Occurrence of the genus Pseudoboletia (Echinoidea: Toxopneustidae) in the South-Western Atlantic: morphological and molecular approaches Elinia M. Lopes1, Carlos Eduardo L. Ferreira2 & Carlos Renato R. Ventura1 1. Departamento de Invertebrados, Laboratório de Echinodermata, Museu Nacional/Universidade Federal do Rio de Janeiro, Quinta da Boa Vista s/no, Rio de Janeiro, Brazil; [email protected]/[email protected] 2. Departamento de Biologia Marinha, Universidade Federal Fluminense, Outeiro São João Batista s/nº, Niterói, Brazil; [email protected] Received 21-V-2017. Corrected 22-VI-2017. Accepted 10-VIII-2017. Abstract: The genus Pseudoboletia was recently reported off coast of Brazil (Trindade-Martin Vaz insular com- plex). This study reports the first record of this genus to southern of Brazil in Rio de Janeiro and Santa Catarina coasts. Morphological and molecular data showed that sea urchins from Brazil and São Tomé are the same species, genetically distinct of individuals from Indo-Pacific. However, taxonomic identity of Brazilian species remains as a challenge. Two hypotheses to explain the recent records of this species on Brazilian coast are dis- cussed: a recent natural invasion by long-distance dispersal and a recent population expansion in the Brazilian coast after absence or low density period. Rev. Biol. Trop. 65(Suppl. 1): S299-S308. Epub 2017 November 01. Key words: sea urchin; Brazilian coast; new record; population expansion; long distance dispersal. The sea urchin genus Pseudoboletia Tros- Catarina) extending its range in to subtropical chel, 1869 is distributed from shallow-water waters from southeastern to southern coast. zones to 100 meters depth through tropical and The taxonomic status and phylogenetic subtropical regions of the Atlantic and Indo- affinities among Pseudoboletia species still Pacific oceans (Mortensen, 1943; Pawson, have many gaps and uncertainties. Mortensen 1978; Turner & Graham, 2003). The species (1943) highlights the difficulty in sharply occur over sand or rock bottom and normally delimiting these species, mainly because few cover the test with algal and shells. Pseudobo- and poorly preserved individuals were used letia belongs to the family Toxopneustidae in the original description. The morphologi- Troschel, 1872, which includes eight living cal differences among species pointed out genera. In the Brazilian coast, two toxopneus- by Mortensen (1943) were basically: (1) the tid species are commonly found: Lytechinus number of the pore-pairs in pore-arcs and (2) variegatus (Lamarck, 1816) and Tripneustes the presence of dark spots (macula) on the test ventricosus (Lamarck, 1816). Recently, the and spines. Four species of the genus are rec- genus Pseudoboletia has been reported to ognized by Mortensen (1943) and are currently shallow-water of Trindade Archipelago (1 200 considered valid in Word Register of Marine km east of the coast, the most eastward point of Species database (WoRMS): Pseudoboletia Brazilian territory 20° 31’ 29” S - 29° 19’ 29” maculata Troschel, 1869 from Indo-Pacific and W) (Martins, Souto, Braga, & Tavares, 2016). North-Western Atlantic, Pseudoboletia indiana The present study indicates two new records (Michelin, 1862) from Indo-Pacific, Pseudobo- for Pseudoboletia (Rio de Janeiro and Santa letia atlantica Clark, 1912 from South Atlantic Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65(Suppl. 1): S299-S308, November 2017 S299 Ocean (Ascension and St. Helena Islands) and The present study reports two new Pseudoboletia occidentalis Clark, 1921 from records of the genus Pseudoboletia in the North Atlantic (Barbados and Antigua Island). South and Southeastern Brazilian coast Despite that, synonyms and subspecies were addressing morphological, molecular and described posteriorly. Pawson (1978) synony- biogeographic discussions. mized P. occidentalis and P. maculata consid- ering morphological characters. Additionally, MATERIAL AND METHODS Pawson (1978) described two subspecies of P. maculata differentiated by the number of pore- Collections: The sea urchins were collect- pair of podia: P. maculata maculata Troschel, ed using scuba diving in the Cabo Frio Island, 1869 from Indo-Pacific oceans and North- Arraial do Cabo, Rio de Janeiro (22°58’50’’S; Western Atlantic (with four pore-pair of podia) 41°59’99’’W), in 2016. The habitat consists of and Pseudoboletia maculata atlantica Clark, the interface of rocky boulders with sand flats 1912 from St. Helena and Ascension Islands at about 8-10 meters depth. Substratum cover- (with predominantly five pore-pair of podia). age includes sparse turf algae and abundant The species P. indiana and P. maculata were crustose coralline algae (Fig. 1). At this specific designated as synonyms by Liao and