ESCENARIOS DE ESPECIACIÓN DE ORGANISMOS SESILES EN EL MAR CARIBE: EL GENERO Agelas (Porifera: Demospongiae) UN CASO DE ALTA DIVERSIDAD EN EL

AREA

SPECIATION SCENARIOS OF SESSILE ORGANISMS IN THE CARIBBEAN SEA: THE GENUS Agelas (Porifera: Demospongiae), A CASE OF HIGH DIVERSITY IN THE AREA

FERNANDO JOSÉ PARRA-VELANDIA CÓDIGO 1190805

TESIS PRESENTADA PARA OPTAR AL TÍTULO DE DOCTOR EN CIENCIAS - BIOLOGÍA

DIRIGIDO POR:

SVEN ZEA PhD

Profesor Titular, Departamento de Biología (Sede Bogotá) y Centro de Estudios en Ciencias del Mar – CECIMAR (Sede Caribe, Santa Marta)

Universidad Nacional de Colombia

ROB W.M. van SOEST Dr.rer.nat

Curador de la sección de Invertebrados Museo Zoológico de Amsterdam. Universidad de Amsterdam

UNIVERSIDAD NACIONAL DE COLOMBIA FACULTAD DE CIENCIAS (Sede Bogotá) y

CENTRO DE ESTUDIOS EN CIENCIAS DEL MAR – CECIMAR (Sede Caribe, Santa Marta)

En convenio con el Instituto de Investigaciones Marinas y Costeras – INVEMAR (adscrito al Ministerio del Ambiente, Vivienda y Desarrollo Territorial)

Bogota, Colombia, 2011

ii

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Titulo Escenarios de especiación de organismos sésiles en el Mar Caribe: el genero Agelas (Porifera: Demospongiae) un caso de alta diversidad en el área. Title Speciation scenarios of sessile organisms in the Caribbean Sea: the genus Agelas (Porifera: Demospongiae), a case of high diversity in the area. Resumen El genero Agelas (Duchassaing and Michelotti, 1864; orden Agelasida) es un grupo de esponjas tropicales coralinas distribuidas a lo largo del Atlántico Tropical, Atlántico Norte Templado e Indopacífico Central y Occidental. En este trabajo, primero se perfiló una filogenia molecular a nivel de género y familia. Luego, se realizo una cuidadosa revisión taxonómica de las especies Caribeñas. Finalmente, con el fin de inferir la historia de la distribución y especiación del grupo, comparamos una filogenia basada en el ADN y una descripción general de la biogeografía marina del fanerozoico. Abstract The genus Agelas (Duchassaing and Michelotti, 1864; order Agelasida) is a group of tropical and subtropical rocky and coral reef sponges distributed throughout the Tropical Atlantic, Temperate Northern Atlantic, Western and Central Indo-Pacific Realms. Firstly, a family and genus-level molecular phylogeny was drawn. Then, a thorough taxonomic revision of Caribbean species was undertaken. Finally, in order infer the history of speciation and distribution of the group, we compared the DNA-phylogeny against a general outline of phanerozoic marine biogeography. Palabras clave Astrosclera, Ceratoporella, acantoestilo, biogeografía, Panthalassa Keywords Astrosclera, Ceratoporella, acanthostyle, biogeography, Panthalassa FIRMA DEL DIRECTOR:_________________________________ Fernando José Parra Velandia (1971)

iv

Agradecimientos

Cuando se termina un trabajo como el que se presenta, escribir la lista de personas a las cuales

agradecer es una tarea emocionante y evocadora. A todos los que se sientan identificados en los

textos que siguen, reciban un calido abrazo y mi reconocimiento sincero.

A Sven y Rob, las palabras carecen de sentido para expresarles la profunda gratitud que merecen;

tengo la esperanza de que este trabajo sea digno de sus esfuerzos y desvelos. For Rob and in his

behalf to the Netherlands Kingdom peoples, who gave me great lessons of toughness, tolerance

and humility.

A las entidades financieras, instituciones académicas y laboratorios de investigación:

COLCIENCIAS (proyectos 30003-1-33-81, 30003-154-83, 2105-09-023-93, 2105-09-120-97

para Sven Zea y 1101-09-11241 para Zea y Parra), Fundación Holandesa para el Avance de la

Investigación Tropical - WOTRO (WB 82-261, para van Soest, Zea y Parra), Universidad

Nacional de Colombia y su División de Investigaciones Sede Bogotá (DIB), Universiteit van

Amsterdam e IBED, Smithsonian Institution – CCRE (Belice), U.S. National Science Foundation

(proyectos a J. Pawlik) y tripulacion del R/V Seward Johnson, Oceanario CEINER (Islas del

Rosario), Smithsonian Institution’s Carrie Bow Cay Field Station (Belice), Discovery Bay

Marine Laboratory (Jamaica), Bellairs Research Institute de la McGill University (Barbados),

CARMABI (Curaçao) y personal administrativo, logístico y científico del INVEMAR

(Colombia), nuestro agradecimiento por el generoso e invaluable soporte financiero, educativo y

logístico proporcionado.

Leonor, Julio, Juanita, Judith, Janethe, Yesid y Edwin, mi Familia, me enseñaron entre muchas

cosas que la bondad es gratificante en si misma; que el mundo es ancho y ajeno; arriesgarse es

divertido siempre y peligroso a veces. Por ustedes entendí que la vida es valiosa y desarrollarla lo

más digno. Son la gran constante de mi vida. Para mi tigrecito, primer bebe de una nueva

generación, te amo. Il mio caro drago rosso, sei persino il mio timone, la mia vela, la mia ancora

v

ed il mio mare, grazie. Para Myriam Ortiz Hurtado, la Fundación AprendES, sus hijas e hijos y a

todo el círculo de amor que siempre los rodea, gracias, muchas gracias.

En mi niñez y adolescencia tuve los “malos” amigos que un padre responsable evita y un hijo

irresponsable busca. Todos ellos hoy ausentes, su presencia brilla aun en mi corazón y de alguna

manera están aquí, pues a veces las cosas salen bien a pesar de nuestro mejor esfuerzo.

A lo largo de mi vida académica y profesional he conocido gente valiosa que me brindo

generosamente su amor, lealtad, pasión, cariño, amistad y sabiduría. De ustedes recibí hermosas

experiencias de vida, no importa si dolorosas, divertidas, extrañas e inclasificables pero todas,

independientemente de su significado, perdurables.

Si has llegado hasta este punto del texto, estoy plena y absolutamente seguro que en algún

momento me abriste no solo las puertas de tu corazón, también las puertas de tu familia, que me

recibió como un hijo (o el amigo descarriado). En tu mesa disfrute, discutí, canté, reí, lloré,

compartí y especialmente recibí cantidades enormes e inmerecidas de afecto, cariño y amor. Para

ti, en especial si no supiste qué hacer conmigo en tiempos recientes y para mi familia por todos

sus sobresaltos, vayan estas palabras:

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Este trabajo te lo dedico con todo mi amor y cariño

vii

Table of Contents

Agradecimientos ______________________________________________________________ iv

Table of Contents ____________________________________________________________ vii

FOREWORD _______________________________________________________________ xiii

RESUMEN ___________________________________________________________________1

ABSTRACT __________________________________________________________________2

Chapter 1. AN INTRODUCTORY APPROACH TO EVOLUTIONARY ISSUES IN THE GENUS Agelas (PORIFERA: DEMOSPONGIAE)____________________________________3

Chapter 2. PHYLOGENETIC RELATIONSHIPS OF FOUR GENERA OF THE ORDER AGELASIDA (PORIFERA: DEMOSPONGIAE) _____________________________________5

ABSTRACT ________________________________________________________________5

INTRODUCTION____________________________________________________________6 The Agelasida families _____________________________________________________6 Agelasida fossil record _____________________________________________________8 Origin of the calcareous skeleton____________________________________________10 Systematic classification of recent Agelasida __________________________________10

METHODS ________________________________________________________________11 Sampling________________________________________________________________11 DNA extraction and amplification___________________________________________12 Sequencing ______________________________________________________________13 Data analysis ____________________________________________________________13

RESULTS _________________________________________________________________14

DISCUSSION ______________________________________________________________15

ACKNOWLEDGEMENTS ___________________________________________________18

Chapter 3. REEF SPONGES OF THE GENUS Agelas (PORIFERA: DEMOSPONGIAE) FROM THE CARIBBEAN SEA _______________________________________________________19

ABSTRACT _______________________________________________________________19

INTRODUCTION___________________________________________________________20 Order Agelasida (Hartman, 1980)___________________________________________23 The Caribbean Sea and the genus Agelas _____________________________________24

viii

METHODS ________________________________________________________________25 Collecting _______________________________________________________________25 Microscopy______________________________________________________________25 Museum deposits _________________________________________________________26 Macroscopy _____________________________________________________________26

SYSTEMATIC DESCRIPTIONS_______________________________________________26 Genus Agelas Duchassaing & Michelotti, 1864 ________________________________27 Remarks ______________________________________________________________27

Agelas dispar Duchassaing & Michelotti, 1864_________________________________29 Material and distribution ________________________________________________30 Description ____________________________________________________________32 Remarks ______________________________________________________________33

Agelas cervicornis (Schmidt, 1870)___________________________________________35 Material and distribution ________________________________________________35 Description ____________________________________________________________37 Remarks ______________________________________________________________37

Agelas wiedenmayeri Alcolado, 1984 _________________________________________38 Material and distribution ________________________________________________38 Description ____________________________________________________________39 Remarks ______________________________________________________________40

Agelas sceptrum (Lamarck, 1815) ___________________________________________42 Material and distribution ________________________________________________42 Description ____________________________________________________________44 Remarks ______________________________________________________________44

Agelas dilatata Duchassaing & Michelotti, 1864________________________________45 Material and distribution ________________________________________________45 Description ____________________________________________________________46 Remarks ______________________________________________________________47

Agelas conifera (Schmidt, 1870)_____________________________________________48 Material and distribution ________________________________________________48 Description ____________________________________________________________50 Remarks ______________________________________________________________50

Agelas tubulata Lehnert & van Soest, 1996____________________________________51 Material and distribution ________________________________________________52 Description ____________________________________________________________54 Remarks ______________________________________________________________55

Agelas repens Lehnert & van Soest, 1998 _____________________________________56 Material and distribution ________________________________________________56 Description ____________________________________________________________56 Remarks ______________________________________________________________57

