifigorgia sp4 and Pacifigorgia sp5, and completed by P. bayeri, P. catedralensis, P. ferruginea and P. firma (in mtMutS) was poorly supported (Fig. 3), and an important note to mention is that this clade was unsupported in the other individual genes trees. In fact, polytomies in Pacifigorgia and Leptogorgia were more frequent when the individual gene trees were analysed, especially in the case of mitochondrial genes.

Figure 4. Phylogenetic relationship based on mitochondrial coxI+Igr+coxII (referred to as Cox). For symbols explanation see legend of Figure 2.

The Clade I.B was constant through the individual genes, also formed by P. exilis and P. samarensis (in mtMutS, Cox and ITS) (Figs. 3, 4, 5) but it only had strong support for mtMutS; in 28S these species did not form a clade (Fig. 6). Finally, the Clade I.C was well supported in the four genes concatenated and by mtMutS information, but not for each of the other individual genes.

The results showed some contrasting patterns. The specimens of Clade II.A.1 and II.A.2 were grouped following their respective species, having their own mtDNA 241

haplotypes (see mtMutS and Cox individual trees) (Figs. 3 and 4, Appendices 1 and 2). Nevertheless, the same individuals showed differences in the nuclear DNA markers (see ITS and 28S individual trees). In the ITS tree, the clade II.A.2 was found to be the same as when it was formed in the concatenated tree, except for L. flexilis (1) (first clade), where it ceased to be sister group and instead formed a clade with L. alba (3) and L. alba (4). In the 28S tree the relationships changed, the sample L. flexilis (1) became part of the second subclade, together with L. cuspidata, L. alba (5) and L. flexilis (2). The case of this second subclade (II.A.2) will be discussed in detail later.

Figure 5. Phylogenetic relationship based on nuclear ITS. For symbols explanation see legend of Figure 2.

As in the case of L. ramulus, which split into two separate clades in mtMutS, there were other species in the mitochondrial genes that appeared in different clades according to the origin of the samples (type locality or material from elsewhere). This occured for the samples P. firma (1) (type material from MZC (51918)) and P. firma (2) and P. firma (3), who appeared at two independent lineages (Cox, Appendix 2). Similarly, P. cairnsi (type material from MCZ (51915)) and one of the P. cf. cairnsi sequences (in mtMutS) were also found in different lineages.

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Figure 6. Phylogenetic relationship based on nuclear 28S. For symbols explanation see legend of Figure 2.

4.3.5. Discussion

Molecular markers

A large number of phylogenetic studies still rely on markers of limited utility to provide phylogenetic inferences. Many of these studies have noted the limitations of solely using individual genes as this method seldom recovers the optimal topology (Rokas et al. 2003; Salichos & Rokas 2013). Due to this limitation, the current advancement of phylogenetic analysis is based on the idea of “more is better” (Winkler et al. 2015). In previous studies, mtMutS, both alone and in combination with other mitochondrial genes, has been used to examine inter- and intra-generic relationships among shallow-water Caribbean holaxonians (Sánchez et al. 2003b; Vargas et al. 2010b; Wirshing & Baker 2015), Indo-Pacific alcyoniid soft corals (McFadden et al. 2006) and deep-sea gorgonians (France 2007; Thoma et al. 2009). Indeed, mtMutS and ITS2 are 243

one of the commonly used regions and also are the most variable and available for analysis. Hence, they represent important genetic markers for phylogenetic reconstructions (Aguilar & Sánchez 2007; Herrera et al. 2010; McFadden et al. 2010; Yasuda et al. 2015). Similarly, the incorporation of 28S and Cox has further potentiated such phylogenetic analyses (Brockman & McFadden 2012; France & Hoover 2002; McFadden & van Ofwegen 2013b). It is also important to note at mitochondrial genes ND2 and ND6 (also evaluated in this study however not shown) show equal or minor resolution at species level in comparison to other mitochondrial genes. This has been highlighted previously (McFadden et al. 2004, 2006; Sánchez et al. 2003b).

In this study, the analysed phylogenetic resolution at the species-level indicated that nuclear regions of the octocoral genome (ITS and 28S) showed the greatest number of informative characters (53.9%), with ITS being the principle contributor (34.57%, even including the conserved 5,8S). As well, the mitochondrial region was shown to greatly contribute to the phylogenetic signal in the concatenated analysis (almost 46%), with a seen equal contribution of the two genes mtMutS (22.3%) and Cox (23.79%). Previous studies showed the suitable utility of ITS sequences in octocoral species comparison cases, and as an indicator of hybridisation events (Aguilar & Sánchez 2007; Calderón et al. 2006; McFadden & Hutchinson 2004; McFadden et al. 2001). These results further demonstrate the benefits of incorporating nuclear genes alongside the traditional concatenated mitochondrial genes in phylogenetic analyses (Aguilar & Sánchez 2007; McFadden & van Ofwegen 2013a; McFadden et al. 2004). As stated above, as the number of characters per region increases it follows that the total number of variable sites and the number of gene trees with resolved relationships increases, and hence too the confidence level in the relationships increases (Herrera et al. 2010; Winkler et al. 2015). Unfortunately, with the combination of the four nuclear genes, the variation at the species-level for depicting the relationships of some complexes was found to be insufficient.

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Phylogenetic relationships and evolutionary patterns

As indicated above, the phylogenetic relationships within each genus in Gorgoniidae supported the monophyly of Pacifigorgia, but also showed the genera Leptogorgia and Eugorgia to be of non-monophyletic groups, where different lineages of both genera are involved. Clearly this disagrees with the current taxonomic designations that are exclusively based on morphological characters (Bayer et al. 1983; Breedy & Guzmán 2007; Breedy et al. 2009). These characters are the presence of modified sclerites (disc-spindles, double-disc) in Eugorgia principally, but disc- spindles were also found in some species of Leptogorgia from the Atlantic (Breedy et al. 2009). Moreover, the external morphology in these two genera is quite similar, as well as the rest of characters used in their diagnostic descriptions, such as: colony shape, branching type, polyp mound and colony colour (Breedy & Guzmán 2007; Breedy et al. 2009). Reasons as to why previous phylogenetic studies found Leptogorgia and Eugorgia as monophyletic, where Eugorgia is the sister group to Leptogorgia (Vargas et al. 2014), is certainly due to the source of characters or species included in the analyses. When the representation of these two supposed genera was increased, mainly through the addition of other Eugorgia species, the existence of different lineages and the lack of monophyly of both genera were demonstrated. Thus, if a taxonomy based on evolutionary units is desired, at least the Eastern Pacific Leptogorgia and Eugorgia assigned species should be considered as belonging to the same generic unit. However, it is also probable that the whole worldwide-represented genus Leptogorgia is polyphyletic.

In the combined analysis, certain relationships among species within each genus could not yet be resolved. This was also found to be the case when analysing each individual gene, where incongruent patterns were found mostly for Leptogorgia and Pacifigorgia, pointing to two different not mutually exclusive, evolutionary scenarios.

The first scenario represented probable hybridisation processes in the genus Leptogorgia. These processes have been suggested to have played an important role in the coral evolution (van Oppen et al. 2001; Vollmer & Palumbi 2004; Willis et al. 2006). 245

Indeed, the obtained data hinted this possible hybridisation through the inconsistencies observed among individual gene trees (particularly between mtDNA and nDNA) involving the species L. alba, L. cuspidata, L. flexilis and probably L. cofrini (Clade II.A). These inconsistencies could have arisen from gene and/or organism evolution. In the first case, the evolutionary histories of each marker can proceed from incomplete lineage sorting, and/or their different evolutionary rates can coalesce in different moments of the organism’s history. In the second case, the inconsistencies might be explained by hybridisation/reticulate evolution (Seehausen 2004; Vollmer & Palumbi 2004; Wirshing & Baker 2015). Evolutionary outcomes of formation of hybrids could be dependant on the strengths of reproductive barriers (Li et al. 2016). Assuming that the reproductive isolation between two species coming into contact is incomplete, the first generation hybrids may mate with parental species thereby affecting species history and delimitation (Li et al. 2016; Petit & Excoffier 2009). The complex interspecific gene introgression clues found may involve not only “pure” parental species but also hybrids. This demonstrates hybrid fertility. In fact, cross-fertilisation in mass spawning processes have been reported in Acropora and Madracis (Diekmann et al. 2001; Hatta et al. 1999), where such fertilisations may produce a significant number of hybrids (Diekmann et al. 2001; Fukami et al. 2003; Hatta et al. 1999; van Oppen et al. 2001). This scenario results in a sort of species complex or syngameon (Lotsy 1925) that should lack effective reproductive barriers, and result in repeated rounds of separation and fusion of species, which forms a reticulate pattern of many interconnecting lineages (Veron 1995). Thus, difficulties may arise in distinguishing between origin from single pure species and origin from hybrid swarms when one parental phenotype happens to become fixed (Seehausen 2004).

The second scenario related to a conspicuous morphological differentiation with a minimal or null molecular differentiation. A polytomy was observed among certain Pacifigorgia species (Clade I.A) such as P. irene, P. machalilla n. sp., Pacifigorgia sp1, Pacifigorgia sp 2 or Pacifigorgia sp3, and failed to find any phylogenetic signal that clustered closer specimens from a determinate species than specimens from different taxa, mainly due to the inexistence of informative positions. However, these species that grow sympatrically (often side by side), could be identified by consistent 246

morphological characters (Breedy & Guzmán 2002, 2003; Williams & Breedy 2004). The same situation could be observed in the individual genes, where species such as P. adamsi, P. agassizii, P. irene or P. machalilla n. sp. did not present any genetic differentiation.

Different possible explanations to the lack of genetic differentiation can be considered for these cases. The first one would consist of a process of explosive radiation (Henning & Meyer 2014; Kocher 2004; Winkler et al. 2015), where each species has a fixed species-specific morphology, and is reproductively isolated from a single common ancestral species that was morphologically highly polymorphic (Hatta et al. 1999). Here, however, it is difficult to conceive how several species could have arisen from the ancestral species sympatrically, at the same time and without apparent ecological diversification but instead fixed and different morphologies. Also this case would consist of the phylogenetic relationships of taxa resulting from a rapid radiation over a very short time period. These relationships are difficult to ascertain, since there would be little difference between the forms of successive common ancestors (Hoelzer & Melnick 1994). Pacifigorgia irene, P. machalilla n. sp. or P. rubripunctata, would have given rise from a rapid radiation, and each species would show clear diagnostic characters related to their morphology, but lack molecular evidence of their distinctiveness due to the rapid and recent radiation. Moreover, if the time since speciation is too short, the common ancestral alleles will remain in the species at some loci, and incomplete lineage sorting will be observed, which therefore hinders the reciprocal monophyly (Yasuda et al. 2015).

The species in this study share a common ecological niche, thus it is necessary to know the contribution of other biotic and abiotic factors present in the divergence and speciation of these organisms. Sympatric speciation with reproductive isolation may be a resultant of different situations (Bolnick & Fitzpatrick 2007; Levitan et al. 2004). However, explosive radiation is generally seen as an adaptive radiation, that is the rapid adaptation to different ecological niches, with this being seen in the classic example of the cichlid fish. These fish exploit a broad range of ecological niches accompanied by the evolution of a suite of morphological specialisations, such as

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colour patterns related to sexual selection and variations in body and head shape depending on foraging behaviours (Fryer & Iles 1972; Henning & Meyer 2014). The scenario of adaptive radiation should consider the differentiation in peri-, para- or allopatric areas followed by a secondary contact. Determining the possibility of having had occurred would require several types of new experiments and evidences. For example, a deeper knowledge of the gametogenic cycle and the duration of spawning events, as well as hybridisation experiments that reveal if (even in overlapping spawn) pre- or post-zygotic reproductive incompatibility exists in order to verify if such incompatibility could be associated with the recent localised evolution of life-history differences (see the case of Cryptasterina (Puritz et al. 2012)).

Unfortunately, the relative frequency of sympatric speciation is difficult to resolve because biogeographic changes can obscured underlying geographical patterns of past speciation events (Bolnick & Fitzpatrick 2007). In fact, taxa with good fossil records indicate that any reconstruction of divergence across the tropical Eastern Pacific should consider the possibility that modern-day patterns have been shaped by different processes such as extinction (Guzmán & Cortés 1993; Lessios 2008). Pulses of extinction and species origination of corals in the Western Atlantic since the Pliocene are well documented, but in comparison, the record from the Eastern Pacific is poorer (Lessios 2008).

An ancient hybridisation between differentiated species, provoking a re- homogenisation of the genomes, might also have shaped this pattern of morphological differentiation versus molecular identity. If the hybrids were fertile and could backcross with their parental species, then the transmission of genes from one species to another served as a channel for interspecific gene flow (Hatta et al. 1999). The hybrids could not necessarily express intermediate phenotypes, as it occurs, for example, in some Acropora hybrids that might exhibit unexpected morphologies, being recognised as independent species with their own morphology (Hatta et al. 1999; Wolstenholme et al. 2003).

Other inconsistencies were revealed in our phylogenetic analyses. Species named L. ramulus, P. firma and P. cairnsi, appeared split in different clades (see 248

mtMutS tree and Cox, Appendices 1, 2), pointing to misidentification. The inclusion of type material for the two last species clearly indicated that the specimens studied by Vargas et al. (2014) do no belong to such species or, since only mitochondrial genes were analysed, consist in hybrids with morphological appearance P. firma and P. cairnsi, respectively, but paternal contribution from species as, for instance, P. irene, P adamsi or P. elegans, and P. bayeri, P. catredalensis or P. ferruginea, respectively. However, in the study by Vargas et al. (2014), there were differences between P. cairnsi and P. cf. cairnsi. In the case of L. ramulus, the correct taxonomic designation has yet to be confirmed with the type material.

Finally, it should be put into consideration that the derived consequences of these phylogenetic reconstructions may have arisen from multiple and possibly overlapping causes, because hybridisation, rapid radiation, sympatric differentiation and secondary contacts can model these patterns.

In conclusion, the phylogenetic relationships within each genus supported the monophyly of Pacifigorgia, but showed the genera Leptogorgia and Eugorgia as no- monophyletic and therefore should be considered as belonging to the same genus inside the species analysed. Furthermore, the different relationships found in the species of both genera (Leptogorgia and Pacifigorgia) showed the possibility of unusual speciation processes that occurred in Eastern Pacific waters. Different evolutionary events such as a recent radiation (explosive speciation), an ancient hybridisation or a sympatric speciation, may have generated the evolutionary history of the genus Pacifigorgia. Complex speciation histories, including a reticulate process, may have shaped the evolution of genus Leptogorgia and produce a complex of hybrid individuals, with a possible constant gene flow connecting them.

In addition, these results highlight both the role of phylogenetic analysis in understanding the homoplastic and continuous characters used for the morphological taxonomy of octocorals (Dueñas & Sánchez 2009; Sánchez 2007; Vargas et al. 2010a), and the need for a major revision of the morphological descriptions.

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This study also suggests the need of combined genetic analysis based on concatenation of genes and the search of more informative molecular markers in the detection of boundaries between closely related species. This type of combined analysis is necessary in effectively testing morphologically defined species in octocorals in general, and gorgonians in particular (Vargas et al. 2014; Yasuda et al. 2015).

Lastly, this paper highlights that we are still far from depicting or being able to predict common evolutionary patterns and processes, which could explain adaptation and speciation in some organisms such as corals. It is most likely that as more species studies are conduct, these "unusual" speciation events will be considered more as key- processes (Bilewitch et al. 2014; Brockman & McFadden 2012; van Oppen et al. 2001; Wirshing & Baker 2015) and hence these key-processes will help clarify and solve evolutionary histories of species groups.

Acknowledgements

This research received support from the SYNTHESYS Project http://www.synthesys.info/, which is financed by European Community Research Infrastructure Action under the FP7 Capacities Program. This research was also partially supported by a grant from the Spanish Ministry of Economy and Competitiveness (CTM2014-57949-R). Our appreciation to Gonzalo Giribet and Adam J. Baldinger (Museum of Comparative Zoology, Harvard University; in the program MCZ Ernst Mayr Grant); Aude Andouche (Muséum National d'Histoire Naturelle, Paris); Andrew Cabrinovic and Miranda Lowe (Natural History Museum (NHM), London); Eric Lazo-Wasem and Lourdes Rojas (Yale Peabody Museum of Natural History, New Haven), and Stephen Cairns and Herman H. Wirshing (National Museum of Natural History, Smithsonian (MNH), Washington), for helping in our museum work. We thank to Santiago Villamarín (Museo Ecuatoriano de Ciencias Naturales), Machalilla National Park and Ministerio del Ambiente of Ecuador (Manabí) for permits to collect. Michel Guerrero and his team (Exploramar Diving) are grateful

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for their special interest and collaboration since we began our research. Special thanks to Micaela Peña for her help on the collection expeditions.

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4.4 – Sexual reproductive cycle of two representatives of the families

Gorgoniidae and Plexauridae (Octocorallia) in Ecuadorian waters.

4.4.1. Abstract

Octocorals present a number of known reproductive strategies, some of which have been described for gorgonians. These strategies could vary according to local conditions over the geographical and bathymetrical distribution of the species. However, in the Eastern Pacific and especially in the littoral of Ecuador, biological research concerning reproductive patterns in these organisms is still scarce. In the present study, we analysed the reproductive patterns of two gorgonian species, Leptorgorgia alba (Duchassaing and Michelotti, 1864) and Muricea plantaginea (Valenciennes, 1846), as the most frequent representatives of the families Gorgoniidae and Plexauridae in the study area (Machalilla National Park, Ecuador). The histological study based on a 14-month period showed that both species are gonochoric and broadcast spawners, suggesting a continuous reproductive cycle too. However, there were apparent differences in the size of reproductive products between the two species. Finally, different observed peaks of possible emission did not correspond with the slight changes in the sea water temperature registered. A possible correlation with the prevalence of the Humboldt Current System is suggested.

4.4.2. Introduction

Anthozoans, especially octocoral gorgonians, are one of the best represented taxonomic groups in sublittoral marine ecosystems, and are particularly common in benthic communities of tropical areas (Breedy & Guzmán 2007; Yoshioka & Yoshioka 1989). They play an important role as ecosystem engineers (Cerrano et al. 2000; Jones et al. 2004), providing a diversity of micro-habitats and modifying the environment

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and the structure of the community (Buhl-Mortensen & Mortensen 2004; Plaisance et al. 2011; Wendt et al. 1985).

