Italian Journal of Zoology, 2016, 1–13 http://dx.doi.org/10.1080/11250003.2016.1150525

A DNA-barcoding approach to the phenotypic diversity of Mediterranean species of Felimare Ev. Marcus & Er. Marcus, 1967 (: ), with a preliminary phylogenetic analysis

G. FURFARO1*, M. V. MODICA2, M. OLIVERIO2, & P. MARIOTTINI1

1Dipartimento di Scienze, Università degli Studi di “Roma Tre”, Roma, Italy, and 2Dipartimento di Biologia e Biotecnologie “Charles Darwin”, Università di Roma “La Sapienza”, Roma, Italy

(Received 4 November 2015; accepted 26 January 2016)

Abstract The chromodorid nudibranchs Felimare tricolor (Cantraine, 1835), F. fontandraui (Pruvot-Fol, 1951) and F. picta (Shultz in Philippi, 1836) typically show highly variable colour patterns that are misleading for species identification. Their has long been confused, and their identification can be difficult if based only on morphological characters. We carried out molecular genetic analyses using the mitochondrial cytochrome c oxidase subunit I (COI) and 16S rDNA markers on Mediterranean samples of these three taxa representing a large part of their range of chromatic variation. Results proved the usefulness of the DNA-barcoding approach in assessing species delimitation in this group, and in identification of specimens with body colours not clear for species recognition. We inferred preliminary phylogenetic relationships among one third of the currently recognised species of Felimare: they indicated with good support sister species relationship among Felimare tricolor and F. fontandraui, and suggested (with low support) potential relationships among F. picta, F. verdensis, F. californiensis and F. agassizi.

Keywords: Mollusca, Nudibranchia, Felimare, colour morphotypes, Mediterranean Sea

Introduction the 20th century by Ortea et al. (1996). Among these, specimens referable to Felimare picta (Shultz in Chromodorid nudibranchs (family Philippi, 1836) have been described in the past under Bergh, 1891) are brightly coloured sea slugs, very different names (Trainito 2005;Trainito&Doneddu common in tropical and subtropical coastal waters, 2014) due to their extreme colour variability. In parti- mostly in the Indo-West Pacific region, and, due also cular, the dark blue morphotype was considered an to their aposematic brilliant chromatism, they are very Atlantic species [as Hypselodoris webbi (d’Orbigny, attractive subjects for diving photographers. 1839), Canary Islands as the type locality], with the Downloaded by [Universita Studi la Sapienza] at 05:25 29 February 2016 Chromodorididae is the most diverse family of light blue/yellowish form as its Mediterranean counter- opisthobranchs, and according to the most complete part [as Hypselodoris valenciennesi (Cantraine, 1841), molecular phylogenetic analysis of this group (Johnson Sicily as the type locality]. Their taxonomy has been &Gosliner2012) they are classified into 17 genera. In reviewed by Ortea et al. (1996), who considered a the new classification of chromodorids proposed by single widespread species, with several subspecies, of these authors, the Felimare Ev. Marcus & Er. which three are currently (WoRMS: Bouchet & Marcus, 1967 is represented by 36 species in the World Caballer 2016) considered as valid: F. picta picta, F. Register of Marine Species (WoRMS: Bouchet & picta azorica (Ortea et al. 1996) (Azores as the type Caballer 2016), of which nine occur in the locality), F. picta verdensis (Ortea et al. 1996)(Cabo Mediterranean Sea. All of the eastern Atlantic- Verde as the type locality). Mediterranean members of the genus Felimare,includ- Felimare tricolor (Cantraine, 1835), one of the most ing the species analysed in this work, belong to the so- common Mediterranean species of the genus, has called “blue chromatic group”,asdefined at the end of

*Correspondence: G. Furfaro, Dipartimento di Scienze, Università degli Studi di “Roma Tre”, Viale Marconi 446, I-00146 Rome, Italy. Tel: +39 0657336359. Fax: +39 0657336321. Email: [email protected]

© 2016 Unione Zoologica Italiana 2 G. Furfaro et al.

