Molluscan Studies · October 2012 DOI: 10.1093/Mollus/Eys022

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Two cryptic and sympatric species of Philinopsis (Cephalaspidea: Aglajidae) in the Bahamas distinguished using molecular and anatomical data

Article in Journal of Molluscan Studies · October 2012 DOI: 10.1093/mollus/eys022

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Journal of Molluscan Studies (2012) 78: 313–320. doi:10.1093/mollus/eys022

TWO CRYPTIC AND SYMPATRIC SPECIES OF PHILINOPSIS (CEPHALASPIDEA: AGLAJIDAE) IN THE BAHAMAS DISTINGUISHED USING MOLECULAR AND ANATOMICAL DATA Downloaded from

ELYSSE ORNELAS-GATDULA AND A´NGEL VALDE´S Department of Biological Sciences, California State Polytechnic University, 3801 West Temple Avenue, Pomona, CA 91768, USA

Correspondence: A´. Valde´s; e-mail: [email protected] http://mollus.oxfordjournals.org/ (Received 20 March 2012; accepted 13 June 2012)

ABSTRACT Sequence data of nuclear and mitochondrial genes obtained from specimens of Philinopsis collected in the Bahamas revealed two genetically distinct groups. Further examination of the shell and male reproductive morphology revealed consistent anatomical differences between them, corroborating that they are distinct species. The two species also display consistent differences in external morphology and colour pattern that became evident only after the molecular information was analysed. A review of the literature suggests that one of the species is Philinopsis petra, a widespread species in the at California State Polytechnic U Pomona on October 29, 2012 Caribbean and northern Brazil, whereas the other is described here as a new species. Although both are sympatric in the Bahamas, they appear to be ecologically analogous, suggesting that they might have speciated allopatrically.

INTRODUCTION (based on external morphological traits) and the two appear to be ecologically analogous (i.e. they occupy the same niche). Philinopsis is a group of aglajid opisthobranchs (Cephalaspidea: Differences between C. berolina and C. normani include a differ- Aglajidae) containing about 19 described species. Like most ent developmental mode and penial anatomy, as well as a aglajids, species of Philinopsis are carnivorous hunters lacking a number of molecular apomorphies in mitochondrial and radula, usually found crawling or burrowing on sand or sandy nuclear genes. mud. Rudman (1972) distinguished two groups of Philinopsis In this paper, we examine a similar case, this time with with different digestive anatomies, the ‘typical’ group includes species of Philinopsis (Spinoaglaja sensu Ortea et al., 2007). The species with a large, partially eversible buccal bulb that is used objectives of this paper are to describe a new cryptic species to catch bubble shells and other opisthobranchs upon which of Philinopsis and to provide further data on the intriguing they prey. The ‘atypical’ group includes species with a long, pattern of speciation in the Bahamas first detected by muscular, tubular buccal bulb. Rudman (1972) suggested that Ornelas-Gatdula et al. (2011). species of this second group could feed using the buccal bulb as a long eversible tube that can catch prey, possibly poly- chaete worms. Ortea, Moro & Espinosa (2007) recognized a third group of Atlantic species assigned to Philinopsis, including animals with a noneversible buccal bulb and conical spines on MATERIAL AND METHODS the fully calcified shell. They named this third group Source of specimens Spinoaglaja, but provided no evidence of monophyly. In recent years, we have been sequencing a large collection Thirteen specimens initially identified as Philinopsis petra and of opisthobranch specimens collected around Stocking Island, two specimens of P. pusa (Ev. Marcus & Er. Marcus, 1976) Exumas, Bahamas, and comparing the sequences with those of were used in this study (Table 1). Most specimens were col- other western Atlantic populations of the same species. The lected at Stocking Island, Exumas, Bahamas (Fig. 1). first published paper (Ornelas-Gatdula, DuPont & Valde´s, Specimens were photographed alive, narcotized using a 1 M 2011) revealed the existence of a new cryptic species of solution of MgCl2, and preserved in 70% ethanol. Two add- Chelidonura among the Bahamas material. This new species itional specimens were collected in Tobago and Martinique (C. normani Ornelas-Gatdula, DuPont & Valde´s, 2011) was and preserved in 10% formalin. All the specimens examined almost indistinguishable from the common and widespread are deposited at the Natural History Museum of Los Angeles western Atlantic Chelidonura berolina Marcus & Marcus, 1970 County, USA (abbreviated LACM).

