Molecular evaluation of the phylogenetic position of the ... - BioOne

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Invertebrate Systematics, 2015, 29, 215–222 http://dx.doi.org/10.1071/IS15002

Molecular evaluation of the phylogenetic position of the enigmatic species Trivettea papalotla (Bertsch) (Mollusca : Nudibranchia) Ryan E. Hulett A, Jermaine Mahguib B, Terrence M. Gosliner A and Ángel Valdés B,C A

Department of Invertebrate Zoology and Geology, California Academy of Sciences, 55 Music Concourse Drive, San Francisco, CA 94118, USA. B Department of Biological Sciences, California State Polytechnic University, 3801 West Temple Avenue, Pomona, CA 91768, USA. C Corresponding author. Email: [email protected]

Abstract. Tritoniid sea slugs are specialised predators that feed on a variety of octocorals, including soft corals, gorgonians and sea pens. Trivettea papalotla is a recently described species found in Baja California and mainland Mexico that is unusual in its morphology and feeding behaviour. It is the first tritoniid nudibranch known to feed on zoanthid anthozoans, specifically on an undescribed species of the genus Epizoanthus. Trivettea papalotla also has retractable respiratory structures, prominent dorsal vessels and several other traits not found in any other species of the Tritoniidae. In its original description these unique features of T. papalotla were considered autapomorphies, and the species was tentatively placed within Tritonia based on a morphological phylogenetic analysis. Subsequently, the monotypic genus Trivettea was erected for T. papalotla based on unpublished molecular data. In the present study, the phylogenetic placement of Trivettea is investigated based on molecular data. These phylogenies show T. papalotla is not nested within Tritonia or Tritoniidae and instead appears to be a basal, distinct cladobranch. However, the analyses conducted resulted in poorly resolved basal relationships, suggesting additional markers are probably necessary to fully resolve the phylogeny for the Cladobranchia. Received 25 July 2014, accepted 19 March 2015, published online 30 June 2015

Introduction Tritoniidae (Nudibranchia, Cladobranchia, Dendronotidae) is a family of sea slugs known to feed on a variety of octocorals, including soft corals, gorgonians and sea pens (McDonald and Nybakken 1999). Tritoniidae is currently divided into eight genera, including Tritonia Cuvier, 1798, Tritoniopsis Eliot, 1905, Tritoniella Eliot, 1907, Marionia Vayssiere, 1877, Marianina Pruvot-Fol, 1931, Marioniopsis Odhner, 1934, Paratritonia Baba, 1949, Tochuina Odhner, 1963, and Trivettea Bertsch, 2014 (Bertsch et al. 2009; Pola and Gosliner 2010; Bertsch 2014). Members of Tritoniidae include some of the largest species of nudibranchs and some have been utilised extensively in neurophysiology and behavioural studies (Willows and Hoyle 1967; Katz 1998). Despite the biomedical importance of this group, the taxonomy of the Tritoniidae has long been mired in controversy (Odhner 1963; Gosliner and Ghiselin 1987; Smith and Gosliner 2003). In part, this is due to a lack of standardisation in the use of morphological traits, both external and internal, in the estimation of evolutionary relationships among established species and newly discovered ones (Ballesteros and Avila 2006). Pola and Gosliner (2010) reconstructed the phylogeny Journal compilation CSIRO 2015

of Cladobranchia, a larger clade that includes Tritoniidae and other related nudibranchs, and concluded that Tritoniidae appeared to be monophyletic but weakly supported. Trivettea papalotla (Bertsch et al. 2009) is a recently described species found in Baja California and mainland Mexico that is unusual in several ways, including its morphology and feeding behaviour. Trivettea papalotla is the first tritoniid nudibranch found feeding on a zoanthid anthozoan, an undescribed species of the genus Epizoanthus, rather than on octocorals (Bertsch et al. 2009). Trivettea papalotla also has retractable respiratory structures, prominent dorsal vessels (Fig. 1), a uniseriate radula and several other traits not found in other species of Tritoniidae (Bertsch et al. 2009). Bertsch et al. (2009) concluded that the unique features of T. papalotla are autapomorphies, and that this species should be tentatively placed within Tritonia based on a morphological phylogenetic analysis. Subsequently, Bertsch (2014) proposed the new generic name Trivettea for this species based on our unpublished molecular data and without presenting a formal phylogenetic analysis. The purpose of the present study was to test the phylogenetic placement of Trivettea papalotla within Tritoniidae based on three molecular markers (mitochondrial 16S rRNA and www.publish.csiro.au/journals/is

