The genus Babakina was erected by Roller in 1973, for the species Babakina festiva (Roller, 1972), as a replacement name for BabainaRoller, 1972 (a junior homonym of the dorid nudibranch genus BabainaOdhner, 1968). Miller (1974) described a second species, B. caprinsulensis, from New Zealand, while Ortea (1979) described Rioselleolis anadoni from northern Spain, which was later considered to be a Babakina (Rolán, Rolán-Alvarez & Ortea, 1991). Although Gosliner (1990) questioned whether these taxa were distinct based on the similarity of the colour pattern, a recent review of the genus (Gosliner, Garcia-Duarte & Cervera, 2007) supported the validity of all three species and described an additional one, B. indopacifica from the Philippines, Hawaii, Japan and Madagascar.

The systematic position of Babakina has been the subject of controversy. Roller (1972) erected the family Babainidae for the type species B. festiva, which was later changed to Babakinidae (Roller, 1973) due to homonymy with BabainaOdhner, 1968 a genus of Chromodorididae. The family Babakinidae has been accepted by several authors (Roller, 1973; Behrens, 1991; Rolán et al., 1991; Baba, 2000; Gosliner et al., 2007; Gosliner, Behrens & Valdés, 2008), but Babakina has also been classified in Glaucidae (Miller, 1974; Redfern, 2001; Padula & Absalão, 2005), Facelinidae (Coleman, 2001; Valdés & Bouchet, 2005; Debelius & Kuiter, 2007; CLEMAM, 2011) and Favorinidae (Pérez Sánchez & Moreno, 1990).

The objectives of this study are to test the monophyly of the genus Babakina and systematic status of the family Babakinidae, based on molecular phylogenetic analyses and to infer relationships within this genus.

Samples were obtained by fieldwork using standard Scuba-diving sampling techniques for opisthobranchs as well as from the Museo Nacional de Ciencias Naturales, Madrid (MNCN), the California Academy of Sciences, San Francisco (CASIZ) and the Museu Nacional, Universidade Federal do Rio de Janeiro, Brazil (MNRJ). Collection data and GenBank accession numbers are listed in Table 1.

Table 1.

Specimens used for molecular analyses, collection sites, dates, vouchers and GenBank accession nos.

Family Species Locality Collection dates Voucher GenBank accession nos.
 