Table 1. Material of Philinopsis examined in this study including locality information, collection voucher numbers and GenBank accession numbers.

Species Locality Date Collection number GenBank accession numbers

16S COI H3

P. petra Monument Beach, Stocking I. 9 Mar 2005 LACM 172268 JN825137 – JN825064 P. petra Monument Beach, Stocking I. 9 Mar 2005 LACM 172269 JN825138 JN825192 JN825065 P. petra* Monument Beach, Stocking I. 9 Mar 2005 LACM 172270 JN825139 JN825193 JN825066 P. petra Monument Beach, Stocking I. 9 Mar 2005 LACM 172271 JX188004 – JX188013 P. petra* Hole 1, Stocking I. 8 Feb 2010 LACM 178469 JX188009 – – P. petra* Hole 1, Stocking I. 19 Jan 2010 LACM 178470 JX188007 – – P. petra* Hole 1, Stocking I. 8 Feb 2010 LACM 178471 JX188010 – – P. petra* Store Bay, Tobago – LACM 178472 – – – P. petra* Cave Point, Martinique Jun 1986 LACM 178473 – – –

P. anneae* Sand Dollar Beach, Stocking I. 21 Jan 2004 LACM 3227 JX188005 – JX188014 Downloaded from P. anneae* Sand Dollar Beach, Stocking I. 21 Jan 2004 LACM 3226 JX188006 JX188012 JX188015 P. anneae* Sand Dollar Beach, Stocking I. 26 Dec 2003 LACM 3225 – JX188011 – P. anneae* Hole 1, Stocking I. 8 Feb 2010 LACM 3224 JX188008 – – P. pusa Gaviata Bay, Stocking I. 20 Dec 2005 LACM 173220 JN825144 JN825199 JN825072 P. pusa Hurricane Hole, Stocking I. 11 Jan 2006 LACM 173221 JN825145 JN825200 JN825073 http://mollus.oxfordjournals.org/

*Specimens examined morphologically. at California State Polytechnic U Pomona on October 29, 2012

Figure 1. Map of localities where the specimens included in this study were collected. CRYPTIC PHILINOPSIS IN BAHAMAS

Morphological examination fragments less than 500 bp and BigDye v. 3.1 for fragments larger than 500 bp. Ten specimens were dissected and the features of the male reproductive system were examined and photographed using a dissecting microscope Leica EZ4D. The shell of each specimen was dissected and the surrounding tissue dissolved with Analyses NaOH. Shells were rinsed in water, dried, mounted and Sequences for each gene were assembled and edited using sputter coated for examination with a scanning electron micro- Geneious Pro v. 4.7.4 (Drummond, Ashton & Cheung, 2009). scope (SEM) Hitachi S-3000N at the LACM. Geneious was also used to extract the consensus sequence between the primer regions, to construct the alignment for each gene using the default parameters and to concatenate the DNA extraction aligments. The sequences were not trimmed after alignment. A total of 328 bp for H3, 409–410 bp for 16S and 658 bp for DNA extraction was performed using either a hot Chelexw w COI were used for the phylogenetic analyses. protocol or the DNeasy Blood & Tissue Kit (Qiagen). C. 1– To assess whether H3, 16S and COI have signiﬁcantly con- 3 mg of the foot was cut into small pieces for extraction for

w ﬂicting signals, the incongruence length difference (ILD) test Downloaded from both protocols. For the Chelex extraction, the foot tissue was (Mickevich & Farris, 1981; Farris et al., 1994), implemented in rinsed and rehydrated using 1.0 ml of TE buffer (10 mM Tris, w PAUP* v. 4.0 as the partition homogeneity test (Swofford, 1 mM EDTA, pH 8.0) for 20 min. A 10% (w/v) Chelex 100 2002), was conducted for all genes combined. (100–200 mesh, sodium form, Bio-Rad) solution was prepared The levels of saturation for each gene and for the ﬁrst and using TE buffer. After rehydration, the tissue mixture was cen- second versus third codon positions of COI and H3 were inves- trifuged, 975.00 ml of the supernatant was removed and