Agelas cerebrum Assmann, van Soest & Köck, 2001 ____________________________58 Material and distribution ________________________________________________58 Description ____________________________________________________________59 Remarks ______________________________________________________________60

ix

Agelas clathrodes (Schmidt, 1870) ___________________________________________61 Material and distribution ________________________________________________61 Description ____________________________________________________________63 Remarks ______________________________________________________________64

Agelas schmidti Wilson, 1902 _______________________________________________64 Material and distribution ________________________________________________65 Description ____________________________________________________________65 Remarks ______________________________________________________________67

Agelas citrina Gotera & Alcolado, 1987 ______________________________________68 Material and distribution ________________________________________________68 Description ____________________________________________________________69 Remarks ______________________________________________________________71

Agelas sventres Lehnert & van Soest, 1996 ____________________________________73 Material and distribution ________________________________________________73 Description ____________________________________________________________75 Remarks ______________________________________________________________76

DISCUSSION ______________________________________________________________77

ACKNOWLEDGEMENTS ___________________________________________________83

Chapter 4. PHYLOGENY AND BIOGEOGRAPHY OF THE PAN-TROPICAL MARINE SPONGE GENUS Agelas (PORIFERA: DEMOSPONGIAE) __________________________84

ABSTRACT _______________________________________________________________84

INTRODUCTION___________________________________________________________85

METHODS ________________________________________________________________88 Sampling________________________________________________________________88 DNA extraction and amplification___________________________________________90 Sequencing ______________________________________________________________91 Data analysis ____________________________________________________________91

RESULTS _________________________________________________________________92

DISCUSSION ______________________________________________________________96

ACKNOWLEDGEMENTS __________________________________________________104

Chapter 5. CONCLUSIONS ____________________________________________________106

REFERENCES ______________________________________________________________109

APPENDIX A. Fossil non-calcareous Agelas_______________________________________120

Genus Ropalospongia Mostler, 1994 ‡ __________________________________________120 Remarks _______________________________________________________________120

Genus Fluegelispongites Mostler, 1994 ‡________________________________________121 Remarks _______________________________________________________________122

x

DISCUSSION _____________________________________________________________122

APPENDIX B. Additional publications ___________________________________________124

1- Speculation with Spiculation? - Three Independent Gene Fragments and Biochemical Characters versus Morphology in Demosponge Higher Classification. Erpenbeck, D. Breeuwer, J. A. J., Parra-Velandia, F. J., van Soest, R.W.M.. 2006. Molecular Phylogenetics

and Evolution 38 (2), 293-305. ________________________________________________124

2- New records of the genus Agelas Duchassaing & Michelotti, 1864 (Porifera, Agelasida) off the Amazon River mouth, Brazil, Southwestern Atlantic. Mothes, B.; Campos, M.; Lerner, C.; Carraro, J.L. Parra-Velandia , F.J. 2007, Biota Neotrop. 7 (3). _______________________124

3- A new Agelas (Demospongiae: Agelasida: Agelasidae) from the Thousands Islands, West-Java, Indonesia De Voogd, N.J., Parra-Velandia, F.J. & Soest, R.W.M. Van. 2008. Zool. Med.

Leiden 82 (22), 235-243. _____________________________________________________124

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Table of Figures

Figure 1. Putative Agelasid spicules. _______________________________________________8

Figure 2. Land masses paleopositions during the Ordovician. ____________________________9

Figure 3. Phylogenetic reconstructions of four genera of Agelasida. ______________________15

Figure 4. SEM images of Agelas. _________________________________________________21

Figure 5. Photographs of A. dispar. _______________________________________________31

Figure 6. Photographs of A. cervicornis.____________________________________________36

Figure 7. Photographs of A. wiedenmayeri. _________________________________________39

Figure 8. Photographs of A. sceptrum. _____________________________________________43

Figure 9. Photographs of A. dilatata. ______________________________________________46

Figure 10. Photographs of A. conifera. _____________________________________________49

Figure 11. Photographs of A. tubulata. _____________________________________________54

Figure 12. Photographs of A. repens. ______________________________________________57

Figure 13. Photographs of A. cerebrum. ____________________________________________59

Figure 14. Photographs of A. clathrodes. ___________________________________________62

Figure 15. Photographs of A. schmidti. _____________________________________________66

Figure 16. Photographs of A. citrina. ______________________________________________70

Figure 17. Photographs of A. sventres. _____________________________________________74

Figure 18. Scanned sections of dry specimens shiwing skeletal details. ___________________79

Figure 19. Depth range for each species from the authors database, ______________________81

Figure 20. ITS and CO1 ML phylogram obtained from combined conservative matrix _______93

Figure 21. CO1 ML phylogram obtained from matrix _________________________________94

Figure 22. ITS ML phylogram obtained from conservative matrix._______________________95

Figure 23. Early-Middle Ordovician global plate tectonic distribution (472 ma). ___________100

Figure 24. Silurian global plate tectonic distribution (417 ma). _________________________100

Figure 25. Silurian global plate tectonic distribution (408 ma). _________________________101

Figure 26. Carboniferous global plate tectonic distribution (341 ma). ____________________101

Figure 27. Jurassic global plate tectonic distribution (145.5 ma). _______________________102

Figure 28. Cretaceous global plate tectonic distribution (71.5 ma). ______________________102

xii

Table of Tables

Table 1. Specimen locality, museum accession and GenBank accession numbers. ___________13

Table 2. Characters comparison between Hadromerida, Halichondrida and Agelasida. _______15

Table 3. Main morphological features of Caribbean Agelas. ____________________________28

Table 4. Spicule measurements of Agelas species from the Caribbean Sea. ________________33

Table 5. Specimen locality, voucher museum and sequence GenBank accession numbers. ____88

Table 6. Main phanerozoic tropical and subtropical marine events._______________________97

xiii

FOREWORD

When a starter like the first author read for the first time the old descriptions of Agelas, it was

evident to him that all the foundational fathers of sponge science have worked this genus and, as

a result, a great deal of taxonomical work was already done. It was also evident to him that all

this workers had something in common in the manner to treat species definition: when some

species had not enough support, they almost always had been prudent and confessed the self

ignorance, preventing them to go for new species. But on the other hand, he wants to express his

respect for the same hard workers that could see in the different color, spicule, habit, etc., the key

to uncover a new species. Observation, patience and prudence are the advices that all this great

men told us.

1

RESUMEN

Este trabajo pretendió hacer una revisión detallada de las especies del genero Agelas presentes en

el Caribe y establecer las relaciones filogenéticas entre ellas, utilizando herramientas

taxonómicas clásicas en conjunto con las técnicas moleculares más actuales. De acuerdo a

nuestros resultados, el genero Agelas en el Caribe tiene al menos trece especies validas; cuya

identificación se logra por combinación de caracteres. Encontramos que características

microscópicas como arreglo esqueletal, numero de verticilos o longitud de las espículas están

sujetos de una variación tal que solo pueden ser usadas como herramientas taxonómicas por

comparación con otras especies en el contexto de una misma área, requiriendo aun así algún

grado de familiaridad con la variación regional del género. La distribución y riqueza de estas

especies en el área del Caribe muestra un notorio gradiente de norte a sur, lo que puede estar

indicando la relativa estabilidad geológica de la plataforma bahamense frente a la dinámica

relativamente mayor del Caribe. Los resultados moleculares (secuencias ITS y CO1 del ADN)

permitieron negar la hipótesis de monofília de las especies caribeñas y mediterráneas del genero

Agelas, adelantada inicialmente por nosotros, al aparecer las especies del Indo pacífico

intercaladas entre las especies del Caribe. Los árboles filogenéticos mostraron la existencia de

cuatro grandes clados que incluyen especies de distribución amplia en el Caribe como A. dispar,

A. conifera, A. citrina y A. clathrodes, todos de origen previo a la formación del Caribe. Sin

embargo, con las herramientas moleculares usadas no se logro suficiente resolución para elucidar

la genealogía dentro de estos grupos de especies; esto puede estar indicado un fenómeno de

especiación reciente en el Caribe sin que se fijen aun variaciones en las secuencias usadas aquí,

pero la resolución no permite asociar esta especiación con fenómenos geológicos del neógeno

como el levantamiento del istmo de Panamá y subsecuentes cambios del nivel del mar durante las

glaciaciones; así mismo, tampoco es suficiente para escudriñar la hipótesis general de posible

existencia de evolución reticulada en el género. Sin embargo, en combinación con los resultados

taxonómicos, los árboles filogenéticos y la historia geológica del planeta fue posible plantear un

modelo evolutivo para el género que involucra especiación vicariante y dispersión durante

segregación y agregación de supercontinentes y apertura y cerramiento de vías marinas durante la

deriva continental. Juntos, estos resultados se constituyen en la base sobre la que se pueden

plantear eventos cruciales del origen y la diversificación de faunas arrecifales.

2

ABSTRACT

This work tried to do a detailed review of Agelas species presents in the Caribbean Sea and to

establish the phylogenetic relations among them, using classic taxonomic tools together with the

state-of-the-art molecular techniques. According to our results the genus Agelas in the Caribbean

Sea has at least thirteen valid species; nevertheless, its identification is obtained by combination

of characters. We found that microscopic characteristics like skeleton arrangement, number of

verticills or spicules length are subject of such variation that they can only be used as taxonomic

tools by comparison with other species in the context of the same area, requiring even so some

degree of familiarity with the genus regional variation. The distribution and richness of these

species in the Caribbean area shows a gradient north to south, which can be indicating the relative

geologic stability of the Bahamian platform against to the relatively greater dynamics of the

Caribbean. The sorting of Indopacific species between Caribbean species in the recovered

phylogeny (from ITS and CO1 AND sequences) allowed the rejection of an hypothesis for the

monophyletic origin of Caribbean and Mediterranean species of genus Agelas, initially put

forward by us. The phylogenetic trees show the existence of four great clades including species

of ample Caribbean distribution like A. dispar, A. conifera, A. citrina and A. clathrodes; all of

them with previous origin to the formation of the Caribbean Sea. However, the molecular tools

did not have enough resolution to unravel the genealogy within these species groups; this

phenomenon can be indicative of recent speciation events in the Caribbean that have not yet

incorporated variation in the studied sequences. Unfortunately, the low resolution does not allow

to associate recent speciations with any neogene geologic event like the rise of the Panama

isthmus and subsequent sea level changes during glaciations; also, the molecular variation is not

enough to test for reticulate evolution within the genus. In brief, with the tools that we chose is

not possible to establish clear patterns for the origin of biodiversity at the Caribbean Sea level.