Octocorals can develop different sexual reproductive strategies (see a review by Kahng et al. 2011), although the gonochorism is the most usual (e.g., Fitzsimmons-Sosa et al. 1981; Orejas et al. 2007; Ribes et al. 2007; Barbosa et al. 2014). Some octocoral species are hermaphrodites (e.g., Achituv and Benayahu 1990), or gonochoric with a low percentage of hermaphrodite colonies (Gori et al. 2007; Kahng et al. 2008). Moreover, two basic sexual reproduction modes are known, namely brooding and broadcast spawning, which could vary among species (see a review by Kahng et al. 2011). In broadcast spawners fertilization, further embryo and larval development is external and independent of the parental individuals (Benayahu et al. 1990; Fitzsimmons-Sosa et al. 1981; Harrison 2011; Richmond & Hunter 1990). Brooding could be external (surface brooders) or internal (internal brooders). In the former, oocytes are retained on the surface of the parental colony, under the tentacular crown or on the surface of branches on a mucous secretion (Babcock 1990; Coma et al. 1995a; Garrabou 1999; Gutiérrez-Rodríguez & Lasker 2005; Weinberg 1986). Each of these modes may confer certain reproductive advantages in different environments (Alino & Coll 1989; Babcock 1990). Different authors have proposed that the reproductive strategy and success of fertilization are closely related to the spatial distribution pattern of these marine organisms (Coma & Lasker 1997; Leviton 1982; Orejas et al. 2012; Yund 2000).

Moreover, previous studies have argued that temperature is one of the major factors determining the characteristics of sexual cycles in corals, and therefore in the development of their reproductive products (Dahan & Benayahu 1997; Fautin 2002; Gori et al. 2012; Harrison & Wallace 1990; Ribes et al. 2007). Many authors have suggested that other environmental factors (e.g. lunar cycles, solar insolation or irradiance cycles, upwelling processes, wind regimes) may also influence reproductive patterns, because they act as long-term agents exerting selective pressure on sexuality and reproductive modes (Babcock 1990; Benayahu & Loya 1986; Coma et al. 1995b; Glynn et al. 1991; Van Woesik et al. 2006).

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In particular, gorgonians exhibit low population dynamics (slow growth rates) with low recovery and recruitment rates, and high longevity (low mortality rates) (Coma et al. 1998, 2004; Gotelli 1988; Lasker 1991; Linares et al. 2007). Data on the sexual reproduction of these invertebrates is essential for the study of the effects on population dynamics of natural or anthropogenic disturbances (e.g., stress factors as exposure to high temperatures and/or contamination) (Linares et al. 2008; Rossi & Gili 2009). Thus, under environmental perturbations, these organisms are highly vulnerable and their reproductive rates may be affected, leading to a reduction in the production of sexual outputs (Guzmán & Holst 1993; Linares et al. 2008), and a decrease in the recovery and the re-colonization capacity of the populations at local scale (Cupido et al. 2012; Linares et al. 2007; Quintanilla et al. 2013). In addition, changes in the demographic structure of gorgonians could lead to significant cascade effects on the marine communities in general (Cupido et al. 2012; Lejeusne et al. 2010; Linares et al. 2007, 2008).

In recent decades, studies on octocoral reproduction have especially focused on species from the Great Barrier Reef (e.g. Babcock et al. 1986; Alino and Coll 1989; Babcock 1990) and the Red Sea (e.g. Benayahu and Loya 1986; Benayahu et al. 1990; Dahan and Benayahu 1997; Ben-Yosef and Benayahu 1999). Moreover, a number of reproductive studies have focused on octocorals from the Caribbean or close tropical areas (e.g. Fitzsimmons-Sosa et al. 1981; Brazeau and Lasker 1989; Brazeau and Lasker 1992; Bastidas et al. 2005), the Mediterranean (e.g. Coma et al. 1995b; Gori et al. 2007; Linares et al. 2007; Rossi and Gili 2009; Gori et al. 2012a; Topçu and Öztürk 2016), or polar areas (e.g. Brito 1993; Orejas et al. 2012; Orejas et al. 2007). In particular, the number of studies that have provided reproductive data from gorgonian species in the Eastern Pacific is scarce (Kahng et al. 2011), and data concerning the biology of these organisms in terms of reproductive development is no-existent in the continental littoral of Ecuador, with only a few studies on scleractinians from the Galapagos Islands (Glynn et al. 1991, 1996, 2000, 2011, 2012).

However, the development of studies in a poorly explored geographic area is important in order to obtain a better knowledge of the biology of key marine benthic

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species such as corals (s. l.). In addition, some coral species could show different reproductive patterns according to their distribution, increasing the need to better know local reproductive expressions. For example, the soft coral Heteroxenia elizabethae is gonochoric on the Great Barrier Reef, but it is hermaphrodite in the Red Sea (Hwang & Song 2007), and the scleractinian coral Cladocora caespitosa, is recorded as gonochoric in the Western Mediterranean Sea, but hermaphrodite in the Adriatic Sea (Kersting et al. 2013; Kružić et al. 2008).

From a morphological point of view, octocorals have been the subject of intense taxonomical study in the tropical and subtropical Eastern Pacific. A large number of gorgonians have been reviewed, mainly from the families Gorgoniidae and Plexauridae (Gray 1859), as the most frequent and abundant species (e.g., Prahl et al. 1986; Williams and Breedy 2004; Breedy and Guzmán 2005, 2011, 2013; Breedy and Cortés 2015; Breedy and Guzmán 2015, 2016). However, despite the fact that the Ecuadorian littoral is known to host a high biodiversity (Beck et al. 2008), and to provide a habitat to a great number of invertebrate marine organisms (Bo et al. 2012; Cruz et al. 2003; Guzmán & Cortés 1993), available studies about their biological processes are limited in this area.

In such a scenario, the main objective of this paper was to describe the reproductive biology of the Ecuadorian gorgonians Leptogorgia alba (Duchassaing and Michelotti, 1864) and Muricea plantaginea (Valenciennes, 1846) (this latter species according to the recent description by Breedy and Guzmán 2016), as representatives of the Gorgoniidae and Plexauridae families. In order to achieve this goal, the annual gametogenic cycle was studied during a 14-month period. In addition, we discuss the reproductive output, and compare our results with the patterns described for other species of octocorals from similar and different latitudes.

4.4.3. Materials and Methods The gorgonian material here studied was collected by SCUBA diving from rocky bottoms located in Punta Mala, Machalilla National Park (Manabí, Ecuador), 1°33'41.37"S 80°50'8.79"W. Thirteen sampling trips were carried out between November

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2011 and December 2012, usually on similar monthly dates. Two species were selected due to their being the most abundant and widespread representatives of the families Gorgoniidae and Plexauridae in this area. Twenty colonial fragments of Leptogorgia alba and Muricea plantaginea were collected in each sampling trip, yielding a total of 260 samples in the whole sampling period, for each species. The samples were collected at 15 m in depth. Samples were taken by removing 3-4 cm in lengh apical branches (primary branches) per colony. All samples were immediately preserved in 4 % sea-water-buffered formalin.

Figure 1. Water temperature (°C) at 15 m depth, by HOBO data logger, in Punta Mala (Machalilla National Park, Ecuador.

The temperature was measured by a HOBO Pendant® Temperature Data Logger, which was placed in the study area at the beginning of the study. The maximum and minimum temperature values oscillated between 17ºC and 28ºC in this period (Fig. 1). However, the average values oscillated from 19-20ºC to 24-25ºC. The temperature achieved a value of 24.9 ± 0.97ºC (mean ± SD) in January, decreasing in February and March (19.8 ± 1.93ºC). Then, in April-June, there was an increase in the temperature (up to 24.34 ± 1.36ºC), remaining more stable between July (22.3 ± 1.64 ºC) and November (23.1 ± 1.54 ºC). The temperature rose to 25 ± 0.23 ºC at the end of November and early December (Fig. 1). Sex identification and gamete development stage determination were carried out by histological sections (Fitzsimmons-Sosa et al. 1981). Gorgonian fragments (about 1.5 cm in length) were decalcified in a solution of 10% formic acid for a few minutes, 265

rinsed in running tap water to remove acid excess in the tissues, dehydrated in an alcohol gradient in a vacuum pump, cleared in xylene and then embedded in paraffin. Histological sections, 6 to 8 μm thick, were stained with Ramón y Cajal’s Triple Stain (Gabe 1968). Oocytes were characterized (based on Alvarado et al. 2003; Quintanilla et al. 2013) as: stage I, previtellogenic oocyte (ooplasm reduced, without yolk materials), and stage 2, vitellogenic oocytes (ooplasm enlarged, with drops of yolk). Not enough morphological differences were observed to determine a “mature oocytes” category. Spermatic cysts were identified as: stage 1, without distinct lumen as disorganized spermaries, stage 2, with developed lumen and rounded sperm heads radially arranged with incipient sperm flagella, and stage 3, mature spermatic cysts, with tailed sperm with condensed sperm heads distinctly triangular. A light microscope with an ocular scale was used to measure the diameters of the oocytes and spermatic cysts. Mean diameters of reproductive products were calculated using the Feret’s diameter (see Walton 1948; Servetto et al. 2013; Waller et al. 2014). In all cases where observation possible, the observed number of sexual products was about 300 (n) (per sex, month, and species). The results were presented as the means and standard deviations (mean ± SD).

4.4.4. Results Leptogorgia alba and Muricea plantaginea are gonochoric at polyp and colony level, and current data suggests that they are continuous broadcast spawners. No larvae were observed during the 14-month observation period in the gastrovascular cavities of the polyps, nor on the external surface of the colonies, in any of these species.

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Leptogorgia alba (Fig. 2) Oocytes were present throughout the year (Fig. 3, 5). The maximum monthly (mean ± SD) oocytes diameter observed was 140.55 ± 74.43 µm, a value occurring in May, while the minimum was 45.66 ± 19.78 µm, in July (Fig. 3).

Figure 2. Leptogorgia alba. Longitudinal histological sections along a terminal branch of a colony showing retracted polyps with reproductive products in their gastrovascular cavities; (A) female colony; (B) male colony. Details: (C) Stage I previtellogenic oocytes; (D) Stage 2, vitellogenic mature oocyte; (E) Spermatic cyst in stage 1; (F) Spermatic cyst in stage 2; (G) Spermatic cyst in stage 3; and (H) Spermatic cysts in stage 1, 2 and 3 in the same polyp. Abbreviations: V= vitellogenic, pV= previtellogenic, N= nucleus, n= nucleolus, mc= mesogleal capsule, L= lumen, ts= spermatic tails, and hs= spermatic heads.

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Figure 3. Leptogorgia alba. Diameter (µm) (mean ± SD) of oocytes (solid line) and spermatic cysts (dotted line) in samples collected between November 2011 and December 2012.

Figure 4. Percentages of oocytes (A) and spermatic cysts (B) along to studied reproductive cycle in Leptogorgia alba. Grey bars are stage I, while white with black diagonal lines are stage II, and dotted bars are stage III. 268

In stage I (previtellogenic oocyte) the mean diameter was 63.48 ± 23.34 µm (n=3167), and they were found in all months (Fig. 4A, 6), the relative maximum proportion of oocytes in this stage being achieved from June to October (> 75% of oocytes in these months) (Fig. 4A), while the minimum values were between March and May (Fig. 4A, 5).

Figure 5. Seasonal frequency distribution in oocytes number of Leptogorgia alba ordered by diameter size-classes. (n) is the total number of oocytes found in all the female colonies by each month. Dark grey bars are previtellogenic oocytes (stage I), while light grey bars are vitellogenic ones (stage II).

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In stage II (vitellogenic oocytes) the maximum observed diameter was 301 µm, in December; the maximum proportions of oocytes in this stage were achieved between March and May (~ 35%), and the minimums in July, August and October (Figs. 4A, 5). In the examined material there are no clear oocyte cohorts (in the form of a distinct Gaussian bell curve moving along the sampled months, but different small emission events (e.g. February 2012, October-2012) and other more important ones (e. g. June-August 2012) could be detected (Fig. 5). Throughout the sampled period the percentage of oocytes in stage I was never lower than 44 % (sampling study mean percentage 83 ± 14.9), while the percentage of oocytes in stage II was never higher than 55 % (sampling study mean percentage 16 ± 14) (Fig. 4A). No larvae were observed in the polyps in any of the sampled months. Spermatic cysts were present throughout the year (Figs. 3, 4B, 6). The maximum monthly spermatic cyst mean diameter observed was 148.2 ± 40.43 µm, in August, while the minimum was 107.16 ± 21.25 µm, in February (Fig. 3). In stage I, the mean diameter was 90 ± 36.46 µm (n=999), and they were found every month, the maximum relative proportion being achieved in August (> 50% of spermatic cysts in this month) (Figs. 4B, 6). In stage II, the mean diameter was 134.94 ± 28.46 µm (n=1145), with the maximum relative proportion in May (> 50%), and the minimum (14.28 %) in August (Figs. 4B, 6). In stage III, the mean diameter was 133.6 ± 26.36 µm (n=1543), reaching a maximum diameter value of 236 µm in September, with the maximum relative proportion in March (> 60%) (Figs. 4B, 6). During the whole sampling period, spermatic cysts of all stages (I-III) were present, mature spermatic cysts (stage III) existing in a percentage varying from ~30% (Dic-2011, May and July) to 60% (March) (Figs. 4B, 6). In neither of the sexes, throughout the whole study period, did any distinct cohorts appear, but there was apparently a constant flow of input and emission of reproductive products (Figs. 3, 4A,B), so in this case it is possible that different cohorts could be accumulated in similar size classes, and this fact obscures a more correct interpretation of the duration of the gametogenic cycle. For this reason, a continuous reproductive cycle is proposed for Leptogorgia alba.

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Figure 6. Seasonal frequency distribution in spermatic cyst number of Leptogorgia alba ordered by diameter range. (n) is the total number of spermatic cysts found in all the male colonies by each month. Dark grey bars are stage I, while light grey bars are stage II, and intermediate grey with black diagonal lines are stage III.

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Muricea plantaginea (Fig. 7) Oocytes were present throughout the year (Fig. 8, 10). The maximum monthly (mean ± SD) oocyte diameter observed was 177.6 ± 115 µm, a value that occurred in November 2012, while the minimum was 144.32 ± 83.97 µm, in February (Fig. 8).

Figure 7. Muricea plantaginea. (A) Longitudinal histological sections of a branch in female colony with the image of some retracted polyps with oocytes in the gastrovascular cavity; (B) stage 2, vitellogenic mature oocyte; (C) Stage I previtellogenic oocyte; (D) Previtellogenic oocyte between two vitellogenic mature oocytes; (E) Spermatic cyst in stage 1, with disorganized spermaries; (F) Some spermatic cysts in stage 2 in the same polyp; and (G) Spermatic cyst in stage 3 (mature Spermatic cysts). V= vitellogenic, pV= previtellogenic, N= nucleus, n= nucleolus, mc= mesogleal capsule, L= lumen, ts= spermatic tails, and hs= spermatic heads.

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Figure 8. Muricea plantaginea. Diameter (µm) (mean ± SD) of oocytes (solid line) and spermatic cysts (dotted line) in samples collected between November 2011 and December 2012.

Figure 9. Percentages of oocytes (A) and spermatic cysts (B) along to studied reproductive cycle in Muricea plantaginea. Grey bars are stage I, while white with black diagonal lines are stage II, and dotted bars are stage III. 273

In stage I (previtellogenic oocyte) the mean diameter was 91.22 ± 27.22 µm (n=2249), and they were found every month (Figs. 9A, 10), the relative maximum proportion being achieved in November´11 (~ 70% of oocytes in this month), and a minimum in January (~ 40%) (Figs. 9A, 10).

Figure 10. Seasonal frequency distribution in oocytes number of Muriceaplantaginea ordered by diameter range. (n) is the total number of oocytes found in all the female colonies by each month. Dark grey bars are previtellogenic oocytes (stage I), while light grey bars are vitellogenic ones (stage II).

In stage II (vitellogenic oocytes) the maximum observed diameter was 614 µm, in October, and the maximum proportions of oocytes in this stage were achieved in 274

January (~ 60% of oocytes in this month), and a minimum in November 2011 (< 30%) (Figs. 9A, 10). No clear oocyte cohorts were observed during the sampled period (Figs. 9A, 10). There was a nearly constant relative percentage of oocytes in stage I (56.46 ± 7.6 %) throughout the examined cycle, never lower than 40 %. The relative percentage of oocytes in stage II was never lower than 28 % (Fig. 9A). No larvae were observed in the gastrovascular cavities of polyps in any of the sampled months. Spermatic cysts were present throughout the year (Figs. 8, 9B, 11). The maximum monthly spermatic cyst diameter observed was 195.27 ± 76.97 µm, in November 2012, and the minimum was 146.02 ± 39.57 µm in March for male colonies (Fig. 8). In stage I, the mean diameter was 105.5 ± 26.6 µm (n=982), and they were found every month (except in January), the maximum relative proportions being achieved in June and September (~ 38 % of spermatic cysts in these months), and the minimum (~ 4%) in February (Figs. 9B, 11). In stage II, the mean diameter was 182.22 ± 42.3 µm (n=1635), with the maximum relative proportion in February and September (~ 60% of spermatic cysts in these months), and the minimum June (~ 25%) (Figs. 9B, 11). In stage III, the mean diameter was 196.12 ± 56.94 µm (n=896), with maximums in January and November´12 (~ 40% of spermatic cysts in these months), and the minimum in September (where they practically disappeared) (Figs. 9B, 11). Throughout the sampling period, spermatic cysts of all stages (I-III) were present, mature spermatic cysts (stage III) making up an average relative percentage of 26.3 ± 11.2 %. The presence of spermatic cyst (stage II) was usually higher than 45% (except in May’12, June’12 and November´12). An apparent disappearance of spermatic cysts (stage I) in January, and a distinct reduction in mature spermatic cysts (stage III) in September, could be detected (Figs. 9B, 11). In neither of the sexes did there seem to be distinct cohorts (Figs. 5-11) at any time during the study period, but these could be accumulated in similar size classes, which may indicate that Muricea plantaginea has apparently a continuous reproductive cycle.

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Figure 11. Seasonal frequency distribution in spermatic cyst number of Muricea plantaginea ordered by diameter range. (n) is the total number of spermatic cysts found in all the male colonies by each month. Dark grey bars are stage I, while light grey bars are stage II, and intermediate grey with black diagonal lines are stage III.