tigated using the substitution saturation test developed by Xia http://mollus.oxfordjournals.org/ m w 175.00 l of the Chelex solution was added. Samples were et al. (2003) and Xia & Lemey (2009) implemented in the 8 then heated in a water bath at 56 C for 20 min, heated in a program DAMBE (Xia & Xie, 2001). 8 100 C heating block for 8 min and the supernatant was used The Akaike information criterion (Akaike, 1974) was exe- for polymerase chain reaction (PCR). The DNeasy protocol cuted in MrModeltest (Nylander, 2004) to determine the supplied by the manufacturer was followed, with some modifi- best-fit model of evolution (Table 2). Maximum likelihood cations. The elution step was modified such that the first analyses were conducted with the program GARLI v0.96b8 m elution was collected using 100.00 l of Buffer AE and was (Zwickl, 2006). Default parameters were used to run three dif- allowed to incubate at room temperature for 5 min before cen- ferent GARLI searches of 100 replicates each and a total of trifugation. In a new test tube, a second elution step was con- 2000 bootstrap replicates were performed to assess the robust- m ducted using 200.00 l of Buffer AE and was also allowed to ness of each clade (Felsenstein, 1985). Bayesian analyses were incubate at room temperature for 5 min before centrifugation. executed in MrBayes v. 3.2.1 (Huelsenbeck & Ronquist, at California State Polytechnic U Pomona on October 29, 2012 The first elution was used for PCR. 2001), partitioned by gene (unlinked). The Markov-chain Monte Carlo analysis was run with two runs of six chains for 10 million generations, with sampling every 100 generations. PCR amplification and sequencing The default 25% burn-in was applied before constructing Histone-3 universal primers (H3AF 50-ATGGCTCGTACCA majority-rule consensus trees. AGCAGACGGC-30,H3AR50-ATATCCTTGGGCATGAT Diagnostic nucleotides for each clade were identified visually GGTGAC-30 developed by Colgan et al., 1998), 16S rRNA in the alignments after collapsing identical haplotypes using universal primers (16S ar-L 50-CGCCTGTTTATCAAAAA the program Collapse v. 1.2 (Posada, 2004). CAT-30, 16S br-H 50- CCGGTCTGAACTCAGATCACGT-30 The Species Delimitation plugin (Masters, Fan & Ross, developed by Palumbi, 1996) and COI universal primers 2011) for Geneious was used to provide a statistical framework (LCO1490 50-GGTCAACAAATCATAAAGATATTGG-30, to help determine whether clades obtained in the phylogenetic HCO2198, 50-TAAACTTCAGGGTGACCAAAAAATCA-30 developed by Folmer et al., 1994) were used to amplify the regions of interest for all specimens. Table 2. Summary of each dataset used for the Bayesian and The master mix was prepared using 34.75 mlHO, 5.00 ml maximum likelihood analysis of all species, with the best-fit 2 evolutionary models and estimated parameters. Buffer B (ExACTGene, Fisher Scientific), 5.00 ml25mM MgCl2, 1.00 ml 40 mM dNTPs, 1.00 ml 10 mM primer 1, Parameters H3 16S COI H3+16S+COI 1.00 ml primer 2, 0.25 ml 5 mg/ml Taq and 2.00 ml extracted DNA. Reaction conditions for H3 and 16S were as follows: an No. of included characters 328 413 658 1399 initial denaturation of 948C for 2 min, 30 cycles of 948C for Best-fit model HKY+I HKY GTR+I GTR+I+G 30 s, 508C for 30 s and 728C for 1 min, followed by a final Frequency A 0.2445 0.3137 0.2437 0.2651 elongation of 728C for 7 min. Reaction conditions for COI Frequency C 0.3202 0.1510 0.1554 0.1933 consisted of an initial denaturation of 958C for 3 min, 35 cycles Frequency G 0.2559 0.2118 0.1862 0.2103 of 948C for 45 s, 458C for 45 s and 728C for 2 min, followed by Frequency T 0.1794 0.3236 0.4146 0.3312 a final elongation step of 728C for 10 min. PCR products yielding bands of appropriate size (c. 375 bp R-matrix [A-C] – – 0.0000 0.6074 for H3, 475 bp for 16S and 700 bp for COI) were purified R-matrix [A-G] – – 9.4090 5.5083 using the Montage PCR Cleanup Kit (Millipore). Cleaned R-matrix [A-T] – – 3.1954 2.2476 PCR samples were quantified using a NanoDrop 1000 R-matrix [C-G] – – 0.0000 0.2661 Spectrophotometer (Thermo Scientific). Each primer was R-matrix [C-T] – – 21.2143 8.3427 diluted to 2.0 pmol/ml to send out for sequencing with the R-matrix [G-T] – – 1.0000 1.0000 PCR products. PCR products were diluted to 6.0, 7.5 and G shape (G) – – – 0.6382 11.5 ng/ml for H3, 16S and COI, respectively. Samples were Proportion of invariant 0.8446 0.6836 0.6043 sequenced at the City of Hope DNA Sequencing Laboratory sites (I) (Duarte, CA) using chemistry types BigDye v. 1.1 for