COI 16S H3 
Tritoniidae Tritonia nilsodhneri Marcus, 1983 Cape Province, South Africa (ATL, GB) 04 Jun 2008  HM162716 HM162641 HM162548 
Dendronotidae Dendronotus venustus MacFarland, 1966 Santa Monica, California (GB) Dec 2007  HM162709 HM162630 HM162536 
Proctonotidae Janolus mirabilis Baba & Abe, 1970 Philippines (GB) 19 May 2009  HM162750 HM162674 HM162583 
Babakinidae Babakina anadoni (Ortea, 1979Bahamas 29 Jun 1999 MNCN 15.05/46706 — — HQ616805 
  Brazil May 2006 MNRJ 10890 — HQ616742 HQ616806 
  Brazil May 2006 MNRJ 10891 — HQ616743 — 
  Brazil Feb 2006 MNRJ 10893 HQ616746 HQ616709 HQ616775 
  Canary Is (ATL) 30 Oct 2004 MNCN 15.05/46705 HQ616747 HQ616710 HQ616776 
  Galicia, Spain (ATL) 25 May 2005 MNCN 15.05/46704 HQ616767 HQ616730 HQ616796 
  Galicia, Spain (ATL) 31 Mar 2006 MNCN 15.05/46979 — HQ616744 HQ616807 
  W Andalusia, Spain (ATL) 5 Sep 2003 MNCN 15.05/46702 HQ616748 HQ616711 HQ616777 
 Babakina festiva (Roller, 1972California 1 Nov 2009 CASIZ 182204 — HQ616735 HQ616801 
  California 10 Jan 2010 CASIZ 182205 — HQ616736 HQ616802 
  Japan May 2005 MNCN 15.05/46741 — — HQ616803 
 Babakina indopacifica Gosliner, González-Duarte & Cervera, 2007 Philippines (GB) 20 Mar 2008  HM162754 HM162678 HM162587 
Aeolidiidae Aeolidiella alderi (Cocks, 1852) France (MED) 26 Jul 2002 ZSM Mol 20020982 HQ616765 HQ616728 HQ616794 
  Italy (MED) 18 Apr 2001 ZSM Mol 20012341 HQ616766 HQ616729 HQ616795 
 Berghia major (Eliot, 1903) Hawaii 07 May 2008 MNCN 15.05/54987 HQ616771 HQ616734 HQ616800 
  Philippines 16 Apr 2008 CASIZ 177602 HQ616770 HQ616733 HQ616799 
 Berghia verrucicornis (Costa, 1864) Agadir, Morocco (ATL) 30 Mar 2009 MNCN 15.05/53686 HQ616749 HQ616712 HQ616778 
  W Andalusia, Spain (ATL) 05 Apr 2008 MNCN 15.05/53687 HQ616750 HQ616713 HQ616779 
 Limenandra nodosa Haefelfinger & Stamm, 1958 Menorca, Spain (MED) Sep 2007 MNCN 24.923 HQ616768 HQ616731 HQ616797 
  Philippines 21 May 2009 CASIZ 181280 HQ616769 HQ616732 HQ616798 
 Spurilla neapolitana (Delle Chiaje, 1823) Portugal 08 Jul 2002 CASIZ 175756 HQ616764 HQ616727 HQ616793 
  Trieste, Italy 29 Sep 2008 MNCN 15.05/54988 HQ616763 HQ616726 HQ616792 
Facelinidae Caloria elegans (Alder & Hancock, 1845) Menorca, Spain (MED) 03 May 2008 MNCN 15.05/53689 HQ616751 HQ616714 HQ616780 
  W Andalusia, Spain (ATL) 05 Sep 2003 MNCN 15.05/53690 — HQ616738 — 
 Caloria indica (Bergh, 1896) Kaneohe Bay, Hawai (GB) —  DQ417325 DQ417273 — 
 Cratena peregrina Gmelin, 1791 Senegal 30 May 2005 MNCN 15.05/53691 HQ616752 HQ616715 HQ616781 