However, a combination of the taxonomic results, the phylogenetic trees and the Earth geologic

history allows drawing a hypothetical evolutionary scenario for the genus which involves

vicariant speciation and dispersal during segregation and aggregation of supercontinents and

opening and closing of seaways from continental drift. Together, these results constitute a

framework from which crucial events in the origin and diversification of reef faunas can be

drawn and contrasted.

3

Chapter 1. AN INTRODUCTORY APPROACH TO EVOLUTIONARY ISSUES IN THE

GENUS Agelas (PORIFERA: DEMOSPONGIAE)

The dynamics of ancient paleoenvironments is an intriguing field with likely high significance

for Present day biogeography (Hallman, 1994). In the next decade, it may be possible to explain

the results of a growing phylogenetic database of marine and terrestrial organisms using paleo-

events such as supercontinent formation and ocean closures, seaway openings and tectonic drift,

among others, are geological and oceanographic phenomena linked to continental-level marine

vicariance phenomena and to dispersal and distance isolation, whose impact is expected to

become visible in, for example, the phylogenetic tree of a given group of marine organisms

(Nance et al, 1988; Vermeij, 1995). In this dissertation, the sponge genus Agelas was used to

draw upon a wide framework of paleogeographical phenomena to explain the evolutionary

history of tropical marine sessile organisms.

The genus Agelas (Duchassaing and Michelotti, 1864; order Agelasida) is a group of tropical and

subtropical rocky and coral reef sponges distributed throughout the Tropical Atlantic, Temperate

Northern Atlantic, Western and Central Indo-Pacific Realms. This group is defined by the

presence of a unique type of regularly verticillated acanthostyle-like megasclere spicule. These

spicules have been detected in the Permian of Texas (Mostler, 1994) and related ones in the

Ordovician (470 ma) Eastern Panthalassa tropical paleocoasts of the Laurentia Paleocontinent

(Present day Nevada, USA; Kozur et al, 1996), extending the genus Agelas fossil record 200 ma

before previously estimated.

Firstly, a family and genus-level molecular phylogeny was drawn. Then, a thorough taxonomic

revision of Caribbean species was undertaken. Finally, in order infer the history of speciation and

distribution of the group, we compared the DNA-phylogeny against a general outline of

phanerozoic marine biogeography.

The 2nd chapter of this work deals with suprageneric issues, proposing a new system for the

order Agelasida. We suggest a synapomorphy between Agelas, a fleshy sponge, and Astrosclera,

a “living fossil” having a basal calcareous exoskeleton in addition to the regular siliceous spicules

endoskeleton. Even if that closeness is proven valid, as molecular phylogenetic analysis and

4

spicule morphology point out, other issues remain obscure, such as the presence or absence of a

calcareous skeleton in the last common ancestor of Agelasidae.

The 3rd chapter unravels the classical morphological taxonomy of the recent Caribbean species

on the basis of extensive sampling and analysis of museum material, clarifying some

controversial species definitions and defining a number of species groups that share certain

morphological similarities such as spicule architecture, skin color and spongin fiber disposition.

According to our results, the genus Agelas in the Caribbean has at least thirteen valid species, viz.

Agelas sceptrum (Lamarck, 1815), A. dispar Duchassaing & Michelotti, 1864, A. dilatata

Duchassaing & Michelotti, 1864, A. clathrodes (Schmidt, 1870), A. cervicornis (Schmidt, 1870),

A. conifera (Schmidt, 1870), A. schmidti Wilson, 1902, A. tubulata Lehnert & van Soest, 1996, A.

wiedenmayeri Alcolado, 1984, A. citrina Gotera & Alcolado, 1987, A. sventres Lehnert & van

Soest, 1996, A. repens Lehnert & van Soest, 1998, and A. cerebrum Assmann, van Soest & Köck,

2001.

In the 4th chapter, through molecular phylogenetic analyses of ITS and CO1, the hypothesis of a

monophyletic origin for the Caribbean-Mediterranean Agelas was rejected, as all the Caribbean

groups turned out to have sister representatives in other distributional areas apart from the

Mediterranean Sea. The reconstructed phylogeny also shows that each major speciation event has

left representatives in all the recent distributional areas of Agelas (Tropical Atlantic, Temperate

Northern Atlantic, Western and Central Indo-Pacific Realms).

From the results above, it was possible to infer the probable place and approximate date for the

presence of Agelas and a subsequent speciation and distribution history. Indeed, major geological

events in Earth history (drawn from the literature and available state-of-the-art paleomaps) and a

few arbitrary but logic assumptions, suggest a scenario for the main phenomena behind the

speciation process of Agelas that involves supercontinent aggregations and segregations, ocean

closures, seaway openings and continental drift. These results constitute a framework from which

crucial events in the origin and diversification of reef faunas can be drawn and contrasted.

5

Chapter 2. PHYLOGENETIC RELATIONSHIPS OF FOUR GENERA OF THE ORDER

AGELASIDA (PORIFERA: DEMOSPONGIAE)

FERNANDO J. PARRA-VELANDIA 1, 2, DIRK ERPENBECK2,3, SVEN ZEA1 AND ROB

W. M. van SOEST2 1 Universidad Nacional de Colombia, Departamento de Biología (Sede Bogotá) and Centro de Estudios en Ciencias

del Mar –CECIMAR (Sede Caribe); INVEMAR, Cerro Punta de Betín, A.A. 10-16, Santa Marta, Colombia. 2Institute for Biodiversity and Ecosystem Dynamics (IBED) and Zoological Museum, University of Amsterdam,

P.O. Box 94766 1090 GT Amsterdam, The Netherlands. 3 Department of Earth- and Environmental Sciences, Ludwig - Maximilians University Munich & GeoBio-

CenterLMU, Richard-Wagner-Str. 10, 80333 Munich, Germany.

Keywords: Sponges, Agelas, Astroscleridae, Astrosclera, Ceratoporellidae, Ceratoporella

Stromatospongia

ABSTRACT

The order Agelasida is a group of tropical to sub-tropical rocky and coral reef demosponges

distributed throughout the Tropical Atlantic, Temperate Northern Atlantic, Western and Central

Indo-Pacific Realms. This order comprises two recent families, one with calcareous basal

skeleton with entrapped spicules and cryptic habit and the other one with an exposed fleshy body

supported by spongin fibres and spicules. The fossil record suggests that this order has been

present in the tropics since the Ordovician. We will test the currently suggested monophyly of

calcareous Agelasida using a genus-level molecular phylogeny. We carried out a phylogenetic

analysis using nuclear (ITS1, 5.8S, ITS2) and mitochondrial (CO1) sequences including both

calcareous (Stromatospongia vermicola, Ceratoporella nicholsoni) and fleshy (Agelas tubulata

and Agelas citrina) specimens, collected in Jamaica, Barbados and the Bahamas, together with

data of species from secondary sources (Astrosclera willeyana, Axinella corrugata and

Prosuberites laughlini, the latter two for the purpose of outgroup comparison). Our results

contrast with the current phylogeny of Agelasida as A. willeyana appeared as more closely related

to the family Agelasidae than other Astroscleridae. Although this phylogeny could be supported

by a subtle acanthostyle character, it does not clarify the ancestral skeletal microstructure and

suggests a paraphyletic condition for this character. Thus, the current non-calcareous deposition

in Agelas could be reached by, at least, four ways, which are discussed.

6

INTRODUCTION

Agelasida Hartman, 1980, is an order of demosponges defined by the presence of monactine

style-type megascleres with spines in rows or verticils (van Soest and Hooper, 2002). Two

families are currently recognized within this order, which have a quite different structure (Hooper

& van Soest, 2002): a) Astroscleridae Lister, 1900 with an aragonitic basal exo-skeleton covered

by an encrusting layer of living tissue. b) Agelasidae Verrill, 1907 with fleshy body supported by

a reticulate endo-skeleton of spongin fibers and spicules. The taxonomic position of Agelasidae

has been quite variable through time, being assigned to three different orders, vis. Poecilosclerida

(as in Wiedenmayer, 1977), "Axinellida" (from its similarities to species of the family

Axinellidae, see Bergquist, 1978), and its own order Agelasida (as in Hartman, 1980). Despite

Astroscleridae calcareous massive basal skeleton, recent multiple and independent

morphological, biochemical and molecular evidence supported a closer relationship between

Agelasidae and Astroscleridae than between Agelasidae and Axinellidae (Williams and Faulkner,

1996; Chombard et al., 1997; Alvarez et al., 2000; Borchiellini, 2004; Nichols, 2005). This ended

the somewhat contorted history of the taxonomy of the two currently recognized families of order

Agelasida (Reitner, 1992; Wörheide, 1998, van Soest and Hooper, 2002). In the framework of a

close relationship recognized between Agelasidae, Astroscleridae and Axinellidae (in part:

Hooper and van Soest, 2002), an early Paleozoic basal splitting suggested for Agelasida (Reitner,

1992; Mostler, 1994) has support from 18S rDNA (Holmes and Blanch, 2008) and CO1

phylogenies (Erpenbeck et al, 2008). In this introduction, we draw a space and time limits for the

later split-off of Agelasidae-Astroscleridae.

The Agelasida families

The recent representatives of family Agelasidae belong to the monotypic genus Agelas

Duchassaing and Michelloti, 1864, with 28 extant tropical and subtropical species: 1 from the

Temperate Northern Atlantic, 13 from the Tropical Atlantic (Parra et al. Chapter 3), and at least

14 from the Western and Central Indo-Pacific (de Voogd et al, 2008). Agelas acanthostyles are

between 60 to 400 µm in length; the body skeletal structure is made up of primary fibers cored

and echinated by acanthostyles, secondary fibers normally echinated and tertiaries far less

echinated, so far with no evidence of a basal calcareous skeleton (Parra et al. Chapter 3).

7

The recent members of family Astroscleridae include five tropical genera, all with an aragonite

basal skeleton. They are Astrosclera Lister, 1900, Ceratoporella Hickson, 1911, Goreauiella

Hartman, 1969, Hispidopetra Hartman, 1969 and Stromatospongia Hartman, 1969 (Vacelet,

2002).