been reported in association with sponges of the gen- & Johnson 1999; Haber et al. 2010). However, era Dysidea Johnston, 1842, Scalarispongia Cook & Chemello and Tumbiolo (1992), dealing with the Bergquist, 2000 and Spongia Linnaeus, 1759 and dis- change of colour pattern during the development of plays a quite variable aposematic chromatism (Ortea F. fontandraui, reported the appearance of MDF in et al. 1996;Trainito&Doneddu2014). There is a Italian specimens of F. fontandraui 8 mm long, raising long-unresolved issue about the valid name of this doubts about the validity of this character for species species (Gosliner & Johnson 1999), mostly due to identification. The high level of intraspecificchro- the original description of Doris tricolor (Cantraine, matic variation in both F. tricolor and F. fontandraui 1835) from Sicily, reporting morphological features, has been well documented in the literature (Trainito like the respiratory system being completely blue, that 2005; Calado & Silva 2012; Trainito & Doneddu did not describe the intraspecific variability of this 2014), as well as depicted in the Sea Slug Forum taxon. Hypselodoris midatlantica Gosliner, 1990 was (Rudman 2001), where several specimens were not introduced with the aim of partly stabilising the identified and/or not clearly distinguishable. nomenclature. However, WoRMS (Bouchet & Specimens of Felimare from a Tyrrhenian locality, Caballer 2014) adopted F. tricolor as the valid name found associated with the poriferan Spongia lamella for this taxon (with F. midatlantica as a synonym). (Schulze, 1879) for feeding and laying eggs, dis- According to Ortea et al. (1996) and Calado and played a range of colour patterns often difficult to Silva (2012) F. tricolor and F. fontandraui (Pruvot- assign unambiguously to either F. tricolor or F. fon- Fol, 1951), albeit very similar, can be diagnosed by tandraui (Figure 1a, b), including individuals with the uniformly blue-coloured rhinophores of F. tricolor, the median line of the notum frayed and sometimes whilst a white/yellow spot is positioned at their base in discontinued; most of these specimens showed a line F. fontandraui (Calado & Silva 2012). F. fontandraui on both sides of the notum that was patchy and was originally described by Pruvot-Fol on specimens larger than the ones normally observed in typical from Banyuls. The absence in F. fontandraui of repug- F. tricolor (Figure 1c); some peculiar morphotypes natorial glands, definedasmantledermalformations showed the median line expanded behind the (MDF), present in F. tricolor, was also considered a rhinophores and anchor-shaped (Figure 1a), which good diagnostic character (Ortea et al. 1996;Gosliner is normally considered a diagnostic sketch of Downloaded by [Universita Studi la Sapienza] at 05:25 29 February 2016

Figure 1. Phenotypic variability of Felimare tricolor (Cantraine, 1835). (a, b) Individuals from “Secche di Tor Paterno” MPA, Italy, 41° 36ʹ18ʹʹN, 12°20ʹ20ʹʹE; 25 m depth; showing the atypical “anchor shape” on the head or the enlarged dorsal line. (c) The typical colour pattern of F. tricolor from Sant’Agostino, Italy, 42°07ʹ00.7ʹʹN, 11°45ʹ51.9ʹʹE. (d) Specimen BAU2051 from Ischia Is., Italy, 40°43ʹ50ʹʹN, 13° 58ʹ04ʹʹE; 30 m depth; with the lateral yellow line fragmented and discontinuous. DNA-barcoding and phylogeny of Mediterranean Felimare nudibranchs 3

F. fontandraui. Individuals (marked with * in agradedseries(50–70-90–95-100% EtOH), critical Table I) displaying chromatic features intermediate point-dried in a Balzer Union CPD 030 unit, gold between typical F. tricolor and F. fontandraui have coated in an Emitech K550 unit, and finally exam- been observed also at other central Tyrrhenian local- ined using the fieldemissionSEMcolumnofthe ities (Figures 1d, 2a). Dualbeam (FIB/SEM) Helios Nanolab (FEI DNA characters have proven very useful in disen- Company, Eindhoven, The Netherlands) at the tangling taxonomic problems in nudibranchs (see LIME (Electron Microscopy Interdepartmental e.g. Pola et al. 2006, 2007, 2012; Turner & Wilson Laboratory, RomaTre University, Rome, Italy), 2008; Wilson et al. 2009; Padula et al. 2014; Hirose with secondary electrons and an operating voltage et al. 2015). Given the lack of morphological diag- of 5 kV. All EtOH-fixed were deposited as nostic characters (from either radula or body colour vouchers at the Museum of the Department of pattern), we employed an integrated approach for Biology and Biotechnologies “Charles Darwin” the taxonomic delimitation of Mediterranean species (LaSapienzaRomeUniversity:cataloguecode of Felimare, with a focus on F. picta, F. tricolor and F. BAU). DNA was extracted using a standard protei- fontandraui. We used partial sequences of two mito- nase K phenol/chloroform method with ethanol chondrial markers, the barcode cytochrome c oxi- precipitation, as reported in Oliverio and dase subunit I (COI) and the 16S rDNA, from Mariottini (2001). A fragment of the mitochondrial specimens of the different colour morphotypes of F. 16S rDNA was amplified by polymerase chain reac- tricolor and F. fontandraui, and we included tion (PCR) using the universal primers 16Sar-L and sequences (newly produced and retrieved from 16Sbr-H (Palumbi et al. 2001), while the barcode GenBank) of Felimare spp. (one third of the recog- fragment of the mitochondrial Cytochrome Oxidase nised species) to check the phylogenetic support to I(COI)wasamplified using the universal primers the distance-based delimitation of species. The main LCO1490 and HCO2198 (Folmer et al. 1994). For aims of this work were to provide an integrated PCR conditions we followed Prkić et al. (2014); in taxonomic assessment of intermediate tricolor-fontan- particular the PCR profile was: 5 min denaturation draui specimens and variable Felimare picta speci- step at 94°C; 35 cycles of 94°C/30 s, 45–50°C/60 s, mens, as well as a preliminary inference on 72°C/60 s; 7 min final extension at 72°C. All ampli- phylogenetic relationships among Atlantic- cons were sequenced either by Genechron Centre Mediterranean Felimare species. of Sequencing, ENEA (La Casaccia, Rome, Italy) or by the European Division of Macrogen Inc. (Amsterdam, The Netherlands), using the same Materials and methods PCR primers. Forward and reverse sequences Collection data, voucher IDs and accession numbers were assembled and edited with Sequencher (ver- are listed in Table I. Specimens of Felimare ssp. from sion 4.1; Gene Codes Corporation, Ann Arbor, different Italian localities were collected by hand MI). BLAST search (at http://blast.ncbi.nlm.nih. during SCUBA diving. Sampling at the Marine gov) was conducted for each sequence to check for Protected Area (MPA) “Secche di Tor Paterno” contamination. COI sequences were readily aligned was authorised by “RomaNatura” (under Art. 4, by hand, whilst 16S were first aligned with Clustal comma 3 g, Decreto Istitutivo, 29 November 2000: X(version2.1:Larkinetal.2007), then the align-