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Fig. 1. Photograph of a live animal of Trivettea papalotla.

cytochrome c oxidase 1, COI, and nuclear histone 3, H3) obtained from newly collected specimens of T. papalotla and additional sequences of representative tritoniids and other cladobranchs. Materials and methods

heat block for exactly 8 min, after which the supernatant was used for polymerase chain reaction (PCR). Extraction of DNA from specimens from the California Academy of Sciences was performed using tissue taken from the foot of the animals and a standard DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, U.S.A.) for animal tissues.

Specimens and sequence data used in study Specimens of Trivettea papalotla were obtained from Baja California and preserved in ethanol. These specimens were then sent to the California State Polytechnic University for DNA extraction, gene amplification and purification. All specimens utilised for this study are shown in Table 1, along with catalogue numbers for specimens used from the California Academy of Sciences invertebrate collection sequenced specifically for this study and accession numbers for sequences taken from GenBank. DNA extraction Extraction of DNA from specimens of Trivettea papalotla was performed using ~1–3 mg of tissue taken from the foot of the animals and a standard hot Chelex extraction protocol with minor modifications. Tissue samples were placed into 1.7 mL tubes containing 1.0 mL TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and allowed to incubate at room temperature on a rotator overnight to rehydrate the tissue and allow cells to begin dissociating. Samples were then vortexed using a touch mixer followed by centrifugation for 3 min at 23 897.25g. After samples were centrifuged, 975 mL of the original 1 mL of TE buffer in each tube was carefully removed without disturbing the pellet of tissue formed at the bottom. After buffer removal, 175 mL of Chelex solution was added. Samples were then vortexed and placed in a 56C water bath for at least 20 min. Additional vortexing was done after samples were removed from the water bath. Lastly, samples were placed in a 100C

Primers For the 16S rRNA gene, universal primers were used (Palumbi 1996); for the COI gene, Folmer’s universal COI primers were used (Folmer et al. 1994); and for the H3 gene, Colgan’s universal H3 primers were used (Colgan et al. 1998). All primer sequences are shown in Table 2. Polymerase chain reaction The master mix apportioned for each DNA sample that was amplified for Trivettea papalotla consisted of 34.75 mL H2O, 5 mL Buffer B (ExACTGene, Fisher Scientific, Inc., Fair Lawn, New Jersey, U.S.A.), 5 mL 25 mM MgCl2, 1 mL 40 mM dNTPs, 1 mL 10 mM primer 1, 1 mL 10 mM primer 2, 0.25 mL 5 mg mL–1 Taq, and 2 mL extracted DNA. The master mix for each DNA sample consisted of 14.5 mL of H2O, 2.5 mL of 10 USB buffer, 1 mL of 25 mM MgCl2, 1 mL mM primer 1, 1 mL mM primer 2, 0.5 mL 10 mM dNTPs, 1.5 mL 10 mg mL–1 BSA, 1 mL 1.25 mL–1 HotStart Taq, and 2 mL of extracted DNA. Polymerase chain reaction conditions for COI were an initial denaturation for 3 min at 95C, then 35 cycles of denaturation for 45 s at 94C, annealing for 45 s at 45C, and elongation for 2 min at 72C, and a final elongation for 10 min at 72C. Reaction conditions for 16S and H3 were an initial denaturation for 2 min at 94C, then 30 cycles of denaturation for 30 s at 94C, annealing for 30 s at 50C, and elongation for 1 min at 72C, and a final elongation for 7 min at 72C.