  Spain (MED, GB) —  AF249786 — — 
 Dicata odhneri Schmekel, 1967 W Andalusia, Spain (ATL) 02 Aug 2006 MNCN 15.05/53692 HQ616773 HQ616739 — 
 Dondice banyulensis Portmann & Sandmeier, 1960 W Andalusia, Spain (ATL) 26 May 2009 MNCN 15.05/53693 — HQ616740 HQ616804 
 Facelina bostoniensis (Couthouy, 1838) Clachan Seil, Scotland —  AY345031 — — 
 Facelina punctata Alder & Hancock, 1845 Spain (MED, GB) —  AF249816 — — 
 Favorinus branchialis (Rathke, 1806) Azores 11 Jun 2002 MNCN 15.05/53694 — HQ616741 — 
  W Andalusia, Spain (ATL) 26 Jun 2007 MNCN 15.05/53695 HQ616761 HQ616724 HQ616790 
 Favorinus elenalexiarum García & Troncoso, 2001 Guanacaste, Costa Rica (PAC, GB) 17 Apr 2007  HM162755 HM162679 HM162588 
 Godiva quadricolor (Barnard, 1927) Cape Province, South Africa (ATL, GB) 09 Jan 2008  HM162692 HM162602 HM162508 
 Phyllodesmium horridum (Macnae, 1954) Cape Province, South Africa (ATL, GB) 03 Jan 2008  HM162757 HM162681 HM162590 
 Pruvotfolia longicirrha (Eliot, 1906) Cape Verde Is Mar 2010 MNCN 15.05/53703 HQ616760 HQ616723 HQ616789 
 Pruvotfolia pselliotes (Labbé, 1923) Cap Ferret, France (ATL) 05 Sep 2004 MNCN 15.05/53705 HQ616762 HQ616725 HQ616791 
 Sakuraeolis enosimensis (Baba, 1930) San Francisco Bay, California (GB) 13 Dec 2007  HM162758 HM162682 HM162591 
Flabellinidae Calmella cavolini (Vérany, 1846) Otranto, Italy Jul 2004 MNCN 15.05/53688 HQ616772 HQ616737 — 
 Flabellina affinis (Gmelin, 1791) Menorca, Spain (MED) 14 Jul 2007 MNCN 15.05/53696 HQ616753 HQ616716 HQ616782 
 Flabellina babai Schmekel, 1972 Chafarinas Is (MED) 25 Feb 2007 MNCN 15.05/53698 HQ616754 HQ616717 HQ616783 
 Flabellina baetica García-Gómez, 1984 W Andalusia, Spain (ATL) 14 Jan 2005 MNCN 15.05/53699 HQ616755 HQ616718 HQ616784 
 Flabellina ischitana Hirano & Thompson, 1990 Temara, Morocco 07 Mar 2008 MNCN 15.05/53700 HQ616756 HQ616719 HQ616785 
  W Andalusia, Spain (ATL) 14 Jun 2008 MNCN 15.05/53701 — HQ616745 HQ616808 
  W Andalusia, Spain (ATL) 26 Mar 2009 MNCN 15.05/53697 HQ616757 HQ616720 HQ616786 
 Flabellina pedata (Montagu, 1815) Malaga, Spain (MED) 13 Oct 2007 MNCN 15.05/53702 HQ616758 HQ616721 HQ616787 
 Flabellina verrucosa (M. Sars, 1829) USA, NW Atlantic (GB) —  AF249790 AF249245  
Piseinotecidae Piseinotecus gaditanus Cervera, García-Gómez & García, 1987 W Andalusia, Spain (ATL) 20 Jun 2007 MNCN 15.05/53704 HQ616759 HQ616722 HQ616788 
 Piseinotecus sp. Philippines (GB) 22 Apr 2008  HM162694 HM162604 HM162510 
Family Species Locality Collection dates Voucher GenBank accession nos.
 