315 E. ORNELAS-GATDULA AND A´.VALDE´S analysis have identity as distinct species. The statistics imple- substitutions unique for each clade in all three genes (3 in H3, mented were the ratio between the mean distance within the 11 in 16S and 69 in COI) provided a strong phylogenetic members of the clade and the mean distance of those indivi- signal. duals to the nearest clade (the larger the value of this ratio is, the more distinct is the identity of the group) and the p ID, which represents the mean probability, 95% conﬁdence inter- Morphology val, for a member of the putative species to ﬁt inside (strict p ID), or at least to be the sister group (liberal p ID) of the The observation of the shell and penial morphology, as well as clade made up by the other individuals belonging to this the external coloration and body shape, revealed consistent dif- species. ferences between the two clades distinguished using molecular data (Fig. 2). Clade 1 includes animals generally yellow– orange with elongate, smooth bodies and a series of denser con- RESULTS centrations of white pigment on the head (where it forms three Molecular data large spots arranged on a transverse line), the posterior end of the cephalic shield, the posterior end of the visceral mass

The saturation analysis showed insigniﬁcant levels of saturation (where it forms an inverted triangle) and the edge of the para- Downloaded from for all three genes: COI: Iss (0.4351) , Iss.c (0.7738), podia. In contrast specimens in clade 2 vary from dark brown P ¼ 0.000; 16S: Iss (0.0665) , Iss.c (0.7123), P ¼ 0.000; H3: to light yellow–orange, their bodies are wider and have a Iss (0.3091) , Iss.c (0.7363), P ¼ 0.000, even when the visible transverse lateral expansion of the body on each side, third codon positions of COI: Iss (0.4167) , Iss.c (0.7409), near the end of the cephalic shield; the white pigment concen- P ¼ 0.000) and H3: Iss (0.0103) , Iss.c (0.7141), P ¼ 0.000 trations are located on the head, the posterior end of the ceph-

were analysed independently. The ILD test showed no signifi- alic shield, the posterior end of the visceral mass and on the http://mollus.oxfordjournals.org/ cant conflicting signals between the three genes combined lateral expansions of the body. All specimens examined in (P ¼ 1), COI vs.H3(P ¼ 0.688), COI vs. 16S (P ¼ 0.229) and clade 1 have a well-calcified shell with a single conical spine on 16S vs.H3(P ¼ 0.606). the apex, whereas all animals of clade 2 examined have two The combined analysis of the three genes (H3, 16S and conical spines on the apex of the shell (Fig. 2). The male re- COI) produced consensus bootstrap and Bayesian trees in productive organs of members of clade 1 have a prostate much which two main clades are well supported (Fig. 2). One of the larger than that of specimens in clade 2 and a comparatively clades (clade 1), with a posterior probability of 1 and boot- smaller penis (Fig. 3). Additional specimens collected from strap value of 100, includes four specimens. The other clade Tobago and Martinique were preserved in formalin, thus they (clade 2), with a posterior probability of 0.98 and a bootstrap were unsuitable for molecular work and the shells were dis- value of 72, includes seven specimens. A number of solved. However, examination of the male reproductive at California State Polytechnic U Pomona on October 29, 2012

Figure 2. Bayesian tree of Philinopsis specimens examined, including posterior probabilities and bootstrap values from the maximum likelihood analysis. The shell apical morphology and male reproductive anatomy for the main clades is illustrated as well as the external morphology of all specimens.