4.4.5. Discussion Reproductive cycle/breeding According to the results, in Ecuador Leptogorgia alba and Muricea plantaginea are gonochoric and continous broadcast spawners, suggesting an external fertilization

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and development of planula larvae. Massive release could not be observed at the site during the monitored time. Both species appeared to exhibit a continuous gametogenesis. This hypothesis is supported by the presence of male and female reproductive products in all stages of development throughout the year (Cordes et al. 2001; Pires et al. 2009). Moreover, L. alba and M. plantaginea harbour a constant amount of previtellogenic and vitellogenic oocytes, as well as internal brooder gorgonian species such as Acabaria biserialis (Ben-Yosef & Benayahu 1999), and Tripalea clavaria (Excoffon et al. 2004). In addition, the present observed pattern of continuous gametogenesis with external fertilization, may be similar to that found in other tropical octocorals such as Carijoa riisei (Hawaii, Puerto Rico) (Kahng et al. 2008), and Dendronephthya hemprichi (Red Sea, subtropical) (Dahan & Benayahu 1997), and also in temperate waters as for Spinimuricea klavereni in the Mediterranean (Topçu & Öztürk 2016), or even from cold waters, as for Primnoa resedaeformis (Canada) (Mercier & Hamel 2011). Also, in the sea pens Pennatula aculeata (Gulf of Maine) (Eckelbarger et al. 1998) and Kophobelemnon stelliferum (south-west of Ireland) (Rice et al. 1992), the possibility was suggested that these species could present a continuous spawning due to there being no evidence for seasonality in gametogenic development. Other octocoral species with continuous reproduction are the external surface brooder Sarcothelia edmondsoni (Hawaii) (Davis 1977 in Kahng et al. 2011) and the internal brooder Anthomastus ritteri (Baja California) (Cordes et al. 2001). Broadcast spawning is considered to be the most common sexual reproductive strategy found in octocorals in general (Kahng et al. 2011) and in alcyonaceans in particular (Coll et al. 1995). Fertilization is predominantly external in tropical areas, where the gametes are directly broadcast to the water column (Excoffon et al. 2011). However, octocoral species are prone to internal fertilization and brooding in cold or temperate waters (see Excoffon et al. 2011; Gori et al. 2012b; Waller et al. 2014). The two species in the present study are gonochoric, which is the dominant sexuality mode among octocorals (Cordes et al. 2001; McFadden et al. 2001; Orejas et al. 2012; Pires et al. 2009). According to Kahng et al. (2011), of 159 octocoral species in

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which sexuality is reported, 89% are gonochorics and 9% are simultaneously hermaphroditic. Specifically, quite a few studies of gorgonian reproduction also showed gonochorism as the dominant trait (Ben-Yosef & Benayahu 1999; Brazeau & Lasker 1989; Coma et al. 1995b; Excoffon et al. 2011; Ribes et al. 2007; Rossi & Gili 2009; Sánchez 2009). In an evolutionary framework, according to the study by McFadden et al. (2001) on soft corals, it seems that external brooding may represent an intermediate transitional stage between internal brooding and broadcast spawning. At the same time, these authors suggest that gonochorism is a plesiomorphic character in the genus Alcyonium, a statement probably applicable to the main stem of Octocorallia.

Maximum size of reproductive sexual products The maximum observed diameter of female and male reproductive products in Leptogorgia alba was rather small compared to other gorgonian species, in contrast with Muricea plantaginea, which showed relatively larger reproductive products, similar to other tropical and temperate plexaurid species (see Table 1). A number of factors have been proposed to account for the inter-specific variation in the maximum size of reproductive products, especially for oocytes. Some previous studies related the oocyte size to the level of energy allocated to reproduction in each species (Shlesinger et al. 1998). For example, it was hypothesized that some species, e.g. Leptogorgia species, could invest a high part of their energy reserves in other secondary production processes, such as growth, using only part of their reserves for reproduction (Rossi & Gili 2009; Rossi 2002).

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Table 1. Comparative summary among studied species bold and some previous studies about maximum oocyte and spermatic cysts size in gorgonians and other octocorals species. The study location and reference paper are indicated. G= gonochoric, E= external surface brooder, B= internal brooder, S= broadcast spawner, ? = no available data.

max. max. spermatic Family Species Location Reference oocyte size cysts size

Primnoidae Fannyella spinosa (G) (B) ∼ 310 µm ∼ 310 µm Weddell Sea Orejas et al. 2007

Gorgoniidae Leptogorgia hebes (G) (S) ∼ 340 µm ∼ 350 µm Gulf of Mexico Beasley and Dardeau 2003

Gorgoniidae Leptogorgia alba (G) (S) ∼ 350 µm ∼ 248 µm Ecuador Present paper

Primnoidae Fannyella rossii (G) (B) ∼ 450 µm ∼ 200 µm Weddell Sea Orejas et al. 2007

Dendronephthya hemprichi Nephetheidae 500 µm 400 µm Red Sea Dahan and Benayahu 1997 (G) (S)

Leptogorgia sarmentosa Gorgoniidae 500 µm 550 µm Mediterranean Sea Rossi and Gili 2009 (G) (S o E?)

Paramuricea clavata (G) Coma et al. 1995; Gori et al. Plexauridae 550 µm 700 µm Mediterranean Sea (E) 2007

Antillogorgia elisabethae Gutierrez-Rodriguez and Lasker Gorgoniidae 580 µm 390 Bahamas (G) (E) 2004

Plexaura kuna Plexaura A Plexauridae 600 µm Panama Brazeau and Lasker 1989 (G) (S) ∼

Antillogorgia americana Gorgoniidae 600 µm Florida Fitzsimmons-Sosa et al. 1981 (G) (S) ∼

Clavulariidae Carijoa riisei (G) (?) ∼600 µm ∼515 µm Puerto Rico Kahng et al. 2008

Muricea plantaginea (G) Plexauridae 614 µm 420 µm Ecuador Present paper (S) ∼ ∼

Plexaura homomalla (G) Fitzsimmons-Sosa et al. 1981; Plexauridae 640 µm 405 µm Mexico, FLorida (S) ∼ ∼ Martin 1982

Plexauridae Muricea fructicosa (G) (B) ∼650 µm ∼450 µm California Grigg 1977

Fitzsimmons-Sosa et al. 1981; Plexauridae Eunicea flexuosa (G) (S) 667 µm 450 µm Panama, Florida ∼ ∼ Beiring and Lasker 1996

Muricea californica (G) Plexauridae 750 µm 600 µm California Grigg 1977 (B) ∼ ∼

Spongiodermid Mar del Plata, Tripalea clavaria (G) (B) 752 µm 900 µm Excoffon et al. 2004 ae ∼ ∼ Argentina

Pseudoplexaura porosa Kapela and Lasker 1999; De Plexauridae 860 µm 500 µm Bermuda, Panama (G) (S) ∼ ∼ Putron and Ryland 2009

Eunicella singularis (G) Gori et al. 2007; Ribes et al. Gorgoniidae 900 µm 830 µm Mediterranean Sea (B) ∼ 2007

Coralliidae Corallium rubrum (G) (B) ∼900 µm ∼900 µm Mediterranean Sea Tsounis et al. 2006

Dasystenella acanthina (G) Primnoidae 1200 µm 780 µm Weddell Sea Orejas et al. 2007 (?) ∼ ∼

Primnoidae Ainigmaptilon antarcticum 700 µm 1000 µm Weddell Sea Orejas et al. 2012

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Furthermore, the relatively large diameter of oocytes in species such as Muricea plantaginea in our study, could define the duration of the maturation period, as large oocytes may indicate a prolonged oogenesis period (see Harrison & Wallace 1990). Indeed, species with oogenesis >1 yr tend to have larger oocytes (Kahng et al. 2011; Orejas et al. 2012) than species with shorter oogenesis (Quintanilla et al. 2013), although some exceptions exist (see Benayahu & Loya 1986, 22-23 months for mature oocytes of 500-750 µm in Sacophyton glaucum). In fact, according to our results, and despite several cohorts seem to overlap along the 14-month sampled period in both examined species, Leptogorgia alba with maximum oocytes diameter of ~ 350 µm has more than probably a shorter oocyte maturation period than Muricea plantaginea (~614 µm).

Environmental and biological factors

Although annual seasonal and inter-annual variation of temperature have to be considered in order to elucidate the timing of reproductive patterns (Dahan & Benayahu 1997; Glynn et al. 1991, 2012; Rossi & Gili 2009), the local or regional environmental conditions may also act as additional or alternative controls on sexual reproduction (Harrison & Wallace 1990; Quintanilla et al. 2013; Simpson 1985). Thus, besides temperature, there are other factors that may affect the reproduction of gorgonians (Gori et al., 2012; Rossi and Gili, 2009). Among spawner octocoral species, the release of reproductive products may coincide with lunar cycles, for instance, around or following full moons (Babcock 1990; Benayahu & Loya 1986; Coma et al. 1995b), or rainfall and wind velocity/regimes (Acosta & Zea 1997; Alvarado et al. 2003; Babcock et al. 1986). In the same way, annual cycles of insolation could also predict spawning times, due to the fact that ultimately, there is an interdependent relationship between solar radiation phenomena and sea surface temperature (Van Woesik et al. 2006). In relation to the rise of temperature and the trophic crisis in the Mediterranean summer, Alcyonium coralloides (with one of the shorter gametogenesis cycles in octocorals) releases its larvae before its gorgonian host Paramuricea clavata is ready to do so, conferring on the Alcyonium species a certain colonization advantage (Coma et

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al. 1995a; Quintanilla et al. 2013). Another factor is the depth: as previous studies showed significant differences in the reproductive cycle, especially in reproductive product volume, between shallow and deep water in the case of Eunicella singularis populations in the Mediterranean Sea (Gori et al. 2012) and in the tropical gorgonian Briareum asbestinum (West et al. 1993). Maybe, these differences with the depth could be indirect and directly related with the differences in temperature and availability and quality of nutrients. Even, environments subject to seasonal upwelling (phytoplankton availability) could influence the broadcast spawning moment (Glynn et al. 1991, 1996; Ben-Yosef and Benayahu 1999; Glynn et al. 2000, 2011, 2012), or the constant availability of trophic resources could support a continuous gametogenesis (see Spinimuricea klavereni in Topçu and Öztürk 2016). According to our data, no assumptions can be made about the possibility that spawning occurs in a certain phase of the lunar cycle. In our study area, the mean monthly temperature fluctuated by ±4ºC, and the continuous broadcast spawning suggested for Leptogorgia alba and Muricea plantaginea does not seem to correspond to an apparent variation in the water temperature. However, both species presented some fluctuation in their reproductive activity, such as possible pulses of emissions of mature reproductive products and reduced production of previtellogenic oocytes and spermatic cysts stage I, between the months of June and September, which was especially apparent in L. alba. At this time of the year, the littoral of Ecuador is strongly influenced by the nutrient rich Humboldt Current System (Cucalón 1996; Thiel et al. 2007). This period promotes a variety of ecological processes due to the oceanographic effects of upwellings (Alheit & Niquen 2004; Carrasco & Santander 1987; Thiel et al. 2007), such as the increase in primary production both in the plankton and in the benthos (Iriarte & González 2004; Thiel et al. 2007), and it also influences larvae dispersal of some organisms (Poulin et al. 2002). One possible hypothesis may be that the increased availability of nutrients during this period benefits benthic organisms such as the studied gorgonians, increasing the broadcast of mature gametes and facilitating the generation of new reproductive products.

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In conclusion, this study shows that L. alba and M. plantaginea have interspecific differences with respect to the maximum size of their reproductive products, but similarities regarding their sexuality and reproductive patterns in that, they are gonochoric species and continuous broadcast spawners, with external fertilization. These patterns in these species seem not to be related to temperature changes, but may undergoa possible fluctuation at certain times associated with the increase of Humboldt Current System. The strategies of these gorgonians and a comparison with other octocorals can help to gain a better understanding of the reproductive biology in these species in the little-known littoral of Ecuador. Finally, this kind of information is of special interest with reference to the local or regional management of marine protected areas, as well as providing an insight into the possible repercussions for the benthic communities of those changes in the different phenomena associated with the currents systems occurring in the Central Eastern Pacific.

Ackwoledgements Our appreciation to Santiago Villamarin (Museo Ecuatoriano de Ciencias Naturales), Machalilla National Park and Ministerio del Ambiente of Ecuador (Manabí) for permits to collect and support. We thank Michel Guerrero and his team (Exploramar Diving) for his special interest and collaboration from the beginning of our research. Special thanks to Micaela Peña and Eva Andrés Marruedo for their essential support throughout the sampling period.

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4.5- Biodiversity patterns of gorgonians epifaunal community in Ecuador (Eastern Pacific).

4.5.1. Abstract

The high biodiversity of the coral reef is widely acknowledged, as well as the role it plays as an ‘ecosystem engineer’ providing different microhabitats for a wide number of associated organisms. However, only a few studies have examined the diversity and community structure of coral-dwelling invertebrates. In particular, Ecuador (Eastern Pacific) is an area recognized for its high biodiversity, but at the time of writing, the ecological processes concerning the relationship between the gorgonian host and its associated epifaunal are unknown in this area. The present study aimed to examine the patterns of diversity, abundance and structure of the epifaunal assemblage associated with the gorgonians of Ecuador. Two gorgonian species with different morphological characteristics were selected, Leptogorgia sp. and L. obscura, during two different seasons, June (rainy season) and November (dry season), in two consecutive years. Multivariate and univariate analyses were performed using PERMANOVA statistical treatments. The living space of host species was considered as covariable in each analysis. According to the results of this study, the gorgonians from Ecuador showed a hyper-abundant epifaunal community in comparison with other coral hosts in other regions, and with other ecosystem engineers. This has demonstrated their high effectiveness as bio-constructors, and illustrates the need to preserve gorgonian gardens in this region. In addition, different patterns related to the structure of the community, abundance and diversity were observed according to different host gorgonian species and their morphology. Moreover, the two seasonal periods, which were affected by different regional oceanographic processes, may significantly contribute to abundances of epifaunal assemblages. This was especially notable in the increase of abundance related to the Humboldt Current System.

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4.5.2. Introduction

Since the last century, one of the major objectives of ecology has been to understand how interspecific interactions influence community organization (Idjadi & Edmunds 2006; Law 2016). In particular, coral reefs (gorgonian and scleractinian species) have an extraordinary associated biodiversity and provide support for a wide variety of different taxa, making them one of the most diverse and complex marine environments (Idjadi and Edmunds 2006; Knowlton et al. 2010; Stella et al. 2010). It has been estimated that the total number of species living on coral reefs around the world may be about 93,000 (Knowlton et al. 2010; Reaka-Kudla 1997).

Corals in general and gorgonians in particular are considered ecosystem engineers (Cerrano et al. 2000; Jones et al. 2004), which provide a diversity of microhabitats for small invertebrates (Wendt et al. 1985; Buhl-Mortensen and Mortensen 2004; Plaisance et al. 2011), as well as shelter, food and protection (Kumagai 2008; Patton 1974). The particular characteristics of a specific coral host, such as its size, morphology or its chemical ecology (secondary metabolites) determine the diversity, composition and abundance of the associated fauna, this relationship being susceptible to environmental changes (Greene 2008; Stella et al. 2010). The associated invertebrates may also have an important influence on hosts, connect corals with other reef organisms, or influence the hosts’ health, sometimes being fundamental to their persistence and resilience (Mokady et al. 1998; Stella et al. 2011).

The current degradation of coral habitats is causing a parallel unavoidable decline in the diversity of their associated biota (Munday 2004; Pandolfi 2011; Pratchett et al. 2008). Knowledge about the relationships between the epifaunal community and corals, as well as the identification of the functional roles of the former and the susceptibility of this relationships to environmental changes, are fundamental for science-based conservation of biodiversity and use of these habitats (Carvalho et al. 2014; McKinney 1997; Stella et al. 2011).

Many authors have recognized the importance of gorgonians as characteristic members of benthic communities in polar, tropical and temperate ecosystems (Arntz

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W 1999; Coma et al. 1995; Kinzie 1973; Tsounis et al. 2006). The family Gorgoniidae is one of the most diverse, and includes some of the genera with the highest number of species in shallow temperate and tropical waters (Soler-Hurtado & López-González 2012). This family is widely distributed along the Eastern Pacific, from Baja California to Peru, including the oceanic islands (Breedy et al. 2009). Four gorgoniid genera have been previously reported in the Eastern Pacific, among which the genus Leptogorgia Milne-Edwards and Haime 1857, with about 60 valid species (27 species in the Eastern Pacific), is one of the most frequent (Breedy & Guzmán 2007). Within the Eastern Pacific region, the coast of Ecuador is recognized for its high biodiversity, possessing 3 of the 34 "hotspots" around the world (Beck et al. 2008; Myers et al. 2000). The Ecuadorian littoral hosts a high biological richness, and provides habitat to a great number of marine organisms, although the available information is scarce and incomplete (Bo et al. 2012; Cruz et al. 2003; Soler-Hurtado & Guerra-García 2016; Soler- Hurtado & López-González 2012). Studies concerning marine biodiversity in this area would be of high interest.

The littoral of Ecuador is strongly influenced by several complex oceanographic processes (Glynn 2003). From June to October, the nutrient rich and cold Humboldt Current is constantly flowing from the south, enhancing the coastal upwelling phenomena; from November to May, the poor and warm tropical waters from the Bay of Panama, in the north, dominate the coastal areas (Cucalón 1996; Thiel et al. 2007). The Humboldt Current System (HCS), which extends along the west coast of South America from southern Chile up to Ecuador and the Galapagos Islands, is the most productive eastern boundary upwelling system of the world’s oceans in terms of fisheries (Chavez et al. 2008; Echevin et al. 2012; Thiel et al. 2007). In addition, in this area there is the notable presence of periodic climatic events, i.e. El Niño and La Niña (Bo et al. 2012; Glynn 2003). How these processes affect marine biodiversity on the Ecuadorian littoral is mostly unknown.

In Ecuador, Machalilla National Park is one of the most important marine- terrestrial reserves of the country (Glynn et al. 2003), which covers an area of 56.184 ha of protected lands, coastlines and some volcanic islands (Zambrano 1998). This area is

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known for its high diversity of terrestrial and marine fauna, and the notable presence of the only coral reef in the continental region of the country (Cruz et al. 2003; Guzmán & Cortés 1993). Two species of the genus Leptogorgia, L. obscura Bielschowsky 1929 and Leptogorgia sp. (still not assigned to any nominal species) are among the most common species in this area and form important gorgonian gardens between 3- 30 meters in depth (pers. obs.). These two species show different morphological characteristics which may potentially affect their associated epifaunal community.