Downloaded by [Universita Studi la Sapienza] at 05:25 29 February 2016 Ministry of the Environment and Protection of Land ment was slightly optimised according to secondary and Sea). structure homology, based on a secondary structure All specimens analysed in this work were photo- model for mollusks (Lydeard et al. 2000). To define graphed in situ and/or in an aquarium (pictures of preliminary species hypotheses (PSH), we used the living animals are available upon request), then criteria of divergence and reciprocal monophyly fixed in 100% ethanol. A piece of tissue was dis- supported by mitochondrial genetic markers sected from the foot for DNA extraction. The buc- (Knowlton 2000; Wheeler & Meier 2000;Reid cal masses of some individuals of typical F. et al. 2006; Malaquias & Reid 2009). We employed fontandraui and F. tricolor (§ labelled in Table I) the Automatic Barcode Gap Discovery (ABGD, were utilised for both anatomical and molecular available at http://wwwabi.snv.jussieu.fr/public/ analyses. Tissues from dissected animals were abgd/), a distance-based method designed to detect placed in a 10% sodium hydroxide (NaOH) solu- the so-called “barcode gap” in the distribution of tion for radula and buccal armature extraction. For pairwise distances calculated in a sequence align- the high-resolution scanning electron microscopy ment (Puillandre et al. 2012a, 2012b). The COI (SEM) images, the dissected radulae were dehy- and 16S alignments were submitted and processed drated to 100% ethanol (EtOH) by passing through in ABGD (excluding the outgroups) using the Downloaded by [Universita Studi la Sapienza] at 05:25 29 February 2016 4 Table I. Voucher ID, collection localities and sequence accession numbers of Felimare specimens: in the last column is reported the nomenclature used in the cited reference and/or in the GenBank accession if different from ours. The symbol * indicates specimens with intermediate phenotype F. tricolor/F. fontandraui, while the § symbol indicates animals used for anatomical analyses (see text). al. et Furfaro G.