316 CRYPTIC PHILINOPSIS IN BAHAMAS

morphology of the reproductive male anatomy (Marcus, 1976) and the external body shape and coloration (Ortea & Espinosa, 2001; Ortea et al., 2007). The possible synonymy between P. petra and P. aeci and the taxonomic placement of the two nominal species are discussed below. On the contrary, there are no published records of any western Atlantic species of Aglajidae with the characteristics of members of clade 1, thus a new species name (Philinopsis anneae) is here introduced for this species. Valde´s et al. (2006) illustrated specimens of P. petra from Martinique, Cayman Islands and Honduras, all matching the colouration and body morphology of P. petra. Additional photos from Florida provided by Anne DuPont also match the P. petra colour pattern and body shape. No illustrations of specimens with the colour pattern of P. anneae appear to exist from

locations other than the Bahamas. This suggests that P. anneae Downloaded from is likely endemic to the Bahamas.

SYSTEMATIC DESCRIPTION Philinopsis anneae new species (Figs 3C, 4) http://mollus.oxfordjournals.org/ Figure 3. Light microscope photographs of the penial anatomy of Philinopsis species. A. P. petra from Tobago (LACM 178472). B. P. petra 8 0 00 8 0 00 from the Bahamas (LACM 178469). C. P. anneae from the Bahamas Type material: Holotype: Hole 1 (23 31 20 N, 75 45 20 W), (LACM 3224). Stocking I., Exumas, Bahamas, 8 February 2010, leg. Anne DuPont (LACM 3224). Paratypes: Sand Dollar Beach (2383005500N, 7584403600W), Stocking I., Exumas, Bahamas, Table 3. Species delimitation results from the Bayesian tree of 26 December 2003, 1 specimen, leg. Anne DuPont (LACM Philinopsis species. 3225); 21 January 2004, 2 specimens, leg. Anne DuPont (LACM 3226, LACM 3227). Species Closest Monophyly Intra Inter Intra/ pID PID Etymology: This species is named in honour of our friend and

species dist dist Inter (strict) (liberal) at California State Polytechnic U Pomona on October 29, 2012 colleague Anne DuPont, who collected all the specimens closest studied in this paper. P. pusa P. petra No 0.005 0.468 0.01 0.59 0.98 Diagnosis: Body cylindrical, elongate, smooth (Fig. 4A). P. petra P. anneae Yes 0.01 0.138 0.07 0.91 0.98 Cephalic shield slightly longer than visceral mass when animal P. anneae P. Petra Yes 0.035 0.138 0.25 0.7 0.92 is in motion. Posterior end of body with two triangular exten- sions (tails) similar in size and shape. Posterior edge of cephalic shield with a low crest. Parapodia short, covering sides of body. Background colour varies from dark orange to yellow, morphology (Fig. 3) and external coloration and body shape with white pigment distributed over entire body. Denser con- confirmed that they belong to clade 1. centrations of white pigment occur consistently on several parts of body including head, where it forms three large spots arranged on a transverse line; tapering crest at posterior end of Taxonomy cephalic shield; posterior end of visceral mass, where it forms an inverted triangle; and edge of parapodia. Shell calcified Because of the presence of molecular and morphological syn- (Fig. 4B), elongate with a conspicuous conical spine on apical apomorphies in each of the two clades recovered in the phylo- end. Digestive system with large, non-eversible buccal bulb. genetic analysis, these clades are regarded as two distinct Penis conical, directly connected to elongate prostate species, based on contemporary approaches to species level tax- (Fig. 3C). onomy (Ferguson, 2002). This conclusion is substantiated with results from the Species Delimitation analyses (Table 3). From Biology: Specimens were found crawling on sand during the these data, it was concluded that all the groups are monophy- day. When disturbed they released an orange substance. letic, except for the outgroup, P. pusa, for which mononophyly was not tested. The values of the ratio between the average distance between the samples of one group (Intra Dist) and the DISCUSSION average distance between those samples and the closest clade Taxonomic status of Philinopsis petra and Spinoaglaja (Inter Dist) are small, below 0.25 (Lo´pez-Lo´pez, Hudson & Galiań, 2012), meaning that genetic differences within all Philinopsis petra was originally described from northern Brazil clades are small relative to the differences between clades, so (Pernambuco) by Marcus (1976) as Chelidonura petra. The ori- that there is a well-defined separation of clades. The probabil- ginal description was based on a preserved specimen and there ity (p ID) of a new sequence fitting inside (strict) or at least as is no information on the colour of the live animal. However, sister group (liberal) of its clade is high in all cases. Marcus (1976) described the internal anatomy in detail, in- The morphology of members of clade 2 is consistent with cluding drawings of the shell, nervous system and male and that of descriptions of western Atlantic Philinopsis assigned to female reproductive systems. The main diagnostic characteris- P. petra (Marcus, 1976) and P. aeci (Ortea & Espinosa, 2001; tic of this species is the presence of a well-calcified shell with Ortea et al., 2007), including the presence of two spines on the two spines on the apex. Additionally, the penis is wider and shell apex (Marcus, 1976; Ortea & Espinosa, 2001), the about as long as the prostate. Ortea & Espinosa (2001)

