Mar Biodiv DOI 10.1007/s12526-016-0507-0 ORIGINAL PAPER First record of the Aeolid Anteaeolidiella fijensis (Nudibranchia, Aeolidiidae) from India Leila Carmona 1,3 & Juan Lucas Cervera 1 & Appukuttannair Biju Kumar 2 & Balachandran Komalam Snehachandran 2 Received: 31 August 2015 / Revised: 9 May 2016 / Accepted: 11 May 2016 # Senckenberg Gesellschaft für Naturforschung and Springer-Verlag Berlin Heidelberg 2016 Abstract The aeolid Anteaeolidiella fijensis Carmona, Bhave, Salunkhe, Pola, Gosliner and Cervera, 2014 is reported for the first time from India. Differences in the external colouration compared with the original description hampered its identification, and therefore, a molecular approach was needed. Maximum-likelihood and Bayesian analyses of partial DNA sequences of the mitochondrial cytochrome c oxidase subunit I and 16S rRNA genes, and the nuclear gene histone-3 were used to infer phylogenetic trees. ABGD species delimitation analyses and morphological study complemented this contribution. This is the first record of Anteaeolidiella fijensis outside the type locality and extends its distribution range remarkably. Here, we also complete the original description with new data regarding the external colouration and internal anatomy. Finally, the instraspecific variation may be related with an ontogenetic process or be a populationally distinctive feature. Keywords Mollusca . Aeolidida . India . Biodiversity . New record Communicated by V. Urgorri * Leila Carmona leila.carmona.barnosi@marine.gu.se 1 Departamento de Biología, Facultad de Ciencias del Mar y Ambientales, Ampus de Excelencia Internacional del Mar (CEI · MAR,) Universidad de Cádiz, Polígono Río San Pedro, s/n, Ap.40, 11510 Puerto Real (Cádiz), Spain 2 Department of Aquatic Biology and Fisheries, University of Kerala, Thiruvananthapuram 695581, Kerala, India 3 Present address: Department of Marine Sciences, University of Gothenburg, Box 460, 40530 Gothenburg, Sweden Introduction At the beginning of this century, Miller (2001) erected the aeolidiid genus Anteaeolidiella, which was lately reviewed by Carmona et al. (2014a) that diagnosed the members of this genus as aeolids with predominant orange and/or white colouration and large oral glands covered by giant secretory cells. Carmona et al. (2014a) pointed out the relevance of colour patterns within Anteaeolidiella, despite the fact these markings have been considered as an intraspecific variation of the type species A. indica Bergh 1888 (e.g. Gosliner and Griffiths 1981). In addition, Carmona et al. (2014a) stated that in terms of biogeography, all the species of the genus Anteaeolidiella present a quite restricted distribution, with the exception of A. lurana (Er. Marcus and Ev. Marcus, 1967), which can be found from the Mediterranean to Brazil as well as in the Eastern Pacific. On the other hand, the knowledge about the Indian fauna of Opisthobranchia (here, used as an informal group, since its monophyly has been rejected, e.g. Wägele et al. 2014) is quite incomplete and irregular. The most comprehensive study was that conducted in the 70s by Narayanan (1969, 1970, 1971a, b), although, in the last seven years, several contributions have increased the total knowledge of opisthobranch diversity in India (Apte 2009; Apte et al. 2010; Ramakrishna et al. 2010; Bhave and Apte 2011; Sankar et al. 2011; Sreeraj et al. 2012; Apte and Bhave 2014; Poriya et al. 2015; Prasade et al. 2015). In this contribution, we present the first record of Anteaeolidiella fijensis Carmona, Bhave, Salunkhe, Pola, Gosliner and Cervera, 2014 from India, the second species of this genus reported in this country so far. In addition, we complete the original description of this species based on the Indian specimen. Table 1 List of specimens used for phylogenetic analyses. Abbreviations: EA, Eastern Atlantic; EP, Eastern Pacific; GB, GenBank Family Tritoniidae Lamarck, 1809 Aeolidiidae Gray, 1827 Species name Tritonia challengeriana Bergh, 1884 Anteaeolidiella cacaotica (Stimpson, 1855) Anteaeolidiella chromosoma (Cockerell & Eliot, 1905) Anteaeolidiella fijensis Carmona, Bhave, Salunkhe, Pola, Gosliner & Cervera, 2014 Anteaeolidiella ireneae Carmona, Bhave, Salunkhe, Pola, Gosliner & Cervera, 2014 Anteaeolidiella lurana (Marcus & Marcus, 1967) Anteaeolidiella oliviae (MacFarland, 1966) Anteaeolidiella poshitra Carmona, Bhave, Salunkhe, Pola, Gosliner & Cervera, 2014 Babakinidae Roller, 1973 Anteaeolidiella saldanhensis (Barnard, 1927) Anteaeolidiella takanosimensis (Baba, 1930) Babakina indopacifica Gosliner, GonzálezDuarte & Cervera, 2007 Locality Collection dates Voucher GenBank accession nos. COI 16S H3 Bouvetoya (EA, GB) Eastern Australia Japan Line Is. Mexico (EP) 30Jun04 14Feb10 10Aug10 28Sep06 26Feb06 MNCN/ADN: 51922 MNCN/ADN: 51924 CASIZ 174212 CASIZ 173060 HM162718 JX087528 – JQ997030 JQ997018 HM162643 JX087455 JX087457 JQ996825 JQ996812 HM162550 JX087590 – JQ996926 JQ996911 Peru Fiji 04Dic06 20Aug06 ZSM Mol 20090868 ZSM Mol 20070193 – JQ997020 JQ996813 JQ996815 JQ996912 JQ996914 India Clipperton Is. DABF-UOK/GAS 4 1998 CASIZ 115686 KX276653 JQ997028 KX276652 JQ996823 KX276655r JQ996924 Clipperton Is. Eastern Australia 1998 05Aug06 CASIZ 115686 C.477129.003 JQ997029 KF273324 JQ996824 KF273321 JQ996925 KF273328 Eastern Australia Bermuda Brazil Brazil Italy Mexico California India 05Aug06 20Jun09 19Dic07 14Dec08 Oct11 Aug06 26May09 11Dec09 C.477129.002 ZMBN82992 MZSP97638 MZSP97574 CASIZ 186820 CNMO 2996 CASIZ 181315 BNHS-opistho-38 KF273325 – JQ997027 – JQ997031 KF273326 JQ997034 KF273327 KF273322 JQ996821 JQ996822 – JQ996826 KF273323 JQ996829 – KF273329 JQ996922 – JQ996923 JQ996927 – JQ996930 – India South Africa (EA) Japan Philippines (GB) 2Jan14 06Jan08 19Mar06 20Mar08 DABF-UOK/GAS 5 CASIZ 176313 MNCN/ADN: 51923 – KX276654 JQ997032 JX087529 HM162754 – JQ996827 JX087456 HM162678 KX276656 JQ996928 JX087591 HM162587 Mar Biodiv Mar Biodiv One specimen of Anteaeolidiella fijensis was studied. The remaining species of the genus Anteaeolidiella were those included by Carmona et al. (2014a), plus an additional specimen of A. poshitra from India (see Table 1 for a full list of samples, localities and vouchers). Tritonia challengeriana Bergh, 1884 and Babakina indopacifica Gosliner, GonzalezDuarte and Cervera, 2007 were chosen as outgroups based on the contribution of Carmona et al. (2013). Tissue samples were taken from the foot. The DNeasy blood & tissue kit of Qiagen (Qiagen, Valencia, CA, USA; 09/2001) was used for DNA extraction. Partial sequences of cytochrome c oxidase subunit I (COI), 16S and H3 were amplified by polymerase chain reaction (PCR) using the primers: LCO1490 (5′GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAATCA-3′) (Folmer et al. 1994) for COI; 16S ar-L (5′CGCCTGTTTATCAAAAACAT-3′) and 16S br-H (5′CCGGTCTGAACTCAGATCACGT-3′) (Palumbi et al. 1991) for 16S rRNA; and H3AD5′3′ (5′ATGGCTCGTACCAAGCAGACVGC-3′) and H3BD5′3′ (5′-ATATCCTTR GGCATRATRGTGAC-3′) (Colgan et al. 1998) for H3. These three gene regions are commonly used in systematic studies of heterobranchs (e.g. Ohnheiser and Malaquias 2013; Ortigosa et al. 2014; Pola et al. 2014a, b; Eilertsen and Malaquias 2015; Hallas and Gosliner 2015; Shipman and Gosliner 2015). PCRs were conducted in 25-μl volume reactions containing 1 μl of both forward and Fig. 1 Phylogenetic hypothesis for the genus Anteaeolidiella based on the combined dataset (H3 + COI + 16S) inferred by Bayesian inference (BI) analysis. Numbers above branches represent posterior probabilities from BI. Numbers below branches represent bootstrap values from ML. Different species delimitation methods results are also plotted. Specimens sequenced in this study are in bold. Abbreviations: A, ABGD, based on COI data set, with both models (Jukes-Cantor and Kimura); B, number of species based on SplitsTree results; green rectangle = Anteaeolidiella chromosoma; red rectangle = Anteaeolidiella oliviae; dashed rectangle = Anteaeolidiella fijensis; orange rectangle = Anteaeolidiella lurana; blue rectangle = Anteaeolidiella saldanhensis; yellow rectangle = Anteaeolidiella cacaotica; grey rectangle = Anteaeolidiella poshitra; pink rectangle = Anteaeolidiella takanosimensis; purple rectangle = Anteaeolidiella ireneae Material and methods Molecular work DNA