The aim of the present study is to describe the patterns of diversity, abundance and structure of the epifaunal community associated with the gorgonian gardens of Ecuador, using as examples two of the most representative species of this area, Leptogorgia sp. and L. obscura. Specific questions addressed in this research were: are the observed patterns different in communities associated with different gorgonian hosts that show different morphological characteristics? Do these patterns fluctuate seasonally throughout the year, under the influence of the dominant oceanographic processes, over and above the year-to-year variability? Is there any specific group that characterizes each host species or each season?

4.5.3. Material & Methods

Samplings

Gorgonian colonies were collected by SCUBA diving from rocky bottoms located in Los Frailes, Machalilla National Park (Manabí, Ecuador) (1°30'14"S 80°48'33”W), between 10 and 15 m in depth. Samplings were carried out in November 2012 and 2013, and June 2013 and 2014 to cover the two main equatorial environmental conditions over the year. Ten colonies from each of the two species, Leptogorgia sp. and Leptogorgia obscura, were collected in each sampling time. We selected colonies of similar height (150-200 mm). The entire colonies were enclosed into individual plastic bags underwater to prevent faunal loss. We left in place a holdfast with its coenenchyme, to allow the colony to grow up again. Samples were sieved through a 100

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μm mesh sieve. All specimens associated with each colony were preserved in 70% ethanol. Due to the lack of taxonomic information on the epifaunal assemblages species from this area and the extraordinary abundances observed, the speciemens were sorted and separated into general groups with clearly different morphologies and, potentially, different ecological characteristics and life histories, thus avoiding problems associated with taxa misidentifications (Guerra-García et al. 2011; Kelaher et al. 2001; Klitgaard 1995; Nogueira et al. 2015; Sánchez-Moyano et al. 2006).

Morphometry of the colonies

To analyse the characteristics of each of the two gorgonian species as a habitat for the associated epifauna, the available living space (LS) (based on Stella et al. 2010) was compared between them with a Wilcoxon rank-sum test (WRS) (Wilcoxon 1946). In order to do this, we measured the total surface area of each fan on lateral view (A), using the Image J software (Rasband 2007), and the colony thickness (H). The volume displaced by the colony was also measured in a known volume of water (1,000 cm3). The LS was calculated by multiplying A x H and subtracting the displaced water volume (based on Stella et al. 2010). In addition, the dry weight of each colony was also measured, and the density (number of associated individuals per unit of LS) was calculated. The results were presented as the means and the standard errors (mean ± SE).

Structure of the community

The number of individuals of each group were organized in a taxa/sample abundance matrix, and a Bray–Curtis similarity matrix was calculated on fourth-root- transformed data, as a distance measure among samples (Bray & Curtis 1957). The differences in the multivariate structure of the fauna associated with gorgonians were analysed in a distance-based permutational multivariate analysis of variance

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(PERMANOVA) (Anderson 2001; McArdle & Anderson 2001). The experimental design included two crossed fixed factors: “host species” (with 2 levels, Leptogorgia obscura and Leptogorgia sp.), and “season” (with 2 levels, June and November). One additional random factor was included, “year”, to account for the interannual variability in the observed patterns; this factor (“year”) was nested in seasons. The LS could have an effect on the abundances of the different epifaunal general groups, which would not necessarily be equal for all of them, and it could potentially have an effect on the community structure (Anderson et al. 2005); the LS was included in the model as a continuous predictor. Thus, Type I (sequential) sums of squares was used in this analysis, fitting the continuous predictor (covariate) first. The analysis was re-run swapping the order of factors in the model to check whether this affected any essential interpretations of the results (Anderson et al. 2008). The complete model generated a negative estimate of a component of variation in a three way interaction, and this term was excluded from the final model (Anderson et al. 2008). When the number of total possible permutations to obtain the P values was low, we used the estimation obtained by Monte Carlo sampling (Anderson & Robinson 2003). The same analysis was performed with standardized data to check which differences were mainly due to total abundances. The homogeneity of multivariate dispersion was tested using PERMDISP (Anderson 2006). The distances were represented by non-metric multi-dimensional scaling ordinations (nMDS; Clarke 1993). SIMPER (Clarke 1993) was used to identify the percentage contribution that each taxon made to the similarity within and dissimilarity among the different levels of the fixed factors for which significant differences were found. The ratio “average similarity/standard deviation” (SIM/SD) and “average dissimilarity/standard deviation” (DISS/SD) are useful measures of how consistently a component of the assemblage typifies a group or discriminates between groups (Clarke & Warwick 2001). We tabulated the taxa with DISS/SD or SIM/SD larger than or equal to 1.4 (González-Duarte et al. 2014; Megina et al. 2013). Multivariate analyses were performed using the software PRIMER v6.1.11 and PERMANOVA v1.0.1 statistical package (Clarke & Gorley 2006).

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Abundance and Diversity

We examined the significant differences in the abundance (N) and diversity (Shannon-Wiener Index, H´) using PERMANOVA tests on Euclidean distance matrices, in an approach similar to parametric ANOVA (Anderson 2001; Schultz et al. 2012; Scyphers et al. 2011), using the same design explained in the previous section. N, H’ and LS were log transformed. We tested the homogeneity of the slopes by including the interactions with the covariable (LS). The sums of squares used were also Type I, and the effect of the order in which the terms were fitted was also checked. A model selection was then performed; firstly excluding non-significant interactions with the covariable (hence, assuming homogeneity of the slopes among the levels of the factor in turn). Secondly, if the complete model generated negative estimates of components of variation, the affected terms were excluded, one at a time, beginning with the term having the smallest mean square (Anderson et al. 2008). When significant interactions among categorical predictors were detected, they were further explored within the levels of the interacting factors separately.

When the number of total possible permutations was low, we used the estimation of P values obtained by Monte Carlo sampling (Anderson & Robinson 2003). Homogeneity of dispersions was also tested with PERMDISP routine, which performs a Levene’s-type test using the group means, but obtaining the p-values by permutations (Anderson et al. 2008). Boxplots and plots were made in Rstudio (Team 2015).

4.5.4. Results

A total of 50,284 individuals belonging to 10 phyla and 20 epifaunal general groups were identified from the 80 colonies of Leptogorgia (Table 1). The individuals (N) per colony, the average densities (ind/cm3 of displaced volume and ind/g of colony dry mass), per host species and per season, and their range of values, are shown in

Table 2.

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Table 1. Total number of individuals per Pyllum and per OTUs (presence or absence in Bryozoa and Porifera) in each gorgonian host species (Leptogorgia obscura and Leptogorgia sp.) and each season time (November and June).

Gorgonian host species Season

L. obscura L. sp. Dry (Nov.) Rainy (June)

Aracnida (Acari) 9 5 4 10 Bryozoa 0 3 0 3 Caprelloidea 81 43 20 104 Cirripedia (cipris) 128 124 64 188 Copepoda 2723 1634 1103 3254

Decapoda 395 26 95 326 Echinoidea 1 4 0 5

Gammaridea 14314 780 3158 11936 Gastropoda (Opisthobranchia) 4 12 13 3 Gastropoda (Prosobranchia) 335 137 116 356 Isopoda 483 491 143 831 Mollusca (no Gastropoda) 91 36 30 97

Nematoda 22 15 8 29 Ophiuroidea 9627 18227 6310 21544

Ostracoda 38 24 26 36 Polychaeta 313 96 149 260 Porifera 7 0 1 6 Pycnogonida 16 16 5 27 Sipuncula 3 2 0 5

Tanaidacea 10 9 3 16

At high level groups, crustaceans and equinoderms dominated the epifauna of the two gorgonian hosts, comprising together more than 90%: 63.54% of crustaceans and 33.66% of equinoderms in L. obscura, and 14,44% of crustaceans and 84,08% of equinoderms in Leptogorgia sp.

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Living Space

Leptogorgia obscura had a significantly larger living space (W = 1236, p < 0.0001), almost 3 times higher (mean±sd= 363.8±180.7) than Leptogorgia sp. (mean±sd= 123.7±88.2).

Table 2. The means with either the standard error (mean ± SE) of the number of individuals per colony (ind/col), the density of displaced volume (ind/cm3) and colony dry mass (ind/gr), per species and per season, with their range of values.

Individuals per colony Individual per displaced Individual per dry

(ind/col) volume (ind/cm3) mass (ind/g)

Total = 50,284 ind. 674.5 ± 69.57 37 ± 2.76 60 ± 4.64

Leptogorgia obscura 715 ± 116.45 31,4 ± 4.12 42 ± 6.08

range (min-max) 21 - 3068 1 - 112 1 - 187

Leptogorgia sp. 637 ± 56.68 42.67 ± 3.16 80 ± 5.72

range (min-max) 146 - 1434 14 - 95 27 - 146

June 975,9 ± 105 47 ± 3.69 73 ± 5.9

range (min-max) 102 - 3068 7 - 112 12 - 187

November 330 ± 39.29 24.4 ± 3.27 44 ± 6.64

range (min-max) 21 - 898 1 - 78 1 - 136

Structure of the community

Once the variability due to LS that was fitted first was taken into consideration, there was a significant effect of the “gorgonian host species” in the community structure (Table 3). No significant effect of the “season” as a main factor was observed, but the interaction between “season” and LS was marginally significant. The multivariate dispersion did not show significant differences between the levels of the factor “host species” (PERMDISP F1,72: 1.9324; P(perm): 0.2005).

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Table 3. PERMANOVA table of results, Multivariate analyses. LS= living space; S= species; Se= season; T= time (year). (*) P estimation obtained by Monte Carlo sampling.

df SS MS Pseudo-F P(perm)

LS 1 3928.1 3928.1 12.358 0.0001 S 1 3250.1 3250.1 5.8212 0.0469

Se 1 4603.3 4603.3 1.6699 0.2222(*)

T(Se) 2 5361.2 2680.6 8.4744 0.0001

LSxS 1 472.82 472.82 1.4926 0.1875

LSxSe 1 632.74 632.74 2.0003 0.0607

SxSe 1 797.65 797.65 2.2099 0.2284

LSxT(Se) 2 860.36 430.18 1.36 0.1938

SxT(Se) 2 663.63 331.81 1.049 0.4186

LSxSxSe 1 407.52 407.52 1.2883 0.2752

Residual 60 18979 316.32

Total 73 39957

Given that the two studied host gorgonians had different LS, the variability in the community structure due to LS overlaped with that of “host species”. However, when we changed the order in which the terms of the model were fitted, the results remained constant for either of the significant differences between the two hosts species and the non-significant differences between the main factor “seasons” (Table 3, Fig. 1A).

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Figure 1. Non-metric multidimensional scaling ordination of the invertebrates associated assemblages on the basis of the Bray–Curtis dissimilarity. (A) Host gorgonian species assemblages: ■ Leptogorgia obscura, ▽ Leptogorgia sp. (B) Season assemblages: ▼ June, ○ November. Stress = 0.22.

The differences due to LS, in contrast, were only significant when this term was fitted in first place (the usual protocol when using continuous predictors) (Table 3, Fig. 1B). We did not eliminate the LS from the model because of the marginaly significant interaction with "season" (Table 3). This interaction was clearly not significant in the second model where the data were standardized, removing the effect of the abundances (P(perm) for LSxSe: 0.5845).

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Table 4. SIMPER analysis. OTUs significantly contributing to the dissimilarity between Leptogorgia obscura and Leptogorgia sp. (average dissimilarity/SD ≥1.4). ordered from higher to lower contributors. (*) OTUs significantly contributing to the similarity within each host species (average similarity/SD ≥1.4).

Leptogorgia obscura Leptogorgia sp.

Abundance mean ± SD Abundance mean ± SD

Gammaridea 14314 357.85 ± 507.89 * 780 22.94 ± 24.88 *

Copepoda 2723 68.07 ± 99.03 * 1634 48.05 ± 52.92 *

Isopoda 483 12.07 ± 17.81 * 491 14.44 ± 16.33 *

Decapoda 395 9.87 ± 14.48 * 26 0.76 ± 1.37

Polychaeta 313 7.82 ± 7.13 * 96 2.82 ± 3.23 *

Ophiuroidea 9627 240.67 ± 374.13 * 18227 536.08 ± 301.81 *

Gastropoda 335 8.37 ± 9.1 * 137 4.02 ± 5.12 (Prosobranchia)

According to SIMPER analyses, seven epifaunal groups quantitatively characterized the fauna associated with Leptogorgia obscura (average similarity [SIM] = 71.62 %) (Table 4). Five taxa contributed significantly (SIM/ SD 1.4) to quantitatively characterize the epifauna of Leptogorgia sp. (average similarity [SIM] = 74.55 %) (Table 4). Four taxa contributed to differentiate between the two species: Gammaridea, Copepoda, Decapoda and Ophiuroidea (average dissimilarity ([DIS] = 33.9 %; Table 4), the Gammaridea, Copepoda and Decapoda specimens for L. obscura and Ophiuroidea for Leptogorgia sp., being more abundant. In Leptogorgia sp. the dominance of Ophiuroidea was very noticeable (Table 4). Leptogorgia obscura showed dominance of Gammaridea, but with more equitable values of abundance.

Abundace

After fitting LS, there was still a significant difference in the total abundance associated with the “host species”, this being larger in Lsp (Table 5, Fig. 2). The

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densities (N / LS) were 6.24±0.44 and 2.18±0.42 for Lsp and Lo respectively. The absolute average abundance, in contrast, showed similar values (Lsp = 637±56.68, Lo = 715±116.45). The total abundance showed a different range of variation between the two studied gorgonies, which was not homogenised with transformation (PERMDISP: F1,72: 22.4, P(perm) < 0.001), being larger in L. obscura. Still, the differences due to average density were very clear (Pseudo-F: 10.84, P(perm) < 0.001) and can be confidentially interpreted (Fig. 3; see Discussion). Neither the main effect of LS nor the main effect of “season” was significant, but their interaction was significant (Table 5). Density tended to be larger in June (d=5.32±0.53) than in November (d=2.55±0.52) (the main effect of season is marginally significant, Table 5), but the effect of the LS is not homogeneous between the two seasons (Fig. 4).

Figure 2. Graphical representation about the correlation between abundance (Log N) and living space (Log LS), for each host gorgonian species (Lo= Leptogorgia obscura, Lsp= Leptogorgia sp.).

When we analysed these relationships separately in the two studied seasons, the living space had a significant effect on the total abundances only in June (PERMANOVA: Pseudo-F: 15.51, P(perm) < 0.001), while it had no significant effect in November (PERMANOVA: Pseudo-F: 1.54, P(perm): 0.225). There also were random differences between different sampling years (not explored further due to their random nature). 307

Table 5. Abundance PERMANOVA table of results, Univariate analyses. LS= living space; S= species; Se= season; T= time (year). (*) P estimation obtained by Monte Carlo sampling.

df SS MS Pseudo-F P(MC)

LS 1 0.14026 0.14026 1.6557 0.2038

Se 1 4.441 4.441 12.029 0.0765

S 1 1.0001 1.0001 10.836 0.0008

T(Se) 2 0.71157 0.35579 4.2086 0.0186

LSxSe 1 0.91909 0.91909 10.872 0.0012

Residual 67 5.6641 8.4539E-2

Total 73 12.876

Figure 3. Plot representation about the Density (Log N / Log LS) (mean and dispertion) for each host gorgonian species (Lo= Leptogorgia obscura, Lsp= Leptogorgia sp.).

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Figure 4. Graphical representation about the correlation between abundance (Log N) and living space (Log LS), for each season time (June, November).

Diversity

There was no seasonal effect in H’ (all p-values larger than 0.65, either for the main effect and its interactions), and these terms were excluded from the final model, considering the effect of sampling times as a random annual variability (Table 6). H’ showed a significant negative covariation with LS (log transformed) (Fig. 5), whose slope did not change among the levels of the other factors analysed (Table 6). The gorgonian host, in its turn, had a global influence on H’, with L. obscura showing higher diversity values than Leptogorgia sp. (Fig. 4; PERMDISP: F1,72= 1.04; P(perm)= 0.315). The annual variability was not globally significant but, among different sampling years, there was a significant random variation in the strength of the differences between the two gorgonian hosts (Table 6).

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Figure 5. Graphical representation about the correlation between diversity of Shannon (H´) and living space (Log LS), for each host gorgonian species (Lo= Leptogorgia obscura, Lsp= Leptogorgia sp.).

Table 6. Diversity (H’) PERMANOVA table of results, Univariate Analyses. LS= living space; S= species; Se= season; T= time (year).

Pseudo- Unique df SS MS P(perm) F perms

LS 1 1.5167 1.5167 18.848 0.0002 9851

S 1 3.8002 3.8002 20.907 0.0178 8698

T(year) 3 0.33475 0.11158 1.387 0.2525 9966

SxT(Se) 3 0.72935 0.24312 3.0219 0.0361 9950

Residual 65 5.2294 8.0452E-2

Total 73 11.61

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4.5.5. Discussion

Epifauna associated with the gorgonians Leptogorgia sp. and Leptogorgia obscura present in shallow waters of Ecuador were surprisingly abundant (50,284 total individuals in 80 colonies, 715 ind/col in L. obscura and 637 ind/col in Leptogorgia sp., Table 2), in comparison with other coral hosts in other regions: 22.4 ind/colony (254 and 194 ind/col in rainy and dry season respectively) in Pocillopora damicornis in Costa Rica (Alvarado & Vargas-Castillo 2012); 12.40 ind/colony (1 mm2 mesh sieve), in four scleractinian corals in the Great Barrier Reef (Australia) (Stella et al. 2010); Mortensen and Mortensen (2005) estimated approximately 22 and 46 individuals per colony (250 μm mesh sieve) or fragment in Paragorgia arborea and Primnoa resedaeformis (deep- sea gorgonians) respectively, in Atlantic Canada. Carvalho et al. (2014) obtained a range of 8–737 and 6–358 ind/colony (100 μm mesh sieve) in Leptogorgia lusitanica and Eunicella gazella, respectively, in Atlantic waters.

Many species that inhabit coral reefs depend on the cover and structure of live corals, which have a recognised fundamental function as bioconstructors (Almany 2004; Rodríguez-Zaragoza & Arias-González 2015). They also provided protected “cryptic habitats”, i.e. the undersides of corals and the interior crevices and caves, that sometimes harbour a more abundant biota community than that of the open surfaces (Buss & Jackson 1979; Jackson et al. 1971; Zuschin & Mayrhofer 2009). Other organisms such as algae, bryozoans, sponges and hydrozoans, act also as bioconstructors (Cocito 2004). The gorgonian species in this study were particularly effective as bioconstructors, in comparison with these other groups, included in previous studies (see the comparative summary Tables 2 and 7). Bryozoan have been cited as hosting a high diversity of associated biota, although the available information is scarce, and their role as reef-constructors in tropic areas is usually not considered due to their low biovolume (Cocito 2004).