317 E. ORNELAS-GATDULA AND A´.VALDE´S

preparation) shows that P. petra and P. anneae appear to be sister species (which is also supported by the presence of the unique shell and cephalic shield features mentioned above), but Spinoaglaja (P. petra þ P. anneae) is nested within Philinopsis and is therefore considered a synonym of the latter.

Cryptic species We consider P. petra and P. anneae to be cryptic species because, even though P. anneae can be distinguished from P. petra by some external traits, these differences only became evident in light of molecular data. The main differences between these two species are molecular and internal (shell and reproductive morphology) and therefore not visible in live animals.

Mode of speciation and biogeography Downloaded from One of the most interesting aspects of the biology of P. anneae is the fact that it is completely sympatric with the more widespread P. petra. Coyne & Orr (2004) and Bolnick & Fitzpatrick (2007) proposed that sister species with overlapping ranges,

inhabiting areas in which there is no evidence of present or http://mollus.oxfordjournals.org/ past biogeographic barriers to dispersal, could be potential cases of sympatric speciation. Friesen et al. (2007) listed various sympatric models in which reproductive isolation could evolve in the absence of geographic isolation through polyploidization (such as in flowering plants), nonrandom mating driven by ecological isolation (e.g. host preference, differential migratory route, allochrony) and disruptive selection (e.g. frequency- dependent competition, divergent sexual selection). Thus, evidence of competitive exclusion (character displacement), divergent sexual selection or allochrony between the two Figure 4. Philinopsis anneae new species. A. External morphology of the at California State Polytechnic U Pomona on October 29, 2012 living holotype (LACM 3224). B. SEM of the shells of a paratype species should be necessary to identify cases of sympatric speci- (LACM 3225). ation in animals. Field observations suggest that P. petra and P. anneae in the Bahamas are ecologically analogous. Although the specific diet described P. aeci from Costa Rica, based only on external char- of these two species is unknown, adult animals of both species acteristics. They compared their new species with P. pusa Ev. were found in the same environment, at the same time of the Marcus & Er. Marcus, 1967 but not with P. petra. Valde´s et al. day and sometimes together (Table 4) and display a similar (2006) suggested that P. petra should be transferred to behaviour. This seems to reject any hypotheses of niche parti- Philinopsis and P. aeci should be synonymized with it, as these tioning or allochrony. two names probably refer to the same species. In a subsequent Differences in the male reproductively anatomy between paper, Ortea et al. (2007) redescribed P. aeci including add- P. petra and P. anneae, particularly the larger prostate size of itional specimens from the Bahamas and Cuba and provided a P. anneae in relation to the penis, would suggest the action of drawing and description of the shell. The shell of P. aeci is very sexual selection. Anthes et al. (2008) found that reproductive similar to that described for P. petra and Ortea et al. (2007) trait variation in Aglajidae is mediated by sexual coevolution recognized the possibility that these two species could be syno- and low gregariousness was associated with long, reciprocal nyms, but preferred to maintain them as different species until copulations. It is conceivable that a larger prostate could be an new material became available, particularly specimens with adaptation to longer copulations or sperm competition in the shells of intermediate size between 4.5 3mm(P. petra holo- less common, geographically restricted P. anneae, but because type) and 1.6 1 mm (specimen of P. aeci from Cuba). One of this scenario requires initial populations with different degrees the specimens from Bahamas here examined and assigned to of gregariousness it is unlikely it could occur sympatrically. All P. petra has a shell measuring 2.5 1.7 mm and its morphology available evidence suggests that sympatric speciation is not a is very similar to that of the shells of P. petra and P. aeci, includ- likely explanation for the morphological and behavioral traits ing the presence of two apical spines. Thus we consider observed in P. petra and P. anneae. these two species to be synonymous. In addition, Ortea et al. Ornelas-Gatdula et al. (2011) found a similar situation in (2007) synonymized Melanochlamys wildpetri Ortea, Moro & another aglajid group. Chelidonura normani is a Bahamas Bacallado, 2003, an eastern Atlantic species, with P. aeci. endemic closely related to two other widespread tropical Evidence from molecular studies of other species of Aglajidae Atlantic species, C. berolina (also found in the Bahamas) and (Ornelas-Gatdula et al., 2011) suggests the absence of gene C. africana Pruvot-Fol, 1953 (from the eastern Atlantic). flow across the Atlantic Ocean. The synonymization of Chelidonura normani and C. berolina differ in their developmental M. wildpetri with P. aeci or P. petra could have been premature modes, suggesting that the nonplanktotrophic C. normani is until molecular data for the former become available. adapted to recruit locally, maintaining a restricted range. Ortea et al. (2007) introduced the new genus name Chelidonura normani and C. berolina are likewise ecological analo- Spinoaglaja for species of Aglajidae previously included in gues and were often found together in the same environment Philinopsis with apical spines on the shell and lacking a raised at the same time of the day; however no evidence of gene flow posterior edge of the cephalic shield (P. petra and P. aeci). (hybridization) between them was found, which could have A preliminary molecular phylogeny of the Aglajidae (in suggested reinforcement.

318 CRYPTIC PHILINOPSIS IN BAHAMAS

Table 4. Date and time of observation, habitat and behaviour of specimens of Philinopsis petra and P. anneae found in the Bahamas.

