Universidade Federal Do Rio De Janeiro BÁSLAVI MARISBEL CÓNDOR LUJÁN BIODIVERSITY and CONNECTIVITY of CALCAREOUS SPONGES

Universidade Federal do Rio de Janeiro

BÁSLAVI MARISBEL CÓNDOR LUJÁN

BIODIVERSITY AND CONNECTIVITY OF CALCAREOUS SPONGES

(PORIFERA: CALCAREA) IN THE WESTERN TROPICAL ATLANTIC

Rio de Janeiro 2017 ii

Biodiversity and Connectivity of calcareous sponges (Porifera: Calcarea) in the Western Tropical Atlantic

Báslavi Marisbel Cóndor Luján

Tese apresentada ao Programa de Pós-Graduação em Biodiversidade e Biologia Evolutiva, Instituto de Biologia, Universidade Federal do Rio de Janeiro, como parte dos requisitos necessários à obtenção do título de Doutor em Ciências Biológicas.

Orientadora: Dra. Michelle Klautau

Rio de Janeiro Fevereiro/2017 iii

Biodiversity and Connectivity of calcareous sponges (Porifera: Calcarea) in the Western Tropical Atlantic

Báslavi Marisbel Cóndor Luján Orientadora: Dra. Michelle Klautau

Banca examinadora:

Prof. Dra. Carla Zilberberg, IB/UFRJ ______

Prof. Dr. Cristiano Valentim da Silva Lazoski, IB/UFRJ ______

Prof. Dr. Eduardo Carlos Meduna Hajdu, MN/UFRJ ______

Prof. Dr. Guilherme Ramos da Silva Muricy, MN/UFRJ ______

Prof. Dr. Marcelo Weksler, MN/UFRJ ______

Dr. Fernando Moraes, JBRJ (Suplente) ______

Prof. Dr. Paulo Cesar de Paiva, IB/UFRJ (Suplente) ______

Rio de Janeiro Fevereiro/2017 iv

Trabalho realizado no Laboratório de Biologia de Porifera (LaBiPor) Departamento de Zoologia, Instituto de Biologia Universidade Federal do Rio de Janeiro – UFRJ Orientadora: Profa. Dra. Michelle Klautau

Capa: Centro: Rede filogeográfica de Leucetta floridana (modificada). Sentido horário: Leucetta floridana, Leucilla uter, Leucascus luteoatlanticus sp. nov., Ascandra torquata sp. nov., Sycon conulosum sp. nov., Nicola tetela, triactina âncora de Amphoriscus hirstutus sp. nov., triactina atrial de Grantessa tumida sp. nov., esqueleto de Leucandrilla pseudosagittata sp. nov., esqueleto de Ernstia citrea. Créditos: A. Padua, E. Hajdu, F. Azevedo e T. Pérez. v

Ficha catalográfica

CÓNDOR LUJÁN, Báslavi Marisbel

Biodiversity and Connectivity of calcareous sponges (Porifera: Calcarea) in the Western

Tropical Atlantic / Báslavi Marisbel Cóndor Luján. Rio de Janeiro: UFRJ, IB, 2017.

xiii, 259f. il; 29.7 (cm)

Orientador: Michelle Klautau.

Tese (Doutorado). UFRJ/IB/Programa de Pós-graduação em Biodiversidade e Biologia

Evolutiva, 2017.

Referências bibliográficas: 236-256f.

1. Esponjas Calcareas. 2. Mar do Caribe. 3. Costa do Brasil. 4. Conectividade genética. I.

Klautau, Michelle. II. Universidade Federal do Rio de Janeiro, Instituto de Biologia, Programa de Pós-graduação em Biodiversidade e Biologia Evolutiva. III. Tese. vi

Dedico esta tesis a mis queridos padres quienes, estando lejos, están siempre a mi lado. vii

Huk llactapi maypi llapan maskhay runakuna mamaqocha hatun yachaypi, Llapan maskhay

runakuna ruanku anchoveta challway, ñoqa huk punchay ruwayramuni qochayuyo sasa llachayta, soqta huata llanqay tukukuy manan llakikamunichu (In Quechua by E. Pezo Zegarra)

Translation: In a country where almost all marine science is focused on the fishery of

Engraulins ringens (Peruvian anchoveta), I took the risky decision to study sponges. After six

carioca years, I do not regret. viii

AGRADECIMENTOS

Esta tesis es el resultado de un maravilloso viaje a través de las primeras descripciones de las esponjas del Mar del Caribe y del Brasil, colectas en lugares en los que nunca imaginé bucear, interesantes diálogos con especialistas en Porifera y muchas horas dedicadas al microscopio, a la amplificación de secuencias de ADN y a las simulaciones bayesianas. Todo esto sólo fue posible gracias a la colaboración, apoyo e incentivo de muchísimas personas e instituciones.

Muchas gracias a todos y en especial a:

A minha orientadora Michelle Klautau por ter aceito a minha proposta de doutorado e ter me mostrado o caminho para ser uma boa pesquisadora.

Aos membros do projeto MARRIO, em especial ao Eduardo Hajdu e ao Thierry Pérez por terem organisado as maravilhosas expedições que geraram os resultados apresentados nesta tese.

Aos membros do LaBiPor, Andrézinho, Bernardo, Bárbara, Gabi, Fernanda, Mattheus,

Tayara, Taynara, Pedro e Raissa pela boa disposição para trabalhar em équipe e me auxiliar com os pedidos de último momento pelo whatsapp (rsrs). Às meninas que alguma vez fizeram parte do LabiPor, a Carol e Malena pelo trabalho de bancada e a Natalia e Vivi pela agradável companhia.

Aos pesquisadores (alemḿ do LaBiPor) que dedicaram um tempo durante os mergulhos para procurar e coletar Calcarea: A. Bispo, A. Ereskovsky, B. Thacker, C. Leal, C. Ruiz, C. Castello-

Branco, C. Diaz, G. Lôbo-Hajdu, H. Fortunato, J. García-Hernández, J. Vacelet, J. Carraro, L.

Van Bostal, M. Carvalho, O. Thomas, P. Chevaldonné, S. Chenesseau, S. Zea and S. Salani.

Aos pesquisadores espongólogos brasileiros e estrangeiros que conheci ao longo destes anos

(Simpósio de Porifera na Bahia, “La Martinique” Sponge Course, Pacotilles Expedition,

MARRIO Workshops). Todos eles, certamente, contribuiram na minha formação como pesquisadora. ix

Aos profesores do Museu Nacional e do Instituto de Biologia. Um especial “gracias” à

Ghennie Rodriguez pela orientação nas ańalises demográficas da Leucetta floridana.

Às agencias de fomento CAPES, CNPQ, CNRS, COFECUB, FAPERJ e Grupo Boticário de

Proteção à Natureza pelo financiamento das expedicçõs de coleta e os projetos do laboratório.

Ao programa PEC-PG e à CAPES pela concessão da bolsa de doutorado.

A mi mamá, papá y hermano por el apoyo incondicional y la confianza depositada en mi.

A Helmunt quien no sólo ha sabido darme su amor a lo largo de estos años, si no, también me ha ayudado en momentos computacionales muy críticos.

Muchas Gracias! Muito obrigada! x

ABSTRACT

BIODIVERSITY AND CONNECTIVITY OF CALCAREOUS SPONGES (PORIFERA: CALCAREA) IN THE WESTERN TROPICAL ATLANTIC

Báslavi Marisbel Cóndor Luján Supervisor: Dr. Michelle Klautau

Abstract of the thesis submitted to the Graduate Program Biodiversity and Evolutionary Biology, Institute of Biology, Federal University of Rio de Janeiro – UFRJ, as part of the requirements to obtain the title of Doctor in Biological Sciences.

The Western Tropical Atlantic (WTA) is a wide region that comprises the Tropical Northwestern Atlantic (TNA), the North Brazil Shelf (NBS) and the Tropical Southwestern Atlantic (TSA) provinces. Although it harbours a high diversity of marine species, sponges of the class Calcarea have been poorly studied. This lack of knowledge hinders our understanding of the biogeographical affinities of the calcareous sponges in the WTA and the role of the Amazon River as an effective barrier to dispersal of these sponges. In the present study, the diversity of the class Calcarea in the WTA was investigated using morphological and molecular approaches and the population connectivity of a calcareous sponge, Leucetta floridana, was assessed. With this contribution, the number of calcareous sponges from the WTA raised from 67 to 86, including the descriptions of 15 new species and four new records. Among them, 21 species are endemic to the TNA, three to the NBS and 22 to the TSA. The 14 species shared between the TNA and TSA support a Caribbean-Brazilian affinity for calcareous sponges. Genetic analyses evidenced five structured populations in L. floridana: one widespread population maintaining gene flow between the TNA and TSA provinces and four other isolated populations within the Caribbean Sea. The panmitic population suggested that the outflow of the Amazon River in the Atlantic is not an effective barrier to the maintenance of gene flow among trans-Amazonian populations of L. floridana.

Keywords: Caribbean Sea, NE Brazilian coast, phylogeography, demography, Amazon River.

Rio de Janeiro

February, 2017 xi

RESUMO

BIODIVERSIDADE E CONECTIVIDADE DE ESPONJAS CALCAREAS (PORIFERA, CALCAREA) NO ATLÂNTICO TROPICAL OCIDENTAL

Báslavi Marisbel Cóndor Luján Orientadora: Dra. Michelle Klautau

Resumo da Tese submetida ao Programa de Pós-Graduação em Biodiversidade e Biologia Evolutiva, Instituto de Biologia, Universidade Federal do Rio de Janeiro – UFRJ, como parte dos requisitos necessários à obtenção do título de Doutor em Ciências Biológicas.

O Atlântico Tropical Ocidental (WTA) é uma vasta região que compreende as províncias Atlântico Noroeste Tropical (TNA), Plataforma Norte do Brasil (NBS) e Atlântico Tropical Sudoeste (TSA). Embora abrigue uma grande diversidade de espécies marinhas, as esponjas da classe Calcarea têm sido pouco estudadas. Essa falta de conhecimento dificulta nossa compreensão das afinidades biogeográficas das esponjas Calcarea no WTA e do papel do Rio Amazonas como uma barreira efetiva para a dispersão dessas esponjas. Neste estudo, foi investigada a diversidade da classe Calcarea no WTA por meio de abordagens morfológicas e moleculares e também foi avaliada a conectividade populacional da esponja Calcarea Leucetta floridana. Com a presente contribuição, o número de esponjas da classe Calcarea no WTA aumentou de 67 para 86 espécies, incluindo descrições de 15 espécies novas e 4 novos registros. Entre elas, 21 espécies são endêmicas do TNA, três ocorrem só NBS e 22 são exclusivas do TSA. As 14 espécies compartilhadas entre o TNA e o TSA sugerem uma afinidade Caribe-Brasil para as esponjas calcareas. As análises genéticas evidenciaram cinco populações estruturadas em L. floridana: uma população amplamente distribuida e mantendo fluxo gênico entre as províncias TNA e TSA e quatro outras populações isoladas no Mar do Caribe. A população panmítica sugeriu que a descarga do Rio Amazonas no Atlântico não é uma barreira efetiva à manutenção do fluxo gênico entre populações trans-amazônicas de L. floridana.

Palavras-chave: Mar do Caribe, costa NE do Brasil, filogeografia, demographia, Rio Amazonas.

Rio de Janeiro Fevereiro de 2017 xii

INDEX

1. INTRODUCTION …...... 1

1.1 General introduction …...... 2

1.2 General Aim …...... 5

1.3 Specific Aims …...... 5

2. LITERATURE REVIEW …...... 6

2.1. Geographic Scenario …...... 7

2.1.1 The Western Tropical Atlantic …...... 7

2.1.2 The Caribbean Sea …...... 8

2.1.3. The Tropical Brazilian Shelf and the Oceanic Islands …...... 11

2.2. Connectivity within the Western Tropical Atlantic …...... 13

2.2.1 Estimating connectivity …...... 13

2.2.2 General patterns of connectivity …...... 14

2.2.3 Connectivity patterns in the Western Tropical Atlantic …...... 15

2.3. Porifera in the Western Tropical Atlantic …...... 16

2.3.1 Generalities of the Phylum Porifera …...... 16

2.3.2 Generalities of the Class Calcarea …...... 17

2.3.3 Diversity of Calcarea in the Western Tropical Atlantic …...... 20

3. RESULTS …...... 21

3.1 Nicola gen. nov. with redescription of Nicola tetela (Borojevic & Peixinho, 1976) (Porifera:

Calcarea: Calcinea: Clathrinida) …...... 22

3.2 Calcareous sponges (Porifera: Calcarea) from Curaçao including Brazilian shared species and phylogenetic remarks …...... 34

3.3 New records of calcareous sponges (Porifera: Calcarea) from the Northeastern Brazilian coast including three new species …...... 132

3.4 Evolutionary history of the calcareous sponge Leucetta floridana in the Western Tropical

Atlantic …...... 176 xiii

4. GENERAL DISCUSSION …...... 215

5. CONCLUSIONS AND PERSPECTIVES …...... 233

6. REFERENCES …...... 236

APPENDIX …...... 257

Appendix 1 …...... 257

Appendix 2 …...... 258

Appendix 3 …...... 259 Chapter 1

INTRODUCTION 2

1.1 General Introduction

For many decades, the zoogeographical affinities within the Western Tropical Atlantic, specifically between the Caribbean and the Brazilian coast, have been discussed and several biogeographic divisions have been proposed. Both areas were once considered a single biogeographic province, the Caribbean Province, which extended from the coast of Florida

(United States of America) down to Cabo Frio in Rio de Janeiro (Brazil) (Ekman, 1953). Some years later, based on the Brazilian endemism of corals, hydrozoans, molluscs and fishes, Briggs

(1974) proposed a division in Caribbean, West Indian (Lesser Antilles) and Brazilian provinces.

The latter included also the Brazilian Oceanic Islands. The discovery of “Caribbean” reef fishes under the Amazon freshwater plume (Collete & Rützler, 1977) made Helfman et al., (1997) reconsider the presence of a unique province in this region. Palacio (1982) did not refuse this but restricted the Tropical Province (Caribbean) down to the 23°C isotherm, between Espírito

Santo and Rio de Janeiro. Nonetheless, as knowledge on Brazilian endemic species increased, the division in Caribbean and Brazilian provinces became more accepted (Briggs, 1995; Floeter

& Gasparini, 2000; Boschi, 2000).

In 2007, Spalding et al. introduced a new biogeographic classification, the Marine

Ecoregions of the World (MEOW) integrating previous biogeographic divisions, geomorphological and oceanographic features and new data of species distribution and dominant habitats. In that classification, the Western Tropical Atlantic (WTA) comprised three biogeographic provinces: the Tropical Northwestern Atlantic (TNA), the North Brazil Shelf

(NBS) and the Tropical Southwestern Atlantic (TSA). More recently, modifications to those provinces have been proposed by Floeter et al., (2008) and Briggs & Bowen (2012) based on new studies on fish endemism and phylogeography.

The existence of different biogeographic provinces in the WTA relies on the possibility of barriers that can prevent the dispersal of organisms within this broad area, such as the Amazon 3 or Orinoco rivers. According to Rocha (2003), the freshwater and sediment outflow from the

Amazon river would act as a strong barrier to some reef fishes and might be responsible for the endemism observed in the Brazilian coast. Nonetheless, the effectiveness of this barrier might be restricted to shallow-water organisms and highly influenced by the sea-level fluctuations.

Therefore, larval dispersal (gene flow) between Caribbean and Brazilian populations of widespread species would occur during interglacial periods and would be facilitated by the sponge assemblages found underneath the Amazon river that would act as a connectivity corridor.

Sponges are exclusively aquatic, sessile, filter-feeding, multicellular organisms. They are the oldest extant metazoan group (Antcliffe et al., 2014) and present many adaptations to different habitats, including marine and freshwater environments. They play an important ecological role in marine ecosystems as they contribute to the recycling of nutrients from the water column into the benthic communities (de Goeij et al., 2013). Moreover, they constitute one of the principal marine invertebrate sources for the isolation of bioactive compounds of pharmaceutical interest

(Blunt, 2006, Perdicaris et al., 2013)

As all sponges have lecithotrophic larvae (Ereskovsky, 2010), low dispersal capabilities and restricted geographic distribution ranges are expected. However, some studies revealed that widespread species do occur along the WTA (Lazoski et al., 2001; Valderrama et al., 2009,

Zilberberg, 2006). These species constitute good models to evaluate the effectiveness of the

Amazon River as a real barrier to gene flow, nonetheless, up to date not such study was performed. The knowledge on sponge connectivity within the WTA is fragmentary or restricted to some species within one single biogeographic province (De Biasse et al., 2010, 2016;

Chaves-Fonnegra et al., 2015; López-Legentil & Pawlik, 2009; Richards et al., 2016; de Bakker et al., 2016; Padua, 2012). 4

Among the four extant classes of Porifera (Demospongiae, Homoscleromorpha,

Hexactinellida and Calcarea), Calcarea is the only with skeleton exclusively composed of calcium carbonate). The knowledge on the diversity of calcareous sponges is somehow restricted to geographical areas to which specialized taxonomists have access. For instance, the

Mediterranean Sea and the Caribbean Sea have comparable coastal extension (Coll et al., 2010;

Miloslavich et al, 2011); however, the number of calcareous sponges reported in the former is ca. 65 whereas in the latter, it is only 23. This possibly reflects the lack of taxonomic effort in the Caribbean Sea, a region considered a diversity hotspot for many other taxa.

Within this context, this study is intended to present the diversity and population connectivity of calcareous sponges in the Western Tropical Atlantic, with a major emphasis on the Caribbean Sea and the Northeastern Brazilian coast.

This thesis is organised in five chapters. The first chapter includes this general introduction and the aims of this work. The second chapter sets the theoretical framework of this study through a brief review on the characterization of the WTA, population connectivity and calcareous sponges. The third chapter includes the results of this study and it is divided in four articles. The first article proposes the new Calcinean genus Nicola based on the redescription of the type material of “Guancha” tetela and the description of new specimens collected in

Curaçao. In the second and third articles, I described the calcareous sponges from Curaçao and from the NE Brazilian coast, respectively, using morphological and molecular approaches. The fourth article is the first study on population connectivity and demography of a calcareous sponge (Leucetta floridana) in the WTA. The fourth chapter comprises a general discussion. The fifth chapter presents the main conclusions and perspectives of this study. 5

1.2 General Aim

To know the biodiversity and connectivity of the class Calcarea in the Western Tropical Atlantic

1.3 Specific Aims

1.3.1 To identify and describe the sponges of the class Calcarea from the Caribbean Sea.

1.3.2 To identify and describe the sponges of the class Calcarea from the Northeastern Brazilian coast.

1.3.4 To assess the genetic structure of calcareous sponges in the Western Tropical Atlantic.

1.3.3 To test if the Amazon River is an effective barrier to connectivity among populations of calcareous sponges. Chapter 2

LITERATURE REVIEW 7

2.1. Geographic Scenario

2.1.2 The Western Tropical Atlantic

The Atlantic Ocean emerged after the break-up of the supercontinent Pangaea in the late

Jurassic Period (163.5 -145 million years BP). Two important geological events occurred: (1) the separation of the Pangaea in Laurasia and Gondwana, which resulted in the formation of the

Central Atlantic Ocean, and (2) the gradual south-to-north split of West Gondwana in South

America and Africa during the Early Cretaceous (145-100.5 million years BP) that originated the South Atlantic Ocean (Seton et al., 2012). During the next million years, the Atlantic Ocean continued spreading until its current configuration.

According to the latest marine biogeographic classification system (MEOW - Spalding et al.,

2007), the Tropical Atlantic Realm (TA, Figure 1A) comprises six biogeographic provinces

(Figure 1B): Tropical Northwestern Atlantic (TNA, n°12), North Brazil Shelf (NBS, n°13),

Tropical Southwestern Atlantic (TSA, n°14), St. Helena and Ascension Islands (n°15), West

African Transition (n°16) and Gulf of Guinea (n°17).

Figure 1. Biogeographical regionalization according to Spalding et al. (2007). A. Realms in the Atlantic Ocean. B. Biogeographic provinces in the Tropical Atlantic Realm. C. Ecoregions in the Western Tropical Atlantic. 8

The Western Tropical Atlantic (WTA) comprises the TNA, NBS and TSA biogeographic provinces and includes 16 ecoregions (Figure 1C). The TNA comprises nine ecoregions:

Southern Gulf of Mexico (n°69), Western Caribbean (n°68), Southwestern Caribbean (n°67),

Southern Caribbean (n°66), Greater Antilles (n°65), Eastern Caribbean (n°64), Bahamiam

(n°63), Bermuda (n°62), and Floridian (n°63). The NBS includes the Guianian (n°71) and

Amazonia ecoregions (n°72). The TSA Province comprises São Pedro and São Paulo Islands

(n°73), Fernando de Noronha and Atoll das Rocas (n°74), Northeastern Brazil (n°75), Eastern

Brazil (n°76) and Trindade and Martin Vaz Islands (n°77). This study is focused on the TNA and

TSA provinces, specifically, the Caribbean Sea and the northeastern Brazilian coastal shelf.

2.1.2 The Caribbean Sea

The Caribbean Sea is a semienclosed basin within the TNA Province. It is bounded by Central

America on the west, Greater Antilles on the north, Lesser Antilles on the east and the northern coast of South America on the south. It includes five ecoregions: Western Caribbean,

Southwestern Caribbean, Southern Caribbean, Greater Antilles and Eastern Caribbean. The

Caribbean basin comprises an area of approximately 2,754,000 km2, a volume of about 6.5x106 km3 and its coastline has an extension of more than 13,500 km (Miloslavich et al., 2011).

The geological age of the Caribbean Sea is related to the formation of the Caribbean Plate. In the Middle Cretaceous (100.5–80 million years BP), a flood basalt event produced an oceanic crust with a thickness of about 15-20 km (Burke, 1978). According to the model of Meschede &

Frisch (1998), this event took place somewhere between North and South America. An alternative hypothesis (Pindell, 1994) suggested that the flood basalt occurred in the Pacific

Ocean, in the Galapagos hotspot, which afterwards, drifted eastwards (Figure 2). 9

Figure 2. Hypotheses to explain the origin of the Caribbean plate. A. Origin in an inter- American position. B. Pacific origin. Taken from Meschede & Frisch (1998).

The Caribbean Sea is divided into five deep water regions: the Yucatan Basin, the Cayman

Trough, the Colombian Basin, the Venezuelan Basin and the Grenada Basin, which are separated by underwater ridges - the Cayman Ridge, the Nicaraguan Rise, the Beata Ridge and the Aves Ridge, respectively (Draper et al., 1994, Figure 3). The average seafloor depth is approximately 2,400 m and the deepest area (more than 7,500) is comprised between Cuba and

Jamaica, in the Cayman Trough (Matthews & Holcombe, 1985).

Figure 3. General geography and topography of the Caribbean Sea indicating the political divisions and basins mentioned in the text. Source: Encyclopædia Britannica, Inc. 10

The present oceanographic configuration of the Caribbean Sea was established only after the uplift of the Isthmus of Panama (2.8 million years BP - O'dea et al., 2016). Previously, the

Caribbean Sea experienced increased upwelling as it was connected to the Pacific Ocean (O'dea et al., 2007). Nowadays, the Caribbean waters are clear, oligotrophic and with warm temperatures ranging from 22 to 29°C (Kinder et al., 1985).

The Caribbean Sea receives the Atlantic inflow from the North Brazil current through several passages located in the Lesser Antilles (Windward Islands Passages and Leeward Islands

Passages) and the Greater Antilles (Johns et al., 2002). The Caribbean current is formed mainly by the masses of water that pass through the Windward Island Passages, specially through the

Grenada Passage located at the south of Grenade (near 11.51 oN, Cherub, 2007). Within the

Caribbean basin, this current flows westward at 13–16°N and between Nicaragua and Jamaica it turns northwest into the Gulf of Mexico through the Yucatan Channel. In the southernmost part of the Caribbean Sea, one of the branches of the Caribbean Current forms the counterclockwise

Panama Gyre (Figure 4, Molinari et al., 1981).

Figure 4. Currents in the Caribbean Sea (as represented by the Mariano Global Surface Velocity Analysis). Downloaded from http://oceancurrents.rsmas.miami.edu. Abbreviations: P=passage. 11

Although the Caribbean waters are mainly clear, they are influenced by the freshwater outflow from major rivers. The freshwater and sediments from the Amazon (Brazil) and

Orinoco (Venezuela) plumes enter through the North Brazil Current and the discharge of the

Magdalena river (Santa Marta, Colombia) directly interacts with the Caribbean current in the

Southwestern Caribbean (Cherub, 2007).

The Caribbean Sea is considered a global-scale hotspot for marine biodiversity (Roberts et al., 2002). It encompasses a high diversity of flora and fauna distributed in different ecosystems including coral reefs, mangroves and seagrasses (Miloslavich et al., 2010).

2.1.3 The Tropical Brazilian Shelf and the Oceanic Islands

The vast extension of the Brazilian coast (7,500 km) includes two major biogeographic realms, the Tropical Atlantic (TA) and the Temperate South America (Spalding et al., 2007, Figure 1A).

In the present work only the TA was studied.

The Tropical Brazilian Shelf and the Oceanic Islands comprise seven ecoregions, one in the

NBS and six in the TSA (Figure 1C) . This area has been shaped by the Quaternary sea-level changes over the past 5000 years, with the exposure of coastal sediments, river load, and coastal drift (Dominguez et al., 1983).

The northern Brazilian coast, which corresponds to the Amazonian ecoregion of the NBS

(Figure 1C, n°72), is characterised by a combination of freshwater, estuarine and marine ecosystems as a consequence of the outflow of the Amazon river. Benthic environments comprise muddy-bank-shorelines (Anthony et al., 2010), rhodolith beds (Moura et al., 2016), coral and sponge reefs (Colette & Rützler, 1976; Moura et al., 1999; 2016).

The northeastern and part of the southern Brazilian Coast (3°S - 20°S), which correspond to the Northeastern Brazil (n°75), Eastern Brazil (n°76) and Trindade and Martin Vaz Islands

(n°77) ecoregions of the TSA, have an oligotrophic environment with little influence of river runoff and only subjected to local upwelling (Soares et al., 2016). This area is characterised by a 12 variety of ecosystems, including mangrove forests, seagrass beds, coral reefs, sandy beaches, rocky shores, lagoons and estuaries (Miloslavich et al., 2011; Moura et al., 2013).

The Brazilian oceanic islands include Fernando de Noronha Archipelago, Rocas Atoll (the only atoll in the South Atlantic) and São Pedro and São Paulo Archipelago (ecoregions n° 73 and 74). These islands harbour tropical environments similar to those of the Caribbean Sea and constitute very important hotspots of biodiversity (Soares et al., 2016). Mesophotic reefs and rhodolith beds are also common within these islands (Magalhães et al., 2015; Amado-Filho et al., 2016).

The entire coast of Brazil is under the influence of warm and temperate marine currents, freshwater discharge from several rivers and upwelling events (Coelho-Souza et al., 2012;

Soares et al., 2016). In the Tropical Brazilian continental margin, the principal surface currents are the Brazilian Current (BC) and the North Brazilian Current (NBC), originated from the

South Equatorial Current at about 5–6°S (Stramma, 1991; Silveira et al., 1994). The BC flows to the south whereas the NBC runs to the north and northwest (Figure 5).

Figure 5. Surface currents in the Tropical Brazilian coast (as represented by the Mariano Global Surface Velocity Analysis): A. North Brazil Current (NBC) and B. Brazilian Current (BC). Downloaded from http://oceancurrents.rsmas.miami. 13

2.2 Connectivity within the Western Tropical Atlantic

2.2.1 Estimating connectivity

Population connectivity is understood as the exchange of individuals among populations or subpopulations (Gagnaire et al., 2015) and it can be divided into genetic and demographic connectivity. Genetic connectivity refers to the degree to which gene flow affects evolutionary processes within populations, whereas demographic connectivity refers to the degree to which population growth and vital rates (survival, reproduction, emigration) are affected by dispersal

(Lowe & Allendorf, 2010).

The estimation of connectivity among marine populations is based on the assessment of larval dispersal through indirect methods. These methods include the use of geochemical tags from calcified structures (otoliths, statoliths and shells) or from artificial sources (fluorescent compounds inserted in larvae (Thorrold et al., 2007), coupled biophysical models which integrate oceanographic factors and larvae biological traits (e.g. Cowen et al., 2006; Paris et al.,

2007) and genetic approaches that allow the calculation of migration rates (e. g. Selkoe et al.

2010), among other parameters,

Molecular approaches include the amplification of neutral “frequency” or “sequence” markers. Frequency markers such as microsatellites are adequate to answer questions in more ecological timescales, whereas sequence markers such the cytochrome oxidase I (COI) aid to resolve the history of divergence among populations. However, both types of markers present some disadvantages. On the one hand, the isolation of microsatellites can be time-consuming and consistent results may require large samplings and sophisticated analyses. In some cases, it is even necessary to test the Mendelian condition of the loci before genotyping (Hellberg, 2009).

On the other hand, duplication or insertions from nuclear genome (pseudogenes) can alter mtDNA patterns (Williams & Knowlton, 2001; Schizas, 2012). 14

Once chosen the adequate marker, further analyses to estimate connectivity comprise the computation of indirect estimators (indexes) as the fixation index (FST) and the application of direct methods such as parentage assignment or clustering analyses.

2.2.2 General patterns of connectivity

Marine population connectivity is driven by two main integrated components: (1) physical processes and (2) biological traits (Cowen & Sponaugle, 2009). The former includes the particular oceanographic processes occurring in the connectivity area (currents, tides, upwelling, hurricanes), whereas, the latter corresponds to the life history of the target species including its larval behaviour (mechanisms of defense against predators, vertical migrations, horizontal swimming), chemical signals for settlement and adult spawning (Cowen et al., 2000; Cowen et al, 2005). Other dispersal factors non-mediated by larvae such as adult rafting also influence population connectivity (Kinlan et al., 2005; Cowen & Sponaugle, 2009).

In the last 20 years, several studies have notably contributed to the understanding of marine population connectivity. Herein, I point out which I consider the most relevant conclusions:

1. Not all marine populations are open. Coastal marine populations were previously considered open populations which would exchange individuals from very distant localities as a consequence of the passive larval dispersal driven by water currents. Using Eulerian and

Lagrangian flow models, Cowen et al. (2000) found higher levels of larvae retention near the natal localities than in distant areas. These results were also observed in further studies (Swearer et al. 2002, Jones et al., 2005). Nowadays, studies suggest that populations range from fully open to fully closed (Cowen & Sponaugle, 2009).

2. Larval dispersal capability may not be a good indicator of connectivity. Species with low larval dispersal capabilities are believed to present structured populations or high rates of self- recruitment (local replenishment); however, several studies on connectivity of reef fishes did not evidence this. Bowen et al., (2006) found that Myripristis jacobus, a fish with short lifespan had 15 a population much less structured than Holocentrus ascensionis, a species with longer larval duration. Almany et al. (2007) evidenced that fishes with different larval duration (orange clownfish - Amphiprion percula and vagabond butterflyfish – Chaetodon vagabundus) presented equal high levels of self-recruitment (60%). These studies indicated that larval duration was a poor predictor of connectivity.

3. Genetic analyses should include oceanographic input. As traditional genetic analyses based on the correlation between geographic distance (Eclidean distance) and genetic dissimilarity failed to explain the connectivity patterns observed in several studies (e.g. Brandbury &

Bentzen, 2007), environmental information, such as ocean currents, started to be incorporated in genetic calculations (White et al., 2010). That approach greatly improved the predictive value of population genetics studies on small spatial scales (Selkoe et al., 2010). Within this context, two adjacent localities are not necessarily connected nor two distant localities are genetically isolated (White, 2010).

4. Still dealing with limited knowledge. Despite the considerable advances on marine connectivity due to studies integrating different approaches, our knowledge is still fragmented or restricted to specific taxa (mainly, fish and coral species). As pointed out by White (2010), even with perfect simulations, the obtained results may refer to potential population connectivity.

2.2.3 Connectivity patterns in the Western Tropical Atlantic

Our knowledge of population connectivity in the Tropical Western Atlantic is mainly based on studies of reef fish connectivity using mitochondrial DNA sequences and with focus on the

Caribbean Sea and the Northeastern Brazilian coast.

Rocha et al. (2002) evaluated the role of the Amazon river as an effective barrier to the dispersal of surgeonfishes (Acanthurus) and found levels of connectivity that correlated with habitat preference. The species A. bahianus, which rarely settles outside shallow reefs, revealed 16 highly structured Brazilian and Caribbean populations, whereas A. chirurgus, collected on soft sponge bottoms under the mouth of the Amazon River, presented genetically homogeneous populations. This indicated that the Amazon outflow acted as a semi-permeable barrier. Rocha

(2003) suggested that the sponge assemblages discovered under the mouth of the Amazon river

(Colette & Rützler, 1976) would act as a corridor for species that require clear waters with normal salinity but not for species strictly dependent on shallow reef habitats.

In the Caribbean Sea, Cowen et al. (2006) defined four broad regions of connectivity: (1) the eastern Caribbean (Puerto Rico to Aruba); (2) the western Caribbean (Cuba to Nicaragua); (3)

Bahamas, Turks and Caicos Islands; and (4) the peripheral area of the Colombia-Panama Gyre.

Within these regions, population isolation and connectivity would be related to oceanographic conditions such as the strong currents at the Mona Passage between Cuba and Puerto Rico and the Colombia-Panama counter-gyre or to topographical features e.g. the shallow shelves that remained exposed during the Pleistocene sea-level fluctuations.

2.3. Porifera in the Western Tropical Atlantic

2.3.1 Generalities of the Phylum Porifera

The phylum Porifera is composed of exclusively aquatic, sessile and multicellular organisms, commonly known as sponges (Hooper et al., 2002). They are the oldest metazoan group still extant and they present many ecological adaptations to different habitats including marine and freshwater environments.

These animals are also characterised by the presence of a skeleton composed of spicules

(mineral aggregations of calcium carbonate or silica) or spongin (a protein from the family of the collagen) and of an aquiferous system, principally used for filtering-feeding (Hooper et al.,

2002).1

1 Porifera also includes species without any type of skeleton (e.g. Hexadella and Oscarella spp.) or aquiferous system (carnivorous sponges). 17

Sponges present cells with high mobility and totipotency, which facilitate cellular differentiation and transdifferentiation (Klautau, 2016). Except in species of the class

Homoscleromorpha, these cells do not form true tissues, however, the choanoderm (composed of choancytes) and the pinacoderm (formed by pinacocytes), important components of the aquiferous system, act as functional epithelia (Leys & Riesgo, 2012).

Sponges have short-lived lecithotrophic larvae (Ereskovsky, 2010) with a maximum lifespan of two weeks (Fry, 1971; Maldonado, 2006). This may constrain their dispersion and consequently, restrict their geographic distribution. Nonetheless, they present asexual processes such as budding, gemmulation and fragmentation (Maldonado & Riesgo, 2008) that may contribute to expand their distribution range.

According to the World Porifera Database (Van Soest et al., 2016), the phylum Porifera currently comprises 8781 species distributed in four classes: Calcarea Bowerbank, 1862,

Demospongiae Sollas, 1885, Hexactinellida Schmidt, 1870 and Homoscleromorpha Gazave et al, 2012. Among them, Calcarea is the only class that reunites the species whose skeleton is exclusively composed of spicules made of calcium carbonate.

2.3.2 Generalities of the class Calcarea

The class Calcarea comprises marine sponges with a skeleton exclusively composed of calcium carbonate, consisting of free, rarely linked or cemented spicules, to which a solid calcitic skeleton can be added. They are viviparous and present blastula larvae (Manuel et al., 2002). In this class, all the known types of aquiferous system in Porifera (asconoid, syconoid, sylleibid, solenoid and leuconoid) are present (Figure 6, Cavalcanti & Klautau, 2011).

Figura 6. Aquiferous system of Porifera: asconoid (a), syconoid (b), sylleibid (c), leuconoid (d) and solenoide (e). Grey lines represent the choanoderm. Taken from Cavalcanti & Klautau (2011).

Calcarean sponges commonly reach small sizes (measured in mm or a few cm), however, species of more than 20 cm in length were reported e.g. Leucandra multiformis, Leucetta avocado, Pericharax heteroraphis, Sycon ciliatum (Poléjaeff, 1883; Koechlin, 1977; Van Soest et al., 2012). Although most of the known species are white or beige; some yellow, red and pink species have also been described e.g. Clathrina clathrus, C. rubra, Leucascus roseus. They can be found in shallow tropical waters as well as in very deep waters in the Antarctic (Janussen &

Rapp, 2011; Rapp et al., 2011) and North Atlantic (Greenland, Rapp et al., 2013), in cryptic environments protected of light such as overhangs, roofs of caves, crevices or underneath boulders. Associated microorganisms have been found within the mesohyl of calcareous sponges (Fromont et al., 2016) as well as benthic fauna (Padua et al., 2013) among the tubes or atrium of these sponges.

The class Calcarea is divided into two monophyletic subclasses Calcinea and Calcaronea

Bidder, 1898(Figure 7). Calcaroneans are characterised by presenting sagittal spicules, apinucleated choanocytes, amphiblastula larvae and diactines as the first spicules to be produced, whereas calcineans have a skeleton mainly composed of regular spicules, basinucleated choanocytes, calciblastula larvae and triactines as the first spicules to be secreted

(Manuel et al. 2002). 19

Figure 7. Calcaronea: A. Amphiblastula larvae (Lanna & Klautau, 2016), B. Apinucleated choanocyte (Borojevic et al., 2000), C. Sagittal spicules and diactines (Van Soest et al., 2012). Calcinea: D. Calciblastula larvae (Ereskovsky & Willenz, 2008). E. Basinucleated choanocytes (Borojevic et al, 1990). F. Regular spicules and diactines (Van Soest et al., 2012).

Phylogenetic relationships within these two subclasses remain unclear as many orders, families and even genera are paraphyletic or polyphyletic (Voigt et al., 2012; Voigt & Wörheide,

2016). However, in the subclass Calcinea, certain skeletal traits have evidenced phylogenetic signal in some genera, e.g. Clathrina and Borojevia (Rossi et al., 2011; Klautau et al., 2013).

The total number of described calcareous species (ca. 680) represents only about 8% of all described extant sponges (Van Soest et al, 2012) and most of these records are restricted to areas where the taxonomic effort on calcareous sponges has been intense (e. g. Australia, Japan and

Mediterranean Sea). Furthermore, the plasticity of a few morphological characters in some

Calcarean taxa difficults the species identification and sometimes require integrative approaches

(e.g. Valderrama et al., 2009; Imeseck et al., 2014; Azevedo et al., 2015; Klautau et al., 2016). 20

2.3.3 Diversity of Calcarea in the Western Tropical Atlantic

In general terms, the sponge diversity (including all the classes) is concentrated in the tropics

(Soares et al., 2016). However, the first attempt to elucidate the distribution patterns within the class Calcarea (Van Soest et al., 2012) revealed very low values of diversity in that area. As pointed out in the same study, those results did not reflect the real diversity but the need of describing the sponge fauna from poorly explored areas.

The number of valid species known for the Western Tropical Atlantic, compiled from the

World Porifera Database (Van Soest et al., 2016; Van Soest, 2017), unpublished literature

(Azevedo, 2013) and submitted manuscripts (Azevedo et al., submitted) is 67 species. Some species are shared between two or three biogeographic provinces and other species are endemic to a province and in some cases, to an ecoregion. Chapter 3

RESULTS 22

Nicola gen. nov. with redescription of Nicola tetela (Borojevic & Peixinho, 1976) (Porifera: Calcarea: Calcinea: Clathrinida) CÓNDOR-LUJÁN, B. & KLAUTAU, M.

Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Av. Carlos Chagas Filho, 373, 21941-902, Rio de Janeiro, RJ, Brasil. [email protected]; [email protected] Corresponding author: [email protected] Article published in ZOOTAXA 4103(3): 230-238

Abstract Guancha tetela was originally described as a species having a peduncle and a skeleton exclusively composed of sagittal triactines. Therefore, according to the most recent phylogeny of Clathrinida, it should be placed in the genus Clathrina. This species was collected on the Northeastern Brazilian coast in 1968 and it was not collected again until 2011 in Curaçao. In this study, we reanalyzed the type material and the new specimens from Curaçao under a morphological-molecular approach. Morphological analysis revealed the presence of tetractines in the skeleton of all the studied specimens, including a slide of the holotype. In the molecular phylogeny G. tetela grouped with genera containing tetractines, but as an independent new lineage, different from all the other genera of Clathrinida. Based on these results, we propose the erection of a new genus, Nicola gen. nov., to include species whose body is composed of tubes without anastomosis nor branches but that run in parallel and coalesce at the apical and basal regions. Moreover, the skeleton is exclusively composed of sagittal triactines and tetractines. Key words: Atlantic Ocean, Northeastern Brazilian Coast, Caribbean Sea, Curaçao, molecular systematics, new genus, Guancha tetela.

Introduction The genus Guancha was originally proposed by Miklucho-Maclay (1868) in order to describe Guancha blanca, a species from the Canary Islands (Lanzarote) that presented a clathroid cormus with a stalk. Although Miklucho-Maclay (1868) considered that the presence of a stalk was sufficient to separate this species in a new genus, subsequent authors did not agree with this point of view and placed G. blanca in different genera: Ascetta (Haeckel 1872; Vosmaer 1881; Lendenfeld 1891), Leucosolenia (Lackschewitsch 1886; Vosmaer 1887; Breitfuss 1896, 1898; 23

Dendy & Row 1913; Arndt 1928; Hôzawa 1929; Burton 1930; Brøndsted 1931; Breitfuss 1932, 1935; Topsent 1936; Arndt 1941; Tanita 1943), and Clathrina (Minchin 1896; Jenkin 1908). Only in 1976 the genus Guancha became accepted, with the description of G. tetela by Borojevic and Peixinho (1976), and had its first diagnosis: "Clathrinidae à cormus constitué d'un pédoncule et d'un corps clathroïde. Spicules réguliers et parassagittaux, ou uniquement parasagittaux, orientés parallèlement dans les parois des tubes, au moins dans la partie basale de l'éponge avec l'actine impaire basipète" (Borojevic & Peixinho 1976) (Translation: Clathrinidae with a cormus composed of a stalk and a clathroid body. Spicules are regular and parasagittal or only parasagittal, organised in parallel in the tubes wall, at least at the basal part of the sponge with the unpaired actine basipetally oriented). Later on, in the Systema Porifera (Borojevic et al. 2002), the diagnosis proposed was: "Clathrinidae with a cormus composed of a peduncle and a clathroid body. The peduncle may be formed by true tubes with a normal choanoderm, or may be solid with a special skeleton. The skeleton is composed of regular (equiangular and equiradiate) spicules to which parasagittal spicules are added, at least in the peduncle. In some species only parasagittal spicules are present. The unpaired actine of parasagittal spicules is always basipetally oriented." Since the publication of the Systema Porifera and this new diagnosis of Guancha, four new species were described within this genus: Guancha arnesenae Rapp, 2006, Guancha camura Rapp, 2006, Guancha pellucida Rapp, 2006, and Guancha ramosa Azevedo et al., 2009. More recently, however, molecular studies showed that Guancha was not a monophyletic genus, and the authors proposed its synonymisation with Clathrina (Rossi et al. 2011; Klautau et al. 2013). This synonymisation was confirmed when the type species of the genus (G. blanca) was included in a molecular tree (Imešek et al. 2014). Currently, we follow what was proposed by Klautau et al. (2013), that all Guancha species with only triactine spicules should be transferred to Clathrina, and that Guancha species with triactines and tetractines should be assigned to any of the other genera proposed or rediagnosed in the same article (namely Arthuria, Ascandra, Borojevia, Brattegardia, and Ernstia). According to this proposal, the species Guancha tetela, originally described as having a skeleton exclusively composed of sagittal triactines, should be placed in Clathrina. However, a more detailed revision of this species revealed the presence of tetractines in its skeleton, which precludes its classification as a Clathrina. As G. tetela presents more triactines than tetractines, it should then be considered Arthuria, however, the organisation of the cormus of G. tetela is 24 different of that of other Arthuria. Hence, molecular and detailed morphological analyses were performed in the present work to verify the correct classification of G. tetela.

Material and Methods The holotype of Guancha tetela is deposited in the Muséum Nationale d'Histoire Naturelle de Paris under the number MNHN-LBIM-C-1975-1 and there are also two slides from the holotype (one spicule slide and one section slide) deposited in the collection of the Museu Nacional do Rio de Janeiro (MNRJ 40). In the present work we analysed the slides MNRJ 40. The holotype of G. tetela was collected by dredging during a survey of the oceanographic vessel “Almirante Saldanha” along the Northeastern Brazilian Continental Shelf. Recently (2011), five specimens were collected from Curaçao by SCUBA diving and were deposited in the Porifera Collection of the Biology Institute of the Universidade Federal do Rio de Janeiro (UFRJPOR 6714, UFRJPOR 6723, UFRJPOR 6724, UFRJPOR 6746, and UFRJPOR 6767). Species names, voucher numbers, and GenBank accession numbers of the DNA sequences used for a phylogenetic analysis are listed in Table 1. Morphological analyses For the preparation of spicule slides, fragments of the sponge were dissolved in sodium hypochlorite (commercial bleach) in a test tube. After digestion, the spicules were washed five times in distilled water and three times in absolute ethanol. They were then transferred to slides and the ethanol was heat-evaporated. The mounting medium used was Entellan (Merck). For the scanning electron microscopy (SEM), the spicules were placed on a cover-slip mounted on a stub with double-sided carbon tape and sputter-coated with gold. The analysis was performed in a JSM-6510 scanning electron microscope at the Institute of Biology of the Universidade Federal de Rio de Janeiro. For the preparation of the slides sections, small fragments of the sponge were stained with a 5% acid Fuchsin alcoholic solution for 10 min. The excess of Fuchsin was removed with absolute ethanol for 5 min and the fragments were transferred to the slides, covered with some drops of xylene and mounted with Entellan. Spicules measurements were made using an ocular micrometer. The length and the width at the base of each actine were measured for every spicule category. The results are presented in tabular form, featuring length and width (minimum, mean, standard deviation [s], and maximum), and sample size (n). Photographs were taken with a Zeiss AxioCam ERc5s coupled to a ZEISS Stemi 2000C stereoscope and with a digital camera connected to a Zeiss Axioscop microscope. 25

Molecular phylogenetic analyses Genomic DNA was extracted with the guanidine/phenol-chloroform protocol (Sambrook et al. 1989) and stored at –20°C until amplification. The region comprising the partial 18S and 28S, the spacers ITS1 and ITS2 and the 5.8S ribosomal DNA was amplified by PCR with the following primers: 18S (5`-TCATTTAGAGGAAGTAAAAGTCG-3`) and 28S (5`- GTTAGTTTCTTTTCCTC CGCTT -3`) (Lobo-Hajdu et al. 2004). Each PCR amplification reaction mixture contained: 1X buffer (5X GoTaq R Green Reaction Buffer Flexi, PROMEGA),

Table 1. Analyzed specimens with voucher numbers and GenBank accession numbers.

Species Voucher number Genbank accession number Ascaltis reticulum UFRJPOR6258 HQ588973 Ascandra falcata UFRJPOR5856 HQ588962 Ascandra contorta UFRJPOR6327 HQ588970 Arthuria hirsuta ZMAPOR07061 KC843431 Arthuria spiralatta MNRJ12860 KC985139 Borojevia cerebrum UFRJPOR6322 HQ588964 Borojevia brasiliensis UFRJPOR5214 HQ588978 Brattegardia nanseni UFRJPOR6332 HQ588982 Clathrina antofagastensis MNRJ 9289 HQ588985 Clathrina blanca PMR-14307 KC479087 26

Clathrina clathrus UFRJPOR6315 HQ588974 Clathrina lacunosa UFRJPOR6334 HQ588991 Clathrina ramosa MNRJ 10313 HQ588990 Ernstia tetractina UFRJPOR5183 HQ589000 Ernstia sp. UFRJPOR6621 KC843433 Guancha tetela UFRJPOR 6723 KU568492 Leucaltis clathria UFRJPOR 6944 KU568493 Leucaltis nodusgordii QMG316050 AJ633857 Leucettusa nuda MNRJ 10804 KC843453 Leucascus simplex BMOO16283 KC843454 Leucetta floridana PTL09.P100 KC843456 Leucetta potiguar UFPEPOR547 EU781986

Results Class Calcarea Bowerbank, 1864 Subclass Calcinea Bidder, 1898 Order Clathrinida Hartman, 1958 emend. Nicola gen. nov. Etymology: For Nicole Boury-Esnault in recognition of her dedicated work on the taxonomy of sponges, including calcareous sponges. Type species: Nicola tetela (Borojevic & Peixinho, 1976) Diagnosis: Clathrinida with a globular to ovoid body composed of parallel tubes that coalesce at the apical and basal regions. They do not anastomose nor ramify. The skeleton exclusively contains sagittal spicules: triactines and tetractines. The paired actines are rudimentary and they form a straight angle (180°s). The unpaired actine is always the longest actine. Diactines including trichoxeas may be added. The aquiferous system is asconoid.

Nicola tetela comb. nov. Synonyms: Guancha tetela, Borojevic & Peixinho 1976: 998 Material examined: Slide of the holotype (MNRJ 40), Station 1966, Northeastern Brazilian Continental Shelf (Southern coast of Bahia State) (17°55'S, 37°30'W), collected by dredging by the “Almirante Saldanha” (SAL) vessel, 17th August 1968, 47 m deep; UFRJPOR 6714, UFRJPOR 6723, UFRJPOR 6724, Playa Kalki, Westpunt, Curaçao (12°22'29.86ʺ N, 69°09'30.63ʺ W), collected by E. Hajdu and B. Cóndor-Luján, 21st August 2011, 5.6 m deep; UFRJPOR 6746, Sunset Waters, Soto, Curaçao (12°16'01.58ʺ N, 69°07'44.85ʺ W), collected by E. Hajdu, 20th August 2011, 8.9 m deep; 27

UFRJPOR 6767, Sunset Waters, Soto, Curaçao (12°16'01.58ʺ N, 69°07'44.85ʺ W); collected by B. Cóndor-Luján; 22nd August 2011, 9–12 m deep. Colour: bright orange in life and white in ethanol. Description The specimens have a globular (Figure 1A) to ovoid body (Figure 1B), with apical osculum and a peduncle at the base. The peduncle is formed by coalescent tubes with choanoderm. Above the stalk, each tube is divided into two tubes which do not anastomose nor ramify; instead, they run in parallel and then converge to form the osculum (Figure 1C). The aquiferous system is asconoid, with choanocytes, intercalated by porocytes, covering the interior of the tubes (Figure 1D). The surface is smooth and bright and the consistency is fragile. The skeleton is composed of triactines and tetractines arranged in parallel, the triactines being more abundant than the tetractines. The unpaired actine of the spicules is always basipetally oriented (Figure 1E). The apical actine of the tetractines is projected into the lumen of the tubes (Figure 1F). Triactines are equally distributed all over the sponge body, whereas tetractines seem to be more concentrated in the apical region, near the osculum (at least in UFRJPOR 6723). The size of the spicules is very variable and although the unpaired actine is frequently much longer than the paired ones (Figures 1G, H), it sometimes can be only a little longer (Figure 1I). Spicules (Table 2, Figures 1G-K). Triactines. Sagittal. Actines are straight, conical, with sharp tips. The unpaired actine presents a constriction near its base. They present very variable size and are the most abundant spicules (Figures 1G-I). Size: 75.0-440.0/5.0-10.0 µm (unpaired actine); 17.5-60.0/5.0-7.5 µm (paired actine). Tetractines. Sagittal. Actines are straight, conical with sharp tips. The unpaired actine has a constriction near its base (Figures 1G, J). The apical actine is smooth and can be straight or curved. It is longer and, generally, narrower than the paired actines (Figure 1K). They present very variable size. Size: 80.0-370.0/5.0-10.0 µm (unpaired actine); 12.5-45.0/5.0-8.7 µm (paired actine); 17.5-75.0/2.5-6.2 µm (apical actine). Ecology: The Brazilian specimen was collected at 47 m deep in a calcareous-algae bottom, whereas the specimens from Curaçao were found underneath broken corals in shallow waters down to 12 m. No organisms were found on the surface or among the tubes of the studied specimens. 28

Figure 1. Nicola tetela comb. nov. A-B: Live specimens: UFRJPOR 6714 (A) and UFRJPOR 6746 (B) (photos taken by E. Hajdu). C. Specimens after fixation (UFRJPOR 6714, 6723, 6724). D. Tangential section of the skeleton showing choanocytes and porocytes (pc). E. Detail of the apical region of the body. F. Apical actines projected into the lumen of a tube (arrow pointing to one apical actine). G. Spicules: Triactine (left) and tetractine (right). H – K: SEM images of spicules: H. Large triactine. I. Small triactine. J. Tetractine. I. Apical actine of a tetractine. All skeleton and spicule images were taken from the specimen UFRJPOR 6723. 29

Table 2. Spicule measurements of Nicola tetela comb. nov., including the original measurements of the holotype (MNRJ 40) by Borojevic & Peixinho (1976).