Accession numbers

Species Voucher ID Locality COI 16S References Reported as

Felimare agassizi CASIZ 175441 Punta Carbon, Costa Rica JQ727883 JQ727764 Johnson and Gosliner (2012) Hypselodoris agassizii Felimare agassizi CASIZ 175429 Punta Carbon, Costa Rica JQ727884 JQ727765 Johnson and Gosliner (2012) Hypselodoris agassizii Felimare bayeri CASIZ 175461 Bocas del Toro, Salt Creek, Panama ––– JQ727768 Johnson and Gosliner (2012) Hypselodoris bayeri Felimare bilineata ––– Madeira, Portugal EF535125 EF534052 Turner and Wilson (2008) Hypselodoris bilineata Felimare CASIZ 175442 Baja California, Mexico EU982744 EU982796 Johnson (2011) Hypselodoris californiensis californiensis Felimare elegans ––– Spain AF249787 AF249238 Wollscheid-Lengeling et al. Hypselodoris elegans (2001) Felimare BAU 2047§ Ischia Is., Italy, 40°43ʹ50ʹʹ N, 13°58ʹ04ʹʹE, 30 m depth LN715202 LN715181 Present study fontandraui Felimare BAU 2048.1 * Giglio Is., Italy, 42°22ʹ27ʹʹN, 10°52ʹ47ʹʹE, 30 m depth LN715203 LN715182 Present study fontandraui Felimare BAU 2048.2 * Giglio Is., Italy, 42°22ʹ27ʹʹN, 10°52ʹ47ʹʹE, 30 m depth LN912979 LN912985 Present study fontandraui Felimare BAU 2049 Bosa Marina, Sardinia, 40°12ʹ54ʹʹN, 008°13ʹ33ʹʹE, 35 m depth LN912981 LN715183 Present study fontandraui Felimare BAU 2366 Tarifa, Spain, 36° 0ʹ14.16ʹʹ N, 5°36ʹ21.32ʹʹW, 10 m depth LN912980 LN912986 Present study fontandraui Felimare kempfi ––– Florida Keys, USA EF535121 EF534047 Turner and Wilson (2008) Mexichromis kempfi Felimare kempfi CASIZ 185129b Los Huecos, Punta Uva, Costa Rica ––– JQ727800 Johnson and Gosliner (2012) Mexichromis kempfi Felimare kempfi CASIZ 185129a Los Huecos, Punta Uva, Costa Rica ––– JQ727799 Johnson and Gosliner (2012) Mexichromis kempfi Felimare marci CASIZ 175545 Bocas del Toro, Crawl Cay, Panama ––– JQ727787 Johnson and Gosliner (2012) Hypselodoris marci Felimare orsinii ––– Cadiz, Spain AJ223265 AJ225189 Thollesson (2000) Hypselodoris orsinii Felimare picta picta BAU 2050.1 Capo Palinuro, Italy, 40°02ʹ00ʹʹN, 15°16ʹ00ʹʹE, 5 m depth LN715204 LN715184 Present study Felimare picta picta BAU 2050.2 Capo Palinuro, Italy, 40°02ʹ00ʹʹN, 15°16ʹ00ʹʹE, 5 m depth LN912982 LN715185 Present study Felimare picta picta BAU 2050.3 Capo Palinuro, Italy, 40°02ʹ00ʹʹN, 15°16ʹ00ʹʹE, 5 m depth LN912983 LN912987 Present study Felimare picta picta BAU 2050.4 Capo Palinuro, Italy, 40°02ʹ00ʹʹN, 15°16ʹ00ʹʹE, 5 m depth LN912984 LN912988 Present study Felimare picta NHMUK Senegal KF992179 KJ022810 Oskars et al. (2015) 20030798 Felimare picta CASIZ179384 Principe Is., São Tomé and Principe HM162685 HM162594 Pola and Gosliner (2010) Hypselodoris picta verdensis verdensis Felimare porterae ––– Palos Verdes, CA, USA EF535139 EF534067 Turner and Wilson (2008) Mexichromis porterae Felimare ruthae CASIZ 175458 Bocas del Toro, Hospital Point, Panama EU982747 EU982799 Johnson (2011) Hypselodoris ruthae Felimare tricolor CASIZ 175,443 Islotes do Martinha, Algarve, JQ727898 JQ727789 Johnson and Gosliner (2012) Felimare midatlantica Portugal Felimare tricolor BAU 2052 Ponza Is., Italy, 40°52ʹ52ʹʹN, 012°58ʹ02ʹ”E, 25 m depth ––– LN715187 Present study Felimare tricolor BAU 2053 Circeo, Italy, 41°12ʹ41ʹʹN, 13°00ʹ57ʹʹE 25 m depth LN715205 ––– Present study Felimare tricolor BAU2054.4§ “Secche di Tor Paterno” MPA, Italy 41°36ʹ18ʹʹN, 12°20ʹ20ʹʹE, LN715208 ––– Present study 25 m depth (Continued) Downloaded by [Universita Studi la Sapienza] at 05:25 29 February 2016

Table I. (Continued).

Accession numbers

Species Voucher ID Locality COI 16S References Reported as

Felimare tricolor BAU2054.8 “Secche di Tor Paterno” MPA, Italy 41°36ʹ18ʹʹN, 12°20ʹ20ʹʹE, LN715212 LN715194 Present study 25 m depth Felimare tricolor BAU2054.11§ “Secche di Tor Paterno” MPA, Italy 41°36ʹ18ʹʹN, 12°20ʹ20ʹʹE, LN715215 LN715197 Present study 25 m depth Felimare tricolor BAU 2055 Tavolara Is., Sardinia, 40°53ʹ52ʹʹN, 9°40ʹ35ʹʹE LN715216 LN715198 Present study Felimare tricolor BAU 2051 * Ischia Is., Italy, 40°43ʹ50ʹʹN, 13°58ʹ04ʹʹE, 30 m depth ––– LN715186 Present study Felimare tricolor BAU2054.1 * “Secche di Tor Paterno” MPA, Italy 41°36ʹ18ʹʹN, 12°20ʹ20ʹʹE, ––– LN715188 Present study 25 m depth Felimare tricolor BAU2054.2 * “Secche di Tor Paterno” MPA, Italy 41°36ʹ18ʹʹN, 12°20ʹ20ʹʹE, LN715206 LN715189 Present study 25 m depth