Species Date and time Habitat and behaviour Locality

P. petra 2 Apr 2003: unknown Crawling on sand Highbourne Cay 28 Jan 2005: 14:00–16:00 Crawling on sand Hole 1, Stocking I. 9 Mar 2005: 12:00–13:30 Crawling on sand Monument Beach, Stocking I. 9 Mar 2005: 12:00–13:30 Crawling on sand Monument Beach, Stocking I. 9 Mar 2005: 12:00–13:30 Crawling on sand Monument Beach, Stocking I. 9 Mar 2005: 12:00–13:30 Crawling on sand Monument Beach, Stocking I. 19 Jan 2010: 8:00 Crawling on sand Hole 1, Stocking I. 8 Feb 2010: 14:00–14:30 Crawling on sand Hole 1, Stocking I. 8 Feb 2010: 14:00–14:30 Crawling on sand Hole 1, Stocking I. P. anneae 26 Dec 2003: 11:00–13:00 Crawling on sand Sand Dollar Beach, Stocking I. 21 Jan 2004: 15:00–18:00 Crawling on sand Sand Dollar Beach, Stocking I. Downloaded from 21 Jan 2004: 15:00–18:00 Crawling on sand Sand Dollar Beach, Stocking I. 8 Feb 2010: 14:00–14:30 Crawling on sand Hole 1, Stocking I. 8 Feb 2010: 14:00–14:30 Crawling on sand Hole 1, Stocking I. http://mollus.oxfordjournals.org/ Several studies have investigated the genetic structure a subsequent recolonization of the Bahamas by P. petra and (Mitton, Berg & Orr, 1989; Dıáz-Ferguson et al., 2010, 2012) C. berolina. Hypotheses on the isolation of the Bahamas based and patterns of diversification (Lee & O´Foighil, 2004, 2005) on low connectivity or postrecruitment ecological factors do of coastal tropical western Atlantic marine molluscs. Some of not fully explain the pattern of genetic differentiation and these studies revealed the existence of genetically distinct clades range overlap here observed. At present we can only speculate in the Bahamas (Lee & O´Foighil, 2004, 2005; Dıáz-Ferguson as to the causes of this pattern, although the hypothesis of et al., 2010), which have also been detected in reef fishes and formation of a barrier between the Bahamas and the rest of the corals (Taylor & Hellberg, 2003; Galindo, Olson & Palumbi, Caribbean for long enough to result in speciation, followed by 2006). However, other Western Atlantic molluscs show little to the subsequent relaxation of the barrier allowing recolonization no population structure (Mitton, Berg & Orr, 1989)orpat- by widespread species seems a good fit for our observations. terns of genetic differentiation not including distinct Bahamas A much broader sampling effort in the western Atlantic includ- at California State Polytechnic U Pomona on October 29, 2012 clades (Dıáz-Ferguson et al., 2012). Most authors agree that it ing other species with a similar ecology is necessary to test this is difficult to make generalizations about connectivity and di- hypothesis properly. versification patterns of marine organisms in the tropical western Atlantic (Lee & O´Foighil, 2005; Vollmer & Palumbi, 2007; Dıáz-Ferguson et al., 2012). The complex patterns