The genus Astrosclera has one extant Indo-Pacific species occurring from the Suez channel to the

Great Barrier Reef (GBR) of Australia (Wörheide, 1998). Its body consists of a thin layer of

tissue covering a calcareous skeleton divided in a cavernous external cortex (5-7 mm thick) and a

solid inner core. The calcareous skeleton is spherulitic in microstructure (sclerodermites size 10–

60 µm), stromatoporoid-like in building-grade and aragonitic in its mineralogy; this skeleton set

will be called herein astrosclerid-like. Its acanthostyles, ranging in dimension from 42 to 150 µm

long by 1.7 to 11 µm wide, protrude from the calcareous matrix, sometimes even supporting the

dermis; there is no evidence of spicule tracts or fibre coring (Wörheide, 1998).

The genus Ceratoporella Hickson, 1911 has one extant species from the Caribbean Sea, C.

nicholsoni. Ceratoporella has long acanthostyles from 200 to 300 µm, arranged intramural and

subparallel to the microstructure of calcareous columns; the body consists of a thin layer of tissue

(3-4 mm) over a basal skeleton chaetetid-like in building-grade, aragonitic in its mineralogy and a

fascicular fibrous microstructure (also know as clinogonal, elongate spherulitic, water-jet or

penicillated) to be called herein ceratoporellid-like (Hartman and Goreau, 1970; Gray, 1980;

Finks and Rigby, 2004).

The genus Stromatospongia Hartman, 1969 has two extant species from the Caribbean Sea and

one from the tropical Western Pacific. Stromatospongia forms a thin layer of tissue over a

ceratoporellid-like skeleton; the length of its acanthostyles ranges from 75 to 500 µm in S.

vermicola and 195 to 215 µm in S. norae, and they are arranged intramural and subparallel to the

microstructure skeletal fibres (Hartman and Goreau, 1970; Gray, 1980). So far there is no fossil

record.

The last two less known Astroscleridae extant genera, with no fossil record and each with one

species from the Caribbean Sea are: Goreauiella Hartman, 1969 (G. auriculata) with

acanthostrongyles 35-124 µm arranged intramural and subparallel to the microstructure fibres

and a tissue veneer over a thin ceratoporellid-like skeleton (Hartman and Goreau, 1970; Gray,

8

1980); Hispidopetra Hartman, 1969, with H. miniana which has smooth styles 270 – 300 µm

long, arranged intramural and subparallel to the microstructure fibres and a tissue veneer over a

thin ceratoporellid-like skeleton (Hartman and Goreau, 1970; Gray, 1980).

Agelasida fossil record

Although the chances to fossilize for both families are quite different due to their different

skeletal compositions and materials, a general overview of their fossil record allows pointing out

some boundaries in their evolutionary history. So far 25 families and 144 genera of

hypercalcified fossil Demospongiae with aragonite basal astrosclerid- or ceratoporellid-like

(Astroscleridae) skeletons have been described (Finks and Rigby, 2004; Alroy, 2008). From the

Ordovician (490 Ma) to the Paleogene (65 Ma), the Astroscleridae fossil record follows the

Paleozoic marine biodiversity trends, with richness peaks during the Ordovician and Permo-

Triassic boundary (Droser and Finnegan, 2003; Finks and Rigby, 2004; Sala and Knowlton,

2006; Alroy, 2008).

A

B

C

Figure 1. Putative Agelasid spicules. A) “discorhabd” from the Lower Ordovician of Nevada 360 x 33 µm (Kozur et al, 1996). B) Ropalospongia fluegeli acanthostyle from the Permian of Texas 470 x 47 µm (Mostler, 1994). C) Fluegelispongites trettoensis acanthostyle from the Middle Triassic of Italy 213 x 13 µm (Mostler, 1994). See descriptions appendix A.

The Astrosclera fossil record begins in the Late Triassic (210 Ma) of South Europe and Turkey

and a related fossil genus is Cooperaria (type species C. getawayensis Finks, 1995) from the

Middle Permian of Texas (270 Ma ; Alroy, 2008). The fossil A. cuifi Wörheide, 1998, had scarce

9

spicules, sometimes, partially incorporated into the basal skeleton, with lengths of about 100 µm

and thickness of 10-15 µm. Its spherulite size ranges from 60 µm to 150 µm (Wörheide, 1998;

Vacelet, 2002). Ceratoporella fossil record begins in the Middle Permian (264 Ma) of Tunisia

and the Late Triassic (222 Ma) of Italy. The fossil genus related to Ceratoporella is

Preceratoporella (type species P. tunisiana Termier and Termier, 1977) from the Middle

Permian of Tunisia (265 Ma; Alroy, 2008).

Spicules with arguable Agelasidae architecture have been found in the USA Lower Ordovician

(Nevada, Laurentian paleo-continent western coast; 490-452 m.y.; Kozur et al, 1996: 207, 219, pl

4, fig 10; Kiessling, 2002: Pag 193, fig. 3; see Figs. 1A, 2 and Appendix A herein), Ireland

Lower Carboniferous (366-356 m.y.; Mostler, 1994), USA Permian (Texas; 270 Ma;

Ropalospongia sp Mostler, 1994 Fig 1B) and Italy and Austria Middle and Upper Triassic (204

Ma; Fluegelispongites sp Mostler, 1994).

Figure 2. Land masses paleopositions during the Ordovician. The black star marks the location of Kozur et al (1996) material from what is now Nevada, USA. South Pole azimuthal projection. From Mac Niocaill et al (1997). Note that there are other areas equally suitable for reef development around Laurentia and Siberia Plates, the implicit paleoceanography suggests biogeographically testable links

The paleoenvironments, where those spicules where deposited and Agelasidae current ecology

suggest that since the Ordovician, the family's evolutionary scenario has been restricted to

tropical and subtropical coastal and reefal environments (Reitner, 1992; Mostler, 1994; Kozur et

al, 1996; Hooper and van Soest, 2002; Finks et al, 2004). Predictions from the state-of-the-art

paleomaps and phanerozoic circulation models (Kiesgling, 2002) and Plate tectonics, does not

reject that such an origin (Laurentian Ordovician) could lead to the actual distribution (Tropical

Atlantic, Temperate Northern Atlantic, Western and Central Indo-Pacific).

10

Although the older spicules should be discussed (Appendix A), up to this point it can be

concluded that: a) by the Lower Silurian (440 Ma) both aragonitic and fleshy groups of Agelasida

probably were already established: b) by the Middle Permian (270 Ma) the Agelas, Ceratoporella

and Astrosclera ancestors/relatives already existed (Reitner, 1992; Mostler, 1994; Wörheide,

1998; Finks and Rigby, 2004; Alroy, 2008).

Origin of the calcareous skeleton

It has been suggested that an increase in the dissolved calcium concentration in the seawater

triggered the appearance of calcareous biodeposition as a Ca2+ detoxification strategy with

concomitant appearance of fossilizable remains and skeleton fossils in many Poriferan groups

(Simkiss, 1977).

Likewise it is believed that variations in both oxygen atmospheric content and dissolved calcium

concentration during the Cambrian and the Ordovician have played a facilitating role by making

the calcium-deposited skeleton physiologically available (Vermeij, 1989; Stanley, 2006). The

selective role of detoxification has been challenged, and instead a major role of vagile predators

linked to the appearance of hard defensive structures in the Cambrian has been proposed

(Butterfield, 2007). However, both hypotheses could be true in a scenario where the evolutionary

trend of calcareous deposition involved first of all a detoxification strategy, then it became a

structural element, and finally it was used as a defensive strategy (Wood, 1990; Cook, 2002).

The fossil record of the calcareous demosponges perhaps supports part of this hypothesis as the

‘older’ Cambrian calcareous skeletons are simplest, slender, delicate deposition forms, such as

archaeocyaths and sphinctozoans, whereas the ‘younger’ Ordovician forms are solid and hard

forms, with stromatoporid-like and chaetetid-like skeletons (Wood, 1990; Cook, 2002; Wörheide,

2002).

Systematic classification of recent Agelasida

After Hooper & van Soest (2002), the systematic classification of the extant members of this

group is as follows:

Order Agelasida Hartman, 1980

11

Family Agelasidae Verril, 1907

Genus Agelas Duchassaing and Michelloti, 1864

28 Agelas spp. Western Atlantic, Mediterranean Sea, Indian and Pacific

Family Astroscleridae

Genus Astrosclera

A. willeyana Lister, 1900 Indian and Pacific

Genus Ceratoporella

C. nicholsoni Hickson, 1911 Western Atlantic

Genus Hispidopetra

H. miniana Hartman, 1969 Western Atlantic

Genus Stromatospongia

S. vermicola Hartman, 1969 Western Atlantic

S. norae Hartman, 1969 Western Atlantic

S. micronesica Hartman and Goreau, 1976 Pacific

Genus Goreauiella

G. auriculata Hartman, 1969 Western Atlantic

The present classification establishes that the synapomorphy of Astroscleridae is the possession

of aragonite mineralogy; leaving spherulitic, stromatoporoid-like skeletal morphology for

Astrosclera and ceratoporellid-like skeletal morphology for Ceratoporella, Stromatospongia,

Hispidopetra and Goreauiella as autapomorphies (van Soest, 2002), although the fascicular

fibrous microstructure character could be a derivation of a former spherulitic mode (Finks and

Rigby, 2004). In this work, we will test the monophyly of calcareous Agelasida using a genus-

level molecular phylogeny and contrast it with the available fossil record information.

METHODS

Sampling

Complete length ITS1-5.8S-ITS2, and partial CO1 mtDNA sequences were amplified from

Stromatospongia vermicola, Ceratoporella nicholsoni, Agelas tubulata and Agelas citrina

collected by F. Parra-Velandia and S. Zea in 2002 in Jamaica, Barbados and the Bahamas (Table

1). Astrosclera willeyana was included in the analysis thanks to its ITS1-5.8S-ITS2, and partial

CO1 mtDNA fragments were made available through Genbank. As outgroup, Axinella corrugata

12

(order Halichondrida) and Prosuberites laughlini (order Hadromerida) were used, also using

Genbank sequences (Table 1). Specimens of Goreauiella and Hispidopetra were not included as

DNA fragments from them were not available.

To avoid contamination from external sources after collection, as soon as possible deep portions

(2-3 cm3) were cut from the choanosome and then, if necessary, carefully cleaned from any non-

sponge tissue present (e.g. worms, arthropods); then the clean tissue was cut into small pieces.

The samples were preserved either in Silica Gel (as described in Alvarez et al., 2000), and then

stored at 4 ˚C, or in 96% ethanol and kept at room temperature until time of extraction. Voucher

specimens have been deposited in the Museo de Historia Natural Marina de Colombia

(MHNMC) at INVEMAR (Santa Marta) and the Museo de Historia Natural –Instituto de

Ciencias Naturales (MHN-ICN) at the Universidad Nacional de Colombia (Bogotá). Fragments

of the specimens have been deposited at the Zoological Museum of the University of Amsterdam

(ZMA).