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Table 1. Nudibranch species used in the study Voucher numbers for invertebrate collections are also listed (CASIZ, California Academy of Sciences, Invertebrate Zoology; CPIC, California State Polytechnic University; MNCN, Museo Nacional de Ciencias Naturales, Madrid; LACM, Natural History Museum of Los Angeles County; FSBC, Florida Fish and Wildlife Conservation Commission; MZUCR, Museo de Zoología, Universidad de Costa Rica; ZSM, Zoologische Staatssammlung, Munich; MZSP, Museu de Zoologia, Universidade de São Paulo); accession numbers for gene sequences taken from GenBank are shown as well Taxon Outgroups Pleurobranchoidea Berthella martensi Nudibranchia Doridacea Bathydoris aioca Felimida fentoni Tambja marbellensis Ingroups Nudibranchia Cladobranchia Aeolidia papillosa Aeolidiella alderi Armina semperi Armina sp. E Babakina anadoni Bornella calcarata Bornella stellifer Caloria elegans Caloria indica Cuthona divae Dendronotus frondosus Dendronotus regius Dermatobranchus sp. 21 Dirona albolineata Doto coronata Doto koenneckeri Favourinus branchialis Favourinus elenalexiarum Flabellina exoptata Flabellina pedata Hancockia californica Leminda millecra Lomanotus sp. E Marianina rosea Marionia arborescens Marionia arborescens Marionia blainvillea Marionia distincta Marionia distincta Marionia levis Marionia levis Marionia sp. 3 Marionia sp. 5 Marionia sp. 6 Marionia sp. 10 Marionia sp. 10 Marionia sp. 10 Marionia sp. Marionia sp. Melibe digitata Melibe viridis Notobryon thompsoni Notobryon wardi

Voucher no. 16S

GenBank accession numbers COI

H3

MZUCR 6982

HM162592

HM162683

HM162498

CPIC 01053 FSBC I67095 CASIZ 180379

KP153249 KP153250 HM162599

KP153283 GU815540 HM162689

KP153316 GU815542 HM162505

CPIC 00717 ZSM Mol20020982 CASIZ 177534 CASIZ 177535 – MZSP 84448 CASIZ 167989 MNCN15.05/53690 – CASIZ 174495 LACM 174860 CASIZ 179493 CASIZ 177375 CASIZ 174466 CASIZ 176278 CASIZ 178247 MNCN15.05/53694 CASIZ 178875 LACM 153895 MNCN15.05/53702 CASIZ 175722 CASIZ 176348 LACM 174962 CASIZ 175746 CASIZ 177735 CASIZ 177578 CASIZ 176812 CASIZ 110364 CASIZ 173317 CASIZ 192357A CASIZ 192357B CASIZ 182818 CASIZ 177513 CASIZ 182857 CASIZ 173349 CASIZ 182864A CASIZ 182864B CASIZ 181242 CASIZ 166891 CASIZ 177478 CASIZ 176981 CASIZ 176277 CASIZ 177540

JQ699475 HQ616728 HM162606 HQ010539 HQ616743 HM162627 HM162623 HQ616738 DQ417273 JQ699479 JN869406 JN869407 HM162616 HM162668 HM162657 HM162658 HQ616741 HM162679 JQ699485 HQ616721 HM162621 HM162669 HM162640 HM162656 KP226859 HM162646 HM162645 KP226860 HM162648 KP153251 KP153252 KP153253 HM162650 KP153254 HM162651 KP153255 KP153256 KP153257 HM162649 HM162617 JX306068 JN869412 JN869411

JQ699565 HQ616765 HM162696 HQ010504 HQ616767 HM162707 HM162703 HQ616751 DQ417325 JQ699569 JN869450 JN869451 HM162698 GQ292058 HM162734 HM162735 HQ616761 HM162755 JQ699572 HQ616758 HM162702 HM162745 HM162715 HM162733 KP226855 HM162722 HM162721 KP226856 HM162725 KP153284 KP153285 KP153286 HM162727 KP153287 HM162728 KP153288 KP153289 KP153290 HM162726 HM162699 JX306075 JN869456 JN869454

JQ699385 HQ616794 HM162512 HQ010473 HQ616796 HM162533 HM162529 HQ616780 JQ699389 JQ699391 JN869429 JN869430 HM162522 HM162577 HM162566 HM162567 HQ616790 HM162588 JQ699397 HQ616787 HM162527 HM162578 HM162547 HM162565 KP226857 HM162554 HM162553 KP226858 HM162557 KP153317 KP153318 KP153319 HM162559 KP153320 HM162560 KP153321 KP153322 KP153323 HM162558 HM162523 JX306083 JN869438 JN869437 (continued next page )