COI 16S H3 
Tritoniidae Tritonia nilsodhneri Marcus, 1983 Cape Province, South Africa (ATL, GB) 04 Jun 2008  HM162716 HM162641 HM162548 
Dendronotidae Dendronotus venustus MacFarland, 1966 Santa Monica, California (GB) Dec 2007  HM162709 HM162630 HM162536 
Proctonotidae Janolus mirabilis Baba & Abe, 1970 Philippines (GB) 19 May 2009  HM162750 HM162674 HM162583 
Babakinidae Babakina anadoni (Ortea, 1979Bahamas 29 Jun 1999 MNCN 15.05/46706 — — HQ616805 
  Brazil May 2006 MNRJ 10890 — HQ616742 HQ616806 
  Brazil May 2006 MNRJ 10891 — HQ616743 — 
  Brazil Feb 2006 MNRJ 10893 HQ616746 HQ616709 HQ616775 
  Canary Is (ATL) 30 Oct 2004 MNCN 15.05/46705 HQ616747 HQ616710 HQ616776 
  Galicia, Spain (ATL) 25 May 2005 MNCN 15.05/46704 HQ616767 HQ616730 HQ616796 
  Galicia, Spain (ATL) 31 Mar 2006 MNCN 15.05/46979 — HQ616744 HQ616807 
  W Andalusia, Spain (ATL) 5 Sep 2003 MNCN 15.05/46702 HQ616748 HQ616711 HQ616777 
 Babakina festiva (Roller, 1972California 1 Nov 2009 CASIZ 182204 — HQ616735 HQ616801 
  California 10 Jan 2010 CASIZ 182205 — HQ616736 HQ616802 
  Japan May 2005 MNCN 15.05/46741 — — HQ616803 
 Babakina indopacifica Gosliner, González-Duarte & Cervera, 2007 Philippines (GB) 20 Mar 2008  HM162754 HM162678 HM162587 
Aeolidiidae Aeolidiella alderi (Cocks, 1852) France (MED) 26 Jul 2002 ZSM Mol 20020982 HQ616765 HQ616728 HQ616794 
  Italy (MED) 18 Apr 2001 ZSM Mol 20012341 HQ616766 HQ616729 HQ616795 
 Berghia major (Eliot, 1903) Hawaii 07 May 2008 MNCN 15.05/54987 HQ616771 HQ616734 HQ616800 
  Philippines 16 Apr 2008 CASIZ 177602 HQ616770 HQ616733 HQ616799 
 Berghia verrucicornis (Costa, 1864) Agadir, Morocco (ATL) 30 Mar 2009 MNCN 15.05/53686 HQ616749 HQ616712 HQ616778 
  W Andalusia, Spain (ATL) 05 Apr 2008 MNCN 15.05/53687 HQ616750 HQ616713 HQ616779 
 Limenandra nodosa Haefelfinger & Stamm, 1958 Menorca, Spain (MED) Sep 2007 MNCN 24.923 HQ616768 HQ616731 HQ616797 
  Philippines 21 May 2009 CASIZ 181280 HQ616769 HQ616732 HQ616798 
 Spurilla neapolitana (Delle Chiaje, 1823) Portugal 08 Jul 2002 CASIZ 175756 HQ616764 HQ616727 HQ616793 
  Trieste, Italy 29 Sep 2008 MNCN 15.05/54988 HQ616763 HQ616726 HQ616792 
Facelinidae Caloria elegans (Alder & Hancock, 1845) Menorca, Spain (MED) 03 May 2008 MNCN 15.05/53689 HQ616751 HQ616714 HQ616780 
  W Andalusia, Spain (ATL) 05 Sep 2003 MNCN 15.05/53690 — HQ616738 — 
 Caloria indica (Bergh, 1896) Kaneohe Bay, Hawai (GB) —  DQ417325 DQ417273 — 
 Cratena peregrina Gmelin, 1791 Senegal 30 May 2005 MNCN 15.05/53691 HQ616752 HQ616715 HQ616781 
  Spain (MED, GB) —  AF249786 — — 
 Dicata odhneri Schmekel, 1967 W Andalusia, Spain (ATL) 02 Aug 2006 MNCN 15.05/53692 HQ616773 HQ616739 — 
 Dondice banyulensis Portmann & Sandmeier, 1960 W Andalusia, Spain (ATL) 26 May 2009 MNCN 15.05/53693 — HQ616740 HQ616804 
 Facelina bostoniensis (Couthouy, 1838) Clachan Seil, Scotland —  AY345031 — — 
 Facelina punctata Alder & Hancock, 1845 Spain (MED, GB) —  AF249816 — — 
 Favorinus branchialis (Rathke, 1806) Azores 11 Jun 2002 MNCN 15.05/53694 — HQ616741 — 
  W Andalusia, Spain (ATL) 26 Jun 2007 MNCN 15.05/53695 HQ616761 HQ616724 HQ616790 
 Favorinus elenalexiarum García & Troncoso, 2001 Guanacaste, Costa Rica (PAC, GB) 17 Apr 2007  HM162755 HM162679 HM162588 
 Godiva quadricolor (Barnard, 1927) Cape Province, South Africa (ATL, GB) 09 Jan 2008  HM162692 HM162602 HM162508 
 Phyllodesmium horridum (Macnae, 1954) Cape Province, South Africa (ATL, GB) 03 Jan 2008  HM162757 HM162681 HM162590 
 Pruvotfolia longicirrha (Eliot, 1906) Cape Verde Is Mar 2010 MNCN 15.05/53703 HQ616760 HQ616723 HQ616789 
 Pruvotfolia pselliotes (Labbé, 1923) Cap Ferret, France (ATL) 05 Sep 2004 MNCN 15.05/53705 HQ616762 HQ616725 HQ616791 
 Sakuraeolis enosimensis (Baba, 1930) San Francisco Bay, California (GB) 13 Dec 2007  HM162758 HM162682 HM162591 
Flabellinidae Calmella cavolini (Vérany, 1846) Otranto, Italy Jul 2004 MNCN 15.05/53688 HQ616772 HQ616737 — 
 Flabellina affinis (Gmelin, 1791) Menorca, Spain (MED) 14 Jul 2007 MNCN 15.05/53696 HQ616753 HQ616716 HQ616782 
 Flabellina babai Schmekel, 1972 Chafarinas Is (MED) 25 Feb 2007 MNCN 15.05/53698 HQ616754 HQ616717 HQ616783 
 Flabellina baetica García-Gómez, 1984 W Andalusia, Spain (ATL) 14 Jan 2005 MNCN 15.05/53699 HQ616755 HQ616718 HQ616784 
 Flabellina ischitana Hirano & Thompson, 1990 Temara, Morocco 07 Mar 2008 MNCN 15.05/53700 HQ616756 HQ616719 HQ616785 
  W Andalusia, Spain (ATL) 14 Jun 2008 MNCN 15.05/53701 — HQ616745 HQ616808 
  W Andalusia, Spain (ATL) 26 Mar 2009 MNCN 15.05/53697 HQ616757 HQ616720 HQ616786 
 Flabellina pedata (Montagu, 1815) Malaga, Spain (MED) 13 Oct 2007 MNCN 15.05/53702 HQ616758 HQ616721 HQ616787 
 Flabellina verrucosa (M. Sars, 1829) USA, NW Atlantic (GB) —  AF249790 AF249245  
Piseinotecidae Piseinotecus gaditanus Cervera, García-Gómez & García, 1987 W Andalusia, Spain (ATL) 20 Jun 2007 MNCN 15.05/53704 HQ616759 HQ616722 HQ616788 
 Piseinotecus sp. Philippines (GB) 22 Apr 2008  HM162694 HM162604 HM162510 