Felimare tricolor BAU2054.3 * “Secche di Tor Paterno” MPA, Italy 41°36ʹ18ʹʹN, 12°20ʹ20ʹʹE, LN715207 LN715190 Present study Mediterranean of phylogeny and DNA-barcoding 25 m depth Felimare tricolor BAU2054.5 * “Secche di Tor Paterno” MPA, Italy 41°36ʹ18ʹʹN, 12°20ʹ20ʹʹE, LN715209 LN715191 Present study 25 m depth Felimare tricolor BAU2054.6 * “Secche di Tor Paterno” MPA, Italy 41°36ʹ18ʹʹN, 12°20ʹ20ʹʹE, LN715210 LN715192 Present study 25 m depth Felimare tricolor BAU2054.7 * “Secche di Tor Paterno” MPA, Italy 41°36ʹ18ʹʹN, 12°20ʹ20ʹʹE, LN715211 LN715193 Present study 25 m depth Felimare tricolor BAU2054.9 * “Secche di Tor Paterno” MPA, Italy 41°36ʹ18ʹʹN, 12°20ʹ20ʹʹE, LN715213 LN715195 Present study 25 m depth Felimare tricolor BAU2054.10 * “Secche di Tor Paterno” MPA, Italy 41°36ʹ18ʹʹN, 12°20ʹ20ʹʹE, LN715214 LN715196 Present study 25 m depth Felimare villafranca CASIZ 185127 Algarve, Portugal ––– JQ727793 Johnson and Gosliner (2012) Hypselodoris villafranca Felimare villafranca BAU 2056.1 Split, Croatia ––– LN715199 Present study Felimare villafranca BAU 2056.2 Split, Croatia ––– LN715200 Present study Felimare cf. BAU 2057 “Secche di Tor Paterno” MPA, Italy 41°36ʹ18ʹʹN, 12°20ʹ20ʹʹE, LN715217 LN715201 Present study villafranca 25 m depth Felimare villafranca MNCN 15.05/ Taghazout, Morocco KJ911288 KJ911268 Ortigosa et al. (2014) 70681 Felimare villafranca MNCN 15.05/ Taghazout, Morocco KJ911289 KJ911269 Ortigosa et al. (2014) 70682 Felimare Felimare villafranca MNCN 15.05/ Menorca, Cap Cavalleria, Spain KJ911290 KJ911270 Ortigosa et al. (2014) 70683 Felimare villafranca MNCN 15.05/ Santa Marıa Beach, Cadiz, Spain KJ911291 KJ911271 Ortigosa et al. (2014) 70684 Felimare villafranca MNCN 15.05/ San Garcıa, Cadiz, Spain (Strait of Gibraltar) KJ911292 KJ911272 Ortigosa et al. (2014) nudibranchs 70695 Felimare villafranca ––– Spain ––– AF249237 Wollscheid-Lengeling et al. Hypselodoris villafranca (2001) Felimare villafranca ––– Cadiz, Spain AJ223266 AJ225190 Thollesson (2000) Hypselodoris villafranca Hypselodoris CASIZ 139599 Tulamben, Bali, Indonesia JQ727904 JQ727796 Johnson and Gosliner (2012) zephyra 5 Thorunna australis CASIZ 069910 Onna Village, Okinawa, Ryukyu Islands, Japan JQ727912 JQ727816 Johnson and Gosliner (2012) 6 G. Furfaro et al.

Figure 2. Phenotypic variability of Felimare fontandraui (Pruvot-Fol, 1951). (a) BAU2048.1 and BAU2048.2, Giglio Is., Italy, 42°22ʹ27ʹʹN, 10°52ʹ47ʹʹE; 30 m depth. (b) BAU2047, Ischia Is., Italy, 40°43ʹ50ʹʹN, 13°58ʹ04ʹʹE; 30 m depth. (c) BAU2049, Bosa Marina, Sardinia, 40° 12ʹ54ʹʹN, 008°13ʹ33ʹʹE; 35 m depth. (d) BAU 2366, Tarifa, Spain, 36°0ʹ14.16ʹʹN, 5°36ʹ21.32ʹʹW; 15 m depth.