Specimen Spicule Actine Length (µm) Width (µm) N Min Mean SD Max Min Mean SD Max UFRJPOR Triactine Unpaired 75.0 223.6 91.7 440.0 5.0 7.5 0.9 10 30 6723 Paired 27.5 31.3 4.3 47.5 5.0 6.4 1.0 7.5 30 Tetractine Unpaired 102.5 202.2 54.9 305.0 5.0 8.0 1.3 10 30 Paired 25.0 31.4 3.2 37.5 5.0 6.1 1.2 8.7 30 Apical 30.0 50.4 10.4 62.5 2.5 3.9 1.0 5 20 UFRJPOR Triactine Unpaired 75.0 231.6 95.3 435.0 5.0 6.5 1.2 8.7 30 6746 Paired 17.5 29.6 5.0 40.0 5.0 5.3 0.7 7.5 30 Tetractine Unpaired 80.0 215.0 110.5 370.0 5.0 6.7 1.0 7.5 6 Paired 25.0 32.5 8.1 45.0 5.0 5.2 0.5 6.3 6 Apical 35.0 58.5 15.5 75.0 2.5 3.3 1.1 5 5 UFRJPOR Triactine Unpaired 102.5 213.5 69.2 375.0 5.0 7.1 1.1 8.7 30 6767 Paired 20.0 33.4 6.7 50.0 5.0 6.3 1.2 7.5 30 Tetractine Unpaired 100.0 173.7 40.4 255.0 5.0 7.0 1.1 8.7 30 Paired 12.5 29.8 7.1 40.0 5.0 5.6 1.0 7.5 30 Apical 17.5 37.5 8.9 47.5 2.5 2.7 0.7 5 12 MNRJ 40 Triactine Unpaired 150.0 - - 400 7.0 - - 10 - (original) Paired 30.0 - - 60 - - - - - MNRJ 40 Triactine Unpaired 100.0 229.3 64.2 375.0 6.3 7.4 0.4 7.5 20 (present Paired 32.5 36.9 3.0 42.5 5.0 6.4 1.0 7.5 20 work) Tetractine Unpaired 145.0 222.3 51.3 315.0 5.0 7.4 0.7 8.7 20 Paired 30.0 35.0 5.0 45.0 5.0 7.1 0.8 8.7 20 Apical 32.5 32.5 0.0 32.5 5.0 5.6 0.9 6.2 2

Remarks: Borojevic & Peixinho (1976) originally described the skeleton of this species as being exclusively composed of triactines. Reanalysing the type material, we also found tetractines, although those spicules were outnumbered by triactines (Figures 2A, B). Because of the scarce quantity of tetractines in the holotype slide, they might have neglected them. Moreover, Borojevic & Peixinho (1976) characterised the spicules as parasagittal, pointing out the straight angle (90°s) formed by the unpaired and paired actines. We do recognize the referred angle, however, we consider that it would be more correct to characterise these spicules as sagittal, according to Boury-Esnault & Rützler (1997). The molecular analysis produced the same tree topology with both phylogenetic methods (ML and BI) and recovered the lineages found by Klautau et al. (2013) (Figure 3). Nicola tetela comb. nov. did not cluster with any of the already known genera, not even Arthuria, confirming that it is a new genus. 30

Figure 2. Photographs taken from the original slide of the holotype (MNRJ 40). A. Apical region. B. Basal region. In each photo a tetractine is indicated by an arrow.

Figure 3. Bayesian 50% majority rule consensus tree (106 trees sampled; burn-in =1000 trees) inferred from the ITS rDNA sequences under the GTR model. Bayesian posterior probabilities (BI) and bootstrap (ML) are given on the branches. 31

Discussion The anastomosis of the cormus is an important taxonomic character in the order Clathrinida. For example, all species of the genus Ascandra show a different anastomosis, with tubes free at least at the apical region, while Arthuria, Borojevia, Brattegardia, Clathrina, and Ernstia have well anastomosed tubes. In the new genus Nicola, tubes are not anastomosed but run in parallel and are reconnected at the base and at the osculum. Another remarkable morphological character of Nicola gen. nov. is its skeleton composed exclusively of sagittal spicules, which has not been found in any other Calcinean genus. The triactines with reduced paired actines present in Nicola gen. nov. are even similar to the “nail-spicules” found in the Calcaronean genera Kebira and Grantiopsis (Calcaronea: Lelapiidae), most probably as a result of convergence. Our results point that spicule shape and composition together with the organisation of the body are very important characters for the taxonomy of Calcinea. Considering only spicule composition we would expect to find a closer proximity of Nicola gen. nov. with Arthuria, however, we observed in our phylogenetic tree that they are not sister genera. This means that the presence of only sagittal spicules and the differentiated organisation of the cormus in Nicola gen. nov. are strong characters that define well this new genus as separated from all the others known up to date.

Acknowledgments We are indebted to E. Hajdu and G. Lôbo-Hajdu for assistance and photographing during the sample collections. Mark Vermeij and CARMABI are acknowledged for providing logistical support in Curaçao. B. C. L. received scholarship from the Brazilian National Research Council (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES). M.K. is funded by fellowships and research grants from the CNPq, CAPES, and the Rio de Janeiro State Research Foundation (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro - FAPERJ). This paper is part of the DSc. requirements of Báslavi Cóndor Luján at the Biodiversity and Evolutionary Biology Program of the Federal University of Rio de Janeiro.

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Calcareous sponges (Porifera: Calcarea) from Curaçao including Brazilian shared species and phylogenetic remarks

Báslavi Cóndor-Luján1, Taynara Louzada1,2, Fernanda Azevedo1, André Padua1, Eduardo Hajdu3 & Michelle Klautau1

1Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Av. Carlos Chagas Filho, 373, CEP 21941-902, Rio de Janeiro, RJ, Brasil. 2 Universidade Federal do Estado do Rio de Janeiro, Instituto de Biociências, Av. Pasteur, 458, Urca, CEP 22290-240, Rio de Janeiro, RJ, Brasil. 3Universidade Federal do Rio de Janeiro, Museu Nacional, Departamento de Invertebrados, Quinta da Boa Vista, São Cristóvão, CEP 20940-040, Rio de Janeiro, RJ, Brasil.

Corresponding author: M. Klautau, [email protected] Journal: Journal of the Marine Biological Association of the United Kingdom.

ABSTRACT This is the first inventory of calcareous sponges from Curaçao, Caribbean Sea. The studied material comprised specimens sampled along the Curaçaoan Island as well as some Brazilian specimens previously collected. They were analysed using morphological and molecular (ITS and C-LSU) approaches. A total of 18 species were found and are described here: 11 calcineans and seven calcaroneans. Eleven of these species are new to science; nine are provisionally endemic to Curaçao, Amphoriscus micropilosus sp. nov., Arthuria vansoesti sp. nov., Clathrina curaçaoensis sp. nov., Grantessa tumida sp. nov., Leucandra caribea sp. nov., Leucandrilla pseudosagittata sp. nov., Leucilla antillana sp. nov., Sycon conulosum sp. nov., Sycon magniapicalis sp. nov., and two are shared between Curaçao and Brazil, Ascandra torquata sp. nov. and Clathrina aspera sp. nov. The formerly Brazilian endemic species C. lutea, C. insularis, C. mutabilis and Borojevia tenuispinata have their distribution widen to the Caribbean Sea. Clathrina cf. blanca, C. hondurensis and Leucetta floridana are new records for Curaçaoan waters. The new phylogenetic affinities in Calcaronea as well as already reported Calcinean relationships recovered in the molecular analyses are discussed.

Key words: Biodiversity, Calcinea, Calcaronea, Caribbean Sea, Southern Caribbean ecoregion, Systematics, Western Tropical Atlantic. 35

INTRODUCTION The Caribbean Sea is considered a global-scale hotspot of marine biodiversity (Roberts et al., 2002). In its 2,754,000 km2 and over 13,500 km of coastline, it encompasses a high diversity of flora and fauna distributed in different ecosystems including coral reefs, mangroves, seagrasses and other environments (Miloslavich et al., 2010). The sponges (Phylum Porifera) constitute one of the most diverse benthic faunal groups in the subtidal habitats of coral reefs and mangroves (Diaz & Rützler, 2011), however, the studies on sponge diversity are mostly restricted to the conspicuous species of the class Demospongiae. As species of the class Calcarea are usually small and devoid of colour (Wörheide & Hooper, 1999; Rapp, 2006) and inhabit light protected or cryptic environments (e.g. overhangs, caves, crevices), they are easily neglected in sponge fauna inventories. Furthermore, the plasticity of a few morphological characters within some Calcarean taxa makes the identification of these species difficult, sometimes requiring complementary approaches such as molecular analyses (e.g. Valderrama et al., 2009; Imešek et al., 2014; Azevedo et al., 2015; Klautau et al., 2016). The class Calcarea comprises species with skeleton composed exclusively of calcium carbonate. It is divided in two monophyletic subclasses, Calcinea and Calcaronea Bidder, 1898. Phylogenetic relationships within these two subclasses are still not well-understood as many orders, families and even genera are polyphyletic (Voigt et al., 2012; Voigt & Wörheide, 2016). However, certain skeletal traits have evidenced phylogenetic signal in some Calcinean genera (Rossi et al., 2011; Klautau et al., 2013). Up to date, only 24 calcareous sponges have been reported from the Caribbean Sea (Cóndor- Luján & Klautau, 2016; Van Soest et al., 2016;) including representatives of both subclasses. In a similar geographical range such as the Mediterranean Sea, whose basin comprises 2,969,000 km2 (Coll et al., 2010), the number of recorded calcareous species is more than doubled; including ca. 65 species (Van Soest et al., 2016). Among the Caribbean coral reefs, the Curaçaoan reefs are considered some of the healthiest reefs, harbouring a great diversity of organisms including endemic species (Vermeij, 2012). Although the sponge diversity within this island has been intensively assessed for many years, very few calcareous species were reported for Curaçao. The pioneering study of Arndt (1927) included the first record of the subclass Calcarea, Leucilla amphora Haeckel, 1872. Further studies compelling shallow-water sponges (Van Soest, 1978, 1980, 1981, 1984; Hajdu & Van Soest, 1992; Alvarez et al., 1998; De Weerdt, 2000), sponges from cryptic habitats (Van Soest, 2009) and deep-water sponges (Van Soest, 2014) did not report any other Calcarean species, although one of them did mention the presence of calcareous sponges (Van Soest, 1981). More 36 recently, a former Brazilian endemic species, Nicola tetela (Borojevic & Peixinho, 1976) was recorded for this island (Cóndor-Luján & Klautau, 2016). This very low number of records may just reflect the lack of taxomomic expertise in this region, reinforcing the necessity of faunistical studies on Calcarea. The present work aimed to start filling the gap on the knowledge of Calcarea from the Caribbean Sea through the description of the calcareous sponge fauna of Curaçao, integrating morphological and molecular information. We also discuss the phylogenetic relationships within Calcarea considering the species described here.

MATERIALS AND METHODS Abbreviations BMNH = The Natural History Museum, London, United Kingdom GW = Gert Wörheide IRB = Institut Ruđer Bošković, Zagreb, Croatia MM = Michel Manuel MNRJ = Museu Nacional do Rio de Janeiro, Brazil PMJ = Phyletisches Museum Jena, Germany PMR = Prirodoslovni Muzej Rijeka, Croatia QM = Queensland Museum, Australia SAM = South Australian Museum, Australia UFRJPOR = Porifera collection of the Biology Institute of Universidade Federal do Rio de Ja- neiro (UFRJ), Brazil WAMZ = Zoological collection of the Western Australian Museum, Perth, Australia ZMAPOR = Zoölogisch Museum, Instituut voor Systematiek en Populatiebiologie, Amsterdam, The Netherlands

Study area: Curaçao Curaçao is a volcanic island located in the Leeward Antilles Ridge, a boundary zone between the Caribbean and the South American Tectonic Plates. It was originated in the Cretaceous Period and received different Miocenic sediment depositions. Its current geology has been shaped during the Quaternary (Hyppolyte & Mann, 2011). It is localised in the southern Caribbean Basin, at 60 km north from Venezuela (Figure1A) and it is included in the Southern Caribbean ecoregion of the Tropical Northwestern Atlantic 37

Province (TNA - Spalding et al., 2007). This island has a total area of 444 km2 and it is surrounded by a fringing reef with 7.85 km2 situated 20-250 m from the coast (Vermeij, 2012).

Fig. 1. Study area. (A) location of Curaçao in the Caribbean Sea; (B) localities sampled along the coast of Curaçao.

Analysed Material In this study, a total of 50 specimens were analysed. The material included specimens collected in Curaçao in 2011 as well as Brazilian specimens already deposited in the Porifera Collection of the Universidade Federal do Rio de Janeiro, Brazil (UFRJPOR) and that were conspecific with the Curaçaoan specimens. In Curaçao, eight localities along the western coast were surveyed (Figure 1B). The collections were performed by SCUBA down to 20 m of depth. The specimens were photographed in situ and carefully removed from the substrate with the aid of forceps and small knives. At the CARMABI (Caribbean Research and Management of Biodiversity) Marine Research Station, they were fixed in 96% ethanol. Brazilian specimens were previously collected by SCUBA at depths ranging from four to 15 m, in localities within the southeastern coast (Rio de Janeiro State) and in a southern island (Arvoredo Island in the Santa Catarina State). All the samples are preserved in 96% ethanol and deposited in the UFRJPOR Collection.

Morphological analyses The external morphology was examined through the observation of macroscopic characters on the fixed specimens and complemented with information from the in situ pictures. The anatomy was assessed through the analysis of the skeleton composition obtained from microscopy slides. The preparation of section and spicule slides as well as the spicule measurements followed 38 standard procedures (Wörheide & Hooper, 1999; Klautau & Valentine, 2003; Cóndor-Luján & Klautau, 2016). The spicule measurements are presented in tabular form, featuring length and width (minimum, mean, standard deviation [SD] and maximum). Spicule measurements of the species used for comparative purposes were obtained from the original descriptions or from more detailed recent descriptions of the type material. The species identifications followed the Systema Porifera (Hooper & Van Soest, 2002) and additional literature (Klautau & Valentine, 2003; Klautau et al., 2013; Azevedo et al., submitted). To illustrate the species descriptions, photographs were taken with a digital Canon camera coupled to a Zeiss Axioscop microscope. Scanning electron microscopy (SEM) micrographs of particular spicule ornamentations were taken at the Biology Institute of the UFRJ using a JSM- 6510 SEM microscope. The spicule preparation for SEM images followed Azevedo et al. (2015).

Molecular analyses The total genomic DNA was extracted using the guanidine/phenol-chloroform protocol (Sambrook et al., 1989) or with a QIAamp DNA MiniKit (Qiagen) and stored at –20°C until amplification. Two DNA regions were amplified. The C-region of the 28S (C-LSU) was amplified using the primers fwd: 5'GAAAAGCACTTTGAAAAGAGA-3' (Voigt & Worheide, 2015) and rv: 5'-TCCGTGTTTCAAGACGGG-3' (Chombard et al., 1998) and the region containing the partial genes 18S and 28S, the spacers ITS1 and ITS2 and the 5.8S ribosomal DNA (named herein as ITS) was amplified with the primers: fwd: 5`- TCATTTAGAGGAAGTAAAAGTCG-3` and rv: 5`-GTTAGTTTCTTTTCCTCCGCTT-3`) (Lôbo-Hajdu et al., 2004). The ITS region was only amplified for calcinean species. The PCR mixture included 1x buffer (5x GoTaq® Green Reaction Buffer Flexi,

platform (Katoh & Standley, 2013) using the strategy Q-INS-i (Katoh & Toh, 2008) which provides a better alignment as it considers the secondary structure of the amplified region. The nucleotide substitution model that best fit the alignment was GTR+G+I for both DNA regions, as indicated by the Bayesian Information Criterion in MEGA 6 (Nei & Kumar, 2000; Tamura et al., 2013). Phylogenetic reconstructions were performed under Maximum Likelihood (ML) and Bayesian Inference (BI) approaches. The ML analyses were conducted on MEGA 6 using an initial NJ tree (BIONJ) and a 1000 pseudo-replicates bootstrap. The BI reconstructions were obtained with MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003) under 3.5 x 106 generations and a burn-in of 3,500 sampled trees, yielding a consensus tree of majority.

Table 1. Species used in the phylogenetic analyses with locality, voucher number and GenBank (GB) accession number. *Sequences generated in the present study.

Species Locality Voucher number GB accession number CALCINEA LSU ITS Ascandra contorta Mediterranean UFRJPOR6327 - HQ588970 Ascandra coralicolla Norway UFRJPOR6329 - HQ588994 Ascandra falcata Mediterranean UFRJPOR5856 - HQ588962 Ascandra sp. Polynesia BMOO16290 - KC843446 Ascandra spalatensis Adriatic Sea UFRJPOR7540 - KP740024 Ascandra torquata sp. nov.* Brazil UFRJPOR6080 - This study Ascandra torquata sp. nov. Brazil UFRJPOR6084 - KC843448 Ascandra torquata sp. nov.* Curaçao UFRJPOR6738 - This study Borojevia aff. aspina Brazil UFRJPOR5245 - HQ588998 Borojevia aff. aspina Brazil UFRJPOR5495 HQ589017 - Borojevia brasiliensis Brazil UFRJPOR5214 HQ589015 HQ588978 Borojevia cerebrum Mediterranean UFRJPOR6322 HQ589008 HQ588964 Borojevia croatica Adriatic Sea IRB-CLB6 - KP740023 Borojevia sp. QMG313824 JQ272287 - Borojevia tenuispinata Brazil UFRJPOR6484 - KX548916 Borojevia tenuispinata Brazil UFRJPOR6492 - KX548917 Borojevia tenuispinata* Curaçao UFRJPOR6700 This study This study Borojevia trispinata Brazil UFRJPOR6487 - KX548919 Clathrina aspera* Brazil UFRJPOR5531(P) - This study Clathrina aspera* Brazil UFRJPOR6346 This study Clathrina aspera* Brazil UFRJPOR6472 This study Clathrina aspera* Brazil UFRJPOR6510 This study Clathrina aspera* Curaçao UFRJPOR 6758 This study - Clathrina aurea Brazil MNRJ 8998 HQ589005 HQ588968 Clathrina clathrus Mediterranean UFRJPOR6315 HQ589009 HQ588974 Clathrina conifera Brazil UFRJPOR8991 HQ589010 HQ588959 Clathrina coriacea Norway UFRJPOR6330 HQ589001 HQ588986 40

Clathrina curaçaoensis sp. Curaçao UFRJPOR6734 This study This study nov.* Clathrina cylindractina Brazil UFRJPOR 5206 HQ589007 HQ588979 Clathrina fjordica Chile MNRJ8143 HQ588984 Clathrina fjordica Chile MNRJ9964 HQ589016 - Clathrina helveola Australia QMG313680 JQ272291 HQ588988 Clathrina insularis Brazil UFRJPOR6527 - KX548920 Clathrina insularis* Brazil UFRJPOR6530 - This study Clathrina insularis Brazil UFRJPOR6532 - KX548921 Clathrina insularis* Brazil UFRJPOR6533 - This study Clathrina insularis Brazil UFRJPOR6536 - KX548922 Clathrina insularis* Brazil UFRJPOR6537 - This study Clathrina insularis* Curaçao UFRJPOR6737 This study KC843435 Clathrina lutea Brazil UFRJPOR5172 HQ589004 HQ588961 Clathrina lutea Brazil UFRJPOR5173 - HQ588976 Clathrina lutea Brazil UFRJPOR 6543 - KX548923 Clathrina lutea Brazil UFRJPOR 6545 - KC843442 Clathrina lutea* Curaçao UFRJPOR6761 - KC843445 Clathrina lutea Virgin Islands ZMAPOR08344 - KC843444 Clathrina lutea Florida, USA UFRJPOR5818 - KC843443 Clathrina luteoculcitella Australia QMG313684 JQ272283 HQ588989 Clathrina mutabilis* Brazil UFRJPOR6525 - This study Clathrina mutabilis Brazil UFRJPOR6526 - KX548925 Clathrina mutabilis Brazil UFRJPOR6528 - KX548926 Clathrina mutabilis* Brazil UFRJPOR6539 - This study Clathrina mutabilis* Brazil UFRJPOR6540 - This study Clathrina mutabilis* Curaçao UFRJPOR6704 - This study Clathrina mutabilis* Curaçao UFRJPOR6719 - This study Clathrina mutabilis Curaçao UFRJPOR6733 - KC843436 Clathrina mutabilis* Curaçao UFRJPOR 6735 - This study Clathrina mutabilis* Curaçao UFRJPOR6740 - This study Clathrina mutabilis* Curaçao UFRJPOR6741 This study KC843437 Clathrina mutabilis* Curaçao UFRJPOR6743 - This study Clathrina mutabilis* Curaçao UFRJPOR6744 - This study Clathrina mutabilis* Curaçao UFRJPOR6745 - This study Clathrina mutabilis* Curaçao UFRJPOR6747 - This study Clathrina sp. Polynesia UF:Porifera:1600 - KC843438 Clathrina sp. Polynesia UFRJPOR6461 - KC843439 Clathrina wistariensis Australia QMG313663 JQ272303 HQ588987 Clathrina zelinhae* Brazil UFRJPOR 6627 This study - Leucetta chagosensis - BMOO16210 -- - Leucetta floridana Panama PTL09.P100 - KC843456 Leucetta floridana* Curaçao UFRJPOR6726 - This study Leucetta floridana* Curaçao UFRJPOR6765 - This study Leucetta antarctica Antarctic MNRJ13798 - KC849700 Leucetta microraphis Australia QMG313659 - AJ633874 Leucetta pyriformis Antarctic MNRJ13843 - KC843457 Leucetta potiguar Brazil UFPEPOR569 - EU781987 Nicola tetela Curaçao UFRJPOR6723 KU568492 - 41

CALCARONEA LSU Amphoriscus micropilosus sp. nov. * Curaçao UFRJPOR6755(P) This study - Anamixilla torresi - - AY563636 - Aphroceras sp. SAM-PS0349 JQ272273 - Eilhardia schulzei QMG316071 JQ272256 - Grantessa tumida sp. nov.* Curaçao UFRJPOR6701(P) This study - Grantessa tumida sp. nov.* Curaçao UFRJPOR6695(P) This study - Grantessa aff. GW979 JQ272278 - intusarticulata Grantia compressa - - AY563538 - Grantiopsis heroni Australia QMG313670 JQ272261 - Grantiopsis cylindrica Australia GW973 JQ272261 - Leucandra aspera - - AY563535 - Leucandra falakra Adriatic Sea UFRJPOR8349 KT447560 - Leucandra nicolae QMG313672 JQ272268 - Leucandra sp. QMG316285 JQ272265 - Leucandra spinifera Adriatic Sea UFRJPOR8348 KT447561 Leucandrilla pseudosagittata sp. nov.* Curaçao UFRJPOR6696 This study - L. pseudosagittata sp. nov.* Curaçao UFRJPOR6705 This study - Leucascandra caveolata QMG316057 JQ272259 - Leucilla antillana sp. nov.* Curaçao UFRJPOR6768 This study - Leuconia nivea - - AY563534 - Paraleucilla dalmatica Adriatic Sea UFRJPOR8346 KT447566 - Paraleucilla magna - GW824 KT447564 - Petrobiona massiliana - GW1729 JQ272307 - Sycettusa aff. hastifera Red Sea GW893 JQ272267 - Sycettusa cf. simplex Western India ZMAPOR11566 JQ272279 - Sycettusa sp. - MM-2004 AY563530 - Sycettusa tenuis Australia QMG313685 JQ272281 - Sycon ancora Adriatic Sea UFRJPOR8347 KT447568 - Sycon capricorn - QMG316187 JQ272272 - Sycon carteri Australia SAM-PS0143 JQ272260 - Sycon ciliatum - - AY563532 - Sycon conulosum sp. nov.* Curaçao UFRJPOR 6707 This study - Sycon cf. villosum - GW51115 KR052809 - Sycon magniapicalis sp. Curaçao UFRJPOR6748 This study - nov.* Sycon magniapicalis sp. Curaçao UFRJPOR6763 This study - nov.* Sycon raphanus - - AY563537 - Syconessa panicula Australia QMG313672 AM181007 - Synute pulchella - WAMZ1404 JQ272274 - Teichonopsis labyrinthica - SAMPS0228 JQ272264 - Utte aff. syconoides - QMG323233 JQ272269 - Utte aff. syconoides - QMG313694 JQ272271 - Ute ampullacea - QMG313669 JQ272266 - Vosmaeropsis sp. - MM-2004 AY026372 - 42

RESULTS Taxonomy Integrating traditional morphological examination with molecular phylogenetic analyses, we recognised 18 species including 11 calcineans and seven calcaroneans. Within the subclass Calcinea, the richest genus was Clathrina with seven species: C. aspera sp. nov., Clathrina cf. blanca, C. curaçaoensis sp. nov., C. hondurensis, C. insularis, C. lutea, and C. mutabilis. In Calcaronea, Sycon was the richest genera with two species: Sycon conulosum sp. nov and Sycon magnapicalis sp. nov. The calcinean genera Ascandra and Borojevia as well as the calcaronean Leucandrilla and Grantessa constitute not only new records for Curaçao, but also for the Caribbean Sea.

Systematic Index Class CALCAREA Bowerbank, 1862 Subclass CALCINEA Bidder, 1898 Order CLATHRINIDA Hartman, 1958 Genus Arthuria Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013 Arthuria vansoesti sp. nov. Genus Ascandra Haeckel, 1872 Ascandra torquata sp. nov. Genus Borojevia Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013 Borojevia tenuispinata Azevedo et al., submitted Genus Clathrina Gray, 1867 sensu Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013 Clathrina aspera sp. nov. Clathrina cf. blanca Miklucho-Maclay, 1868 Clathrina curaçaoensis sp. nov. Clathrina hondurensis Klautau & Valentine, 2003 Clathrina insularis Azevedo et al., submitted Clathrina lutea Azevedo et al., submitted Clathrina mutabilis Azevedo et al., submitted Genus Leucetta Haeckel, 1872 Leucetta floridana Haeckel, 1872 Subclass CALCARONEA Bidder, 1898 Order LEUCOSOLENIIDA Hartman, 1958 43

Family AMPHORISCIDAE Dendy, 1893 Genus Amphoriscus Haeckel, 1872 Amphoriscus micropilosus sp. nov. Genus Leucilla Haeckel, 1872 Leucilla antillana sp. nov. Family GRANTIIDAE Dendy, 1893 Genus Leucandra Haeckel, 1872 Leucandra caribea sp. nov. Genus Leucandrilla Borojevic, Boury-Esnault & Vacelet, 2000 Leucandrilla pseudosagittata sp. nov. Family HETEROPIIDAE Dendy, 1893 Genus Grantessa Lendenfeld, 1885 Grantessa tumida sp. nov. Family SYCETTIDAE Dendy, 1892 Genus Sycon Risso, 1827 Sycon conulosum sp. nov. Sycon magniapicalis sp. nov.

Description of taxa

Genus Arthuria Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013 TYPE SPECIES Arthuria hirsuta (Klautau & Valentine, 2003). DIAGNOSIS "Calcinea in which the cormus comprises a typical clathroid body. A stalk may be present. The skeleton contains regular (equiangular and equiradiate) triactines and tetractines. However, tetractines are more rare. Diactines may be added. Asconoid aquiferous system" (Klautau et al. 2013).

Arthuria vansoesti sp. nov. (Figure 2, Table 2) ETIMOLOGY Named after Rob van Soest in recognition of his dedicated work on the taxonomy of the sponges, including those from Curaçao. 44

TYPE LOCALITY Daai Booi, St. Willibrordus, Curaçao. TYPE MATERIAL Holotype: UFRJPOR 6720 (specimen in ethanol and slides); Daai Booi, St. Willibrordus; 12°12'43.12"N, 69°05'8.42''W; 5.2 m deep; coll. B. Cóndor-Luján, 19 August 2011. COLOUR Yellow in life and white to beige in ethanol. MORPHOLOGY AND ANATOMY This species has a massive and smooth cormus. The holotype is 0.7 x 0.6 x 0.2 cm (Figures 2A- B). The cormus is composed of irregular and loosely anastomosed tubes. A water-collecting tube (2 x 1 mm) was present in the centre of the cormus of the holotype (arrow in Figure 2A). The aquiferous system is asconoid. The skeleton has no special organization (Figure 2C) and it is composed of abundant triactines (two shape categories) and rare tetractines. SPICULES Triactines I. Regular (equiangular and equiradiate). Most abundant spicules. Actines are cylindrical, slightly undulated at the distal part and with rounded tips (Figure 2D). They resemble the triactines of Clathrina aurea. Size: 72.5-87.5/3.8-5 µm. Triactines II. Regular (equiangular and equiradiate). Rare. Actines are straight, slightly conical with blunt to sharp tips (Figure 2E). Size: 52.5-82.5/2.5-5 µm. Tetractines. Regular (equiangular and equiradiate). Basal actines are straight, cylindrical, with rounded to blunt tips (Figure 2F). The apical actine is the shortest actine. It is straight and smooth; however, some curved actines were found (Figure 2G). It has sharp or blunt tip. Size: 60-87.5/3.8-5 µm (basal actine) and 25/3.8-5 µm (apical actine). ECOLOGY This sponge was found in a cryptic habitat, underneath coral boulders. GEOGRAPHIC DISTRIBUTION Southern Caribbean (provisionally endemic to Curaçao, present study). REMARKS The genus Arthuria now comprises 11 valid species, A. africana (Klautau & Valentine, 2003), A. alcatraziensis (Lanna et al., 2007); A. darwinii (Haeckel, 1870); A. dubia (Dendy, 1891); A. hirsuta (Klautau & Valentine, 2003); A. spirallata Azevedo et al., 2015; A. sueziana (Klautau & Valentine, 2003); A. tenuipilosa (Dendy, 1905); A. trindadensis Azevedo et al., submitted, A. tu- buloreticulosa Van Soest & de Voogd, 2015 and A. vansoesti sp. nov. Most of them possess a skeleton mainly composed of triactines with conical actines except for A. dubia, A. sueziana, A. 45 tubuloreticulata and A. vansoesti sp. nov. whose skeletons also include triactines with cylindrical actines. Arthuria vansoesti sp. nov. can be easily distinguished from A. dubia and A. sueziana because they have different spicule width (Table 2), being thinner in the new species (2.5–5.0 µm) than in A. dubia (13.7 – 17.0 µm) and A. sueziana (8.0 – 11.8 µm). Besides, C. dubia has granular cells which are absent in the new species. The species whose skeleton more resembles A. vansoesti sp. nov. is the Indonesian A. tubuloreticulata, however, they present some differences. A. tuboloreticulata is orange in live and its cormus has several oscula whereas the new species is yellow in life and presents water-collecting tubes. Moreover, the spicules of the Curaçaoan species have rounded tips and in the Indonesian species, they are sharp or blunt.

Table 2. Spicule measurements of Arthuria vansoesti sp. nov. (UFRJPOR 6720), A. dubia (BMNH 1891.9.19.2) taken from Klautau & Valentine (2003), A. sueziana (BMNH 1912.2.1.3) and A. tuboreticulata (RMNH Por. 5547). H=holotype, L=lectotype.. B=basal and A=apical actines.

Length (µm) Width (µm) N Specimen Spicule Actine Min Mean SD Max Min Mean SD Max UFRJPOR Triactine I 72.5 79.8 4.9 87.5 3.8 4.6 0.6 5.0 30 6720 (H) Triactine II 52.5 70.4 7.8 82.5 2.5 3.9 0.8 5.0 30 Tetractine B 60.0 75.3 8.1 87.5 3.8 4.8 0.5 5.0 15 A 25.0 25.0 0.0 25.0 3.8 4.4 0.7 5.0 6 BMNH Diactine 77.5 256.0 97.0 418.2 - 13.7 5.0 - 30 1891.9. Triactine 92.5 151.5 14.0 170.0 - 15.5 1.8 - 30 19.2 (L)* Tetractine B 120.0 140.3 9.5 155.0 - 16.0 1.0 - 9 A 100.0 110.0 6.8 117.5 - 9.8 0.5 - BMNH Trichoxea 250.0 - - - <0.3 - - - 1912.2. Triactine 75.0 91.3 10.9 137.5 - 10.3 1.5 - 30 1.3 (H) Tetractine B 70.0 86.0 6.1 97.5 - 9.4 1.4 - 30 A 50.0 56.3 4.5 62.5 - 5.0 0.0 - 4 RMNH Triactine 63 112.1 - 138 4 5.3 - 6.5 - Por. 5547 Tetractine B 62 119.5 - 156 4 5.4 - 6 - (H) A 39 - - 132 3.5 - - 6.5 - 46

Fig. 2. Arthuria vansoesti sp. nov. (UFRJPOR 6720): (A) specimen in vivo; (B) specimen after fixation; (C) tangential section of the body (cormus); (D) triactine I; (E) triactine II; (F) tetractine; (G) apical actine of tetractine. 47

Genus Ascandra Haeckel, 1872 TYPE SPECIES Ascandra falcata Haeckel 1872 DIAGNOSIS " Calcinea with loosely anastomosed tubes. Tubes are free, at least in the apical region. The skeleton contains regular (equiangular and equiradiate) or sagittal triactines and tetractines. The apical actine is very thin (needle-like) or very thick at the base. Diactines may be added. Asconoid aquiferous system" (Klautau et al., 2016, emend).

Ascandra torquata sp. nov. (Figures 3 & 4, Table 3) ETIMOLOGY From the Latin torquere (= twist), for the twisted tip of the apical actine of the tetractines. TYPE LOCALITY Ponta do Vidal, Arvoredo Island, Reserva Biológica Marinha (REBIOMAR) do Arvoredo, Santa Catarina, Brazil. TYPE MATERIAL Holotype (specimen in ethanol and slides) UFRJPOR 6084, Ponta do Vidal, REBIOMAR Arvoredo, Santa Catarina, Brazil; 27°17’52.3’’S, 48°21’33.4’’W; 10-15 m deep; coll. F. Azevedo, J. Carraro and A. Padua, 11 December 2009. Paratypes (specimens in ethanol and slides) UFRJPOR 6080 and UFRJPOR 6082, Ponta do Vidal, REBIOMAR Arvoredo, Santa Catarina, Brazil; 27°17'52.3"S, 48°21'33.4"W; 10-15 m deep; coll. F. Azevedo, J. Carraro and A. Padua, 11 December 2009. UFRJPOR 6738, Playa Jeremi, Soto, Curaçao; 12°19'43.73"N, 69°09'07.80"W; 15 m deep; coll. B. Cóndor-Luján and E. Hajdu, 22 August 2011. COLOUR White in life and beige to light brown in ethanol. MORPHOLOGY AND ANATOMY The holotype (UFRJPOR 6084) measures 4.0 x 1.5 x 1.5 cm (Figure 3A). Cormus varying from encrusting to massive, fragile in consistency and composed of irregular and loosely anastomosed tubes at the base and free at the apical region. In fact, at the apical region, there is no anastomosis as large vertical tubes arise and connect themselves culminating in oscula with variable diameters (1-3 mm, Figure 3B). At the base, the surface is smooth while at the apical region, it is very hispid due to diactines protruding the surface. Few diactines were also found in the basal region of some specimens. The aquiferous system is asconoid. The skeleton has no 48 special organization and it is composed of abundant tetractines differentiated in two size categories and of less frequent triactines and diactines (Figure 3C-D). The specimen from Curaçao (UFRJPOR 6738, Figure 4A) is partitioned as shown in Figure 4B. The largest fragment is 5 mm long and the free tubes can reach 5 mm high. In this specimen, diactines were not observed (Figure 4C). SPICULES (Table 3) Diactines. Slightly sinuous with sharp tips (Figure 3E). Very variable size (length/width): 100.0- 395.0/5.0-17.5 µm. Triactines. Regular (equiangular and equiradiate). Actines are conical, straight, with blunt to sharp tips (Figures 3F-G and 4D). Subregular triactines were also found. Size (length/width): 45.0-197.5/7.5-15.0 µm. Tetractines I. Regular (equiangular and equiradiate). The basal actines are slightly conical, straight, with blunt to sharp tips (Figures 3H and 4E). The apical actine is smooth and conical. Most apical actines are characteristically twisted, however, some straight ones with sharp tips were also observed. Size (length/width): 57.0-225.0/5.0-22.5 µm (basal actine) and 50.0- 167.5/6.2-14.0 µm (apical actine). Tetractines II. Regular (equiangular and equiradiate). Larger than tetractines I. The basal actines are conical, straight, with blunt to sharp tips (Figure 3I and 4F). The apical actine is thinner than the basal ones. It is smooth, straight, conical, with sharp tips. Some twisted apical actines were also observed (Figure 3J). Size (length/width): 170.0-487.5/10.0-37.5 µm (basal actine) and 52.5-205.0/12.5-32.5 µm (apical actine). ECOLOGY Specimens inhabited sunlight protected environments. Brazilian specimens were collected in vertical walls of large boulders while the Curaçaoan specimen was found underneath small boulders. Amphipods, bryozoans, ophiuroids, polychaets and hydrozoans were found living in association with the Brazilian specimens. GEOGRAPHIC DISTRIBUTION Disjunct distribution in the Western Atlantic: Southern Caribbean (Curaçao) and Southern Brazil ecoregion of the Warm Temperate Southwestern Atlantic Province (Santa Catarina State). REMARKS Ascandra is currently composed of 15 species. Ascandra torquata sp. nov. can be easily distinguished from all them by the twisted apical actine of the tetractines which is absent in all the other 14 known species. This new species is the third Ascandra registered for the Atlantic Ocean 49

- the other two are A. ascandroides (Borojević, 1971) from Brazil and A. corallicola (Rapp, 2006) from Norway. A previous study evidenced that trichoxeas were not good taxonomic characters for identifying Clathrina species as they could be present or not in C. mutabilis (Azevedo et al., submitted). The same seems to be valid for the diactines of A. torquata sp. nov. as they were present in the specimens from Brazil but not in the one from Curaçao. Despite having this morphological difference, the molecular analysis confirmed their conspecificity (Figure 31). Therefore, at least for A. torquata sp. nov., diactines are not a reliable taxonomic character. The clade of Ascandra was well supported in our ITS phylogenetic tree (pp=1, b=99, Figure 31), confirming the validity of this genus with the present diagnosis. In that tree, A. torquata sp. nov. is a sister species of the type species of the genus A. falcata and of A corallicola. The ITS sequence of A. torquata sp. nov. (UFRJPOR 6084) had already been published by Klautau et al. (2013) as Clathrina sp. nov. 11.

Table 3. Spicule measurements of Ascandra torquata sp. nov. H=holotype; P=paratype.

Specimen Spicule Actine Length (µm) Width (µm) N Min Mean SD Max Min Mean SD Max UFRJPOR Diactine - 100.0 208.9 90.7 382.5 7.5 12.0 3.0 17.5 30 6084 (H) Triactine - 45.0 138.5 40.4 197.5 7.5 11.2 1.9 15 30 Tetractine I basal 120.0 163.2 22.5 212.5 12.5 15.2 1.4 17.5 30 apical 72.5 112.0 30.8 167.5 7.5 8.8 1.7 14 30 Tetractine II basal 195.0 256.1 38.8 337.5 10 26.6 6.6 37.5 22 apical 52.5 101.2 31.8 150.0 12.5 19.6 2.8 22.5 12 UFRJPOR Diactine - 115 245.5 53.5 335.0 5 10.8 2.4 15 30 6080 (P) Triactine - 67.5 116.5 24.2 157.5 7.5 9.8 1.5 12.5 30 Tetractine I basal 57.5 124.5 28.4 165.0 5 12.7 3.5 22.5 30 apical 62.5 112.2 26.5 162.5 7.5 9.6 1.8 12.5 25 Tetractine II basal 207.5 360.7 81.6 487.5 21.2 32.1 6.3 32.1 30 apical 80.0 139.9 32.3 190.0 17.5 25.2 3.5 32.5 30 UFRJPOR Diactine - 142.5 255.8 73.1 395.0 5.0 14.3 2.8 15.0 17 6082 (P) Triactine - 97.5 150.3 23.9 192.5 7.5 12.3 2.1 12.5 30 Tetractine I basal 100.0 159.8 27.9 225.0 5.0 13.7 2.5 22.5 30 apical 65.0 118.5 19.9 150.0 7.5 10.5 2.2 12.5 30 Tetractine II basal 170.0 272.5 47.7 375.0 21.3 29.6 3.8 45.0 30 apical 87.5 141.8 30.6 205.0 17.5 25.7 2.8 32.5 30 UFRJPOR Triactine - 75.0 116.4 20.0 150.0 8.8 11.6 1.6 15.0 30 6738 (P) Tetractine I basal 85.0 122.5 22.0 192.5 10.0 12.4 2.1 17.5 30 apical 50.0 66.5 25.3 107.5 6.2 7.8 1.4 10.0 5 Tetractine II basal 205.0 261.8 36.7 325.0 25 27.3 2.7 35 20 apical - 125.0 - - - 15.0 - - 1 50

Fig. 3. Holotype of Ascandra torquata sp. nov. (UFRJPOR 6084). (A-B) specimens after fixation; (C-D) tangential section of the body indicating diactines (white arrows); (E) diactine; (F-G) triactines; (H) tetractine I; (I) tetractine II; (J); apical actines of tetractines I. 51

Fig. 4. Paratype of Ascandra torquata sp. nov. (UFRJPOR 6738). (A) specimen in vivo; (B) specimen after fixation; (C) tangential section of the body (cormus); (D) triactine; (E) tetractine I; (F) tetractine II. 52

Genus Borojevia Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013 TYPE SPECIES Borojevia cerebrum (Haeckel, 1872) DIAGNOSIS "Calcinea in which the cormus comprises tightly anastomosed tubes. The skeleton contains regular (equiangular and equiradiate) triactines, tetractines, and tripods. The apical actine of the tetractines has spines. Aquiferous system asconoid" (Klautau et al., 2013).

Borojevia tenuispinata Azevedo et al., submitted (Figure 5, Table 4) SYNONYMS Borojevia tenuispinata: Azevedo et al., submitted TYPE LOCALITY Cabeço da Tartaruga, São Pedro e São Paulo Archipelago, Brazil. MATERIAL EXAMINED UFRJPOR 6700 and UFRJPOR 6708 (specimens in ethanol and slides); Daai Booi, St. Willibrordus, Curaçao; 12°12'43.12"N, 69°05'8.42"W; 3-5 m deep; coll. B. Cóndor-Luján, 18 August 2011. COMPARATIVE MATERIAL EXAMINED Borojevia tenuispinata. Holotype (specimen in ethanol and slides) UFRJPOR 6484; Cabeço da Tartaruga, São Pedro e São Paulo Archipelago, Brazil; 0o54'57''N, 29o20'46''W; coll. G. Rodríguez & F. Azevedo, 16 June 2011. COLOUR White in life and ethanol. MORPHOLOGY AND ANATOMY The largest specimen (UFRJPOR 6700) measures 0.7 x 0.4 x 0.3 mm. Cormus massive, rough and compressible, composed of irregular and (mostly) tightly anastomosed tubes (Figure 5A). The tubes in contact with the substrate are less tightly anastomosed than the most superficial ones. The aquiferous system is asconoid. No water-collecting tubes were observed. The skeleton has no special organization and it comprises triactines, tripods and tetractines (Figure 5B). SPICULES Triactines. Regular (equiangular and equiradiate). Actines are straight, conical, with blunt tips (Figure 5C). They are the most abundant spicules. Size: 62.5-100/6.3-11.3µm. 53

Tripods. Regular (equiangular and equiradiate). Actines are straight, conical, with blunt tips. They do not have the raised centre of typical tripods, instead they look like stout conical triactines (Figure 5D). Size: 72.5-162.5/12.5-22.5 µm. Tetractines. Regular (equiangular and equiradiate). The basal actines are straight, conical, with blunt tips (Figure 5E). The apical actine is shorter and thinner than the basal ones and present spines. The most common pattern of spine distribution observed consisted of spines arranged in rows spreading from about two-thirds of the actine up to the tip (Figure 5F). Some few tetractines with curved paired actines were also found. Size: 62.5-95.0/7.5-11.3 µm (basal actine), 25-40/5-7.5 µm (apical actine). ECOLOGY This sponge was found in a cryptic habitat, underneath boulders. GEOGRAPHIC DISTRIBUTION São Pedro and São Paulo Islands ecoregion of the Tropical Southwestern Atlantic (São Pedro e São Paulo Archipelago, Azevedo et al., submitted) and Southern Caribbean (Curaçao, this study). REMARKS The genus Borojevia comprises eight species. Among them, six have a skeleton composition (one category of tripods, triactines and tetractines) similar to the specimens from Curaçao. These species are the Brazilians B. aspina (Klautau et al., 1994), B. brasiliensis (Solé-Cava et al., 1991), B. tenuispinata and B. trispinata Azevedo et al., submitted, B. cerebrum (Haeckel, 1872) from the Mediterranean Sea and B. croatica Klautau et al., 2016 from the Adriatic Sea. However, only B. brasiliensis, B. croatica and B. tenuispinata, have tetractines with apical actines bearing a spine distribution pattern similar to the Curaçaoan specimens and share comparable spicule size range (Table 4). Different from the species B. brasiliensis and B. croatica, whose skeletons present more triactines than tetractines, in B. tenuispinata and in the specimens from Curaçao, triactines and tetractines occur in the same proportion. The only difference observed between the Caribbean specimens and the holotype of B. tenuispinata is that the spicules of the former can attain larger sizes. This difference, however, can be perhaps attributed to plasticity. Besides, in the ITS phylogenetic tree (Figure 31), the specimen UFRJPOR 6700 clustered within the clade of B. tenuispinata (pp=0.98, b=100), indicating its co-specificity. 54

Table 4. Spicule measurements of Borojevia tenuispinata from Curaçao (UFRJPOR6700 and UFRJPOR6708) and of the holotype (UFRJPOR 8464), B. brasiliensis (MNHN-LBIM.C. 1989.2) taken from Klautau & Valentine (2003) and B. croatica (UFRJPOR6865). H=holotype.

Length (µm) Width (µm) N Specimen Spicule Actine Min Mean SD Max Min Mean SD Max UFRJPOR Triactine 62.5 79.5 9.7 100.0 6.3 8.8 1.6 11.3 36 6700 Tripod 87.5 109.4 16.6 162.5 12.5 13.9 2.9 22.5 27 Tetractine basal 67.5 78.8 7.1 92.5 7.5 8.5 1.0 10.0 30 apical 25.0 33.6 4.5 40.0 5.0 7.2 0.8 7.5 9 UFRJPOR Triactine 62.5 75.3 5.9 85.0 7.5 9.8 1.1 11.3 30 6708 Tripod 72.5 99.5 12.4 125.0 12.5 13.4 1.1 15.0 30 Tetractine basal 62.5 75.7 7.9 95.0 7.5 9.3 1.2 11.3 30 apical 25.0 - - - - 7.5 - - 1 UFRJPOR Triactine 59.4 65.3 4.9 75.6 5.4 7.2 0.7 8.1 30 8464 (H) Tripod 56.7 80.9 11.9 102.6 8.1 10.1 1.4 12.2 30 Tetractine basal 51.3 64.1 5.3 72.9 5.4 7.2 0.8 8.1 30 apical 27.0 40.0 8.6 56.7 4.1 4.9 0.8 6.8 20 MNHN- Triactine 60.9 78.2 10.6 102.2 - 10.8 1.5 - 20 LBIM.C. Tripod 67 81 8.2 95.7 - 11 1.7 - 20 1989.2 Tetractine basal 56.5 75.3 10.0 91.3 - 10.4 1.3 - 20 (H) apical 17.4 36.4 9.1 50.0 - 8-0 2.2 - 20 UFRJPOR Triactine 57.5 66.6 6.7 82.5 7.5 7.5 0.0 7.5 20 6865 Tripod 85.0 102.6 10.0 115.0 10.0 11.9 1.5 15.0 20 (H) Tetractine basal 60.0 70.0 6.3 77.5 7.5 8.3 1.2 11.3 10 apical - 20 . . . 5.0 - - 1 55

Fig. 5. Borojevia tenuispinata (UFRJPOR 6700): (A) specimen after fixation; (B) tangential section of the body (cormus); (C) triactine; (D) tripod; (E) tetractine; (F) detail of the spined apical actine of a tetractine. 56

Genus Clathrina Gray, 1867 TYPE SPECIES Clathrina clathrus (Schmidt, 1864) DIAGNOSIS "Calcinea in which the cormus comprises anastomosed tubes. A stalk may be present. The skeleton ontains regular (equiangular and equiradiate) and/or parasagittal triactines, to which diactines and tripods may be added. Asconoid aquiferous system" (Klautau et al., 2013).

Clathrina aspera sp. nov. (Figures 6 & 7, Table 5) ETIMOLOGY From the Latin asper (= rough), after the species rough surface. TYPE LOCALITY Water Factory, Willemstadt, Curaçao. TYPE MATERIAL Holotype: UFRJPOR 6758 (specimen in ethanol and slides); Water Factory, Willemstadt, Curaçao; 12°06'30.88"N, 68°57'13.53"W; 13.2 m deep; coll. B. Cóndor-Luján and E. Hajdu, 23 August 2011. Paratypes: UFRJPOR 5487 (specimen in ethanol and slides); Ilhas Botinas, Angra dos Reis, Rio de Janeiro, Brazil; 23º03'19.36''S, 44º19'44.98''W; 1-3 m deep; coll. F. Azevedo & M. Klautau, 25 May 2007 and UFRJPOR 5531 (specimen in ethanol and slides); Praia do Bonfim, Angra dos Reis, Rio de Janeiro, Brazil; 23°01'14.26''S, 44°19'48.18''W; 1-2 m deep; coll. M. Klautau, 27 May 2007. ADDITIONAL ANALYSED MATERIAL UFRJPOR 6345 (specimen in ethanol and slides); UFRJPOR 6472 (specimen in ethanol and slides); Enseada dos Cardeiros, Arraial do Cabo, Rio de Janeiro, Brazil; 7m deep; coll. F. Azevedo and G. Rodríguez; 22 May 2011 and UFRJPOR 6510 (specimen in ethanol and slides); Praia do Forno, Arraial do Cabo, Rio de Janeiro, Brazil; 1.5 m deep; coll. E. Lanna and A. Padua, April 2011. COLOUR White in life and beige to light brown in ethanol. MORPHOLOGY AND ANATOMY Cormus massive (Figure 6A) to almost spherical (Figure 7A), composed of thin, irregular to regular and tightly anastomosed tubes finishing in large apical oscula. The holotype measures 0.8 x 0.6 x 0.2 cm (Figure 6B). It has rough surface due to the presence of large triactines on the 57 external tubes. Water-collecting tubes were not observed. The aquiferous system is asconoid. The skeleton has no special organization and it is composed of two categories of triactines (Figures 6C and 7B). The smaller triactines are the most abundant spicules whereas the large triactines are less frequent and located on the surface. Near the oscular region, the triactines become more sagittal. SPICULES (Table 5) Triactines I. Regular (equiangular and equiradiate). Actines are slightly conical to conical, with blunt to sharp tips (Figures 6D, 6F and 7C-D). Some subregular triactines were also found (Figure 7E). Size: 50.0-152.5/6.8-12.5 µm. Triactines II: Regular (equiangular and equiradiate). Actines are conical and robust with sharp tips (Figure 6E). Size: 127.5-325.0/17.5-37.5 µm. ECOLOGY The specimens inhabited shaded environments, underneath boulders or attached to an oyster farming rope. They were found associated to macroalgae and to other invertebrates (octocorals, bryozoans) or growing on oyster shells. GEOGRAPHIC DISTRIBUTION Southern Caribbean (Curaçao) and Eastern Brazil ecoregion of the Tropical Southwestern Atlantic Province (Rio de Janeiro, Southern Brazilian Coast). REMARKS Within Clathrina, six species whose colour alive is white (or unknown) present two or more spicule categories (Klautau & Valentine, 2003; Klautau et al. 2016; Azevedo et al., submitted); however, only three have a cormus composed of tightly anastomosed tubes and large triactines (including tripods) in the external tubes: C. clara Klautau & Valentine, 2003 from Christmas Islands, C. laminoclathrata Carter 1886 from southern Australia and C. rotunda Klautau & Valentine, 2003 from South Africa. Clathrina aspera sp. nov. can be easily distinguished from those species as its skeleton comprises triactines whose actines are slightly conical to conical and with blunt to sharp tips whereas the skeletons of the other three species only comprise triactines whose actines are conical with sharp tips. Moreover, they differ in spicule size (Table 5) and external morphology. Clathrina clara presents slightly thinner triactines II (21.8±3.5 µm) than those of C. aspera sp. nov. (28.5±5.6 µm) and its cormus have water-collecting tubes which are absent in the new Curaçaoan species. Clathrina laminoclathrata has three categories of triactines and even the largest category (triactine III) is much thinner (18.0±3.0 µm). In this species, the large triactines are present in the external tubes and can also be found at the base of the sponge (“basal lamina” 58 according to Carter, 1886) whereas in C. aspera sp. nov, they are restricted to the external tubes. Clathrina rotunda has smaller triactines (triactines I: 52.6±3.6/5.8±0.7 µm and tripods: 59.0±9.8/9.6±2.4 µm) compared to C. aspera sp. nov. (triactine I: 115.4±12.9/9.7±1.1 µm and triactine II: 227.5±50.8/28.5±5.6 µm – holotype measurements) and their actines are slightly undulated whereas in the new Curaçaoan species, they are straight. Besides, water-collecting tubes are also present in C. rotunda. In the ITS phylogenetic tree, the sequences of C. aspera obtained from several specimens (UFRJPOR 5531, UFRJPOR 6346, UFRJPOR 6472 and UFRJPOR 6510) clustered together. However, as sequences of C. clara, C. laminoclathrata and C. rotunda were not available in the Genbank databse, it was not possible to infer the phylogenetic affinities among them.

Fig. 6. Holotype of Clathrina aspera sp. nov. (UFRJPOR 6758): (A) specimen in vivo; (B) specimen after fixation; (C) tangential section of the body (cormus); (D and E) triactine I; (F) triactine II. 59

Fig. 7. Paratype of Clathrina aspera sp. nov. (UFRJPOR 5487): (A) specimen after fixation; (B) tangential section of the body (cormus) indicating triactines II (white arrows); (C-E) triactine I.

Table 5. Spicule measurements of Clathrina aspera sp. nov. (UFRJPOR 6758, UFRJPOR 5487 and UFJPOR 5531), C clara (BMNH 1927.2.14.152), C. laminoclathrata (BMNH 1887.7.12.42) taken from Klautau & Valentine (2003) and C. rotunda (BMNH 1935.10.21.50). H=holotype, P=paratype, L=lectotype.