DNA extraction and amplification

Samples were extracted using the DNEasy Tissue Kit® following the manufacturer instructions.

Some DNA samples were extracted following the Marmur (1969) technique. A DNA nuclear

region, reaching about 680 bp, that contains the ribosomal ITS regions 1 and 2 and the 5.8S

rDNA region, was amplified with universal primers ITS1 (TCC GTA GGT GAA CCT GCG) and

ITS4 (TCC TCC GCT TAT TGA TAT GC) designed by White et al. (1990). The mitochondrial

Cytochrome c oxidase subunit I (CO1) region, reaching about 690 bp, was amplified with the

universal primers LCO1490 (GGT CAA CAA ATC ATA AAG ATA TTG G) and HCO2198

(TAA ACT TCA GGG TGA CCA AAA AAT CA) designed by Folmer et al. (1994).

We performed our PCR amplifications with 1.5 µl of genomic DNA in a volume of 51.4 µl, that

contained 30 µl H20 (Merck), 5 µl of buffer, 8 µl dNTP (10mM), 3.2 µl BSA (0.1mg/ml), 3.2 µl

MgCl (25 mM), 0.4 µl each primer (10 µM) and 0.2 µl Promega Taq polymerase. Some PCR

reactions were made in total volume of 25.7 µl with the same proportions. After an initial

denaturation step at 95 ˚C for 3 min, the DNA region of interest was amplified during 36 cycles

of 94 ˚C for 30 s, 52 ˚C for 45 s, and 72 ˚C for 60 s; finally, the mixture was incubated at 72 ˚C

for 10 min; PCR reactions for both ITS and CO1 were made under the same temperature profile

13

and mixture proportions. Due to lower yield of DNA, in some cases better results were obtained

with four to five volumes of primer.

Table 1. Specimen locality, museum accession and GenBank accession numbers. ITS1-5.8S-ITS2 rDNA and Cytochrome Oxidase I cox1 sequences. INV-POR = Invemar – Porifera; ZMA = Zoölogisch Museum Amsterdam; ICN-MHN-PO = Instituto de Ciencias Naturales, Museo de Historia Natural, Porífera; MNHN = Muséum national d’Historie naturelle.

Species name Locality ITS1-5.8S-

ITS2 cox1 Museum accession Field number

Agelas tubulata holotype Jamaica DQ075810 DQ075697 ZMA Por 11323 ZMA Por 11323 Agelas citrina Barbados DQ075817 DQ075704 INV-POR 916 BB303 Ceratoporella nicholsoni Bahamas DQ075884 DQ075775 INV-POR 829 PBH72 Ceratoporella nicholsoni Jamaica DQ075857 DQ075747 ICN-MHN-PO-0214 JMA201 Stromatospongia vermicola Jamaica DQ075864 DQ075754 ICN-MHN-PO-0216 JMA504 Astrosclera willeyana GenBank AF338348 AY561969 Axinella corrugata GenBank AF300463 AY791693 Prosuberites laughlini GenBank AJ633936 AY561960

Sequencing

The resulting PCR products were purified in Agarose TAE 2% gels using a silica-based method

(Boyle and Lew, 1995). For several PCR products, the QIAquick PCR Purification Kit® was

used. All clean PCR products were sequenced in both directions using the above primers and the

ABI® Big Dye 3.1 cycle sequencing kit following this protocol: 1.5 µl clean PCR product (about

10 ng/µl), 0.5 µl ready reaction mix, 1.75 µl buffer 5x, 1 µl primer (1 µM) and 5.25 µl water, for a

total volume of 10 µl. After the sequencing cycle, 10 µl of water were added to each microtube.

The cycle-sequence products were analyzed using an ABI® 3770 automated DNA sequencer

(Applied Biosystems).

Data analysis

The sequences were assembled using BioEdit Software, automatically aligned using the ClustalW

option; they were then inspected and optimized by eye using MacClade 4.0. The regions with

uncertain alignment and insertion/deletion positions were excluded for the ITS region data

matrix; this conservative data matrix was used to recover the phylogeny. Likelihood settings from

best-fit model (HKY+G) were selected by hLRT in Modeltest 3.7 (Posada and Crandall, 1998).

Maximum likelihood (ML), Maximum Parsimony (MP), Minimum Evolution (ME) were

performed using PAUP* 4.0b10 (Swofford, 1999). The transition/transversion (ts/tv) ratios were

estimated from the data matrix using Paup 4.0. Bootstrap analyses were performed with 1,000

14

replicates whenever possible; MrBayes settings for the best-fit model (GTR+G) were selected by

hLRT in MrModeltest 2.2 (Nylander, 2004). Bayesian Analyses (BA, 10 million generations, 2

runs, 4 chains, default temperature) were carried out with MrBayes 3 (Ronquist and Huelsenbeck

2003), starting with random initial trees, sampling every 10.000 generations and constructing 50

% majority-rule consensus of the trees retained after burning 25% of initial trees (Huelsenbeck

and Ronquist, 2001). To analyze the consistency of the recovered phylogeny, it was compared

with the current morphology-based systematics.

RESULTS

Coming from 6 ingroup and 2 outgroup taxa (Table 1) were analyzed: for COI matrix, 442

positions were constant, 68 positions were variable parsimony-uninformative and 129 positions

were parsimony-informative. For ITS matrix, 480 positions were constant, 54 positions were

variable parsimony-uninformative and 111 positions were parsimony-informative. For the

combined matrix a total of 1284 positions (639 bp of COI, 645 bp of ITS), 915 positions were

constant, 130 variable positions were parsimony-uninformative and 239 positions were

parsimony-informative. The mitochondrial and nuclear sequences are deposited in Genbank [see

table 1 for Accession numbers, Sequences are published upon manuscript acceptance].

A. corrugata was selected as outgroup in MrBayes; P. laughlini and also A. corrugata were

selected as outgroup in PAUP. The phylogenetic reconstructions from separated (COI and ITS)

and combined gene matrices consistently recovered the same tree structure (Fig. 3) irrespective

outgroup. In the three reconstructions, Clade I comprises Agelas tubulata and A. citrina with

Astrosclera (represented by A. willeyana) as their sister genus; whereas Clade II that comprises

the genera Ceratoporella and Stromatospongia, was recovered only in the COI and combined

matrices reconstructions.

15

Figure 3. Phylogenetic reconstructions of four genera of Agelasida. Combined data strict-consensus tree is shown with Bayesian posterior probabilities and Maximum Likelihood, Maximum Parsimony, Minimum Evolution bootstrap values. Axinella corrugata, Prosuberites laughlini, Stromatospongia vermicola, Ceratoporella nicholsoni, Astrosclera willeyana, Agelas tubulata and Agelas citrina. Note the paraphyletic position of A. willeyana respect to C. nicholsoni and S. vermicola.

The current classification thus contrasts with the recovered phylogeny as A. willeyana is more

closely related to Agelasidae species studied than to the other Astroscleridae, with good bootstrap

and Bayesian support (>98 %), making this family paraphyletic.

DISCUSSION

The current Agelasida systematics implies that aragonite deposition is an Astroscleridae

synapomorphy and the microstructure and skeleton type are autapomorphies for Astrosclera and

Ceratoporella–Stromatospongia (and probably other Ceratoporellid-like genera). However, this

proposed phylogeny suggests that aragonite deposition is a paraphyletic feature present in two

different microstructures and construction-grades; but it did not clarify if the last common

ancestor of Agelasida had an aragonite basal skeleton, or its microstructure nor construction-

grade as the phylogenetic sequence of skeletal arrangements (deposition vs. non deposition;

16

astrosclerid-like vs. ceratoporellid-like; Table 2) cannot be clearly traced from this phylogeny

alone.

Table 2. Characters comparison between Hadromerida, Halichondrida and Agelasida. Shaded cells links equal character pattern. Hadromerida,

Suberitiidae Prosuberites

laughlini (Díaz, Álvarez, & van Soest, 1987)

Halichondrida, Axinellidae, Axinella

corrugata (George & Wilson, 1919)

Agelasida, Agelasidae, Agelas citrina

Gotera & Alcolado, 1987, and A. tubulata Lehnert and van Soest, 1996

Agelasida, Astroscleridae, Astrosclera

willeyana Lister, 1900

Agelasida, Astroscleridae, Ceratoporella

nicholsoni (Hickson, 1911)

Agelasida, Astroscleridae, Stromatospongia

vermicola Hartman, 1969

Basal skeleton Mineralogy Mg Calcite Aragonite

Absent Absent Absent Aragonite Aragonite Aragonite

Basal skeleton Microstructure Lamellar Penicillated Spherulitic Absent

Absent Absent Absent Spherulitic Penicillated Penicillated

Basal skeleton morphology Absent Absent Absent Astrosclerid-like Ceratoporellid-like

Ceratoporellid -like

Biomineralization IC = intracellular EC = extracellular

? ? ? IC-EC EC-EC EC-EC

Reproductive mode Oviparity and Viviparity

Oviparity Oviparity Viviparity, parenchymella larvae

?viviparity ?oviparity

Spicule shape oxeas subtylostyles tylostyles derivatives

oxeas anisoxeas styles sinuous strongyles

Acanthostyle long

Acanthostyle short

Acanthostyle long

Acanthostyle long

Spicule/spongin head embedded spicule tracts head embedded spicule tracts

head embedded

head embedded Intramural (?spicule tracts)

head embedded Intramural (?spicule tracts)

The recovered phylogeny is congruent with the possession by Stromatospongia and

Ceratoporella of a chaetetid-like basal skeletal framework and a penicillate microstructure. If

aragonite deposition is a paraphyletic feature then construction-grade and microstructure in turn

become the synapomorphies of Astroscleridae as spherulitic stromatoporoid-like and

Ceratoporellidae (Hartman and Goreau, 1972) as penicillated chaetetid-like. It would confirm the

suggestion of Vacelet (2002) about the validity of the Ceratoporellidae family including the

genera Ceratoporella, Stromatospongia, Hispidopetra and Goreauiella, leaving the family

Astroscleridae only with the genus Astrosclera.

From the spicule perspective, a character which seems to support the recovered phylogeny is the

orientation of the terminal spicule spines. In Agelas and Astrosclera spicules, terminal spines at

both ends in general are oriented in the direction of the head, while in Ceratoporella and

17

Stromatospongia, head (proximal) spines are oriented downward (towards the point of the

spicule) and apical end spines are directed upward.