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Table 1. (continued ) Taxon Phyllodesmium macphersonae Phyllodesmium parangatum Piseinotecus gaditanus Piseinotecus sp. Pruvotfolia longicirrha Pruvotfolia pselliotes Sakuraeolis enosimensis Scyllaea pelagica Trivettea papalotla Tritonia antarctica Tritonia festiva Tritonia hamnerorum Tritonia hamnerorum Tritonia nilsodhneri Tritonia nilsodhneri Tritonia nilsodhneri Tritonia nilsodhneri Tritonia pickensi Tritonia sp. 1 Tritonia sp. 2 Tritonia sp. 4 Tritonia sp. F Tritonia sp. Tritonia sp. Tritonia sp. Tritonia sp. Tritonia sp. Tritoniid Tritoniid Tritoniid Tritoniid Tritoniid Tritoniid Tritoniopsis frydis Tritoniopsis sp. Tritoniopsis sp. Tritonopsilla alba Tritonopsilla alba

Voucher no. CASIZ 177493 CASIZ 174440 MNCN15.05/53704 CASIZ 177740 MNCN15.05/53703 MNCN15.05/53705 CASIZ 178876 2 CASIZ 175651 CPIC 01291 CASIZ 171177 CASIZ 186478 CASIZ 181095 CASIZ 181090 CASIZ 176218A CASIZ 176218B CASIZ 176218C CASIZ 176219 CASIZ 175718 CASIZ 191597 CASIZ 189246 CASIZ 177668 CASIZ 179495 CASIZ 191401 CASIZ 190807 CASIZ 191476 CASIZ 173748 CASIZ 181055 CASIZ 189311A CASIZ 189311B CASIZ 189311C CASIZ 189459 CASIZ 189262A CASIZ 189419 CASIZ 181156 CASIZ 191453A CASIZ 191453B CASIZ 69928 CASIZ 69980

16S

GenBank accession numbers COI

H3

HQ010522 JQ699560 HQ616722 HM162604 HQ616723 HQ616725 HQ010538 HM162633 KP153264 HM162643 KP153258 KP153259 KP153260 KP153261 KP153262 KP153263 HM162641 HM162642 KP153265 KP153266 HM162655 HM162644 KP153267 KP153268 KP153269 KP153270 KP153271 KP153272 KP153273 KP153274 KP153275 KP153276 KP153277 KP153278 KP153279 KP153280 KP153281 KP153282



Table 2. Primer sequences for genes of interest Forward (F) and reverse (R) sequences are shown for mitochondrial 16S rRNA (Palumbi), mitochondrial cytochrome c oxidase 1 (Folmer) and nuclear histone 3 (Colgan) Primers 16S 16Sar-L (F) 16Sbr-H (R) COI LCO1490 (F) HCO2198 (R) H3 HexAF (F) HexAR (R)

Sequence 50 –30

Source

CGC CTG TTT ATC AAA AAC AT CCG GTC TGA ACT CAG ATC ACG T

Palumbi (1996) Palumbi (1996)

GGT CAA CAA ATC ATA AAG ATA TTG G TAA ACT TCA GGG TGA CCA AAA AAT CA

Folmer et al. (1994) Folmer et al. (1994)

ATG GCT CGT ACC AAG CAG ACG GC ATA TCC TTG GGC ATG ATG GTG AC

Colgan et al. (1998) Colgan et al. (1998)