The results of this study reinforce the idea that gorgonians play an important role as bioconstructors, particularly in tropical communities. Firstly, in these regions, predation is particularly intense and gorgonian colonies provide within their interstices an excellent refuge (Austin et al. 1980; Patton 1994). Secondly, the 311

arborescent structure of the gorgonian colonies and their orientation with respect to dominant currents, maximises the volume of water passing through the polyps, providing enhanced feeding opportunities that are also passed on to any filter-feeding epibiontic organisms (Buhl-Mortensen & Mortensen 2004; Wainwright & Dillon 1969; Wainwright & Koehl 1976). Finally, gorgonians can secrete a mucus in which it is possible to find both microorganisms and decaying organic matter, used by detritivores and filter-feeding organisms (McCloskey 1970; Patton 1972; Reed et al. 1982).

Table 7. Comparative summary of previous studies about other bioconstructor organisms.

Bioconstructor Species Density Reference Location Observation

Macroalgae Asparragosis 7 ind/cm3 Guerra-García Iberian 500 µm mesh armata et al. (2011) Peninsula sieve Corallina 6.63 ind/cm3 Guerra-García Iberian 500 µm mesh elongata et al. (2011) Peninsula sieve Dictyota spp. 21.7 - 41.5 ind/g Cunha et al. Brazil 100 µm mesh (2013) sieve Ptilota 16 ind/g Lippert et al. Artic gunneri (2001) Ocean Laminaria 0.32 ind/g Lippert et al. Artic digitata (2001) Ocean Sponge Agelas 1.02 ind/cm3 - 6.8 Koukouras et North 500 µm mesh oroides ind/g al. (1996) Aegea Sea sieve Aplysina 2.9 ind/cm3 - 21.5 Koukouras et North 500 µm mesh aerophoba ind/g al. (1996) Aegea Sea sieve Axinella 1.21 ind/cm3 - Koukouras et North 500 µm mesh cannabina 7.72 ind/g al. (1996) Aegea Sea sieve Verongia 0.09 ind/cm3 Voultsiadou- Greece 1 mm mesh aerophoba Koukoura sieve (1987) Paraleucilla 0.91 ind/cm3 Padua et al. Brazil magna (2013) Halichondria 0.0027 - 26.80 Ávila and Mexico 500 µm mesh melanadocia ind/cm3 Ortega-Bastida sieve (2014) Hydrocoral Millepora 0.01 - 0.27 Garcia et al. Brazil alcicornis ind/cm3 (2008) Bryozoan Flustra 1 ind/cm2 on the Stebbing South foliacea flat surfaces (1971) Wales

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Sessile associated fauna which lives attached to the surface of the colony, can sometimes cause damage to the gorgonian at different levels, competing for trophic resources, reducing growth and reproductive success, and ultimately, inducing partial or complete colony death (Cúrdia et al. 2015; Kim & Lasker 1997). Injuries to the colonies can be colonized by pioneer epibiont species such as some hydrozoans, bryozoans and algae, which in turn facilitate the colonization by subsequent epibiota (Bavestrello et al. 1996; Cerrano et al. 2000). Our results showed a small density of sessile epibionts and modular organisms (hydrozoans, bryozoans and Porifera) in comparison with similar studies (Cúrdia et al. 2015). Coral reefs are usually exposed to anthropogenic stressors, such as pollution, overfishing, sedimentation, eutrophication etc., that can make a reef more susceptible to the proliferation and development of opportunistic epibiont organisms, which take advantage of the weakening or disease of corals (Contreras et al. 2011; Szmant 2002). Our study area is located in a National Park with a protection law and a more restrictive use, which could be related with the low level of physical damage observed in these gorgonian species, and the low level of epibiosis observed.

Some authors have observed that there are specific associations among particular coral hosts and their associated invertebrates (Abele & Patton 1976; Sin 1999; Stella et al. 2010), which could be related to differences in the morphology, distribution and abundance of the different host species (Cocito 2004; Stella et al. 2011). This relationship works in either direction, since the associated biota can participate in the formation, shape and growth of the host colonies (Cocito 2004; Moissete & Pouyet 1991). Stella et al. (2010) found a significant variation in the abundance of epifauna across some coral hosts that could be partly attributable to differences in their morphology. Thus, pocilloporid corals had a more complex branch growth form, which provided greater protection against predation. In our study, the two host species showed clear differences in their morphology that could give rise to different microhabitats. Leptogorgia sp. showed an open fan in a single plane while Leptogorgia obscura has a distinct three-dimensional morphology (bushy), with a higher mean living space and a higher proportion of more protected inner gaps. This was correlated

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with a difference in total abundance and diversity of the associated fauna, as well as the structure of this community.

In the studied species, the epifaunal density was higher in Leptogorgia sp., if we take into account that it provided a significantly lower living space. This is a robust result even if there was heterogeneity of dispersions between the two species of gorgonians. On the one hand, the differences were very clear (see PseudoF and P values in Table 5) and, on the other hand, PERMANOVA, as well as ANOVA, is conservative when variances are correlated with sample sizes in unbalanced designs; that is, when the group with larger dispersion is also the group with larger sample sizes (Anderson & Walsh, 2016), as in this case. Within each of the two host species, we observed an increase in abundance with increasing living space, as would be expected. There is a general consensus that the size of a host-coral colony is positively correlated with abundance of associated fauna (Abele & Patton 1976; Abele 1976), due to increased resource availability on larger colonies (Huber & Coles 1986). This fact is not clear in the community associated with gorgonians, and studies such as Carvalho et al. (2014) could not demonstrate a significant increase of either abundance or diversity (H′) with colony size, although an increasing tendency was observed. In our study, diversity (H´) showed the opposite trend, higher in the bushy L. obscura but decreasing with increasing LS within each of the two host species. This is in apparent opposition to the habitat heterogeneity hypothesis (MacArthur & Wilson 1967) which would predict an increase of habitat complexity with increasing colony size (Anderson et al. 2005), as this would make available a higher number of different niches. In this scenario, diversity would increase if niche partitioning is a major force in community structure. Carvalho et al. (2014) hypotethised that, for gorgonian gardens in South Portugal, an aggregation of some species could occur with increasing colony size, and these species could become dominant. This may explain, at least partially, the observed pattern. However, this was a first step in a still little known region, so futures studies with a higher taxonomic resolution would be necessary.

We observed a dominance of amphipods (Gammaridea and Caprelloidea) in L. obscura. The dominance of amphipods among the fauna associated with gorgonians

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had been widely reported for different regions such as Atlantic waters (Buhl & Mortensen 2004; Buhl-Mortensen & Mortensen 2004; Carvalho et al. 2014; Greene 2008) and Indo-Pacific waters (Goh et al. 1999; Kumagai & Aoki 2003). In Leptogorgia sp., instead, there was a notable dominance of ophiuroids. Echinoderm is one of the most abundant groups on coral reefs and can play a “keystone” role in controlling coral community structure (Glynn & Enochs 2011). This association has been related to the different trophic uses that the ophiuroids can make of the gorgonian host, such as stealing its food or its mucus secretion, or to use these "hosts" to reach a higher position in the water column for suspension feeding (Emson & Woodley 1987; Fujita & Ohta 1988). On soft bottoms, mobile ophiuroids have been shown to function like epibenthic disturbers (Peterson 1979). Indeed, Ambrose (1993) showed that ophiuroids affected species diversity (Shannon-Wiener Index), which was higher in their absence. In our study, the dominance of ophiuroids in Leptogorgia sp. was also correlated with a significant lower diversity.

With regard to the seasonal variability, total abundances (N) in our study tended to be globally higher in June than in November, although this trend was more apparent in colonies with more available living spaces. The period of higher intensity of the cold and nutritious Humboldt Current system (HCS) lasts from June to September and it gradually disappears during November (Duxbury et al. 2002; Peixoto & Oort 1992) due to the incursion of the Panama Current (Coello et al. 2001). The HCS promotes a variety of ecological processes due to the oceanographic effects of upwelling (Alheit & Niquen 2004; Carrasco & Santander 1987; Thiel et al. 2007), increasing primary production in both plankton and benthos (Iriarte & González 2004; Thiel et al. 2007). These processes also influence zooplankton community composition (Escribano et al. 2004), fish population dynamics (Halpin et al. 2004) or larvae dispersal (Poulin et al. 2002). In our study, the abundance showed a positive relationship with the living space in June, while in November this was not observed (Table 5, Fig. 3). Provided that enough living space is available within a host colony, the cold and rich HCS apparently led to a sharp increase in abundances but did not affect diversity. The seasonal changes in epifauna associated with corals have seldom been studied but, for instance, Alvarado and Vargas-Castillo (2012) found a significant 315

effect of the season on Pocillopora damicornis in Costa Rica, where the abundance was higher in rainy than in dry seasons.

In conclusion, the high effectiveness of the gorgonians from Ecuador as bioconstructor has been verified, given that they host a hyperabundant epifaunal community in comparison with other coral hosts in other regions, and with other ecosystem engineers such as algae, bryozoans, sponges and hydrozoans. This highlights the need for conservation of gorgonian gardens in this region. A more complex pattern of variability has also been observed with respect to the abundance, the diversity and the structure of the community living on these aggregations in Ecuador, in comparison with other areas. On the one hand, different host species can harbour associated communities with significantly different characteristics. On the other hand, regional oceanographic processes may significantly affect benthic communities, with alternating cold-rich and warm-poor currents temporally associated with different community structures, and specifically with a notable increase in abundance related to the Humboldt Current System.

Acknowledgements

Our appreciation to Santiago Villamarin (Museo Ecuatoriano de Ciencias Naturales), Machalilla National Park and Ministerio del Ambiente of Ecuador (Manabí) for permits to collect and support. We thank to Michel Guerrero and his team (Exploramar Diving) for his special interest and collaboration since we began our research. Special thanks to Micaela Peña for her hard support.

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4.6- Aspergillus sydowii and other potential fungal pathogens in

gorgonian octocorals of the Ecuadorian Pacific.

4.6.1 Abstract

Emerging fungal diseases are threatening ecosystems and have increased in recent decades. In corals, the prevalence and consequences of these infections have also increased in frequency and severity. Coral reefs are affected by an emerging fungal disease named aspergillosis, caused by Aspergillus sydowii. This disease and its pathogen have been reported along the Caribbean and Pacific coasts of Colombia. Despite this, an important number of coral reefs worldwide have not been investigated for the presence of this pathogen. In this work, we carried out the surveillance of the main coral reef of the Ecuadorian Pacific with a focus on the two most abundant and cosmopolitan species of this ecosystem, Leptogorgia sp. and Leptogorgia obscura. We collected 60 isolates and obtained the corresponding sequences of their Internal Transcribed Spacers (ITS) of the ribosomal DNA. These were phylogenetically analyzed, indicating the presence of two isolates of the coral reef pathogen A. sydowii, as well as 16 additional species that are potentially pathogenic to corals. Although the analyzed gorgonian specimens appeared healthy, the presence of these pathogens, especially of A. sydowii, alert us to the potential risk to the health and future survival of the Pacific Ecuadorian coral ecosystem under the current scenario of increasing threats and stressors to coral reefs, such as habitat alterations by humans and global climate change.

4.6.2. Introduction

Coral reefs are considered one of the most biologically diverse ecosystems in the marine realm (Enochs & Hockensmith 2008). They maintain a high biomass and abundance of varied organisms (Sanchez & Dueñas 2012) and provide a plethora of micro-habitats to support enormous biodiversity (Buhl & Mortensen 2004; Gates & Ainsworth 2011; Thrush & Dayton 2002; Tsounis et al. 2006). In recent decades, coral

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reefs have experienced increasing pressures, and are disturbed by a combination of direct human impacts, e.g., habitat fragmentation and reduction of functional diversity (Nyström et al. 2000), and global climate change, e.g., increasing ocean acidification and temperature, coral bleaching, etc. (Wilkinson 2008). These conditions make reefs more susceptible to the proliferation and development of opportunistic organisms, which take advantage of the weakened corals (Contreras et al. 2011; Szmant 2002).

The coral disease aspergillosis has produced significant deterioration and partial and massive mortalities of coral communities in the Caribbean Sea (Smith et al. 1996; Nagelkerken et al. 1997a, 1997b; Ward et al. 2006; Toledo-Hernández et al. 2008; Sánchez et al. 2011). The responsible pathogen is the ascomycetous fungus Aspergillus sydowii (Bainier and Sartory, 1926). The first report of this disease in gorgonians dates back to 1995 (Nagelkerken et al. 1997a; b), although similar symptoms and outbreaks had been previously reported in the 1980s (Garzón-Ferreira & Zea 1992). The ascomycete fungus A. sydowii is globally distributed and occurs in diverse environments where it survives as a soil decomposing saprotroph (Klinch 2002; Raper & Fenell 1965). It is essentially a terrestrial fungus, but it is salt tolerant and capable of growing in the sea (Hallegraeff et al. 2014). Moreover, A. sydowii has been reported as a food contaminant (Zhang et al. 2013), and a human pathogen in immune-compromised patients (de Hoog et al. 2000; Nagarajan et al. 2014). In marine ecosystems, A. sydowii has been isolated from some gorgonian communities of the Caribbean (Alker et al. 2001; Smith et al. 1996), Colombian Pacific coasts (Barrero-Canosa et al. 2013), and environmental samples of the Australian coastal waters (Hallegraeff et al. 2014).

Aspergillosis causes selective mortality of large sea fans (Kim & Harvell 2004), and suppression of reproduction in infected individuals (Petes et al. 2003). As a consequence, coral population levels decrease (Rypien 2008a). The symptoms include purpling of the tissue, galling, and lesions (Smith et al. 1996), associated with necrotic sea fan tissue (Nagelkerken et al. 1997b). Prevalence (percentage of fans infected) and disease severity (mean percentage of fan tissue affected by disease) are positively correlated with water depth, and large sea fans are more likely to be infected than small fans (Kim & Harvell 2002; Nagelkerken et al. 1997a). Although the origin of this

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disease and its epidemiology is unknown, microsatellites and phylogenetic studies reveal a pattern of global panmixia among isolates. Moreover, sea isolates are interspersed with those isolated from environmental samples (Rypien et al. 2008). The incidence of this pathogen can be similar to other fungal species, i.e., Fusarium keratoplasticum and F. falciforme, in other animals and ecosystems (Sarmiento- Ramirez et al. 2014), exacerbated by the effects of global climate change and habitat alteration by humans.

In the Ecuadorian Pacific, there are no records of A. sydowii and coral reefs appear to be healthy. Due to the current trend of expansion of fungal infections and the endangered situation of coral reefs, we performed a survey in the Machalilla gorgonian gardens, which includes the most representative gorgonian species in a hot spot of marine biodiversity in Ecuador. We investigated the presence of A. sydowii in these organisms.

4.6.3. Material and methods

Sampling

Gorgonian octocoral colonies were collected by SCUBA diving from rocky bottoms located in The Frailes, Machalilla National Park (Manabí, Ecuador) (1°30'14"S 80°48'33”W). Due to the absence of symptoms, we randomly selected 40 colonies from the two most abundant and cosmopolitan gorgonian species of this area (pers. obs.), Leptogorgia Milne-Edwards and Haime 1857: Leptogorgia obscura Bielschowsky 1929, and Leptogorgia sp. The colonies were collected within a range of 10 to 15 m in depth. Samples were kept in individual sterile plastic bags and processed in the laboratory under axenic conditions.

Fungal isolation

From each colony, fragments of 3 cm wide from randomly selected areas were excised using a sterile scalpel. To remove fungi not associated with the octocorals, the selected fragments were surface-cleaned with 70% ethanol for 30 s (Barrero-Canosa et 331

al. 2013). For fungal isolations, the selected fragments were transferred onto a peptone glucose agar media (PGA) (Söderhäll et al. 1978) supplemented with penicillin (100 mg/l). A glass-ring technique was used for isolation following the methodology described in Sarmiento-Ramirez et al. (2014). Resulting pure cultures were maintained in PGA at 4ºC. Cultures were labeled as 001ASP through 058ASP in the culture collection of the Real Jardín Botánico (RJB-CSIC), Madrid, Spain (Table 1).

DNA extraction, PCR amplification, sequencing, and species identification

DNA was extracted from 20 mg of the fungal isolate tissues using the DNeasy extraction kit (Qiagen, Inc.) according to the manufacturer’s protocol. DNA fragments containing internal transcribed spacers ITS1 and ITS2, including 5.8S, were amplified and sequenced with primer pair ITS5/ITS4 (White et al. 1990). The PCR profile was: 2.5

µl 10 x buffer, 1.4 µl 50 mM MgCl2, 1.6 µl 25 mM dNTPs, 0.5 µl of each 10 mM primer (forward and reverse), 1 µl 1 mg/ml BSA, 1 µl DNA, 0.3 µl 5 U/µl Taq polymerase, and

16.2 µl ddH2O. The PCR conditions were 1 min at 95ºC, 35 cycles of 1 min at 95ºC, 45 s at 58ºC and 1 min at 72ºC, and finally 10 min at 72ºC. The amplicons were sequenced for both strands using BigDye Terminator in an ABI 3730 genetic analyzer (Applied Biosystems).

The sequences were edited and primers trimmed using the Sequencher v.4.9 program (Gene Code Corporation, Ann Arbor, MI, USA). BLAST (Altschul et al. 1990) was used to compare the sequences against those existing in the National Center of Biotechnology Information (NCBI) nucleotide databases. This first screening was useful to obtain the sequences of reference that were used in posterior data treatment.

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Table 1. Fungal isolates from gorgonians Leptogorgia obscura and Leptogorgia sp. from the Eastern Pacific of Ecuador and the resulting molecular identification based on phylogenetic analysis. RJB = Real Jardín Botánico (Madrid).

RJB number Isolate number Gorgoniidae Species Fungus Species GenBank Acc. Num.

ASP001 GORG01 Leptogorgia sp. Pyrenochaetopsis leptospora

ASP002 GORG02 Leptogorgia sp. Nigrospora sp.

ASP003 GORG03 Leptogorgia sp. Penicillium chrysogenum

ASP004 GORG04 Leptogorgia sp. Penicillium chrysogenum

ASP005 GORG05 Leptogorgia sp. Penicillium chrysogenum

ASP006 GORG07 Leptogorgia obscura Tritirachium sp.