of connectivity in the tropical western Atlantic and the existence of ACKNOWLEDGMENTS oceanographic barriers to gene flow (Cowen et al., 2006)area We are extremely grateful to Anne DuPont and Jeff Hamann possible explanation for the maintenance of allopatric distribu- for providing specimens that were essential for the completion tions of genetically distinct species or populations over evolu- this project. Lindsey Groves from the Natural History Museum tionary timescales. The Bahamas are largely isolated from the of Los Angeles County helped with access to the collection and rest of the Caribbean, except for a minor exchange from the curation of specimens. Hooman Borhani and Jermaine Mahguib north coast of Cuba and Haiti (Cowen et al., 2006), which assisted with the dissection of specimens, extraction of shells would predict a certain level of genetic isolation in Bahamas and image processing with Adobe Photoshopw.Thispaper populations. Galindo et al. (2006) developed a genetic model was supported by a Cal Poly Pomona Research, Scholarship that uses connectivity estimates from oceanographic models. and Creative Activity grant to A. Valde´s. Additional material This model predicts empirical genetic patterns resulting from and travel support was provided by student grants from larval dispersal in a western Atlantic coral, including the isola- Conchologists of America and the Southern California Academy tion of the Bahamas and an east–west divergence near Puerto of Sciences to E. Ornelas-Gatdula. Finally, E. Ornelas-Gatdula Rico. On the contrary, other authors have argued that postre- was supported by an NIH MBRS Research Initiative for cruitment ecological factors rather than limited connectivity Scientific Enhancement (RISE) grant to Cal Poly Pomona are the primary cause of allopatric ranges of genetically dis- (2 R25 GM061190-05A2). The SEM work was conducted at tinct species or populations (Lee & O´Foighil, 2005). Much the Natural History Museum of Los Angeles County, supported less attention has been paid to historical biogeography and the by the National Science Foundation under MRI grant potential role of the formation and disappearance of barriers to DBI-0216506 to A. Valde´s et al., with the assistance of Giar-Ann gene flow in the divergence of allopatric clades over geological Kung. timescales. Our data seem to indicate that the Bahamas endemics P. anneae and C. normani have evolved allopatrically from their sister taxa P. pusa and C. berolina. Because the geographic range REFERENCES of the widespread species P. petra and C. berolina include the AKAIKE, H. 1974. A new look at the statistical model identifications. western tropical Atlantic from Brazil to the Bahamas, whereas IEEE Transactions on Automatic Control, 19: 716–723. 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