extraction, amplification and sequencing Mar Biodiv reverse primers (10 μM), 2.5 μl of dNTP (2 mM), a genedependent amount of magnesium chloride (25 mM), 0.25 μl of Qiagen DNA polymerase (5 units/μl), 5 μl of BQ-solution^ (5x), 2.5 μl of Qiagen buffer (10x; Qiagen Taq PCR core kit cat. no. 201225) and 2 μl of genomic DNA. Magnesium chloride amounts were 3.5 μl for COI and 16S, and 2 μl for H3. Amplification of COI was performed with an initial denaturation for 5 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 30 s at 44 °C (annealing temperature) and 1 min at 72 °C with a final extension of 7 min at 72 °C. The 16S amplification began with an initial denaturation for 5 min at 95 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 44 °C (annealing temperature), 1 min at 72 °C with a final extension of 7 min at 72 °C. H3 amplification was performed with an initial denaturation for 3 min at 95 °C, followed by 40 cycles of 45 s at 94 °C, 45 s at 50 °C (annealing temperature), 2 min at 72 °C, with a final extension of 10 min at 72 °C. Sequence reactions were run on a 3730XL DNA sequencer, Applied Biosystems. All new sequences have been deposited in GenBank (Table 1). Sequence alignment and phylogenetic analyses Sequences were assembled and edited with Geneious Pro v. 4.7.6 (Drummond et al. 2009) and aligned in MAFFT (Katoh et al. 2009). The alignments were further checked using MacClade v. 4.06 (Maddison and Maddison 2005). Proteincoding sequences were translated into amino acids for confirmation of alignment. The most variable regions from the 16S rRNA alignment were removed using the default settings in Gblocks (Talavera and Castresana 2007). Excluding Bindelrich^ regions, the tree was in general very similar when Fig. 2 Neighbour-Net graph of the COI sequences obtained with SplitsTree including the variable regions. Therefore, final analyses were performed including all bases. Sequences of COI, 16S and H3 were trimmed to 658, 438 and 327 base pairs, respectively. Single gene analyses and a concatenated analysis were performed. The best-fit evolutionary model was determined in MrModeltest v. 2.3 (Nylander 2004), using the Akaike information criterion (Akaike 1974). The GTR + G was selected for the first and third position (H3 and COI), GTR + I for the second codon position of H3 and COI, and HKY + I + G for the 16S. Maximum likelihood (ML) analyses were performed using the software RAxML v7.0.4 (Stamatakis et al. 2008). Node support was assessed with non-parametric bootstrapping (BS) with 5000 replicates, random starting trees, and parameters estimated from each dataset under the model selected for the original dataset. MrBayes v. 3.1.2 (Ronquist and Huelsenbeck 2003) was used for Bayesian inference (BI) analysis and to estimate posterior probabilities (PP) for node support with two runs of 5,000,000 generations each. Convergence was checked in TRACER v. 1.5 (Drummond and Rambaut 2007) with a burn-in of 25 %. Only nodes supported by BS ≥ 70 (Hillis and Bull 1993) and PP ≥ 0.95 (Alfaro et al. 2003) are discussed. Species delimitation analyses To define species, we used an integrative approach including tree topologies, Carmona et al. (2014a) cut-off values, automatic barcode gap discovery (ABGD), the external colouration of the living animals and the morphological data. Pairwise uncorrected p-distance values between each taxon were calculated for the COI gene using PAUP* 4.0b 10.0 (Swofford 2002). We also applied the ABGD method Mar Biodiv (Puillandre et al. 2012). This method was run for COI and for all the specimens of the genus Anteaeolidiella included in this study. ABGD settings were the following: Pmin = 0.001, Pmax = 0.1, Steps = 10, X = 1.0, Nb bins = 20, and Jukes Cantor (JC69) and Kimura (K80). Additionally, SplitsTree 4 (Huson and Bryant 2006) was used to construct the NeighborNet network (Bryant and Moulton 2004). The resulting tree was converted to graphics in FigTree v1.4.0 and final adjustments were done in Adobe Illustrator CS5. corresponded with each species Anteaeolidiella of the genus included in this study. Finally, the SliptsTree network depicts a small level of conflict (contradictory