Specimen Spicule Length (µm) Width (µm) N Min Mean SD Max Min Mean SD Max UFRJPOR Triactine I 75.0 115.4 12.9 152.5 7.5 9.7 1.1 12.5 40 6758 (H) Triactine II 132.5 227.2 50.8 325.0 17.5 28.5 5.6 37.5 30 UFRJPOR Triactine I 65.0 91.1 13.5 117.5 6.3 7.2 0.9 10 40 5487 (P) Triactine II 232.5 242.5 9.0 250.0 27.5 31.7 3.8 35.0 3 UFRJPOR Triactine I 57.5 95.0 15.7 127.5 6.3 7.6 1.2 10 40 5531 (P) Triactine II 212.5 235.0 31.8 257.5 27.5 28.7 1.8 30 2 BMNH 1927. Triactine I 67.5 84.5 8.8 102.5 - 9.8 0.8 - 30 2.14.152 (H) Triactine II 102.5 164.5 34.3 245.0 - 21.8 3.5 - 30 BMNH 1887. Triactine I 50.0 72.0 15.0 113.0 - 8.0 2.0 - 30 7.12.42 (L) Triactine II 88.0 132.0 16.0 168.0 - 13.0 2.0 - 30 Triactine III 125.0 188.0 31.0 235.0 18.0 3.0 30 BMNH 1935. Triactine I 45.6 52.6 3.6 57.6 - 5.8 0.7 - 20 10.21.50 (H) Tripod 45.6 59.0 9.8 79.6 - 9.6 2.4 - 20 60

Clathrina cf. blanca (Miklucho-Maclay, 1868) (Figure 8, Table 6) SYNONYMS Ascetta blanca, Haeckel, 1872: 38; Hansen, 1885: 20; Lendenfeld, 1891: 34; Arnesen, 1901: 9; Mello-Leitão et al., 1961: 3. Clathrina blanca: Minchin, 1896: 359; Jenkin, 1908: 438; Borojevic, 1971: 525; Ereskovsky, 1995: 730; Imešek et al., 2014: 23 Klautau et al., 2016: 37,38. Clathrina cf. blanca: Rapp, 2015: 9. Guancha blanca: Miklucho-Maclay, 1868: 221; Borojevic & Boury-Esnault, 1987: 14; Barthel & Tendal, 1993: 84; Janussen et al., 2003: 17; Rapp, 2006: 352. Leucosolenia blanca: Lackschewitsch, 1886: 300; Breitfuss, 1896: 426; Breitfuss, 1898a: 13; Breitfuss, 1898: 105; Breitfuss, 1911: 224; Derjugin, 1915: 289; Breitfuss, 1927: 27; Arndt, 1928: 19; Hôzawa, 1929: 282; Brøndsted, 1931: 12; Breitfuss, 1930: 275; Breitfuss, 1932: 240; Breitfuss, 1935: 7; Breitfuss, 1936: 5; Topsent, 1936: 9; Arndt, 1941: 45; Tanita, 1942b: 75. TYPE LOCALITY Lanzarote Islands, Canary Islands, Atlantic Ocean. MATERIAL EXAMINED UFRJPOR 6753 and UFRJPOR 6759 (specimens in ethanol and slides); Tug Boat, Caracasbaai, Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W; 10 m deep; coll. B. Cóndor-Luján, 23 August 2011. COLOUR White in life and in ethanol. MORPHOLOGY AND ANATOMY This species has a globular clathroid body with an apical osculum and a stalk (Figure 8A). The surface is smooth and the texture is soft. The consistency is compressible, even the stalk. In the largest specimen (UFRJPOR 6759), the clathroid body measures 0.5 x 0.5 x 0.1 cm and the stalk is 0.5 x 0.1 x 0.1 cm. The cormus is formed by irregular and tightly anastomosed tubes, which converge at the centre of the sponge forming a single apical osculum. The stalk is formed by true tubes with choanoderm (arrow in Figure 8A), except for the portion that attaches it to the substrate which is solid. The aquiferous system is asconoid. The skeleton of the clathroid body has no special organisation and it is composed of two categories of regular triactines (Figure 8B). The skeleton of the stalk is composed exclusively of parasagittal triactines, whose unpaired actine is basipetally oriented. These spicules are more numerous and more closely disposed in the median part of the stalk. 61

SPICULES Triactines I of the clathroid body. Regular (equiangular and equiradiate) or subregular. Most frequent spicules. Actines are cylindrical with blunt tips (Figure 8C). Some of them are slightly undulated at the distal part. Size: 67.5-108.0/4.1-5.4 µm. Triactines II of the clathroid body. Regular (equiangular and equiradiate). Actines are conical and straight with sharp tips. Shorter and thicker than triactine I (Figure 8D). Size: 45.9-62.1/5.4- 8.1 µm. Triactines of the stalk. Parasagittal (equiangular). The paired actines are slightly conical and very short (sometimes they seem to be rudimentary). The unpaired actine is straight and cylindrical to slightly conical with sharp tips (Figure 8F). Size: 40.5-67.5/5.4-8.1 µm (paired actine) and 94.5-199.8/5.4-8.1 µm (unpaired actine). ECOLOGY This sponge was collected in a light protected environment, underneath coral boulder. GEOGRAPHIC DISTRIBUTION This species has an allegedly cosmopolitan distribution (Van Soest et al., 2016) including the Caribbean Sea (Southen Caribbean, Curaçao, present study). REMARKS The external morphology of the specimens from Curaçao, a clathroid body attached to a stalk, resembles the species formerly considered guanchas and now allocated in Clathrina (Klautau et al., 2013). Among them, only two species have a clathroid body composed of tightly anastomosed tubes, a stalk formed of true tubes and a skeleton composed of regular and parasagittal triactines, as observed in the Curaçaoan specimens. These species are Clathrina blanca (Miklucho-Maclay, 1868) from the Lanzarote Islands and C. pellucida (Rapp 2006) from Norway. Clathrina pellucida has a short stalk (less than 1/3 of the body) and a skeleton exclusively composed of triactines with undulated actines whereas the Curaçaoan specimens have a longer stalk (half the length of the body) and its skeleton also comprises triactines with straight actines. Besides, the smaller triactines observed in the Curaçaoan specimens (Triactines II of the clathroid body) are absent in C. pellucida. Clathrina blanca is the species that most resembles the specimens from Curaçao. As spicule sizes were not provided in the original description (Miklucho-Maclay, 1868), we used the measurements provided by Haeckel (1872) for the parasagittal triactines (length/width): 50- 70/3-4 µm (paired actines) and 80-100/3-4 (unpaired actine). Compared to them, the analised Curaçaoan specimens have thicker actines (5.4-8.1 µm). Compared to more detailed 62 descriptions of C. blanca reported from different localities, namely, Brazil (Borojevic, 1971), Bay of Biscaye (Borojevic & Boury-Esnault, 1987), Norway (Rapp, 2006) and Adriactic Sea (Imešek et al., 2014), we observed certain dissimilarities. In none of the referred descriptions, spicules comparable to the small triactines observed in the specimens from Curaçao were mentioned or illustrated. Moreover, the parasagittal triactines described for C. blanca sensu Borojevic (1971) and C. blanca sensu Borojevic & Boury-Esnault (1987) are different to the ones observed in our material (the paired actines are shorter in the Curaçaoan specimens). The cormus of C. blanca sensu Imešek et al. (2014) presents several water-collecting tubes whereas in our specimens, the tubes gather together in a single water-colllecting tube as also described for C. blanca sensu Rapp (2006). As shown above, the specimens named after C. blanca show a great range of morphological variability and it is possible that they constitute a species complex, as already suggested by some authors (Muricy et al., 2011; Rapp, 2015). The DNA amplification of the Curaçaoan specimens was not successful and its relationship with the only available sequence of C. blanca from the Adriatic Sea (Imešek et al., 2014) remains unknown. We name the specimens collected in Curaçao and analised in this study as Clathrina cf. blanca until a molecular study considering the putative populations of Clathrina “blanca” is done. 63

Fig. 8. Clathrina cf. blanca (UFRJPOR 6759): (A) specimen after fixation (arrow indicates the stalk); (B) tangential section of the clathroid body; (C) triactine I; (D) triactine II; (E) triactine of the stalk.

Table 6. Spicule measurements of Clathrina cf. blanca from Curaçao (UFRJPOR 6759).

Length (µm) Width (µm) N Specimen Spicule Actine Min Mean SD Max Min Mean SD Max UFRJPOR Triactine 67.5 87.2 11.6 108.0 4.1 5.3 0.4 5.4 30 6759 I (body - Triactine 45.9 54.8 4.6 62.1 5.4 6.4 0.9 8.1 30 II (body) - Triactine Unpaired 94.5 119.8 21.6 151.2 5.4 7.9 1.8 9.4 7 (stalk) Paired 40.5 51.8 5.7 62.1 8.1 8.1 0.0 8.1 10

Clathrina curaçaoensis sp. nov. (Figure 9, Table 7) ETIMOLOGY Named after the country of the type locality. TYPE LOCALITY Sunset Waters, Soto, Curaçao. TYPE MATERIAL Holotype: UFRJPOR 6734 (specimen in ethanol and slides); Sunset Waters, Soto, Curaçao; 12°16'01.58''N, 69°07'44.85''W; 3-10 m deep; coll. B. Cóndor-Luján, 20 August 2011. COLOUR Yellow in life and light beige in ethanol. MORPHOLOGY AND ANATOMY The analysed specimen has a massive cormus (0.7 x 0.4 x 0.3 mm) with a smooth and delicate surface. The cormus is composed of irregular and loosely anastomosed tubes (Figure 9A). No water-collecting tubes were observed. The aquiferous system is asconoid. The skeleton has no special organization (Figure 9B) and it is exclusively composed of triactines. The tubes that attach the sponge to the substrate are composed of parasagittal triactines (Figure 9C). SPICULES (Table 7) Triactines I. Regular (equiangular and equiradiate). Actines are slightly conical with blunt to sharp tips (Figure 9D). Size: 87.5-130.0/7.5-10.0 µm. Triactines II. Parasagittal (equiangular). Actines are slightly conical with blunt to sharp tips (Figure 9E). Size: 57.5-80/7.5 µm (paired actine) and 99.9-137.7/8.1-8.8 µm (unpaired actine). ECOLOGY The specimen was found underneath boulders. 64

GEOGRAPHIC DISTRIBUTION Southern Caribbean (provisionally endemic to Curaçao, present study). REMARKS Within the genus Clathrina, seven species are yellow as C. curaçaoensis sp. nov.: C. aurea Solé-Cava et al., 1991; C. chrysea Borojevic & Klautau, 2000, C. clathrus (Schmidt, 1864), C. insularis Azevedo et al., submitted, C. lutea Azevedo et al., submitted, C. luteoculcitella Wörheide & Hooper, 1999 and C. mutabilis Azevedo et al., submitted. However, compared to C. curaçaoensis sp. nov, these species do not possess tubes differentiated for substrate attachment supported by parasagittal triactines (triactines II) nor the same external morphology . Among the non-yellow clathrinas with stalk (former “guanchas”), the species whose external morphology most resembles that of C. curaçaoensis sp. nov. is C. arnesenae (Rapp, 2006). Nonetheless, its skeleton does not comprise regular triactines as is the case of the new species; instead, it is only composed of parasagittal triactines with cylindrical actines. Regarding the triactines of the body of C. curaçaoensis sp. nov. (triactines I), they resemble those of C. lutea. However, both species differ in their external morphology (as already mentioned above). The cormus of C. lutea is formed by regular and tightly anastomosed tubes while the cormus of C. curaçaoensis sp. nov. is composed of irregular and loosely anastomosed tubes. Furthermore, in the ITS and C-LSU phylogenetic trees (Figures 31 and 32), C. curaçaoensis sp. nov. (UFRJPOR 6734) did not cluster with C. lutea nor with any other yellow clathrina. It appeared as a different lineage. Thus, based in morphological and DNA evidence, we recognise it as a new species of Clathrina.

Table 7. Spicule measurements of Clathrina curaçaoensis sp. nov. (Holotype=UFRJPOR 6734).

Length (µm) Width (µm) N Spicule Actine Min Mean SD Max Min Mean SD Max Triactine I 87.5 102.8 13.6 130.0 7.5 8.5 1.0 10.0 20 Triactine II Unpaired 99.9 119.9 13.1 137.7 8.1 8.2 0.3 8.8 10 Paired 57.5 68.5 6.3 80.0 7.5 7.5 0 7.5 10 65

Fig. 9. Clathrina curaçaoensis sp. nov. (UFRJPOR 6734): (A) specimen after fixation; (B) tangential section of the body (cormus); (C) tangential section of an attachment tube; (D) triactine I; (E) triactine II (parasagittal).

Clathrina hondurensis Klautau & Valentine, 2003 (Figure 10, Table 8) SYNONYMS Clathrina hondurensis: Klautau & Valentine, 2003: 46; Non Clathrina hondurensis: Rützler et al., 2014:101 Non Clathrina cf. hondurensis: Imešek et al., 2014:25; Klautau et al., 2016: 21. TYPE LOCALITY Turneffe, British Honduras, Caribbean Sea. MATERIAL EXAMINED UFRJPOR 6732 (specimens in ethanol and slides); Porto Mari, St. Willibrordus; 12°13'6.62"N, 69°05'13.26"W; 7.9 m deep; coll. B. Cóndor-Luján, 20 August 2011. 66

COMPARATIVE MATERIAL EXAMINED Clathrina hondurensis. Holotype (slide) BMNH 1938.3.28.4; Turneffe, British Honduras, Caribbean Sea; coll. J.H. Borley, 20–22 March 1935. COLOUR White in life and light brown in ethanol. MORPHOLOGY AND ANATOMY The specimen is massive and it measures 1.4 x 1.0 x 0.2 cm (Figure 10A). The surface is smooth and the consistency is compressible. The cormus is composed of irregular and tightly anastomosed tubes (Figure 10B). No water-collecting tubes were observed. The aquiferous system is asconoid. The skeleton has no special organization (Figure 10C) and it is composed of triactines and rare trichoxeas (Figure 10C, arrow). SPICULES (Table 8) Trichoxeas. Straight and very slender (Figure 10D). Mostly broken. The size of the unique whole trichoxea found is 325/2.5 µm. Triactines. Regular (equiangular and equiradiate). Actines are conical with sharp tips (Figure 10E). Size: 100.0-212.5/13.6-25 µm. ECOLOGY This specimen was collected underneath boulders. GEOGRAPHIC DISTRIBUTION Southwestern Caribbean (Belize, Klautau & Valentine, 2003) and Southern Caribbean (Curaçao, present study) ecoregions. REMARKS The external morphology and the shape of the triactines observed in the specimen from Curaçao are similar to C. hondurensis. However, in the original description of that species, trichoxeas were not reported and after the re-examination of the slides of the holotype, we did not find them. As seen in other species, the presence of trichoxeas may not constitute a diagnostic character, as they apparently constitute plastic characters. Regarding the spicule size, although the triactines of the specimen from Curaçao can attain slightly larger sizes compared to the specimen from Turneffe (Table 11), they are in the same size range. It is important to point out that C. hondurensis was originally described based on one single specimen. Considering this, the presence of trichoxeas and of slightly larger triactines in the specimen from Curaçao can be attributed to intraspecific variation. Rützler et al. (2014) recorded C. hondurensis from another Belizean locality (Belize barrier reef near Carrie Bow), however, the skeleton of their specimen was composed of triactines 67 whose size range was 85-100 μm in length and 8-12 μm in width. Based on the original description of C. hondurensis and the specimens from Curaçao, we believe that the specimen reported by Rützler et al. (2014) does not correspond to C. hondurensis. Recently, Klautau et al. (2016) considered the possibility of synonymy between C. hondurensis and C. primordialis (Haeckel, 1872). In fact, both species present similar morphology and their spicule size range do overlap, however, it is possible to recognize thicker spicules in C. hondurensis (Table 6). As we could not successfully amplify any DNA region from UFRJPOR 6732, we cannot affirm that the difference in width reflects plasticity and that C. hondurensis and C. primordialis are synonyms. Therefore, we decided to maintain C. hondurensis as a valid species and to identify the Curaçaoan specimen as C. hondurensis.

Fig. 10. Clathrina hondurensis (UFRJPOR 6732): (A) specimen in vivo; (B) specimen after fixation; (C) tangential section of the body (cormus) skeleton; (D) detail of trichoxea indicated by an arrow; (E) triactine. 68

Table 8. Spicule measurements of Clathrina hondurensis from Curaçao (UFRJPOR 6732) and of the holotype (BMNH 1938.3.28.4) and of C. primordialis taken from * Klautau & Valentine (2003) and ** Haeckel (1872).

Length (µm) Width (µm) N Specimen Spicule Min Mean SD Max Min Mean SD Max UFRJPOR Triactine 100.0 149.6 30.0 212.5 13.6 19.1 3.1 25 30 6732 Trichoxea 325.0 - - - 2.5 - - 1 BMNH 1938. Triactine 120.0 142.9 15.5 175.0 12.5 16.8 2.4 20 20 3.28.4 BMNH 1938. Triactine 105.6 133.4 17.0 156.0 - 15.6 1.7 - 20 3.28.4* C. primordialis Triactine 100.0 - - 150.0 8 - - 12.0 - **

Clathrina insularis Azevedo et al, submitted (Figure 11, Table 9) SYNONYMS Clathrina insularis: Azevedo et al, submitted Clathrina sp. nov. 3 - UFRJPOR 6737a=UFRJPOR 6737: Klautau et al., 2013, 449 and 451. TYPE LOCALITY Cagarras, Fernando de Noronha Archipelago, Pernambuco, Brazil MATERIAL EXAMINED UFRJPOR 6737 (specimen in ethanol and slides); Playa Jeremi, Soto; 12°19'43.73''N,69°09'07.80''W; 14.9 m; coll. B. Cóndor-Luján, 22 August 2011. ADDITIONAL MATERIAL RE-ANALYZED Specimens in ethanol and slides: UFRJPOR 6533; Cagarras, Fernando de Noronha Archipelago, Pernambuco, Brazil; 3°48'34.59''S, 32°23'27.91''W; 15 m; coll. F. Azevedo and G. Rodríguez, 27 June 2011. UFRJPOR 6530 and 6537; Ilha do Meio, Fernando de Noronha Archipelago, Pernambuco, Brazil; 03°49'5.88''S,32°23'36.6''W;15 m ; coll. F. Azevedo and G. Rodríguez, 27 June 2011. MATERIAL STUDIED FOR COMPARISON Clathrina insularis. Holotype (specimen in ethanol and slides) UFRJPOR 6532; Cagarras, Fernando de Noronha Archipelago, Pernambuco, Brazil; 3°48'34.59''S, 32°23'27.91''W; 15 m; coll. F. Azevedo and G. Rodríguez, 27 June 2011. COLOUR Pale yellow in life and beige in ethanol. 69

MORPHOLOGY AND ANATOMY The specimen is finely encrusting (0.4 x 0.4 x 0.1 mm, Figure 11A). The surface is smooth and the texture is soft. The cormus is composed of irregular and loosely anastomosed tubes. Water- collecting tubes are not present. The aquiferous system is asconoid. The skeleton has no organization (Figure 11B) and it is composed of two categories of triactines. SPICULES Triactines I. Regular (equiangular and equiradiate). Actines are conical with sharp tips (Figure 9C). Size: 52.5-77.5/3.8-6.3 µm. Triactines II. Regular (equiangular and equiradiate) or subregular (equiangular). Most abundant spicules. Actines are cylindrical to slightly conical, distally undulated and with sharp tips (Figure 9D-E). Size: 102.7-145.0/3.7-6.6 µm. ECOLOGY This sponge was found in a cryptic habitat, underneath boulder. GEOGRAPHIC DISTRIBUTION Fernando de Noronha and Atoll das Rocas ecoregion of the Tropical Southwestern Atlantic Province (Fernando de Noronha Archipelago, Azevedo et al., submitted) and Southern Caribbean (Curaçao, present study). REMARKS The external morphology and the skeleton composition of the Curaçaoan specimen match the description of C. insularis. In the ITS phylogenetic tree (Figure 31), it clustered in the clade of that species (pp=1, b=100), corroborating its morphological identification. The specimens UFRJPOR 6530, UFRJPOR 6533 and UFRJPOR 6537 were already analysed in Azevedo et al., submitted, and herein, we provide their ITS sequences.

Table 9. Spicule measurements of Clathrina insularis (UFRJPOR 6737) from Curaçao and of the holotype (UFRJPOR 6532).

Length (µm) Width (µm) N Specimen Spicule Min Mean SD Max Min Mean SD Max UFRJPOR Triactine I 52.5 62.6 6.7 77.5 3.8 5.2 0.5 6.3 30 6737 Triactine II 102.7 119.0 8.6 145.0 4.1 5.4 0.7 6.8 50 UFRJPOR Triactine I 47.5 76.4 12.4 97.5 5.0 6.1 0.7 7.5 30 6532 Triactine II 98.8 121.1 11.0 145.0 5.7 6.3 0.8 8.2 60 70

Fig. 11. Clathrina insularis (UFRJPOR 6737): (A) specimen in vivo; (B) tangential section of the body (cormus), (C) triactine I, (D-E) triactine II.

Clathrina lutea Azevedo et al., submitted (Figure 12, Table 10) SYNONYMS Clathrina primordialis: Lehner & Van Soest, 1998: 97? Clathrina sp. nov. 8 - UFRJPOR 6761 and ZMAPOR 08344: Klautau et al., 2013, 449 and 451. TYPE LOCALITY Pedra Lixa, Abrolhos Archipelago, Caravelas, Bahia, Brazil. MATERIAL EXAMINED UFRJPOR 6761 (specimen in ethanol and slides); Porto Mari, St. Willibrordus, Curaçao; 12°13'6.62"N, 69°05'13.26"W; 10.6 m deep; coll. Giselle Lôbo-Hajdu, 20 August 2011. COMPARATIVE MATERIAL EXAMINED Clathrina lutea. Holotype (specimen in ethanol and slides) UFRJPOR 5173; Pedra Lixa, Abrolhos Archipelago, Caravelas, Bahia, Brazil; 17°41'S, 38°59'W; 7 m deep; coll. C. Zilberberg & L. Monteiro, 21 March 2005. 71

COLOUR Dark yellow in life and white in ethanol. MORPHOLOGY AND ANATOMY This specimen has a rough massive cormus (1.0 x 0.4 x 0.1 cm, Figure 12A) composed of regular and tightly anastomosed tubes (Figure 12B). Water-collecting tubes were observed. The aquiferous system is asconoid. The skeleton has no organization and it is composed of one category of triactines (Figure 12C). Some broken trichoxeas were found in the spicule slide. SPICULES Triactines. Regular. Actines are cylindrical to slightly conical, slightly undulated and with blunt tips (Figure 12D). Size: 75.0-97.5/7.5-10 µm. ECOLOGY This sponge was collected in a light protected environment, inside a small crevice. GEOGRAPHIC DISTRIBUTION Within the Tropical Southwestern Atlantic includes the Floridian (Florida, United States of America: UFRJPOR 5818, Klautau et al., 2013), Eastern Caribbean (Virgin Islands: ZMAPOR 08344, Klautau et al., 2013) and Southern Caribbean (Curaçao, present study) ecoregions. In the Tropical Northwestern Atlantic includes Eastern Brazil (Abrolhos Archipelago) and Fernando de Noronha and Atoll das Rocas (Rocas Atoll) ecoregions (Azevedo et al., submitted). REMARKS Compared to the type material of C. lutea, our specimen has slightly larger triactines and actines are not very undulated. Besides, some broken trichoxeas were found in the spicule slide of the Curaçaoan material whereas in the Brazilian specimens they were not observed. Despite these slight differences, the Curaçaoan specimen grouped in the clade of C. lutea (pp=1, b=100, Figure 31), which included Brazilian and Caribbean specimens. In 1998, Lehner & Van Soest reported C. primordialis to Jamaica. After revising the description of that record, including the in vivo figure (Figure 23, page 97), we rather consider it as C. lutea based principally on its external morphology.

Table 10. Spicule measurements of Clathrina lutea from Curaçao (UFRJPOR 6761) and of the holotype (UFRJPOR 5173).

Length (µm) Width (µm) N Specimen Spicule Min Mean SD Max Min Mean SD Max UFRJPOR 6761 Triactine 75.0 88.8 5.6 97.5 7.5 9.4 0.7 10.0 30 UFRJPOR 5173 Triactine 69.3 78.5 3.8 84.0 6.5 7.5 0.4 8.3 30 72

Fig. 12. Clathrina lutea (UFRJPOR 6761): (A) specimen in vivo (photo taken by G. Lôbo- Hajdu); (B) specimen after fixation; (C) tangential section of the body (cormus); (D) triactine.

Clathrina mutabilis Azevedo et al., submitted (Figure 13, Table 11) SYNONYMS Clathrina mutabilis: Azevedo et al., submitted Clathrina sp. nov. 4 - UFRJPOR 6733 and UFRJPOR 6741: Klautau et al., 2013, p: 449 and 451. TYPE LOCALITY Cagarras, Fernando de Noronha Archipelago, Pernambuco, Brazil. MATERIAL EXAMINED Specimens in ethanol and slides: UFRJPOR 6704 and UFRJPOR 6719; Playa Kalki, Westpunt, Curaçao; 12°22'29.86"N, 69°09'30.63"W; 6.7 m deep; coll. E. Hajdu and B. Cóndor-Luján, 21 August 2011; UFRJPOR 6717; Water Factory, Willemstadt, Curaçao; 12°06'30.88"N, 73

68°57'13.53"W; coll. B. Cóndor-Luján, 19 August 2011; UFRJPOR 6733 and UFRJPOR 6735, Porto Mari, St. Willibrordus, Curaçao; 12°13'6.62"N, 69°05'13.26"W; 7.9 m deep; coll. B. Cóndor-Luján, 20 August 2011; UFRJPOR 6740; Sunset Waters, Soto, Curaçao; 12°16'01.58"N, 69°07'44.85"W, 9-12 m deep; coll. B. Cóndor-Luján, 22 August 2011; UFRJPOR 6741, UFRJPOR 6743 and UFRJPOR 6744; Sunset Waters, Soto, Curaçao; 12°16'01.58"N, 69°07'44.85"W, 8.9 m, 9.8 m and 7.2 m deep, respectively; coll. B. Cóndor-Luján, 22 August 2011. Slides: UFRJPOR 6699; Hook’s Hut, Willemstadt, Curaçao; 12°07'18.94"N, 68°58'11.46"W; < 10 m deep; coll. B. Cóndor Luján and G. Lôbo-Hajdu, 17 August 2011; UFRJPOR 6712; Daai Booi, St. Willibrordus, Curaçao; 12°12'43.12"N, 69°05'8.42"W; 4.9 m deep; coll. B. Cóndor-Luján, deep, 18 August 2011; UFRJPOR 6736 (specimens in ethanol and slides); Playa Jeremi, Soto, Curaçao; 12°19'43.73ʺ N, 69°09'07.80ʺ W; 4.9 m deep; coll. B. Cóndor-Luján, 22 August 2011; UFRJPOR 6747; Sunset Waters, Soto, Curaçao; 12°16'01.58"N, 69°07'44.85"W, 4.9 m deep; coll. B. Cóndor-Luján, 22 August 2011; UFRJPOR 6750; Tug Boat, Caracasbaai, Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W, coll. B. Cóndor-Luján, 23 August 2011. COMPARATIVE MATERIAL EXAMINED Clathrina mutabilis. Holotype (specimen in ethanol and slides) UFRJPOR 6526; Cagarras, Fernando de Noronha Archipelago, Pernambuco, Brazil; 3°48'34.59''S, 32°23'27.91''W; 15 m; coll. F. Azevedo and G. Rodríguez, 27 June 2011. COLOUR Pale yellow in life and white to beige in ethanol. MORPHOLOGY AND ANATOMY The specimens have a massive cormus composed of irregular and loosely anastomosed tubes (Figure 11A) which form water-collecting tubes. When tubes are contracted, this sponge has a more encrusting growth form (UFRJPOR 6743, Figure 11B). The largest fragment deposited in our collection belongs to the specimen UFRJPOR 6735 (Figure 11C) which measures 0.8 x 0.6 x 0.2 cm. The aquiferous system is asconoid. The skeleton has no organization (Figure 11D) and it is composed of two categories of triactines. SPICULES (Table 11) Triactines I. Regular (equiangular and equiradiate) but some subregular spicules (equiangular but not equiradiate) were also found. Actines are conical, straight, with sharp tips (Figure 13E). Smaller than triactines II. Size: 60-112.5/7.5-10 µm. 74

Triactines II. Regular (equiangular and equiradiate), subregular (equiangular but not equiradiate) (Figure 13F) or parasagittal (Figure 13G). Frequent. Actines are slightly conical to cylindrical, slightly undulated, with blunt tips. Size: 100.0-195.0/5-11.3 µm. ECOLOGY The specimens were found underneath boulders and broken corals. GEOGRAPHIC DISTRIBUTION Fernando de Noronha and Atoll das Rocas (Fernando de Noronha Archipelago , Azevedo et al., submitted) and Southern Caribbean (Curaçao , present study) ecoregions. Clathrina mutabilis was found in all Curaçaoan sampled localities. REMARKS In the ITS phylogenetic tree, these specimens clustered within the clade of C. mutabilis (pp=0.81, b=91%, Figure 31) and they match its morphological description. However, some slight differences were found. Compared to the holotype, the skeleton of the Curaçaoan specimens has more parasagittal triactines II and does not present trichoxeas (or at least, they were not observed). This may be the result of plasticity. It is important to point out that in the analised specimens, it was possible to observe water-collecting tubes, and thus, we complement the original description of this species providing one more diagnostic character.

Table 11. Spicule measurements of Clathrina mutabilis from Curaçao (UFRJPOR 6741, UFRJPOR 6704 and UFRJPOR 6745) and of the holotype (UFRJPOR 6526). U=unpaired and P=paired actines.

Length (µm) Width (µm) N Specimen Spicule Actine Min Mean SD Max Min Mean SD Max UFRJPOR Triactine I 100.0 123.8 10.4 145.0 7.5 8.9 0.9 10 30 6741 Parasagittal U 125.0 166.0 17.3 195.0 5 8.2 1.1 10 30 Triactine I P 100.0 116.1 11.2 132.5 5 7.5 1.1 10 30 Triactine II 67.5 82.4 8.5 95.0 6.3 8.0 0.8 8.8 30 UFRJPOR Triactine I 107.5 135.5 15.9 162.5 7.5 10.3 1.0 11.3 20 6704 Parasagittal U 122.5 142.4 15.6 177.5 7.5 8.3 1.0 10 30 Triactine I P 75.0 98.7 12.8 127.5 6.3 7.6 0.5 8.8 30 Triactine II 60.0 95.5 12.6 112.5 7.5 8.8 1.2 10 20 UFRJPOR Triactine I 100.0 126.0 18.1 160.0 7.5 9.0 1.1 10 20 6745 Parasagittal U 150.0 169.5 13.1 195.0 7.5 9.1 1.0 10 30 Triactine I P 97.5 121.0 12.9 140.0 7.5 8.3 0.9 10 30 Triactine II 60.0 81.6 13.4 102.5 7.5 8.6 1.1 10 20 UFRJPOR Triactine I 94.5 124.7 14.4 148.5 6.8 7.9 0.6 9.5 40 6526 Triactine II 56.7 69.8 7.9 91.8 8.1 8.4 0.6 9.5 20 75

Fig. 13. Clathrina mutabilis: (A) specimen UFRJPOR 6704 in vivo; (B) specimen UFRJPOR 6743 in vivo; (C) specimen UFRJPOR 6735 after fixation; (D) tangential section of the body (cormus); (E) triactine I; (F) triactine II; (G) parasagittal triactine II. Skeleton images were taken from slides of the specimen UFRJPOR 6741. 76

Genus Leucetta Haeckel, 1872 TYPE SPECIES Leucetta primigenia Haeckel, 1872. DIAGNOSIS "Leucettidae with a homogeneous organisation of the wall and a typical leuconoid aquiferous system. There is neither a clear distinction between the cortex and the choanoskeleton, nor the presence of a distinct layer of subcortical inhalant cavities. The atrium is frequently reduced to a system of exhalant canals that open directly into the osculum" (Borojevic et al., 2002).

Leucetta floridana Haeckel, 1872 (Figure 14, Table 12) SYNONYMS Amphoriscus floridanus: Haeckel, 1872: 144. Dyssycus floridanus: Haeckel, 1872: 144. Leucaltis floridana: Haeckel, 1872: 144. Leucaltis impura: Haeckel, 1872: 144. Leucaltis pura: Haeckel, 1872: 144. Leucetta aff. floridana: Lehnert & van Soest, 1998: 99. Leucetta floridana: de Laubenfels, 1950: 146; Moraes et al., 2006: 167; Muricy et al., 2008: 132; Valderrama et al., 2009: 9; Lanna et al., 2009: 7; Muricy et al., 2011: 36-37; Rützler et al., 2014: 102; Azevedo et al, submitted. Leucetta microraphis: Tanita, 1942: 111; Borojevic & Peixinho, 1976: 1003. Leucetta sp. : Moraes et al., 2003: 17. Leucilla floridana: Jenkin, 1908: 453. Lipostomella floridana: Haeckel, 1872: 144. TYPE LOCALITY Coast of Florida, United States of America. MATERIAL EXAMINED UFRJPOR 6726 (specimen in ethanol and slides); Water Factory, Willemstadt; 12°06'30.88"N, 68°57'13.53"W; 17.8 m deep; coll. E. Hajdu, 19 August 2011; UFRJPOR 6757 (specimen in ethanol and slides); Tug Boat, Caracasbaai, Willemstadt; 12°04'08.20"N, 68°51'44.40"W; 6.2 m deep; coll. B. Cóndor-Luján, 23 August 2011; UFRJPOR 6765 (specimen in ethanol); Hook’s Hut, Willemstadt, Curaçao; 12°07'18.94"N, 68°58'11.46"W; 13.3 m deep; coll. E. Hajdu, 18 August 2011. 77

COLOUR White to light blue in life and grayish white to brown in ethanol. MORPHOLOGY AND ANATOMY This species has a massive growth form (Figure 14A). The largest specimen measures 1.6 x 2.6 cm (Figure 14B). The body is ridged and hispid. The three analised specimens presented a single apical osculum (largest diameter=0.5 cm). In the specimen UFRJPOR 6757, the osculum is particularly elongated and bears a very delicate margin. The atrial cavity is wide and also very hispid. The aquiferous system is leuconoid. The consistency is very rough and incompressible. SKELETON The skeleton is typical of the genus. It does not have a special organization and it is composed of two size categories of triactines (I and II) and tetractines (I and II). The cortex and atrial wall are thin whereas the choanosome is thick. Triactines II and tetractines II, which are the largest spicules, are found in in the cortex and in the choanosome, tangentially disposed. Tetractines II are rare. Triactines I and tetractines I are spread in the choanosome and in the atrium. The apical actine of tetractines I penetrates the exhalant canals and the atrial cavity. Near the atrium, triactines I and tetractines I become sagittal. SPICULES Triactines I. Regular. Actines are conical, straight, with blunt tips (Figure 14C). Frequent. Sagittal triactines I were also observed. Size: 87.5-175.0/10.0-22.5 µm. Triactines II. Regular. Actines are conical, straight, with blunt tips (Figure 14D). Very variable size: 378.4-2378.4/54.1-389.2 µm. Tetractines I. Regular. Actines are slightly conical, straight, with blunt to sharp tips (Figure 14E). The apical actine is smooth, thinner than the basal actines and has a sharp tip. Sagittal tetractines I were also observed. Size: 102.5-200.0/11.2-20.0 µm (basal actine) and 25-50/7.5-10 µm (apical actine). Tetractines II. Regular. Rare. Actines are conical, straight, with blunt tips (Figure 14F). Very variable size: 464.9-2162.2/108.1-270.3 µm. ECOLOGY This species was found underneath boulders close to some incrusting and massive (cf. Clathria) demospongias. No associated organism was found on the surface of the analised specimens. GEOGRAPHIC DISTRIBUTION Widespread in the Western Atlantic: Tropical Southwestern Atlantic Province including the Floridian (Haeckel, 1872), Bermuda (Bermudas, de Laubenfels, 1950), Greater Antilles (Jamaica, Lehner & van Soest, 1998) and the Southern Caribbean (Urabá and San Andrés, 78

Valderrama et al., 2009 and Curaçao, present study) ecoregions, North Brazil Shelf Province (Pará, Borojevic & Peixinho, 1976) and Tropical Northwestern Atlantic Province comprising the Northeastern Brazil, Eastern Brazil, Fernando de Noronha and Atoll das Rocas ecoregions (Borojevic & Peixinho, 1976; Valderrama et al., 2009). REMARKS Our specimens match the original description of L. floridana as well as the redescription provided by Valderrama et al. (2009). Haeckel's measurements are provided here: Triactines and tetractines I: 150-250/10-15 µm and triactines and tetractines II: 700-1500/100-150 µm and those of Valderrama et al., (2009) are presented in Table 12. Leucetta floridana is not only one of the few species of Calcarea already reported for the Caribbean Sea but it is also one of the most widespread species along the Western Tropical Atlantic.

Table 12. Spicule measurements of Leucetta floridana from Curaçao (UFRJPOR 6726 and UFRJPOR 6757) and of its redescription (UFRJPOR 5360. Valderrama et al., 2009).

Length (µm) Width (µm) N Specimen Spicule Actine Min Mean SD Max Min Mean SD Max UFRJPOR Triactine I - 121.5 143.6 14.6 172.8 10.8 13.9 1.6 16.2 20 6726 Triactine II - 972.0 1431.3 354.9 2378.4 129.6 214.5 52.5 345.6 21 Tetractine I B 105.0 139.1 23.3 200.0 12.5 15.2 2.2 20.0 20 A 25.0 33.4 8.4 45 7.5 8.1 1.2 10 8 Tetractine II B 675.7 1228.2 293.6 1621.6 108.1 171.8 49.7 216.2 9 UFRJPOR Triactine I - 87.5 126.7 22.2 175.0 10.0 15.5 3.2 22.5 30 6757 Triactine II - 378.4 1383.3 673.9 2378.4 54.05 207.7 98.3 389.2 24 Tetractine I B 102.5 138.3 19.6 200.0 11.2 15.3 2.8 20.0 30 A 40.0 44.4 4.3 50.0 10 10.0 0.0 10 4 Tetractine II B 464.9 1294.9 568.7 2162.2 108.1 172.0 59.1 270.3 9 UFRJPOR Triactine I - 105.6 143.3 28.7 217.8 9.9 17.1 4.9 33,0 30 5360 Triactine II - 257.4 696.2 279.7 1181.5 33.0 102.1 46.2 194.6 30 Tetractine I - 105.6 137.4 24.1 224.4 9.9 15.4 3.6 26.4 30 Tetractine II - 278.0 665.5 301.0 1042.5 48.7 102.5 51.3 180.7 8 79

Fig. 14. Leucetta floridana (UFRJPOR 6757): (A) specimen in vivo; (B) specimen after fixation; (C) triactine I; (D) triactine II; (E) tetractine I; (F) tetractine II.

Genus Amphoriscus Haeckel, 1872 TYPE SPECIES Amphoriscus chrysalis (Schmidt, 1864) DIAGNOSIS "Amphoriscidae with syconoid organization of the aquiferous system. Scattered spicules in the choanosome are always absent" (Borojevic et al., 2002). 80

Amphoriscus micropilosus sp. nov. (Figures 15 & 16, Table 13) ETIMOLOGY From the Latin pilosus (= hairy), for the presence of microdiactines crossing the cortex. TYPE LOCALITY Sunset Waters, Soto, Curaçao. TYPE MATERIAL Holotype: UFRJPOR 6739 (specimens in ethanol and slides); Sunset Waters, Soto, Curaçao; 12°16'01.58"N, 69°07'44.85"W; 13.1 m deep; coll. B. Cóndor-Luján, 22 August 2011. Paratypes: UFRJPOR 6755 and UFRJPOR 6756 (specimens in ethanol and slides); Tug Boat, Caracasbaai, Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W; 8.6 m deep; coll. B. Cóndor-Luján, 23 August 2011. COLOUR White in life and in ethanol. Transparent and bright. MORPHOLOGY AND ANATOMY This sponge has a very variable external morphology (Figure 15A-F), which can be tubular (Figures 15A and 15D) to flatened sac-shaped (Figures 15C and 15F) but always with an apical osculum. The surface is smooth, although microdiactines protrude through the surface. The consistency is rough. The largest specimen (UFRJPOR 6756) measures 1.2 x 0.4 cm and it presents a short peduncle (arrow in Figure 15D). The osculum has a margin sustained by T- shaped triactines and it is surrounded by short trichoxeas. The aquiferous system is syconoid. SKELETON The skeleton is typical of the genus (Figure 15G). The cortical skeleton is composed of perpendicular microdiactines spread through the surface (Figure 15H) or organized in tufts (only in UFRJPOR 6739, Figure 15I), triactines and the paired actines of the subcortical tetractines. The triactines are distributed tangentially to the surface (arrow in Figure 15J). The choanosomal skeleton is inarticulated, composed of the apical actines of the subcortical tetractines and by rare subatrial triactines. The apical actine of the tetractines crosses the choanosome and occasionally reaches the atrium (black arrow in Figure 15K). The unpaired actine of the subatrial triactines points toward the cortex (white arrow in Figure 15K). The atrial skeleton is exclusively composed of tetractines with their apical actine projected into the atrial cavity (asterisk in Figure 15K). SPICULES Microdiactines. Fusiform, straight, with sharp tips. Size: 27.0-94.5/1.1-1.4 µm 81

Cortical triactines. Sagittal. Actines are smooth, conical, with sharp tips (Figure 16A). Sometimes the paired actines are curved. Size: 102.6-310.5/8.1-18.9 µm (paired actines), 89.1- 310.5/6.7-18.9 µm (unpaired actine). Subcortical tetractines. Sagittal. Actines are straight, smooth, conical, with sharp tips (Figure 16B). The apical actine is very large. They are the largest spicules in this species. Size: 172.8- 572.4/21.6-64.8 µm (paired actine), 183.6-540/30-64.8 µm (unpaired actine), 162.0- 1036.8/21.6-64.8 µm (apical actine). Subatrial triactines. Sagittal. Actines are conical, smooth, straigth, with sharp tips (Figure 16C). The paired actines are shorter than the unpaired ones and some are slightly curved. Sometimes, one paired actine is longer than the other one. Size: 81.0-240.3/8.1-21.6 µm (paired actine), 91.8-610/8.1-18.9 µm (unpaired actine). Atrial tetractines. Sagittal. Actines are conical, straight, smooth and have sharp tips. The unpaired actine is slightly longer than the paired ones (Figure 16D). The apical actine is the shortest actine. Size (length/width): 100.0-264.6/10-24.3 µm (paired actine), 83.7-297/10-21.6 µm (unpaired actine), 27-115/8.1-13.5 µm (apical). ECOLOGY Specimens were found underneath broken coral boulders. No associated organism was found. Some balls of sediment were found inside the atrial cavity of the holotype. GEOGRAPHIC DISTRIBUTION Southern Caribbean (provisionally endemic to Curaçao present study). REMARKS The genus Amphoriscus comprises 16 species (Van Soest et al., 2016, Van Soest, 2017). Among them, four species have been reported for the Caribbean Sea: Amphoriscus oviparus (Haeckel, 1872), A. perforatus (Haeckel, 1872), A. testiparus (Haeckel, 1872), and A. urna Haeckel, 1872, however, none of them present a skeleton composition similar to A. micropilosus sp. nov. Different from the new species, whose subatrial skeleton is exclusively composed of triactines, all the referred species have subatrial tetractines and lack cortical microdiactines. Moreover, A. perforatus presents atrial triactines, which are absent in A. micropilosus sp. nov., and A. urna lacks the cortical triactines present in the Curaçaoan species. Considering the species recorded out of the Caribbean Sea, the species that most resembles the new species is A. elongatus (Poléjaeff, 1883), originally described from the Indian Ocean. Nonetheless, these two species differ in spicule size (Table 13), mainly in the width of the apical actine of the atrial tetractines. which is thinner in the new species (8.1-13.5 µm) than in A. elongatus (16-20 µm). Besides, in A. elongatus the radial tubes “meet in threes, in fours, or in 82 larger numbers around the same shallow invagination of the gastric cavity” (Poléjaeff, 1883), which was not observed in the new species. 83

Fig. 15. Amphoriscus micropilosus sp. nov.: (A-C) UFRJPOR 6756, UFRJPOR 6739 and UFRJPOR 6755 in vivo; (D-F) UFRJPOR 6756, UFRJPOR 6739 and UFRJPOR 6755 after fixation; (G) cross section of the skeleton; (H) trichoxeas along the surface in UFRJPOR 6755; (I) tufts of trichoxeas in UFRJPOR 6739; (J) cortical skeleton showing triactines (arrow); (K) choanosomal and atrial skeletons and the apical actine of cortical tetractines (black arrow), unpaired actine of a subatrial triactine (white arrow) and apical actine of an atrial tetractine (*). Abbreviations: cx=cortex; at=atrium. All photos were taken from the holotype slide (UFRJPOR 6739) except when indicated.

Fig. 16. Spicules of Amphoriscus micropilosus sp. nov. (Holotype=UFRJPOR 6739): (A) cortical triactine; (B) subcortical tetractine; (C) subatrial triactine; (D) atrial tetractine.

Table 13. Spicule measurements of Amphoriscus micropilosus sp. nov. (UFRJPOR 6739, UFRJPOR 6755 and UFRJPOR 6756) and of A. elongatus . H=holotype. P=paired, U=unpaired, A=apical and B=basal actines.

Length (µm) Width (µm) N Specimen Spicule Actine Min Mean SD Max Min Mean SD Max UFRJPOR Micro- 6739 (H) diactine - 51.3 75.6 16.8 94.5 1.1 1.3 0.1 1.4 7 Cortical P 102.6 204.0 51.7 310.5 8.1 14.4 2.8 18.9 14 Triactine U 110.7 229.5 54.5 283.5 6.7 14.1 3.0 18.9 15 Subcortical P 356.4 475.2 71.1 572.4 32.4 52.9 9.2 64.8 20 Tetractine U 216.0 392.0 127.2 540.0 48.6 54.5 4.7 64.8 10 A 162.0 449.6 158.8 658.8 21.6 49.7 12.4 64.8 19 Subatrial P 81.0 177.5 46.7 240.3 8.1 13.7 3.7 21.6 20 Triactine U 91.8 186.3 50.5 243.0 8.1 12.4 2.6 16.2 20 84

Atrial P 167.4 205.2 29.8 264.6 12.2 17.1 3.6 24.3 9 tetractine U 140.4 228.6 45.2 297.0 12.2 15.6 3.0 21.6 9 A 51.3 65.1 11.4 81.0 8.1 11.5 1.9 13.5 8 UFRJPOR Micro- 6755 diactine - 27.0 51.3 22.7 81.0 1.1 1.2 0.2 1.4 4 Cortical P 113.4 174.2 32.3 240.3 8.1 14.0 2.6 18.9 20 triactine U 89.1 221.1 64.9 310.5 8.1 14.4 3.2 18.9 20 Subcortical P 172.8 223.2 62.6 367.2 21.6 30.5 7.5 43.2 14 Tetractine U 183.6 229.0 54.8 324.0 32.4 40.0 9.0 54.0 5 A 291.6 491.8 217.8 1036.8 21.6 35.7 11.4 54.0 13 Subatrial P 86.4 165.4 41.8 232.2 8.1 14.0 2.5 18.9 20 triactine U 135.0 242.2 61.6 351.0 9.5 13.7 2.4 18.9 20 Atrial P 113.4 173.3 34.1 229.5 10.8 14.0 1.7 16.2 20 tetractine U 83.7 200.0 56.6 283.5 10.8 15.6 2.5 21.6 19 A 27.0 49.7 16.2 78.3 8.1 11.1 1.8 13.5 19 UFRJPOR Micro- 6756 diactine - 45.0 61.7 16.7 87.5 1.0 1.2 0.1 1.2 6 Cortical P 107.5 157.3 23.6 212.5 10 12.0 1.5 17.5 27 triactine U 157.5 195.4 23.0 230.0 10.0 12.2 0.8 12.5 26 Subcortical P 250.0 363.9 67.9 510.0 25.0 39.3 5.9 50 23 Tetractine U 250.0 279.0 23.3 315.0 30.0 37.0 4.5 40.0 5 A 325.0 555.0 110.4 740.0 30.0 42.3 7.0 55.0 30 Subatrial P 125.0 160.8 23.5 207.5 12.5 12.9 0.9 15 9 triactine U 255.0 346.3 110.2 610.0 10.0 13.8 2.4 18.8 15 Atrial P 100.0 142.3 25.4 212.5 10.0 11.9 1.7 15 27 tetractine U 125.0 184.7 43.4 250.0 10.0 12.4 1.8 17.5 22 A 55.0 78.8 17.8 115.0 10.0 10.3 0.8 12.5 10 A. Micro- elongatus diactine - - 100 - - - 2.5 - - - Cortical P - - - 250 - 15 - - - triactine U - - - 450 - 15 - - - Cortical B - 600 - - - 70 - - - tetractine A 600 ------Atrial P - 250 - - 16 - - 20 - tetractine U - - - 450 16 - - 20 -

Genus Leucilla Haeckel, 1872 TYPE SPECIES Leucilla amphora Haeckel, 1872 DIAGNOSIS "Amphoriscidae with sylleibid or leuconoid organization. The choanoskeleton is formed primarily by the apical actines of giant cortical tetractines and the unpaired actines of subatrial triactines or tetractines. It may contain dispersed spicules, but a typical articulated choanoskeleton is always absent" (Borojevic et al., 2002, emend). 85

Leucilla antillana sp. nov. (Figures 17 & 18, Table 14) ETIMOLOGY From its distribution in Curaçao located in the Leeward Antilles Ridge. TYPE LOCALITY Water Factory, Willemstadt, Curaçao. TYPE MATERIAL Holotype: UFRJPOR 6768 (specimen in ethanol and slides); Water Factory, Willemstadt, Curaçao; 12°06'30.88"N, 68°57'13.53"W; 9.9 m deep; coll. B. Cóndor-Luján, 23 August 2011. COLOUR White in life and in ethanol. MORPHOLOGY AND ANATOMY This species has an irregular tubular shape being wider at the base (Figure 17A). It measures 1.0 x 0.8 x 0.1 cm. The surface is slightly hispid due to some protruding spicules. Its texture is rough. The osculum is apical and has a delicate crown of trichoxeas (arrown in the Figure 17B). The aquiferous system is leuconoid. SKELETON The skeleton is characteristic of the genus (Figure 17C). The cortical skeleton is exclusively formed by tetractines (Figure 17D) with the basal actines tangentially disposed on the surface. The choanosomal skeleton is inarticulated, composed of the apical actine of the cortical tetractines (arrow in Figure 16E), which occasionally crosses the atrial skeleton, and of the unpaired actine of the subatrial triactines (white arrow in Figure 16F). The subatrial triactines do not form a continuous layer, instead, they are irregularly scattered at this region. The atrial skeleton is composed of tetractines with the apical actine projected into the atrium (black arrow in Figure 16F). SPICULES (Table 14) Cortical tetractines. Sagittal. Actines are conical with sharp tips. The paired actines are frequently curved. The apical actine is straight and it is the longest actine (Figure 17A). Some undulated apical actines were also observed. Size: 350-485/35-60 μm (paired actine), 75- 200/35-60 μm (unpaired actine) and 285-550/35-55 μm (apical actine). Subatrial triactines. Sagittal. Actines are conical with sharp tips. The paired actines are straight and smaller than the unpaired one (Figure 17B-C). Some slightly curved paired actines were also observed. Very variable size: 175-460/15-60 μm (paired actine) and 225-490/15-55 μm (unpaired actine). 86

Atrial tetractines. Sagittal. Actines are conical with very sharp tips. The unpaired actine is slightly longer than the paired ones (Figure 17D). The apical actine is the thinnest and shortest actine. Size: 120-270/10-12.5 μm (paired actine), 185-355/10-12.5 μm (unpaired actine) and 15- 50/5-10 μm (apical actine). ECOLOGY This species was found underneath coral boulders. GEOGRAPHIC DISTRIBUTION Southern Caribbean (provisionally endemic to Curaçao, present study). REMARKS The genus Leucilla comprises 13 valid species distributed in all oceans (Van Soest et al., 2016). Among these species, none of them has the same skeleton composition as Leucilla antillana sp. nov. The species that most resemble our new species are Leucilla uter Poléjaeff, 1883 (type locality: Bermudas) and L. sacculata Carter, 1890 (type locality: Fernando de Noronha Archipelago, Brazil). However, their skeletons include subatrial tetractines, which are absent in L. antillana sp. nov. Besides, the size range of the other spicule categories does not match our new species (Table 14). The apical actine of the cortical tetractines of L. uter is almost twice longer (400--1200 μm) than that of L. antillana sp. nov. (285-550 μm) and its atrial tetractines are thicker as well (20 μm against 10-12.5 μm). Comparing to L. sacculata, L. antillana sp. nov. has thinner spicules (84.7 μm against 15-60 μm). Leucilla sacculata and L. uter also differ from L. antillana sp. nov. in the ornamentation of the osculum. In L. antillana sp. nov., the osculum is surrounded by a crown of trichoxeas whereas in L. sacculata it is surrounded by a crown of microdiactines and in L. uter it is naked. However, this last difference should be taken with caution as the presence of a crown of trichoxeas is not consistent among individuals of the same species within some calcaronean genera. 87

Fig. 17. Leucilla antillana sp. nov. (UFRJPOR 6768): (A) specimen in vivo; (B) specimen after fixation with the crown of trichoxea (arrow); (C) cross section of the skeleton; (D) tangential section of the cortex; (E) choanoskeleton with the apical actine of a tetractine (arrow); (F) atrial skeleton with a subatrial triactine (white arrow) and an apical actine of an atrial tetractine (black arrow). Abbreviations: ct=cortex, at=atrium. 88

Fig. 18. Spicules of Leucilla antillana sp. nov. (UFRJPOR 6768): (A) cortical tetractine; (B) large subatrial triactine; (C) small subatrial triactine; (D) atrial tetractine.

Table 14. Spicule measurements of Leucilla antillana sp. nov. (H=holotype=UFRJPOR 6768), L. uter and L. sacculata. P=paired, U=unpaired, A=apical and B=basal actines.