ASP007 GORG08 Leptogorgia sp. Penicillium chrysogenum

ASP008 GORG09 Leptogorgia obscura Penicillium chrysogenum

ASP009 GORG10 Leptogorgia sp. Penicillium chrysogenum

ASP010 GORG11 Leptogorgia obscura Penicillium chrysogenum

ASP011 GORG12 Leptogorgia obscura Fusarium longipes

ASP012 GORG13 Leptogorgia sp. Penicillium chrysogenum

ASP013 GORG16 Leptogorgia sp. Nigrospora sp.

ASP014 GORG17 Leptogorgia obscura Lasiodiplodia pseudotheobromae

ASP015 GORG18 Leptogorgia obscura Phoma sp.

ASP016 GORG21 Leptogorgia obscura Penicillium chrysogenum

ASP017 GORG22 Leptogorgia sp. Penicillium chrysogenum

ASP018 GORG24 Leptogorgia obscura Fusarium longipes

ASP019 GORG25 Leptogorgia obscura Cladosporium dominicanum

ASP020 GORG26 Leptogorgia obscura Aspergillus sydowii

ASP021 GORG27 Leptogorgia obscura Aspergillus sydowii

ASP022 GORG29 Leptogorgia obscura Penicillium chrysogenum

ASP023 GORG30 Leptogorgia sp. Penicillium chrysogenum

ASP024 GORG31 Leptogorgia obscura Nigrospora sp.

ASP025 GORG32 Leptogorgia sp. Aspergillus wentii

ASP026 GORG33 Leptogorgia sp. Penicillium chrysogenum

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Table 1. Continuation.

ASP027 GORG34 Leptogorgia sp. Penicillium chrysogenum

ASP028 GORG35 Leptogorgia obscura Penicillium chrysogenum

ASP029 GORG36 Leptogorgia sp. Penicillium chrysogenum

ASP030 GORG37 Leptogorgia sp. Penicillium chrysogenum

ASP031 GORG38 Leptogorgia sp. Cladosporium sphaerospermum

ASP032 GORG40 Leptogorgia obscura Nigrospora sp.

ASP033 GORG42 Leptogorgia obscura Fusarium longipes

ASP034 GORG43 Leptogorgia obscura Penicillium chrysogenum

ASP035 GORG44 Leptogorgia obscura Penicillium chrysogenum

ASP036 GORG45 Leptogorgia sp. Penicillium chrysogenum

ASP037 GORG46 Leptogorgia obscura Penicillium chrysogenum

ASP038 GORG47 Leptogorgia sp. Penicillium chrysogenum

ASP039 GORG48 Leptogorgia obscura Nigrospora sp.

ASP040 GORG49 Leptogorgia obscura Fusarium longipes

ASP041 GORG50 Leptogorgia sp. Penicillium chrysogenum

ASP042 GORG51 Leptogorgia sp. Penicillium chrysogenum

ASP043 GORG53 Leptogorgia sp. Aspergillus ochraceopetaliformis

ASP044 GORG54 Leptogorgia obscura Nigrospora sp.

ASP045 GORG55 Leptogorgia obscura Penicillium chrysogenum

ASP046 GORG63 Leptogorgia sp. Cladosporium sphaerospermum

ASP047 GORG68 Leptogorgia sp. Penicillium chrysogenum

ASP048 GORG71 Leptogorgia obscura Capnobotryella sp.

ASP049 GORG76 Leptogorgia sp. Cladosporium sphaerospermum

ASP050 GORG77 Leptogorgia sp. Cladosporium sphaerospermum

ASP051 GORG78 Leptogorgia sp. Curvularia sp.

ASP052 GORG80 Leptogorgia sp. Alternaria sp.

ASP053 GORG83 Leptogorgia obscura Aspergillus sclerotiorum

ASP054 GORG84 Leptogorgia obscura Aspergillus sclerotiorum

ASP055 GORG85 Leptogorgia sp. Nigrospora sp.

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Table 1. Continuation.

ASP056 GORG87 Leptogorgia sp. Cladosporium sphaerospermum

ASP057 GORG88 Leptogorgia sp. Penicillium mallochii

ASP058 GORG90 Leptogorgia obscura Cladosporium dominicanum

ASP059 GORG92 Leptogorgia sp. Tritirachium sp.

For species identification of the isolates, the corresponding ITS sequences were phylogenetically analyzed with a number of selected ITS sequences of reference of closely related fungal species obtained from the NCBI (see Table 2, Fig. 1). To perform the phylogenetic analyses, a GTR + G + I substitution model was first obtained using the jModelTest v2.1.5 (Darriba et al. 2012) program. This model was selected based on the Akaike Information Criterion (AIC). Bayesian inference and Maximum Likelihood analyses were performed using MrBayes v3.2.5 (Huelsenbeck & Ronquist 2001) and RaxML v8.0.0 (Stamatakis 2014), respectively. The Bayesian inference analysis was implemented with three runs of 20 million generations sampling one tree per 1000 replicates. For each run, eight Markov chain Monte Carlo (MCMC) simulations were conducted. These simulations were run until a critical value was reached for the topological convergence diagnostic lower than 0.005. Branch supports were evaluated by posterior probabilities after a burn-in of 25%. The Maximum Likelihood analysis was implemented with a random starting tree and clade support was assessed with 1000 bootstrap replicates. The Maximum Likelihood analysis was implemented in the graphical user interface raxmlGUI v1.5 (Silvestro & Michalak 2012). The clade support was assessed with 1000 bootstrap replicates after selecting the best tree from 100 trees generated.

Morphological characterization

Isolates corresponding to A. sydowii according to phylogenetic analysis were morphologically characterized using scanning electron microscopy, SEM (Hitachi s3000N, Real Jardín Botánico, RJB-CSIC, Madrid, Spain). Mycelia with characteristic features were fixed in 2% glutaraldehyde for 1 h, washed in distilled sterile water, and

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then dehydrated for 1 h in a series of ethanol (30, 50, 70, 80, 90, 95 and 100%) solutions. The isolates dehydrated in absolute ethanol were critical-point dried and the material was sputter coated in a vacuum with an electrically conductive layer of gold to a thickness of about 80 nm. Samples were observed at a beam specimen angle of 45º with an accelerating voltage of 20kV and final aperture at 200 μm.

4.6.4. Results

Fungal isolation and species identification

A total of 60 fungal isolates were obtained and the phylogenetic analyses resulted in 17 phylogenetically supported clusters (Fig. 1). The majority of the isolates could be assigned to a species reference sequence. These species were: Penicillium chrysogenum, P. mallochii, Aspergillus sclerotiorum, A. ochraceopetaliformis, A. sydowii, A. wentii, Lasiodiplodia pseudotheobromae, Cladosporium shpaerospermum, C. dominicanum, Fusarium longipes, and Pyrenochaetopsis leptospora. Five groups of isolates grouped with sequences of references of unknown species: Alternaria sp., Capnobotryella sp. Curvularia sp., Phoma sp., and Tritirachium sp. The sequences grouping into the cluster containing Nigrospora spp. were diverse and could not be assigned to any known ITS sequences (Table 2).

The majority of the isolates belonged to the genera Penicillium and Aspergillus (Fig. 2). The most frequent species was Penicillium chrysogenum (24 out of 60), which occurred in colonies of both L. obscura and Leptogorgia sp., followed by Fusarium longipes and Penicillium granulatum (both 4 out of 60 each), Aspergillus sydowii (3 out of 60, 5%) (Fig. 2). The species A. sydowii and Fusarium longipes were only found in L. obscura.

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Figure 1. Bayesian out-group-rooted cladogram inferred from ITS rRNA gene sequences of fungal isolates from Leptogorgia obscura and Leptogorgia sp. from the Eastern Ecuadorian Pacific. Numbers placed above the internodes are, respectively, PP and BS of the Bayesian and Maximum Likelihood analyses. 337

Table 2. Genbank rDNA ITS reference sequences for fungal species used in phylogenetic analysis to identify fungal isolates from Ecuadorian gorgonians.

Species GenBank number Isolate / strain Type material

Penicillium chrysogenum NR_077145 CBS 306.48 yes

Aspergillus sclerotiorum NR_131294 NRRL 415 yes

Aspergillus KF384187 FJ120 ochraceopetaliformis

Aspergillus sydowii AB267812 CBS 593.65

Penicillium mallochii NR_111674 DAOM 239917 yes

Aspergillus wentii NR_077152 ATCC 1023 yes

Lasiodiplodia NR_111264 CBS 116459 yes pseudotheobromae

Cladosporium NR_111222 CBS 193.54 yes sphaerospermum

Cladosporium dominicanum NR_119603 yes

Nigrospora oryzae HQ607925 ATT291

Nigrospora sp. KP793234 M116

Nigrospora sp. JN207335 P39E2

Fusarium longipes AB820724 IFM 50036

Tritirachium sp. EU497949 F13

Alternaria sp. KM507780 311a

Curvularia sp. HE861836 UTHSC:08-2905

Pyrenochaetopsis leptospora NR_119958 yes

Phoma sp. KF367550 4 BRO-2013

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Figure 2. Pie charts showing isolation frequency of different fungal isolates from Leptogorgia obscura and Leptogorgia sp of the Eastern Ecuadorian Pacific.

Morphological characterization

The isolates molecularly assigned to A. sydowii showed conidiophores with conidia, metulae, and phialides seen in the SEM micrographs (Fig 3). The length of the metulae ranged from 2.5 to 3.5 µm, and the breadth from 4.2 to 6.5 µm. Phialides ranged from 2.2 to 3.0 µm in length and from 3.4 to 6.1µm in breadth. The conidia were globose with a roughened or spinose ornamentation. These conidia had a diameter that ranged from 2.5 to 4.4 μm (Fig 3).

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Figure 3. Scanning electron microscopy showing characteristic features of conidiophores of Aspergillus sydowii isolated from Leptogorgia obscura of the Eastern Ecuadorian Pacific: A. conidiophore structure with metule (m), phialide (p), and globose conidia (c); B. morphological features of conidia head with a vesicle characteristic (v), metulae (m), phialide (p), and globose conidia (c); C. phialide (p) and mature globose conidia (c) with verruculose ornamentation (*); D. globose conidia (c) with verruculose ornamentation (*).

4.6.5 Discussion

The Ecuadorian Pacific coral reefs are one of the most important and unstudied marine ecosystems and biodiversity ‘hot spots’. In this study, we found that the gorgonian communities of these reefs, specifically L. obscura and Leptogorgia sp. colonies, hold a large, diverse, and mostly unknown fungal community. Interestingly, this fungal community includes the coral pathogen A. sydowii. This constitutes the first report of this pathogen in Ecuador and the second in the Eastern Pacific. The gorgonian community appeared to be healthy and showed no symptoms of fungal disease during the sampling period. These results are similar to those of Toledo- Hernández et al. (2008b) and Zuluaga-Montero et al. (2009), who also found isolates of A. sydowii in healthy colonies of Gorgonia ventalina in the Caribbean. In Ecuador, this

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pathogen was only found in the samples of L. obscura but not in those of Leptogorgia sp. This could be due to the limited sampling and the low prevalence of this fungus in these populations (only 2 specimens out of 60). A different susceptibility of the gorgonian species could also explain this result. A previous study by Nagelkerken et al. (1997a) indicated differences in the incidence of aspergillosis between two species of gorgonians, e.g., G. ventalina and G. flabellum.

Moreover, we provide new data on fungal communities of marine environments, particularly in coral reefs. Studies on fungal communities in coral reefs are scarce. In Singapore, an investigation on 10 species of gorgonian corals indicated the presence of 16 fungal genera, including Acremonium, Aspergillus, Chaetophoma, Cladosporium and Penicillium (Koh et al. 2000); among these genera, the species Cladosporium sphaerospermum and Phoma sp. were also found in our study. In a study carried out in colonies of the Caribbean sea fan G. ventalina, Toledo-Hernández et al. (2007) found 15 new fungal species corresponding to 8 genera, including Aspergillus, Cladosporium, Gloeotinia and Penicillium. In 2008, as part of a larger sampling effort, the same authors identified 35 fungal species corresponding to 15 genera, including Aspergillus, Cladosporium, Nectria, Penicillium and Stachybotrys. Among these genera, the species A. sydowii and C. sphaerospermum were also found in our study.

In the Pacific, the only sampled coral reef area studied for fungal community composition was at Chocó, Colombia (Barrero-Canosa et al. 2013). This area is located near the coral reef investigated in our study. They found 60 fungal species in the sea fan Pacifigorgia spp. corresponding to 13 fungal genera (e.g., Aspergillus, Penicillium). In our work, we only found A. sydowii and A. sclerotiorum, two species common to those found by Barrero-Canosa et al. (2013). Thus, all other fungal species identified in our study represent the first report of this species in the Pacific gorgonians. We also found species that had been previously reported in gorgonians. These included Penicillium chrysogenum (Barrero-Canosa et al. 2013; Moree et al. 2014; Toledo- Hernández et al. 2008) and Cladosporium sphaerospermum (Toledo-Hernández et al. 2007). However, the role of pathogens of these species remains unknown.

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We found some fungal species that had never been described in gorgonians, including Aspergillus fumigatus (previously described in soil, air, water, food, plants and organic matter), Capnobotryella sp. (previously described in lichens and pumpkins), Fusarium longipes (previously described in soil of tropical regions), Lasiodiplodia theobromae (previously described causing damage in vascular plants), Phoma sp. (described from soil, as saprophytes on various plants, and as pathogens in plants and humans) and Pyrenochaetopsis leptospora (soil borne and mainly associated with gramineous plants). The pathogenicity of these fungal species to gorgonians is unknown. However, whether these species are also characteristic of marine environments or originate from land as agricultural runoff and move to marine environments via discharges from rivers is unknown.

Other species found in this work have been previously found in marine environments but not in gorgonians or coral reefs. For example, Aspergillus ochraceopetaliformis and Alternaria sp. were found in deep-sea environments (Landy & Jones 2006; Zhang et al. 2014), Nigrospora sp. in sea anemones (Yang et al. 2012), or saline environments in general, such as Penicillium mallochii was before isolated from the guts of tropical leaf-eating caterpillars in Costa Rica (Rivera et al. 2012).

In spite of the presence of known pathogens, such as A. sydowii, and a wide diversity of fungal pathogens in the Ecuadorian coral reefs, we did not observe any damage or mortality. The factors that lead to aspergillosis or the development of other fungal pathogens on coral reefs are unknown. In phylogenetically related fungal pathogens, some environmental stressors influence the development of disease (Diéguez-Uribeondo et al. 2011; Sarmiento-Ramirez et al. 2014). Green and Bruckner (2000) suggested that coral aspergillosis could be enhanced by abiotic factors (Rypien 2008b) and speculated that this disease could be the result of specific combinations of environmental factors (e.g., UV, temperature, aerosol concentrations) or of large-scale climate patterns (oceanic currents). Specifically, the microclimatic parameter of temperature has been shown to be involved in the gorgonia-A. sydowii interaction by promoting the growth and activity of the pathogen and reducing the efficacy of host defenses (Alker et al. 2001; Barrero-Canosa et al. 2013).

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Thus far, studies on coral diseases, such as aspergillosis, are limited by the lack of isolates from marine sources prior to epidemics (Rypien et al. 2008). The detection of these pathogens in healthy Ecuadorian gorgonian gardens is, therefore, of key importance since it indicates the presence of A. sydowii and other potential coral pathogens in asymptomatic colonies. This can help in further investigations aiming to decipher the factors leading to gorgonian disease and an eventual deterioration of this ecosystem. Alert models on diverse environmental stressors and the monitoring of the health of coral reefs are crucial for a better understanding of the development of aspergillosis disease. Future studies on this pathogen and the factors triggering disease require the comparison of healthy coral reefs with the presence of A. sydowii to those affected by this pathogen.

Understanding where and to what extent potentially harmful organisms exist is of crucial importance to the design of adequate conservation plans. Fungal diseases currently represent one of the main threats to biodiversity worldwide (Fisher et al. 2012) and corals are no exception. The results presented here contribute to a better understanding of the biodiversity of fungal communities in coral reefs and the construction of data-baselines for the presence and incidence of these fungal pathogens in natural systems and in particular coral reefs.

Acknowledgements

We thank the Museo Ecuatoriano de Ciencias Naturales, Machalilla National Park and Ministerio del Ambiente of Ecuador (Manabí) for participation and collection permits. Thanks to Michel Guerrero and his team (Exploramar Diving) for his special interest from the early stages of our research. Special thanks to Micaela Peña for unconditional help and support. This research was partially supported by a grant from the Spanish Ministry of Economy and Competitiveness (CTM2014-57949-R). Javier Diéguez- Uribeondo and Jose V. Sandoval-Sierra were supported by a grant of the MINECO Spain (CGL2012-39357).

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4.6.6. References

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5. DISCUSIÓN FINAL

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Discusión final

El objetivo global de la presente Tesis fue mejorar el conocimiento sobre las comunidades de gorgonias de la familia Gorgoniidae, en un área como el litoral de Ecuador, donde es importante destacar la enorme falta de información existente sobre la diversidad y el papel ecológico de estas especies de gorgonias. Para ello se abordó de una forma conjunta el estudio de estos organismos, desde varias disciplinas, con el fin de abarcar la máxima comprensión acerca de los mismos. El trabajo desarrollado proporciona nuevos datos, a través de un enfoque multidisciplinario, sobre la taxonomía morfológica, morfométrica y molecular, las relaciones filogenéticas, la biología reproductiva, las interacciones ecológicas y posibles patógenos asociados (Fig. 1).

Figura 1. Esquema de las diferentes disciplinas abordadas y trabajos realizados a los largo de la presente Tesis en las comunidades de gorgonias.

Para desarrollar estos enfoques, se emplearon diferentes metodologías y procedimientos, desde la fase de muestreo, el trabajo de laboratorio, la obtención y 352

análisis de datos y la presentación de los resultados. El estudio fue desarrollado en un contexto comparativo con los congéneres más cercanos de las especies estudiadas, sobre todo en el ámbito del Pacífico oriental, para trabajar dentro un marco global y no sólo a escala local. Como es preceptivo, todos los resultados obtenidos han sido complementados con la revisión y discusión de estudios previos y relevantes en cada una de las áreas de investigación tratadas. En esta sección se presenta una visión general de los principales resultados obtenidos y discutidos a lo largo del desarrollo de los capítulos de la presente Tesis, así como sus implicaciones en futuras líneas de investigación.