Abbreviations: ATL, Atlantic Ocean; GB, GenBank; MED, Mediterranean; PAC, Pacific Ocean.

Twelve individuals of three species of Babakina and 41 individuals representing 27 species of Flabellinidae, Piseinotecidae, Facelinidae and Aeolidiidae were used for the phylogenetic reconstruction. A total of 29 specimens were sequenced for the cytochrome c oxidase subunit I (COI), 37 for the 16S rRNA and 34 for the histone-3 (H3) genes. An additional 30 sequences of 12 nudibranch species were obtained from GenBank (Table 1). Tritonia nilsodhneri, Dendronotus venustus and Janolus mirabilis were chosen as outgroups, since they represent three different clades from Aeolidida within Cladobranchia (Pola & Gosliner, 2010).

DNA was extracted from foot tissue of specimens preserved in 70–100% ethanol, except in the case of small animals where the whole specimen was used, employing DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA). Partial sequences of COI, 16S rRNA and H3 were amplified by PCR, conducted in a 50 μl volume. Reactions contained 2 μl of a forward and reverse PCR primer, 5 μl of dNTP, a gene-dependent concentration of magnesium chloride, 0.5 μl of Qiagen DNA polymerase (Qiagen Taq cat. no. 201205), 10 μl of “Q-solution” (5×) and 5 μl of Qiagen buffer (10×). Magnesium chloride concentrations were 7 μl for COI and 16S, and 4 μl for H3. COI amplification 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 as annealing temperature, 1 min at 72°C, with a final extension of 7 min at 72°C. The partial 16S amplification followed the same conditions, except for a shorter denaturation time (30 s). 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 annealing temperature (50°C), 2 min at 72°C, with a final extension of 10 min at 72°C. For sequencing, purified PCR products were sent to MACROGEN (www.macrogen.com). All new sequences have been deposited in GenBank.

DNA sequences were assembled, edited and checked by eye using Geneious Pro 4.7.6 (Drummond et al., 2009) and aligned with Clustal X (Thompson et al., 1997). The alignments were optimized by eye using MacClade v. 4.06 (Maddison & Maddison, 2005). In order to study poorly aligned positions and variable regions of the alignments, additional analysis were conducted with Gblocks (Castresana, 2000). The partition-homogeneity test (Swofford, 2002) was performed to test if the three genes could be combined in a single dataset. Analyses were conducted with a full dataset (all taxa even when sequences were not available for all genes) and short dataset (only taxa with sequence data for all three genes). Phylogenetic analyses were conducted by maximum likelihood (ML) and Bayesian inference (BI). The best-fit model of evolution for each dataset was determined using the Akaike information criterion (Akaike, 1974) implemented in Modeltest v. 3.7 (Posada & Crandall, 1998). The GTR + I + G model was chosen for H3 and COI, the TVM + I + G model for 16S, the GTR + I + G model for the combined full dataset and the TIM + I + G model for the combined short dataset.