Length (µm) Width (µm) N Species Spicule Actine Min Mean SD Max Min Mean SD Max UFRJPOR Cortical P 305.0 404.4 62.0 485.0 35 50.1 6.4 60.0 17 6768 (H) Tetractine U 75.0 131.0 45.3 200.0 35 48.8 7.4 60.0 10 A 285.0 405.0 64.6 550.0 35 49.2 7.1 55.0 19 Subatrial P 175.0 276.0 91.3 460 15.0 32.8 13.1 60.0 20 Triactine U 225.0 389.4 71.2 490.0 15.0 34.4 13.4 55.0 18 Atrial P 120.0 224.0 34.8 270.0 10 10.3 0.8 12.5 20 Tetractine U 185.0 265.0 41.9 355.0 10 10.5 1.0 12.5 20 A 15.0 30.0 8.9 50.0 5 8.0 1.7 10.0 20 Leucilla Micro- uter diactine - - 400 - - - 2.5 - - - Cortical B 400.0 - - 600.0 - - - 50 - Tetractine A 400.0 - - 1200. - - - 50 - 0 Subatrial U - - - 600.0 30 - - 50 - Triactine P - - - 420.0 21 - - 35 - 89

Atrial P - - - 400.0 - 20 - - - Tetractine U 250.0 - - 350.0 - 20 - - - A - - - 200.0 - 20 - - - Leucilla Micro- sacculata diactine - - 84.7 ------Cortical - - 564.4 - - - - - 84.7 - Tetractine Subatrial - - 564.4 - - - - - 84.7 - Triactine Subatrial - - 564.4 - - - - - 84.7 - Tetractine

Genus Leucandra Haeckel, 1872 TYPE SPECIES Leucandra egedii (Schmidt, 1870) DIAGNOSIS "Grantiidae with sylleibid or leuconoid organization. Longitudinal large diactines, if present, are not restricted to the cortex, but lie obliquely across the external part of the sponge wall and protrude from the surface of the sponge" (Borojevic et al., 2002).

Leucandra caribea sp. nov. (Figures 19-21, Table 17) ETIMOLOGY Named after its distribution in the Caribbean Sea. TYPE LOCALITY Tug Boat, Caracasbaai, Willemstadt, Curaçao. TYPE MATERIAL Holotype: UFRJPOR 6754 (specimen in ethanol and slides); Tug Boat, Caracasbaai, Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W; 13.9 m deep; coll. B. Cóndor-Luján, 23 August 2011. COLOUR White in life and beige in ethanol. MORPHOLOGY AND ANATOMY This species has a sac-shaped external morphology: it is wide at the base and becomes narrower near the apical osculum (Figure 19A). It measures 0.7 cm length and 0.3 cm width (Figure 19B). This sponge is quite smooth and compressible. Near the suboscular region, scattered short trichoxeas protrude through the surface (Figure 19C). Although some trichoxeas do protrude the 90 surface, it is not very hispid. The osculum is supported by triactines, tetractines and has a discrete crown of trichoxeas (Figures 19D and 19E). The aquiferous system is leuconoid with subspherical choanocytary chambers ranging from 28 to 34 µm (Figure 19F). SKELETON The skeleton is typical of the genus (Figure 20A). As mentioned before, the oscular margin has a differentiated skeleton. It is composed of T-shaped triactines and tetractines tangentially disposed and short trichoxeas perpendicular to the cortex. The cortical skeleton is composed of tangential triactines (Figure 20B). The unpaired actine can point either to the surface or to the choanosome. The choanosomal skeleton does not have a special organization and it is formed by triactines of variable size (as shown in Figures 18F and 19A). Several exhalant choanosomal canals with a diameter varying from 140 to 300 µm were observed within this region. They are surrounded by tetractines with the apical actine projected inside them (Figure 20C). Some triactines lining the canals were also found (Figure 20D). No subatrial skeleton was observed. The atrial skeleton is formed by triactines (Figures 20E) and rare tetractines (Figure 20F). The apical actine of the tetractines protrudes into the atrial cavity (arrow in Figure 19F). SPICULES Cortical triactines. Subregular or sagittal. Actines are slightly conical with sharp tips. The paired actines are frequently curved and slightly longer than the unpaired one which is always straight (Figure 21A). Compared to the atrial triactines, the cortical triactines are thicker. Size: 110-340/7.5-16.3 μm (paired actine) and 105-325/7.5-16.3 μm (unpaired actine). Choanosomal triactines. Regular or subregular. Actines are conical with sharp tips (Figure 21B). They are largest spicules in L. caribea sp. nov. Very variable size: 370-960/25-75 μm. Triactines and tetractines of the canals. Sagittal. Actines are conical with sharp tips. The paired actines are curved, following the shape of the choanosomal canals (Figure 21C). The apical actine of the tetractines is smooth and it is thinner than the basal ones (as shown in Figures 20C- D). The size of the triactines is similar to that of the tetractines. Size of tetractines: 112.5- 220.0/7.5-12.5 μm (paired), 62.5-210.0/7.5-12.5 μm (unpaired) and 45.0-110.0/5.0-10.0 μm (apical). Atrial triactines (shown in Figure 20E): Sagittal. Actines are conical with sharp tips. Compared to the cortical triactines, the angle formed by the paired actines of the atrial triactines is more open. Size: 132.3-253.8/8.1-13.5 μm (paired actine) and 164.7-253.8/5.4-13.5 μm (unpaired actine). 91

Atrial tetractines. Sagittal. Actines are conical with sharp tips (Figure 21D). The apical actine is smooth. Size: 150-262.5/7.5-15.0 μm (paired actine), 162.0-297.0/8.1-16.2 μm (unpaired actine) and 35.0-132.5/7.5-12.5 μm (apical actine). ECOLOGY This specimen was found underneath a coral boulder. GEOGRAPHIC DISTRIBUTION Southern Caribbean (provisionally endemic to Curaçao, present study). REMARKS Among the species of Leucandra reported from the Caribbean Sea, namely L. crustacea (Haeckel, 1872), L. barbata (Duchassaing & Michelotti, 1864), L. curva (Schuffner, 1877), L. multiformis Poléjaeff, 1883, L. rudifera Poléjaeff, 1883, and L. typica (Poléjaeff, 1883) (Van Soest et al., 2016), only L. typica possess a similar skeleton composition as that of L. caribea sp. nov. The skeletons of the other Caribbean species include cortical tetractines (L. crustacea and L. curva), diactines (L. barbata, L. multiformis and L. rudifera) and atrial grapnel spicules (also in L. rudifera), all of which are not present in L. caribea sp. nov. Leucandra caribea sp. nov. can be differentiated from L. typica as the former possess an atrial skeleton mainly composed of triactines and few tetractines whereas in the latter triactines and tetractines are in the same proportion (or at least, Poléjaeff did not indicate the opposite). Besides, in the new species, the choanosomal canals are lined by tetractines and triactines and in L. typica, they are only lined by tetractines. In L. typica, trichoxeas (<300/1 μm) are scattered in the choanosome and spindle-shaped microdiactines (100/4 μm) are concentrated in the suboscular region whereas in L. caribea sp. nov., trichoxeas (>100/1.2) were found only in the suboscular region. Within the other species of Leucandra with similar skeleton composition and external morphology, L. falakra Klautau et al., 2016 from the Adriactic Sea is the one that most resembles L. caribea sp. nov. Nonetheless, they have some important differences. The choanosomal skeleton of the Curaçaoan species is composed of one single type of triactine (370-960/25-75) while in the Adriatic species, it is composed of small (paired actine: 94.5- 180.9/8.1-13.5 μm and unpaired actine: 70-143.1/8.1-16.2) and giant triactines (342- 1047.6/48.6-118.8 μm). In addition, although almost all the spicule categories have similar size (Table 17), the unpaired actine of the atrial tetractines is shorter in L. falakra (59.4-126.9 µm) than in the new species (162-297 µm). 92

Table 17. Spicule measurements of Leucandra caribea sp. nov. (H=holotype: UFRJPOR 6754) and Leucandra falakra (H=holotype: UFRJPOR 8349). P=paired, U=unpaired and A=apical actines.

Length (µm) Width (µm) N Specimen Spicule Actine Min Mean SD Max Min Mean SD Max UFRJPOR Trichoxea - - - - >105 1.1 1.2 0.2 1.6 15 6754 (crown) Trichoxea - - - - >108 1.1 1.2 0.2 1.6 10 (cortex) Cortical P 110-.0 218.4 65.2 340.0 7.5 11.6 2.4 16.3 25 triactine U 105.0 219.3 63.7 325.0 7.5 12.6 2.4 16.3 25 Choanosomal - 370.0 576.3 188.8 960.0 25.0 41.3 10.8 75.0 24 triactine Canal P 112.5 179.3 32.0 220.0 7.5 9.8 1.8 12.5 10 tetractine U 62.5 140.6 60.4 210.0 7.5 10.0 2.0 12.5 4 A 45.0 70.9 19.0 110.0 5.0 7.9 1.3 10.0 14 Atrial P 132.3 209.4 41.8 253.8 8.1 11.2 1.9 13.5 12 triactine U 164.7 210.6 35.0 253.8 5.4 10.1 3.4 13.5 6 Atrial P 150.0 206.9 28.7 262.5 7.5 11.6 1.9 15.0 20 tetractine U 162.0 237.6 35.1 297.0 8.1 12.9 2.5 16.2 20 A 35.0 69.0 28.3 132.5 7.5 9.6 1.2 12.5 20 UFRJPOR Cortical P 94.5 136.4 24.0 180.9 8.1 11.1 1.9 13.5 20 8349 triactine U 70.2 106.0 18.8 143.1 8.1 11.4 2.4 16.2 20 Cortical and - 342.0 624.5 192.3 1047. 48.6 81.5 20.6 118. 23 choanosomal 6 8 triactine Choanosomal P 162.0 214.2 39.8 288.9 13.5 18.3 4.0 27.0 20 triactine U 108.0 189.7 58.9 351.0 13.5 19.8 4.1 29.7 20 Canal P 99.9 154.0 26.4 199.8 8.1 12.4 2.4 16.2 19 tetractine U 45.9 143.0 56.5 288.9 9.5 12.4 1.9 16.2 19 A 50 80.6 24.4 137.5 7.5 9.6 1.5 12.5 20 Atrial P 140.4 222.7 33.7 294.3 9.5 15.1 2.5 20.3 30 triactine U 78.3 111.2 24.4 159.3 8.1 12.3 1.7 16.2 30 Atrial P 145.8 191.4 26.0 256.5 10.8 14.9 2.6 18.9 30 tetractine U 59.4 92.0 22.1 126.9 10.8 13.1 1.7 16.2 16 A 67.5 110.3 30.3 162.0 8.1 11.9 2.8 16.2 15 93

Fig. 19. Leucandra caribea sp. nov.: (A) specimen in vivo; (B) specimen after fixation; (C) detail of trichoxeas (arrow) along the surface; (D) tangential section of the oscular region; (E) detail of the oscular trichoxeas (arrow); (F) cross section of the skeleton. Abbreviations: c=choanosomal canals, cc=choanocytary chambers. 94

Fig. 20. Skeleton of Leucandra caribea sp. nov.: (A) cross section of the skeleton; (B) cortex; (C) tetractine of a choanosomal canal; (D) triactine of a choanosomal canal; (E) atrial triactine; (F) atrial tetractine with the apical actine projected into the atrial cavity (arrow). Abbreviations: cx=cortex, c=choanosomal canal, ct=cortical triactines, at=atrium. 95

Fig. 21. Spicules of Leucandra caribea sp. nov: (A) cortical triactine; (B) choanosomal triactine; (C) tetractine of choanosomal canals; (D) atrial tetractine.

Genus Leucandrilla Bojorevic, Boury-Esnault & Vacelet, 2000

TYPE SPECIES Leucilla wasinensis Jenkin, 1908 DIAGNOSIS "Grantiidae with leuconoid organization. In addition to triactines the cortex contains tetractines, with the apical actines turned into the choanoderm. The articulated choanoskeleton is supported by subatrial triactine spicules, and numerous rows of choanosomal triactines and/or tetractines, with apical actines of cortical tetractines in the distal region" (Borojevic et al., 2002).

Leucandrilla pseudosagittata sp. nov. (Figures 22-24, Table 18) ETIMOLOGY Derived from the presence of subcortical tetractines with pseudosagittal shape. 96

TYPE MATERIAL Holotype: UFRJPOR 6752 (specimen in ethanol and slides); Tug Boat, Caracasbaai, Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W; 15.2 m deep; coll. E. Hajdu, 23 August 2011. Paratypes: UFRJPOR 6696 (specimen in ethanol and slides); Sunset Waters, Soto, Curaçao; 12°07'18.94"N, 68°58'11.46"W; <10 m deep; coll. B. Cóndor-Luján and G. Lôbo-Hajdu, 17 August 2011 and UFRJPOR 6705 (specimen in ethanol and slides); Water Factory, Willemstadt, Curaçao; 12°12'43.12"N, 69°05'8.42"W; 3-5 m deep; coll. B. Cóndor-Luján, 18 August 2011. TYPE LOCALITY Tug Boat, Caracasbaai, Willemstadt, Curaçao. COLOUR White in life and in ethanol. MORPHOLOGY AND ANATOMY This species has a cylindrical massive body (Figure 22A-E). The holotype is the largest specimen and it measures 3.8 cm x 2.0 cm (Figures 22A and 22C). In this specimen, the choanosomal wall is thinner at the apical region (0.2 cm) and thicker at the basal region (0.6 cm).The surface is slightly rough and the consistency is hard. The osculum is apical and its diameter is 1.2 mm. The atrial cavity is not hispid and measures 0.4 cm. The aquiferous system is leuconoid with spherical choanocytary chambers. SKELETON In the three analised specimens the oscular margin is composed of T-shaped spicules including triactines and rare tetractines (Figure 23A). The skeleton is not typical of the genus (Figure 23B) as it presents rare tetractines with pseudosagittal shape. The cortical skeleton is composed of triactines (Figure 23B and 23C) tangentially disposed on the surface. The subcortical skeleton is formed by large sagittal tetractines. Some of them have pseudosagittal shape (white arrow in Figure 23D). The longer paired actine of the pseudosagittal tetractines and the apical actine of the sagittal tetractines cross the choanosome and sometimes reach the atrial skeleton. The choanosomal skeleton has no special organization and some subcortical tetractines invade this region. The choanosomal canals (average diameter: 108-238 µm) are surrounded by small tetractines whose apical actine is projected into them (Figure 23E). The poorly developed subatrial skeleton is formed only by tetractines (black arrow in Figure 23D). The atrial skeleton is exclusively composed of tetractines with the apical actine projected into the atrial cavity (Figure 23F). 97

SPICULES Cortical triactines. Sagittal. Actines are conical with blunt to sharp tips. Some paired actines are slightly curved (Figure 24A). Very variable size: 97.2-421.2/8.1-21.6 µm (paired actine), 75.6- 421.2/5.4-21.6 µm (unpaired actine). Subcortical tetractines I. Sagittal. Actines are conical with sharp tips. The paired actines are slightly curved (Figure 24B). The apical actine is generally longer than the paired ones. Size: 248.4-1188.0/27.0-75.6 µm (paired actine), 162.0-564.0/27.0-64.8 µm (unpaired actine) and 345.6-1122.0/21.6-75.6 µm (apical actine). Subcortical tetractines II (indicated in Figure 24D). Pseudosagittal. Rare. Actines are conical with sharp tips. Size: 270.0-529.2/37.8-54.0 µm (shorter paired actine), 237.6-1134.0/32.4-75.6 µm (longer paired actine), 270.0-1036.8/32.4-75.6 µm (unpaired actine) and 162.0-540.0/21.6- 64.8 µm (apical actine). Canal tetractines. Sagittal. Actines are conical with sharp tips. The paired actines are curved following the shape of the canals and they are longer than the other actines (Figure 24C). Size: 118.8-264.6/8.1-17.6 µm (paired actine), 70.2-213.3/8.1-17.6 µm (unpaired actine) and 40.5- 132.3/8.1-13.5 µm (apical actine). Subatrial tetractines. Sagittal. Actines are conical with sharp tips (Figure 24D). The apical actine is shorter than the basal ones. Size: 205.2-993.6/21.6-75.6 µm (paired), 205.2-766.8/21.6- 70.2 µm (unpaired) and 129.6-432.0/16.2-54.0 µm (apical actine). Atrial tetractines. Sagittal. Actines are conical with blunt to sharp tips (Figure 24E). Sometimes the paired actines are slightly curved. The apical actine is conical, shorter than the basal ones and has sharp tips. Size: 67.5-391.5/8.1-21.6 µm (paired actine), 59.4-270.0/8.1-21.6 µm (unpaired actine) and 21.6-108.0/6.4-13.5 µm (apical actine). ECOLOGY The three specimens were collected in light protected habitats. The holotype and one paratype (UFRJPOR 6696) were collected underneath boulders. A polychaete was found inside the holotype. GEOGRAPHIC DISTRIBUTION Southern Caribbean (provisionally endemic to Curaçao, present study). REMARKS Although the skeleton of the analysed specimens from Curaçao presents rare subcortical tetractines with pseudosagittal shape, which would suggest their allocation in one of the genera of the family Heteropiidae, we placed them in the genus Leucandrilla following Borojevic et al., 2000: "In any calcaronean sponge with a strong cortex, some subcortical spicules may be in 98 the position and have the shape of pseudosagittal spicules, due to the restriction of their growth by the rigidity of the cortical skeleton. They should not be interpreted as an indication that the sponge belongs to the family Heteropiidae." Three species are considered within the genus Leucandrilla: L. intermedia (Row, 1909) from the Red Sea, L. lanceolata (Row & Hôzawa, 1931) from Southwestern Australia and L. wasinensis (Jenkin, 1908) from East Africa. Leucandrilla pseudosagittata sp. nov. can be easily differentiated from them by the absence of diactines and choanosomal triactines and by the presence of pseudosagittal subcortical tetractines. The two sequences of L. pseudosagittata sp. nov. formed a monophyletic clade (pp=1, bb=100) within the large clade LEUCII (which did not include any Heteropiidae species, see Phylogenetic section). In both BI and ML phylogenetic trees, L. pseudosagittata nested with other Grantiidae and Amphoriscidae species but with low support. In the BI tree (Figure 30), it grouped with Amphoriscus micropilosus sp. nov., Leucilla antillana sp. nov., Sycon conulosum sp. nov. and S. megapicalis sp. nov. (pp=0.57) and in the ML tree (data not shown), it clustered with Leucandra nicolae, Paraleucilla magna and P. dalmatica (b=44.7).

Fig. 21. Leucandrilla pseudosagittata sp. nov.: (A-B) Specimens in vivo; (C-E) specimens after fixation. 99

Fig. 22. Skeleton composition of Leucandrilla pseudosagittata sp. nov. (paratype UFRJPOR 6705): (A) osculum; (B) cross section of the skeleton; (C) cortex; (D) choanoskeleton indicating the subcortical tetractine (white arrow) and the subatrial tetractine (black arrow); (E) choanosomal canals (c); (F) atrial skeleton. Abbreviations: ct=cortex, at=atrium. 100

Fig. 23. Spicules of Leucandrilla pseudosagittata sp. nov. (paratype UFRJPOR 6705): (A) cortical triactine; (B) subcortical tetractine; (C) tetractine of a canal; (D) subatrial tetractine; (E) atrial tetractine.

Table 18. Spicule measurements of Leucandrilla pseudosagittata sp. nov. (UFRJPOR 6696, UFRJPOR 6705 and UFRJPOR 6752). H=holotype. P=paired, U=unpaired and A=apical actines.

Length Width N Specimen Spicule Actine Min Mean SD Max Min Mean SD Max UFRJPOR Cortical P 97.2 273.2 77.4 399.6 8.1 14.9 3.6 21.6 20 6696 (H) Triactine U 97.2 207.4 70.3 345.6 5.4 14.1 4.3 21.6 20 Subcortical P 248.4 543.8 161.5 810.0 27.0 43.5 10.3 64.8 20 Tetractine I U 162.0 271.1 90.0 399.6 32.4 37.8 5.7 43.2 10 A 367.2 541.8 104.2 702.0 21.6 38.4 10.9 64.8 17 Subcortical P + 356.4 574.4 154.7 896.4 32.4 53.0 11.3 64.8 11 Tetractine P - 313.2 402.8 71.8 529.2 37.8 46.4 5.8 54.0 10 II U 324.0 479.5 105.7 648.0 32.4 49.7 8.0 59.4 10 A 162.0 261.4 78.7 378.0 21.6 32.9 6.5 43.4 10 Canal P 118.8 200.7 39.9 256.5 8.1 12.6 2.8 17.6 20 Tetractine U 97.2 135.8 25.4 189.0 8.1 12.4 2.6 17.6 20 A 54.0 89.4 23.1 124.2 8.1 10.2 1.8 13.5 20 Subatrial P 205.2 522.7 172.1 993.6 21.6 44.8 12.3 75.6 20 101

Tetractines U 205.2 388.3 113.8 637.2 21.6 35.4 7.9 48.6 20 A 129.6 276.9 87.8 432.0 16.2 33.2 8.2 43.2 14 Atrial P 67.5 201.1 57.5 297.0 8.1 13.1 3.2 18.9 19 Tetractine U 59.4 187.5 54.3 240.3 8.1 12.6 3.4 18.9 11 A 32.4 62.9 23.0 105.3 6.4 9.3 2.1 14.9 11 UFRJPOR Cortical P 129.6 307.3 85.0 410.4 9.5 13.8 3.2 21.6 21 6705 Triactine U 129.6 261.4 65.3 367.2 9.5 14.0 3.4 21.6 12 Subcortical P 259.2 639.4 211.0 972.0 27.0 54.3 13.5 70.0 20 Tetractine I U 162.0 350.1 103.3 464.4 27.0 39.6 11.4 64.8 12 A 345.6 511.2 147.7 864.0 37.8 51.3 8.7 70.2 20 Subcortical P + 453.6 680.4 162.5 1134. 43.2 58.4 7.9 70.2 16 Tetractine 0 II P - 237.6 460.1 145.5 648.0 43.2 52.4 3.6 54.0 10 U 270.0 583.2 145.9 756.0 48.6 53.3 4.0 64.8 15 A 270.0 359.1 78.3 486.0 43.2 52.0 7.0 64.8 8 Canal P 143.1 210.7 32.1 264.6 8.1 12.5 2.5 17.6 20 Tetractine U 97.2 143.3 29.1 205.2 9.5 13.1 2.3 17.6 18 A 62.1 86.4 16.7 124.2 8.1 9.6 1.5 13.5 20 Subatrial P 259.2 489.8 207.9 864.0 32.4 48.3 13.2 70.2 20 Tetractines U 216.0 405.0 131.6 669.6 32.4 48.3 11.8 70.2 20 A 129.6 279.0 99.5 432.0 21.6 35.1 9.4 54.0 12 Atrial P 135.0 230.0 69.4 391.5 8.1 13.8 3.5 21.6 20 Tetractine U 94.5 164.4 43.8 264.6 9.5 13.4 3.1 21.6 20 A 27.0 46.4 14.0 64.8 8.1 9.5 2.3 13.5 19 UFRJPOR Cortical P 118.8 276.5 89.3 421.2 10.8 15.0 2.7 20.3 20 6752 Triactine U 75.6 242.5 84.9 421.2 12.2 15.1 2.3 18.9 20 Subcortical P 313.2 689.6 222.4 1188. 32.4 52.9 11.0 75.6 20 Tetractine I 0 U 194.4 360.7 134.3 594.0 32.4 36.7 7.1 54.0 10 A 367.2 693.6 232.2 1122. 32.4 51.3 11.0 75.6 20 0 Subcortical P + 237.6 539.2 197.1 810.0 32.4 45.5 11.2 75.6 14 Tetractine P - 270.0 459.0 121.5 594.0 37.8 47.0 6.3 54.0 10 II U 313.2 548.1 258.1 1036. 32.4 48.6 12.6 75.6 12 8 A 216.0 346.8 93.1 540.0 32.4 39.0 5.2 43.2 9 Canal P 118.8 186.4 46.5 245.7 8.1 12.0 2.4 16.2 20 Tetractine U 70.2 128.5 41.2 213.3 8.1 10.9 2.6 16.2 20 A 40.5 81.7 23.5 132.3 8.1 10.2 1.7 13.5 20 Subatrial P 280.8 518.4 191.1 853.2 32.4 46.5 10.7 59.4 13 Tetractine U 302.4 502.7 167.1 766.8 32.4 44.7 9.4 54.0 11 A 216.0 332.6 66.1 432.0 27.0 40.0 10.6 54.0 10 Atrial P 153.9 205.9 48.0 332.1 9.5 13.8 3.0 21.6 29 Tetractine U 126.9 202.8 40.9 270.0 9.5 13.8 3.1 21.6 24 A 21.6 60.5 23.0 108.0 6.7 9.8 1.9 14.9 30 102

Genus Grantessa Lendenfeld, 1885 TYPE SPECIES Grantessa sacca Lendenfeld, 1885 DIAGNOSIS "Heteropiidae with syconoid organization and an articulated choanoskeleton. A thin cortex is formed by triactines but lacks longitudinal large diactines. The distal part of the radial tubes is frequently decorated by tufts of radially arranged diactines, indicating a close relationship to the genus Syconessa" (Borojevic et al., 2002).

Grantessa tumida sp. nov. (Figures 24 & 25, Table 19) ETIMOLOGY From the Latin tumidus (=swollen), for the presence of subatrial and atrial spicules with distally swollen unpaired actines. TYPE LOCALITY Sunset Waters, Soto, Curaçao TYPE MATERIAL Holotype: UFRJPOR 6766 (specimen in ethanol and slides); Sunset Waters, Soto, Curaçao; 12°16'01.58"N, 69°07'44.85"W; 13.1 m deep; coll. B. Cóndor-Luján, 23 August 2011. Paratypes: UFRJPOR 6701 (specimen in ethanol and slides); Daai Booi, St. Willibrordus, Curaçao; 12°12'43.12"N, 69°05'8.42"W; 3-5 m deep; coll. B. Cóndor-Luján, 18 August 2011 and UFRJPOR 6695 (specimen in ethanol and slides); Hook’s Hut, Willemstadt, Curaçao; 12°07'18.94"N, 68°58'11.46"W; 6.3 m deep; coll. B. Cóndor-Luján & G. Lôbo-Hajdu, 17 August 2011. COLOUR Beige to light brownish in life and beige in ethanol. MORPHOLOGY AND ANATOMY This species has a tubular to sac-shaped body with an apical osculum surrounded by a crown of trichoxeas. The holotype (UFRJPOR 6766) is the largest specimen. It measures 0.5 x 0.2 cm (Figure 24A). The surface is smooth to the touch although diactines protrude through the surface. The consistency is compressible. The aquiferous system is syconoid. SKELETON The osculum is surrounded by a crown of trichoxeas supported by T-shaped triactines and rare tetractines (Figure 24B). The skeleton is typical of the genus. The cortical skeleton is composed of diactines and triactines (Figure 24C). The diactines are perpendicularly arranged in tufts and 103 do not penetrate the choanosome. The triactines are tangentially disposed with the paired actines laying tangentially to the subcortical region (black arrow in Figure 24C). The subcortical skeleton is composed of pseudosagittal triactines with the longest paired actine (actine 1) penetrating the choanosome (white arrow in Figure 24C). The tubar skeleton is articulated (Figure 24D), composed of several rows of triactines with the unpaired actine pointing to the cortex. The subatrial skeleton is composed of triactines with the unpaired actine pointing to the cortex (Figure 24E). The atrial skeleton is composed of triactines and tetractines with the unpaired actine adjacent to the atrium (in the common position of the paired actines). Some atrial tetractines with the unpaired actine disposed in the traditional position were also found. The apical actine of the tetractines penetrates the atrial cavity (Figure 24F). SPICULES Diactines. Fusiform, straight, with tips usually sharp (Figures 25A-B). Size: 108.0-637.2/5.4- 16.2 µm. Cortical triactines. Sagittal. Actines are conical with sharp tips. The paired actines are less straight than the unpaired one (Figure 25C). Size: 48.6-102.6/4.1-8.1 µm (paired actine), 51.3- 175.5/4.1-8.1 µm (unpaired actine). Subcortical triactines. Pseudosagittal. Actines are conical with sharp tips. One of the paired actines is shorter and more curved than the other. The unpaired actine is straight (Figure 25D). Size: 67.5-145.8/4.1-5.4 µm (paired actine 1), 51.3-113.4/2.7-5.4 µm (paired actine 2), 54.0- 129.6/4.1-8.1 µm (unpaired actine). Tubar triactines I. Sagittal. Actines are conical with sharp tips. One of the paired actines is shorter and more curved than the other. The unpaired actine is straight and usually slightly longer than the paired ones (Figure 25E). Size: 62.1-140.4/5.4-8.1 µm (paired actine), 21.6- 72.9/4.1-8.1 µm (short paired actine), 99.9-202.5/5.4-8.1µm (unpaired actine). Tubar triactines II. Sagittal. Actines are conical with sharp tips. The paired actines have almost the same size and are slightly curved. The unpaired actine is straight and generally longer than the paired ones (Figure 25F). Size: 48.6-121.5/5.4-8.1 µm (paired actine), 72.9-205.5/5.4-8.1 µm (unpaired actine). Subatrial triactine. Sagittal. Actines are slightly conical with sharp tips. The unpaired actine is straight and longer than the paired ones. The paired actines are inwardly curved (Figure 25G). Some paired actines with different lengths in the same spicule were also found. Size: 40.5- 108.8/4.1-6.8 µm (paired actine), 35.1-243.0/4.1-8.1 µm (unpaired actine). 104

Atrial triactine. Sagittal. Actines are conical with sharp tips. The unpaired actine is elongated and swollen at the distal part (Figures 25H-I). Size: 89.1-162.0/4.1-8.1µm (paired actine), 159.3-283.5/5.4-8.1 µm (unpaired actine). Atrial tetractine I: Sagittal. Actines are conical with sharp tips. The unpaired actine is longer than the paired ones (Figure 25J). Some apical actines are curved. Size: 78.3-148.5/4.1-5.4 µm (paired actine), 108.0-280.8/5.4-8.1 µm (unpaired actine), 13.5-108.0/4.1-6.8 µm (apical actine). Atrial tetractine II. Sagittal. Actines are conical with sharp tips. The unpaired actine is elongated in tetractines II and, unlike the unpaired actine of the tetractines I, it is swollen at the distal part (Figure 25H). The apical actine is curved and it is the shortest actine of this species. Size: 94.5- 151.2/4.1-8.1 µm (paired actine), 162.0-283.5/5.1-8.1 µm (unpaired actine), 13.5-43.2/4.1-5.4 µm (apical actine). ECOLOGY The specimens were found underneath coral boulders. One of the paratypes (UFRJPOR 6695) was covered with sediment when collected. GEOGRAPHIC DISTRIBUTION Southern Caribbean (provisionally endemic to Curaçao, present study). REMARKS Among the 28 valid species of the genus Grantessa, only G. ramosa sensu Borojevic (1967) from South Africa and G. tenhoveni Van Soest & de Voogd, 2015 from Indonesia present atrial spicules with swollen unpaired actine. Grantessa ramosa sensu Borojevic (1967) is very similar to the Curaçaoan species, however, they present some dissimilarities. In G. tumida sp. nov. all the spicules (but the diactines) are thinner (Table 19) and the apical actine of the atrial tetractines is straight whereas in G. ramosa sensu Borojevic (1967) it is curved. Differently from G. tenhoveni, the skeleton of our new species does not bear subatrial tetractines nor atrial tetractines with a very long apical actine (660-960 µm) as described for the Indonensian species. It is important to point out here that G. ramosa sensu Borojevic (1967) differs greatly from the original description of G. ramosa (Haeckel, 1872), also from South Africa, in spicule size and shape (Table 22). Based on the written descriptions and drawings, they probably belong to two different species. The C-LSU sequences of G. tumida sp. nov. (UFRJPOR6701 and UFRJPOR6695) grouped together in the same cluster (pp=1, b=100) within the large clade LEUCI (which included species of the Grantiidae and Heteropiidae families). Nonetheless, G. tumida sp. nov. did not group with the other Grantessa species, G. aff. intusarticulata (Figure 30). 105

Fig. 24. Grantessa tumida sp. nov. (Holotype=UFRJPOR 6766): (A) specimen after fixation; (B) osculum; (C) distal part of the skeleton indicating a cortical (black arrow) and pseudosagittal subcortical triactine (white arrow); (D) tubar skeleton; (E) subatrial skeleton; (F) atrial skeleton. 106

Fig. 25. Spicules of Grantessa tumida sp. nov (Holotype=UFRJPOR 6766): (A, B) diactine; (C) cortical triactine; (D) subcortical triactine; (E) tubar triactine I; (F) tubar triactine II; (G) subatrial triactine; (H) atrial triactine; (I) detail of the swollen tip of the atrial triactine; (J) atrial tetractine II. 107

Table 19. Spicule measurements of Grantessa tumida sp. nov. (UFRJPOR 6695, UFRJPOR 6701 and UFRJPOR 6766), G. ramosa (Haeckel, 1872) and G. ramosa sensu Borojevic (1967). H=holotype, P=Paired, U=unpaired, A=apical, CP=cortical paired and IP=internal paired actines.

Specimen Spicule Actine Length (µm) Width (µm) N Min Mean S Max Min Mean S Max UFRJPOR Diactine 108.0 250.6 99.6 432.0 5.4 9.7 2.8 16.2 20 6695 Cortical P 48.6 62.1 9.8 81.0 5.4 5.4 0.0 5.4 20 triactine U 51.3 93.9 21.7 121.5 5.4 5.6 0.5 6.8 20 Pseudo P1 67.5 106.7 27.3 140.4 4.1 5.1 0.6 5.4 15 sagittal P2 51.3 73.7 17.9 108.0 4.1 5.1 0.6 5.4 13 Triactine U 54.0 93.4 24.5 124.2 4.1 5.1 0.6 5.4 15 Tubar P1 72.9 110.0 22.2 140.4 5.4 6.4 1.1 8.1 20 Triactine 1 P2 29.7 50.1 14.3 72.9 5.4 5.8 0.6 6.8 20 U 99.9 138.4 21.4 164.7 5.4 6.0 0.8 8.1 20 Tubar P 51.3 91.8 20.8 121.5 5.4 6.4 1.1 8.1 20 Triactine U 99.9 153.4 35.3 207.9 5.4 6.0 0.8 8.1 20 Subatrial P 43.2 83.5 21.8 108.0 4.1 5.2 0.9 6.8 15 Triactine U 70.2 156.2 60.8 229.5 4.1 5.8 0.9 6.8 14 Atrial P 94.5 119.8 17.4 148.5 4.1 5.1 0.6 5.4 14 triactine U 159.3 223.3 32.9 283.5 5.4 6.1 1.0 8.1 20 Atrial P 78.3 117.5 22.7 148.5 4.1 5.1 0.6 5.4 12 Tetractine U 121.5 196.9 53.9 270.0 5.4 6.4 1.2 8.1 12 I A 24.3 56.2 17.8 86.4 4.1 4.9 0.7 5.4 10 Atrial P 94.5 119.2 14.0 148.5 4.1 5.3 0.6 6.8 14 Tetractine U 162.0 222.3 29.5 270.0 5.1 5.6 0.5 6.8 18 II A 24.3 30.2 5.2 37.8 4.1 5.0 0.7 5.4 6 UFRJPOR Diactine - 108.0 360.7 110.0 615.6 5.4 10.8 3.5 16.2 20 6701 Cortical P 56.7 78.4 10.7 102.6 5.4 5.5 0.6 8.1 20 triactine U 86.4 117.5 15.8 140.4 5.4 6.0 0.8 8.1 20 Pseudo P1 78.3 125.1 17.5 145.8 5.4 5.4 0.0 5.4 20 sagittal P2 56.7 92.2 15.6 108.0 4.1 5.2 0.5 5.4 20 Triactine U 78.3 105.3 13.9 129.6 4.1 5.3 0.8 8.1 20 Tubar P1 62.1 89.4 9.1 105.3 5.4 6.2 1.2 8.1 20 Triactine 1 P2 27.0 45.6 12.3 72.9 4.1 5.6 1.6 8.1 20 U 105.3 129.9 20.5 194.4 5.4 6.1 0.9 8.1 20 Tubar P 78.3 88.0 7.9 105.3 5.4 6.1 1.1 8.1 20 Triactine 2 U 72.9 129.2 27.7 189.0 5.4 6.2 1.2 8.1 20 Subatrial P 48.6 74.9 11.8 89.1 4.1 5.1 0.6 5.4 16 Triactine U 35.1 151.6 59.2 243.0 4.1 5.6 1.0 8.1 16 Atrial P 89.1 120.4 19.8 162.0 4.1 5.2 0.5 5.4 20 triactine U 162.0 223.4 27.2 270.0 5.4 6.3 1.2 8.1 20 Atrial P 83.7 115.0 25.9 145.8 4.1 5.1 0.6 5.4 10 Tetractine U 129.6 179.6 49.3 280.8 5.4 5.9 1.0 8.1 8 I A 21.6 46.5 24.4 86.4 4.1 5.1 0.6 5.4 9 Atrial P 94.5 125.3 18.6 151.2 4.1 5.1 0.6 5.4 10 108

Tetractine U 189.0 230.6 28.3 270.0 5.4 6.3 1.3 8.1 12 II A 13.5 29.2 11.7 43.2 4.1 5.1 0.6 5.4 5 UFRJPOR Diactine 194.4 381.2 138.2 637.2 5.4 9.7 2.8 16.2 20 6766 (H) Cortical P 51.3 79.4 12.7 102.6 4.1 5.3 0.4 5.4 20 Triactine U 81.0 128.9 22.3 175.5 4.1 5.5 0.8 8.1 20 Pseudo P1 78.3 110.7 18.1 137.7 4.1 5.3 0.3 5.4 15 sagittal P2 62.1 84.6 14.2 113.4 2.7 5.0 0.8 5.4 15 Triactine U 56.7 101.8 26.9 126.9 4.1 5.3 0.4 5.4 10 Tubar P1 62.1 95.9 15.2 113.4 5.4 6.3 1.2 8.1 20 Triactine 1 P2 21.6 50.2 11.5 72.9 5.4 5.6 1.5 8.1 20 U 124.2 162.9 21.2 202.5 5.4 5.9 0.7 6.8 20 Tubar P 48.6 95.6 13.3 118.8 5.4 6.0 1.0 8.1 20 Triactine 2 U 83.7 145.9 27.9 205.2 5.4 5.9 0.8 8.1 20 Subatrial P 40.5 80.3 17.3 99.9 4.1 5.3 0.6 6.8 16 Triactine U 67.5 162.0 47.6 224.1 4.1 5.7 1.0 6.8 16 Atrial P 97.2 123.1 13.2 148.5 4.1 5.7 1.1 8.1 20 triactine U 189.0 233.1 26.7 283.5 5.4 6.1 1.0 8.1 20 Atrial P 97.2 118.8 17.5 137.7 4.1 5.1 0.7 5.4 4 Tetractine U 108.0 160.7 53.4 234.9 5.4 6.4 1.3 8.1 4 I A 13.5 83.4 27.6 108.0 5.4 5.7 0.6 6.8 10 Atrial P 94.5 121.5 16.9 148.5 5.4 6.2 1.1 8.1 10 Tetractine U 189.0 240.5 28.7 283.5 5.4 6.1 1.1 8.1 11 II A 13.5 21.2 8.4 35.1 5.4 5.4 0.0 5.4 6 Diactine - 60.0 - - 80.0 - - - - Tubar P 40.0 - - 80.0 6.0 - - 8.0 Grantessa Triactine U 80.0 - - 120.0 6.0 - - 8.0 ramosa Subatrial P 40.0 - - 80.0 6.0 - - 8.0 (Haeckel, Triactine U 160.0 - - 200.0 6.0 - - 8.0 1872) Atrial P 50.0 - - 80.0 6.0 - - 8.0 Tetractine U 100.0 - - 120.0 6.0 - - 8.0 A 20.0 - - 30.0 - 8.0 - - Diactine - 150.0 - - 380.0 6.0. - - 12.0 Cortical U 70.0 - - 150.0 8.0 - - 12.0 Triactine* Subcortica CP 80.0 - - 180.0 15.0 - - 18.0 l IP 150.0 - - 240.0 15.0 - - 18.0 Grantessa Triactine U 100.0 - - 200.0 14.0 - - 16.0 ramosa Tubar U 150.0 - - 230.0 14.0 - - 18.0 sensu Triactine Borojevic Subatrial P 80.0 - - 120.0 9.0 - - 11.0 (1967) Triactine U 180.0 - - 250.0 12.0 - - 14.0 Atrial P 60.0 - - 180.0 8.0 - - 10.0 Triactine U 200.0 - - 400.0 8.0 - - 10.0 Atrial P 60.0 - - 180.0 - 25.0 - - Tetractine U <200 - - <400 - 25.0 - - A - - - 140.0 - 25.0 - - * Paired actines slightly longer than unpaired actine. 109

Genus Sycon Risso, 1827 TYPE SPECIES Sycon humboldti Risso, 1827 DIAGNOSIS "Sycettidae with radial tubes partially or fully coalescent; distal cones are decorated by tufts of diactines. The inhalant canals are generally well defined between the radial tubes and are often closed at the distal end by a membrane that is perforated by an ostium, devoid of a skeleton. There is no continuous cortex covering the distal ends of the radial tubes. Skeleton of the atrium and of the tubes composed of triactines and/or tetractines" (Borojevic et al., 2002).

Sycon conulosum sp. nov. (Figures 26 & 27, Tables 20 & 21) ETIMOLOGY Derived from the conulose appearance of the surface. TYPE LOCALITY Daai Booi, St. Willibrordus, Curaçao. TYPE MATERIAL Holotype: UFRJPOR 6707 (specimen in ethanol and slides); Daai Booi, St. Willibrordus, Curaçao; 12°12'43.12"N, 69°05'8.42"W; 3-5m; coll. B. Cóndor-Luján, 18 August 2011. COLOUR White in life and in ethanol. MORPHOLOGY AND ANATOMY This species has a ficiform shape (Figure 26A). It measures 0.9 cm long and 0.7 cm wide. The surface is hispid with tufts of diactines protruding through the body. It has a conulose appearance because of the distal cones, which are very separated. The osculum (diameter=0.9 mm) is apical with no crown and it is supported by sagittal triactines (Figura 26B-C). The atrium measures 2.4 mm. The radial tubes are fully coalescent (Figure 26D). The aquiferous system is syconoid. SKELETON The skeleton is typical of the genus. The skeleton of the distal cones is composed of short diactines, rare trichoxeas and triactines (white arrow in Figure 26E). The tubar skeleton is articulated, composed of several rows of triactines with the unpaired actine pointing towards the distal cones (black arrow in Figure 26E). The subatrial skeleton is composed of triactines whose unpaired actine points to the surface (arrow in Figure 26F). The atrial skeleton is composed of 110 tangential triactines (black arrow in Figure 26G) and fewer tetractines (white arrow in Figure 26G). The apical actine of the tetractines penetrates the atrial cavity. SPICULES (Table 20) Diactines. Fusiform with tips usually sharp (Figure 27A). Size: 100-275/6.3-11.3 µm. Triactines of the distal cones. Sagittal. Actines are conical with sharp tips. The paired actines are frequently curved (Figure 27B). In the distal part of the cone, the unpaired actine is very long. Size: 50-102.5/5-10 µm (paired actine) and 65-175/5-8.8 µm (unpaired actine). Tubar triactines I. Sagittal. Actines are conical with sharp tips. One of the paired actines is shorter and more curved than the other. The unpaired actine is straight and it is generally slightly longer than the paired ones (Figure 27C, left). Size: 32.5-60/5-10 µm (short paired actine), 67.5- 95/5-11.3 µm (long paired actine) and 82.5-125/5-10 µm (unpaired actine). Tubar triactines II. Sagittal. Actines are conical with sharp tips. The paired actines are slightly curved and have equal sizes. The unpaired actine is straight and generally longer than the paired ones (Figure 27C, right). Size: 40-80/3.8-10 (paired actine) and 62.5-155/3.8-10 µm (unpaired actine). Subatrial triactines. Sagittal. Actines are slightly conical with sharp tips. The paired actines are inwardly curved. Some paired actines have different lengths. The unpaired actine is straight and longer than the paired ones (Figure 27D). Size: 42.5-80/5-8.8 µm (paired actine) and 88.5- 172.5/5-10 µm (unpaired actine). Atrial triactines. Sagittal. Actines are conical, straight and have sharp tips. The unpaired actine is usually longer than the paired ones (Figure 27E). Size: 80-130/7.1-14.3 µm (paired actine) and 80-172.5/5-10 µm (unpaired actine). Atrial tetractines. Sagittal. Actines are conical, straight with sharp tips (Figure 27F). The apical actine is the shortest actine of this species. Size: 62.5-90/7.5-11.3 µm (paired actines), 92.5- 130/7.5-10 µm (unpaired actine) and 15-40/5-10 µm (apical actine). ECOLOGY This species was found in a light protected environment. No associated organisms were found. GEOGRAPHIC DISTRIBUTION Southern Caribbean (provisionally endemic to Curaçao, present study). REMARKS Within the 88 accepted species of Sycon (Van Soest et al., 2016), 15 have the same skeleton composition as the analised specimen from Curaçao: S. abyssale Borojevic & Graat-Kleeton, 1965, S. ampulla (Haeckel, 1870), S. arcticum (Haeckel, 1870), S. barbadensis (Schuffner, 1877), S. brasiliense Borojevic, 1971, S. boreal (Schuffner, 1877), S. dunstervillia (Haeckel, 111

1872), S. formosum (Haeckel, 1870), S. humboldt Risso, 1827, S. raphanus Schmidt, 1862), S. scaldiense (Van Koolwijk, 1982), S. schmidti (Haeckel, 1872), S. setosum Schmidt, 1862, , S. tuba Lendenfeld, 1891 and S. villosum (Haeckel, 1870). Sycon conulosum sp. nov. can be easily differentiated from S. abyssale, S. brasiliense, S. dunstervilla and S. formosum because of the shape of their diactines. In the new species, diactines are fusiform whereas in S. abyssale and S. brasiliense, one of the tips is lanceolated and in S. dunstervilla and S. formosum, diactines have distally swollen tips (originally described as “keulen” or “kolb”). The remaining 11 species differ from the new species in spicule size (Table 21). 112

Fig. 26. Sycon conulosum sp. nov. (UFRJPOR 6707): (A) specimen after fixation; (B) osculum; (C) detail of the oscular T-shaped triactines; (D) cross section of the skeleton; (E) skeleton with a tuft of diactines, a triactine of the distal cone (white arrow) and a tubar triactine (black arrow); (F) subatrial skeleton indicating a subatrial triactine (arrow); (G) atrial skeleton indicating atrial triactine (black arrow) and atrial tetractine (white arrow). Abbreviations: at=atrium.

Fig. 27. Spicules of Sycon conulosum sp. nov. (UFRJPOR 6707): (A) diactine of the distal cone; (B) triactine of the distal cone; (C) tubar triactine I (left) and tubar triactine II (right); (D) subatrial triactine; (E) atrial triactine; (F) atrial tetractine. 113

Table 20. Spicule measurements of Sycon conulosum sp. nov. (UFRJPOR 6707).

Length (µm) Width (µm) N Spicule Actine Min Mean SD Max Min Mean SD Max Diactine 100.0 216.5 48.0 275.0 6.3 8.4 1.3 11.3 20 Triactine Paired 50.0 77.4 16.4 112.5 5.0 7.2 1.6 10.0 20 (distal cone) Unpaired 65.0 124.1 28.9 175.0 5.0 6.4 1.3 8.8 20 Tubar Paired (>) 67.5 78.3 7.7 95.0 5.0 8.1 1.6 11.3 20 Triactine 1 Paired (<) 32.5 48.6 6.5 60.0 5.0 8.3 2.2 10.0 20 Unpaired 82.5 64.6 1.6 125.0 5.0 8.3 1.6 10.0 20 Tubar Paired 40.0 63.5 10.5 80.0 3.8 7.4 1.7 10.0 20 Triactine 2 Unpaired 62.5 106.4 21.2 155.0 3.8 7.3 1.7 10.0 20 Subatrial Paired 42.5 65.1 9.9 80.0 5.0 5.8 1.2 8.8 20 Triactine Unpaired 82.5 135.1 20.2 172.5 5.0 7.1 1.6 10.0 20 Atrial Paired 80.0 103.0 11.8 130.0 7.1 10.0 2.2 14.3 14 Triactine Unpaired 80.0 126.6 23.7 172.5 5.0 7.9 1.9 10.0 14 Atrial Paired 62.5 77.5 11.4 90.0 7.5 9.1 1.9 11.3 4 Tetractine Unpaired 92.5 111.3 20.3 130.0 7.5 8.8 1.4 10.0 4 Apical 15.0 27.9 9.6 40.0 5.0 7.3 1.7 10.0 12

Table 21. Original measurements of Sycon ampulla, S. arcticum, S. barbadense, S. boreale, S. humboldti, S. raphanus, S. scaldiense, S. schmidti, S. setosum, S. tuba and S. villosum. Values in micrometers (µm). *Taken from Haeckel (1872).

Species - type Distal cone Tubar Subatrial Atrial Atrial locality triactine triactine triactine tetractine S. ampulla – Diactine: Paired: 50-80/5 - 60-80/5 Unpaired: Atlantic coast 100-500/5 Unpaired: 60-80/5 of South 100-150/5 Apical: 40-60/5 America (rarely 100) S. arcticum Diactine: 1000- Paired: - Paired: Paired: – Arctic 3000/10-40 100-200/10 50-150/10 50-150/10 Ocean Trichoxea: Unpaired: Unpaired: Unpaired: 100-300/1-5 200-300/10 200-250/10 200-250/10 Triactine: Apical: 20- smaller than 40/10 tubar S. barbadense Diactine: 400/9 Unpaired: - ≤ 200/9 Paired: ≤ 150/9 – Barbados ≤ 150 Unpaired: ≤ 200/9 Apical: ≤ 80/13 Sycon boreale Diactine: 500/9 Paired: 500 - Paired: shorter Apical: 50/13 - Norway Unpaired: 180 than unpaired. Unpaired: 180/6.8 114

S. humboldti* Diactine I: 80-120/8-12 - Paired: Paired: – Western 500–2000/20-40 50-120/8 50-20/8 Mediterranean Diactines II: Unpaired: Unpaired: 200-400/5-20 50-200/5-8 50-200/5-8 Triactine: Apical: Paired: 100-120 30-60/ 10-15 Unpaired: 150-250/20-30 S. raphanus* Diactine: Paired: ?/5-8 (Subregular Subregular to – Adriatic Sea 1000–3000/ 100-180 to sagittal) sagittal 20 -24 /10 – 12 150 – 250/8 – Apical: Unpaired: 150 10 60 – 120 – 250/10 – 12 S. scaldiense – Diactine: 80-150/9-11 Paired:80 Paired: 100 - Paired:100- Netherlands 500–700/5-10 -200/5-10 280/5-10 280/5-10 Triactine: Unpaired: Unpaired: 200 Unpaired: 30-60/5-10 180- – 300/5-10 200–300/5-10 350/5-10 Apical: 180–350/5-10 S. schmidti – Diactine I: Paired: - Subregular Subregular Adriatic Sea 100 – 300/10 100-150/10 200-400/10- Basal: Diactine II: Unpaired: 15 200-400/10-15 100–500/20 - 30 200-300/10 Apical: 40-50/12-16 S. setosum* – Diactine: Paired: 80- - Subregular Subregular Adriatic Sea 1000-3000/20 160/5-8 100-115/5 Apical: Diactine: Unpaired: 300-600/5 100-200/1 120-200/5-8 S. tuba– Diactine: Paired: 280/7 - Paired: 280/7 Paired: 280/7 Adriatic Sea 300/10 Unpaired: Unpaired: Unpaired: 220/7 320/7 320/7 Apical: 2 00-260/7 S. villosum– Diactine: 100-300/30 Sagittal Sagittal Paired: North Atlantic 1000-3000/ Paired: Paired: 100/30 100-150/5-8 10-40 100/30 Unpaired: Unpaired: Triactine: Unpaired: 300/10-30 300-400/5-8 100-200/5-8 300/10- Apical: 30 500-800/≤10

Sycon magniapicalis sp. nov. (Figures 28 & 29, Table 22) ETIMOLOGY From the Latin magna (=large), for the long apical actine of the atrial tetractine. TYPE LOCALITY Tug Boat, Caracasbaai, Willemstadt, Curaçao. 115

TYPE MATERIAL Holotype: UFRJPOR 6748 (specimen in ethanol and slides); Tug Boat, Caracasbaai, Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W; 14.9 m deep; coll. B. Cóndor-Luján, 23 August 2011. Paratype: UFRJPOR 6763 (specimen in ethanol and slides); Water Factory, Willemstadt, Curaçao; 12°06'30.88"N, 68°57'13.53"W; 8.4 m deep; coll. E. Hajdu, 23 August 2011. COLOUR White to beige in life and yellowish in ethanol. MORPHOLOGY AND ANATOMY This species has a globular body with an apical osculum (Figure 28A). The consistency is hard, although compressible. The surface is very hispid with long diactines and trichoxeas protruding through the surface. The holotype measures 6.5 cm length x 2.5 cm width (Figure 28B). The osculum has a neck with a delicate crown of trichoxeas (arrows in Figure 28B-C). The radial tubes are fully coalescent. The atrial cavity is hispid and the aquiferous system is syconoid. SKELETON The osculum has a neck composed of T-shaped triactines and tetractines and a crown of trichoxeas (Figure 28D). The triactines and tetractines are arranged parallel to each other (Figure 28E). In the paratype, the crown is more conspicuous as shown in Figure 28C. The skeleton of the body is typical of the genus (Figure 28F). The distal cones have diactines, triactines and rare trichoxeas tangentially disposed (Figure 28G). The diactines comprise two size categories. In the larger diactines, the proximal part crosses the choanosome and occasionally, reaches the atrium. The tubar skeleton is articulated, exclusively formed by triactines with the unpaired actine pointing to the distal cones (Figure 28H). The subatrial skeleton is composed of tetractines (white arrow in Figure 28I) and rare triactines (black arrow in Figure 28I) with the unpaired actine pointing to the surface. The atrial skeleton is formed by tetractines. The basal actines lay tangentially and the very long apical actine is projected into the atrium (Figure 28I). SPICULES Trichoxeas. Straight with tips always broken. Ticker than usually. Size: >1690.2/1.4-5.4 μm. Diactines I. Fusiform and straight. The proximal tip is lanceolated while the distal tip is usually abruptly sharp (Figure 29A). Size: 100.0-864.0/5.4-21.6 μm. Diactines II. Fusiform and straight. The proximal tip is sharp and the distal tip was always found broken. They are the largest diactines in this species. Size: > 2970.0/23.0-32.4 μm. 116

Triactines of the distal cones. Sagittal. Actines are conical with sharp tips. Some paired actines can be slightly longer than the unpaired one (Figure 29B). Size: 54.0-153.9/5.4-10.8 μm (paired actine) and 40.5-175.5/5.4-10.8 μm (unpaired actine). Tubar triactines. Sagittal. Actines are conical with sharp tips (Figure 29C). Some paired actines are curved. Size: 54.0-135.0/5.4-10.8 μm (paired actine) and 35.1-189.0/ 5.4-10.8 μm (unpaired actine). Subatrial triactines. Sagittal. Actines are conical with sharp tips (Figure 29D). The unpaired actine is straight and longer than the paired ones. The paired actines are inwardly curved. In some triactines, one paired actine can be longer than the other. Size: 67.5-126.9/2.7-6.8 μm (paired actine), 27.0-99.9/2.7- 6.8 μm (smaller paired actine) and 67.5-245.7/4.1- 8.1 μm (unpaired actine). Subatrial tetractines. Sagittal. Actines are conical with sharp tips. The unpaired actine is straight and longer than the basal actines. It can be slightly conical. The paired actines are inwardly curved (Figure 29E). The apical actine is the shortest actine in this species Size: 40.0-143.1/4.1- 8.1 µm (paired actine), 143.1-237.6/5.4-10.8 μm (unpaired actine) and 10.8-75.6/2.7-6.8 μm (apical actine). Atrial tetractines. Sagittal. The basal actines are conical with sharp tips (Figure 29F). The apical actine is slightly conical to cylindrical, straight, smooth and very long. It can be straight or curved. Size: 110.7-237.6/5.4-13.5 μm (paired actine), 29.7-151.2/5.4-16.2 μm (unpaired actine) and 124.2 - 496.0/5.4-16.2 μm (apical actine). ECOLOGY Specimens were collected underneath coral boulders, partially covered with sediment. GEOGRAPHIC DISTRIBUTION Southern Caribbean (provisionally endemic to Curaçao, present study). REMARKS Surprisingly, Sycon plumosum Tanita, 1943 from Palau (Carolinas Islands) is the only species of Sycon that presents the same skeleton composition as that of the specimens from Curaçao. However, they greatly differ in spicule size as almost all the spicule categories are smaller in S. magniapicalis sp. nov. (only the atrial tetractines have similar size in both species). Additionally, S. plumosum lacks the oscular neck observed in both Curaçaoan specimens. In the C-LSU phylogenetic tree (Figure 30), the sequences of S. magniapicalis sp. nov. (UFRJPOR6748 and UFRJPOR6763) grouped together with high support (pp=1, b=99.9), confirming their co-specificity. Nonetheless, they did not cluster with the other species of Sycon (S. ancora, S. capricorn, S. cf. vilosum, S. ciliatum, S. conulosum sp. nov. and S. raphanus). 117

Only in the BI tree, S. magniapicalis sp. nov. appeared as a sister taxon (pp=0.99) of the clade formed by Sycon conulosum sp. nov. + L. antillana (pp=1, b=99.9).