patterns) among Anteaeolidiella species due to the presence of parallel edges of equal lengths, especially between A. poshitra and its sister species A. takanosimensis (Fig. 2). In addition, the SplitsTree analysis supports the results obtained by the tree reconstructions and ABGD analysis (Figs. 1 and 2). Systematics Morphological study Source of specimen and morphology The specimen was dissected by dorsal incision. Their internal features were examined and drawn under a stereoscopic microscope with the aid of a camera lucida. Special attention was paid to the morphology of the reproductive system and oral and salivary glands. The buccal mass was removed and dissolved in 10 % sodium hydroxide until the radula was isolated from the surrounding tissue. The radula was then rinsed in water, dried and mounted for examination via scanning electron microscopy (SEM). Voucher specimens are held at the museum collections of the Department of Aquatic Biology and Fisheries, University of Kerala, Thiruvananthapuram 695581, Kerala, India (Accession numbers DABF-UOK/GAS 04 and DABF-UOK/GAS 05). Family Aeolidiidae Gray, 1827 Genus Anteaeolidiella Miller 2001 Anteaeolidiella fijensis Carmona, Bhave, Salunkhe, Pola, Gosliner and Cervera, 2014. Material examined: DABF-UOK/GAS 04, one specimen dissected, 8 mm in length preserved, Kavarathi Island (10033′ 54″N 72037′34″E), India, 2 January 2014, found in Results Molecular results The concatenated dataset generated an alignment of 1423 base pairs. No saturation was observed across genes and codon positions (not shown). Topologies of the ML trees were congruent with the results yielded by BI analysis and, thus, ML trees are not shown. Figure 1 shows the genus Anteaeolidiella phylogenetic hypothesis based on the combined dataset represented by BI. This figure also illustrates the results obtained from different delimitation approaches used in this study. Our results confirmed the monophyly of the genus Anteaeolidiella since all the specimens of this genus included in the analyses clustered together (PP = 1, BS = 93). The genus Anteaeolidiella was constituted by two clades, one with A. olivae and A. chromosoma (PP = 1, BS = 95) and a second clade with the remaining species of this genus included in this study (PP = 1, BS = 100). Within this larger group, both specimens of A. fijensis (one from Fiji, the type locality, and one from India) appeared together (PP = 1, BS = 100) and with a basal position. The ABGD analyses recovered nine groups with P values ranging from 0.001 to 0.06, independently from the chosen model (Jukes and Cantor 1969 and Kimura 1980). Each group Fig. 3 Different morphotypes of Anteaeolidiella fijensis. a–b, Photograph of the living animals and schematic dorsal colour patterns of the specimen from Fiji (taken from Carmona et al. 2014a); c–e, photographs of the living animals and schematic dorsal colour patterns of the specimen from India Mar Biodiv association with the bryozoan Bugula sp., at 0.80-m depth, collected by R. Ravinesh. Type locality: Laucala Bay, Fiji (Carmona et al. 2014a). Geographical distribution: Known from Fiji (Carmona et al. 2014a) and Kavarathi Island, India (present study). External morphology (Fig. 3): Body aeolid form, elongate, moderately slender, tapering gradually towards posterior end of foot. Propodial tentacles stout, slightly protruded. Background colour translucent white with bright orange pigment over dorsum (Fig. 3a–b). Head with orange mark. Chalky white area behind rhinophores, forming a continuous patch over dorsum, less dense over pericardial area (Fig. 3c– e). Rhinophores translucent white, with some orange pigmentation and white tips white. Rhinophores stout. Oral tentacles translucent white, with some orange pigmentation. Oral tentacles conical, slightly longer than rhinophores. Black eyes visible behind rhinophores. Cerata longer than rhinophores, moderately thick, extending from rhinophores to metapodium, leaving bare space over dorsum. Ceratal epithelium diffusely covered with orange pigment and chalky white pigmentation. Ramifications of digestive gland greenish or brownish. Subapical band