Clark site, a dozen of sea urchins could be found at (1995), making Pseudoboletia a monotypic the interface (rocky-sandy bottoms), however genus. According to Liao and Clark (1995) in this region the species is considered rare the test morphology of the P. indiana and P. compared to other four mostly abundant spe- maculata are identical except for differences cies (Cordeiro et al., 2014). In the southeastern in the color pattern (absence of dark spots in coast, one individual was photographed in Deserta Island, Florianópolis, Santa Catarina P. indiana). However, this classification was (27°16’6.28”S; 48°20’9.67”W), in 2012. The not followed by subsequent studies. Moreover, specimen was found at 8 meters depth over the species status of the P. indiana was con- rocky boulders covered by thick turf algae firmed by molecular analysis (Zigler, Byrne, and sharing space with Lytechinus variegatus. M., Raff, Lessios, & Raff, 2012). P. indiana Therefore, the species is also considered rare at and P. maculata maculata from Indo-Pacific Deserta Island (Batista, pers. com.). are distinct species with 7.3% of the genetic divergence (Cytochrome oxidase I sequences) (Zigler et al., 2012). According to Zigler et al. (2012), hybridization between them can occur in sympatric populations at Sydney coast (Southeastern Australia), where individuals with intermediate coloration are reported. The authors also included in the analyses Pseudobo- letia samples from São Tomé Island (South- Eastern Atlantic Ocean). Individuals from São Tomé were distinct species, different from P. indiana and P. maculata of Indo-Pacific region. Therefore, Zigler et al. (2012) referred the indi- viduals from São Tomé Island as Pseudoboletia sp. As a genus occurring globally, from tropi- cal to temperate provinces, the biogeographic scenario involved suggests an interesting case Fig. 1. Alive specimen in subtropical habitats of Arraial do Cabo, southeastern Brazilian coast. for evolutionary and phylogeographic studies, Fig. 1. Espécimen vivo de Pseudoboletia en el hábitat which remains misunderstood. subtropical de Arraial do Cabo, costa sureste de Brasil. S300 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65(Suppl. 1): S299-S308, November 2017 Morphological analyses: The specimens The PCR products were purified and were preserved in 95% ethanol, photographed, sequenced at the Macrogen Inc. (South Korea). measured and deposited in the collection of the The sequences have been deposited in the National Museum of the Rio de Janeiro (Uni- Gen-Bank (accession numbers MG051273 - versidade Federal do Rio de Janeiro, Brazil). MG051274). DNA sequences were aligned Some individuals were treated with sodium using the CLUSTALW algorithm with default hypochlorite solution (NaClO) to remove all parameters in software MEGA version 5.05 organic material, spines and pedicellariae in (Tamura et al., 2011) and were manually order to expose the morphological test char- checked. Final aligned dataset included COI acters. The valves of pedicellariae and spic- sequences of a total length of 561 bp. Maxi- ules were also bleached, dehydrated (air-dried) mum Likelihood (ML) tree were reconstructed and gold-coated in order to be photographed in the software MEGA version 5.05 (Tamura in a scanning electronic microscope (SEM) et al., 2011) using HKY+ gamma model. The (JEOL JSM 6490LV). The morphological iden- best-fit evolutionary model was calculated with tification followed Mortensen (1943), Pawson jModelTest 0.1.1 (Posada, 2008) and selected (1978), and Turner and Graham (2003). by Akaike information criteria. Node support was evaluated using 1 000 rapid bootstrap Molecular analyses: Molecular analy- algorithms (Stamatakis, Hoover & Rougemont, 2008). The genetic distances between the main ses were carried out in order to compare clades were calculated under Kimura’s two- specimens from Brazil and specimens of the parameter model (Kimura, 1980) in MEGA genus Pseudoboletia from other localities. The version 5.10 (Tamura et al., 2011). analyses included sequences obtained from the Haplotype networks for COI sequences Genbank Database for Pseudoboletia macu- were estimated using the PEGAS package lata and Pseudoboletia indiana from Sydney (Paradis, 2010) in the statistical program R (R (Australia), Pseudoboletia indiana from Easter Core Team, 2011) in order to provide an esti- Island (Chile) and Pseudoboletia sp. from mate of possible relationships among species São Tomé. All sequences were provided by and populations. Zigler et al. (2012). The species Lytechinus variegatus and Tripneustes ventricosus were used as outgroups. RESULTS Total DNA of the Brazilian specimens was extracted from preserved tube feet using Taxonomic account: a REDExtract-N-Amp Tissue PCR Kit