Confirmation, purification and sequencing Confirmation of gene amplification of samples from Trivettea papalotla was done using agarose gel electrophoresis with ethidium bromide as the fluorescent tag. Polymerase chain reaction products yielding bands of appropriate size (~695 base pairs (bp) for COI, 560 bp for 16S, and 375 bp for H3) were purified using the Montage PCR Cleanup Kit (Millipore, Belford, MA, USA). Polymerase chain reaction products from all other tritoniid specimens were purified using the ExoSAP-IT kit (Affymetrix, Santa Clara, CA, USA). Nucleotide concentrations were determined using a NanoDrop 1000 spectrophotometer. Purified PCR products from specimens of Trivettea papalotla were sent to Eton Bioscience Inc. (San Diego, CA) for DNA sequencing. Purified PCR products from specimens taken from the California Academy of Sciences underwent cycle-sequencing reactions performed using ABI Prism Big Dye Terminator (Applied Biosystems, Foster City, CA, USA) and analysed using the ABI 3130 Genetic Analyzer in the Center for Comparative Genomics. Digital sequencing chromatographs were assembled and edited using Geneious Pro 4.7.4. Geneious was also used to extract consensus sequences and to construct alignments for each gene using the Geneious alignment option. The alignments were edited manually. Bayesian phylogenetic analyses A phylogenetic analysis was conducted for all genes concatenated. The best-fit models of evolution for the concatenated alignment as well as for the individual gene alignments were determined using the Akaike information criterion (Akaike 1974) implemented in MrModelTest (Nylander 2004). Bayesian analyses were conducted using MrBayes 3.2 (Ronquist et al. 2012), partitioned by gene

Table 3. Summary of datasets used for Bayesian and maximum likelihood analyses and associated parameters Total number of taxa and characters (nucleotide positions) are shown for each alignment. Parameters calculated using the Akaike information criterion, executed with MrModelTest, are as follows: the best-fit model of evolution; nucleotide base frequencies; Ti/Tv ratios; R-matrices; proportion of invariant sites Parameters Number of taxa Number of characters Best-fit model Nucleotide base frequency A C G T R-matrix [A-C] [A-G] [A-T] [C-G] [C-T] [G-T] Proportion of invariant sites

16S

COI

H3

87 498 GTR+I+G

87 664 GTR+I+G

87 328 GTR+I+G

0.3748 0.0816 0.1687 0.3750

0.3003 0.0869 0.1879 0.4249

0.2691 0.2966 0.2494 0.1848

0.9646 3.0008 0.8302 0.7370 5.3050 1.0000 0.2188

5.4166 40.3400 4.5174 13.7713 229.9958 1.0000 0.4515

1.4971 7.1333 2.9471 1.3996 14.0409 1.0000 0.5827

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(unlinked). Each partition was applied to the model of evolution obtained from MrModelltest for that particular gene fragment (Table 3). The Markov chain Monte Carlo analysis was run with two runs of six chains for 10 million generations, with sampling every 100 generations. The default 25% burn-in was applied before constructing the majority-rule consensus tree. Individual Bayesian analyses for each gene alignment were also conducted, in the same manner described above. Maximum likelihood analyses A maximum likelihood analysis was also conducted for the concatenated dataset and for each of the individual gene alignments. Analyses were carried out using GARLI 2.0 (Zwickl 2006). Maximum likelihood analyses were carried out for bootstrap 2000 replicates. Parameters for both the Bayesian and maximum likelihood sets of analyses are summarised in Table 3. Saturation and convergence tests Individual gene sequence alignments were investigated for levels of substitution saturation using the test developed by Xia et al. (2003) and Xia and Lemey (2009) implemented in the program DAMBE (Xia and Xie 2001). The convergence of phylogenetic trees generated in MrBayes was investigated by eye using the ‘Trace’ function in Tracer 1.5 (Rambaut and Drummond 2007). Results Substitution saturation and convergence The results of the substitution saturation test for each gene alignment are summarised in Table 4. Calculated p-values were significant for all gene alignments and Iss (index of substitution saturation) values were less than Iss.c (critical index of substitution saturation) in all cases, indicating little saturation. Bayesian analysis The Bayesian phylogenic analysis conducted on the concatenated dataset resulted in a poorly resolved phylogeny of Cladobranchia (Fig. 2), very similar to that produced by Pola and Gosliner (2010). In this tree, the position of Trivettea papalotla is unresolved, but it is not nested among other species of Tritonia or any of the tritoniid taxa included in the analysis. Species of Tritonia clustered into two separate but strongly supported groups, suggesting that they are not monophyletic (posterior probability = 1 for each group) Table 4. Results of substitution saturation tests for each gene alignment Alignments with a significant p-value (0.05) and Iss (index of substitution saturation) < Iss.c (critical index of substitution saturation) show little saturation. Saturation tests were conducted for fully resolved sites only, in the program DAMBE. OTU = operational taxonomic units; df = degrees of freedom. Results were interpreted for symmetrical trees Datasets 16S COI H3