Fig. 28. Sycon magniapicalis sp. nov.: (A) specimen in vivo; (B) specimen after fixation; (C) paratype after fixation; (D) skeleton of the osculum indicating the neck (asterisk) and the crown; (E) detail of the oscular T-shaped triactine (white arrow) and tetractine (black arrow); (F) cross 118 section of the skeleton; (G) distal cones with diactines II (black arrow) and triactines (white arrow); (H) tubar skeleton; (I) subatrial skeleton with tetractine (white arrow) and triactine (black arrow); (J) atrial skeleton with tetractines. Abbreviations: at=atrium. All pictures correspond to the holotype except when indicated.

Fig. 29. Spicules of Sycon magniapicalis sp. nov. (UFRJPOR 6748): (A) diactine I; (B) triactine of the distal cone; (C) tubar triactine; (D) subatrial triactine; (E) subatrial tetractine; (F) atrial tetractine. 119

Table 22. Spicule measurements of Sycon magniapicalis sp. nov. (UFRJPOR6748 and UFRJPOR6763) and of S. plumosum from Tanita, 1943. P=paired, U=unpaired and A=apical.

Length (µm) Width (µm) N Specimen Spicule Actine Min Mean SD Max Min Mean SD Max UFRJPOR Trichoxea 864.0 - - - 1.3 3.6 1.2 5..4 20 6748 Diactine I 100.0 285.7 121.9 486.0 5.4 12.1 3.6 18.9 16 Diactine II >2835 - - - 23.0 26.4 1.3 27.0 12 Triactine P 64.8 110.6 17.9 145.8 5.4 9.7 1.6 10.8 20 (cone) U 40.5 97.7 34.5 175.5 5.4 8.8 1.8 10.8 20 Tubar P 54.0 94.0 26.9 135.0 5.4 7.2 1.6 10.8 19 Triactine U 35.1 65.5 25.2 118.8 5.4 6.3 1.2 8.1 20 Subatrial P 67.5 88.2 16.6 126.9 4.1 5.5 0.7 6.8 20 Triactine P- 29.7 53.2 16.8 78.3 4.1 5.4 0.6 6.8 13 U 121.5 184.4 29.6 245.7 5.4 6.1 0.9 8.1 20 Subatrial P 75.6 106.0 20.2 143.1 4.1 5.5 1.0 8.1 20 Tetractine U 143.1 194.5 23.5 237.6 5.4 7.4 1.1 8.1 20 A 16.2 28.9 15.1 75.6 2.7 4.7 0.9 6.8 19 Atrial P 116.1 157.0 19.4 183.6 5.4 8.3 1.3 10.8 15 Tetractine U 29.7 67.5 34.3 151.2 5.4 7.5 1.5 10.8 13 A 221.4 313.6 84.4 496.0 5.4 8.8 1.7 10.8 11 UFRJPOR Trichoxea - 216.0 671.7 386.0 1690.2 1.4 3.2 2.0 5.4 20 6763 Diactine I - 100.0 432.3 178.0 864.0 10.8 15.3 3.0 21.6 22 Diactine II - >2970 - - - 24.3 27.4 1.7 32.4 17 Triactine P 54.0 89.1 24.3 153.9 5.4 6.8 1.2 8.1 20 (cone) U 40.5 64.4 21.5 121.5 5.4 5.9 1.0 8.1 20 Tubar P 54.0 104.1 23.2 129.6 5.4 8.5 1.3 10.8 20 Triactine U 94.5 156.6 25.7 189.0 5.4 8.8 1.5 10.8 20 Subatrial P + 72.9 91.0 13.0 113.4 2.7 4.9 1.0 6.8 20 Triactine P - 27.0 57.8 22.0 99.9 2.7 4.8 0.9 5.4 20 U 67.5 169.0 37.6 234.9 4.1 6.1 1.3 8.1 20 Subatrial P 75.6 103.8 14.6 132.3 5.4 5.7 0.8 8.1 20 Tetractine U 148.5 191.3 24.9 232.2 5.4 7.1 1.5 10.8 20 A 10.8 20.9 9.6 51.3 2.7 4.2 1.2 5.4 20 Atrial P 110.7 148.4 25.4 237.6 6.8 9.7 1.8 13.5 20 Tetractine U 32.4 63.5 25.9 113.4 5.4 9.3 2.6 16.2 20 A 124.2 227.0 56.5 345.0 8.1 10.8 2.5 16.2 19 Sycon Diactine - 800 - - 3000 30.0 - - 35 - plumosum Triactine P 200 - - 240 - 16 - - - (cone) U 140 - - 200 16 - - - Tubar P 170 - - 240 15 - - 18 - Triactine U 270 - - 360 15 - - 18 - Subatrial P 120 - - 180 8 - - 10 - Triactine U 250 - - 360 8 - - 10 - Subatrial P 120 - - 180 8 - - 10 - Tetractine U 250 - - 360 8 - - 10 - A 70 - - 100 8 - - 10 - Atrial P 170 - - 200 12 - - 16 - Tetractine U 200 - - 280 12 - - 16 - A 130 - - 350 12 - - 16 - 120

Phylogenetic analyses

Calcaronean phylogeny C-LSU sequences of six new Calcaronean species, Amphoriscus micropilosus sp. nov. (n=1), Grantessa tumida sp. nov. (n=2), Leucilla antillana sp. nov. (n=1), Leucandrilla pseudosagittata sp. nov. (n=2), Sycon conulosum sp. nov. (n=1) and Sycon magniapicalis sp. nov. (n=2) were produced and provided herein. The aligned C-LSU sequences had a total length of 458 bp including gaps. BI and ML approaches recovered similar tree topologies including monophyletic Calcaronean ( pp=1, b=79) and Calcinean with high BI posterior probability (pp:1) and ML bootstrap (b=100) support. The complete C-LSU phylogenetic tree obtained by BI is shown in Figure S1(Supplementary Material). Within the Calcaronean cluster, we recovered two major clades (Figure 30). One of them included all Heteropiidae and some Grantiidae species and it was previously referred as LEUC I (pp=0.89, b=73) by Voigt et al, (2012). The other clade BAE + LEUC II (pp=1, b=90) nested the Baerida species (BAE: Eilhardia schulzei, Leuconia nivea and Petrobiona massiliana) and almost all the other Leucosolenida species but Leucosolenia sp. (LEUC II). In the LEUC I clade, Grantessa tumida sp. nov. did not cluster with G. aff. intusarticulata, instead it clustered with the Grantiidae species, Synute pulchella (pp=0.93, b=58), suggesting the non-monophyly of the genus Grantessa. Interestingly, G. tumida sp. nov. as well as S. pulchella do not present tetractines in the tubar skeleton whereas the specimens identified as G. aff intusarticulata do have this spicule category. Sycon was recovered again as a polyphyletic genus (Voigt et al., 2012; Klautau et al., 2016). The sequences of the new species, S. conulosum sp. nov. and S. magniapicalis sp. nov., as well as S. ancora, S. carteri, S. cf. ciliatum and S. raphanus appeared in different clades within the clade BAE + LEUC II whereas S. capricorn and S. carteri nested in the LEUC I clade (pp=0.89. b=73). The Amphoriscidae species (A. micropilosus sp. nov., L. antillana sp. nov., Paraleucilla dalmatica and P. magna) did not cluster together. Contrary to expected, L. antillana sp. nov. evidenced a high affinity (pp=1, b= 99.9) with S. conulosum sp. nov. These two species are very different, however, their skeletons do not have choanosomal tetractines (tubar or subatrial). Two relationships found by Klautau et al. (2016) using the whole 28S LSU were also recovered in our phylogeny: (1) the genus Paraleucilla (including P. dalmatica and P. magna) formed a highly supported clade with Leucandra nicolae in the BI analyses (pp=1) and (2) Leucandra spinifera and L. aspera clustered together as sister species (pp=0.97, b=54). 121

Calcinean phylogeny We provided ITS and C-LSU sequences for three new species, Ascandra torquata sp. nov. (2 ITS), Clathrina aspera sp. nov. (4 ITS and 1 C-LSU) and C. curaçaoensis sp. nov. (1 ITS and 1 C-LSU), and for five already known calcinean species, Borojevia tenuispinata (1 ITS and 1 C- LSU), C. lutea (1 ITS), C. insularis (1 ITS and 1 C-LSU), C. mutabilis (8 ITS and 1 C-LSU), C. zelinhae (1 C-LSU) and Leucetta floridana (2 ITS). The aligned ITS sequences had a total length of 1200 bp including gaps. BI and ML approaches recovered similar tree topologies. The BI phylogenetic tree is shown in Figure 31. The genera Ascandra (pp=1, b=99), Borojevia (pp=0.7, b=65) and Clathrina (pp=1, b=100) were recovered as monophyletic clades including the type species of each genus and the sequences of the new species described herein. The Calcinean C-LSU clade recovered in the general Bayesian tree is shown in Figure 32. Two important phylogenetic relationships were recovered in both, the ITS and C-LSU phylogenies: (1) the yellow C. aurea, C. clathrus, C. curaçaoensis sp. nov, C. lutea and C. luteoculcitella clustered together in one clade (ITS: pp=1, b=55; C-LSU: pp=0.86, b=65) whereas the other yellow clathrinas C. mutabilis and C. insularis, appeared in separate clades and (2) Clathrina aspera sp. nov. clustered together with the geographically distant Australian species C. helveola and C. wistariensis (ITS and C-LSU: pp=1, b=100). Although several clathrinas from the Tropical Atlantic Ocean were included in the ITS phylogenetic analysis, C. mutabilis appeared closer to a clathrina from the French Polynesia (pp=1, bb=100). Another interesting grouping was observed within the clade of L. floridana. The Curaçaoan specimens evidenced a higher affinity with the Brazilian specimen than with the Panamanian one despite their geographic distance. 122

Figure 30. Calcaronean cluster from the Bayesian phylogenetic tree inferred from the C-LSU sequences. Posterior probabilities and bootstrap values are given on the branches (pp/bootstrap). BAE (Baerida), LEUC I (Leucosolenida I) and LEUC II (Leucosolenida II) refer to clades found by Voigt et al, 2012. *Sequences generated in this study. 123

Figure 31. Bayesian phylogenetic tree inferred from the ITS sequences of the Calcinean species. Posterior probabilities and bootstrap values are given on the branches (pp/bootstrap). *Sequences generated in this study. 124

Figure 32. Calcinean cluster from the Bayesian phylogenetic tree inferred from the C-LSU sequences. Posterior probabilities and bootstrap values are given on the branches (pp/bootstrap). *Sequences generated in this study.

DISCUSSION Biodiversity and Distribution With the present work we increased from two to 20 the number of known species of calcareous sponges from Curaçao. We found a total of 12 representants of the subclass Calcinea and eight of the subclass Calcaronea. With the addition of these Curaçaoan records, the number of calcareous sponges reported for the Caribbean Sea rose from 24 to 40 species, an increase of 66.7% in our knowledge of the biodiversity of Caribbean calcareous sponges. The finding of nine provisionally Curaçaoan endemic species (45%; 9/20) - Amphoriscus micropilosus sp. nov., Arthuria vansoesti sp. nov., Clathrina curaçaoensis sp. nov., Grantessa tumida sp. nov., Leucandra caribea sp. nov., Leucandrilla pseudosagittata sp. nov. Leucilla antillana sp. nov., Sycon conulosum sp. nov. and Sycon magniapicalis sp. nov. - may indicate a high endemism of Calcarea in this island, however, it is also possible that it is just showing how understudied this class is in the Caribbean Sea. Considering the geographic distribution of calcareous sponges species, only six species were previously reported occurring in both, the Brazilian Coast and the Caribbean Sea, Leucetta 125 floridana, Leucaltis clathria, Leucandra barbata, Leucandra rudifera, Nicola tetela and Sycon ampulla (Muricy et al., 2011; Cóndor-Luján & Klautau, 2016). In this study, we expanded the geographical distribution of four former Brazilian endemic species (B. tenuispinata, C. insularis, C. lutea and C. mutabilis, Azevedo et al., submitted) to the Caribbean Sea (Curaçao) and reported two new species (Ascandra torquata sp. nov. and Clathrina aspera sp. nov.) present in both regions. Therefore, 12 species are currently shared between the Brazilian coast and the Caribbean Sea. This result suggests a Brazilian-Caribbean sponge affinity for Calcarea and questions the role of the Amazon and Orinoco Rivers as effective barriers to the dispersal between these two regions. Taxonomy and Phylogeny Important phylogenetic traits with systematic implications can be discussed based on our results. The phylogenetic importance of the skeleton composition in Calcinean systematics has already been demonstrated (Rossi et al., 2011; Klautau et al., 2013), nonetheless, in Calcaronea this has not been tested so far. In this study, we found that the presence of subatrial tetractines clustered Leucilla antillana sp. nov. with Sycon conulosum sp. nov. and the absence of tubar tetractines separated Grantessa tumida sp. nov. from Grantessa aff. intusarticulata. Whether choanosomal tetractines (subatrial or tubar) do have phylogenetical signal across other species within Calcaronea should be analysed in further studies with a larger number of species. The close affinity between Heteropiidae and Grantiidae with large longitudinal diactines was firstly observed in a 28S-phylogeny (Voigt et al., 2012) and it was recently recovered with the shorter C-LSU sequences (Voigt & Worheide, 2016). This relationship was based on two highly supported clades: (1) Sycettusa spp. + Grantessa aff. intusarticulata and Aphroceras sp. and (2) Vosmaeropsis sp. + Sycettusa spp. and Sycon ciliatum. Herein, we recovered that relationship with a third pair of species: G. tumida sp. nov. and Synute pulchella. Azevedo et al., (submitted) suggested that trichoxeas were not reliable characters to differentiate species in Clathrina (as specimens of C. mutabilis with and without trichoxeas clustered together). We not only recovered the same pattern in another Clathrina species (Clathrina lutea) but also in Ascandra as diactines can be present or not in A. torquata sp. nov. Perhaps in the future, not only trichoxeas but also diactines will not be considered as diagnostic characters for all Calcinean sponges. The obtained ITS and C-LSU phylogenies suggested again that the yellow colour appeared more than once in Calcinea (Rossi et al., 2011; Klautau et al., 2013) and that affinities found between Atlantic and Indo-Pacific species (C. aspera sp. nov. and C. wistariensis; C. mutabilis and Clathrina sp. from French Polynesia) should receive attention in further studies. 126

ACKNOWLEDGEMENTS We would like to thank Giselle Lôbo-Hajdu for assistance during the sample collections. Mark Vermeij and CARMABI are acknowledged for providing logistical support in Curaçao. We are thankful to Marcelo Soares for the assistance with the SEM procedures. FINANCIAL SUPPORT This study was funded by the Brazilian National Research Council (CNPq), Coordination for the Improvement of Higher Education Personnel (CAPES), Foundation Grupo Boticário de Proteção à Natureza and Rio de Janeiro State Research Foundation (FAPERJ). B. C. L. received a scholarship from CAPES. T. L. received a scholarship from FAPERJ. F.A. and A.P. are granted with post-doc scholarships from FAPERJ and CAPES. E.H. and M.K. have fellowships from CNPq.

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SUPPLEMENTARY MATERIAL

Figure S1. Bayesian phylogenetic tree inferred from the C-LSU sequences of the Calcaronean and Calcinean species. Posterior probabilities and bootstrap values are given on the branches (pp/bootstrap). BAE (Baerida), LEUC I (Leucosolenida I) and LEUC II (Leucosolenida II) refer to clades found by Voigt et al, 2012. *Sequences generated in this study. 132

New records of calcareous sponges (Porifera: Calcarea) from the Northeastern Brazilian coast including three new species

BÁSLAVI CÓNDOR-LUJÁN1, FERNANDA AZEVEDO1, ANDRÉ PADUA1, EDUARDO HAJDU2 & MICHELLE KLAUTAU1*

1Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Av. Carlos Chagas Filho, 373, Rio de Janeiro, RJ, Brasil, 21941-902. E-mail: [email protected]; [email protected]; [email protected]; [email protected]. 2Universidade Federal do Rio de Janeiro, Museu Nacional, Departamento de Invertebrados, Quinta da Boa Vista, São Cristóvão, Rio de Janeiro, RJ, Brasil, 20940-040. E-mail: [email protected].

*Corresponding author: [email protected] Journal to be submitted: ZOOTAXA

ABSTRACT Despite the efforts done in order to know the diversity of calcareous sponges along the Brazilian coast, several areas still remain unexplored or poorly investigated. In this study, we examined the material collected in recent expeditions along the NE Brazilian coast using morphological and molecular approaches. Sampled localities included three protected areas: Área de Proteção Ambiental dos Recifes de Corais de Maracajaú in Rio Grande do Norte State, Fernando de Noronha Archipelago in Pernambuco State and Abrolhos National Marine Park in Bahia State and other localities in Ceará State and Rio Grande do Norte. Collections were performed by snorkeling and SCUBA down to 20 m deep. A total of 14 species were identified including three new species: Amphoriscus hirsutus sp. nov., Grantia grandisapicalis sp. nov. and Leucascus luteoatlanticus sp. nov. Arthuria vansoesti Borojevia brasiliensis, Clathrina aspera and C. luteoculcitella constitute new records for the NE Brazil. Clathrina mutabilis, Ernstia citrea and E. rocasensis are recorded for the first time from Rio Grande do Norte, Bahia and Pernambuco, respectively. Clathrina aurea, C. lutea, Leucascus roseus and Leucilla uter are recorded from new localities within the Abrolhos Marine National Park.

Keywords: Brazilian continental shelf, Fernando de Noronha and Atoll das Rocas, NE Brazil, Eastern Brazil, Calcaronea, Calcinea. 133

Introduction The Northeastern Brazilian coast comprises a large area of more than 3,300 km, representing almost 45% of the Brazilian coast. It is located in the Tropical Southwestern Atlantic Province and covers three entire marine ecoregions, São Pedro and São Paulo Islands, Fernando de Noronha and Atoll das Rocas and Northeastern Brazil, and part of the Eastern Brazil ecoregion (Spalding et al. 2007). It also includes nine Brazilian political-administrative divisions (States): Maranhão, Piauí, Ceará, Rio Grande do Norte, Paraíba, Pernambuco, Alagoas, Sergipe and Bahia. This vast region encompasses a variety of ecosystems, coral and algal reefs, estuaries, mangroves, rocky shores, sandy beaches, seagrasses, tide pools and coastal and oceanic islands (Moraes 2011; Longo & Amado-Filho 2014; Araújo & Amaral 2016), housing a great diversity of marine taxa, including sponges (Muricy et al. 2011). However, due to its huge dimension and habitat heterogeneity, several areas are still unexplored or badly known concerning its marine biodiversity, especially for some faunistic groups, such as sponges of the class Calcarea. The class Calcarea (Porifera) is composed of sponges whose skeleton is built exclusively of calcium carbonate. They are divided into two monophyletic subclasses: Calcaronea and Calcinea. Calcaroneans are characterised by presenting sagittal spicules, apinucleated choanocytes and amphiblastula larvae and diactines are the first spicules to be produced, whereas calcineans have a skeleton mainly composed of regular spicules, basinucleated choanocytes, calciblastula larvae and triactines are the first spicules to be secreted (Manuel et al. 2002). Within the subclasses of Calcarea, molecular phylogenetic studies have demostrated that high taxonomical categories such as orders, families and even some calcaronean genera are polyphyletic (Voigt et al. 2012; Voigt & Wörheide 2015; Klautau et al. 2016), whereas other studies have evidenced monophyletic genera in Calcinea (Rossi et al. 2011; Klautau et al. 2013). According to the catalogue of Brazilian Porifera, among the 47 species of calcareous sponges recorded from the Brazilian coast, 26 species (55.3%) were reported from the Northeastern region (Muricy et al. 2011). This might suggest that the diversity of Calcarea within this region is well investigated, however, recent studies in this area revealed new species to science (Cavalcanti et al. 2014, 2015; Azevedo et al. submitted), which shows how the diversity of this class is still poorly known. The first records of calcareous sponges from the NE Brazil were published in the 19th century: Amphoriscus synaptum (Schmidt in Haeckel, 1872) and Leucilla sacculata (Carter, 1890). Almost one century later, Borojevic & Peixinho (1976) reported 24 species including four new species to science at that time. In the last decade, the knowledge of calcareous sponges 134 from the NE Brazil increased by studies focusing on specific localities such as the Potiguar Basin (Rio Grande do Norte, Lanna et al. 2009), Bahia State (Hajdu et al. 2011) and oceanic and mid-shelf islands (Azevedo et al. submitted), or in genera as Paraleucilla and Vosmaeropsis (Cavalcanti et al. 2014, 2015, respectively). Despite this considerable increase of descriptions in the last years, the diversity and distribution of calcareous sponges in the NE Brazil is still underestimated. In the present work, using morphological and molecular approaches, we studied material collected in recent expeditions conducted along the NE Brazilian coast including three Protected Areas (Corais de Maracajaú, Abrolhos Marine National Park and Fernando de Noronha Archipelago) and two other localities in Ceará and Rio Grande do Norte States.

Material and Methods Analysed material In this study, we examined the calcareous sponges collected in three expeditions carried out in the NE Brazilian Coast during 2014 and 2016: ExpoCERN (Expedição Poriferos do Ceará e Rio Grande do Norte, April 2014), Abrolhos Expedition (December 2015) and TAXPOmol Biodescoberta 2016 Expedition (April 2016). As some localities are within protected areas, namely, Abrolhos Marine National Park, Fernando de Noronha and Área de Proteção Ambiental dos Recifes de Corais de Maracajaú (APARC- Maracajaú), these expeditions received the adequate support and permissions from the ICMBio (Instituto Chico Mendes de Conservação da Biodiversidade). The sampled localities where calcarean specimens were found are detailed in Table 1 and Figure 1. The collections were conducted by snorkeling and SCUBA diving down to 20 m deep. The specimens were photographed in situ and fixed in 96% ethanol. All the samples are preserved in 96% ethanol and deposited in the Porifera Collection of the Biology Institute of the Universidade Federal do Rio de Janeiro, Brazil (UFRJPOR) or in the Museu Nacional do Rio de Janeiro (MNRJ/UFRJ). Morphological procedures The external morphology and internal anatomy were assessed through the observation of the fixed specimens and the examination of microscopy slides. Sections and spicule slides preparations followed standard protocols (Wörheide & Hooper 1999; Klautau & Valentine 2003). The spicule measurements including length and width (minimum, mean, standard deviation [SD] and maximum) are presented in tabular form. The species identifications followed the Systema Porifera (Hooper &Van Soest 2002) and additional appropriate literature 135 for calcineans (Klautau & Valentine 2003; Klautau et al. 2013; Cavalcanti et al. 2013). To illustrate the species descriptions, photographs were taken with a digital AxioCam MRC5 coupled to a Zeiss Imager A2 microscope. Additionally, spicules were placed on a cover-slip, mounted on a stub with double-sided carbon tape and sputter-coated with gold for scanning electron microscopy. Microphotographs were taken with a JSM-6510 SEM at the Institute of Biology of the Universidade Federal de Rio de Janeiro (Brazil).

Table 1. Sampled localities, ecoregions sensu Spalding et al. (2007) and geographic coordinates. Brazilian States: BA=Bahia, CE=Ceará, PE=Pernambuco, RN=Rio Grande do Norte.

L. Locality Ecoregion Geographic Coordinates 1 Porto do Pecém, São Gonçalo do Northeastern Brazil 03º32.106'S, 38º47.893'W Amarante, CE 2 Ressurreta, Fernando de Noronha Fernando de Noronha and 03°48.817'S, 32°23.483'W Archipelago, PE Atoll das Rocas 3 Farol Tereza Pança, Área de Proteção Northeastern Brazil 05º24.133'S, 35º17.849'W Ambiental dos Recifes de Corais de Maracajaú, Maracajaú, RN 4 Batente das Agulhas, Natal, RN Northeastern Brazil 05°33.841'S, 35°04.367'W 5 Parcel das Paredes, Abrolhos Marine Eastern Brazil 17°54.319'S, 38°56.658'W National Park, BA 6 Mato Verde, Ilha Santa Bárbara, Eastern Brazil 17°57.847'S, 38°41.979'W Abrolhos Marine National Park, BA 7 Portinho Sul, Ilha Santa Bárbara, Eastern Brazil 17°57.876'S, 38°41.877'W Abrolhos Marine National Park, BA 8 Ilha Guarita, Abrolhos Marine Eastern Brazil 17°57.583'S, 38°41.550'W National Park, BA 9 Chapeirão 1, Abrolhos Marine Eastern Brazil 17°58.765'S, 38°41.670'W National Park, BA 10 Chapeirão 2, Abrolhos Marine Eastern Brazil 17°58.244'S, 38°40.263'W National Park, BA

Molecular procedures The total genomic DNA was extracted with the guanidine/phenol-chloroform protocol (Sambrook et al. 1989) or with the QIAamp_DNA MiniKit (Qiagen) and stored at –20°C. For calcinean species, the region containing the partial 18S and 28S, the spacers ITS1 and ITS2 and the 5.8S ribosomal DNA (ITS) was amplified with the primers: fwd: 5`- TCATTTAGAGGAAGTAAAA GTCG-3` and rv: 5`-GTTAGTTTCTTTTCCTCCGCTT-3`) (Lôbo-Hajdu et al. 2004). For calcaroneans, the C-region of the 28S (C-LSU) was amplified using the primers fwd: 5'GAAAAGCACTTTGAAAAGAGA-3' (Voigt & Wörheide 2015) and rv: 5'-TCCGTGTTTCAAGACGGG-3' (Chombard et al. 1998). 136

Figure 1. Map of South America highlighting the Northeastern Brazilian coast and the sampled localities in the present study. (A) Ceará State; (B) Fernando de Noronha Archipelago; (C) Rio Grande do Norte State, and (D) Abrolhos Marine National Park. The numbers refers to the sampled localities in Table 1.

The PCR mixture included 1x buffer (5x GoTaq® Green Reaction Buffer Flexi,

PROMEGA), 0.2 mM dNTP, 2.5 mM MgCl2, 0.5 µg/µL bovine serum albumin (BSA), 0.33 µM of each primer, one unit of Taq DNA polymerase (Fermentas or PROMEGA) and 1 µL of DNA in a volume of 15 µL. The PCR amplification comprised one first cycle of 4 min at 94°C, 1 min at 50°C and 1 min at 72°C, 35 cycles of 1 min at 92°C, 1 min at 50°C and one minute at 72°C, and a final cycle of 6 min at 72°C. Forward and reverse strands were sequenced in an ABI 3500 (Applied Biosystems). The new sequences and the ones retrieved from the GenBank database are listed in Table 2. The set of sequences was aligned through the MAFFT v.7 online platform (Katoh & Standley 2013) using the strategy Q-INS-i (Katoh & Toh 2008). The nucleotide substitution models that best fit the alignment were GTR+G for C-LSU and TN93+G for ITS sequences, as indicated by the Bayesian Information Criterion in MEGA 6 (Nei & Kumar 2000; Tamura et al. 2013). Phylogenetic reconstructions were obtained using Bayesian Inference (BI) and Maximum Likelihood (ML) methods. The BI reconstructions were conducted in MrBayes 3.1.2 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003) under 106 generations and a burn-in of 1000 sampled trees, yielding a consensus tree of majority. As the TN93 model is not implemented in Mr.Bayes, we used the GTR model. The ML analyses were performed on 137

MEGA 6 using an initial NJ tree (BIONJ) and a bootstrap of 1000 pseudo-replicates, yielding a mid-point rooted tree. BI posterior probabilities (pp) values as well as ML bootstrap values (b) are indicated on the branches of the inferred trees. In order to know the genetic intraspecific variability within the sequenced species, we calculated the uncorrected p distance considering complete deletion in MEGA 6.

Table 2. Species used in the phylogenetic analyses with locality, voucher number and GenBank (GB) accession number for the ITS (Calcinea) and C-LSU (mostly Calcaronea) regions. *Sequences generated in the present study. H=holotype, P=paratype.

Species Locality Voucher number GenBank Number CALCARONEA Amphoriscus micropilosus Curaçao UFRJPOR6755 (H) Curaçao paper Amphoriscus hirsutus sp. nov.* NE Brazil UFRJPOR7570 (H) This study Anamixilla torresi - - AY563636 Aphroceras sp. - SAM-PS0349 JQ272273 Grantessa tumida Curaçao UFRJPOR6701 (P) Curaçao paper Grantessa aff. intusarticulata - GW979 JQ272278 Grantia compressa - - AY563538 Grantia grandisapicalis sp. nov.* NE Brazil UFRJPOR7567 (H) This study Leucandra aspera - - AY563535 Leucandra falakra Adriatic Sea UFRJPOR8349 (H) KT447560 Leucandra nicolae - - JQ272268 Leucandra sp. - QMG316285 JQ272265 Leucandra spinifera Adriatic Sea UFRJPOR8348 (H) KT447561 Leucandrilla pseudosagittata Curaçao UFRJPOR 6705 (P) Curaçao paper Leucascandra caveolata - QMG316057 JQ272259 Leucilla antillana Curaçao UFRJPOR 6768 (H) Curaçao paper Leuconia nivea - - AY563534 Paraleucilla dalmatica Adriatic Sea UFRJPOR8346 (H) KT447566 Paraleucilla magna Adriatic Sea IRB-P14 KT447564 Sycettusa aff. hastifera Red Sea GW 893 JQ272282 Sycettusa cf. simplex Western India ZMAPOR11566 JQ272279 Sycettusa tenuis Australia QMG313685 JQ272281 Sycettusa sp. - - AY563530 Sycon ancora Adriatic Sea UFRJPOR8347 (P) KT447568 Sycon carteri Australia SAM-PS0143 JQ272260 Sycon ciliatum - - AY563532 Sycon conulosum Curaçao UFRJPOR6707 (H) Curaçao paper Sycon cf. villosum - GW51115 KR052809 Sycon magniapicalis Curaçao UFRJPOR 6748 (H) Curaçao paper Sycon raphanus - - AY563537 Syconessa panicula Australia QM G313672 AM181007 Utte aff. syconoides - QMG323233 JQ272269 Utte aff. syconoides - QMG313694 JQ272271 Ute ampullacea - QMG313669 JQ272266 Vosmaeropsis sp. - MM-2004 AY026372 138

CALCINEA Ascandra falcata Mediterranean UFRJPOR5856 HQ588962 Ascaltis reticulum Mediterranean UFRJPOR 6260 HQ588977 Borojevia aff. aspina Brazil UFRJPOR 5245 HQ588998 Borojevia brasiliensis SE Brazil UFRJPOR5214 HQ588978 Borojevia brasiliensis SE Brazil UFRJPOR5230 HQ588999 Borojevia brasiliensis SE Brazil UFRJPOR 5406 KX548909 Borojevia brasiliensis* NE Brazil UFRJPOR 7384 This study Borojevia cerebrum Mediterranean UFRJPOR6322 HQ588964 Borojevia croatica Adriatic Sea IRB-CLB6 KP740023 Borojevia tenuispinata Brazil UFRJPOR6484 (H) KX548916 Borojevia trispinata Brazil UFRJPOR6419 (H) KX548918 Clathrina antofagastensis Chile MNRJ 9289 HQ588985 Clathrina aphrodita Peru MNRJ 12994 KC985138 Clathrina aspera SE Brazil UFRJPOR 5531 (P) Curaçao paper Clathrina aspera Curaçao UFRJPOR 6758 (H) Curaçao paper Clathrina aspera* NE Brazil UFRJPOR 7390 This study Clathrina aspera* NE Brazil UFRJPOR 7391 This study Clathrina aurea SE Brazil MNRJ 8998 HQ588968 Clathrina aurea* NE Brazil UFRJPOR7544 This study Clathrina aurea* NE Brazil UFRJPOR7571 This study Clathrina aurea* NE Brazil UFRJPOR7574 This study Clathrina aurea* NE Brazil UFRJPOR7584 This study Clathrina blanca Adriatic Sea PMR-14307 KC479087 Clathrina clathrus Mediterranean UFRJPOR6315 HQ589009 (C-LSU) HQ588974 (ITS) Clathrina conifera Brazil MNRJ8991 HQ588959 Clathrina coriacea Norway UFRJPOR6330 HQ588986 Clathrina curaçaoensis Curaçao UFRJPOR6734 Curaçao paper Clathrina cylindractina Brazil UFRJPOR5206 HQ588979 Clathrina fjordica Chile MNRJ 8143 HQ588984 Clathrina hispanica Mediterranean UFRJPOR 6305 KC843432 Clathrina insularis Brazil UFRJPOR6532 (H) KX548921 Clathrina insularis Brazil UFRJPOR6536 (P) KX548922 Clathrina helveola Australia QMG313680 HQ588988 Clathrina lacunosa Norway UFRJPOR 6334 HQ588991 Clathrina lutea NE Brazil UFRJPOR5172 HQ588961 Clathrina lutea NE Brazil UFRJPOR5173 (H) HQ588976 Clathrina lutea NE Brazil UFRJPOR 6543 KX548923 Clathrina lutea NE Brazil UFRJPOR 6545 KC843442 Clathrina lutea* NE Brazil UFRJPOR 7549 This study Clathrina lutea* NE Brazil UFRJPOR 7561 This study Clathrina lutea* NE Brazil UFRJPOR 7579 This study Clathrina lutea* NEBrazil UFRJPOR 7591 This study Clathrina lutea* NE Brazil UFRJPOR 7592 This study Clathrina lutea* NE Brazil MNRJ 18900 This study Clathrina luteoculcitella Australia QMG313684 HQ588989 Clathrina luteoculcitella Indonesia ZMAPOR08657 KX548906 Clathrina luteoculcitella* NE Brazil UFRJPOR 7551 This study 139

Clathrina luteoculcitella* NE Brazil UFRJPOR 7569 This study Clathrina mutabilis NE Brazil UFRJPOR6526 (H) KX548925 Clathrina mutabilis NE Brazil UFRJPOR 6528 (P) KX548926 Clathrina mutabilis* NE Brazil UFRJPOR 7386 This study Clathrina mutabilis Curaçao UFRJPOR 6741 KC843437 Clathrina nuroensis Peru MNRJ 13032 KC985136 Clathrina peruana Peru MNRJ 12839 (H) KC985135 Clathrina primordialis Adriatic Sea UFRJPOR 6863 KP740016 Clathrina ramosa Chile MNRJ 10313 HQ588990 Clathrina rubra Adriatic Sea PMR 14306 KC479088 Clathrina wistariensis Australia QMG313663 HQ588987 Ernstia tetractina Brazil UFRJPOR 5183 HQ589000 Leucascus luteoatlanticus sp. nov.* NE Brazil UFRJPOR 7582 (H) This study Leucascus simplex Polynesia BMOO16283 KC843454

RESULTS

Taxonomy From a total of 69 specimens analysed, we identified three calcaronean and 11 calcinean species including three new species to science: Amphoriscus hirsutus sp. nov., Grantia grandisapicalis sp. nov. and Leucascus luteoatlanticus sp. nov. Among the already known species, four are reported for the first time from the NE Brazil: Borojevia brasiliensis (Solé- Cava et al., 1991), Clathrina aspera Cóndor-Luján et al., in press, C. luteoculcitella Worheide & Hooper, 1999 and Arthuria vansoesti Cóndor-Luján et al., in press. Seven species already recorded from the NE were also found in this study. Clathrina mutablis Azevedo et al., submitted was originally described from Fernando de Noronha Archipelago and herein, we expand its distribution to Maracajaú. Ernstia citrea Azevedo et al., submitted, was only recorded from Rocas Atoll (Rio Grande do Norte) and now it was found in the Abrolhos Marine National Park. Ernstia rocasensis Azevedo et al., submitted, previously reported from Rocas Atoll and Abrolhos Archipelago, is being reported from Fernando de Noronha Archipelago now. Clathrina aurea Solé- Cava et al., 1991, C. lutea Azevedo et al., submitted, Leucascus roseus Lanna et al., 2007 and Leucilla uter Poléjaeff, 1883 were found in new localities within the Abrolhos Marine National Park. In the next section, the new records from the NE Brazil as well as the allegedly cosmopolitan Leucilla uter are broadly described and illustrated. Calcineans already reported from the NE region and found in the studied material are listed in Table 11. 140

Description of species Class CALCAREA Bowerbank, 1862 Subclass CALCARONEA Bidder, 1898 Order LEUCOSOLENIIDA Hartman, 1958 Family AMPHORISCIDAE Dendy, 1893

Genus Amphoriscus Haeckel, 1872 Amphoriscus hirsutus sp. nov. (Figures 2-3, Table 3) Etimology. From the Latin hirsutus (= full of bristles), for the presence of diactines protruding along the body. Type locality. Ilha Guarita, Abrolhos National Marine Park, Bahia State. Type material. Holotype (ethanol): UFRJPOR 7570; Ilha Guarita, Abrolhos Marine National Park; 10.3 m deep, collected by A. Padua & F. Azevedo, 4/XII/2014. Colour. White in life and yellowish in ethanol. Description. This sponge has a friable, tubular body with an apical osculum (Figure 2A). The holotype measures 22 x 6 mm. The osculum has a margin composed of sagittal tetractines and it is surrounded by a crown of trichoxeas (Figure 2B). The surface is very hispid due to diactines and anchor-like triactines which protrude through the sponge surface. The diactines are distributed along the body (arrows in Figure 2B) and the anchor-like triactines are present at the basal region of the sponge (arrowheads in Figure 2B-C). The aquiferous system is syconoid. Skeleton. The skeleton is typical of the genus (Figure 2D). Diactines cross perpendicularly the cortex (arrow in Figure 2D), which is composed of tetractines. The basal actines of the cortical tetractines lay tangentially to the surface (black arrow in Figure 2E). The apical actine of the tetractines and the diactines cross the choanosome, eventually reaching the atrium. The choanosomal skeleton is inarticulated and it is formed by the apical actines of the cortical tetractines (white arrow in Figures 2E-F) and the unpaired actine of subatrial triactines (black arrows in Figure 2F). Subatrial-like triactines (arrowhead in Figure 2F) and tetractines (arrow in Figure 2G) were found scattered in this region. The atrial skeleton is composed of tetractines with the apical actine projected into the atrial cavity (Figure 2H). The anchor-like triactines are organised in tufts (Figure 2I) with the unpaired actine located in the choanosome and the paired actines protruding the sponge surface. Spicules Diactines (Figure 3A). One tip is sharp and the other, which protrudes the cortex, is lanceolated. Very variable size: 462.5-2150.0/12.5-20.0 µm. 141

Cortical tetractines (Figure 3B). Sagittal. Actines are conical, straight, smooth and have sharp tips. The paired actines can be curved. The apical actine is the largest actine and can be slightly undulated. Size: 150.0-165.0/12.5-17.5 µm (unpaired actine), 200-350/7.5-20.0 µm (paired actine) and 150-450/10-20 µm (apical actine). Choanosomal tetractines (Figure 3C). Very rare. Sagittal. Subatrial-like. Actines are slightly conical and with sharp tips. The paired actines are curved and can have different length. Subatrial triactines (Figure 3D-F). Sagittal. Actines are conical, straight to slightly curved and with sharp tips. The paired actines can have different length. Size: 315.0–565.0/10–15 µm (unpaired actine) and 165.0–365.0/7.5–12.5 µm (paired actine). Atrial tetractines (Figure 3G). Sagittal. Actines are slightly conical, straight, smooth and with sharp tips. The paired actines can be curved. The apical actine is the shortest one. Size: 80- 285/7.5-12.5 µm (unpaired actine), 165.0-400.0/5-12.5 µm (paired actine) and 50-150/5-8.7 µm (apical actine). Anchor-like triactines (Figures 3H-I). Sagittal. The unpaired actine is very long. It is curved at the base and very straight from the median region to the distal part (Figure 3H). The paired actines are rudimentary and centripetally curved (Figure 3I). Size: 1650->1750/10 µm (unpaired actine) and 25.0–40/6.2–7.5 µm. Ecology. This specimen was found covered with sediment, surrounded by algae. Geographic Distribution. Provisionally endemic to the NE Brazil (present study). Remarks. Amphoriscus hirsutus sp. nov. is the second Amphoriscus species bearing anchor-like triactines. The other species is A. ancora Van Soest, 2017 from the Guyana Shelf. Although both species have similar skeletons, they present relevant differences. In A. ancora, diactines and anchor-like triactines are restricted to the base of the sponge, whereas in A. hirsutus sp. nov. the diactines are present along the whole body and the anchor-like triactines are present not only in the base but also in adjacent (basal) regions. The new Brazilian species present triactines and tetractines scattered in the choanosomal skeleton which are absent in A. ancora. Besides, the size (mean length/width) of the atrial tetractines of A. hirsutus (paired actine: 293.2/8.6 µm, unpaired actine: 190.0/9.4 µm and apical actine: 88.4/7.2 µm) exceeds that of A. anconra (paired actine: 99.0/7.7 µm, unpaired actine: 87/8.6 µm and apical actine: 26/5.3 µm). Additionaly, the osculum of A. hirsutus presents a crown of trichoxeas whereas in A. ancora, it is naked, however, as this is a plastic character, it must be treated with caution. Another species of Amphoriscus recorded from the Brazilian coast is Amphoriscus synaptum (Schmidt in Haeckel, 1872), however, it can be easily differentiated from A. hirsutus sp. nov. by the presence of anchor-like tetractines in the former. 142

Not considering the anchor-like spicules, the species that more resemble A. hirsutus sp. nov. in skeleton composition is A. buccichii Ebner, 1887 from the Adriatic Sea, however, the skeleton of the latter also comprises small subcortical tetractines (length: 80-120 µm) which are absent in the new species. Moreover, they differ in spicule size. A. buccichii has smaller diactines (60-200/3-5 µm) and thicker cortical tetractines (width: 30–40 µm) compared to A. hirsutus sp. nov. (size of diactines: 462.5-2150/12-20 µm and width of cortical tetractines: 7.5-2 µm). For additional comparisons, all the measurements of A. ancora and A. buccichii are presented in Table 3.

Table 3. Measurements of Amphoriscus hirsutus sp. nov. (UFRJPOR 7570), A. ancora and A. buccichii. *Taken from the original descriptions. H=holotype. P=paired, U=unpaired and A=apical actines.

Species Spicule Actine Length (µm) Width (µm) N Min Mean SD Max Min Mean SD Max UFRJPOR Diactine 462.5 2150.0 510.3 2150.0 12.5 16.9 2.6 20.0 14 7570 Cortical P 200.0 280.4 46.1 350.0 7.5 12.6 3.4 20-0 25 (H) Tetractine U 150.0 157.5 10.6 165.0 12.5 15.0 3.5 17.5 2 A 150.0 324.8 85.6 500.0 10.0 14.0 2.9 20.0 26 Subatrial P 165.0 266.8 51.6 365.0 7.5 10.1 7.5 12.5 22 Triactine U 315.0 443.8 70.9 565.0 10.0 11.8 1.7 15.0 21 Atrial P 165.0 293.2 64.0 400.0 5.0 8.6 1.6 12.5 24 Tetractine U 80.0 190.0 65.9 285.0 7.5 9.4 1.8 12.5 17 A 50.0 88.4 31.4 150.0 5.0 7.2 1.0 8.7 20 Anchor-like P 25.0 32.1 5.8 40.0 6.2 7.3 0.5 7.5 7 triactine U 1650.0 - - >1750.0 - 10 - - A. Diactine 600.0 1017.0 - 1230.0 3.0 4.2 - 6.5 - ancora* Cortical P 135.0 239.0 - 320.0 8.0 15.4 - 21.0 - Tetractine U 151.0 216.0 - 330..0 12.0 16.1 - 21.0 - A 258.0 311.0 - 366.0 8.0 17.8 - 20.0 - Subatrial P 129.0 218.0 - 324.0 7.0 8.6 - 10.5 - Triactine U 201.0 367.0 - 468.0 7.0 10.1 - 12.0 - Atrial P 57.0 99.0 - 165.0 3.0 7.7 - 11.0 - Tetractine U 42.0 87.0 - 186.0 3.0 8.6 - 12.0 - A 9.0 26.0 - 60.0 3.0 5.3 - 15.0 - Anchor-like P 18.0 24.8.0 - 29.0 5.0 6.8 - 9.0 - triactine U 96.0 386.0 - 660 7.0 8.6 - 10.0 - A. Diactine 60.0 - - 200 3.0 - - 5.0 - buccichii* Cortical P 360.0 - - 420 30.0 - - 40.0 - Tetractine U 360.0 - - 540 30.0 - - 40.0 - A 300 - - 420 30.0 - - 40.0 - Subatrial P 100 - - 120 6.0 - - 7.0 - Triactine U 200 - - 260 6.0 - - 7.0 - Atrial P 150 - - 200 7.0. - - 10.0 - Tetractine U 300 - - 400 7.0 - - 10.0 - A 100 - - 150 6.0 - - 12.0 - 143

Figure 2. Amphoriscus hirsutus sp. nov. (UFRJPOR 7570). A. Specimen in vivo. B. Specimen after fixation showing diactines (arrows) and anchor-like triactine (arrowhead). C. Anchor-like triactine. D. Cross section of skeleton with diactines (black arrow). D. Cortex with the paired actines (black arrow) and apical actine (white arrow) of a tetractine. F. Choanosome showing an apical actine of a cortical tetractine (white arrow), unpaired actines of subatrial triactines (black 144 arrows) and a choanosomal triactine (arrowhead). G. Choanosome with subatrial-like tetractine. H. Atrial skeleton. I. Tuft of anchor-like triactines. Abbreviations: cx=cortex; at=atrium.

Figure 3. Spicules of Amphoriscus hirsutus sp. nov. (UFRJPOR 7570). A. Diactines. B. Cortical tetractines. C. Choanosomal subatrial-like tetractine. D-F. Subatrial triactines. G. Atrial tetractine. H. Anchor-like tetractine. I. Paired actines of the anchor-like tetractine. 145

Genus Leucilla Haeckel, 1872 Leucilla uter Poléjaeff, 1883 (Figures 4-5, Table 4) Synonyms. Amphoriscus chrysalis Burton, 1963: 545. Leucilla australiensis, Borojevic, 1967: 221; Borojevic & Peixinho, 1976: 1031 Leucilla uter Poléjaeff, 1883: 53; Dendy & Row, 1913: 784; Borojevic & Boury-Esnault, 1987: 35; Muricy & Silva, 1999: 160, Muricy et al., 2011: 25 Leucilla? uter, Muricy et al., 1991: 1187. Type locality. Poléjaeff described specimens from Bermudas and Philippines, however the lectotype deposited in the Natural History Museum (London) is from Bermudas. Type specimens. Lectotype (ethanol and dried) BMNH 1884.4.22.21, paralectotypes (slides): BMNH.4.22.30 and BMNH 1884.4.22.31. Material examined. UFRJPOR 7545 (ethanol), Mato Verde, Ilha Santa Bárbara, Abrolhos Marine National Park, 7.1 m deep, collected by A. Padua & F. Azevedo, 3/XII/2014. Colour. Bright white in life and in ethanol. Description. The analysed specimen has a sac-shaped body with an apical osculum (Figure 4A). It measures 9.4 x 3.3 mm (Figure 4B). The osculum has a crown of trichoxeas (2.6 x 1.7 mm) supported by sagittal T-shaped tetractines. In the apical part of the body, diactines protrude through the surface (arrow in Figure 4B). The surface is rough and the consistency is friable. The aquiferous system is leuconoid. Skeleton. The skeleton is typical of the genus (Figure 4C). The cortical skeleton is formed by tetractines and very rare triactines (arrow in Figure 4D). The basal actines of the tetractines are tangentially disposed and the apical actine penetrates the choanosome and can reach the atrium. Some diactines were also observed in this region. The choanosomal skeleton is inarticulated, formed by the apical actine of the cortical tetractines (white arrow in Figure 4E) and by the unpaired actine of the subatrial triactines (black arrow in Figure 4E) and rare subatrial tetractines. Rare tetractines and triactines were found scattered in the choanosome, with their unpaired actine pointing to the surface (as shown in Figure 4C). The atrial skeleton is exclusively composed of tetractines with the apical actine projected into the atrium (Figure 4F). Spicules Diactines (Figure 5A). Fusiform (sharp tips). Size (length/width): >1250/6-10 μm. Cortical triactines (Figures 5B). Sagittal. Very rare. Actines are straight, slightly conical with sharp tips. The paired actines can be slightly curved and longer than the unpaired one. Size: 200.0-205.0/10.0-11.2 μm (paired actine) and 122.5/10.0-12.5 μm (unpaired actine). 146

Cortical tetractines (Figure 5C-D). Sagittal. Actines are conical with sharp tips. The paired actines are frequently inwardly curved. The apical actine is the longest actine. Size: 225.0- 530.0/22.5-40.0 μm (paired actine), 265.0-500.0/30-42.5 μm (unpaired actine) and 255- 1000/22.5-40.0 μm (apical actine). Choanosomal triactines (Figure 5E) and tetractines (Figure 5F-G). Sagittal. Rare. Actines are conical with sharp tips. Different from the cortical tetractines, the actines are always straight. Size of triactines: 250.0/22.5 μm (paired actine) and 237.5/22.5-25 μm (unpaired actine). Size of tetractines: 335-400/15-40 μm (paired actine), 227.5-425.0/27.5-37.5 μm (unpaired actine) and 87.5/25.0 μm (apical actine). Subatrial triactines (Figure 5H). Sagittal. Actines are conical with sharp tips. The paired actines are smaller than the unpaired one and can be slightly curved. Very variable size: 75- 320.0/7.5-22.5 μm (paired actine) and 150-540/7.5-20.0 μm (unpaired actine). Subatrial tetractines (Figure 5I). Sagittal. Rare. Actines are conical with sharp tips, similar to subatrial triactines. The apical actine is shorter and thinner than the basal ones. Size: 112.5- 285.0/7.5-15 μm (paired actine), 175.0-350.0/7.5-20 (unpaired actine) and 50.0-87.5/7.5 μm (apical actine). Atrial tetractines (Figure 5J). Sagittal. Actines are conical with very sharp tips. The paired actines are longer than the unpaired one. The apical actine is the thinnest and shortest actine. Size: 115.0-285.0/5.0-12.5 μm (paired actines), 137.5-187.5/7.5-12.5 μm (unpaired actine) and 62.5-150.0/7.4-10.0 μm (apical actine). Ecology. This specimen was collected underneath boulders. Geographic distribution. Leucilla uter is an allegedly cosmopolitan species. It was originally described from Bermudas and Philippines (Poléjaeff, 1883) and then, recorded from Brazil where it is widely distributed, from Alagoas to Rio de Janeiro States (Borojevic & Peixinho 1976; Muricy et al. 2011). Remarks. The analysed specimen in the present study mostly matches the original description of Leucilla uter (Poléjaeff 1833) as well as the description of its lectotype (Borojevic & Boury- Esnault, 1987). The only difference is the presence of rare diactines and cortical triactines in our specimen. As the validity of those spicules as diagnostic characters within Leucilla species has been questioned (Borojevic & Boury-Esnault, 1987), we decided not to differentiate the studied specimen as a new species and to identify it as L. uter until an update revision of this genus is done. It is worth to mention that the revised specimen slightly differs from the specimens misidentified as L. australiensis by Borojevic & Peixinho (1976) and re-identified as L. uter in 147

Muricy et al. (2011) in the size of the cortical triactines and of the atrial tetractines. The unpaired actines of both spicule categories are larger in the specimens described by Borojevic & Peixinho (cortical triactine=200-400/10-16 μm and atrial tetractine=270-400/10-12 μm) than in our specimen (cortical triactine=122.5/10-12.5 μm and atrial tetractine=137.5-187.5/7.5-12.5 μm). This is the first record of L. uter occurring in very shallow waters (<10 m). Previously, it was reported from depths ranging from 10 to 61 m (Muricy et al. 2011).