Centrándonos en el objeto principal de nuestro estudio, las gorgonias (Alcyonacea: Octocorallia), representan uno de los organismos bentónicos marinos con mayor endemicidad en aguas tropicales en ambas costas del continente americano, tanto en el Atlántico, como en el Pacífico (Bayer 1953; Sánchez 2007; Sánchez et al. 2003b). Como se ha destacado en los primeros capítulos de la presente Tesis, desde el punto de vista de la taxonomía tanto morfológica como molecular, el estudio de estas especies ha representado un importante reto. Históricamente, el grupo ha recibido una gran atención taxonómica morfológica (Bielschowsky 1918, 1929; Duchassaing & Michelotti 1864; Hickson 1928; Horn 1860; Kükenthal 1919, 1924; Valenciennes 1846, 1855; Verrill 1868, 1869, 1870, 1864). Además de los trabajos anteriormente mencionados, es importante resaltar aquellos llevados a cabo por Bayer desde mediados del siglo pasado ( Bayer 1951, 1953, 1961, 1973, 1981, 2000; Bayer et al. 1983). Sin embargo, las revisiones de estas descripciones mostraron que estas eran incompletas, con escasez de ilustraciones o no estar escritas de una manera homogénea, lo que dificultaba su comprensión en detalle (Sánchez 2007). Fue a partir de comienzos del presente siglo, cuando hubo un auge exponencial en la descripción de nuevas especies, así como la publicación de importantes revisiones (Breedy 2001; Breedy & Guzmán 2002, 2003a, b, 2004, 2005, 2007, 2013; Breedy et al. 2013, 2009, 2012; Horvath 2011; Williams & Breedy 2004). Estas aportaciones han sido de gran ayuda a lo largo del estudio de estas especies y las posibles relaciones que existen entre ellas, en un contexto global del Pacífico oriental. Sin embargo, la localización restringida de la mayoría de estos trabajos, sobre todo en California, Costa Rica y Panamá, ha creado la 353

necesidad de ampliar la distribución de las zonas de estudio hacia áreas más inexploradas, como es el caso de Ecuador continental.

Uno de los primeros enfoques a tratar, y desde nuestro punto de vista básico y esencial en cualquier línea de investigación, fue la revisión, descripción y delimitación de las especies de Gorgoniidae, tanto aquellas consideradas como nuevas para la ciencia, como aquellas que significaban nuevos registros para Ecuador. Para ello, se contó con el apoyo del material depositado en las colecciones de referencia de los principales museos visitados, y aquel recogido en Ecuador a lo largo de las fases de muestreo. En este primer objetivo, nos apoyamos en los caracteres de la morfología clásica, utilizados comúnmente en la taxonomía de octocorales y el estudio faunístico de las especies de los tres géneros estudiados dentro de esta familia, Eugorgia, Leptogorgia y Pacifigorgia (Bayer et al. 1983; Breedy & Guzmán 2002, 2003b, 2007; Breedy et al. 2009). Para ello, se utilizaron la combinación de diferentes caracteres asociados con la morfología, como los rangos de tamaño y el color, tanto de la colonia, como de los escleritos de cada uno de los ejemplares examinados. Sin embargo, estos caracteres son a menudo de naturaleza continua y homoplásica (Sánchez 2007), pueden presentar una cierta plasticidad fenotípica (Flot et al. 2008b; Sánchez et al. 2007; Weinbauer & Branko 1995), y algunos de ellos estar influidos, en parte, por la subjetividad del taxónomo. Esto suponía que, en ocasiones, los caracteres fueran difíciles de codificar o polarizar (Vargas et al. 2010), lo que complicaba sobre todo la delimitación de especies muy cercanas morfológicamente, debido a veces a un solapamiento de caracteres. Para ello, se incorporaron nuevos caracteres y se desarrollaron algunos de los ya utilizados comúnmente, tratando de conseguir descripciones más detalladas. Gracias a esto, se reveló la existencia de tres nuevas especies para la ciencia, Eugorgia ahorcadensis, Leptogorgia mariarosae y Pacifigorgia machalilla n. sp., además de la incorporación de nuevos registros y ampliaciones de distribuciones geográficas y batimétricas de otras diez especies de Gorgoniidae presentes en el litoral de Ecuador.

En situaciones de delimitaciones inter-específicas complicadas, se comprobó la utilidad de la búsqueda morfológica, a través de caracteres morfométricos

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cuantificables, en distintos invertebrados (Lumley & Sperling 2010; Mutanen & Pretorius 2007; Schwarzfeld & Sperling 2014), incluidas gorgonias del Mediterráneo como Eunicella (Gori et al. 2012). No obstante, estos caracteres no habían sido utilizados en estos géneros de gorgonias del Pacífico oriental, antes de la realización de la presente Tesis. Gracias a ello, se demostró cómo el uso de nueve caracteres no correlacionados, podían complementar y clarificar de forma estadística la delimitación y descripción de dos especies estrechamente relacionadas, como fue el caso de Leptogorgia alba y Leptogorgia manabiensis (descrita como nueva para la ciencia). Esto abría una nueva puerta a la búsqueda de posibles características morfológicas anteriormente inexploradas, generando nuevas oportunidades al entendimiento de los límites entre estos organismos, evitando posibles casos de enmascaramiento o sinonimización de especies (ver Dincă et al. 2011; Eberle et al. 2016; Hebert et al. 2004; Rocha-Olivares et al. 2001). Por lo tanto, debería profundizarse en la búsqueda de nuevos caracteres diagnósticos, con el objetivo de clarificar la naturaleza de los mismos y poder definir aquellas autapomorfías que ayuden a determinar la posición taxonómica de algunas de estas especies.

Además, todo este enfoque se trató de coordinar en un marco de taxonomía integradora (Dayrat 2005; Padial et al. 2010; Schlick-Steiner et al. 2010), donde la morfología clásica y la morfometría estadística incorporada, fue complementada con la implementación de técnicas moleculares.

Como ya se ha mencionado, en las últimas décadas los trabajos enfocados a la resolución de las relaciones filogenéticas entre estos organismos, han experimentado un aparente desarrollo (McFadden et al. 2010; Sánchez et al. 2003a; Vargas et al. 2014). Sin embargo, en algunas ocasiones, los análisis moleculares no son todavía totalmente efectivos para resolver ciertas relaciones filogenéticas a nivel de especie o incluso en la categoría de género (McFadden et al. 2010; McFadden et al. 2011; McFadden et al. 2014; Sánchez et al. 2003a; Vargas et al. 2014). A lo largo de este trabajo, hemos hablado de cómo una de las características más resaltables a nivel molecular de los octocorales, es que sus marcadores mitocondriales evolucionan inusualmente de forma más lenta, estimándose que poseen una evolución de 10 a 100 veces menor que otros metazoos

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(France & Hoover 2002; Hellberg 2006; McFadden et al. 2004; Shearer et al. 2002). Esto se debe especialmente a la presencia del gen mtMutS, que puede codificar un sistema de reparación de apareamientos erróneos en el ADN mitocondrial (France & Hoover 2002). Sin embargo, paradójicamente, este gen se ha utilizado como uno de los más variables dentro de estas especies y propuesto como posible “barcode” junto con COI. Aún así, el uso exclusivo de marcadores mitocondriales ha determinado que muchos de los taxones así analizados no pudieran ser diferenciados adecuadamente (McFadden et al. 2010; McFadden et al. 2011; Vargas et al. 2014).

Por ello, a lo largo de la presente Tesis nos basamos en el concepto de “más es mejor” (Winkler et al. 2015), con el estudio y la incorporación de forma individual y concatenada de diferentes marcadores mitocondriales típicamente usados como mtMutS, Cox (CoxI-Igr-CoxII), y nucleares como ITS y 28S, previamente utilizados en otros estudios (Aguilar & Sánchez 2007; McFadden et al. 2006; McFadden et al. 2011; McFadden et al. 2014; Sánchez 2007). Los diferentes resultados obtenidos mostraron que cada uno de los genes de forma individual, proporcionaban una determinada resolución filogenética que ayudaba a la resolución de un grupo de especies u otro. En todo caso, a la hora de concatenar los genes, las regiones del genoma nuclear (ITS y 28S) de los octocorales aquí analizados, poseen un mayor número de caracteres informativos totales.

A través de los análisis moleculares, comprobamos cómo las inferencias filogenéticas en los géneros de Gorgoniidae estudiados, apoyaron la monofilia del género Pacifigorgia, pero mostraron a los géneros Leptogorgia y Eugorgia como un clado no monofilético, que alberga las especies de ambos géneros. Por tanto, los resultados basados en unidades evolutivas contrastarían con las designaciones taxonómicas procedentes de caracteres morfológicos aceptadas hasta la fecha (Breedy & Guzmán 2007, 2013; Breedy et al. 2009), basadas principalmente en la presencia de unos escleritos modificados detectados principalmente en Eugorgia (Breedy & Guzmán 2013; Breedy et al. 2009). De hecho, la morfología externa en estos dos géneros es bastante similar, así como el resto de caracteres utilizados en la descripción, tales como la forma de colonia, el tipo de ramificación, la forma del pólipo retraído, color de

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colonia, etc. (Breedy & Guzmán 2007; Breedy et al. 2009). Por las razones descritas, proponemos una revisión exhaustiva de ambos géneros, teniendo en consideración las especies de Leptogorgia en su distribución global, y discutir si deben unificarse en el mismo género, que sería Leptogorgia por su prioridad en antigüedad.

Por otro lado, a lo largo del presente estudio, la heterogeneidad encontrada en las divergencias genéticas entre las especies los géneros Leptogorgia y Pacifigorgia, así como la falta de apoyo en grupos monofiléticos específicos, sugerían la existencia de nuevos linajes y/0 la necesidad de una revisión detallada de los diferentes escenarios evolutivos posibles.

El primer escenario representó una diferenciación morfológica claramente visible con una diferenciación molecular mínima o nula, dentro de una politomía observada entre ciertas especies simpátricas del género Pacifigorgia. En este caso, diferentes posibles explicaciones fueron consideradas. Una de ellas fue un proceso de radiación explosiva con una diversificación muy reciente (Henning & Meyer 2014; Kocher 2004; Winkler et al. 2015), donde las especies poseerían un ancestro común polimórfico morfológicamente y deberían haber quedado reproductivamente aisladas en algún momento de su historia evolutiva (Hatta et al. 1999). Por desgracia, los casos de especiación simpátrica son difíciles de resolver, ya que las distribuciones actuales podrían enmascarar patrones biogeográficos pasados (Bolnick & Fitzpatrick 2007), en los que los taxones podrían haber evolucionado en alopatría, tras lo cual una extensión de su rango de distribución habría llevado a un contacto secundario entre las especies. De hecho, es cierto que estos estudios sobre procesos de radiaciones recientes en especies “jóvenes”, se caracterizan porque las designaciones de las propias especies pueden seguir representando una incertidumbre, y probablemente seguirán considerándose un reto (Eberle et al. 2016). Por otro lado, otra de las posibles explicaciones discutidas ante este evento de especiación, fue la posibilidad de una antigua hibridación entre especies perfectamente diferenciadas, con una posterior re- homogeneización del genoma y segregación de morfotipos, dando lugar a este patrón de diferenciación morfológica vs identidad molecular. En este caso, los híbridos

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habrían resultado fértiles, dándose retrocruzamientos donde se desarrollarían canales para el flujo de genes entre las especies (Hatta et al. 1999).

El segundo escenario representa eventos de poli- y parafilia presentes en el género Leptogorgia, correspondientes a posibles procesos de hibridación reticulada y/o clasificación o resolución incompleta de linajes (“incomplete lineage sorting”, caso en el que las copias de los genes no coalescen en el tiempo de formación de las especies, sino en una especie ancestral). Estos procesos han sido sugeridos como importantes en la evolución de corales (Diekmann et al. 2001; van Oppen et al. 2001; Willis et al. 2006), pudiendo ser particularmente relevantes en especies que divergieron rápida y recientemente (Funk & Omland 2003; Henning & Meyer 2014; Hoelzer & Melnick 1994). De hecho, pueden presentar un problema en la correcta identificación y delimitación de las aquellos organismos estrechamente relacionados, donde podrían producirse resultados contradictorios o incluso engañosos, si no son detectados (Nater et al. 2015; van Velzen et al. 2012). Este planteamiento se realizó debido a las inconsistencias observadas entre los árboles de genes individuales, particularmente entre los marcadores de ADN mitocondrial y ADN nuclear, e implican especialmente a un complejo de especies formado por L. alba, L. manabiensis, L. cuspidata, L. flexilis y probablemente L. cofrini.

Como ya se ha mencionado, estos procesos evolutivos han sido observados en muchas especies de organismos diferentes (Carstens & Knowles 2007; Meyer & Paulay 2005; Pollard et al. 2006; Puritz et al. 2012; Wiemers & Fiedler 2007), donde en el caso de las especies coralíferas parece que dejan de ser posiblemente casos tan “inusuales” como se creía en un principio (McFadden et al. 2010). Entre los numerosos ejemplos de taxones que pueden haber sufrido estos procesos se encuentran los géneros Acropora (van Oppen et al. 2001; Vollmer & Palumbi 2004; Wolstenholme et al. 2003), Madracis (Diekmann et al. 2001), Seriatopora (Flot et al. 2008a), Montipora (Forsman et al. 2010) o especies de abanicos de mar (Pseudopterogorgia y Gorgonia) en el Caribe (Sánchez & Dorado 2008), entre otros.

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Figura 2. Figura obtenida del trabajo de Joly et al. (2009) donde se muestran los diferentes escenarios posibles de árboles de genes que se esperan bajo un linaje clasificación incompleta (A) y un proceso de hibridación (B-D) para dos especies. Las secuencias estudiadas a partir de dos especies se distinguen por cuadrados y círculos. El cuadrado negro representa una secuencia que está más estrechamente relacionada con las secuencias de las otras especies, haciendo que la especie sea no-monofilética. En el escenario de un linaje clasificación incompleta (A), la secuencia incongruente siempre confluye (en la posición del círculo negro) con las secuencias de la otra especie antes del evento de especiación. En un escenario de hibridación, podría unirse después (B) o antes (C) del evento de especiación. Sin embargo, la hibridación no siempre tiene por qué llevar siempre a situaciones de no-monofília (D), en cuyo caso no serán detectados.

En estos casos de no-monofilia detectados en Leptogorgia, como en otros, es difícil diferenciar entre posibles eventos de hibridación o resolución incompleta linajes. Para ello, Joly et al. (2009) (Fig. 2) sugieren el uso de análisis estadísticos para discriminarlos, considerando que las distancias genéticas entre secuencias de dos especies es menor en casos de hibridación. Sin embargo, estos mismos autores admiten que hay determinados casos donde estos dos eventos evolutivos son indiferenciables (Fig. 2C).

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Por otro lado, la obtención de otras evidencias a favor de los procesos evolutivos observados en estas especies, como por ejemplo, la formación de híbridos, dependerá de la permeabilidad que exista en sus barreras reproductivas (Li et al. 2016). Por otra parte el conocimiento de la biología reproductiva de los organismos, es un punto esencial para los futuros planes de gestión y conservación de los mismos. En el planteamiento de este objetivo, comprendimos que, escoger un representante de las dos principales familias de gorgonias presentes en Ecuador, Gorgoniidae y Plexauridae, podría ser la mejor forma de obtener una visión más global de la naturaleza reproductiva de estos organismos en estas localidades. Gracias a este estudio, observamos que en la región de Ecuador las especies seleccionadas, Leptogorgia alba y Muricea plantafinea, presentaban una gametogénesis continua con una difusión permanente de productos reproductivos (con ciertos periodos de mayor emisión o descanso), sin trazas de desarrollo interno o externo de estados planuloides, por lo que asuminos una fertilización externa (Rossi & Gili 2009). Esta hipótesis estuvo apoyada por la presencia de productos reproductivos masculinos y femeninos en todas las fases de desarrollo a lo largo de los 14 meses de seguimiento (Cordes et al. 2001; Pires et al. 2009). Por otro lado, ambas especies eran gonocóricas, la cual parece ser dominante entre los octocorales (Cordes et al. 2001; McFadden et al. 2001; Orejas et al. 2012; Pires et al. 2009), mientras que gran parte de los hexacorales suelen ser hermafroditas (see Harrison 2011; Kahng et al. 2011). Sin embargo, se ha sugerido que los corales escleractinios poseían un antepasado gonocórico, como en el resto de los antozoos (Baird et al. 2009; Harrison 2011). En el desarrollo de la revisión bibliográfica comprobamos la gran variabilidad de estrategias reproductivas (sensu lato) presentes en los octocorales, desde especies gonocóricas, hermafroditas, gonocóricas con un porcentaje de colonias hermafroditas (mixtas), partenogenéticas, con múltiples ciclos de oogénesis continuos o solapados, de fecundación y desarrollo interno, fecundación externa o externa pero con cuidado parental en su superficie, etc. (Kahng et al. 2011). En ocasiones, este tipo de patrones no tiene una relación directa con el área geográfica de la que proceden las especies (Kahng et al. 2011). De hecho, encontramos numerosas similitudes reproductivas de las especies estudiadas, con otros octocorales tropicales como Carijoa riisei (Hawai, Puerto Rico) (Bardales 1981; Kahng et al. 2008),

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Dendronephthya hemprichi (Mar Rojo, subtropical) (Dahan & Benayahu 1997), especies de profundidad como Anthoptilum murrayi (Brasil) (Pires et al. 2009), o incluso de aguas frías, como Primnoa resedaeformis (Canadá) (Mercier & Hamel 2011) y Pennatula aculeata (en el noroeste de Norteamérica) (Eckelbarger et al. 1998).

Por otra parte, Leptogorgia alba y Muricea plantafinea, presentaban diferencias en cuanto a la variación del tamaño de los productos sexuales, especialmente en los ovocitos. Algunos estudios como el de Shlesinger et al. (1998), relacionan el tamaño de los ovocitos con el nivel de energía que asigna cada especie a la reproducción. Se planteó la hipótesis de que algunas especies, como en el caso de L. alba, podrían invertir una gran parte de sus reservas de energía para otros procesos de producción secundarios tales como el crecimiento (Rossi & Gili 2009; Rossi 2002). De manera opuesta, parece que el diámetro de gran tamaño de los ovocitos, como en el caso Muricea plantafinea, podría definir la mayor duración del periodo de maduración o el tipo de la fertilización (Harrison & Wallace 1990; Kahng et al. 2011). Sin embargo, algunos autores consideran que esta relación no siempre se cumple (ver Benayahu 1997; Fadlallah & Pearse 1982; Orejas et al. 2007; Shlesinger et al. 1998). De hecho, en nuestro estudio ambas especies presentaban patrones de reproducción muy similares, pero el tamaño de los productos sexuales fue diferente.