Trees were reconstructed in PAUP* 4.0b10 (Swofford, 2002), RAxML v. 7.0.4 (Stamatakis, Hoover & Rougemont, 2008) and MrBayes v. 3.1.2b (Ronquist & Huelsenbeck, 2003). Support for the nodes in ML analysis was assessed with nonparametric bootstrapping with 10,000 replicates, random starting trees and parameters estimated from each dataset under the model selected for the original dataset. BI was conducted for 8,000,000 generations, with a sampling frequency of 1,000. The models implemented were those estimated with Modeltest or the most similar one available in MrBayes. Convergence was diagnosed graphically by plotting for each run the likelihood against the number of generations using the software Tracer v. 1.4.1 (Drummond & Rambaut, 2007). For each analysis the first 2,000 trees were discarded (‘burn-in’ period). Bayesian posterior probabilities (PP) of 0.90 or higher were considered strongly supported, while in the ML analyses bootstrap (BS) values of 75 or higher were considered strongly supported.

The combined dataset produced an alignment of 1,467 positions, of which 555 were parsimony-informative. The incongruence length differences test showed no significantly conflicting phylogenetic signals between the three dataset (P = 0.01), allowing these markers to be combined in a single analysis (Cunningham, 1997). No significant differences were found between the analyses with and without variable regions. Thus, all positions were included for analyses.

The combined tree provided better resolution than individual gene trees. Details of the analyses for each gene are given in Supplementary material. The topology of the tree obtained by ML (not shown) was the same as the one inferred by Bayesian analysis.

Figures 1 and 2 show, respectively, the phylogenetic hypothesis for full and short combined datasets produced by BI. In both ML and BI analyses Babakina is monophyletic (Figs 1, 2), although this was only weekly supported (BS = 70) in the ML analysis of the full dataset. The fact that one B. anadoni from Bahamas and one B. festiva from Japan did not cluster with other conspecific samples in the analysis of the full dataset is likely an artefact resulting from the inclusion of only H3 sequences for these two specimens (Fig. 1, Table 1). Otherwise, the results from both full and short datasets analyses are consistent with the interpretation that B. anadoni, B. indopacifica and B. festiva are distinct species (Figs 1, 2; Supplementary material), in agreement with the recent morphological revision of Babakina (Gosliner et al., 2007).

Figure 1.

Phylogenetic hypothesis based on full dataset (H3 + COI + 16S; with missing values for some taxa, see Table 1), produced by BI. Numbers above branches are posterior probabilities from BI; numbers below branches are bootstrap values for ML analysis. Abbreviations: EA, Eastern Atlantic Ocean; F, Facelinidae; Flb, Flabellinidae; GB, GenBank; MED, Mediterranean Sea; P, Piseinotecidae.

Figure 1.

Phylogenetic hypothesis based on full dataset (H3 + COI + 16S; with missing values for some taxa, see Table 1), produced by BI. Numbers above branches are posterior probabilities from BI; numbers below branches are bootstrap values for ML analysis. Abbreviations: EA, Eastern Atlantic Ocean; F, Facelinidae; Flb, Flabellinidae; GB, GenBank; MED, Mediterranean Sea; P, Piseinotecidae.

Figure 2.

Phylogenetic hypothesis based on reduced dataset of taxa for which all genes were sequenced (H3 + COI + 16S), produced by BI. Numbers above branches are posterior probabilities from BI; numbers below branches are bootstrap values for ML analysis. Abbreviations: EA, Eastern Atlantic Ocean; Flb, Flabellinidae; GB, GenBank; MED, Mediterranean Sea; P, Piseinotecidae.

Figure 2.