Figure 4. Leucilla uter (UFRJPOR 7545). A. Specimen in vivo. B. Specimen after fixation. C. Cross section of the skeleton. D. Cortex with cortical triactines (arrow). E. Inarticulated 148 choanosomal skeleton with the apical actine of a cortical tetractine (white arrow) and the unpaired actine of a subatrial triactine (arrow). F. Atrial skeleton. Abbreviations: cx=cortex; at=atrium.

Figure 5. Spicules of Leucilla uter (UFRJPOR 7545). A. Diactines. B. Cortical triactine. C-D. Cortical tetractines. E. Choanosomal triactine. F-G Choanosomal tetractines. H. Subatrial triactine. I. Subatrial tetractine. J. Atrial tetractine. 149

Table 4. Measurements of Leucilla uter of the specimen UFRJPOR 7545, a Brazilian specimen by Borojevic & Peixinho (1976) and of the type specimen by Poléjaeff (1893).

Specimen Spicule Actine Length (µm) Width (µm) N Min Mean SD Max Min Mean SD Max UFRJPOR Trichoxea 2250 - - - - 2.5 - - 2 7545 Diactine 1250 - - - 6 8 - 10 3 Cortical Paired 200.0 201.7 2.9 205.0 10.0 10.4 0.7 11.2 3 triactine Unpaired - 122.5 - - 10.0 11.2 1.8 12.5 2 Cortical Paired 225.0 368.5 85.4 530 22.5 32.5 6.1 40.0 20 tetractine Unpaired 265.0 381.7 66.5 500.0 30.0 36.1 4.3 42.5 20 Apical 255.0 535.0 171.3 1000 22.5 31.3 5.7 40.0 26 Choanosomal Paired - 250.0 0.0 - - 22.5 0.0 2 Triactine Unpaired - 237.5 0.0 - 22.5 23.8 1.8 25.0 2 Choanosomal Paired 335.0 365.7 35.4 400.0 15.0 30.7 8.7 40.0 7 Tetractine Unpaired 227.5 370.4 71.8 425 27.5 33.8 4.9 37.5 6 Apical - 87.5 - - - 25 - - 1 Subatrial Paired 75.0 186.9 77.7 320.0 7.5 11.6 4.8 22.5 20 Triactine Unpaired 150.0 370.8 124.5 540.0 7.5 13.9 4.9 20.0 20 Subatrial Paired 112.5 222.3 50.4 285.0 7.5 10.1 2.5 15.0 13 Tetractine Unpaired 175.0 275.9 45.7 350.0 7.5 10.8 3.2 20.0 14 Apical 50.0 73.1 17.2 87.5 - 7.5 0.0 - 4 Atrial Paired 115.0 204.8 50.6 285.0 5.0 7.9 1.7 12.5 15 Tetractine Unpaired 137.5 158.1 21.2 187.5 7.5 10.3 2.5 12.5 4 Apical 62.5 95.2 22.3 150.0 5.0 7.4 1.1 10.0 21 Leucilla Cortical Paired 170.0 - - 400.0 10.0 - - 16.0 - uter sensu triactine Unpaired 200.0 - - 400.0 10.0 - - 16.0 - Borojevic & Cortical Basal 300.0 - - 700.0 20.0 - - 60.0 - Peixinho, Tetractine Apical 180.0 - - 600.0 20.0 - - 60.0 - 1976 Choanosomal 300.0 - - 600.0 30.0 - - 50.0 - Triactine Choanosomal Basal 300.0 - - 600.0 30.0 - - 50.0 - Tetractine Apical - - 200.0 - - - - - Subatrial Paired 170.0 - - 250.0 10.0 - - 16.0 - triactine Unpaired 300.0 - - 400.0 - 16.0 - - - Atrial Paired 200.0 - - 350.0 10.0 - - 12.0 - tetractine Unpaired 270.0 - - 400.0 10.0 - - 12.0 - Apical 40.0 - - 80.0 - - - - - Original Diactine - - 400.0 - 2.5 - - - description Cortical Basal 400.0 - - 600.0 - - - 50.0 - tetractine Apical 400.0 - - 1200 - - - 50.0 - Choanosomal Basal 400.0 - - 600.0 - - - 50.0 - tetractine Apical 400.0 - - 1200 - - - 50.0 - Subatrial Unpaired - - - 600.0 30.0 - - 50.0 - triactine Paired - - - 420.0 21.0 - - 35.0 - Subatrial Unpaired - - - 600.0 30.0 - - 50.0 - tetractine Paired - - - 420.0 21.0 - - 35.0 - Atrial Paired - - - 400.0 12.5 20.0 - - - tetractine* Unpaired 250.0 - - 350.0 12.5 20.0 - - - Apical - - - 200.0 12.5 20.0 - - - 150

Family Grantiidae Dendy, 1893 Genus Grantia Fleming, 1828 Grantia grandisapicalis sp. nov. (Figure 6 and 7, Table 5) Etimology. From the Latin grandis (= large) for the long apical actine of the atrial tetractines. Type locality. Chapeirão 2, Abrolhos Marine National Park, Bahia State, Brazil. Type material. Holotype (ethanol): UFRJPOR 7567, Chapeirão 2, Abrolhos Marine National Park, 15.2 m deep, collected by A. Padua & F. Azevedo, 04/XII/2014. Colour. White in life and beige in ethanol. Description. This sponge has a globular body with an apical osculum (Figures 6A-B). The holotype measures 14 x 6 mm. The surface is very hispid because of diactines protruding through the cortex (arrow in Figure 6B). The osculum (diameter=2.5 mm) is sustained by a margin composed of T-shaped triactines and tetractines and it is surrounded by a crown of trichoxeas (arrowhead in Figure 6B). The atrium is hispid due to the long apical actines of the atrial tetractines. The aquiferous system is syconoid with elongated chambers arranged side by side. Skeleton. The skeleton is typical of the genus (Figure 6C). The cortical skeleton is composed of perpendicular diactines (white arrow in Figure 6D) and tangential triactines (black arrow in Figure 6D). Diactines do not penetrate the choanosome. The tubar skeleton is articulated, composed of several rows of triactines and tetractines (in less proportion) with the unpaired actine pointing to the cortex (Figure 6E). The subatrial skeleton is composed of triactines with the unpaired actine pointing to the choanosome. The atrial skeleton is exclusively composed of tetractines with the apical actine projected into the atrial cavity (Figure 6F). Spicules Trichoxea of the crown (Figure 7A). Slender. Size: More than 500 μm long. Diactines I (Figure 7B). Fusiform and small. Size: 300-580/22.5-30.0 μm. Diactines II (Figure 7C). Fusiform and large. Very variable size: 1000.0 to > 3125.0/35.0-55.0 μm. Cortical triactines (Figures 7D-F). Sagittal. Actines are conical, straight and have sharp tips. The paired actines are frequently longer than the unpaired one and can be slightly curved. Size: 75.0-142.5/7.5-11.3 μm (paired actine) and 47.5-107.5/7.5-12.5 μm (unpaired actine). Tubar triactines (Figures 7G-I). Sagittal. Actines are conical, straight with sharp tips (Figure 7G). The paired actines have different sizes (Figure 7H) and one of them can be curved (Figure 7I). Size: 32.5-67.5/5-10 μm (paired actine 1), 62.5-120.0/5-7.5 μm (paired actine 2) and 119.4- 140/5-8.8 μm (unpaired actine). 151

Tubar tetractines (Figures 7J-K). Sagittal. Actines are conical, straight with sharp tips (Figure 7J). Some paired actines have different sizes and one of them can be curved. The unpaired actine can be longer than the paired ones (Figure 7K). Size: 67.5-142.5/5.0-7.5 μm (paired actines), 80.0-197.5/6.2-8.7 μm (unpaired actine) and 17.5-35.0/5-7.5 μm (apical actine). Subatrial triactines (Figure 7L). Sagittal. Actines are slightly conical, straight with sharp tips. The paired actines can be curved. Size: 50-110/5-7.5 μm (paired actines) and 127.5-207.5/7.5 μm (unpaired actine). Atrial tetractines (Figures 7M-N). Sagittal. Actines are conical with sharp tips (Figure 7M). The apical actine is long, thick and distally curved (Figure 7N). Size: 80.0-175.0/5-8.7 μm (paired actines), 80.0-112.5/5-6.2 μm (unpaired actine) and 100-172.5/5-8.7 μm (apical actine). Ecology. The surface of the analysed specimen was found covered with sediment. Geographic distribution: This species is provisionally endemic to the NE Brazil (present study). Remarks. The genus Grantia comprises 40 valid species (Van Soest et al. 2016). Among them, only G. aculeata Urban, 1908 from the Mediterranean Sea, G. infrequens Carter, 1886 from Australia, G. intermedia Thacker, 1908 from Cape Verde and G. kempfi Borojevic & Peixinho, 1976 from Brazil present triactines and tetractines in the tubar skeleton. Grantia kempfi is the species that more resembles G. grandisapicalis sp. nov., however, they can be distinguished by the choanocytary chambers which are ramified near the atrium in the former and elongated in the latter. Besides, the skeleton of G. kempfi comprises comprises subatrial tetractines and atrial triactines, which are absent in the new species. The other species of Grantia reported from Brazil is G. atlantica Ridley, 1881 (Muricy et al. 2011), which does not present subatrial triactines nor tubar tetractines (Borojevic & Peixinho 1976). 152

Figure 6. Grantia grandisapicalis sp. nov. (UFRJPOR 7567). A. Specimen in vivo. B. Specimen after fixation indicating crown (arrowhead) and diactines (arrow). C. Cross section of the skeleton. D. Cortical skeleton with diactine (white arrow) and cortical triactine (black arrow). E. Tubar skeleton with triactine (white arrow) and tetractine (black arrow). F. Atrial skeleton. Abbreviations: cx=cortex; at=atrium. 153

Figure 7. Spicules of Grantia grandisapicalis sp. nov. (UFRJPOR 7567). A. Broken trichoxea. B. Diactine I. C. Broken diactine II. D-F. Cortical triactines. G-I. Tubar triactines. J-K. Tubar tetractines. L. Subatrial triactine. M. Atrial tetractine. N. Apical actine of an atrial tetractine.

Table 5. Spicule measurements of Grantia grandisapicalis sp. nov. (UFRJPOR 7567).

Length (µm) Width (µm) N Spicule Actine Min Mean SD Max Min Mean SD Max Diactine I - 300.0 440.0 140.0 580.0 22.5 27.5 4.3 30.0 3 Diactine II - 1000.0 >3125.0 35 42.0 6.9 55.0 7 Cortical Paired 75.0 112.1 18.0 142.5 7.5 9.0 1.2 11.3 21 triactine Unpaired 47.5 74.5 14.2 107.5 7.5 10.3 1.4 12.5 20 Tubar Paired - 32.5 48.4 9.8 67.5 5.0 6.2 1.3 10 20 triactine Paired+ 62.5 87.7 14.1 120.0 5.0 6.4 1.2 7.5 21 Unpaired 119.4 90.0 14.3 140.0 5.0 7.3 1.1 8.8 20 154

Tubar Paired 67.5 94.6 17.8 142.5 5.0 7.0 1.0 7.5 30 tetractine Unpaired 80.0 141.6 28.8 197.5 6.2 7.7 0.5 8.7 30 Apical 17.5 26.4 5.0 35.0 5.0 5.4 0.8 7.5 16 Subatrial Paired 50.0 80.5 19.3 110.0 5.0 5.9 1.2 7.5 10 triactine Unpaired 127.5 160.8 27.3 207.5 7.5 7.5 0.0 7.5 10 Atrial Paired 80.0 125.0 29.5 175.0 5.0 6.6 1.1 8.7 20 tetractine Unpaired 80.0 99.4 13.9 112.5 5.0 5.6 0.7 6.2 4 Apical 100 132.2 18.1 172.5 5.0 7.2 0.9 8.7 23

Subclass Calcinea Bidder, 1898 Family Clathrinidae Minchin, 1900

Genus Arthuria Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013 Arthuria vansoesti Cóndor-Luján, Azevedo, Padua, Hajdu, Klautau, in press (Figure 8, Table 6) Synonyms Arthuria vansoesti Cóndor-Luján et al., in press Type locality. Daai Booi, St. Willibrordus, Curaçao, Caribbean Sea. Type specimens. Holotype (ethanol): UFRJPOR 6720 (holotype); Daai Booi, St. Willibrordus; 12°12'43.12"N, 69°05'8.42W; 5.2 m deep; collected by B. Cóndor-Luján, 19/VIII/2011. Material examined. UFRJPOR 7557 (ethanol), Chapeirão2, Abrolhos Marine National Park; 15.2 m deep; collected by F. Azevedo & A. Padua, 4/XII/2014. Colour. Light yellow in life and beige (or yellowish white) in ethanol. Description. The analysed specimen has a massive cormus (6 x 5 x 3 mm) formed by irregular and loosely anastomosed tubes and presents water-collecting tubes (Figures 8A-B). The aquiferous system is asconoid. No granular cells were observed. The skeleton has no special organization and it is composed of abundant triactines (distinguished in two shape categories) and rare tetractines. Spicules (Table 6) Triactines I (Figure 8C). Regular (equiangular and equiradiate). Very frequent. Actines are cylindrical, slightly undulated at the distal part and with rounded tips. Size: 70.0-102.5/2.5-5.0 μm. Triactines II (Figure 8D). Regular (equiangular and equiradiate). Actines are straight, slightly conical with blunt to sharp tips. Size: 52.5-92.5/2.5-5.0 μm Tetractines (Figures 8E-F). Regular (equiangular and equiradiate). Rare. Basal actines are cylindrical, slightly undulated at the distal part and with rounded to blunt tips (Figure 11E). The 155 apical actine is straight, smooth and bear sharp tips (Figure 11F). Size: 80.0-85.0/3.8-5 μm (basal actine) and 25.0-67.5/2.5-5.0 μm (apical actine). Geographic distribution. Curaçao (Cóndor-Luján et al. in press) and Abrolhos Marine National Park (present study) Remarks: This species was originally described from the Caribbean Sea (Cóndor-Luján et al. in press) and we are expanding its distribution to the NE Brazil. The other Arthuria occurring in the NE is A. trindadensis Azevedo et al., submitted. The new species can be easily differentiated from A. trindadensis by the in vivo colour, which is yellow in the former and white in the latter. Besides, the skeleton of A. trindadensis comprises triactines whose actines only have sharp tips whereas A. vansoesti has triactines with rounded (Triactines I) and sharp (Triactines II) tips.

Table 6. Spicule measurements of Arthuria vansoesti from Abrolhos (UFRJPOR 7557) and of the holotype . *Taken from Cóndor-Luján et al. in press.

Specimen Spicule Actine Length (µm) Width (µm) N Min Mean SD Max Min Mean SD Max UFRJPOR Triactine I - 70.0 85.6 7.4 102.5 2.5 3.5 0.7 5.0 25 7557 Triactine II - 52.5 76.5 9.9 92.5 2.5 3.4 0.9 5.0 20 Tetractine basal 80.0 81.7 2.9 85.0 3.8 4.6 0.7 5.0 3 apical 25.0 46.3 30.0 67.5 2.5 3.8 1.8 5.0 2 Holotype* Triactine I 72.5 79.8 4.9 87.5 3.8 4.6 0.6 5.0 30 Triactine II 52.5 70.4 7.8 82.5 2.5 3.9 0.8 5.0 30 Tetractine basal 60.0 75.3 8.1 87.5 3.8 4.8 0.5 5.0 15 apical 25.0 25.0 0.0 25.0 3.8 4.4 0.7 5.0 6 156

Figure 8. Arthuria vansoesti (UFRJPOR 7557). A. Specimen in vivo. B. Specimen after fixation. C. Triactine I. D. Triactine II. E. Tetractine. F. Apical actine of a tetractine.

Genus Borojevia Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013

Borojevia brasiliensis (Solé-Cava, Klautau, Boury-Esnault, Borojevic & Thorpe, 1991) (Figure 9, Table 7) Synonyms Clathrina cerebrum Borojevic 1971: 526 Clathrina brasiliensis Solé-Cava et al. 1991: 382; Klautau et al. 1994: 372; Muricy & Silva 1999: 160; Klautau & Borojevic 2001: 403; Klautau & Valentine 2003: 11; Muricy et al. 2011: 33. Borojevia brasiliensis Klautau et al. 2013: 459 Type locality. Arraial do Cabo, Brazil Type specimens. Holotype (ethanol): MNHN-LBIM.C. 1989.2, Arraial do Cabo (Enseada), Rio de Janeiro, Brazil. Collected by G. Muricy, 16/XII/ 1986. 157

Material examined. UFRJPOR 7384 (ethanol), Porto do Pecém, São Gonçalo do Amarante, Ceará State, collected by E. Hajdu & S. Salani, 2/IV/2014. Colour. White in life and ethanol. Description. This species has a massive, rough and slightly compressible cormus (17 x 6 x 3 mm) composed of irregular and tightly anastomosed tubes (Figures 9A-B). The aquiferous system is asconoid and the oscula are spread through the cormus. No cells with granules were observed. The skeleton has no special organization and it comprises tripods that are in fact large triactines, triactines and tetractines. Triactines are more abundant than tetractines. Spicules (Table 7) Tripods. (Figures 9C-D). Equiangular, subregular or parasagittal. Actines are straight, slightly conical with blunt tips. They do not have the centre raised as in typical tripods. Size: 121.5- 153.9/9.4-12.1 µm. Triactines.. Regular (equiangular and equiradiate). Very abundant. Actines are straight, conical with blunt tips (Figure 9E). Sagittal triactines with curved paired actines were also observed (Figure 8F). Size: 62.1-72.6/8.1-10.8 µm. Tetractines.. Regular (equiangular and equiradiate). Actines are straight, conical with blunt tips (Figure 9G-H). Sagittal tetractines with curved paired actines were also observed (Figure 8I). The apical actine is shorter and thinner than the basal ones and has spines. Spines are located near the tip of the actine (Figure 9J). Some tetractines with no spines were also observed. Size: 62.1-83.7/8.1-10.8 µm (basal actine) and 37.5-65.0/5.0-7.5 µm (apical actine). Geographic distribution. SE Brazil (Klautau & Valentine 2003; Muricy et al. 2011) and NE Brazil (this study). Remarks. Four Borojevia species are known from Brazilian waters: B. aspina (Klautau, Solé- Cava, & Borojevic, 1994), B. brasiliensis, B. tenuispinata and B. trispinata Azevedo et al., submitted. Among these, our specimen more resembles B. brasiliensis as it presents a skeleton with more triactines than tetractines and similar spines distribution along the apical actine of the tetractines, however, the tripods are different. Compared to previous descriptions of B. brasiliensis (Solé-Cava et al. 1991; Klautau & Borojevic 2001; Klautau & Valentine 2003) the specimen from Ceará have tripods with longer and less conical actines (Table 7). Nonetheless, as the variability in the shape of tripods in Borojevia has been assigned to polymorphism or plasticity (Klautau et al. 2016), the observed difference in the analysed specimen does not constitute a diagnostic character. Therefore, we identified UFRJPOR 7384 as B. brasiliensis. Furthermore, in the phylogenetic tree, it clustered within the B. brasiliensis clade with a high support (pp=0.98, b=95, Figure 15), corroborating the morphological identification. 158

Figure 9. Borojevia brasiliensis (UFRJPOR 7384). A. Specimen in vivo. B. Specimen after fixation. C-D. Tripods. E-F. Triactines. G-I. Tetractines. J. Apical actine of a tetractine.

Table 7. Spicule measurements of Borojevia brasiliensis from Ceará (UFRJPOR 7384) and of the holotype. *Taken from Klautau & Valentine (2003).

Specimen Spicule Actine Length (µm) Width (µm) N Min Mean SD Max Min Mean SD Max UFRJPOR Tripod 121.5 138.0 9.4 153.9 9.4 11.2 0.8 12.1 20 7384 Triactine 62.1 68.8 3.8 72.6 8.1 8.7 0.8 10.8 20 Tetractine basal 62.1 71.4 4.8 83.7 8.1 9.2 0.9 10.8 20 apical 37.5 50.0 7.0 65.0 5.0 6.7 0.8 7.5 20 Holotype* Tripod 67 81 8.2 95.7 - 11 1.7 - 20 Triactine 60.9 78.2 10.6 102.2 - 10.8 1.5 - 20 Tetractine basal 56.5 75.3 10.0 91.3 - 10.4 1.3 - 20 apical 17.4 36.4 9.1 50.0 - 8.0 2.2 - 20 159

Genus Clathrina Gray, 1867 sensu Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013

Clathrina aspera Cóndor-Luján, Azevedo, Padua, Hajdu, Klautau, in press (Figure 10, Table 8). Synonyms Clathrina aspera Cóndor-Luján et al., in press. Type locality. Water Factory, Willemstadt, Curaçao, Caribbean Sea. Type specimens. Holotype (ethanol): UFRJPOR 6758, Water Factory, Willemstadt, Curaçao, 12°06'30.88"N, 68°57'13.53"W, 13.2 m deep, collected by B. Cóndor-Luján and E. Hajdu, 23/VIII/2011. Paratypes (ethanol): UFRJPOR 5487, Ilhas Botinas, Angra dos Reis, Rio de Janeiro, Brazil; 23º03'19.36''S, 44º19'44.98''W; 1-3 m deep; collected by F. Azevedo and M. Klautau, 25/V/2007 and UFRJPOR 5531; Praia do Bonfim, Angra dos Reis, Rio de Janeiro, Brazil; 23°01'14.26''S, 44°19'48.18''W; 1-2 m deep; collected by M. Klautau, 27/V/2007. Material examined. UFRJPOR 7390 and UFRJPOR 7391 (ethanol), Farol da Praia de Maracajaú, Área de Proteção Ambiental dos Recifes de Corais de Maracajaú (APARC- Maracajaú), Rio Grande do Norte State, collected by B. Cóndor-Luján, 2 m deep, 8/IV/2014. Colour. White in life and in ethanol. Description. The largest specimen (UFRJPOR 7390) measures 13 x 11 x 4 mm. The cormus is massive and composed of irregular and tightly anastomosed tubes (Figures 10A-B). Because of the presence of large triactines located on the external tubes, the surface is rough. The aquiferous system is asconoid with oscula widespread on the surface. No water-collecting tubes were observed. The skeleton has no special organization and it comprises two categories of triactines. Spicules. Triactines I (Figure 10C). Regular (equiangular and equiradiate). Large and tripod-like. Actines are straight, conical with sharp tip. Size: 225.0-390.0/20.0-35.0 µm. Triactines II (Figure 10D). Regular (equiangular and equiradiate). Actines are straght, cylindrical to slightly conical with sharp tips. Size: 45.0-125.0/5.0-8.7 µm. Ecology. This sponge was found underneath boulders. Adjacent individuals to UFRJPOR 7391 were eaten by fish (field observation). Geographic distribution. Brazil, including the NE (this study) and SE regions, and Curaçao (Cóndor-Luján et al. in press). Remarks. The present record of C. aspera from Rio Grande do Norte contributes to the understanding of its distribution and genetic connectivity in the Western Tropical Atlantic as it 160 was previously reported from two very distant localities, Curaçao (Caribbean Sea) and Rio de Janeiro (Cóndor-Luján et al. in press). In both ITS and C-LSU phylogenetic reconstructions (Figures 14 and 15), the NE specimens clustered with sequences of the type material of C.aspera (UFRJPOR5531 and UFRJPOR6758) with high support (ITS: pp=0.99, b=99 and C- LSU: pp=1, b=91) in concordance with the morphological identification.

Figure 10. Clathrina aspera (UFRJPOR 7390). A. Specimen in vivo. B. Specimen after fixation. C. Triactine I. D. Triactines II.

Table 8. Spicule measurements of Clathrina aspera from Rio Grande do Norte (UFRJPOR 7390 and UFRJPOR 7391) and from the holotype. *Taken from Cóndor-Luján et al. in press

Specimen Spicule Length (µm) Width (µm) N Min Mean SD Max Min Mean SD Max UFRJPOR Triactine I - 245.0 - - - 20 - - 1 7390 Triactine II 50.0 78.9 24.0 112.5 5.0 7.0 0.8 8.7 40 UFRJPOR Triactine I 225.0 317.5 50.0 390.0 20.0 25.6 3.6 35.0 12 7391 Triactine II 45.0 78.6 24.5 125.0 5.0 6.7 1.0 7.5 40 Holotype* Triactine I 132.5 227.2 50.8 325.0 17.5 28.5 5.6 37.5 30 Triactine II 75.0 115.4 12.9 152.5 7.5 9.7 1.1 12.5 40 161

Clathrina luteoculcitella Wörheide & Hooper, 1999 (Figure 11, Table 9) Synonyms Clathrina luteoculcitella Wörheide & Hooper 1999: 868; Klautau & Valentine 2003: 30, Klautau et al. 2013: 12. Clathrina aff. luteoculcitella: Van Soest & de Voogd 2015:13. Type locality. Great Barrier Reef, Australia Type specimen. Holotype (ethanol): QMG 313684, ‘The Patch’, at the N end of the channel between Heron Island and Wistari Reef, Great Barrier Reef, 23°26.6'S, 151°53.4'E, 25 m deep. Material examined. UFRJPOR 7551 (ethanol); Chapeirão1, Parque Nacional Marinho dos Abrolhos; 16.2 m; collected by A. Padua & F. Azevedo, 04/XII/2014; UFRJPOR 7569 (ethanol); Chapeirão2, Parque Nacional Marinho dos Abrolhos; 15.2 m; collected by A. Padua & F. Azevedo, 04/XII/2014. Colour. Beige in life and white in ethanol. Description. The largest specimen measures 21 x 13 x 5 mm. The cormus is massive, rough and slightly compressible. It is composed of thin, regular and tightly anastomosed tubes which form folds (giving an apparent spherical shape, Figure 11A). The aquiferous system is asconoid. The oscula are spread along the surface (Figure 11B), however, in UFRJPOR 7551, structures similar to water-collecting tubes (laterally located) were observed. The skeleton has no special organization and it comprises one single category of triactines and very rare diactines. Diactines were only found in UFRJPOR 7551 and most of them were located tangentially to the surface (only one diactine perpendicularly inserted was observed). Spicules Triactines (Figure 11C). Regular (equiangular and equiradiate). Actines are slightly conical to conical, distally undulated and with sharp tips. Size: 59.5-90.0/5.4-10 µm. Diactines (Figure 11D). Very slender, trichoxea-like. Most of them were broken but it was possible to distinguish two tips: one sharp tip and another lanceolated (wider). Size: >162.5/2.5 µm. Ecology. The specimen UFRJPOR 7551 was found in a crevice, on a red calcareous algae. Balls of sediment were found inside the tubes of UFRJPOR 7569. Geographic distribution: Australia (Wörheide & Hooper 1999), Indonesia (Van Soest & de Voogd 2016) and NE Brazil (present study). Remarks. The analysed specimens resemble the Brazilian endemic species Clathrina angraensis Azevedo et al., 2007 in external morphology and spicule size (48-100/6.8±10 µm), 162 however, the cormus of the latter does not form folds and its triactines are less conical than those observed in the specimens from Abrolhos. The in vivo colour of the studied specimens is beige, nonetheless, in the ITS phylogenetic analysis, they grouped (pp=1, b=99, Figure 15) with the yellow C. luteoculcitella from Australia, indicating its conspecificity. Different from the Australian specimens whose skeleton comprise abundant diactines perpendicularly disposed to the surface of the cormus (Wörheide & Hooper 1999; Klautau & Valentine 2003), in the Brazilian specimens, (thinner) diactines were rarely found and, when observed, they were tangential to the surface. This indicates that diactines may constitute plastic morphological characters in Clathrina, as recently suggested by Azevedo et al. submitted. Clathrina luteoculcitella constitutes the first confirmed record of an Indo-Pacific species present in the Western Atlantic Ocean. This disjunct distribution is puzzling and should receive attention in further studies as it is possible that this species was introduced in the Brazilian coast by anthropogenic means (M. Klautau, pers. Obs.).

Figure 11. Clathrina luteoculcitella (UFRJPOR 7551). A. Specimen in vivo. B. Specimen after fixation. C. Triactines. D. Broken diactine. 163

Table 9. Spicule measurements of Clathrina luteoculcitella from Abrolhos (UFRJPOR 7551 and UFRJPOR 7569) and from the original description (Wörheide & Hooper 1999).

Specimen Spicule Length (µm) Width (µm) N Min Mean SD Max Min Mean SD Max UFRJPOR 7551 Diactine >87.5 - - >162.5 2.5 2.5 0 2.5 6 Triactine 72.5 81.4 5.0 90.0 7.5 7.8 0.7 10 20 UFRJPOR 7569 Triactine 59.5 75.3 8.6 86.5 5.4 6.3 1.2 8.1 20 Original Diactine 90.0 164.4 - 220.0 2.0 3.1 - 6.0 description Triactine 68.0 77.7 - 84.0 8.0 9.4 - 12.0

Family Leucascidae Dendy, 1892 Genus Leucascus Dendy, 1892

Leucascus luteoatlanticus sp. nov. (Figures 12 and 13, Table 10) Etimology. From the Latin lutea (=yellow) for the yellow colour of the cormus and the type locality in the Atlantic Ocean. Type locality. Parcel das Paredes, Abrolhos Marine National Park, Bahia State, Brazil Type material. Holotype: UFRJPOR 7582; Parcel das Paredes, Abrolhos Marine National Park, Bahia State, Brazil, 14. 6 m deep; collected by A. Padua & F. Azevedo, 5/XII/2014. Material used for comparison: Holotype of L. flavus (ethanol): ZMAPOR 13145, Sulawesi, Bone Baku, Indonesia, station BB/NV/120597, collected by N. J. de Voogd, 16/V/1997. Colour. Yellow in life and beige in ethanol. Description: The analysed specimen measured 18 x 10 x 3 mm. This species has a rough, firm and massive cormus composed of regular and tightly anastomosed tubes (Figure 12A). It is possible to recognize thin and delicate cortical and atrial membranes delimiting the cormus and the atrium, respectively. The cortical membrane is perforated by inhalant apertures. The aquiferous system is solenoid. The oscula are located on the top of elevations and do not have ornamentation (Figure 12B). Skeleton. The skeleton comprises triactines and tetractines (Figure 12C). In the cortex, there are more triactines than tetractines (Figure 12D), whereas in the tubes and in the atrium, triactines and tetractines seem to be present in equal proportions (Figure 12F). Most choanocytary tubes are hispid because of the apical actine projected inside of them (Figure 12E). Spicules Triactines (Figure 13A). Regular (equiangular and equiradiate). Actines are slightly conical to conical, straight with sharp tips. Size: 80.0-125.0/7.5-11.3 µm. 164

Tetractines (Figures 13B-C). Regular (equiangular and equiradiate). Actines are slightly conical, straight with sharp tips (Figure 13B). The apical actine is slightly conical, thinner than the basal ones and bear spines. The spines are distributed along the apical actine (Figure 13C). Size: 75-105.0/7.5-10 µm (basal actines) and 40.0-85.0/5-7,5 µm (apical actine). Ecology. A crustacean and a polychaeta were found inside the atrium. Geographic distribution. Provisionally endemic to the NE Brazil (present study). Remarks. The specimen analysed resembles Leucascus flavus Cavalcanti et al., 2013 from Indonesia. Both species are yellow in vivo, present a cortical skeleton mainly composed of triactines and the skeleton of their choanocytary tubes have triactines and tetractines in equal proportions. However, they can be differentiated by their atrial skeletons which present more tetractines than triactines in L. flavus, whereas in the Brazilian specimen, triactines and tetractines are present in similar proportions. As this character (proportion of spicules in the atrial skeleton) has been validated as a diagnostic character in the revision of Leucascus (Cavalcanti et al. 2013), we propose to name the analysed specimen herein as a new species, L. luteoatlanticus sp. nov. Additionally, as Van Soest & de Voogd (2015) provided in situ pictures of L. flavus, it was possible to recognize a wider atrium in L. flavus when compared to L. luteoatlanticus sp. nov. Before this study, only two species of Leucascus were reported for the Brazilian coast, L. roseus Lanna et al., 2007 and L. albus Cavalcanti et al., 2013. Different from L. luteoatlanticus sp. nov., which is yellow in vivo and have spicules with conical actines, L. roseus is pink and possesses spicules with cylindrical actines whereas L. albus is white and its skeleton bears microdiactines.

Table 10. Spicule measurements of Leucascus luteoatlanticus sp. nov. (UFRJPOR 7582) and of the holotype of L. flavus. *Taken from Cavalcanti et al. (2013).

Specimen Spicule Actine Length (µm) Width (µm) N Min Mean SD Max Min Mean SD Max UFRJPOR Triactine 80.0 100.3 11.9 125.0 7.5 9l4 1.1 11.3 20 7582 Tetractine basal 75.0 92.4 9,2 105.0 7.5 8l4 0.9 10 20 apical 40.0 61.9 13.6 85.0 5.0 5l5 1.0 7.5 16 Leucascus Triactine 70.0 100.9 9.5 120.0 7.5 9l7 1.1 12.5 30 flavus* Tetractine basal 75.0 98,7 7.4 115.0 7.5 8l9 1.3 10.0 30 apical 41.3 49.6 5.3 60.7 3.6 4l2 0.6 4.9 30 165

Figure 12. Leucascus luteoatlanticus sp. nov. (UFRJPOR 7582). A. Specimen in vivo. B. Specimen after fixation. C. Cross section of the skeleton. D. Cortical membrane. E. Atrial membrane. F. Choanocytary tube with projected apical actines (arrow). Abbreviations: cx=cortex; ct=choanocytary tube. 166

Figure 13. Spicules of Leucascus luteoatlanticus sp. nov. (UFRJPOR 7582). A. Triactines. B. Tetractine. C. Apical actine of a tetractine covered with spines. 167

Table 11. New locality records of calcareous sponges from the NE coast.

Species - Material New Record Type Locality examined Locality, depth, collectors, collection date Clathrina aurea - UFRJPOR 7544 L6, 7.1 m, coll. A Padua & F. Azevedo, 03/XII/2014. Arraial do Cabo, UFRJPOR 7548 L9, 16.2 m, coll. A. Padua & F. Azevedo, 04/XII/2014. Rio de Janeiro, UFRJPOR 7552 L9, 16.2 m, coll. A. Padua & F. Azevedo, 04/XII/2014. Brazil. UFRJPOR 7553 UFRJPOR 7554 UFRJPOR 7572 L5, 11.7 m, coll. A. Padua & F. Azevedo, XII/2014. UFRJPOR 7571 L5, 14.3 m, coll. A. Padua & F. Azevedo, XII/2014. UFRJPOR 7574 UFRJPOR 7576 UFRJPOR 7580 L5, 14.6 m, coll. A. Padua & F. Azevedo, XII/2014. UFRJPOR 7584 UFRJPOR 7585 MNRJ 18965 L10, 13.1 m, coll. A. Bispo & J. Carraro, 04/XII/2014. Clathrina lutea - UFRJPOR 7549 L9, 16.2 m, coll. A Padua & F. Azevedo, 4/XII/2016. Pedra Lixa, UFRJPOR 7550 Abrolhos Archipelago, UFRJPOR 7555 Bahia, Brazil. UFRJPOR 7561 L10, 15.2 m, coll. A. Padua & F. Azevedo, 4/XII/2016. UFRJPOR 7563 UFRJPOR 7564 UFRJPOR 7565 UFRJPOR 7566 UFRJPOR 7568 UFRJPOR 7577 L5, 14.3 m, coll. T. Pérez & O. Thomas, 05/XII/2014. UFRJPOR 7578 UFRJPOR 7579 L5, 14.6 m, coll. A. Padua & F. Azevedo, 05/ XII /2014. UFRJPOR 7583 UFRJPOR 7589 UFRJPOR 7590 UFRJPOR 7591 L10, 9-15 m, coll. A. Bispo & J. Carraro, 04/ XII /2014. UFRJPOR 7592 Abrolhos Marine National Park, T. Pérez & O. Thomas, UFRJPOR 7593 04/XII/2014. UFRJPOR 7594 UFRJPOR 7595 UFRJPOR 7596 UFRJPOR 7597 UFRJPOR 7598 UFRJPOR 7599 UFRJPOR 7600 UFRJPOR 7601 UFRJPOR 7602 MNRJ 18844 L10, 11.6 m, coll. E. Hajdu, 04/XII/2014. MNRJ 18900 L5, 5 – 16 m, coll. A. Bispo & J. Carraro, , 04/XII/2014.

Clathrina mutabilis - UFRJOR 7386 L4, 20 m, coll. B. Cóndor-Luján, 07/IV/2014. Fernando de Noronha UFRJPOR 7388 L3, 2 m, coll. B. Cóndor-Luján, 08/IV/2014. Archipelago, Brazil 168

Ernstia citrea - UFRJPOR 7547 L7, 7.1 m, coll. A. Padua & F. Azevedo, 03/XII/2014. Rocas Atoll, UFRJPOR 7556 L10, 15.2, coll. A. Padua & F. Azevedo, 04/XII/2014. Rio Grande do Norte, UFRJPOR 7560 Brazil UFRJPOR 7573 L5, 14.3 m, coll. A. Padua & F. Azevedo, 05/XII/2014.

Ernstia rocansensis - UFRJPOR 8547 L2, 4 m, coll. A. Bispo & S. Salani, 23/IV/2016. Rocas Atoll, Rio Grande do Norte, Brazil

Leucascus roseus - UFRJPOR 7558 L10, 15.2 m, coll. A. Padua & F. Azevedo, 04/XII/2014. Alcatrazes Archipelago, UFRJPOR 7559 São Sebastião, UFRJPOR 7575 L5, 14.3 m, coll. A. Padua & F. Azevedo, 04/XII/2014. São Paulo, Brazil. UFRJPOR 7581 L5, 14.6 m, coll. A. Padua & F. Azevedo, 04/XII/2014. UFRJPOR 7586 L5, 14.6 m, coll. A. Padua & F. Azevedo, 05/XII/2014. UFRJPOR 7587 UFRJPOR 7588 MNRJ18884 L10, 10 – 15 m, coll. A. Bispo & J. Carraro, 04/XII/2014. MNRJ 18885

Molecular analyses In this study, 19 DNA sequences were generated. We provided sequences for the new species described herein: C-LSU for Amphoriscus hirsutus sp. nov. and Grantia grandisapicalis and ITS for Leucascus luteoatlanticus sp. nov. Furthermore, new ITS sequences were generated for Clathrina aspera, C. aurea, C. lutea, C. luteoculcitella, C. mutabilis and Borojevia brasiliensis. An additional C-LSU sequence of C. aspera was presented as well. The C-LSU sequences produced an alignment of 457 bp including gaps. Both phylogenetetic methods (BI and ML) recovered the same tree topology (BI tree is shown in Figure 14). The phylogenetic tree yielded similar results to those previously obtained (Voigt & Wörheide 2016; Cóndor-Luján et al. in press). With the addition of the new sequences, Amphoriscus and Grantia appeared as non monophyletic genera. Grantita grandispicalis sp. nov. nested within the clade of Heteropiidae and Grantiidae, instead of clustering with Grantia compressa, which is the type species of the genus. Interestingly, the skeleton of G. grandisapicalis sp. nov. comprises tubar tetractines whereas that of G. compressa does not. Furthermore, A. hirsutus sp. nov. whose choanosomal skeleton is mainly composed of triactines clustered with Sycon conulosum + Leucilla antillana (pp=0.99, b=90) whose tubar and subatrial skeletons (respectively) are exclusively composed of triactines (Cóndor-Luján et al. in press). The intraspecific variation (estimated by the uncorrected p distance) of the ITS Calcinean sequences ranged between 0 and 1.4%. Borojevia brasiliensis, C. lutea and C. aspera showed 169 no intraspecific variation (0%), The values ranged from 0 to 0.5% in C. mutabilis and from 0- 0.7% in C. luteoculcitella. The highest variation was observed in C. aurea (0-1.4%).

Figure 14. Bayesian phylogenetic tree inferred from the C-LSU sequences of the studied species. Bayesian posterior probabilities and bootstrap values (pp/b) are given on the branches. * Sequences generated in this study. 170

The alignment of the ITS sequences had a total length of 1349 bp including gaps. Both the BI and ML methods yielded similar tree topologies (ML tree is shown in Figure 15). The phylogenetic tree (1) recovered the monophyletic genera Clathrina (pp=0.96, b=71) and Borojevia (pp=1 b=100) obtained in previous studies using a similar set of sequences (Klautau et al. 2013, 2016; Azevedo et al. submitted; Cóndor-Luján et al. in press) and (2) revealed a close affinity between Leucascus (L. simplex + L. luteoatlanticus sp. nov.) and Ascaltis (A. reticulum) supported by high values (pp=0.1, b=99).

DISCUSSION With the results of the present study, the list of calcareous sponges from the NE Brazil increased from 42 (Azevedo et al. submitted; Van Soest et al. 2016) to 49 species. Among these, 42.9% (18) are endemic from this region, 23.8% (10) also occur in the Caribbean Sea and 19% (8) have an amphi-Atlantic distribution (being present in South Africa or in the Mediterranean Sea). Whether this apparent high endemism in the NE Brazil reflects a true distribution pattern for Calcarea remains uncertain as formerly NE Brazilian endemic species have been recently recorded from other areas, e.g. Grantia kempfi in the Guyana Shelf (Van Soest 2017) and Borojevia tenuispinata, Clathrina mutabilis, C. insularis and Nicola tetela from Curaçao (Cóndor-Luján & Klautau 2016; Cóndor-Luján et al. in press). Further samplings from adjacent localities are necessary to validate this observed endemic pattern. The new records provided herein bring new insights to understand the distribution patterns of certain Brazilian calcareous sponges and highlight interesting affinities among the northeastern localities. The new record of Ernstia rocasensis from Fernando de Noronha suggests an affinity among three isolated areas: Rocas Atoll, Abrolhos and Fernando de Noronha Archipelagos. Apart from this species, Leucetta floridana is the only species previously reported from these localities (although it has a broader distribution in the Western Tropical Atlantic, Valderrama et al. 2009; Muricy et al. 2011). The occurrence of C. mutabilis in Maracajaú reinforces the calcarean affinity between the Brazilian continental shelf and the oceanic islands, supported by the previous records of C. aurea, Leucaltis clathria, Leucascus roseus, and Leucetta floridana (Muricy et al. 2011). The finding of C. aspera in the NE Brazil (Maracajaú) constitutes an important intermediate record as previously, the known distribution of this species was disjunct: Curaçao and Rio de Janeiro (Cóndor-Luján et al. in press) and therefore, we point out the importance of continuing to preserve this protected area. The new molecular affinities found in this study suggested an apparent phylogenetic signal in the skeleton composition (choanosomal triactines) of some calcaronean species, as already 171 pointed out (Cóndor-Luján et al. in press). However, more species with different skeleton characteristics should be included in the trees to verify this hypothesis. Besides, it is necessary that more detailed morphological descriptions are made to allow mapping the characters on phylogenetic trees.

ACKNOWLEDGEMENTS We are indebted to Marcelo Soares and Ana Luisa Pires Moreira for providing technical support with collecting licenses, Olivier Thomas, Thierry Pérez, Mariana Carvalho, André Bispo, Sula Salani, Cristiana Castello-Branco, Camille Leal, João Carraro and Humberto Fortunato for field assistance and sponge collection, Carol Leite for some slides preparations and the staff of the Laboratório de Protistologia of Prof. Inácio da Silva Neto for providing assistance with microscopy images. The dive centres Atlantis Divers, Mar de Noronha Divers and Natal Divers are acknowledged for proving technical diving assistance. We thank the Brazilian government agencies that authorized the sampling licenses in MPAs; Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio), Instituto de Desenvolvimento Sustentável e Meio Ambiente do Rio Grande do Norte (IDEMA), Reserva Biológica Marinha do Atol das Rocas, Parque Nacional Marinho de Fernando de Noronha, and the Brazilian Navy (Terminal Portuário do Pecém). This work was funded by the Brazilian National Research Council (CNPq), Coordination for the Improvement of Higher Education Personnel (CAPES), Foundation Grupo Boticário de Proteção à Natureza and Rio de Janeiro State Research Foundation (FAPERJ). B.C.L. received a scholarship from CAPES, F.A. has a post-doc fellowship by CAPES and A.P. has a post-doc fellowship by FAPERJ. E.H. and M.K. have fellowships by CNPq. 172

Figure 15. Maximum Likelihood phylogenetic tree inferred from the ITS sequences of the Calcinean species. Bayesian posterior probabilities and bootstrap values (pp/b) are given on the branches. * Sequences generated in this study. FN: Fernando de Noronha. RN: Rio Grande do Norte. 173

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Evolutionary history of the calcareous sponge Leucetta floridana in the Western Tropical Atlantic

Cóndor-Luján, B. 1; García-Hernández, J.2; Schizas, N.2; Pérez, T.3; Zea, S4. Klautau, M. 1

1 Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Av. Carlos Chagas Filho 373, CCS, Bloco A, A0-100, 21941-902, Rio de Janeiro, RJ, Brasil. 2 Caribbean Laboratory of Marine Genomics, University of Puerto Rico-Mayagüez, P.O. Box 9000, Mayagüez, PR, 00681, United States of America. 3Institut Méditerranéen de Biodiversité et d’Ecologie marine et continentale, CNRS, Aix Marseille Univ, IRD, Avignon Univ. Station Marine d’Endoume, rue de la Batterie des Lions, 13007 Marseille, França. 4Centro de Estudios en Ciencias del Mar – CECIMAR, Universidad Nacional de Colombia, Sede Caribe; c/o INVEMAR, Calle 25 2-55, Rodadero Sur - Playa Salguero, Santa Marta, Colombia. *Corresponding author: [email protected] Running title: Evolutionary history of Leucetta floridana

ABSTRACT Sponges have short-lived lecithotrophic larvae, which constrain their dispersion and consequently restrict their geographic distribution. However, some calcareous species as Leucetta floridana are widespread in the Western Tropical Atlantic (WTA). In order to elucidate the historical processes that originated its current distribution and to evaluate the role of the Amazon River as a barrier to the gene flow between Caribbean and Brazilian sponge populations, several genetic analyses using ITS sequences were performed. A total of 162 specimens from different localities (Puerto Rico, Lesser Antilles, Panama, Colombia, Curaçao and Brazil) was studied. Genetic variation was assessed through sequence-type and nucleotidic diversity indexes. Phylogenetic trees and a MJ network were constructed to explore the genealogical relationships among individuals. To determine population structure, F ST comparisons and AMOVA were performed. Neutrality tests, mismatch distributions and Bayesian skyline plots were conducted to infer demography patterns. The phylogenetic trees as well as the networks showed high structured populations consistent with FST and AMOVA results: four populations restricted to the Caribbean Sea and one population widespread in the WTA. The presence of a large widespread population in the WTA rejects the role of the Amazon 177

River as an effective barrier to gene flow between Caribbean and Brazilian localities. Demography analyses indicated an event of population expansion in the Caribbean Sea during the Last Maximum Glaciation (20 millions year BP). The patterns of population connectivity and demography within the WTA are discussed and a hypothetical evolutionary scenario for L. floridana is presented. Key words: Amazon River, Brazilian Coast, Caribbean Sea, Greater and Lesser Antilles, Population connectivity, Porifera

INTRODUCTION In the marine environment, organisms with short-lived larvae and consequent low dispersal capabilities, such as sponges, are not expected to attain widespread geographic distributions. Some morphological and molecular studies on sponges have rejected the putative cosmopolitanism of some species (Solé-Cava et al., 1991; Boury-Esnault et al., 1992; Hajdu & van Soest, 1992; Klautau et al., 1994; Muricy et al., 1996; Klautau et al., 1999; Klautau & Valentine, 2003; Valderrama et al., 2009), while others revealed that species with widespread distribution do occur in the Western Tropical Atlantic Ocean (WTA) (Lazoski et al., 2001; Valderrama et al., 2009). The occurrence of species with this wide distribution represents a good opportunity to assess the species dispersal across biogeographic barriers such as the Amazon River. Up to date, no study on the effectiveness of the Amazon River as a barrier to gene flow was performed within Porifera. Some recent studies evaluated the genetic connectivity of sponges only within the Tropical Northwestern Atlantic (TNA), e.g. Callyspongia vaginalis (De Biasse et al., 2010, 2016), Cliona delitrix (Chaves-Fonnegra et al., 2015) and Xestospongia muta (López-Legentil & Pawlik, 2009; Richards et al., 2016; de Bakker et al., 2016), or exclusively in the Tropical Southwestern Atlantic (TSA) (Padua et al., in prep.: Clathrina aurea) but none considered populations along the entire Western Tropical Atlantic. Different biochemical and molecular approaches have been used to unravel the genetic structure of sponge populations (isozymes, nuclear and mitochondrial DNA regions). In calcareous sponges, as the traditional mitochondrial DNA marker (COI) was not appropriate for phylogeography inferences or population structure studies (Worheide et al., 2000), nuclear intron regions (ITS and ATPSb-Iii) were employed (Wörheide et al., 2002; Bentlage and Wörheide, 2007, Wörheide et al., 2008). Among them, the ITS proved to be the most suitable one (Wörheide et al., 2008). Recently, the mitochondrial gene cox3 was proposed as an alternative phylogeography marker since it evidenced a variability similar to that of nuclear 178 intron data (Voigt et al., 2012). However, due to the substantial variation at both intra- and interspecific levels found in mitochondrial markers (Lavrov et al., 2013), the use of cox3 remains uncertain. Therefore, for the class Calcarea, the ITS region is still a useful marker at population level. The calcareous sponge Leucetta floridana (Calcarea: Calcinea: Leucettidae) has a patchy distribution within reefs and inhabits environments protected from light such as small crevices, overhangs, and vertical slopes (Valderrama et al., 2009). It has been found from -2 to -90 m of depth (Muricy et al., 2011). As a calcinean species, it is assumed to have a lecithotrophic larvae (calciblastula) (Borojevic et al., 1990; Maldonado & Berquist, 2002), however, the reproduction of this species has not been investigated yet. Leucetta floridana is one of the most widespread calcareous sponges in the TWA and thus, constitute a good model for evaluating population connectivity within the Western Tropical Atlantic. In this study, we assessed the genetic structure of this species at phylogeographic, population and demographic levels in order to (1) understand the historical processes that culminated in its current geographic distribution and to (2) evaluate the role of the Amazon River as a barrier to the gene flow between Caribbean and Brazilian populations of sponges.

MATERIALS AND METHODS Analysed material The analysed material comprised 162 specimens preliminary identified as Leucetta floridana from 18 localities within the Tropical Northwestern Atlantic (TNA, n=147) and the Tropical Southwestern Atlantic (TSA, n=15), at depths varying from -2 to -70 m. Localities in the TNA included four ecoregions, Greater Antilles - Puerto Rico (n=12), Eastern Caribbean – Anguilla (n=1), Saint Martin (n=52), Antigua (n=12), Les Saintes (n=8), Guadeloupe (n=1), Martinique (n=23), and Saba (n=3), Southwestern Caribbean - San Andrés (n=4), Panama (n=19), Urabá (n=3) and Santa Marta (n=7) and Southern Caribbean - Curaçao (n=2), whereas in the TSA comprised three ecoregions, Northeastern Brazil – Ceará (n=5) and Rio Grande do Norte (RN, n=4), Fernando de Noronha and Atoll das Rocas – Fernando de Noronha Archipelago (n=4) and Rocas Atoll (n=1) and Eastern Brazil – Abrolhos Archipelago (n=1) (Table 1, Figure 1). Most of the Caribbean specimens (131 specimens) were collected during several campaigns: Expedition PaCoTilles (Patterns of Connectivity among the Lesser Antilles, 2015, n=87), Santa Marta Expedition (2014, n=7), Martinique Expedition (2013, n=10), Puerto Rico Expedition (2015, n=12) and PorToL Project (2012, n=15). After collection, specimens were fixed and 179 stored in ethanol 93 or 96% until DNA extraction. All voucher specimens are deposited in the Porifera Collection of the Universidade Federal of Rio de Janeiro (UFRJPOR) in Brazil, Universidad de Puerto Rico or INVEMAR (Instituto de Investigaciones Marinas y Costeras) in Colombia. The other 31 specimens were already deposited in the Porifera Collection of the UFRJPOR and were analysed in previous studies (Valderrama et al., 2009; Klautau et al., 2013; Cóndor- Luján et al., in press). Detailed information of any specimen can be accessed in Table S1 of the Supplementary Material. Additional sequences of Leucetta species were retrieved from Genbank (Table 2) for comparative phylogenetic analyses described in the corresponding section.

Figure 1. Map indicating the localities considered in this study. Coloured circles represent different ecoregions according to the MEOW (Spalding et al., 2007). Blue: Eastern Caribbean, green: Southwestern Caribbean, yellow: Southern Caribbean, red: Northeast Brazil, orange: Fernando de Noronha and Atoll das Rocas, and pink= Eastern Brazil. The Amazon River is indicated. 180

Table 1. Locality, geographic coordinates, depth, and Genbank accession numbers of the specimens of Leucetta floridana analysed in this study. CE=Ceará, PE=Pernambuco, RN=Rio Grande do Norte, FN=Fernando de Noronha Archipelago. *Sequences generated in the present study.