Por otro lado, a pesar de que las variaciones en la temperatura son uno de los factores más relacionados con la biología de la reproducción de los corales y su estacionalidad (Dahan & Benayahu 1997; Glynn et al. 1991, 2012; Rossi & Gili 2009), en nuestro estudio parece que este factor no tuvo una influencia clara o aparente. Otros autores sugieren la existencia de distintos elementos relacionados con las condiciones ambientales locales o regionales que también pueden actuar como controles adicionales o alternativos en la reproducción sexual de estos organismos (Harrison & Wallace 1990; Quintanilla et al. 2013; Simpson 1985). Entre esos factores se han observado la influencia de los ciclos lunares (Benayahu y Loya 1986; Babcock 1990;. Coma et al 1995), los ciclos anuales de insolación (Van Woesik et al., 2006), o la lluvia y los regímenes de viento (Babcock et al 1986;. Acosta y Zea 1997;. Alvarado et al 2003). En nuestro caso, planteamos la hipótesis de la relación entre los ciclos reproductivos

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de las gorgonias estudiadas y los procesos oceanográficos complejos que influyen en el litoral de Ecuador (Glynn et al. 2003), los cuales han sido comentados y desarrollados en varios capítulos de la presente Tesis. En este caso, se hipotetiza cómo la influencia de la corriente de Humboldt (Cucalón 1996; Iriarte & González 2004; Poulin et al. 2002; Thiel et al. 2007), puede facilitar la difusión de los productos sexuales maduros. De esta forma, a pesar de darse un proceso continuo de reproducción, se explicaría, al menos en parte, esos momentos de intensificación en la emisión de productos reproductivos y los momentos menor intensidad observados. De esta forma, con este estudio se aporta nueva información sobre los procesos reproductivos en los octocorales del Pacífico oriental, contribuyendo a una nueva visión acerca de los factores que pueden controlar dichos eventos biológicos en este área de estudio.

Otro de los objetivos desarrollados en la presente Tesis, fue el estudio de las comunidades de invertebrados asociados a dos especies de gorgonias, Leptogorgia sp. y Leptogorgia obscura, ampliamente distribuidas en este área. Como ya se ha mencionado, los corales en general y las gorgonias en particular, son considerados importantes ingenieros ecosistémicos (Fig. 3) (Cerrano et al. 2000; Jones et al. 2004), los cuales proporcionan una diversidad de microhábitats para una amplia variedad de taxones (Buhl-Mortensen & Mortensen 2004; Plaisance et al. 2011; Wendt et al. 1985), así como refugio, alimento y protección para los mismos (Kumagai 2008; Patton 1974).

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Figura 3. Iconografía de macrofauna asociada a un coral ramificado de Gerlach (1959), ilustrado en el trabajo de Glynn & Enochs (2011). La imagen representa la gran variedad de organismos sésiles, sedentarios y bio-erosionadores, así como diversidad de taxones de invertebrados marinos que pueden ser albergados por los corales.

A través de nuestro trabajo comprobamos la gran abundancia de organismos asociados a las especies de gorgonias estudiadas, en comparaciones de estudios realizados con metodologías semejantes y sobre organismos próximos (Carvaho et al. 2014). Incluso esto sería así frente a otros ingenieros ecosistémicos como algas, esponjas, hidrozoos o briozoos (Ávila & Ortega-Bastida 2014; Cunha et al. 2013; Garcia et al. 2008; Guerra-García et al. 2011; Koukouras et al. 1996), aunque habría que tener en cuenta que en algunos de estos casos se emplearon medios que podrían estar subestimando dicha abundancia. Esto demostraría la particular efectividad de estas gorgonias respecto a otros organismos, reforzando la relevancia de su papel como bio- constructores, sobre todo en áreas tropicales. Además, pone de manifiesto la necesidad

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de planes de conservación de los jardines de gorgonias en el área continental de Ecuador, no sólo por la gorgonia en sí misma, si no por el soporte que ofrecen para la abundante comunidad de epifauna asociada.

En el desarrollo de este estudio, se tuvo en consideración tanto la propia morfología de las gorgonias hospedadoras, considerando el espacio habitable que aportaba cada una de ellas, como la posible variación de la comunidad asociada a lo largo de los dos años de muestreo. Algunos autores han observado que hay asociaciones específicas entre ciertos corales hospedadores y sus invertebrados asociados (Abele & Patton 1976; Sin 1999; Stella et al. 2010), lo que parece estar relacionado con las diferencias en la morfología, distribución y abundancia de las diferentes especies de huéspedes (Cocito 2004; Stella et al. 2011). En nuestro estudio, las dos especies de gorgonias analizadas mostraron claras diferencias en su morfología que podrían dar lugar a diferentes microhábitats disponibles para la epifauna. Leptogorgia sp. mostraba una forma de abanico abierto en un solo plano y L.obscura una morfología tridimensional (arborescente), con un mayor espacio habitable medio disponible y una mayor proporción de espacios interiores, pudiendo esta morfología proporcionar una mayor protección. Esto se correlacionó con una diferencia en la diversidad de la fauna asociada y su abundancia total, así como la estructura de esta comunidad. Por un lado, entre las dos especies hospedadoras, se observó un aumento de la abundancia con el aumento del espacio habitable, debido posiblemente a una mayor disponibilidad de los recursos en las colonias anfitrionas con un mayor espacio habitable disponible (Abele & Patton 1976; Abele 1976; Huber & Coles 1986). Por el contrario, un resultado sorprendente fue la tendencia opuesta (de disminución) que mostró la diversidad respecto al aumento del espacio habitable, dentro de cada una de las dos especies hospedadoras. Esto parece encontrarse en oposición aparente con la hipótesis de heterogeneidad del hábitat (MacArthur & Wilson 1967), que predice un aumento de la complejidad del hábitat con el aumento del tamaño de las colonias (Anderson et al. 2005), lo que daría lugar a un mayor número de diferentes nichos disponibles para la fauna asociada. Algunos trabajos como el de Carvalho et al. (2014) sugirieron que en los jardines de gorgonias, el aumento del tamaño de las colonias podría conllevar la agregación de algunas especies, convirtiéndolas en dominantes, lo 364

que podría explicar quizás en parte el patrón observado de la reducción de la diversidad. Sin embargo, en el presente estudio se ha realizado el análisis a nivel de grandes grupos, a causa de no existir una información taxónomica actualizada disponible para los distintos grupos en este área geográfica. Por ello, los resultados deben considerarse como un primer paso en una región aún poco conocida como es el litoral de Ecuador, donde estudios futuros con una resolución taxonómica superior serían necesarios. Por otro lado, las abundancias totales en nuestro estudio mostraron una variabilidad estacional aparente, las cuales tendían a ser globalmente mayores en junio (estación lluviosa) que en noviembre (estación seca), aunque esta tendencia fue más evidente en las colonias con un mayor espacio habitable (L. obscura). Como hemos mencionado anteriormente, el litoral de Ecuador está fuertemente influenciado por la corriente fría y cargada de nutrientes de Humbolt, cuyo periodo de mayor intensidad está comprendido de junio a septiembre, desapareciendo gradualmente durante noviembre (Duxbury et al. 2002; Peixoto & Oort 1992) por la incursión de la corriente cálida y baja en nutrientes de Panamá (Coello et al. 2001). Acorde a estos resultados, hipotetizamos que cuando la fauna asociada posee suficiente espacio disponible dentro de un colonia hospedadora, los sucesos biológicos asociados a la corriente de Humbolt aparentemente impulsan un fuerte incremento en su abundancia, pero no parece tener un efecto claro respecto a la diversidad. En resumen, el estudio ecológico de las interacciones de epifauna con las especies de gorgonias hospedadoras, muestra un patrón complejo de variabilidad en la abundancia, la diversidad y la estructura de la comunidades presentes en el litoral de Ecuador, respecto a otros organismos hospedadores y a otras áreas geográficas.

Por último, para completar el objetivo final del trabajo realizado durante estos años, era imprescindible indagar sobre una de las amenazas de estas especies a nivel global. Conocer las comunidades fúngicas y posibles consecuentes enfermedades que pudieran generar en los jardines de gorgonias, es uno de los puntos de crucial importancia para diseñar y adecuar de planes gestión de conservación. Una de las amenazas a las que se enfrentan los jardines de gorgonias son las enfermedades producidas por hongos, que lejos de afectar solamente a gorgonias, atacan a muchos otros organismos y suponen uno de los principales peligros a los que se enfrenta la 365

biodiversidad en todo el mundo (Fisher et al. 2012). De hecho, es ampliamente reconocido que en las últimas décadas, los arrecifes de coral han experimentado un deterioro exponencial debido a la combinación de las perturbaciones y presiones antrópicas. Algunas de las más conocidas son aquellas derivadas del cambio climático global, fragmentación del hábitat y reducción de la diversidad funcional (Nyström et al. 2000; Wilkinson 2008). Estas condiciones hacen a los arrecifes más susceptibles a la proliferación y el desarrollo de organismos oportunistas que aprovechan el debilitamiento de las especies de coral (Contreras et al. 2011; Szmant 2002). En este marco, hongos patógenos como Aspergillus sydowii (Bainier y Sartory, 1926) son los responsables de enfermedades conocidas como la aspergilosis, la cual ha producido un deterioro significativo de las poblaciones y mortalidades tanto parciales, como masivas de comunidades de coral (Smith et al. 1996; Nagelkerken et al. 1997a, 1997b; Ward et al. 2006; Toledo-Hernández et al. 2008; Sánchez et al. 2011). Dentro de las especies de Gorgoniidae seleccionadas para este estudio, L. obscura y Leptogorgia sp., poseían una amplia y diversa comunidad fúngica, en su mayoría desconocida. Esta comunidad de hongos patógenos, la cual incluyó a A. sydowii, se encontró en colonias de gorgonias aparentemente sanas y sin mostrar síntomas claros de enfermedad por hongos durante el período de muestreo. Estos resultados eran similares a los descritos previamente por algunos autores (Toledo-Hernández et al. 2008; Zuluaga-Montero et al. 2009). Sin embargo, esta ha sido la primera vez que A. sydowii se ha registrado en Ecuador y la segunda en el Pacífico oriental (Barrero-Canosa et al. 2013). Los factores que conducen a la aspergilosis o al desarrollo de otros hongos patógenos en los arrecifes de coral, siguen siendo poco conocidos. Algunos autores han sugerido que la combinación de factores de estrés ambiental, tales como la temperatura, los rayos UV, la concentración de aerosoles, etc., pueden reducir de la eficacia de las defensas del organismo (Alker et al. 2001; Barrero-Canosa et al. 2013), y ser factores determinantes en el desarrollo de la enfermedad a través de estos patógenos oportunistas (Diéguez-Uribeondo et al. 2011; Green & Bruckner 2000; Rypien et al. 2008; Sarmiento-Ramirez et al. 2014). La detección e identificación de estas comunidades fúngicas en los jardines de gorgonias de Ecuador es de gran importancia para abrir nuevas líneas de investigación con el objetivo de descifrar los factores que conducen a las posibles enfermedades en las

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comunidades de estas especies y a un eventual deterioro del ecosistema. Estudios futuros sobre estas comunidades patógenas y modelos de alerta sobre diversos factores de estrés ambiental, así como un monitoreo periódico sobre la salud de estos parches de gorgonias, son cruciales para poder prevenir posibles eventos de enfermedad y mortalidad de estas comunidades.

En resumen, con los datos presentados en la presente Tesis, hemos incrementado el conocimiento acerca de la diversidad y complejidad que rodea a especies de gorgonias, en un área geográfica tan rica y desconocida como es el litoral continental de Ecuador. Esta contribución ha supuesto no sólo un aporte importante a la taxonomía de estos organismos, ayudando a valorar los caracteres taxonómicos que se emplean para discriminar especies y otros niveles supra-específicos, sino que también se han abordado las relaciones filogenéticas, así como los procesos determinantes de su historia evolutiva y cómo influyen en su biología sus estrategias reproductivas. Los datos obtenidos acerca de sus patrones evolutivos, reproductivos, interacciones ecológicas y susceptibilidad a ciertas amenazas, son especialmente útiles para poder establecer planes adecuados de gestión y conservación en estas áreas, donde las gorgonias tienen un papel trascendental como ingenieros del ecosistema. De esta forma, hemos aportado una visión multidisciplinar e integradora a la hora de resolver las cuestiones planteadas en cada uno de los objetivos de la presente Tesis. Por último, esperamos que para aquellas cuestiones que no hayan podido ser resueltas en su totalidad, se hayan sentado las bases para futuros análisis e investigaciones.

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6. CONCLUSIONES

Los estudios desarrollados en la presente Tesis Doctoral, han permitido extraer las siguientes conclusiones:

1. Se han identificado diez especies de la familia Gorgoniidae en el área de estudio, constituyendo 7 de ellas nuevos registros para la zona.

2. Se comprobó la utilidad de la búsqueda de nuevos caracteres en la delimitación e identificación de las especies de estudio.

3. A través de una taxonomía integradora (incluyendo caracteres morfológicos, morfométricos y moleculares), se describieron cuatro nuevas especies para la ciencia, E. ahorcadensis, L. mariarosae, L. manabiensis y P. machalilla.

4. Los marcadores moleculares utilizados proporcionaron una mayor resolución filogenética, para las especies analizadas, mediante la concatenación de genes nucleares (ITS y 28S) y mitocondriales (mtMutS y Cox), aunque cada uno de ellos aporta resolución a distintos niveles taxonómicos.

5. Dentro de los géneros de Gorgoniidae estudiados, Pacifigorgia mostró un perfil monofilético, mientras que los géneros Leptogorgia y Eugorgiase integran un mismo clado.

6. Diferentes procesos evolutivos fueron propuestos para las especies de Pacifigorgia, como una de radiación explosiva con una diversificación muy reciente o una posible antigua hibridación con una posterior re- homogeneización del genoma.

7. Para el complejo de especies encontrado en el género Leptogorgia, se observaron posibles procesos de evolución reticulada o resolución incompleta de linajes.

8. Se observó que las especies Leptogorgia alba y Muricea plantafinea (gonocóricas con fecundación externa), desarrollan un ciclo de gametogénesis

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con difusión continua, y una variación en el tamaño de sus productos reproductivos.

9. La biología reproductiva encontrada pareció no tener una relación directa con la temperatura ambiental, pero sí con los sistemas de corrientes predominantes de la zona, como la corriente de Humbolt.

10. En dos especies del género Leptogorgia, se observó la mayor abundancia de fauna asociada hasta ahora encontrada en organismos bio-constructores, mayor incluso que la hallada en especies similares de gorgonias y escleratinios, así como en otros ingenieros ecosistémicos.

11. Se encontraron diferencias significativas en la estructura de la comunidad asociada a dos especies de gorgonias, en cuanto a su abundancia y su diversidad. Estas diferencias están aparentemente relacionadas con la propia morfología del organismo hospedador.

12. Las diferencias estacionales encontradas en la fauna asociada parecen estar influenciadas por la intensificación de la corriente de Humbolt y la posterior incursión de la corriente de Panamá.

13. Se observó que con el aumento del espacio habitable en la gorgonia, se producía una disminución de la diversidad de la epifauna, lo que se opone a la hipótesis de heterogeneidad del hábitat.

14. Se identificó una diversa comunidad fúngica asociada a dos especies de gorgonias del área de estudio, con ciertas especies no registradas previamente.

15. Se detectaron hongos patógenos, en individuos aparentemente sanos, que podrían estar latentes en este área, lo que podría favorecer el desarrollo de enfermedades como la aspergilosis.

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7. CONCLUSIONS

From the studies developed in the present PhD thesis, the following conclusions can be drawn: 1. Ten species of the Family Gorgoniidae have been identified in the study area, seven of them constituting new records in the Ecuadorian region. 2. The usefulness of searching for new characters in the delimitation and identification of the studied species was verified. 3. Through an integrative taxonomic framework (including morphological, morphometric and molecular characters), four species new to science, E. ahorcadensis, L. mariarosae, L. manabiensis and P. machalilla, were described. 4. The molecular markers used provided a greater phylogenetic resolution for the species analyzed by concatenating nuclear (ITS and 28S) and mitochondrial genes (mtMutS and Cox), although each of them contributed to the resolution at different taxonomic levels. 5. Within the Gorgoniidae genera studied, Pacifigorgia showed a monophyletic profile, while the genera Leptogorgia and Eugorgia were integrated together int0 the same clade. 6. Different evolutionary processes were proposed for the Pacifigorgia species, such as an explosive radiation with a very recent diversification, or a possible ancient hybridization with a subsequent genomic re-homogenization. 7. Potential reticulated and/or incomplete lineage sorting processes were observed in the Leptogorgia species complex. 8. Leptogorgia alba and Muricea plantafinea (gonochoric with external fertilization) developed a gametogenesis cycle with continuous diffusion, and a variation in the size of their reproductive products. 9. The reproductive biology did not seem to be directly influenced by the water temperature, but it could be by the water currents, predominantly the Humboldt Current Systems.

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10. The greatest abundance of associated fauna ever found was observed in two Leptogorgia species, this being even higher than that found in other gorgonians and scleractinians or in other “ecosystem engineers”. 11. Significant differences were found in the structure of the community associated with the two gorgonian hosts, specifically with respect to their abundance and diversity. These differences are apparently related to the morphology of the host. 12. The seasonal differences found in the associated fauna might be related to the intensification of the Humboldt Current and the subsequent incursion of the Panama Current. 13. It was observed that an increase in the available habitable gorgonian host space was associated with a decrease in the epifauna diversity. This finding would appear to be contrary to “the heterogeneity of habitat” hypothesis. 14. A diverse fungi community, associated with two gorgonian species from the area of study, was identified. Some of these organisms had never been recorded before. 15. Some pathogenic fungi were detected on apparently healthy individuals. Such circumstances could lead to the development of diseases, such as aspergillosis.

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8. Appendices

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390

391

Relaciones filogenéticas basadas en el gen mitocondrial mtMutS (árbol ampliado). Ver

Appendix 1. legenda en figura 2, capítulo 3.

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(árbol

Cox1 - Igr - Cox2

Relaciones filogenéticas basadas en el gen mitocondrial

. ndix 2 Appe ampliado). Ver legenda en figura 2, capítulo 3.

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(árbol ampliado). Ver

ITS

Relaciones filogenéticas basadas en el gen mitocondrial

Appendix 3. legenda en figura 2, capítu lo 3.

397 (árbol ampliado). Ver 28S

Relaciones filogenéticas basadas en el gen mitocondrial

legenda en figura 2, capítulo 3 Appendix 4.