Kimura two-parameter nucleotide substitution nodes with Bps of 0.95–0.98% and bs of 80–89% model (K2p) and the following settings: a prior for were considered “moderately” supported (lower the maximum value of intraspecific divergence support values were considered not significant). between 0.001 and 0.1, 25 recursive steps within Genetic divergence among the barcode COI the primary partitions defined by the first estimated sequences was observed (p distance), and estimated gap, and a gap width of 0.1. Phylogenetic relation- using the K2p distance. ships among the aligned sequences were inferred by maximum likelihood (ML; bootstrapped over 1000 Results replicates) and Bayesian inference (BI; with 5 × 106 generations, and 25% burn-in) using the software Radulae from specimens of Felimare tricolor and F. MEGA 6.0 (Tamura et al. 2013) and MrBayes fontandraui were studied by SEM at various compar- 3.2.2 (Ronquist et al. 2011)onsinglegeneand able sections (anterior, middle and posterior fi Downloaded by [Universita Studi la Sapienza] at 05:25 29 February 2016 combined data sets. Models of molecular evolution regions), but they did not reveal any signi cant mor- were the best fit ones selected under the Bayesian phological differences (Figure 3a–f). information criterion by JModeltest 2.0 (Darriba We obtained 22 new COI sequences and 25 new et al. 2012):HKY+I+G(16S)andTrN+I+G 16S sequences and analysed them along with those (COI). The combined data set was obtained by retrieved from the GenBank (all alignments and joining sequences of specimens for which both mar- distance matrices available from the authors on kers (COI and 16S) were available, with the two request). All 22 recursive steps in the ABGD ana- fragments treated as distinct partitions. Sequences lysis of the COI alignment resulted in the same of Thorunna australis (Risbec, 1928) and sequence repartitions, with 13 PSH, and all Hypselodoris zephyra (Gosliner & Johnson 1999), grouped the F. tricolor sequences in a single PSH retrieved from the GenBank, were used as suitable (including the Tyrrhenian intermediate specimens outgroup, according to the phylogeny of chromo- and CASIZ175443, named F. midatlantica). dorids by Johnson and Gosliner (2012). Nodes in Sequences of specimens of F. villafranca were the phylogenetic trees with Bayesian posterior prob- split into two PSH: one with BAU2057 (from ability values (Bps) ≥ 0.99% and bootstrap values Central Tyrrhenian Sea), the other with all remain- (bs) ≥ 90% were considered “highly” supported; ing sequences including sequence AF249787 DNA-barcoding and phylogeny of Mediterranean Felimare nudibranchs 7

Figure 3. Buccal armature of Felimare species. (a) Buccal armature of F. fontandraui. (b) Buccal armature of F. tricolor. (c, e) Radula of F. fontandraui. (d, f) Radula of F. tricolor. Scale bars: 100 µm (c, d) and 50 µm (e, f). Downloaded by [Universita Studi la Sapienza] at 05:25 29 February 2016

(erroneously ascribed to Hypselodoris elegans). All maximum value of 0.214 (between F. porterae and sequences from specimens ascribed to F. picta F. fontandraui). The intraspecificK2pestimated picta were grouped in a single PSH, regardless of pairwise distances ranged from 0 to 0.021 (F. vil- their chromatic variation. The sequences of the lafranca), with a barcoding gap in our data set two putative subspecies of F. picta (F. picta picta between 2% and 7%. The K2p distances estimated and F. picta verdensis) were considered as repre- among the F. tricolor individuals [including the senting two distinct PSH. Accordingly, the inter- intermediate Tyrrhenian specimens (Figure 1a, b, specific K2p pairwise distances estimated on the d)andthespecimenascribedtoF. midatlantica] COI data set ranged from minimum values of rangedfrom0.0to0.014.All25recursivestepsin 0.074 (between the Tyrrhenian specimen ascribed the ABGD analysis of the 16S alignment resulted to F. villafranca and the two other sequences in the same sequence repartitions, with 14 PSH ascribed to F. villafranca and H. elegans, respec- almost identical with those of the COI analyses. tively) and 0.103 (between F. picta s. str. and the Differences were detected in sequences of F. villa- putative subspecies F. picta verdensis), to the franca comprising a single PSH, including 8 G. Furfaro et al.

sequence AJ225190 (ascribed to F. villafranca)and repartitions, with 13 PSH, again highly congruent AF249238 (erroneously ascribed to Hypselodoris ele- with the single-gene analyses. Sequences of speci- gans), and the two specimens of F. agassizi (CASIZ mens of F. villafranca were placed in two PSH: 175,441 and 175429) split into two distinct PSH. one with BAU2057 (from Central Tyrrhenian Additionally, the sequences from specimens ascribed Sea), the other with all remaining sequences to F. bayeri (CASIZ175461) and F. marci including the specimen erroneously ascribed to (CASIZ175545) were joined in a single PSH. Hypselodoris elegans. All specimens of F. picta Accordingly, the intraspecific K2p estimated pairwise picta weregroupedinasinglePSH,regardless distances ranged from 0 to 0.041 (including all F. their chromatic variation. The specimens ascribed villafranca in a single PSH) or to 0.023 (excluding to two putative subspecies F. picta picta and F. the F. villafranca comparisons), with a barcoding gap picta verdensis were considered as representing two in our data set that was non-detectable (including all distinct PSH. The two specimens of F. agassizi F. villafranca in a single PSH) or between 2.3 and (CASIZ 175,441 and 175429) were considered a 4% (excluding the F. villafranca comparisons). All single PSH. The phylogenetic analyses of the par- 25 recursive steps in the ABGD analysis of the com- tial COI and 16S sequences, yielded similar trees bined alignment resulted in the same sequence (Figures 4 and 5)byMLandBI. Downloaded by [Universita Studi la Sapienza] at 05:25 29 February 2016