Locality Geographic Coordinates Depth (m) Genbank Number Greater Antilles Ecoregion – Puerto Rico CayoTurrumote 17°56'20.13''N, 67° 2'43.95''W 12.9 - 17.4 N=2* Cayo Mario 17°57'16.89''N, 67° 3'53.42''W 09.0 - 17.7 N=4* Cayo Conserva 17°55'56.91''N, 67° 5'35.01''W 12.6 - 15.6 N=2* Veril – Fallen Rock 17°54'5.04''N, 66°55'27.59''W 31.5 N=1* Pinnacles 17°56'3.96''N, 67° 1'49.80''W 9.9 - 17.1 N=2* Veril - El Hoyo 17°52.529'N, 67° 2.671'W 31.5 N=1* Eastern Caribbean Ecoregion – Lesser Antilles Little Scrub 2, Anguilla 18°17.903'N, 62°57.294'W 23.5 N=1* Trou David, Terres Basses, Saint 18°04.402'N, 63°07.149'W 6.1 N=4* Martin Rocher Créole, Saint Martin 18°07.038'N, 63°03.419'W 8.0 – 10.0 N=1* Les Arches1, Saint Martin 18°07.588'N, 62°58.248'W 10 - 20 N=10* Basse Espagnoles, Saint Martin 18°07.821'N, 63°00.270'W 6 - 10 N=16* Chico2, Saint Martin 18°06.501'N, 62°59.005'W < 22 N=11* Les Arches2, Saint Martin 18°07'32.81''N, 62°58'21.49''W < 16 N=7* Southeastern Coast, Saba 17°37.066'N, 63°13.580'W < 27 N=3* Nanton Point, Saint Paul, 16°59'51.60''N, 61°45'37.22''W < 20 N=9* Antigua and Barbuda Five Islands, Antigua and 17°04.990'N, 61°54.840'W 4 N=1* Barbuda Diamond Bank, Saint Paul, 17°12.000'N, 61°52.800'W 7 – 8 N=2* Antigua and Barbuda Cave Cathédrale, Guadeloupe 16°27.740' N, 61°31.837'W 13.7 N=1* Cave 1, Les Saintes, 15°52.984'N, 61°34.25'W 8 - 10.7 N=9* Guadeloupe Grottes des couleurs, Pointe 14°29.787'N, 61°05.351'W <10 KX355573 Burgos, Grande Anse, Anses KX355574 d'Arlet, Martinique N=7* Anses d´Arlet, Martinique 14°30.377'N, 61°05.850'W? <10 KX355575 N=1* Anse de Fortune, Anses d'Arlet, 14°30.377'N, 61°05.850'W 3 – 6 N=9* Martinique Southwestern Caribbean Ecoregion Bocas del Toro, Panama 09º20.914'N, 082º09.394'W? < 17 EU78189 EU78190 EU78191 N=8* Las Cuevas, Bocas del Toro, 09º20.914'N, 082º09.394'W <20? N=7* Panamá Cayo Zapatilla, Bocas del Toro, 09º14.881'N, 82º01.952'W <20? N=2* Panamá 181

La Piscinita, San Andrés, 12°33'N, 81°43'W? 2- 5 EU781972 Colombia EU781973 EU781974 West View, Leward-reef, San 12°33'N, 81°43'W? 5 EU781971 Andrés, Colombia Bajo Agua Viva, Urabá, 07°53''N, 76°38'W? 15 EU781968 Colombia EU781969 EU781970 El Morro, Santa Marta Bay, 11°14'57.50"N, 74°13'54.50"W 11 - 21 N=4* Colombia Punta Venado, Ensenada de 11°16'25.00"N, 74°12'24.00"W 18 – 18.6 N=2* Taganga, Santa Marta, Colombia Punta Gaira, Gaira Bay, Santa 11°13'08.00"N, 74°14'30.00"W 14 N=1* Marta, Colombia Southern Caribbean Ecoregion - Curaçao Water Factory, Willemstadt 12°06'30.88"N, 68°57'13.53"W 17.8 N=1 Hook’s Hut, Willemstadt 12°07'18.94"N, 68°58'11.46"W 13.3 N=1 Northeastern Brazil Ecoregion- Brazil Potiguar Basin, RN 04º37'31.7"S, 36º46'00.7"W 70 EU781985 N=2* Risca das Bicudas, RN 04º57'00.9"S, 36º07'49.7"W 10 EU781978 Urca do Tubarão, RN 04º50'52.7"S, 36º27'02.1"W N=1* Station 30 - CENPES, CE 2º52'S, 39 º10"W 33 EU781980 EU781982 EU781983 EU781984 N=1* Fernando de Noronha and Atoll das Rocas Ecoregion - Brazil Barretinha, Rocas Atoll, RN 3°51'36'S, 33°49'04''W 12 EU781975 Sela Gineta Island, FN, PE 3°48'49''S, 32°23'29''W 7 EU781976 Ressurreta, FN, PE 3°48'49''S, 32°23'29''W 4 – 7.3 EU781977 N=2* Eastern Brazil Ecoregion Abrolhos Marine National Park, Bahia - Brazil Parcel das Paredes 17°58'S, 38°40'W 8 EU781979

Table 2. Species names, voucher numbers, locality and Genbank accession numbers of the sequences used in the phylogenetic analysis.

Species Voucher Number Locality Genbank Number Leucetta antarctica MNRJ 13798 Antarctic KC849700 Leucetta chagosensis BMOO1612 French Polynesia KC843455 Leucetta microraphis QMG313659 Australia AJ633874 Leucetta pyriformis MNRJ13843 Antarctic KC843457 Leucetta potiguar UFPEPOR 547 Brazil EU781986 Leucetta potiguar MNRJ 8474 Brazil EU781981 Leucetta potiguar UFPEPOR 569 Brazil EU781987 Leucetta potiguar UFPEPOR 588 Brazil EU781988 182

DNA extraction, amplification, sequencing and alignment The DNA of 136 specimens was extracted following the guanidine/phenol-chloroform protocol (Sambrook et al., 1993) and stored at –20°C until amplification. The region comprising the partial 18S and 28S, the spacers ITS1 and ITS2 and the 5.8S ribosomal DNA was amplified by PCR with the primers: 18S (5`-TCATTTAGAGGAAGTAAAAGTCG-3`) and 8S (5`- GTTAGTTTCTTTTCCTCCGCTT-3`) (Lôbo-Hajdu et al., 2004). Each PCR amplification reaction mixture contained: 1X buffer (5X GoTaq R Green Reaction Buffer Flexi, PROMEGA),

0.2 mM dNTP, 2.5 mM MgCl2, 0.5 µg/µL bovine serum albumin (BSA), 0.33 µM of each primer, one unit of Taq DNA polymerase (Fermentas) and 1 µL of DNA, summing up to 15 µL with Milli-Q water. PCR steps included one first cycle of 4 min at 94°C, 1 min at 50°C and 1 min at 72°C, 35 cycles of 1 min at 92°C, 1 min at 50°C and 1 min at 72°C, and a final cycle of 6 min at 72°C. Successful amplification was visualized in an 1% agarose electrophoresis gel and purified with the GE Kit. Forward and reverse strands were automatically sequenced in an ABI 3500 (Applied Biosystems). The electropherograms of the sequences generated in this study as well as the 25 sequences of L. floridana retrieved from the Genbank database were visually edited using Chromas Lite. Electropherograms with dubious base attribution (double-peaks) were not considered in any further analyses and are not presented here. All sequences generated in this work were deposited in the GenBank database (www.ncbi.nlm.nih.gov). Phylogenetic analyses In order to verify the identity of the specimens attributed to L. floridana, phylogenetic reconstructions, including sequences from other Leucetta species (Table 2), were performed under maximum-likelihood (ML) and Bayesian Inference (BI). Sequences were aligned through the MAFFT v.7 online version (Katoh & Standley, 2013) using the method Q which considers the secondary structure of the amplified region (Katoh et al., 2005) and consequently, guarantees a better alignment. The nucleotide substitution model that best fit the alignment was determined using jModelTest2 (Darriba et al., 2012; Guindo & Gascuel, 2003) and corresponded to GTR + G. The ML analyses were conducted on MEGA 6 (Tamura et al., 2013) using an initial NJ tree (BIONJ) and 1000 bootstrap pseudo-replicates. The BI reconstructions were obtained with MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003) under 106 generations and a burn-in of 1 000 sampled trees, yielding a consensus tree of majority. 183

The genetic divergence among all the species and the genetic variability among individuals were assessed by the calculus of the uncorrected p distance, considering complete deletion in MEGA 6. Phylogeography analyses Considering only the individuals of L. floridana, sequences were re-aligned as described in the above section. Gaps were considered as informative characters. The ITS sequence-types (referred herein as ST) were calculated by DNASP v. 5.10.01 (Librados & Rozas, 2009). The nucleotide (π) and the ITS sequence-type (STh) diversities were determined using ARLEQUIN v. 3.5.1 (Schneider et al., 2000). To assess the evolutionary and geographical relationships, two analyses were considered: (1) a tree including the STs within each ecoregion and additional Leucetta sequences, rooted on the most distant lineage(s) and (2) an unrooted tree (network) considering only the specimens of L. floridana. Phylogenetic analyses for the rooted tree followed the procedures detailed in the above section (ML and BI approaches). Networks were calculated using the Median Joining algorithm (Bandelt et al., 1999) within the program NETWORK v. 4.6 (Forster et al., 2007). ST- groups (STG) were defined by an analysis of molecular variance (AMOVA) as it maximizes variation among the groups and minimizes variation within the groups. Population structure The population structure across different geographical partitions (provinces, ecoregions and localities) was assessed through pairwise FST comparisons (Weir & Cockerham, 1984) and an analysis of molecular variance (AMOVA) as implemented in ARLEQUIN. The statistical significance of estimates was assessed by 10 000 permutations. Demographic structure In order to test for population expansion across geographical (provinces, ecoregions and localities) and phylogenetic (clades and STGs) groups, Tajima´s D (D) and Fu´s Fs (Fs) neutrality tests were performed. Moreover, the SSD and raggedness index (r) values based on mismatch distribution were calculated. All calculations considered 104 simulations and were computed in ARLEQUIN. To explore the historical demography of L. floridana, Bayesian skyline analyses (Drummond et al., 2005) were performed in BEAST 1.8 (Drummond et al., 2012) for each structured population (n≥10) as suggested by the AMOVA and network results. The nucleotide substitution model was determined using the Bayesian Information Criterion (BIC) implemented in MEGA 6. The chosen model was Jukes Cantor but as it is not implemented in BEAUti 1.8 (Drummond et al., 2012), we used the closest model with the lowest BIC: the HKY model. As the ITS 184 substitution rate within Calcarea is unknown, we used the 1.0% per MY estimated for the demospongiae Prosuberites laughlini (Worheide et al., 2004) which was also employed to estimate phylogeography patterns in other Leucetta species (Worheide et al., 2008). The analyses assumed a strict molecular clock model and the numbers of groups were set to six (previous exploratory tests with the same sequence set showed no difference in the Bayesian Skyline Plot when using six or 10 groups). A MCMC analysis of 108 generations was run with 10% burn-in. The results were summarized in piecewise-constant Bayesian Skyline Plots using TRACER 1.5 (Rambaut & Drummond, 2009).

RESULTS

Genetic Diversity P distance The length of the alignment including all the Leucetta sequences was 764 pb, including 136 variables sites, 74 parsimony-informative sites and 61 singletons. All specimens preliminarily identified as L. floridana clustered in a clade supported by high ML bootstrap and BI posterior probability (b=99, p=1) values, corroborating the initial identification (Figure 2). The genetic divergences among the species of Leucetta were as follows: L. floridana–L. potiguar: 2.5-3.4%, L. floridana–L. microraphis: 4.7-5.3%, L. floridana–L. chagosensis: 7.1- 7.7% , L. floridana–L. antarctica: 8.0-8.6%, and L. floridana–L. pyriformis: 8.5-9.1%. Leucetta potiguar, a species restricted to the TSA Province, appeared as the sister taxon of L. floridana as already reported in previous studies (Valderrama et al., 2009; Klautau et al., 2013). The length of the alignment considering only L. floridana individuals was 733 pb with 708 invariable sites, 23 variable sites and two sites with alignment gaps. The intraspecific variability inferred by the p-distance ranged from 0 to 1.8%. Interestingly, this same range value (0-1.8%) was found (1) between Caribbean and Brazilian individuals and (2) among Caribbean individuals. Among Brazilian individuals, the range varied between 0 and 0.1%. Sequence-type richness A total of 24 STs were found when considering gaps as informative characters. Among them, two STs were shared by the TNA and TSA (ST1 and ST2), 16 were exclusive to the TNA and one to the TSA (Table 3). ST1 was the most frequent ST, found in 98 individuals (60.5%) and present in all the studied ecoregions except the Southern Caribbean (Curaçao). ST2 was restricted to the Southwestern Caribbean and Fernando de Noronha and Atoll das Rocas ecoregions. Besides ST1 and ST2, other five STs were shared among different ecoregions and 185 localities (ST12, ST15, ST18, ST19 and ST14). Seventeen STs were private to one locality or island (ST3, ST5, ST6, ST7, ST8, ST9, ST10, ST11, ST13, ST14, ST16, ST17, ST20, ST21, ST22, ST23, ST24). The ecoregion with more STs was Eastern Caribbean (K=13) followed by Southwestern Caribbean (K=10, Table 3). Greater Antilles, Northeastern Brazil and Fernando de Noronha and Atoll das Rocas presented the same ST contribution (K=2) whereas the Southern Caribbean and Eastern Brazil presented only one ST. Within the Eastern Caribbean, the island with more STs was Saint Martin (K=8) followed by Martinique with five STs. Both localities presented the same number of private STs (P=4). In the Southwestern Caribbean, the localities with more STs were Panama and San Andrés, both with four STs. Within the Brazilian localities, Fernando de Noronha and Ceará were the most diverse localities presenting two STS. Sequence-type (STd) and nucleotidic (π) diversities The overall STd was 0.62±0.04 and the total π was 0.003±0.002. Similar values were obtained for the TNA whereas slightly lower values were observed for the TSA (Table 3). Among the ecoregions, the Southwestern Caribbean presented the highest values: STd=0.83±0.04 and π=0.0078±0.0039. Within this ecoregion, San Andrés had the highest values: STd=1±0.18 and π=0.0075±0.0054, being followed by Panama with STd=0.71± 0.07 and π =0.005±0.003. Greater Antilles (Puerto Rico) presented the lowest values in STd=0.17 ± 0.133 and in π= 0.0002 ± 0.0004. 186

Figure 2. Maximum Likelihood phylogenetic tree inferred from the ITS sequences of Leucetta floridana and other Leucetta species. ML bootstrap and BI posterior probability values are indicated on the branches.

Table 3. Genetic Diversity. N=number of sequences, S/T=Segregating/Total number of sites, K= number of STs, P=number of private STs, STd= ST diversity, π=nucleotide diversity, SD= standard deviation. RN=Rio Grande do Norte State, FN=Fernando de Noronha Archipelago.

Ecoregion Locality N S/N K P STd (SD) π(SD) Tropical Northern Atlantic 147 24/733 23 16 0.638 (0.045) 0.0038 (0.0022) Greater Antilles Puerto Rico 12 1/732 2 1 0.167 (0.134) 0.0002 (0.0004) Eastern Caribbean 100 19/733 13 8 0.558 (0.056) 0.0022 (0.0014) Anguilla 1 0/732 1 0 - - Saint Martin 52 16/733 8 3 0.3167 (0.084) 0.0015 (0.0011) Saba 3 0/732 1 0 0 0 Antigua 12 13/732 4 2 0.454 ( 0.17) 0.003 (0.002) Guadeloupe 1 0/732 1 0 - - Les Saintes 8 1/732 2 0 0.25 (0.18) 0.0003 (0.0005) Martinique 23 4/733 5 3 0.613 (0.104) 0.0016 (0.0012) Southwestern Caribbean 33 14/732 10 7 0.835 (0.042) 0.007 (0.0039) San Andrés 4 10/731 4 3 1.0 (0.177) 0.0075 (0.0055) Panama 19 9/732 4 2 0.713 (0.074) 0.0055 (0.0032) Urabá 3 1/732 2 1 0.667 (0.314) 0.0009 (0.0011) Santa Marta 7 8/732 2 1 0.476 (0.171) 0.0052 (0.0034) Southern Caribbean Curaçao 2 0/732 1 0 0 0 Tropical Southern Atlantic 15 2/732 3 1 0.457 (0.141) 0.0007 (0.0007) Northeastern Brazil 9 1/732 2 1 0.389 (0.164) 0.0005 (0.0006) Ceará 5 1/732 2 1 0.6 ( 0.175) 0.0008 (0.0009) RN 4 0/732 1 0 0 0 FN and Atoll das Rocas 5 1/732 2 0 0.6 (0.175) 0.0008 (0.0009) FN 4 1/732 2 0 0.5 (0.265) 0.0007 (0.0008) Rocas Atoll 1 0/731 1 0 - - Eastern Brazil Abrolhos 1 0/732 1 0 - - Clade 1= STG1+STG2 144 15/733 17 - 0.527 (0.0497) 0.0014 (0.0011) Clade 2= STG3 9 2/733 3 - 0.556 (0.165) 0.0008 (0.0008) Clade 3 9 4/732 4 - 0.694 (0.147) 0.0022 (0.0016) STG1 139 12/733 15 - 0.492 (0.051) 0.0012 (0.0009) STG2 5 1/733 2 - 0.4 (0.237) 0.0005 (0.0007) STG4 6 1/732 2 - 0.333 (0.215) 0.0004 (0.0006) STG5 3 1/732 2 - 0.667 (0.314) 0.0009 (0.0011) All 162 25/733 24 - 0.624 (0.044) 0.0035 (0.0021) 187

Phylogenetic and phylogeographic inferences The ML phylogenetic tree obtained from the STs of L. floridana observed within each ecoregion and the other leucettas is shown in Figure 3A. In both ML and Bayesian analysis, the clade of L. floridana was subdivided into three clusters with high values of ML bootstrap (b) and Bayesian posterior probability (pp): clade 1 (b=91 and pp=0.99, in light green), clade 2 (b=94 and pp=0.99, in green) and clade 3 (b=97 and pp=0.96, dark green).

Figure 3. A. Maximum-Likelihood phylogenetic tree inferred from the ITS sequences of Leucetta floridana and other Leucetta species indicating the sequence-type (STs) found within each ecoregion. Bootstrap and posterior probability values (ML/BI) are indicated on the branches. B. Parsimony Median-Joining tree obtained from the ITS sequences of Leucetta floridana. The size of the circles is proportional to the frequency of the STs. Colours represent different localities. Numbers between STs indicate mutational steps and when not indicated, it refers to one-mutational step. 188

Clade 1 included representatives from all studied ecoregions in the TSA (Greater Antilles, Eastern Caribbean, Southwestern Caribbean and Southern Caribbean, in black) and in the TNA (Northeastern Brazil, Fernando de Noronha and Atoll das Rocas and Eastern Brazil, in red) and from depths varying from -4 to -70 m. This clade clustered 144 individuals grouped in 17 STs. Clades 2 and 3 comprised exclusively Caribbean specimens. Clade 2 included nine individuals of the Southwestern Caribbean representing three STs whereas Clade 3 grouped nine specimens from four STs. The parsimony MJ network yielded additional information to the phylogenetic ML and IB trees, unraveling five ST-groups (Figure 3B) supported by the AMOVA analyses (Tables S2 and S3, Supplementary Material). Clade 1 was divided into STG1 and STG2, Clade 2 corresponded to STG3 and Clade 3 was separated in STG4 and STG5. (Reference to Supplementary Material: STG1 = STGA+ STGB + STGC + STGD, STG2 = STGE, STG3 = STGF, STG4 = STGG and STG5=STGH). STG1 and STG2 are separated by a three-mutational-step distance. STG1 has a star-like shape in which rare STs are one-mutational-step close to the common central ST1, indicating a population expansion pattern. STG4 and STG5 are situated in the opposite side of the network and are separated by two-mutational steps. SGT3 has apparent central position, however, it is composed of few individuals (n=9). Determined STGs have certain geographic correspondence: STG1 is distributed along the TSA and TNA. STG2, STG3, and STG4 are restricted to the Southwestern Caribbean whereas STG5 occurs only in the Eastern Caribbean. Population Structure AMOVA In the analyses of molecular variance (AMOVA), two hypothetical scenarios of population structure yielded the maximum variation among groups (ɸCT=0.549). The first hypothetical scenario (H13) comprised eight groups: (1) Puerto Rico + Anguilla + Saint Martin + Saba + Antigua + Les Saintes + Guadeloupe, (2) Martinique, (3) San Andrés, (4) Panama, (5) Uraba, (6) Santa Marta, (7) Curaçao, and (8) Brazil. The second scenario (H19) split the Brazilian group into (1) Coastal Brazil (Rio Grande do Norte + Ceará + Abrolhos) and (2) Insular Brazil (Rocas Atoll + Fernando de Noronha). Interestingly, in both hypothesis, the only highly structured locality within the Eastern Caribbean was Martinique, whereas all localities in the Southwestern Caribbean represented separated populations. Additionally, Curaçao is well differentiated from the others despite its low sample size. All tested groups and the F-statistics obtained are presented in Table S4.

FST Comparisons 189

Significant FST indexes ranged from moderate (0.14

Caribbean (except for Panama) with the other Caribbean (0.47≤F ST≤1) and Brazilian localities

(0.35≤FST≤0.96). Despite the small number of sequences analysed from Curaçao, high F ST values were observed between this island and other Caribbean localities (Les Saintes and Puerto Rico).

Within the Lesser Antilles, high pairwise FST values (0.47≤

Southwestern Caribbean (FST=0.86) and Southern Caribbean x Northeastern Brazil (FST=0.73) whereas the lowest value was found in Fernando de Noronha and Atoll das Rocas x

Southwestern Caribbean (FST=0.19) (Table 5). When sequences were grouped according to the clades recovered in the phylogenetic analyses, all pairwise comparisons were significant (Table 6). FST values ranged from 0.82 (Clade 2 x Clade 3) to 0.88 (Clade 1 x Clade 3). When the same set of sequences was grouped considering the ST groups, the FST values obtained were even higher (0.85≤FST≤ 0.96) in almost all cases except in STG1 x STG2 (FST=0.779). Demography Structure Sequence-type (ST) and nucleotidic (π) diversity According to Grant and Bowen (1998), historical processes can be inferred for species or populations based on haplotipic and nucleotidic diversity indexes. Herein, we suggest some demographic events in L. floridana based on the observed ST (sequence-type) and π (nucleotidic) diversity indexes. The overall values of STd>0.5 and π <0.005 suggest the occurrence of a recent bottle-neck event within the populations of L. floridana. In the TNA, this must have been followed by a rapid population growth. Within the Eastern Caribbean, the values observed in Martinique (STd=0.61±0.1 and π=0.002 ±0.001) indicate a rapid growth within this island whereas Antigua, Saint Martin and Les Saintes may represent founder populations (STd<0.5 and π <0.005). The high values observed in the Southwestern Caribbean (STd>0.5 and π >0.005) suggest the presence of a large stable population or a secondary contact among different populations in this area or at least in Panama and San Andrés. Differently, Santa Marta, given its diversity indexes (H<0.5 and π >0.005) may have suffered an ancient bottleneck or may represent divergent populations geographically subdivided. In the TNA, the low values of STd and π of the Brazilian population may indicate that they have also experienced a recent bottleneck or represent founder populations. Table 4. Pairwise FST values among sampled localities or islands. Caribbean localities are PR: Puerto Rico, AG: Anguilla, SN: Saint Martin, SAB: Saba, AN: Antigua, GU: Guadeloupe, LS: Les Saintes, MT: Martinique, PAN: Panama, SAN: San Andrés, URA: Uraba, SM: Santa Marta and CUR: Curaçao. Brazilian localities: CE: Ceará, RN: Rio Grande do Norte, RAT: Rocas Atoll and ABR: Abrolhos Archipelago. Significant values are in bold.

PR AG SN SAB AN GU LS MT PAN SAN URA SM CUR CE RN FN RAT ABR AG -1 SN -0.02 -0.96 SAB -0.19 0 -0.18 AN 0 -1 -0.02 -0.19 GU -1 0 -0.96 0 -1 LS 0.01 -1 -0.04 -0.17 -0.03 -1 MT 0.61 0.38 0.52 0.51 0.47 0.38 0.57 PAN 0.25 -0.36 0.31 0.09 0.14 -0.36 0.21 0.48 SAN 0.57 -0.38 0.53 0.23 0.29 -0.37 0.45 0.60 0.16 URA 0.97 0.91 0.85 0.95 0.75 0.91 0.95 0.88 0.39 0.53 SM 0.73 0.333 0.71 0.54 0.51 0.33 0.67 0.75 0.34 0.29 0.52 CUR 0.86 1 0.37 1.0 0.20 1 0.81 0.22 0.21 0.18 0.95 1.0 CE 0.31 -0.5 0.05 0.12 0.001 -0.5 0.23 0.53 0.18 0.36 0.92 0.6 0.67 RN -0.13 0 -0.13 0 -0.13 0 -0.11 0.56 0.13 0.31 0.96 0.58 1 0.19 FN 0.11 -1 -0.06 -0.09 -0.09 -1 0.05 0.53 0.14 0.2 0.92 0.57 0.71 0.15 0 RAT 0.85 1 0.28 1 -0.04 1 0.78 0.59 -0.02 -0.83 0.91 0.43 1 0.57 1 0.33 ABR -1 0 -0.96 0 -1 .0 -1 0.38 -0.36 -0.37 0.91 0.33 1 -0.5 0 -1 1 0 191

Table 5. Pairwise FST values among ecoregions and provinces. Ecoregions within the TNA (Tropical Northwestern Atlantic): GA= Greater Antilles, EC=Eastern Caribbean, SWC=Southwestern Caribbean and SC=Southern Caribbean. Ecoregions within the TSA (Tropical Southwestern Atlantic): NB=Northeastern Brazil, FA=Fernando de Noronha and Atoll das Rocas and EB=Eastern Brazil. Significant values (p<0.05) are in bold.

GA EC SWC SC NB FA EB TSA EC 0.025 SWC 0.863 0.367 SC 0.263 0.138 0.196 NB 0.106 0.036 0.243 0.732 FA 0.310 0.057 0.192 0.674 0.231 EB -1.0 -0.797 -0.258 1.0 -0.75 -0.5 TNA ------0.035

Table 6. Pairwise FST values among Clades and ST-Groups (STG). All values were significant.

Clade 1 Clade2 Clade 3 STG1 STG2 STG4 = STG1+STG2 = STG3 = ST4+STG5 Clade 2 = STG3 0.871 Clade 3 0.883 0.824 STG1 - 0.892 0.901 STG2 - 0.928 0.87 0.779 STG4 0.884 0.908 - 0.904 0.956 STG5 0.907 0.922 - 0.923 0.955 0.847

Neutrality Tests The values obtained in the neutrality tests considering localities, ecoregions, clades and ST- groups are shown in Table 7. In the overall sample, both tests indicated a deviation from neutrality (Tajima's D=-1.21 and Fu's Fs=-9.56), however, Tajima's D test was not significant (p=0.09). Significant Tajima's D and Fu´s Fs values were obtained for the Eastern Caribbean ecoregion (D=-1.78, p=0.012 and Fs=-4.26, p= 0.047), Clade 1 (D=-1.73, p=0.013 and Fs=- 11.96, p=0.0) and STG1 (D=-1.97, p=0.002 and Fs=-13.32, p=0) indicating population expansion within these groups (Aris-Brosou & Excoffier, 1996). Congruent significant tests were not observed among the other groups tested. Tajima's D values were significant for Saint Martin (D=-2.04, p=0.004) and Antigua (D=-2.1, p=0.002) whereas the Fu´s Fs test was significant for the TNA (Fs=-8.068, p=0.012).

Mismatch Distribution Most geographical partitions analysed herein evidenced a multimodal mismatch distribution, except for Martinique which had an approximate unimodal distribution (Figures 4A-F). Among 192 the genetic groups tested, unimodal-like mismatch distributions were observed for Clade1 and STG1 (Figures 4G-H). These unimodal distributions (Poisson-like) are the signature of demographic or spatial expansions (Rogers and Harpending 1992; Bunje and Wirth, 2008). In none of the cases, the SSD and the Harpending's raggedness index were significant (Figure 4, Table S5) and consequently, it was not possible to reject the sudden expansion model. As expected due to the low variability found in Puerto Rico, Northeast Brazil and Fernando de Noronha and Rocas Atoll, Clade 2, Clade 3, STG3, STG4 and STG5, no clear distribution pattern was observed for them.

Table 7. Demography statistics of Leucetta floridana. TNA=Tropical Northern Atlantic, TSA=Tropical Southern Atlantic, RN=Rio Grande do Norte, p=p value.

Province/Ecoregion Locality Tajima´s D (p) Fu´s FS (p) TNA Caribbean Sea -1.0404 (0.1416) -8.0681 (0.0119) Greater Antilles Puerto Rico -1.1405 (0.1683) -0.4757 (0.1334) Eastern Caribbean Lesser Antilles -1.7835 (0.0124) -4.2641 (0.0472) Saint Martin -2.0454 (0.0037) -2.4497 (0.09) Antigua -2.1039 (0.0023) 1.11937 (0.7496) Les Saintes -1.0548 (0.2096) -0.1820 (0.2007) Les Saintes + Guadeloupe Martinique -0.0204 (0.4933) -0.4473 (0.3819) Southwestern Caribbean 1.6576 (0.9596) 0.7867 (0.6623) San Andrés 0.0834 (0.6645) -0.3992 (0.2231) Panama 1.9623 (0.9809) 4.3061 (0.9612) Urabá 0.0 (1.0) 0.2007 (0.3965) Santa Marta 0.8755 (0.8333) 5.1781 (0.9822) TSA Brazil -0.3988 (0.2884) -0.4474 (0.2619) Northeastern Brazil Ceará + RN 0.15647 (0.7512) 0.47744 (0.3977) Ceará 1.2247 (0.9435) 0.6261 (0.5025) Fernando de Noronha FN Archipelago + Rocas 0.0 (1.0) 0.6261 (0.4986) (FN) and Atoll das Rocas Atoll FN Archipelago 0.0 (1.0) 0.1718 (0.3388) Clade 1= STG1+STG2 -1.7347 (0.0133) -11.9645 (0.0) Clade 2= STG3 0.15647 (0.7615) -0.5321 (0.139) Clade 3 0.9867 (0.8481) -0.0611 (0.45050) STG1 (1 A) -1.6636 (0.0177) -11.0736 (0.0001) STG2 (1 B) 0.0 (1.0) 0.0902 (0.2959) STG4 (3 A) 0.0 (1.0) -0.0027 (0.2612) STG5 (3 B) 0.0 (0.9873) 0.2007 (0.3867) All -1.2085 (0.099) -9.5358 (0.0056) 193

Bayesian Skyline Plot Bayesian Skyline Plots (BSP) evidenced a population expansion pattern when considered (1) the complete set of L. floridana sequences (Figure 5A), (2) STG1+STG2 = Clade 2 (Figure 5B), (3) STG1 (Figure 5C) and (4) AMOVA-G1: Puerto Rico + Anguilla + Saint Martin + Saba + Antigua + Les Saintes + Guadeloupe (Figure 5D). In the other groups tested, the BSP did not detect any demographic event probably due to the small number of individuals per group (n<10).

DISCUSSION

Genetic Structure Although the AMOVA results indicated some genetic structure when geographical partitions were considered, the highest FST values were observed among clades (inferred by ML and BI methods) and ST-Groups (obtained in the MJ network). Therefore, we recognize the occurrence of five structured populations of L. floridana within the Tropical Atlantic Ocean: four populations restricted to the Caribbean Sea and a widespread population in the Western Tropical Atlantic. Caribbean restricted populations Cowen et al. (2006) defined four broad regions of connectivity within the Caribbean: (1) the eastern Caribbean (Puerto Rico to Aruba); (2) the western Caribbean (Cuba to Nicaragua); (3) the Bahamas and the Turks and Caicos Islands; and (4) the peripheral area of the Colombia- Panama Gyre. They also considered smaller areas of isolation within each region. In this study, the four Caribbean-restricted populations of L. floridana matched two of the referred connectivity regions and evidenced isolation patterns within them. In the peripheral area of the Colombian-Panama Gyre, which roughly coincides with the Southwestern ecoregion (Spalding et al., 2007), we found three structured populations: SGT3 in the area of direct influence of the Colombian-Panama Gyre (Panama and Urabá), STG4 in distant localities (San Andrés and Santa Marta) and STG2 in closer localities (Panama and San Andrés)

Isolation of STG3 from the Caribbean Sea (FST= 0.89 – 0.93) and connectivity among the individuals within this population can be explained by the cyclonic circulation of the Colombian-Panama Countercurrent (counter-clockwise to the Central Caribbean Current) which may act as a larval retention current. 194

Figure 4. Mismatch distribution of Leucetta floridana for (A) Antigua, (B) Saint Martin, (C) Panama, (D) Martinique, (E) Southwestern Caribbean, (F) Eastern Caribbean, (G) Clade 1 and (H) STG1. Bars show the observed frequency distribution for the number of pairwise differences among all individuals sampled. The solid lines show the expected distribution under each population expansion model. SSD: Sum of Squared Deviations, r: Harpending's raggedness index. 195

Figure 5. Bayesian Skyline Plots for (A) all ITS sequences, (B) Clade1= ST-Group1 + ST- Group2 of Leucetta floridana, (C) ST-Group1 and (D) AMOVA Group 1: Puerto Rico + Anguilla + Saint Martin + Saba + Antigua + Les Saintes + Guadeloupe of Leucetta floridana. The maximum time is the upper 95% HPD of the root height. The median estimate (black solid line) and 95% HPD limits (blue line) are indicated. 196

As the coast of Santa Marta is directly influenced by the Central Caribbean Current (Diaz et al., 2001), this can explain why individuals from this locality are isolated from Urabá and Panama, presenting a private ST (ST6). However, they are somewhat connected with some individuals from San Andrés (FST<0.25 but not significant), as observed in STG4. This can be attributed to the presence of the Colombian-Panama Countercurrent in Santa Marta during raining periods (Diaz et al., 2001). This latitudinal connectivity between San Andrés and Santa Marta (12.6°N and 11.2°N, respectively) has already been reported in other marine taxa e.g. the reef fish Stegastes partitus (Ospina-Guerrero et al., 2008). Different to STG4, STG2 reunites individuals from longitudinally close (82.1 and 81.7°W) but latitudinally distant (9.3 and 12.6°N) localities (Panama and San Andrés) that may be warranting the gene flow through the Colombian-Panama Countercurrent. This scenario has also been suggested for another Caribbean sponge (Cliona delitrix, Chaves-Fonnegra et al., 2015) Besides the barriers cited above, the whole genetic structure observed within this large area (peripheral area of the Colombia-Panama Gyre) suggests the presence of a strong barrier between San Andrés and Urabá as no STG including both localities was found (FST>0.5 but not significant). An alternative explanation to this could relay on past geological events that occurred within this area isolating populations. The last structured population is located in the Eastern Caribbean connectivity region of Cowen et al., (2006). STG5 included individuals from two islands located in the northern part of the Lesser Antilles arc (Saint Martin and Antigua) and no individuals from the southern islands (Martinique nor Guadeloupe). Whether this corresponds to a split of the Eastern Antilles population in northern and southern populations remains uncertain based on our results, however, this should receive more attention in further studies compelling the Lesser Antilles. A Western Tropical Atlantic widespread population The occurrence of a large population of L. floridana with intense genetic exchange between Caribbean and Brazilian localities (STG1) does not only reject the role of the Amazon river as an effective barrier to gene flow in the Western Tropical Atlantic but also suggests the presence of biological and/or physical mechanisms that would facilitate the observed connectivity pattern. In a broad sense, the population connectivity observed in L. floridana may be mediated by (1) the North Brazilian Current enabling the flow of migrants from the Brazilian coast to the Caribbean Sea and (2) the sponge assemblages under the mouth of the Amazon river acting as a connectivity corridor (as proposed by Rocha et al., 2003 for fish species). 197

Within this context, it is suggested that the larva of L. floridana can pass underneath the Amazon freshwater outflow (-30 m; Curtin, 1986). Although knowledge on the ultrastructure and behaviour of calciblastula larva is scarce, we know that this larva is hollow, oval and surrounded by ciliated cells (Amano & Hori, 2001; Ereskovsky & Willenz, 2008), which may be responsible for its motility. In some Demospongiae, the cilia are used for larval rotation along the longitudinal axis, apparently allowing the larvae to re-adjust its depth while drifting (Maldonado et al., 2003). These ciliated cells may play the same role in L. floridana, facilitating its sinking under the Amazon mouth. Alternatively, the larva of L. floridana can undergo a passive mechanism, being dragged to the bottom by masses of water with more salinity (Maldonado, 2006). Similar patterns of high genetic connectivity along the Western Tropical Atlantic had been attributed to efficient larval dispersal in other marine taxa (e.g. Rocha et al., 2002; Rodriguez et al., 2013). In Porifera, this is paradoxal since sponge larvae are lecithotrophic (Ereskovsky, 2010) and remain in the water column for minutes to a few days (usually <2 weeks - Maldonado, 2006; Padua et al., 2013), which most probably restrict their dispersal and colonization capabilities. Some sponge larvae exhibit complex mechanisms that contribute to expand their pelagic lifespan and consequently increase their dispersal capability such as (1) the incorporation of dissolved compounds by pinocytotic activity in Tedania ignis (Jaeckle, 1995), (2) the capacity to phagocyte and digest bacteria and small (<4 μm) unicellular organisms by the ciliated cells of Halichondria parenchymella (Ivanova, 1999) or (3) just being unpalatable to predators (fish), observed in some Caribbean sponges (Lindquist & Hay, 1996). Whether this occurs in L. floridana or in any other calcareous sponge is unknown but should not be discarded. Another mechanism that improve the dispersal capacity of some sponge larvae is the fragmentation of reproductive sponges. A fragment containing larvae can travel for a long time before the larvae are released and this may contribute to maximise the dispersal capacity (Maldonado & Uriz, 1999).

Demographic history Our results evidenced a population expansion pattern for L. floridana, specifically within the widespread population STG1. Diversity indexes, neutrality tests and mismatch distributions indicated that this occurred in the Caribbean Sea. Complementary information provided by the bayesian skyline plot situated the onset of this event ca. 20.000 years BP. This estimated date 198 coincides with the final period of the Pleistocene: the Last Glaciation Maximum (LGM, 30.000 – 19.000 years BP, Lambeck et al., 2001). During the Pleistocene (2.6 million –11.700 years BP), the glacial-interglacial cycles changed the sea level, superficial sea temperatures, current patterns, upwelling intensity and coastal habitats (Rohling et al., 1998; Lambeck et al., 2002). Global sea levels were 115–130 m lower than today (Fairbank, 1989; Lambeck et al., 2002) and tropical sea-surface temperature oscillated between 1 °C and 3 °C (Herbert et al., 2010). During the LGM, the area of coastal habitats was reduced by approximately 92% in the Gulf of Mexico and Caribbean Sea (Bellwood & Wainwright, 2002). Under those conditions, some species became extinct and several others remained as fragmented populations that nowadays are highly genetically structured (Ludt & Rocha, 2015). It is feasible that the shallow overhangs and vertical slopes inhabited by L. floridana during the Pleistocene ended up being exposed due to the low sea levels and consequently, some populations were decimated. We suggest that L. floridana suffered a bottle-neck event during the late Pleistocene and that its populations remained isolated for a long period of time, which is reflected in the high genetic population structure observed in this study. However, after the LGM, one of the isolated population (STG1) expanded within the Caribbean. In the Western Tropical Atlantic, demographic events have been reported as a result of the fluctuating conditions during the Pleistocene. In the Tropical Northwestern Atlantic, population bottlenecks have been evidenced in invertebrates including gastropods (Johnston et al., 2012), decapods shrimps (Cook et al., 2010) and blenny fish (Eytan & Hellberg, 2010) whereas population expansion was observed in penaeid shrimps (McMillen-Jackson & Bert, 2003), spiny lobster (Naro-Maciel et al., 2011) and West Indian manatee (Vianna et al. 2006). Although most of these events have been recorded in the TNA, population expansion has also been reported in the Tropical Southwestern Atlantic (e.g. Rodriguez-Rey et al., 2013). Within sponges, genetic structure due to interglacial-glacial cycles have been observed in the demosponges Hymeniacidon flavia and H. sinapium (Hoshino et al., 2008) from the Northwest Pacific and the Indo-Pacific calcarean Leucetta chagosensis (Wörheide et al., 2002; 2008) as well as population expansion of Pericharax heteroraphis (Bentlage & Worheide, 2007) within the Great Barrier Reef in Australia.

Evolutionary history of L. floridana Based on the phylogenetic, genetic structure and the demographic results obtained in this study and additional (palaeo)geological information, we propose the following evolutionary scenario 199 for L. floridana. Ancestral populations of this species were broadly distributed in the Western Tropical Atlantic. Once the outflow of the Amazon River started to be deposited in the Atlantic in the late Miocene (10.4 million years BP - Hoorn, 1996), populations were isolated and L. floridana must have been restricted to the Tropical Northwestern Atlantic. During the Pleistocene, important changes in marine taxa occurred (Lundt & Rocha, 2015). In L. floridana, we suggest both genetic isolation and connection due to glacial-interglacial cycles. The glacial periods isolated some populations, as evidenced by the high genetic structure observed within the Caribbean-restricted ST-groups (STG2, STG3, STG4 and STG5). According to the demographic bayesian analyses, these ST-groups share a more ancient history when compared to STG1. During the most recent interglacial periods, the distribution of one population of L. floridana (STG1) was expanded to the northeastern Brazilian coast and maintained connectivity (evidenced by the common ST1) through the sponge corridor, as suggested for other trans- Amazonian species (Rocha, 2003). This southward dispersal from the Caribbean to Brazilian localities has been also observed in reef fish (Lima et al., 2005; Rocha et al., 2008) and although it conflicts with today's current patterns, it must respond to past oceanographic conditions (Iturralde-Vinent & MacPhee, 1999). According to Bowen et al., 2001, populations can disperse and potentially coalesce after periods of isolation. Therefore, even if trans- Amazonian individuals of L. floridana underwent restricted genetic flow for some period of time as a result of the fluctuating conditions of the Pleistocene, they were able to restore connectivity later. This must have been facilitated by the large population size of this widespread species. By the end of the last glaciation maximum, the widespread population (STG1) suffered a bottleneck and a posterior sudden expansion event in the Lesser Antilles (most probably, in Martinique). Whether the Lesser Antilles acted as a Pleistocean refugia is uncertain as not palaeogeological information is available, however, the presence of many private ST may support this (Maggs et al., 2008).

Acknowledgements The authors thank Adeline Pouget-Cuvelier, Amandine Vaslet, Julien Chalifour and Philippe (Filipo) Thélamon for providing relevant information on sampling localities in the Lesser Antilles and also Alexander Ereskovsky, Bob Thacker?, César Ruiz, Cristina Diaz, Eduardo Hajdu, Fernanda Azevedo, Jean Vacelet, Laurent Van Bostal, Pedro Leocorny, Pierre Chevaldonné and Sandrine Chenesseau for collecting L. floridana in the Caribbean Sea. Ghennie Rodriguez is acknowledged for providing invaluable support for the demography 200 analyses. This work was partly supported by the Associated International Laboratory ‘MARRIO’. B.C.L. received a scholarship from the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES). J.E.G.H. received grants from the American Museum of National History Lerner-Gray Fund for Marine Research (sponge collection) and the Sea- Grant Puerto Rico (DNA sequencing). M.K. is funded by fellowships and research grants from the Brazilian National Research Council (CNPq), CAPES, and the Rio de Janeiro State Research Foundation (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro - FAPERJ). T.P. is funded by grants from The French National Center for Scientific Research (CNRS).

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Table S1. Voucher number, locality, geographic coordinates and Genbank accession number of the specimens of Leucetta floridana analysed in this study. Abbreviations: Brazilian States: BA=Bahia, CE=Ceará, RN= Rio Grande do Norte, PE=Pernambuco. SM-MNR=Saint Martin Marine Natural Reserve.

Voucher Number Sample Locality Geographic Coordinates Depth (m) Genbank Number Tropical Northwestern Atlantic Greater Antilles ecoregion: Puerto Rico (n=12) PR52 Cayo Mario 17°57'16.89''N, 67°03'53.42''W 9.0 This study PR81 Cayo Mario 17°57'16.89''N, 67°03'53.42''W 10.8 This study PR97 Veril – Fallen Rock (Guanica) 17°54'05.04''N, 66°55'27.59''W 31.5 This study PR99 Cayo Conserva 17°55'56.91''N, 67°05'35.01''W 12.6 This study PR105 Cayo Conserva 17°55'56.91''N, 67°05'35.01''W 15.6 This study PR112 Pinnacles 17°56'03.96''N, 67°01'49.80''W 9.9 This study PR117 Veril - El Hoyo 17°52.0529'N, 67°02.671'W 31.5 This study PR120 Pinnacles 17°56'03.96''N, 67°1'49.80''W 17.1 This study PR136 Turromote 17°56'20.13''N, 67°2'43.95''W 17.4 This study PR141 Turromote 17°56'20.13''N, 67°2'43.95''W 12.9 This study PR147 Cayo Mario 17°57'16.89''N, 67°3'53.42''W 13.2 This study PR153 Cayo Mario 17°57'16.89''N, 67°3'53.42''W 17.7 This study Eastern Caribbean ecoregion: Lesser Antilles (n=100) Anguilla (n=1) This study UFRJPOR 8278 Little Scrub2 18°17.903'N, 62°57.294'W 23.5 This study Saint Martin (n=52) UFRJPOR 7789 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W <20? This study UFRJPOR 7968 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W < 22 This study UFRJPOR 7970 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W < 22 This study UFRJPOR 7971 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W < 22 This study UFRJPOR 7972 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W < 22 This study UFRJPOR 7974 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W < 22 This study UFRJPOR 7985 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This study UFRJPOR 7986 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This study UFRJPOR 7990 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This study UFRJPOR 7992 Les Arches 2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This study UFRJPOR 7993 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This study UFRJPOR 8000 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This study UFRJPOR 8001 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This study UFRJPOR 8022 Trou David, Les Terres Basses 18°04.402'N, 63°07.149'W 6.1 This study UFRJPOR 8025 Trou David, Les Terres Basses 18°04.402'N, 63°07.149'W 6.1 This study UFRJPOR 8028 Trou David, Les Terres Basses 18°04.402'N, 63°07.149'W 6.1 This study UFRJPOR 8029 Trou David, Les Terres Basses 18°04.402'N, 63°07.149'W 6.1 This study UFRJPOR 8030 Rocher Créole, SM-MNR 18°07.038'N, 63°03.419'W 9.8 This study UFRJPOR 8031 Rocher Créole, SM-MNR 18°07.038'N, 63°03.419'W 9.8 This study UFRJPOR 8098 Rocher Créole, SM-MNR 18°07.038'N, 63°03.419'W 8 - 10 This study UFRJPOR 8142 Rocher Créole, SM-MNR 18°07.038'N, 63°03.419'W 8 - 10 This study UFRJPOR 8150 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This study UFRJPOR 8151 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This study UFRJPOR 8152 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This study UFRJPOR 8153 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This study UFRJPOR 8155 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This study UFRJPOR 8157 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This study UFRJPOR 8158 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This study UFRJPOR 8181 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This study UFRJPOR 8182 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This study UFRJPOR 8192 Basses Espagnoles 18°07.821'N, 63°00.270'W 10 This study UFRJPOR 8193 Basses Espagnoles 18°07.821'N, 63°00.270'W 10 This study UFRJPOR 8194 Basses Espagnoles 18°07.821'N, 63°00.270'W 10 This study UFRJPOR 8195 Basses Espagnoles 18°07.821'N, 63°00.270'W 10 This study UFRJPOR 8196 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This study UFRJPOR 8197 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This study UFRJPOR 8198 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This study UFRJPOR 8199 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This study UFRJPOR 8216 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This study UFRJPOR 8217 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This study UFRJPOR 8218 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This study UFRJPOR 8219 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This study UFRJPOR 8221 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This study UFRJPOR 8222 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This study UFRJPOR 8223 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This study UFRJPOR 8224 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This study UFRJPOR 8231 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 20 This study UFRJPOR 8232 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 13.8 This study UFRJPOR 8234 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 13 - 20 This study UFRJPOR 8235 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 13 - 20 This study UFRJPOR 8237 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 13 - 20 This study UFRJPOR 8238 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 13 - 20 This study Saba (n=3) UFRJPOR 8040 SA1, Southeastern Coast 17°37.066'N, 63°13.580'W < 27 This study UFRJPOR 8041 SA1, Southeastern Coast 17°37.066'N, 63°13.580'W < 27 This study UFRJPOR 8042 SA1, Southeastern Coast 17°37.066'N, 63°13.580'W < 27 This study Antigua and Barbuda (n=12) UFRJPOR 7936 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This study UFRJPOR 7938 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This study UFRJPOR 7939 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This study UFRJPOR 7940 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This study UFRJPOR 7942 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This study UFRJPOR 7943 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This study UFRJPOR 7949 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This study UFRJPOR 7950 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This study UFRJPOR 7952 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This study UFRJPOR 8093 Five Islands 17°04.990'N, 61° 54.840'W 4 This study UFRJPOR 8185 Diamond_Bank, Saint Paul 17°12.000'N, 61°52.800'W 7 - 8 This study UFRJPOR 8186 Diamond Bank, Saint Paul 17°12.000'N, 61°52.800'W 7 - 8 This study Guadeloupe (n=9) UFRJPOR 7655 Cave1, Les Saintes 15°52.984'N, 61°34.25'W < 11 This study UFRJPOR 7659 Cave1, Les Saintes 15°52.984'N, 61°34.25'W < 11 This study UFRJPOR 7648 Cave1, Les Saintes 15°52.984'N, 61°34.25'W < 11 This study UFRJPOR 7827 Cave1, Les Saintes 15°52.984'N, 61°34.25'W 8 - 10 This study UFRJPOR 8087 Cave1, Les Saintes 15°52.984'N, 61°34.25'W 8 - 10 This study UFRJPOR 8558 Cave1, Les Saintes 15°52.984'N, 61°34.25'W 8 - 10 This study UFRJPOR 8091 Cave1, Les Saintes 15°52.984'N, 61°34.25'W 8 - 10 This study UFRJPOR 8092 Cave1, Les Saintes 15°52.984'N, 61°34.25'W 8 - 10 This study UFRJPOR 8340 Cave Cathédrale, ? 16°27.740'N, 61°31.837'W 13.7 This study Martinique (n=23) UFRJPOR 7403 Grottes des couleurs, Pointe Burgos, Grande 14°29.787'N, 61°05.351'W <10 m This study Anse, Les Anses d’Arlet UFRJPOR 7404 Pointe Burgos, Grande Anse, Anses d'Arlet <10 m This study UFRJPOR 7405 Grottes des couleurs, Pointe Burgos, Grande 14°29.787'N, 61°05.351'W <10 m This study Anse, Les Anses d’Arlet UFRJPOR 7406 Grottes des couleurs, Pointe Burgos, Grande 14°29.787'N, 61°05.351'W <10 m This study Anse, Les Anses d’Arlet UFRJPOR 7407 Grottes des couleurs, Pointe Burgos, Grande 14°29.787'N, 61°05.351'W <10 m This study Anse, Les Anses d’Arlet UFRJPOR 7408 Grottes des couleurs, Pointe Burgos, Grande 14°29.787'N, 61°05.351'W <10 m This study Anse, Les Anses d’Arlet UFRJPOR 7409 Grottes des couleurs, Pointe Burgos, Grande 14°29.787'N, 61°05.351'W <10 m This study Anse, Les Anses d’Arlet UFRJPOR 7410 Grottes des couleurs, Pointe Burgos, Grande 14°29.787'N, 61°05.351'W <10 m KX355573 Anse, Les Anses d’Arlet UFRJPOR 7411 Grottes des couleurs, Pointe Burgos, Grande 14°29.787'N, 61°05.351'W <10 m KX355574 Anse, Les Anses d’Arlet UFRJPOR 7412 Les Anses d’Arlet <10 m KX355575 UFRJPOR 7423 Grottes des couleurs, Pointe Burgos, Grande 14°29.787'N, 61°05.351'W <10 m This study Anse, Les Anses d’Arlet UFRPOR 7424 Grottes des couleurs, Pointe Burgos, Grande 14°29.787'N, 61°05.351'W <10 m This study Anse, Les Anses d’Arlet SP2 Les Anses d’Arlet <10 m This study UFRJPOR 7777 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This study UFRJPOR 7778 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This study UFRJPOR 7845 Pointe Burgos, Grande Anse, Les Anses 14°29.787'N, 61°05.351'W? This study d’Arlet UFRJPOR 7859 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 3? This study UFRJPOR 7861 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 3 This study UFRJPOR 7862 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 5 This study UFRJPOR 7863 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This study UFRJPOR 7865 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This study UFRJPOR 7871 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This study UFRJPOR 7877 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This study Southwestern Caribbean ecoregion (n=33) Panamá (n=19) UFRJPOR 6389 Bocas del Toro 09º20.914'N, 82º9.394'W? <20? This study PC BT 12 Bocas del Toro 09º20.914'N, 82º9.394'W? <20? EU781989 PC BT 22 Bocas del Toro 09º20.914'N, 82º9.394'W? <20? EU781990 PC BT 23 Bocas del Toro 09º20.914'N, 82º9.394'W <20? EU781991 UFRJPOR 6948 Las Cuevas, Bocas del Toro 09º20.914'N, 82º9.394'W <20? This study UFRJPOR 6951 Las Cuevas, Bocas del Toro 09º20.914'N, 82º9.394'W <20? This study UFRJPOR 6953 Las Cuevas, Bocas del Toro 09º20.914'N, 82º9.394' W <20? This study UFRJPOR 6954 Las Cuevas, Bocas del Toro 09º20.914'N, 82º09.394'W <20? This study UFRJPOR 6956 Las Cuevas, Bocas del Toro 09º20.914'N, 82º09.394'W <20? This study UFRJPOR 6959 Bocas del Toro <20? This study UFRJPOR 6960 Las Cuevas, Bocas del Toro 09º20.914'N, 82º09.394'W <20? This study UFRJPOR 6961 Las Cuevas, Bocas del Toro 09º20.914'N, 82º09.394'W <20? This study UFRJPOR 6975 Bocas del Toro <20? This study UFRJPOR 6976 Bocas del Toro 17 This study UFRJPOR 6977 Bocas del Toro <20? This study =MNRJ 15748B UFRJPOR 6978 Bocas del Toro <20? This study =MNRJ 15754B UFRJPOR 6988 Bocas del Toro <20? This study UFRJPOR 6989 Cayo Zapatilla, Bocas del Toro 09º14.881'N, 82º01.952'W <20? This study UFRJPOR 6989A Cayo Zapatilla, Bocas del Toro 09º14.881'N, 82º01.952'W <20? This study Colombia (n=14) SM01 El Morro, Santa Marta Bay, Santa Marta 11°14'57.50"N, 74°13'54.50"W 21 This study SM02 El Morro, Santa Marta Bay, Santa Marta 11°14'57.50"N, 74°13'54.50"W 19 This study SM03 El Morro, Santa Marta Bay, Santa Marta 11°14'57.50"N, 74°13'54.50"W 20 This study SM04 El Morro, Santa Marta Bay, Santa Marta 11°14'57.50"N, 74°13'54.50"W 11 This study SM06 Punta Venado, Ensenada de Taganga, Santa 11°16'25.00"N, 74°12'24.00"W 18 This study Marta SM07 Punta Venado, Ensenada de Taganga, Santa 11°16'25.00"N, 74°12'24.00"W 18.6 This study Marta SM12 Punta Gaira, Gaira Bay, Santa Marta 11°13'08.00"N, 74°14'30.00"W 14 This study UFRJPOR 5357 Bajo Agua Viva, Urabá 07°53''N, 76°38'W? 15 EU781970 UFRJPOR 5359 Bajo Agua Viva, Urabá 07°53''N, 76°38'W? 15 EU781969 UFRJPOR 5360 “Bajo Agua Viva”, Urabá 07°53''N, 76°38'W? 15 EU781968 UFRJPOR 5363 “West View”, Leward-reef, San Andrés 12°33'N, 81°43'W? 5 EU781971 UFRJPOR 5364 La Piscinita, San Andrés 12°33'N, 81°43'W? 2 - 5 EU781972 UFRJPOR 5366 La Piscinita, San Andrés 12°33'N, 81°43'W? 2 - 5 EU781973 UFRJPOR 5367 La Piscinita, San Andrés 12°33'N, 81°43'W? 2 - 5 EU781974 Southern Caribbean ecoregion: Curaçao (n=2) UFRJPOR 6726 Water Factory, Willemstadt 12°06'30.88"N, 68°57'13.53"W 17.8 Curaçao paper UFRJPOR 6765 Hook’s Hut, Willemstadt 12°07'18.94"N, 68°58'11.46"W 13.3 Curaçao paper Tropical Southwestern Atlantic Northeastern Brazil ecoregion: Brazil (n=9) BPOTPOR 200 Potiguar Basin, RN 04º37'31.7"S, 36º46'00.7"W 70 This study BPOTPOR 202 Potiguar Basin, RN 04º37'31.7"S, 36º46'00.7"W 70 EU781985 BPOTPOR 610 Risca das Bicudas, RGN 04º57'00.9"S, 36º07'49.7"W 8-10? EU781978 BPOTPOR 634 Urca do Tubarao, RGN 04º50'52.7”S, 36º27'02.1"W 8 This study MNRJ 8440 Station 30 - CENPES, CE 02º52'S, 39º10'W 33 EU781983 MNRJ 8445 Station 30 - CENPES, CE 02º52'S, 39º10'W 33 EU781984 MNRJ 8465 Station 30 - CENPES, CE 02º52'S, 39º10'W 33 EU781982 MNRJ 8481 Station 30 - CENPES, CE 02º52'S, 39º10'W 33 This study MNRJ 8488 Station 30 - CENPES, CE 02º52'S, 39º10'W 33 EU781980 Fernando de Noronha and Atoll das Rocas ecoregion: Brazil (n=5) MNRJ 7725 Barretinha, Rocas Atoll, RGN 03°51'36''S, 33°49'04''W 12 EU781975 MNRJ 8609 Sela Gineta Island, Fernando de Noronha 03°48'49''S, 32°23'29''W 7 EU781976 Archipelago, PE MNRJ 8602 Ressurreta, Fernando de Noronha 03°48'49''S, 32°23'29''W 4 EU781977 Archipelago, PE UFRJPOR 6480 Ressurreta, Fernando de Noronha 03°48'49''S, 32°23'29''W 7.3 This study Archipelago, PE UFRJPOR 6481 Ressurreta, Fernando de Noronha 03°48'49''S, 32°23'29''W 7.3 This study Archipelago, PE Eastern Brazil ecoregion (n=1): NE Brazil UFRJPOR_4703 Parcel das Paredes, Abrolhos Archipelago, 17°58'S, 38°40'W 8 EU781979 BA 211

Table S2. ST-Groups and its corresponding STs as suggested by the network topology of Leucetta floridana. (Number of sequences within each STG and ST)

STGA STGD (110) STGB (1) STGC (4) (24) STGE (5) STGF (9) STGG (6) STGH (3) ST1(98) ST21 (1) ST2 (3) ST12 (3) ST7 (1) ST4 (6) ST6 (5) ST17 (1) ST3 (2) ST8 (1) ST14 (14) ST10 (4) ST5 (1) ST9 (1) ST18 (2) ST13 (2) ST15 (4) ST11 (2) ST19 (4) ST16 (3) ST20 (1) ST22 (1) ST23 (1) ST24 (1) Table S3. AMOVA. Hypotheses of ST-groups (STG-Tests) for Leucetta floridana.with the obtained F.statistics: ɸ CT=variation among hypothetical STGs, ɸ SC= variation within STs among hypothetical groups and ɸ ST= variation within STs. All values were significant (10 000 permutations). The chosen hypothese is indicated in bold. (Reference to principal text: STG1=STGA+STGB+STGC+STGD, STG2=STGE, STG3=STGF, STG4=STGG and STG5=STGH).