No. OTU

Iss

Iss.c

T

df

P-value

32 32 32

0.302 0.457 0.283

0.832 0.685 0.707

8.211 5.404 7.949

71 184 67

0.0000 0.0000 0.0000

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Fig. 2. Phylogeny of concatenated mitochondrial 16S rRNA, cytochrome c oxidase 1 and nuclear histone 3 for tritoniid cladobranchs including Trivettea papalotla. Bayesian posterior probabilities are shown above branches and maximum likelihood bootstrap values are shown below branches in parentheses. Members of Tritoniidae in the above phylogeny form two strongly supported monophyletic groups that exclude T. papalotla, supporting the position that it is not a Tritonia. Branch length of the Melibe clade is ~2.3 relative to scale bar.


(Fig. 2). Members of Marionia clustered into one of the two tritoniid clades, while the single included taxon of Marianina clustered into the other. Tritoniidae was not monophyletic in this analysis. Bayesian posterior probability (pp) values in Fig. 1 are shown above respective branches. Values greater than or equal to 0.95 were considered significant (Alfaro et al. 2003). Maximum likelihood analysis The maximum likelihood analysis run on the concatenated dataset produced a phylogeny that was similar to the Bayesian phylogeny described above. Maximum likelihood bootstrap (mlb) values are shown in Fig. 1 in parentheses below their respective branches, below Bayesian pp values. Only mlb values greater than or equal to 70 were considered significant (Hillis and Bull 1993) and are shown. Phylogenies constructed for each of the three individual genes, with Bayesian pp and mlb values above and below branches, respectively, are shown in Fig. S1. Concatenated versus individual gene analyses The concatenated analyses generally resulted in more resolved relationships than the individual gene analyses, and do not fully agree in the position of T. papalotla. In the 16S analyses (both Bayesian and maximum likelihood) Tritoniidae is not monophyletic and T. papalotla appears to be sister to Marionia and other tritoniids. The H3 and COI trees are poorly resolved indicating marker resolution problems, but T. papalotla is not clustering with any of the tritoniid clades recovered. Discussion Bertsch et al. (2009) compared Trivettea papalotla to other tritoniids, particularly those from the eastern Pacific, and identified several unique features to this species. As mentioned above, T. papalotla feeds on an unnamed species of Epizoanthus (Cnidaria, Anthozoa, Zoantharia), while all other known species of Tritoniidae feed on octocorals. As one might expect, given this unique feeding behaviour, the radular morphology of T. papalotla differs substantially from that of other members of Tritoniidae. Tritoniids have tricuspid rachidian radular teeth and numerous lateral teeth, with only one species, Marianina rosea, deviating from this by having a reduced number of lateral teeth (Bertsch et al. 2009). Trivettea papalotla has pectinate rachidian teeth, similar to that of some aeoliids that also feed on zoanthids, and lacks lateral teeth. Externally, T. papalotla possesses prominent dorsal vessels radiating from the cardiac region, a feature completely absent from all other eastern Pacific tritoniids. Moreover, species of Tritoniidae have prominently displayed branched secondary gills, whereas T. papalotla has digitiform secondary gills that line the raised edges of the mantle and are completely retractable. Bertsch et al. (2009) also studied some features of the reproductive system of Trivettea papalotla and commented on larval development of this species. The reproductive system of T. papalotla has both a bursa copulatrix and receptaculum seminis, whereas the bursa copulatrix is absent in other tritoniids. Bertsch et al. (2009) noted that the overall arrangement of the reproductive system in T. papalotla is similar to that found in species of Berthella. Finally, the size of the eggs in T. papalotla