Figure 4. Maximum likelihood tree based on the combined (16S+COI) data set (GTR model of evolution). Numbers at nodes indicate the support by ML (1000 bootstrap replicates, left half) and BI (5 × 106 generations and 25% burn-in, right half); bootstrap values < 80% and Bayesian posterior support < 0.90 are not reported. Specimens of Felimare tricolor and F. fontandraui marked with * indicate intermediate chromatism (see text). Scale is calibrated ML distance. The grey areas indicate the species hypotheses derived after the ABGD analysis (see text). Species nomenclature is that adopted in Table I. DNA-barcoding and phylogeny of Mediterranean Felimare nudibranchs 9

In all retrieved topologies (single-gene vs. com- chromatism: Figure 2a–d). F. tricolor was more closely bined-data set, ML vs. BI) the sequences of F. tricolor related to F. fontandraui than to any other species were monophyletic, including the Tyrrhenian inter- (although with moderate support). mediate specimens and also the sequences from the Other relationships supported (moderately to specimen ascribed to F. midatlantica.Almostallother highly) in the combined data set analyses were F. p. species-level clades were congruent with current picta/F. p. verdensis, F. orsinii/F. villafranca,(F. p. picta/ knowledge, with the exception of the two specimens F. p. verdensis)/F. californiensis,andF. kempfi/F. por- of F. agassizi (CASIZ 175,441 and 175429), which terae. The 16S data set yielded a strongly supported were paraphyletic in the 16S-data set analyses, but relationship between the two sequences ascribed to F. were constantly monophyletic in all COI- and com- marci and F. bayeri (which ABGD was even unable to bined-data set analyses (Figure 6). All putative speci- resolve as distinct PSH), and a closer relationship of F. mens of F. fontandraui were grouped in a single clade p. verdensis with F. marci/ F. bayeri (although without (including specimens with strikingly different support) than with F. picta. Downloaded by [Universita Studi la Sapienza] at 05:25 29 February 2016

Figure 5. Maximum likelihood tree based on the COI data set (GTR model of evolution). Numbers at nodes indicate the support by ML (1000 bootstrap replicates, left half) and BI (5 × 106 generations and 25% burn-in, right half); bootstrap values < 80% and Bayesian posterior support < 0.90 are not reported. Specimens of Felimare tricolor and F. fontandraui marked with * indicate intermediate chromatism (see text). Scale is calibrated ML distance. The histogram shows the distribution of the pairwise estimated genetic distances (K2p) in intraspecific (left, dark grey) and interspecific (right, light grey) comparisons. The grey areas indicate the species hypotheses derived after the ABGD analysis (see text). Species nomenclature is that adopted in Table I. 10 G. Furfaro et al.

Figure 6. Maximum likelihood tree based on the 16S data set (GTR model of evolution). Numbers at nodes indicate the support by ML (1000 bootstrap replicates, left half) and BI (5 × 106 generations and 25% burn-in, right half); bootstrap values < 80% and Bayesian posterior support < 0.90 are not reported. Specimens of Felimare tricolor and F. fontandraui marked with * indicate intermediate chromatism (see text). Scale is calibrated ML distance. The histogram shows the distribution of the pairwise estimated genetic distances (K2p) in intraspecific (left, dark grey) and interspecific (right, light grey) comparisons. The grey areas indicate the species hypotheses derived after the ABGD analysis (see text). Species nomenclature is that adopted in Table I. Downloaded by [Universita Studi la Sapienza] at 05:25 29 February 2016