This DNA-hypothesis implies that the current non-deposition in Agelas could be reached by at

least four hypothetical ways:

1. The last common ancestor (LCA) of Agelasida had aragonite deposition, it was the

plesiomorphic condition, and then aragonite deposition was lost in the ancestor of Agelas.

a) Here we cannot distinguish which microstructure was plesiomorphic and which was

derived

If this case is true, present day Agelas should have hypothetical vestigial genomic

machinery for calcareous deposition, similar to that in Astrosclera and

Ceratoporella–Stromatospongia.

2. The LCA of Agelasida lacked aragonite deposition; non-deposition was the plesiomorphic

condition.

a) It evolved independently in the Ceratoporella–Stromatospongia and Astrosclera–

Agelas ancestors and then aragonite deposition was lost in Agelas.

If this is true, the aragonite deposition mechanism must be different and unrelated

in both Astrosclera and Ceratoporella–Stromatospongia as suggested by Wood

(1990) and the hypothetical vestigial Agelas genomic machinery for calcareous

deposition must be similar to that of Astrosclera.

b) It evolved independently in Ceratoporella–Stromatospongia ancestor and in

Astrosclera–ancestor after the splitting up of Agelas.

In this case, Agelas would have no evidence of any hypothetical vestigial genomic

machinery for calcareous deposition. This implies that the aragonite-deposition

character evolved twice in Agelasida with two different microstructures.

c) It evolved independently in the ancestors of Ceratoporella–Stromatospongia,

Astrosclera and Agelas, and then it was lost in the ancestor of Agelas.

In this case, Agelas would have evidence of hypothetical vestigial genomic

machinery for calcareous deposition different from that of Astrosclera,

Ceratoporella and Stromatospongia. This implies that the aragonite-deposition

character evolved three times with two known different microstructures (in the

18

ancestor of Ceratoporella–Stromatospongia and in the ancestor of Astrosclera)

and one unknown microstructure in Agelasidae.

Its is clear that hypotheses 2.a and 2.c are the less parsimonious paths; however taking into

account the amount of time involved and the growing knowledge body about the early Paleozoic,

is advisable do not preclude yet their occurrence. Additional evidence will be necessary to

understand the phylogenetic value of the echinating and coring spicules of Agelas against the

partially entrapped spicules into the basal skeleton of Astrosclera willeyana, which may have a

tissue supporting function as they penetrate the basopinacoderm (Wörheide, 1998). For the time

being, we refrain from suggesting formal taxonomic changes in the contents of the families until

further evidence is gathered to test the above hypotheses.

ACKNOWLEDGEMENTS

We would like to give a deep acknowledgement to the Parra-Velandia family, Hurtado-Cuellar

family, P. Rachello-Dolmen, K. Rützler, P. Alcolado, J. Pawlik, E. Hajdu, M. Pansini, J. Hooper,

O. Tendal, R. Bak, C. Valentine, A. Debrot, G. Wörheide, I. Domart-Coulon, A. Baker Johnston,

M. Nestlerode, M. Carpenter, C. Piantoni, M. López-Victoria, L. Barrios, , E. Bellinger, S. Lange

and S. Marijnissen. This work was funded by grants from the Colombian Science Fund-

COLCIENCIAS (earlier collections and observations during grants 30003-1-33-81, 30003-154-

83, 2105-09-023-93, 2105-09-120-97; Agelas systematics and biogeography: 1101-09-11241),

from the Netherlands Foundation for the Advancement of Tropical Research WOTRO (WB 82-

261), and matching funds from Universidad Nacional de Colombia-División de Investigaciones

Sede Bogotá (DIB) and Universiteit van Amsterdam-IBED. Sampling at Belize was partly

funded by Smithsonian Institution’s Caribbean Coral Reef Ecosystem Program – CCRE. The use

of the R/V Seward Johnson during sampling in the Bahamas was made possible through financial

support from the U.S. National Science Foundation (several grants to J. Pawlik). Extraordinary,

generous and helpful logistic support was provided by R. Vieira’s CEINER Oceanarium (Rosario

Islands), Smithsonian Institution’s Carrie Bow Cay Field Station (Belize), Discovery Bay Marine

Laboratory (Jamaica), Bellairs Research Institute from McGill University (Barbados) and

Caribbean Research and Management of Biodiversity – CARMABI (Curaçao). This is

contribution ___ of Centro de Estudios en Ciencias del Mar – CECIMAR and the graduate

program in marine biology, Universidad Nacional de Colombia, 895 of Smithsonian Institution’s

CCRE program and ___ of Instituto de Investigaciones Marinas y Costeras — INVEMAR.

19

Chapter 3. REEF SPONGES OF THE GENUS Agelas (PORIFERA: DEMOSPONGIAE)

FROM THE CARIBBEAN SEA

FERNANDO J. PARRA-VELANDIA1, 2, SVEN ZEA1 AND ROB W. M. VAN SOEST2

1Universidad Nacional de Colombia, Departamento de Biología (Sede Bogotá) and Centro de Estudios en Ciencias del Mar –CECIMAR (Sede Caribe); INVEMAR, Cerro Punta de Betín, A.A. 10-16, Santa Marta, Colombia.

2Institute for Biodiversity and Ecosystem Dynamics (IBED) and Zoological Museum, University of Amsterdam, P.O. Box 94766 1090 GT Amsterdam, The Netherlands

Keywords: Sponges, Agelas, Coral reefs, Caribbean

ABSTRACT

The genus Agelas comprises a group of tropical and subtropical reef sponges that contains large,

long-lived, often brightly colored and conspicuous species, distributed throughout the Tropical

Atlantic, Temperate Northern Atlantic, Western and Central Indo-Pacific Realms. Among

tropical sponge genera, Agelas is one of those with similar species richness in the Greater

Caribbean in comparison to the Indo Pacific. The presence of verticillated acanthostyle spicules

and a fibroreticulate skeleton of spongin fibres cored and/or echinated by spicules characterize

this group. Taxonomic identification relies in a combination of characters, where external

morphology and color play a key role, owing to the paucity of microscopical characters. Thus,

there is still a great deal of taxonomic confusion, even for the more common species. We carried

out a detailed revison of Agelas species throughout the Caribbean Sea using classic taxonomic

tools. Samples and observation covered Colombia, Belize, Jamaica, The Bahamas, Barbados,

Curaçao and Venezuela, and type material from major museum collections. According to our

results, the genus Agelas in the Caribbean has at least thirteen valid species, viz. Agelas sceptrum

(Lamarck, 1815); A. dispar Duchassaing & Michelotti, 1864; A. dilatata Duchassaing &

Michelotti, 1864; A. clathrodes (Schmidt, 1870); A. cervicornis (Schmidt, 1870); A. conifera

(Schmidt, 1870); A. schmidti Wilson, 1902; A. tubulata Lehnert & van Soest, 1996; A.

wiedenmayeri Alcolado, 1984; A. citrina Gotera & Alcolado, 1987; A. sventres Lehnert & van

Soest, 1996; A. repens Lehnert & van Soest, 1998; and A. cerebrum Assmann et al., 2001. We

found that variation of microscopic characteristics like skeleton arrangement, number of verticills

and their spines, spicule length and width, can be used as taxonomic tools, but with a thorough

comparison with other species in the same sub-regional context. Thus, a certain degree of

familiarity with the genus regional variation is often required. The richness and distribution of

20

these species in the Caribbean area show north/south differences and other ecological patterns are

evident.

INTRODUCTION

The genus Agelas Duchassaing and Michelotti (1864) comprises a group of tropical and

subtropical reef sponges that contains long-lived species distributed throughout the

Mediterranean Sea, the Indo-Pacific Ocean, and the Caribbean Sea. This group is defined by the

presence of an almost unique type of regularly verticillated acanthose style-like megasclere

(acanthostyle; see Fig. 4A, 4B). In addition, verticillated acanthoxeas are present, but they are not

abundant. Additional features include having a fibroreticulate spongin skeleton of primary

ascending fibres invariably cored and echinated by spicules, and interconnecting secondary fibres

profusely echinated by spicules and rarely cored (see Figs. 4C, 4D, 4E, 4F and 4G). These

sponges typically have a thin skin, supported by tracts of spicules protruding from the

perpendicular ends of ascending main fibres. Excepting their particular spicule architecture and

the collagen bundles, which build the fibres, transversely arranged (van Soest, 2002), several

morphological features of this genus resemble other Poriferan groups which include sponges of

diverse growth forms, e.g., thickly encrusting, massive, globular, branching, fan shaped and

tubular. From the information stored in the Zoological Museum of Amsterdam (ZMA) Database

(Data downloaded on January-2005) recent species of Agelas are found at depths from 2 m to

deeper than 1000 m, being common between 20 - 125 m (more than 60% of the specimens are in

that range).

Described formally by Duchassaing & Michelotti in 1864, species belonging to Agelas have been

classified under genera Ectyon Gray, 1867; Chalinopsis Schmidt, 1870; Siphonochalinopsis

Schmidt, 1880 and Pachychalinopsis Schmidt, 1880 all of which have been synonimized to

Agelas (see Wiedenmayer, 1977; van Soest, 2002). The oldest references for species which

belong to Agelas are from Lamarck (1813, 1815) who used the generic denominations Spongia

and Alcyonium.

21

A

B

C

D

E

F

G

Figure 4. SEM images of Agelas. A) Spicules of A. conifera holotype. B) Spicules of A. clathrodes holotype, C) Skeleton of A. citrina, primary and secondary fibres echinated, D) Detail of skeleton of A .dispar, showing fibres echination, E) Skeleton of A. conifera, primary and secondary fibres echinated, F) Detail of skeleton of A.

conifera, coring spicules in a broken primary fibre, G) Detail of skeleton of A. citrina, long spicules.

22

Apparently, no complete or partial fossil Agelas specimen has been described. However, agelasid

–like spicules have been isolated from insoluble residues of cherty limestone at the Lower

Ordovician from USA, Lower Carboniferous from Ireland and the Permian from USA; also

similar spicules but with continuous spine spirals instead of verticills, have been recovered from

Triassic deposits in Salzburg (Austria) and Veneto (Italy) regions (Reitner, 1992; Mostler, 1994;

Kozur et al, 1996: 207, 219, pl 4, Fig 10; Finks et al, 2004; see Fig. 1). Their discovery confirms

the suggestion by Reitner (1992) of the ancient origin of the group (Mostler, 1994) and allows

establishing time and space boundaries in order to explain speciation and dispersion processes in

the genus. Agelasids have occupied tropical deep reef zones since the Lower Carboniferous

(Mostler, 1994).