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suggests that this species is likely a lecithotrophic or intracapsular ‘direct’ developer, which contrasts with the planktotrophic development of most tritoniids found in the eastern Pacific. Bertsch et al. (2009) included T. papalotla in Tritonia because in their morphological phylogenetic analysis, this species was nested within a clade containing species of Tritonia, Marionia, Tritoniella and Tochuina. Despite the fact that these groups were poorly supported and the tree generally lacked robustness (Bertsch et al. 2009), T. papalotla was tentatively placed within Tritonia due to the absence of stomach plates. This tentative classification suggested that the unique traits of T. papalotla are autopomorphies, possibly derived from its unique diet. In contrast, Bertsch (2014) reinterpreted the taxonomic value of the unique characters of T. papalotla, and referring to our unpublished molecular data, introduced the genus name Trivettea for this species. Bertsch (2014) did not place Trivettea into any higher order taxonomic grouping within the Cladobranchia. The results of the present molecular phylogenetic analysis contradict the results of the morphological phylogenetic analysis by Bertsch et al. (2009), which placed Tritonia papalotla nested within Tritonia, and instead provide support for the classification of T. papalotla into its own supraspecific taxon as proposed by Bertsch (2014). Historically there has been disagreement among systematists as to whether or not it is appropriate to recognise the validity of monotypic genera. Platnick (1976) argued that from a cladistic perspective, evolution is a process in which genetically unified populations are divided into sister groups, usually dichotomously. He argued that any existing taxon must have or have had at least one sister taxon and thus cannot be a part of a monotypic genus without also being paraphyletic. On the contrary, Wiley (1977) argued that if a monotypic genus is considered paraphyletic because it excludes some descendants of the most recent ancestor, then those excluded descendants, even if they formed a monophyletic group before the classification of the monotypic genus, would also have to be considered paraphyletic, because they exclude a descendent of the most common ancestor. Also, a species in a monotypic genus could potentially be the common ancestor of future cladogenesis that might result in a more speciose genus (Wiley 1977). Scotland and Sanderson (2004) discussed biodiversity in the context of species richness and indicated most taxonomic groups are species poor, and few are species rich. They developed a novel predictive model, called the ‘simultaneous broken tree’, for use as a comparative tool against real taxonomic species richness data. Their model suggested there might actually be much higher percentages of monotypic groups than what has been observed. Scotland and Sanderson (2004) postulated that the discrepancies between their model and the real data could be taxonomic rather than evolutionary, given that taxonomists tend to avoid studying too small or too large genera. Based on the results of the present study and review of relevant literature, Trivettea likely constitutes a monotypic higher taxon, although the matter of its proper placement within Cladobranchia is still in question. At present, the cladobranch clade suffers from a general lack of resolution. A full reconstruction of the phylogeny of Cladobranchia based on additional molecular markers would likely be necessary to begin to resolve the issues in this group. It seems, however, that larger datasets derived from next generation

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sequencing techniques will be necessary to fully address this problem. Acknowledgements We would like to acknowledge two anonymous reviewers and the associate editor (Nerida Willson) for constructive comments on the manuscript that greatly improved its quality. The NIH supported MBRS-RISE program provided us with funding for laboratory supplies and sequencing. The California Academy of Sciences allowed us to take tissue samples from nudibranch specimens in their invertebrate zoology collection. Finally, we would like to acknowledge Joshua Hallas from the California Academy of Sciences for his guidance and training.

References Akaike, H. (1974). A new look at the statistical model identifications. IEEE Transactions on Automatic Control 19, 716–723. doi:10.1109/ TAC.1974.1100705 Alfaro, M. E., Zoller, S., and Lutzoni, F. (2003). Bayes or bootstrap? A simulation study comparing the performance of Bayesian Markov chain Monte Carlo sampling and bootstrapping in assessing phylogenetic confidence. Molecular Biology and Evolution 20, 255–266. doi:10.1093/ molbev/msg028 Ballesteros, M., and Avila, C. (2006). A new tritoniid species (Mollusca: Opisthobranchia) from Bouvet Island. Polar Biology 29, 128–136. doi:10.1007/s00300-005-0059-4 Bertsch, H. (2014). Biodiversity in La Reserva de la Biósfera Bahía de los Ángeles y Canales de Ballenas y Salsipuedes: naming of a new genus, range extensions and new records, and species list of Heterobranchia (Mollusca: Gastropoda), with comments on biodiversity conservation within marine reserves. The Festivus 46, 158–175. Bertsch, H., Valdés, A., and Gosliner, T. M. (2009). A new species of tritoniid nudibranch, the first found feeding on a zoanthid anthozoan, with a preliminary phylogeny of the Tritoniidae. Proceedings of the California Academy of Sciences 60, 431–446. Colgan, D. J., McLauchlan, A., Wilson, G. D. F., Livingston, S. P., Edgecombe, G. D., Macaranas, J., Cassis, G., and Gray, M. R. (1998). Histone H3 and U2 snRNA DNA sequences and arthropod molecular evolution. Australian Journal of Zoology 46, 419–437. doi:10.1071/ ZO98048 Folmer, O., Black, M., Hoeh, W., Lutz, R., and Vrijenhoek, R. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3, 294–299. Gosliner, T. M., and Ghiselin, M. T. (1987). A new species of Tritonia from the Caribbean Sea. Bulletin of Marine Science 40, 428–436. Hillis, D. M., and Bull, J. J. (1993). An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Systematic Biology 42, 182–192. doi:10.1093/sysbio/42.2.182 Katz, P. S. (1998). Neuromodulation intrinsic to the central pattern generator for escape swimming in Tritonia. Annals of the New York Academy of Sciences 860, 181–188. doi:10.1111/j.1749-6632.1998.tb09048.x