STGS within Clade 1 Clade2 Clade3 ɸCT ɸSC ɸST STG-Test1 STGA STGB STGC STGD STGE STGF STGG STGH 0.679 0.943 0.982 STG-Test2 STGA STGB STGC STGD STGE STGF STGG STGH 0.759 0.931 0.983 STG-Test3 STGA STGB STGC STGD STGE STGF STGG STGH 0.767 0.929 0.983 STG-Test4 STGA STGB STGC STGD STGE STGF STGG STGH 0.757 0.922 0.981 STG-Test5 STGA STGB STGC STGD STGE STGF STGG STGH 0.768 0.919 0.981 STG-Test6 STGA STGB STGC STGD STGE STGF STGG STGH 0.667 0.939 0.980 STG-Test7 STGA STGB STGC STGD STGE STGF STGG STGH 0.689 0.910 0.972 STG-Test8 STGA STGB STGC STGD STGE STGF STGG STGH 0.752 0.882 0.971 STG-Test9 STGA STGB STGC STGD STGE STGF STGG STGH 0.729 0.922 0.979 STG-Test10 STGA STGB STGC STGD STGE STGF STGG STGH 0.724 0.894 0.971 STG-Test11 STGA STGB STGC STGD STGE STGF STGG STGH 0.749 0.883 0.970 STG-Test12 STGA STGB STGC STGD STGE STGF STGG STGH 0.692 0.903 0.970 STG-Test13 STGA STGB STGC STGD STGE STGF STGG STGH 0.704 0.926 0.978 STG-Test14 STGA STGB STGC STGD STGE STGF STGG STGH 0.719 0.895 0.970 STG-Test15 STGA STGB STGC STGD STGE STGF STGG STGH 0.757 0.886 0.972 Table S4. AMOVA. Hypotheses of population structure (H) and the obtained F-statistics: ɸCT=variation among hypothetical groups, ɸSC= variation within localities among hypothetical groups and ɸST= variation within localities. Significant values are indicated in bold (10 000 permutations). Ecoregions: SC= Southern Caribbean, NE Brazil=Northeastern Brazil, FN and RA=Fernando de Noronha and Atoll das Rocas and EB=Eastern Brazil. Localities: PR=Puerto Rico, AG=Anguilla, SN=Saint Martin, SB=Saba, AN=Antigua, SNT=Les Saintes, GU=Guadeloupe, MT=Martinique, SAN=San Andrés, URA=Uraba, SM=Santa Marta, CUR=Curaçao, RN=Rio Grande do Norte, CE=Ceará, AT= Rocas Atoll, FN=Fernando de Noronha Archipelago, ABR=Abrolhos Archipelago.

Tropical Northern Atlantic Tropical Southern Atlantic F- statistics

Greater Eastern Caribbean Southwestern SC NE Brazil FN and EB ɸCT ɸSC ɸST Antilles Caribbean RA H1 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR -0.054 0.433 0.402 H2 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR -0.061 0.433 0.398 H3 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.251 0.318 0.489 H4 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.170 0.347 0.458 H5 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.319 0.271 0.503 H6 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.317 0.271 0.502 H7 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.270 0.305 0.492 H8 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.262 0.308 0.490 H9 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.377 0.223 0.516 H10 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.399 0.201 0.520 H11 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.388 0.209 0.516 H12 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.378 0.218 0.514 H13 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.5488 -0.1148 0.4970 H14 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.421 0.081 0.468 H15 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.436 0.061 0.471 H16 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.532 -0.121 0.475 H17 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.538 -0.148 0.470 H18 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.532 -0.170 0.452 H19 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.5494 -0.1192 0.496 H20 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.541 -0.157 0.469 H21 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.429 0.096 0.483 H22 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.423 0.102 0.482 H23 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.376 0.130 0.457 H24 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.341 0.204 0.476 H25 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.362 0.177 0.476 H26 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.354 0.184 0.474 214

Table S5. Mismatch distribution values. SSD=sum of squared deviation, r=Harpending's raggedness index.

SSD SSD P value r r p value Groups Antigua 0.0280 0.18 0.1717 0.5357 Les Saintes 0.2791 0.071 0.3125 0.4099 Les Saintes + Guadaloupe 0.3067 0.0889 0.3580 0.2832

Saint Martin 0.0059 0.4314 0.2760 0.5558 Panama 0.1336 0.0666 0.2870 0.0246 San Andrés 0.1243 0.3188 0.2778 0.7281 Santa Marta 0.4535 0 0.7279 0.9399 Uraba 0.0898 0.6033 0.5555 0.8964 Puerto Rico 0.0229 0.2384 0.4722 0.4358 Fernando de Noronha 0.02194 0.6456 0.25 0.9243 Ceará 0.0542 0.5037 0.4 0.3706 Eastern Caribbean 0.4050 0.0003 0.1161 0.9996 Southwestern Caribbean 0.0515 0.0201 0.0829 0.054 Northeast Brazil 0.0062 0.452 0.2006 0.4564 FN and Rocas Atoll 0.0542 0.5042 0.4 0.3863 Tropical Southwestern Atlantic 0.0116 0.3149 0.1619 0.4790 Tropical Northwestern Atlantic 0.4707 0.0001 0.0677 1.0 Clade 1 0.3638 0.0 0.0931 1.0 Clade 2 = STG3 0.0275 0.2272 0.2037 0.3274 Clade 3 0.0330 0.487 0.1088 0.6829 STG1 0.3337 0 0.1130 0.9996 STG2 0.0072 0.7642 0.2 0.9411 STG4 0.003 0.745 0.2222 0.9268 STG5 0.0898 0.5988 0.5556 0.8925 All 0.4539 0.0001 0.0629 1.0 Chapter 4

GENERAL DISCUSSION 216

This study contributed to the knowledge of the biodiversity of calcareous sponges from the

Western Tropical Atlantic (WTA). Nineteen species, including 15 new species and four new records, were added to the 67 previously known species from this area (Van Soest et al., 2016;

Van Soest, 2017; Azevedo et al., submitted), summing up a total of 86 calcareous sponges in the

WTA (Table 1).

Compared to regions with coastline extensions similar to the study area (ca. 29 000 km) such as Australia and Japan, whose coastlines cover 25,670 and 29,751 km, respectively (CIA, 2013), the richness of Calcarea is, unexpectedly, higher in the WTA. Sixty-seven species have been reported from Australia (Leocorny et al., 2016; Van Soest et al., 2016) and 79 from Japan (Van

Soest et al., 2016). This is very surprising as the calcareous sponges from those Indo-Pacific regions have been thoroughly studied (e.g. Japan: Hôzawa, 1916, 1918, 1929, 1933, 1940;

Hôzawa & Tanita, 1941; Tanita, 1942, 1943 and Australia: Lendenfeld, 1885; Carter, 1886;

Dendy, 1892, 1893; Dendy & Row, 1913; Dendy & Frederick, 1924; Row & Hôzawa, 1931;

Wörheide & Hooper, 1999, 2003).

Considering the biogeographical provinces within the study area, the major contribution was to the TNA (Tropical Northwestern Atlantic), as the previous number of reported species from this province was 23 and it was raised to 45. Twelve new species (Amphoriscus micropilosus sp. nov., Arthuria vansoesti sp. nov., Ascandra torquata sp. nov., Clathrina aspera sp. nov., C. curaçaoensis sp. nov., Ernstia sp. nov. , Leucandra caribea sp. nov., Leucandrilla pseudosagittata sp. nov., Leucilla antillana sp. nov., Grantessa tumida sp. nov., Sycon conulosum sp. nov., Sycon magniapicalis sp. nov.) and nine new records (Arthuria hirsuta,

Borojevia tenuispinata, Clathrina aurea, C. blanca, C. cylindractina, C. insularis, C. lutea, C. mutabilis, and Nicola tetela) were reported herein.

The new 21 records from the TNA have certainly changed the distribution of the species richness within the ecoregions of this province. Before this study, the Greater Antilles presented 217 the highest number (8) of species (Haeckel, 1870, 1872; Lehnert & Van Soest, 1998) and it was followed by Bermuda, with six species (Poléjaeff, 1883; de Laubenfels, 1950). Now, the richest ecoregion is the Southern Caribbean, with 23 species (Arndt, 1927; Haeckel, 1872; this study), followed by the Eastern Caribbean, with 13 species (Duchassaing & Michelotti, 1864;

Schuffner, 1877; this study) and the Southwestern Caribbean, with nine species (Rozemeijer &

Dulfer, 1987; Valderrama et al., 2009; Klautau et al., 2013; this study). Additionally, all these new records are from localities that once had very few or no valid records of Calcarea.

Previously, three species were recorded from Colombia (Rozemeijer & Dulfer, 1987;

Valderrama et al., 2009), two from Panama (Valderrama et al., 2009; Klautau et al., 2013), one from Curaçao (Arndt, 1927) and none from Martinique. Nowadays, those numbers have increased and there are five, seven, eight and 20 calcareous sponges from Colombia, Panama,

Curaçao and Martinique, respectively.

In the Tropical Southwestern Atlantic (TSA), the richness of calcareous sponges has increased from 48 (Muricy et al., 2011; Cavalcanti et al., 2014, 2015; Azevedo et al., submitted) to 54 species. Although known species were found again in the Abrolhos Archipelago (e.g. C. lutea, C. aurea, L. roseus), three new species (Amphoriscus hirsutus sp. nov., Leucascus luteoatlanticus sp. nov. and Grantia grandisapicalis sp. nov.) and two new records (C. luteoculcitella and Ernstia vansoesti) were described from there. This evidenced that sampling in unexplored sites within “known” localities can still reveal novelties in species richness and distribution. Furthermore, the six records raised the number of valid species from the Eastern

Brazil ecoregion from 25 to 30, and now it is the richest ecoregion of the TSA (Borojevic, 1971;

Borojevic & Peixinho, 1976; Cavalcanti et al., 2014, 2015; Azevedo et al., submitted; this study). Nonetheless, in some areas or localities within this ecoregion, such as the littoral of

Espírito Santo State, no Calcarea has ever been recorded (Muricy et al., 2011). 218

In the North Brazilian Shelf (NBS), only 11 species have been reported so far (Borojevic &

Peixinho, 1976; Cavalcanti et al., 2013; Van Soest, 2017). This apparent poor diversity of

Calcarea may be an artefact of the lack of investigation on sponge diversity in this province.

Future samplings in this region may provide important insights for understanding former disjunct species distribution in the Caribbean Sea and NE Brazilian coast.

Comparative information on Porifera2 richness within the WTA is available across some ecoregions. Considering the Southern Caribbean (SC) and the Eastern Brazil (EB),

Demospongiae is accounted as the richest class with 201 (88.2%, Van Soest et al., 2012; 2014;

Alvizu et al., 2013) and 121 (79.6%, Van Soest et al., 2016) species, respectively. Within

Calcarea, 23 species were recorded from the SC (10.1%) and 30 from the EB (19.7%, this study). Homoscleromorpha has four species from the SC (1.7%) and one single record from the

EB (0.7%, Domingos et al., 2016). Interestingly, in these “well-sampled” ecoregions, the observed percentages are close to the ones obtained for global diversity of Porifera:

Demospongiae, 83%; Calcarea, 8% and Homoscleromorpha: 1% (Van Soest et al., 2012).

Despite the limited knowledge on calcareous sponges within certain localities in the WTA, it is possible to elucidate some general distribution patterns. Likewise Demospongiae (Van Soest

& Hajdu, 1997) and Homoscleromorpha species (Domingos et al., 2016), most of the calcareous sponges (65.1%=56 species) presented herein are endemic from the WTA. Among the non-WTA endemic species, 13 species (15.3%) are amphi-Atlantic3, eight (9.4%) are restricted to the

2 Hexactinellida and certain Demospongiae recorded at more than 200 m (published in Van Soest et al., 2014; Hestetum et al., 2016 for the SC) were not considered as the depth boundary for MEOW is 200 m deep (Spalding et al., 2007). 3 Five from the West African Transition (Tropical Atlantic realm; Borojevic & Peixinho, 1872; Thacker, 1908), three from the Temperate Southern Africa realm (Klautau & Valentine, Urban 1908; Brøndsted, 1931) and five from the Temperate Northern Atlantic realm (Topsent, 1892, Miklucho-Maclay, 1868; Borojevic & Boury-Esnault, 1987, Sarà & Gaino, 1971; Klautau et al., 2016). 219

Western Atlantic4, four are Atlanto-Pacific5 and the other four are cosmopolitan6. Although the amphi-Atlantic distribution of certain calcareous sponges proved to be the result of overconservative systematics (Solé-Cava et al., 1991; Klautau et al., 1994); the presence of

Arthuria hirsuta, originally described from South Africa (Klautau & Valentine, 2003), was confirmed in the Caribbean (La Martinique, Pérez et al., submitted, Appendix: Table 1). In this study, the occurrence of the Australian Clathrina luteoculcitella in NE Brazil was corroborated by molecular methods and whether or not this correspond to a natural distribution should be investigated. Unpublished results from the Pacotilles campaign revealed the presence of this species also in Bequia (Eastern Caribbean), which would give some cues for the understanding these unexpected distribution patterns. The records from the WTA of the cosmopolitan species should be revised, except for those of Vosmaeropsis sericata as its type locality is included in the WTA (Cavalcanti et al., 2015).

A higher number of Calcaroneans was recorded from the WTA (46 spp.) compared to

Calcineans (39 spp.); however, the proportion of trans-Amazonian species is higher in Calcinea

(33.3%=13 spp.7) than in Calcaronea (15.2%=7 spp.8). It is possible that there is some kind of bias in this result, but the possibility that the calciblastula larva (from Calcinea) can disperse better than the amphiblastula (from Calcaronea) underneath the Amazon plume should not be discarded. However, this hypothesis requires further investigation.

4 Seven that can also be found in SE or S Brazil (Warm Temperate Southwestern Atlantic; Muricy et al., 2011; Azevedo et al., submitted) and one also recorded in the Northern Gulf of Mexico (WTNA; Haeckel, 1872). 5 Three with records in the Indo-Pacific (Wörheide & Hooper, 1999; Van Soestet al., 2015; Poléjaeff, 1883; Kelly et al., 2009) and one in the SE Pacific (Azevedo et al., 2015). 6 As the type locality of Vosmaeropsis levis is unclear, its distribution was not considered herein. 7 Arthuria vansoesti, Ascandra torquata, Borojevia tenuispinata, Clathrina aspera, C. aurea, C. cylindractina, C. insularis, C. lutea, C. luteoculcitella (considering material from Pacotilles), C. mutabilis, Leucaltis clathria, Leucetta floridana and Nicola tetela. 8 Grantia kempfi, Leucandra barbata, L. rudifera, L. serrata, Leucilla uter, Sycettusa flamma, and Vosmaeropsis complanatispinifera. 220

A biogeographic province is a large geographic area characterised by the presence of endemic species and delimited by particular geomorphological, hydrogeographic and geochemical factors (Spalding et al., 2007). The >10% endemism criterion proposed by Briggs

(1974) has been widely employed for defining biogeographic provinces within the Tropical

Atlantic (e.g. Floeter & Soares-Gomez, 1999; Floeter & Gasparini, 2000; Briggs & Bowen,

2012). In this study, the percentage of endemic species in both the TNA and TSA surpassed the

10%, being 46.7% (21 out of 45 species) in the former and 40.7% (22 out of 54 species) in the latter. Therefore, it is possible to say that the existence of the TNA and TSA provinces is supported by the observed high endemism of calcareous sponges within these areas.

Nonetheless, there are several species that occur in more than one province. Among the 86 species from the WTA, 16 (18.6%) species are shared between the TNA, NBS and/or TSA.

Leucaltis clathria and Leucetta floridana have been recorded from the three provinces

(Borojevic & Peixinho, 1976; Haeckel, 1872; Klautau et al., 2013; Valderrama et al., 2009) and they are considered species with a wide distribution. Grantia kempfi, Sycettusa flamma and

Vosmaeropsis complanatispinifera are shared between the NBS and the TSA (Borojevic &

Peixinho, 1976; Cavalcanti et al., 2015; Van Soest, 2017). Eleven species, Arthuria vansoesti,

Borojevia tenuispinata, Clathrina aspera, C. aurea, C. insularis, C. lutea, C. mutabilis,

Leucandra barbata, L. rudifera, Leucilla uter and Nicola tetela were recorded from the TNA and TSA (Azevedo et al., submitted; Cóndor-Luján & Klautau, 2016, Muricy et al., 2011; this study) but not in the NBS.

The occurrence of 13 species in the TNA and TSA provinces suggests a Caribbean-Brazilian affinity within the class Calcarea. Although this affinity constitutes a novelty for Calcarea, it was already observed in Demospongiae some decades ago (Hechtel, 1976; Colette & Rützler,

1977) and indicated in more recent studies (Moraes, 2011; Muricy et al., 2011; Moura et al.,

2016; Soares et al., 2016). 221

Hechtel (1976) reported numerous Demospongiae from both the West Indies and Brazil and suggested that larvae of those species would cross the Orinoco and Amazon barriers, considering the assumption of hard substrate availability close to the river's discharge which would permit sponge settlement. One year later, Colette & Rützler (1977) not only reported

“Caribbean” sponges underneath the mouth of the Amazon River but also mentioned that there was no apparent effect of freshwater and that there was abundant hard substrata on the bottom of the Amazon mouth. Recently, Moura et al. (2016) reported the existence of a calcium carbonate system underneath the Amazon river plume, which would act as a connectivity corridor for wide depth–ranging reef-associated species, such as sponges.

The presence of calcareous species in both provinces also suggests a possible connectivity between the TNA and TSA populations, which could be explained by a stepping-stone model of population structure in the North Brazil Shelf Province (NBS) (in the Venezuelan Southeastern

Coast, Guyanas or in the Northern Brazilian Coast) facilitating the gene flow between these two provinces. Therefore, it is quite probable to find several of the 13 shared species in the NBS (up to date, only Leucetta floridana and Leucaltis clathria had been reported from the three provinces).

In this study, L. floridana, one of the most widespread species in the WTA, was used as a model to assess connectivity between TNA and TSA populations. The results evidenced five structured populations: one widespread population maintaining gene flow between the TNA and

TSA provinces and four other isolated populations within the Caribbean Sea. The finding of a panmitic widespread population in the WTA was really unexpected as it rejects the hypothesis that the discharge of the Amazon River is an effective barrier to the maintenance of gene flow among trans-Amazonian populations of calcareous sponges. Interestingly, a similar genetic 222 pattern was observed in the Demospongiae Chondrilla aff. nucula collected in the Caribbean and in the Brazilian coast (Boury-Esnault et al., 20169).

Cryptic species are not unusual in Calcarea because of the plasticity of some characters in certain genera and species. Studies assessing the genetic identity of former Brazilian populations of Clathrina clathrus, C. primordialis and Borojevia cerebrum revealed that they were cryptic species and a posterior discovery of diagnostic morphological characters justified the erection of new species: C. aurea, C. cylindractina, C. conifera, and B. brasiliensis (Klautau et al., 1994; Solé-Cava et al., 1991). In L. floridana, the observed genetic variability (0-1.8%) is within the expected intraspecific range estimated for other leucettas (Valderrama et al., 2009) and other Calcinean species (Rossi et al., 2011). Moreover, the high morphological variability of

L. floridana did not match the structured populations found in this study. Individuals with different morphotypes (based on the shape of the oscula) and/or colour-morphs (light blue, pink and white) were found in the same population or ST-group. Likewise, several individuals collected in different habitats (completely protected from light, such as underneath boulders or inside crevices, and semi-exposed to light, such as vertical slopes), depths (- 2 and - 40 m) or localities (adjacent or distant) grouped in the same population. Therefore, there is no evidence to support an ongoing cryptic speciation in L. floridana.

The high intraspecific variation within Leucetta floridana is comparable to that of other two species with similar distribution in the WTA: C. lutea with 0-1.6% and C. mutabilis with 0-2%

(values taken from Chapter 3.4). An exploratory ITS sequence-type network of these two

Clathrina species (Appendix: Figures 2 and 3) showed that each species presented an ITS sequence-type shared by Caribbean and Brazilian individuals (Brazil-Saint Martin and Brazil-

Curaçao-La Martinique-Panama, respectively). Furthermore, genetic analyses10 with C. lutea sequences evidenced a similar phylogeographic and population pattern as those of L. floridana.

9 Results recently presented by N. Boury-Esnault in the II Workshopt ABC/CNRS (LIA MARRIO). 10 Results I recently presented in the II Workshop ABC/CNRS (LIA MARRIO). 223

According to Avise (2000), phylogeographic breaks shared among taxa with varying life histories provide a rational basis for determining where the effective gene flow is restricted or totally absent and thus, facilitate the detection of physical features that may act as important barriers. Consequently, further studies should be addressed to verify the congruence of the phylogeographic and population patterns of C. lutea and L. floridana as they might reveal important traits for understanding connectivity and species evolutionary history in the WTA.

Leucetta floridana and L. potiguar are morphologically very similar species but present strong diagnostic morphological characters (Lanna et al., 2009). Different from L. floridana, L. potiguar is a NE Brazilian endemic species. In all known phylogenetic trees (Valderrama et al.,

2009; Klautau et al., 2013; Azevedo et al., submitted; this study), they appear as sister species.

Given their current geographic distribution and their phylogenetic position (Figure 2 of Chapter

3.4 of this study), it is possible to infer that the ancestral species of these pair of eucettas had a widespread distribution in the WTA (including the Caribbean Sea and the NE Brazilian coast).

In this study, the genetic divergence estimated by the uncorrected p distance between L. floridana and L. potiguar varied from 2.5 to 3.4%. If considered the mutation rate of 1% per

MY calculated for Demospongiae (Wörheide et al., 2004), it is possible to infer that the cladogenetic event occurred ~4 MY BP, during the Pliocene (~5.3 – 2.6 MY BP). Interestingly, the Amazon River became fully established at ~7 MY BP (Figuereido et al., 2009) and during the early Pliocene, it started to drain water and sediments on a large scale to the Atlantic Ocean

(Latrubesse et al., 2010). As the discharge of freshwater and sediments from the Amazon River has been recognised as responsible for the formation of pairs of sister species of reef fishes in the WTA (Rocha, 2003 and references therein), this can also be suggested for this pair of sisters species of Leucetta. Table 1. List of recorded species from the Western Tropical Atlantic. Abbreviations: TL=type locality. Brazilian States - AL: Alagoas, BA: Bahia, CE: Ceará, ES: Espírito Santo, MA: Maranhão, PB: Paraíba, PA: Pará, PE: Pernambuco, RN: Rio Grande do Norte, RJ: Rio de Janeiro, SC: Santa Catarina, SP: São Paulo. FN: Fernando de Noronha. Provinces in the Tropical Atlantic (TA) – NBS: North Brazil Shelf, TNA: Tropical Northwestern Atlantic, TSA: Tropical Southwestern Atlantic, WAT: West African Transition. Other provinces - WTNA: Warm Temperate Northwest Atlantic, WTSA: Warm Temperate Southwestern Atlantic, WTSEP: Warm Temperate Southeastern Pacific. Realms - CIP: Central Indo-Pacific, TAA: Temperate Australasia, TNA: Temperate Northern Atlantic, TNP: Temperate Northern Pacific, TSAfrica: Temperate Southern Africa, TSAmerica: Temperate South America. E: Eastern, N: Northern, NE: Northeastern, S: Southern, SE: Southeastern, SW: Southwestern, W:Western. USA: United States of America. ** Indicates new species and * new records obtained in this study.

Species Distribution Ecoregion, References Province, Realm Subclass Calcaronea 1 Amphoriscus ancora Van Soest, 2017 Guyana Shelf (TL) Guianan, NBS Van Soest, 2017 2 Amphoriscus hirsutus sp. nov.** Abrolhos Archipelago (BA), NE Brazil Eastern Brazil, TSA This study (TL) 3 Amphoriscus micropilosus sp. nov.** Curaçao (TL) S Caribbean, TNA This study 4 Amphoriscus synapta (Schmidt in Haeckel, BA, NE Brazil (TL) NE or E Brazil, TSA Haeckel, 1872 1872) 5 Amphoriscus testiparus (Haeckel, 1872) Cuba (TL) Greater Antilles, TNA Haeckel, 1872 6 Amphoriscus urna Haeckel, 1872 Venezuela (TL) S Caribbean, TNA Haeckel, 1872 7 Grantessa anisactina Borojevic & Peixinho, Paraíba, NE Brazil (TL) E Brazil, TSA Borojevic & Peixinho, 1976 1976 8 Grantessa tumida sp. nov. ** Curaçao (TL) S Caribbean, TNA This study 9 Grantia atlantica Ridley, 1881 Vitória Bank (ES), SE Brazil E Brazil, TSA Ridley, 1881 10 Grantia grandisapicalis sp. nov. ** Abrolhos Archipelago (BA), NE Brazil E Brazil, TSA This study (TL) 11 Grantia kempfi Borojevic & Peixinho, 1976 Guyana Shelf Guianan, NBS Van Soest, 2017 AP, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976 PE (TL), NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976 Off Abrolhos, BA, NE Brazil E Brazil, TSA Borojevic & Peixinho, 1976 12 Leucandra amorpha Poléjaeff, 1883 Bermuda (TL) Bermuda, TNA Poléjaeff, 1883 13 Leucandra armata (Urban, 1908) Off Marajó Island, PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976 CE, PE, AL, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976 Off Abrolhos, BA, NE Brazil and ES, E Brazil, TSA Borojevic & Peixinho, 1976 SE Brazil Brazil Francis Bay (TL), South Africa Natal, Agulhas, Brøndsted, 1931; Urban, 1908, TSAfrica (realm) 1909 14 Leucandra barbata (Duchassaing & Dry Tortugas Floridian, TNA de Laubenfels, 1936 Michelotti, 1864) Virgin Islands (TL) E Caribbean, TNA Duchassaing & Michelotti, 1864 Colombia SW Caribbean, TNA Rozemeijer & Dulfer, 1987 PE, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976 Off Abrolhos, BA, NE Brazil and ES, E Brazil, TSA Borojevic & Peixinho, 1976 SE Brazil 15 Leucandra caribea sp. nov.** Curaçao (TL) S Caribbean, TNA This study 16 Leucandra crassior Ridley, 1881 PE, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976 Vitória Bank (TL), ES, SE Brazil E Brazil, TSA Ridley, 1881 17 Leucandra crosslandi Thacker, 1908 PB, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976 Boa Vista Island, Cape Verde (TL) Cape Verde, WAT Thacker, 1908 18 Leucandra crustacea (Haeckel, 1872) Bermuda Bermuda, TNA De Laubenfels, 1950 Venezuela (TL) S Caribbean, TNA Haeckel, 1872 19 Leucandra curva (Schuffner, 1877) Barbados E Caribbean, TNA Schuffner, 1877 20 Leucandra hentschelii Brøndsted, 1931 NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976 Simonstown, South Africa (TL) Agulhas Bank, Agulhas, Brøndsted, 1931 TSAfrica (realm) 21 Leucandra multiformis Poléjaeff, 1883 Bermuda (TL) Bermuda, TNA Poléjaeff, 1883 22 Leucandra rudifera Poléjaeff, 1883 Bermuda (TL) Bermuda, TNA Poléjaeff, 1883 Trindade Island Trindade and Martin Moraes et al.., 2006 Vaz Islands, TSA Arraial do Cabo, RJ, SE Brazil SE Brazil, WTSA, Muricy et al., 1991, Muricy & TSAmerica Silva, 1999. Cape Verde Cape Verde, WAT Thacker, 1908 23 Leucandra serrata Azevedo & Klautau, 2007 RN, NE Brazil NE Brazil, TSA Lanna et al., 2009 RJ, SE Brazil (TL) SE Brazil, WTSA, Azevedo & Klautau, 2007 TSAmerica 24 Leucandra typica (Poléjaeff, 1883) Bemuda (TL) Bermuda, TNA Poléjaeff, 1883 25 Leucandrilla pseudosagittata sp. nov.** Curaçao (TL) S Caribbean, TNA This study 26 Leucilla amphora Haeckel, 1872 Puerto Rico (TL) Greater Antilles, TNA Haeckel, 1872 Curaçao S Caribbean Arndt, 1927 Senegal, West Africa Sahelian Upwelling, Borojevic & Boury-Esnault, WAT 1987 27 Leucilla antillana sp. nov.** Curaçao (TL) S Caribbean, TNA This study La Martinique* E Caribbean, TNA Pérez et al., submitted: Leucilla sp. nov. 28 Leucilla sacculata (Carter, 1890) FN (TL), NE Brazil FN and Atoll das Carter, 1890 Rocas, TSA 29 Leucilla uter Poléjaeff, 1883 Bermuda (TL) Bermuda, TNA Poléjaeff, 1883 Curaçao S Caribbean, TNA Arnd, 1927 CE, RN, PB, PE, AL, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976 Abrolhos Archipelago*, Off Abrolhos E Brazil, TSA Borojevic & Peixinho, 1976, and Off Belmonte, BA, NE Brazil and this study ES, SE Brazil Arraial do Cabo, RJ, SE Brazil SE Brazil, WTSA, Muricy et al., 1991; Muricy & TSAmerica Silva, 1999; Borojevic & Peixinho, 1976. Philippines E Philippines, Western Poléjaeff, 1883 Coral Triangle, CIP 30 Leucosolenia horrida (Schmidt in Haeckel, Florida. USA (TL) Floridian, TNA Haeckel, 1872 1872) 31 Leucosolenia salpinix Van Soest, 2017 Guyana Shelf (TL) Guianan, NBS Van Soest, 2017 32 Paraleucilla incomposita Cavalcanti et al., Arraial d'Ajuda (TL), BA, NE Brazil E Brazil, TSA Cavalcanti, et al., 2014 2014 33 Paraleucilla oca Cavalcanti et al., 2014 Itaparica Island (TL), BA, NE Brazil E Brazil, TSA Cavalcanti, et al., 2014 34 Paraleucilla solangeae Cavalcanti et al., Guarajuba, BA, NE Brazil (TL) NE Brazil, TSA Cavalcanti, et al., 2014 2014 35 Paraleucilla sphaerica Lanna et al., 2009 Potiguar Basin, NE Brazil (TL) NE Brazil, TSA Lanna et al., 2009 36 Sycettusa flamma (Poléjaeff, 1883) AP, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976 Off Bahia (TL), NE Brazil NE or E Brazil, TNA Poléjaeff, 1883 37 Sycon ampulla (Haeckel, 1872) Venezuela (TL?) S Caribbean, TNA Haeckel, 1972 RJ, SE Brazil and Florianópolis, SC, S SE Brazil, WTSA, Haeckel, 1972; Mello-Leitão Brazil (TL?) TSAmerica et al., 1961; Muricy & Silva, 1999. Azores, Portugal Azores Canaries Topsent, 1892 Madeira, Lusitanian, TNA (realm) 38 Sycon barbadense (Schuffner, 1877) Barbados (TL) E Caribbean, TNA Schuffner, 1877 39 Sycon brasiliense Borojevic, 1971 Cabo São Tomé, RJ, SE Brazil (TL) E Brazil, TSA Borojevic, 1971 40 Sycon conulosum sp. nov.** Curaçao (TL) S Caribbean, TNA This study 41 Sycon formosum (Haeckel, 1870) Cuba (TL) Greater Antilles, TNA Haeckel, 1870 42 Sycon frustulosum Borojevic & Peixinho, PE (TL), NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976 1976 43 Sycon magniapicalis sp. nov.** Curaçao (TL) S Caribbean, TNA This study 44 Sycon vigilans Sarà & Gaino, 1971 Punta Manara (TL), Italy Mediterranean Sea, Sarà & Gaino, 1971 TNA (realm) PE, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976 Urca, RJ (SE) and SC (S), Brazil SE Brazil, WTSA Borojevic &Peixinho, 1976; Mothes & Lerner, 1994. 45 Vosmaeropsis complanatispinifera Cavalcanti AP, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976 et al., 2015 RN (TL), NE Brazil E Brazil, TSA Borojevic & Peixinho, 1976; Cavalcanti et al., 2015 46 Vosmaeropsis levis Hozawa, 1940 Mexico (TL) Atlantic or Pacific TNA? Hôzawa, 1940 Atlantic? Cabo de São Tomé, RJ E Brazil, TSA Borojevic, 1971 47 Vosmaeropsis sericata Ridley, 1881 Vitória Bank, SE Brazil (TL) E Brazil, TSA Ridley 1881 Cosmpolitan Cape Verde Cape Verde, WAT Thacker, 1908 Chile WTSEP, TSAmerica Breitfuss, 1898 SUBCLASS CALCINEA 48 Arthuria hirsuta Klautau & Valentine, 2003* Stil Bay (TL), Cape Town, South Agulhas Bank, Agulhas, Klautau & Valentine, 2003 Africa TSAfrica (realm) La Martinique* E Caribbean, TNA Pérez et al., submitted

49 Arthuria trindadensis Azevedo et al., Trindade Island Trindade and Martin Azevedo et al., submitted submitted Vaz Islands, TSA 50 Arthuria vansoesti sp. Curaçao (TL) S Caribbean, TNA This study nov.** La Martinique* E Caribbean, TNA Pérez et al., submitted: Arthuria sp. nov. Abrolhos Archipelago, BA, NE E Brazil, TSA This study Brazil* 51 Ascaltis agassizii Haeckel 1872 Florida (TL), United States of America Floridian, TNA Haeckel, 1872 Gulf of Mexico TNA? or TA? (realms) Haeckel, 1872 52 Ascalits panis (Haeckel, 1870) Florida, United States of America Floridian, WTNA Haeckel, 1870 Carrier Bow Cay, Belize W Caribbean, TNA Rutzler et al., 2014 Cape Verde, West Africa Cape Verde, WAT Thacker, 1908 53 Ascaltis poterium (Haeckel, 1872) PE, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976 Cosmopolitan Australia (TL) CIP? or TAA? (realms) Haeckel, 1872 Arctic Artcic (realm) Breitfuss, 1932 Chile TSA (realm) Ridley, 1881; Breitfuss, 1898 54 Ascaltis reticulum (Schmidt, 1862) PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976 Cosmopolitan PE, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976 ES, SE Brazil E Brazil, TSA Borojevic & Peixinho, 1976 Banyuls-sur-Mer (TL) and Adriatic Mediterranean Sea, Schmidt, 1862; Klautau et al., Sea, Mediterranean Sea TNA (realm) 2016 (more references in it) Arctic Artcic (realm) Breitfuss, 1932 Japan TNP (realm) Hôzawa, 1940; Tanita, 1943 55 Ascandra ascandroides (Borojevic, 1971) PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976 Cabo São Tomé (TL), RJ, SE Brazil E Brazil, TSA Borojevic, 1971 Arraial do Cabo, RJ (SE) and SC (S) SE Brazil, WTSA Klautau & Borojevic, 2001; Mothes & Lerner, 1994 Bay of Biscay S European Atlantic Borojevic & Boury-Esnault, Shelf, Lusitanian 1987 56 Ascandra atlantica (Thacker, 1908) NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976 Vitoria, SE Brazil E Brazil, TSA Borojevic & Peixinho, 1976 Boa Vista (TL), Cape Verde Cape Verde, WAT Thacker, 1908 Klautau & Valentine 57 Ascandra torquata sp. nov.** Bocas del Toro, Panama* SW Caribbean, TNA Personal observation Santa Marta, Colombia* SW Caribbean, TNA Personal observation Curaçao S Caribbean, TNA This study Arvoredo Archipelago (TL), SC, S S Brazil, WTSA, This study Brazil TSAmerica 58 Borojevia brasiliensis (Solé-Cava et al., CE, NE Brazil* NE Brazil, TSA This study 1991) Cabo de São Tomé, RJ, SE Brazil E Brazil, TSA Borojevic, 1971 Arraial do Cabo, RJ, SE Brazil (TL) SE Brazil, WTSA, Solé-Cava et al., 1991 TSAmerica Muricy et al., 2011 (and references in it) 59 Borojevia tenuispinata Azevedo et al, Curaçao* S Caribbean, TNA This study* submitted São Pedro e São Paulo Archipelago São Pedro e São Paulo Azevedo et al, submitted (TL), NE Brazil Islands, TSA 60 Borojevia trispinata Azevedo et al, submitted São Pedro e São Paulo Archipelago São Pedro e São Paulo Azevedo et al, submitted (TL), NE Brazil Islands, TSA Arraial do Cabo, RJ, SE Brazil SE Brazil, WTSA Azevedo et al, submitted 61 Clathrina aspera sp. nov.** Curaçao (TL) S Caribbean, TNA This study RN, NE Brazil NE Brazil, TSA This study RJ, SE Brazil SE Brazil, WTSA This study* 62 Clathrina aurea (Solé-Cava et al., 1991) Bocas del Toro, Panama* SW Caribbean, TNA Personal observation Martinique* Eastern Caribbean, Pérez et al., submitted* TNA FN, NE Brazil FN Archipelago and Muricy et al., 2001 (more Atoll das Rocas, TSA references in it) Potiguar Basin, NE Brazil NE Brazil, TSA Lanna et al.., 2009 Abrolhos Archipelago*, BA, NE Brazil E Brazil, TSA Azevedo et al., in press; this study RJ (TL) and SP, SE Brazil SE Brazil, WTSA Solé-Cava et al., 1999; Muricy et al., 2011 (references in it) S Peru WTSEP, TSAmerica Azevedo et al., 2015 63 Clathrina blanca Miklucho-Maclay, 1868* Curaçao* S Caribbean, TNA This study Cosmopolitan SP, SE Brazil and SC, S Brazil SE Brazil, WTSA Borojevic, 1971 Canary Islands (TL) and Norway Lusitanian and N Miklucho-Maclay, 1868; Rapp, European Sea, TNA 2006 (references in it) (realm) Antarctic Southern Ocean (realm) Brøndsted, 1931 Japan TNP (realm) Hôzawa, 1929; Tanita, 1943 64 Clathrina conifera (Klautau & Borojevic, NE Brazil NE Brazil, TSA Borojevic & Pexinho, 1976; 2001) Klautau & Valentine, 2003 Arraial do Cabo, RJ (TL), SE brazil SE Brazil, WTSA, Borojevic & Pexinho, 1976; TSAmerica Klautau & Valentine, 2003 Adriatic Sea Mediterranean Sea, Klautau et al., 2006 TNA (realm) 65 Clathrina curaçaoensis sp. nov.** Curaçao (TL) S Caribbean, TNA This study 66 Bocas del Toro, Panama* SW Caribbean, TNA Personal observation Clathrina cylindractina (Klautau et al., Arraial do Cabo, RJ, SE Brazil (TP) SE Brazil, WTSA Klautau et al., 1994 1994)* 67 Clathrina hondurensis Klautau & Valentine, Belize (TP) W Caribbean, TNA Klautau & Valentine, 2003 2003 Carrie Bow Cay, Belize W Caribbean, TNA Rützler et al., 2014 Bocas del Toro, Panama* SW Caribbean, TNA Personal observation Curaçao* S Caribbean, TNA This study 68 Clathrina insularis Azevedo et al., submitted Curaçao* S Caribbean, TNA This study Martinique* E Caribbean, TNA Pérez et al., submitted: Clathrina sp. nov. 1 FN (TL), NE Brazil FN and Atoll das Azevedo et al., submitted Rocas, TSA 69 Clathrina luteoculcitella Wörheide & Abrolhos, NE Brazil* Eastern Brazil, TSA This study Hooper, 1999* Great Barrier Reef (TL), Australia NE Australian Shelf, Wörheide & Hooper, 1999 CIP 70 Clathrina lutea Azevedo et al., submitted Florida, USA Floridian, TNA Klautau et al., 2013; Zea et al., 2014 Little San Salvador, Bahamas Bahamian, TNA Zea et al., 2014 Jamaica* Greater Antilles, TNA Lehner & Van Soest, 1998 (C. primordialis ), this study Curaçao* S Caribbean, TNA This study Virgin Islands E Caribbean, TNA Klautau et al., 2013 Abrolhos Archipelago (TL) E Brazil, TSA Azevedo et al.., submitted; this study* 71 Clathrina mutabilis Azevedo et al., submitted Panamá and Colombia* SW, TNA Personal observation* Curaçao* S Caribbean, TNA This study* Martinique* E Caribbean, TNA Pérez et al., submitted: Clathrina sp. nov. 2* FN Archipelago, NE Brazil (TL) FN and Atoll das Azevedo et al., submitted Rocas, TSA RN, NE Brazil* NE Brazil, TSA This study 72 Ernstia citrea Azevedo et al., submitted Rocas Atoll, NE Brazil FN and Atoll das Azevedo et al., submitted Rocas, TSA Abrolhos Archipelago, NE Brazil* Eastern Brazil, TSA This study 73 Ernstia multispiculata Azevedo et al., Rocas Atoll, NE Brazil FN and Atoll das Azevedo et al., submitted submitted Rocas, TSA 74 Ernstia santipauli Azevedo et al., submitted São Pedro e São Paulo Archipelago São Pedro and São Azevedo et al., submitted (TL), NE Brazil Paulo Islands 75 Ernstia sp. nov. ** La Martinique (TL) E Caribbean, TNA Pérez et al., submitted; Fontana et al., in prep. 76 Ernstia rocasensis Azevedo et al., submitted Rocas Atoll (TL) and FN Archipelago, FN and Atoll das Azevedo et al., submitted; this NE Brazil Rocas, TSA study Abrolhos Archipelago, BA, NE Brazil* E Brazil, TSA Azevedo et al., submitted; this study 77 Ernstia sulphurea Miklucho-Maclay, 1868 FN Archipelago, NE Brazil FN and Atoll das Azevedo et al., submitted Rocas, TSA Trindade Island, SE Brazil Trindade and Martin Azevedo et al., submitted Vaz Islands, TSA Lanzarote Beach, Canary Islands (TL) Azores Canaries Miklucho-Maclay, 1868 Madeira, Lusitanian 78 Leucaltis clathria Haeckel, 1872 Florida, USA (TL) Floridian, TNA Haeckel, 1872 Bocas del Toro, Panama SW Caribbean, TNA Klautau et al., 2013; Zea et al., 2014; personal observation* Guyana Shelf Guianian, NBS Van Soest, 2017 PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976; CE, RG, AL, SE, NE BraziL NE Brazil, TSA Borojevic & Peixinho, 1976; Lanna et al., 2009 ES, SE Brazil E Brazil, TSA Borojevic & Peixinho, 1976. 79 Leucascus luteoatlanticus sp. nov.** Abrolhos Archipelago (TL), BA, NE E Brazil, TSA This study BraziL 80 Leucascus roseus Lanna et al., 2007 Potiguar Basin, RN, NE Brazil NE Brazil, TSA Lanna et al., 2009 Abrolhos Archipelago, BA, NE Brazil Eastern Brazil, TSA Cavalcanti et al., 2013; this study* RJ and SP (TL), SE Brazil Southeastern Brazil, Cavalcanti et al., 2013; Lanna WTSA et al., 2007; 81 Leucascus aff. simplex sensu Cavalcanti et PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976; al., 2013 Cavalcanti et al., 2013 82 Leucetta floridana (Haeckel, 1872) Bermuda Bermuda, TNA de Laubenfels, 1950 Bahamas Bahamian, TNA Zea et al., 2014 Florida (TL) Floridian, TNA Haeckel, 1872 Jamaica and Cayman Islands Greater Antilles, TNA Lehner & van Soest, 1998; Milaslovich et al., 2011 Panama and Colombia (Santa Marta*) SW Caribbean, TNA Klautau et al., 2013; this study (Santa Marta)*; Valderrama et al., 2009; Zea et al., 2014 Curaçao* S Caribbean, TNA This study Lesser Antilles* including St. Eustatius E Caribbean, TNA García-Hernández et al., 2016; Pérez et al., submitted; this study; MA and PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976 MA, CE, RN, PA, PE and AL, NE NE Brazil, TSA Borojevic & Peixinho, 1976; Brazil Lanna et al, 2009; Valderrama et al., 2009 FN Archipelago and Rocas Atoll FN and Atoll das Borojevic & Peixinho, 1976; Rocas, TSA Moraes et al., 2006; Valderrama et al., 2009 Abrolhos, BA, NE Brazil and ES, SE E Brazil, TSA Borojevic & Peixinho, 1976; Brazil. Valderrama et al., 2009 83 Leucetta imberbis (Duchassaing & Virgin Islands (TP) E Caribbean, TNA Duchassaing & Michelotti, Michelotti, 1864) 1864 Santa Marta, Colombia SW Caribbean, TNA Rozemeijer & Dulfer, 1987 Jamaica Greater Antilles, TNA Lehner & Van Soest, 1998 84 Leucetta potiguar Lanna et al., 2009 Potiguar Basin (TL), RN and CE, NE NE Brazil, TSA Lanna et al., 2009; Valderrama Brazil et al., 2009 85 Leucettusa corticata (Haeckel, 1872) Cuba Greater Antilles, TNA Haeckel, 1872 New Zealand S New Zealand?, TAA Kelly et al., 2009 86 Nicola tetela (Borojevic & Peixinho, 1976) Curaçao* S Caribbean, TNA This study Sint Eustatius E Caribbean, TNA García-Hernández et al., 2016 Abrolhos (TL), BA, NE Brazil E Brazil, TSA Borojevic & Peixinho, 1976 Chapter 5

CONCLUSIONS AND PERSPECTIVES 234

This study certainly contributed to our comprehension of the marine biodiversity of the Western

Tropical Atlantic, filling in the gap of knowledge on calcareous sponges from poorly studied areas such as the Caribbean Sea. The 86 species now reported from the WTA represents approximately

12% of all known Calcarea (ca. 730 species; Van Soest et al., 2012; Rapp, 2013; Cavalcanti et al.,

2013, 2014, 2015 ; Azevedo et al., 2015, submitted; Van Soest & Voogd, 2015; Klautau et al., 2016;

Leocorny et al., 2016; Van Soest, 2017; Azevedo et al., submitted). However, the richness of this region is still underestimated as new species are still being discovered from new localities in the

Caribbean Sea (Lesser Antilles, Pacotilles campaign, pers. obs.) and in the NE Brazil (Bahia, F.

Cavalcanti, pers. comm.). Besides, new species of the subclass Calcaronea from Colombia and

Panama (not included in this study) are being described.

As suggested for other marine taxa (Ludt & Rocha 2005), the current distribution of calcareous sponges may be related to the major changes occurred during the Pleistocene in the WTA. It would be interesting to compare phylogeographic patterns across different genera in Calcarea (e.g.

Clathrina and Leucetta) to see if they corroborate this hypothesis.

As mentioned before, the systematics of calcareous sponges is considered particularly difficult because of the plasticity of some of the characters (Van Soest et al., 2012). In this study, molecular approaches played an important role in providing useful information for the identification of species whose morphological characters were very plastic (e.g., diactines in Ascandra torquata) and for the allocation of certain species in its correct genus (e.g. Nicola tetela). In the latter case, I should point out the importance of revising the type material and not to limit to the examination of the original description.

The Amazon River outflow may constitute a semi-permeable barrier to calcareous sponges. It would allow the dispersal of some species such as Leucetta floridana, Clathrina aurea, C. insularis,

C. lutea, and C. mutabilis (whose occurrences in the TNA and TSA were corroborated by morphological and molecular methods) but also, restrict the dispersal of other species (the TNA and 235

TSA endemic species). At least, for one calcareous species, L. floridana, the discharge of freshwater and sediments from the rivers of the TWA (Orinoco and Amazonas) does not constitute an effective barrier to gene flow. Studies using other molecular markers are recommended to give more support to this finding.

In order to fully understand marine connectivity, it is also very important to perform studies on the reproduction of the target species (e.g. larval behaviour) as it provides quite useful information to explain the observed genetic patterns. Non-physical barriers such as chemical defenses and adult ecology should be considered as well. As most of the mentioned information is scarce or even inexistent in Calcarea, it is crucial to begin investigating these aspects if a more precise understanding of calcareous sponges connectivity in the Western Tropical Atlantic is desired. Chapter 6

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Calcareous sponge species recorded in Martinique . C= Cave (dark, semi-dark caves, tunnels, overhangs), M= Mangrove, R= Reef (hard bottoms in general, including coral reefs). Species marked by an * indicate new records for the Eastern Caribbean. Modified from Pérez et al., submitted.

Order Family Species External traits Genbank Habitat Accesion Numbers Clathrinida Clathrinidae Arthuria hirsuta (Klautau & White, massive to semi-spherical KX355564 C Valentine, 2003) * clathroid body. Hispid. Arthuria sp. nov. Yellow, clathrate with water- C collecting tube. Soft. Clathrina aurea Solé-Cava, Light yellow, clathrate with KX355565 - C Klautau, Boury-Esnault, several oscula. KX355567 Borojevic & Thorpe, 1991* Clathrina sp. nov. 1* Light yellow, clathrate with KX355568 - C water-collecting tube. Soft. KX355571 Clathrina sp. nov. 2* Light yellow, clathrate and thin KX355572 C encrusting. Soft. Clathrina sp. White clathrate. C Ernstia sp. nov. Lemon yellow clathrate. Soft. C Leucosolenida Amphoriscidae Leucilla sp. nov. Bright white, tubular with apical C osculum. Delicate. Leucilla sp. 1 C Leucilla sp. 2 C Amphoriscus sp. C Grantiidae Leucandra sp. C Leucettidae Leucetta floridana (Haeckel, White, massive with apical KX355573 - C, R 1872)* osculum. Rough. KX355575 Sycettidae Sycon sp. Beige, globular with apical M osculum surrounded by crown. APPENDIX 2

Median-Joinig network of Clathrina lutea. ST2 is shared between Caribbean (Saint Martin) and Brazilian specimens of C. lutea. Taken from presentation at II Workshop MARRIO. APPENDIX 3 Preliminary Median-Joining network of Clathrina mutabilis. ST1 is shared between Caribbean (Curaçao, Martinique and Panama) and Brazilian specimens of C. mutabilis.