Discussion et al. 2014). However, as in previous works (e.g. Pola et al. 2006, 2007, 2012; Turner & Wilson 2008; The lack of significant morphological differences in Wilson et al. 2009; Padula et al. 2014; Hirose et al. the radular apparatus (Figure 3a–f) is not surprising. 2015), a DNA-barcoding approach helped remark- As depicted also by Ortea et al. (1996) for other ably in assessing the taxonomic status of our sam- species, intraspecific and interspecific variations of ples, and apparently COI (large barcoding gap, the radula often overlap each other in Felimare, con- robust and unequivocal PSH) performed better firming this character as a poor diagnostic tool. On than 16S in delimiting species in this group. Both the contrary, DNA sequences proved optimal mar- F. fontandraui and F. tricolor are blue chromodorids kers to disentangle complicated patterns in this with strikingly variable (and sometimes misleading group. Only mitochondrial sequences were for their identification) chromatic patterns (Figures 1 employed, and, admittedly, further support from an and 2). Some adult individuals lacking the spots at independently transmitted and evolving data set the base of the rinophores (as in typical F. tricolor) (nuclear DNA) is desirable (as done by e.g. Padula DNA-barcoding and phylogeny of Mediterranean Felimare nudibranchs 11

also lacked the MDF (Figure 2a). All specimens Atlantic subspecies F. picta verdensis proved to be a lacking MDF that we sequenced proved to belong distinct species, with an unequivocally high genetic to F. fontandraui. Therefore, molecular analyses of divergence of 9–10% at COI, and we formally pro- specimens observed by Chemello and Tumbiolo pose to raise it to species rank (Felimare verdensis). (1992) could be very useful to clarify the value of COI vs. 16S sequences from specimens ascribed MDF for diagnosis of F. fontandraui. Our molecular to F. villafranca yielded apparently inconclusive study confirmed that both F. tricolor and F. fontan- results: while phylogenetic analyses on COI and draui are valid species; furthermore, we demon- combined data sets supported distinction of the strated the conspecificity between H. midantlantica Tyrrhenian specimen (BAU2057) from the Western and F. tricolor, preferring to adopt the latter name Mediterranean-Atlantic F. villafranca, as in the according to WoRMS (Bouchet & Caballer 2014). ABGD analysis of COI (c. 7% divergence), this Felimare picta also shows a very variable colour was not confirmed by the 16S data set. However, pattern, ranging from a light blue background den- this was not surprising given the lower resolution sely yellow striped or spotted to a dark blue back- power of the 16S in the present case (smaller bar- ground sparsely yellow striped or spotted; we coding gap), and the overall pattern addresses a analysed four morphotypes from the same locality complex needing further analyses. PSH as desumed (Table I) representing this extremely divergent chro- by the analysis of the genetic divergences were matic variability (Figure 7a–d). almost all confirmed in the phylogenetic framework, Various such morphotypes were in the past con- the PSHs comprising highly supported monophyletic sidered distinct species, subspecies or mere morphs. clades. Preliminary phylogenetic relationships as We have analysed in this work specimens of the dark inferred by the combined (COI + 16S) data set blue morphotype [corresponding to Hypselodoris suggested that the two species F. tricolor and F. webbi (d’Orbigny, 1839)], of the light blue/yellowish fontandraui are more closely related to each other form [corresponding to Hypselodoris valenciennesi than to any other species. Low node supports ham- (Cantraine, 1841)] and of two intermediates, which per conclusive assessment of phylogenetic relation- all proved to be conspecific, confirming the assess- ships for several lineages. However, F. picta, F. ment by Ortea et al. (1996). However, the putative verdensis, F. californiensis, F. bilineata and F. agassizi Downloaded by [Universita Studi la Sapienza] at 05:25 29 February 2016

Figure 7. Phenotypic variability of Felimare picta picta (Schultz in Philippi, 1836); from Capo Palinuro, Italy, 40°02ʹ00ʹ’N, 15°16ʹ00ʹ’E; 5 m depth. (a) The light form [corresponding to Hypselodoris valenciennesi (Cantraine, 1841)]. (b, c) The intermediate morphs. (d) The dark morphotype [corresponding to Hypselodoris webbi (d’Orbigny, 1839)]. 12 G. Furfaro et al.

were in the same clade in all analyses (COI, 16S and Haber M, Cerfeda S, Carbone M, Calado G, Gaspar H, Neves R, combined) although always with low support, a rela- Maharajan V, Cimino G, Gavagnin M, Ghiselin MT, Mollo E. tionship to be tested within a larger data set, as 2010. Coloration and defense in the gastropod Hypselodoris fontandraui. The Biological Bulletin 218:181–188. should the close relationship among F. verdensis, F. Hirose M, Hirose E, Kiyomoto M. 2015. Identification of five marci and F. bayeri as suggested by the 16S data set. species of Dendrodoris (Mollusca: Nudibranchia) from Japan, using DNA barcode and larval characters. Marine Biodiversity 45:769–780. doi:10.1007/s12526-014-0288-2. Acknowledgements Johnson RF. 2011. Breaking family ties: Taxon sampling and molecular phylogeny of chromodorid nudibranchs (Mollusca, The authors gratefully acknowledge Juan Lucas Gastropoda). Zoologica Scripta 40:137–157. 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