Phylogenetic hypothesis based on reduced dataset of taxa for which all genes were sequenced (H3 + COI + 16S), produced by BI. Numbers above branches are posterior probabilities from BI; numbers below branches are bootstrap values for ML analysis. Abbreviations: EA, Eastern Atlantic Ocean; Flb, Flabellinidae; GB, GenBank; MED, Mediterranean Sea; P, Piseinotecidae.

The amphiatlantic distribution of Babakina anadoni is supported (Fig. 1, BS = 64, PP = 0.99; Fig. 2, BS = 100, PP = 1), with a genetic divergence of 4% (uncorrected p-distance) between specimens from the eastern and western Atlantic.

The results suggested a closer relationship between B. festiva and B. anadoni than of either with B. indopacifica. However, relationships might change with the inclusion of the fourth, unsampled, species B. caprinsulensis. Gosliner et al. (2007) hypothesized instead a sister relationship between B. festiva and B. indopacifica.

Not much can be said about the sister relationships of Babakinidae. Further resolution of the phylogenetic position of Babakina will require inclusion of additional species of aeolidids and facelinids, and sequences of Babakina caprinsulensis. Nevertheless, our results support a clade (PP = 1) formed by three subclades: one of Babakinidae, one of Aeolidiidae (including Facelina punctata; Fig. 1), and one with some species of facelinids (Figs 1, 2). The relationships between these three subclades were not resolved. A clade containing babakinids, aeolidids and facelinids is supported by at least one synapomorphy, the presence of a uniseriate radula (Gosliner et al., 2007).

In the full combined analysis (Fig. 1) the family Aeolidiidae was not monophyletic, because the clade of aeolids included Facelina punctata (Facelinidae) (BS = 58, PP = 0.99). This outcome could again be an artefact resulting from the availability of only H3 sequence for F. punctata (Fig. 1, Table 1).

Two specimens provisionally identified as Limenandra nodosa from the Mediterranean and the Philippines showed a genetic divergence of 9% (uncorrected p-distance), suggesting these two specimens are not conspecific.

On the other hand Facelinidae is not retrieved as monophyletic in our analyses. The clade composed of Dondice banyulensis, Dicata odhneri, Godiva quadricolor and Phyllodesmium horridum (Fig. 1, BS = 63, PP = 1) or only Godiva quadricolor and Phyllodesmium horridum (Fig. 2, BS = 100, PP = 1) does not cluster together with the other facelinids.

Pruvotfolia pselliotes was retrieved as sister to Pruvotfolia longicirrha (uncorrected p-distance = 10%), supporting the assignment of the latter to the genus Pruvotfolia as suggested by Ortea & Moro (1997) based on morphological characters.

The monophyly of Flabellina and Flabellinidae are not recovered, since Flabellina babai does not cluster together with the other species of Flabellina and Caloria indica is included within the latter clade. However, because Caloria indica is a variable species (Gosliner et al., 2008) and the molecular sequences used here were obtained from GenBank, we cannot entirely discard the possibility of misidentification. In addition, the two Piseinotecus species clustered together with most Flabellina species in different subclades, breaking the monophyly status of both Flabellinidae and Piseinotecidae, supporting a hypothesis proposed earlier by Gosliner et al. (2007).

This study includes only a small representation of the diversity of aeolid opisthobranchs, but almost doubles the number of species included by Wägele, Vonnemann & Wägele, (2003) in the largest aeolid dataset yet analysed. It highlights a prevalent view that much work is still required to understand the phylogeny of most families and genera of aeolid nudibranchs (Pola & Gosliner, 2010).

SUPPLEMENTARY MATERIAL

Supplementary material is available at Journal of Molluscan Studies online.

ACKNOWLEDGEMENTS

Several individuals provided us with specimens of Babakina and other aeolids, including: C. Megina, V. Padula, J. Goddard, N. Tamsouri, M.M. González-Duarte, M. Poddubetskaia, L. Moro and P. Wirtz. To these we express our sincere appreciation as well as to Manuel A.E. Malaquias for his valuable comments and support. This contribution was supported by a Spanish Ministry of Science and Innovation grant CGL2006-05182/BOS and an Institut Menorquí d'Estudis grant to J.L.C. and a National Science Foundation DEB 0329054 PEET grant to T.M.G.

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