They are distributed mainly in tropical reefs localities but with some subtropical incursions. The

highest species richness is found in the Indo-Pacific area with fifteen species (de Voogd et al,

2008), followed closely by the Caribbean Sea with thirteen species, and finally by the

Mediterranean Sea with one species. The tropical Brazilian coast has populations of at least eight

Caribbean species (Mothes et al, 2007); however their status as species or populations needs to

be established, as they likely have experienced a recent isolation.

About the etymology and reasoning behind the word Agelas, we can only guess, as the original

authors (Duchassaing & Michelotti, 1864) said nothing. In his work about Bahamian sponges,

Widenmayer (1977) discussed the feminine gender Agelas and established that it does not exist as

a Greek noun but only as a root; unfortunately, he did not give a meaning for that root. A recent

work of Greek names in Porifera science (Voultsiadou & Gkelis, 2005) established that άγελάς

means “belonging to a herd”. A Spanish-Greek dictionary (Pabón, 1967) gives additional

meanings for the word, although with a different spelling that means, at least in Spanish, a

masculine gender: άγελη ς: crowd, band, section or group. The Greek mythology names several

Agelao, the most relevant being the shepherd/slave commanded by Priamo, King of Troy, to kill

his son Paris due the dream-prophecy of his wife Hecuba. In his mother’s dream, Paris puts Troy

on fire. Finally, the history remembers at least two Corinth kings under that name (Heffner, 1924;

Dunbabin, 1948)

23

Order Agelasida (Hartman, 1980)

As already stated by Mostler (1994), during the late 70’s the Agelasids were assigned to three

different orders and two different subclasses. For a long time, the monogeneric family Agelasidae

(Verrill, 1907), was considered member of the order Poecilosclerida (as in Wiedenmayer, 1977).

Later on, Bergquist (1978), on the grounds of its free amino acid pattern (Bergquist and Hartman,

W. D. 1969), sterol composition, and apparent oviparity (Reiswig, 1976), changed its position to

the order Axinellida (van Soest, 2002). Despite the biochemical and reproductive evidence, this

new position was not generally accepted. Based mainly in the possession of acanthostyles,

Hartman (1980) proposed the creation of the new order Agelasida with a family Agelasidae

(Agelas).

In lieu of biochemical (Williams and Faulkner, 1996, see discussion in Wörheide, 1998: 79) and

molecular evidence (Chombard et al., 1997; Alvarez et al., 2000; Nichols, 2005), a closer

relationship between Agelasidae and Astroscleridae (Astrosclera, Ceratoporella, Goreauiella,

Hispidopetra and Stromatospongia) than between Agelasidae and Axinellidae is supported. This

point of view is accepted in the most comprehensive systematic work on Porifera (Hooper & van

Soest, 2002). Nowadays the affinity of Agelasida with Axinella and Stylissa and an agreement on

their taxonomic ranks are recognized (Erpenbeck et al., 2004; Erpenbeck et al., 2005; Erpenbeck

& Wörheide, 2007).

This closeness between Agelasidae and Astroscleridae (Lister, 1900) is surprising as this latter

family possesses acanthostyles but also a basal aragonite skeleton, a feature that misled authors

until the middle twentieth century to classify them within the Cnidaria (Wörheide, 1998). At least

two different construction microstructures under the same aragonite mineralogy are present in

this family (Vacelet, 2002; Kaesler, 2004):

Astrosclera (Lister, 1900) with stromatoporid skeletal morphology and spherulitic

construction microstructure, where the aragonite is secreted intracellularly and then

extracellularly enlarged;

Ceratoporella (Hickson, 1911) with chaetid-like skeletal morphology and penicillate

construction microstructure, where the aragonite is both secreted and enlarged

extracellularly.

24

A rich fossil record has been described for Agelasida with basal calcareous skeletons (Alroy,

2008). The most comprehensive work in invertebrate palaeontology (Kaesler, 2004) describes 25

families (some with 2 to 6 sub-families) and 144 genera of hypercalcified fossil Demospongiae

under the order Agelasida, all of them with the same microstructure of Astrosclera or

Ceratoporella, but with different morphologies and most of the times without acanthostyles.

A parallel work in progress (Chapter 2) supports a family level reorganization for the Agelasida

that follows partially the suggestion of Vacelet (2002): a separation of astrosclerids in two

families on the grounds of skeletal morphology and construction microstructure: Astroscleridae

including Astrosclera, and Ceratoporellidae (Hartman & Goreau, 1972) including at least the

genera Ceratoporella and Stromatospongia (Hartman, 1969).

The Caribbean Sea and the genus Agelas

Given its conspicuousness in Caribbean reefs, almost all major taxonomic works for this area

include the common species of Agelas (Wiedenmayer, 1977; Pulitzer-Finali, 1986; Zea, 1987;

Lehnert & van Soest ,1996; 1998; 1999). In addition, also some recent detailed works have added

new species (Alcolado, 1984; Gotera & Alcolado, 1987; van Soest & Stentoft, 1988; Lehnert &

van Soest, 1996; Assmann et al., 2001). However, there is still a great deal of taxonomic

confusion, even for the more common species, owing to:

- Reliance on live external features and on too few internal characters, with little

congruence and whose variation is not yet clear to distinguish among species.

- Existence of geographically distinct morphotypes within some species.

- Difficulty in defining species from old, fixed, sometimes unaccounted for, type

specimens.

In the Caribbean Sea, the genus Agelas would comprise between 21 (Assmann et al. 2001) to 16

species (van Soest et al., 2008), including important components of the reef biota (Zea, 1994). In

this chapter, we present a taxonomic revision the genus for the Caribbean Sea, using

morphological characters, ascribing its richness to thirteen species.

Samples and observations covered Colombia, Belize, Jamaica, Bahamas, Barbados, Curaçao and

Venezuela. Only through widespread collecting efforts and detailed morphological studies,

25

together with revision of museum material and molecular analyses (Chapter 4) it has been

possible to begin understanding the extent of local and regional variation and to have a more

clear definition of species.

METHODS

Collecting

Material of each species and morphotypes of Agelas was collected between 2001 and 2003 in San

Andres Island (Colombia, Southwestern Caribbean), Belize (Western Caribbean), Rosario Islands

and Santa Marta (Colombia, Southwestern Caribbean), Curaçao (Southern Caribbean), Barbados

(Eastern Caribbean), Jamaica (Greater Antilles), and the Bahamas (Bahamian). In each reef area,

several dives were performed at different locations and depths. From each specimen the color,

form, size, habitat and other characteristics were recorded; the color was recorded at the surface

following the Naturalist’s Color Guide (NCG; Smithe, 1975). In the description of each species,

these colors are referred by name and NCG number. Macroscopic features and other suiTable

characteristics were recorded following, as far as possible, the Thesaurus of Sponge Morphology

(Boury-Esnault & Rützler, 1997). Underwater photographs were taken by the authors with a

Nikonos V film camera and with a Nikon CoolPix 5000 digital camera on Ikelite housing. Only a

fragment was collected from each specimen, which was fixed in 96 % etOH and later stored in 70

% EtOH.

Microscopy

To obtain spicules, small pieces of sponges were boiled in 2 ml fumic nitric acid, washed in

distilled water, then boiled again in 2 ml nitric acid and finally washed twice in distilled water;

the supernatant was removed after centrifugation in a bench centrifuge. For light microscopy,

spicules were then suspended in 96 % EtOH, poured on slides with Pasteur pipettes, allowed to

dry and mounted in Canada Balsam or Entellan®. The same acidic procedure was followed for

SEM studies but the spicules were poured and dried on a 12 mm stub covered by a thin circular

glass, then sputtered with gold or gold-palladium (4:1) (Boury-Esnault & Rützler, 1997). To

obtain thick sections of tissue to study three-dimensional arrangement of skeleton and spicules,

blocks of about 5 mm per side were cut from each specimen, including the surface. The blocks

were then air dried. Sections as thin as possible were cut by hand with a razor blade, both

tangential and perpendicular to the surface, and mounted on a slide in Canada balsam or

26

Permuount, to be examined under light microscopy (Boury-Esnault & Rützler, 1997). Skeleton

SEM samples were prepared from the remains of DNA extractions carried out in a parallel study.

To eliminate detergents and other DNA extraction reagents, samples were washed and stirred

four times in 5 ml double distilled water, then washed and stirred twice in ethanol 96%, and

finally let to air dry on a SEM stub. They were then covered with gold or gold-palladium (4:1)

powder. For electron microscopy, JEOL JSM-35C or FEI QUANTA 200 microscopes were used.

Museum deposits

Voucher specimens were deposited in the Museo de Historia Natural Marina de Colombia

(MHNMC, coded INV-POR) at INVEMAR (Santa Marta, Colombia) and Instituto de Ciencias

Naturales-Museo de Historia Natural (ICN-MHN) at Universidad Nacional de Colombia in

Bogotá. Several fragments from the former specimens were also deposited in the Zoölogisch

Museum Amsterdam (ZMA) at the University of Amsterdam in The Netherlands. Secondary

material from the collections of MHNMC and ZMA was used. Specimens starting with SZ, were

collected during previous expeditions by S. Zea, are housed at MHNMC and are not yet

catalogued.

Macroscopy

Skeleton scanner samples were prepared from a small dried sponge collection kindly provided by

D. Valderrama (Old Providence, Colombia). From each dried specimen, a thin section was cut-

off with a scalpel following the main fibers direction; then the sections were washed with

commercial soap during three weeks; next, samples were washed with tap water until no foam

was detected; finally, they were let to air dry. These sections were scanned with a HP Scanjet

4070 Photosmart Scanner using the slide transparency adapter provided by the manufacturer,

placing a thick glass over the section in order to flatten it. The voucher material and sections,

none formally catalogued yet, are under the attention of S. Zea and housed at MHNMC.

SYSTEMATIC DESCRIPTIONS

For synonyms and taxonomic treatment, see Wiedenmayer (1977), Zea (1987), Hooper and van

Soest (2002), van Soest (2002) and remarks herein. For higher taxonomic positions, see

discussions in Erpenbeck (2004) and Erpenbeck et al. (2005).