McDonald, G., and Nybakken, J. (1999). A worldwide review of the food of nudibranch mollusks. Part II. The suborder Dendronotacea. The Veliger 42, 62–66. Nylander, J. A. A. (2004). ‘MrModeltest v2.’ Available at https://www.abc. se/~nylander/mrmodeltest2/mrmodeltest2.html [Verified June 2013]. Odhner, N. H. (1963). On the taxonomy of the family Tritoniidae. The Veliger 6, 48–52. Palumbi, S. R. (1996). Nucleic acids II: the polymerase chain reaction. In ‘Molecular Systematics’. (Eds D. M. Hillis, C. Moritz and B. K. Mable.) pp 205–247. (Sinauer Associates: Sunderland, Massachussetts.) Platnick, N. I. (1976). Are montypic genera possible? Systematic Zoology 25, 198–199. doi:10.2307/2412749 Pola, M., and Gosliner, T. M. (2010). The first molecular phylogeny of cladobranchian opisthobranchs (Mollusca, Gastropoda, Nudibranchia). Molecular Phylogenetics and Evolution 56, 931–941. doi:10.1016/j. ympev.2010.05.003 Rambaut, A., and Drummond, A. J. (2007). ‘Tracer v1.5.’ Available at http:// beast.bio.ed.ac.uk/Tracer. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M. A., and Huelsenbeck, J. P. (2012). MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61, 539–542. Scotland, R. W., and Sanderson, M. J. (2004). The significance of few versus many in the tree of life. Science 303, 643. doi:10.1126/science. 1091483 Smith, V. G., and Gosliner, T. M. (2003). A new species of Tritonia from Okinawa (Mollusca: Nudibranchia), and its association with a gorgonian octocoral. Proceedings of the California Academy of Sciences 54, 255–278. Wiley, E. O. (1977). Are montypic genera paraphyletic? A response to Norman Platnick. Systematic Zoology 26, 352–355. doi:10.2307/ 2412683 Willows, A. O. D., and Hoyle, G. (1967). Correlation of behavior with the activity of single identifiable neurons in the brain of Tritonia. In ‘Neurobiology of Invertebrates’. (Ed. J. Salánki.) pp. 443–461. (Akadémiai Kiadó: Budapest.) Xia, X., and Lemey, P. (2009). Assessing substitution saturation with DAMBE. In ‘The Phylogenetic Handbook: a Practical Approach to DNA and Protein Phylogeny, 2nd Edn’. (Eds P. Lemey, M. Salemi and A. M. VanDamme.) pp. 615–630. (Cambridge University Press: Cambridge, UK.) Xia, X., and Xie, Z. (2001). DAMBE: data analysis in molecular biology and evolution. The Journal of Heredity 92, 371–373. doi:10.1093/jhered/ 92.4.371 Xia, X., Xie, Z., Salemi, M., Chen, L., and Wang, Y. (2003). An index of substitution saturation and its application. Molecular Phylogenetics and Evolution 26, 1–7. doi:10.1016/S1055-7903(02)00326-3 Zwickl, D. J. (2006). Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion. Ph.D. Thesis, University of Texas at Austin, Austin, Texas.

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