translucent. Cnidosac white. Cerata in up to 13 rows, each with 2–15 cerata. Anus cleioproctic, between second and third rows of first ceratal group of right posterior digestive branch. Genital pore ventral to first right row. Anatomy (Fig. 4): Masticatory border of jaws smooth (Fig. 4a). Radular formula 18 x 0.1.0 (DABF-UOK/GAS Fig. 4 Internal anatomy of Anteaeolidiella fijensis. a detailed view of the masticatory border of the jaws, scale bar = 100 μm; b radular teeth, scale bar = 50 μm; c reproductive system, scale bar = 0.5 mm. Abbreviations: am ampulla, fgm female gland mass, ps penial sac, rs receptaculum seminalis, s spermoviduct, va vagina, vd vas deferens 04). Teeth wide, bilobed. Central cusp triangular, elongate, with 28–30 elongate and acutely pointed denticles on each side (Fig. 4b). Outermost denticles larger than innermost. Oral glands conspicuous, large, fusiform, dorso-laterally to buccal bulb. Each oral gland with rows of giant secretory cells. Salivary glands are absent. Reproductive system diaulic (Fig. 4c). Hermaphroditic duct widening into moderately long ampulla. Spermoviduct dividing into oviduct and vas deferens. Vas deferens elongate, entering wider proximal portion of penial sac with unarmed penial papilla. Receptaculum seminis pyriform, inserting into short oviduct, before latter forms female glands. Vagina ventral to penis. Discussion Carmona et al. (2014a) first described this species from the Fiji Republic. The specimens studied by the authors presented a less complex colouration, being predominantly orange, with some white pigmentation (Fig. 3a–b). Therefore, the chalky white appearance of the specimen reported herein clearly differs from the holotype of A. fijensis, but our molecular approach as well as the ABGD species delimitation analyses support that both specimens belong to the same species. Despite difference in the chalky white pigmentation, our specimen from India and those studied by Carmona et al. (2014a) Mar Biodiv have some external similarities. For instance, the colouration of the rhinophores and oral tentacles are the same, as well as the orange mark over the head matches. In addition, with regard to anatomical features, the only difference was the number of the radular denticles, more abundant in the Indian specimen (30 against 20 denticles in the Holotype) but probably because of its larger size. This intraspecific variation in terms of external colouration could be ontogenetic, since those specimens studied by Carmona et al. (2014a) were 4 mm long, while the specimen from India reaches 8 mm. This variability due to size has been also reported in the aeolidiid genus Spurilla, in particular within Spurilla braziliana MacFarland, 1909 (Carmona et al. 2014b). Another hypothesis is that each morphotype is particular for each population, which was also observed in those specimens of Spurilla braziliana from the Pacific (Carmona et al. 2014b). However, in order to confirm any of these hypotheses more material of A. fijensis is needed. Carmona et al. (2014a) pointed out that the diversity of the genus Anteaeolidiella was far from being completed. Actually, these authors suggested Bin India more than one Anteaeolidiella species probably cohabit^, based on the pictures published on websites. This study confirms that theory, contributes to a better knowledge of the Bopisthobranch^ diversity in India, and extends the distribution range of A. fijensis notably. Acknowledgments This work was supported by the research grant (CGL2010-17187), Spanish Ministry of Economy and Competitiveness (includes the early Ministry of Sciences and Innovation) to J. L. Cervera. This work was supported by the Department of Biotechnology (Government of India) as well by the Department of Science and Technology (Government of India) with the INSPIRE fellowship. The assistance of Mr R. Ravinesh in field collection is gratefully acknowledged. We would like to thank to Deneb Ortigosa for helping with the SEM, and to Vanessa González Ortiz for her support with the illustrations of this manuscript. This is CEI · MAR journal publication 130. 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