27

The species distribution terminology follows Spalding et al. (2007), although when a species was

assigned to Tropical Northwestern Atlantic Province (TNAP, ‘wider Caribbean’), it does not

include the Bermuda Ecoregion, as we neither collected nor reviewed any material from there.

Unidentified specimens of the genus have also been reported (Logan et al. 1984) along with

Miocene and Eocene fossil evidence of acanthostyles known in the vicinity (Bukry, 1978;

McCartney, 1987).

Classification Phylum Porifera Grant, 1835 Class Demospongiae Sollas, 1875 Order Agelasida Hartman, 1980 Family Agelasidae Verrill, 1907

Genus Agelas Duchassaing & Michelotti, 1864

Type species Agelas dispar Duchassaing & Michelotti, 1864

Remarks

The Agelas of larger sizes were observed in Rosario Islands (Colombia), i.e., 2-3 m in diameter

fan-shaped Agelas clathrodes (Fig. 14E). The smaller from Rio Bueno mouth (Jamaica) A. repens

a 50 cm long but 0.25 cm thin branch (Fig. 12). The species belonging to this genus have several

main forms: branching (Fig. 7E, 12E), tubular (Fig. 11E), globular or ball-shaped (Figs. 5B,

17D), lobate (Fig. 11B, 11C, 14E), fan-shaped (Figs. 14D, 16H), columnar, clavate or thickly

encrusting (Fig. 14C).

The choanosome is typically cavernous although sometimes this feature is not apparent as

channels can be narrow (<0.02 cm) resulting in a dense aspect, as in A. sceptrum, A. conifera or

A. cervicornis. The ectosome is a thin skin supported by tracts of spicules protruding from the

perpendicular main fibres; when dry the skin retracts on its supporting skeleton, the ectosome

takes on an acne-like aspect, remarkable in A. conifera and not so evident in A. clathrodes. The

channels and some apertures can have a lining skin rather smooth and brilliant.

This genus possesses characteristic acanthostyles with regularly spaced verticils of thorns (Figure

4A-B). It is not unusual to find acanthoxeas and, as far we have looked, they seem not to be

restricted to any particular geographical area, but are found more frequently in oceanic islands

28

than in continental areas (Parra-Velandia pers. obs.); their phylogenetic value could be important

explaining the relation between fossil and recent species.

Table 3. Main morphological features of Caribbean Agelas. Primary fibers: st=straight, un=undulated. Color in spirit: b=brown, y=yellowish, o=orange. Tertiary fibers: r=rare, a=absent, p=present. Spicule architecture, length, width, verticils; spines number; vertical completeness: c=complete, i=incomplete; direction: sc=slightly curved, mc=medium curved, tr=straight. Skeletal arrangement: o=ortho, i=iso, a=aniso. Form, frame: m=massive, r=ramose, t=tube, b=barrel. Choanosome: c=cavernous, s=solid. Ecology, biogeography: g=Great Caribbean, c=Caribbean, r=restricted distribution; habitat: x=exposed, c=cryptic, m=mixed, body filling crevices with exposed parts; depth: s=shallow, m=medium, d=deep, v=very deep.

Spicule architecture Form Eco Agelas

Primary fibers

Color Tertiary fibers ~length ~width ~verticils s c d

Sk frame

predominant shape

thickness Choan.

b h d

dispar st b r 130 10 11 5-8 c sc o m nc ear-like thin c g x m

cervicornis st b r 100 9 10 4-5 c tr o r fc even openings

thin s r x d

wiedenmayeri st b r 140 9 12 3-9 c cu o t fc tube very thin s g x d

sceptrum st y a 140 9 >13 6-8 c sc i r fc even openings

thick s g x d

dilatata st y a 110 6 >13 4-7 c tr i m nc fan shape medium s r c d

conifera st y a 140 10 <13 5-9 c tr i r fc elevated openings

medium s g x v

tubulata st y a 110 6 >15 5-8 c tr i t fc tube medium s c x m

repens st y a 180 8 16 5-6 c tr i r fc even openings

thin s r c d

cerebrum st y r 115 7 <13 5-7 c tr i b fc barrel thick s r x s

clathrodes un o p 110 8 10 3-8 c tr a m nc fan shape thick c g x d

schmidti un o p 100 6 <9 3-6 i mc a t fc tube-like thin s g m m

citrina un o p 180 12 13 3-8 c tr a m nc fan shape, barrels

medium c c x d

sventres un o p 130 9 >9 4-7 i sc a t fc tube-like thin c c m m

A network of ascending primaries, interconnected by secondary and tertiary fibres (Fig. 1C-D),

composes the spongin skeleton. The skeleton architecture (Fig. 15) in all species reviewed here

correspond to an axinellid skeleton (Boury-Esnault & Rützler, 1997, p. 28, Fig. 159), or

variations of it, with the fibers forming a plumoreticulate (ibid, p. 34, Fig. 191) mesh, as in A.

conifera and A. spectrum (Fig. 15A-B).

Typically, the primaries ascend while radiating from the main axis of growth toward the skin; in

some species (A. conifera Fig. 11A-D, Fig 15A; A. dispar Fig. 2A-H, Fig 15E) this arrangement

is evident enough to be perceived through the naked eye, while in others this is evident only in

dried specimens (A. clathrodes Fig. 5A-F, Fig 15D; A. dilatata Fig. 6A-H). Primary fibres are

29

echinated and cored (Figure 1D-E); secondary fibers usually echinated and normally not cored

(see A. cervicornis description); tertiary fibers are echinated but never cored.

At the species level and following morphological characteristics, it is possible to arrange species

sets (see Table 2), with features such as skeleton, colour, spicules and dermis architecture,

determining three main Species Groups. Below the group level, the main differences between

species involve, in a minor degree, spicule architecture (length, verticil completeness, number of

spines), and in a greater degree, external morphology. Each Species Group was named after a

representative species and their characters are defined as follows:

- dispar-like Species Group: more or less straight cored fibres with secondary, rarely

tertiary fibres, color dark brown alive and in spirit, short spicules with few but complete

verticils, each one with few spines, and a dense dermis. This Species Group includes A.

dispar, A. wiedenmayeri and A. cervicornis.

- conifera-like Species Group: straight cored fibres, secondary fibres present, but never

tertiary fibres, color yellowish light orange alive and light brown in spirit, long spicules

with lots of spines in many verticils, and a loose dermis. This Species Group includes A.

conifera, A. tubulata, A. cerebrum, A. sceptrum, A. repens and A. dilatata.

- clathrodes-like Species Group: convoluted cored fibres, secondary and tertiary fibres

present, color bright orange alive and orange in spirit, medium size spicules, few verticils,

few spines per verticil and a dense dermis sometimes ornamented by conules. This species

group covers a Species sub-Group called schmidti-like, which exhibits a closer

resemblance to the clathrodes-like Species Group; however the main differences are a

reddish color in spirit, looser skeletal meshes and spicules with incomplete verticils. To

clathrodes-like Species Group belong A. clathrodes, A. citrina, A. schmidti and A.

sventres; the latter two belong to the schmidti-like Species sub-Group.

The species described below are listed by Species Group and then sorted by date of original

description.

Agelas dispar Duchassaing & Michelotti, 1864

Fig. 5

Etymology from Latin, meaning dissimilar, different. Agelas dispar Duchassaing & Michelotti, 1864: 76, pl. 15 Fig. 1.

30

For synonymy and taxonomic treatment, see Wiedenmayer (1977: 128), Zea (1987: 207), and remarks herein. In addition: Agelas dispar; Boury-Esnault, 1973: 285, Fig. 45, pl. 1, Fig. 8; Solé-Cava et al., 1981: 131, Figs.

7, 20; Pulitzer-Finali, 1986: 107, Fig. 32A; Lehnert, 1993: 50, Figs. 22, 79-82 (but Figure 22 does not seem to match); Gammill, 1997: 43, Figs. 41, 45, 50, 52; Lehnert & van Soest, 1998: 81; Lehnert & van Soest, 1999: 154; Assmann, 2000: 38, pl. 6 (in part, brown morphotype only, Figs. B and C; Figs. A and D are A. sventres); Valderrama, 2001: 49; Erpenbeck et al. 2007: 1564; Mothes et al., 2007: 85.

Agelas sparsus; de Laubenfels, 1936: 73; Alcolado, 1976: 4. Agelas sparsus var. clavaeformis; Alcolado, 1976: 4. Agelas sp. 2; Pulitzer-Finali. 1986: 113, Fig. 32G. [Non: A. dispar; van Soest, 1981: 10, 34; Alvarez & Díaz, 1985: 81, Fig. 23; Green et al., 1986:

130, Figs. 5-6; Kobluk & van Soest, 1989: 1216 (=Agelas sventres); Wintermann-Kilian & Kilian, 1984: 132 (unknown identity)].

Material and distribution

Holotype (ZMA-POR 607) revised at the Zoölogisch Museum Amsterdam, collected in Saint

Martin. Duchassaing & Michelotti (1864) mention that this species is also present in Guadeloupe,

St. Croix, St. Thomas and Vieques. There is no mention of depth. The material reviewed here

includes (but is not restricted to) specimens from Barbados (INV-POR 919), Belize (INV-POR

948 and INV-POR 953), Rosario Islands (INV-POR 962, see also Zea, 1987), San Andres Island

(INV-POR 976, see also Zea, 1987 from Old Providence), Jamaica (INV-POR 1002), and the

Bahamas (INV-POR 824 and INV-POR 933). F. Parra-Velandia observed this species in Curaçao

at the island shelf eastern border off Boca Tabla, and S. Zea also observed this species at La

Parguera, SW Puerto Rico. At the ZMA collection there are specimens from Puerto Rico (ZMA-

POR 3318), St. Eustatius (ZMA-POR 3955), Brazil (ZMA-POR 17312) and Martinique (ZMA-

POR 3582).

Previous works (including accounts by Wiedenmayer, 1977 and Zea, 1987) have reported the

species also from Virgin Islands, St. Martin, Guadeloupe (Duchassaing & Michelotti, 1864),

several areas of Brazil (Boury-Esnault, 1973; Solé-Cava et al., 1981; Mothes et al., 2007; G.

Muricy, pers. comm.), Dominican Republic (Pulitzer-Finali 1986), Florida and the Bahamas

(Gammill, 1997), other areas of Colombia (San Bernardo Islands, Urabá, see Valderrama, 2001)

and Dominican Republic (Weil, 2006). It is absent from Santa Marta rocky and coral reefs,

perhaps because of the upwelling there. From the above, we consider A. dispar a Tropical