Open Access

Burnaia Miller, 2001 (Gastropoda, Heterobranchia, Nudibranchia): a facelinid genus with an Aeolidiidae’s outward appearance

  • Leila Carmona1Email author,
  • Marta Pola2,
  • Terrence M. Gosliner3 and
  • Juan Lucas Cervera1
Helgoland Marine Research201569:437

https://doi.org/10.1007/s10152-015-0437-4

Received: 12 March 2015

Accepted: 16 June 2015

Published: 3 July 2015

Abstract

In recent years, several morphological and molecular analyses have been undertaken to study the phylogenetic systematics of Aeolidiidae members. The monospecific genus Burnaia could not be included in the previous analysis, due to the lack of material. This study includes two specimens of Burnaia helicochorda from Australia and places them in their systematic position using two mitochondrial and one nuclear genes (COI and 16S, and H3, respectively). A description of its anatomy is also included with colour pictures of the animal and scanning electron micrographs of radula and jaws. Based on our results, B. helicochorda does not belong to Aeolidiidae since it appears nested among some facelinids.

Keywords

Molluscan diversity Molecular phylogeny Taxonomy Cladobranchia Morphological convergence

Introduction

Burnaia helicochorda (Miller 1987), originally ascribed to Aeolidia Cuvier, 1798, was considered a member of Aeolidiidae although the author did not provide any reason for that systematic position. According to Miller (1987, 2001), this species was of easy identification by its external colouration, large body, lamellate rhinophores, and cerata in arches. Actually, because of all these morphological aspects, Miller (2001) decided to create a new genus, Burnaia, for this species. Since it was first described, B. helicochorda has not been reported many times (Rudman 2001; Grove 2015), and for this reason, Carmona et al. (2013), who conducted a molecular phylogeny of the family Aeolidiidae, could not include material of this not very common aeolid.

Recently collected material form Victoria, Australia, permitted us to study the systematic position of this species. This fresh material allowed us to use a molecular approach as well as to re-examine the morphology and anatomy of B. helicochorda, providing for the first time scanning electron photographs of the radula and jaws.

Materials and methods

Molecular work

DNA extraction, amplification, and sequencing

Two specimens of B. helicochorda were studied. One hundred twenty-four additional sequences were obtained from GenBank (see Table 1 for full list of samples, localities, and vouchers). Tritonia challengeriana Bergh, 1884, was chosen as out-group because of its basal phylogenetic position within Cladobranchia (Pola and Gosliner 2010). Tissue samples were taken from the foot. Extraction, amplification, purification, and sequencing of portions of the COI, 16S rRNA, and H3 genes followed the methods described in Carmona et al. (2013, 2014a, b). Sequence reactions were run on a 3730XL DNA sequencer (Applied Biosystems). Sequences were verified by forward and reverse comparisons and have been deposited in GenBank (Table 1).
Table 1

List of specimens used for phylogenetic analyses

Family

Species

Locality

Collection dates

Voucher

GenBank accession Nos.

COI

16S

H3

Tritoniidae Lamarck, 1809

Tritonia challengeriana Bergh, 1884

Bouvetoya (EA, GB)

30 Jun 04

 

HM162718

HM162643

HM162550

Dendronotidae Allman, 1845

Dendronotus venustus MacFarland, 1966

Santa Monica (California, GB)

Dec 07

 

HM162709

HM162630

HM162536

Proctonotidae Gray, 1853

Janolus mirabilis Baba and Abe, 1970

Philippines (GB)

19 May 09

 

HM162750

HM162674

HM162583

Aeolidiidae Gray, 1827

Aeolidia papillosa (Linnaeus, 1761)

Sweden

14 Sep 11

MNCN/ADN: 51929

JX087534

JX087462

JX087596

 

Aeolidiella alderi (Cocks, 1852)

France (MED)

26 Jul 02

ZSM Mol 20020982

HQ616765

HQ616728

HQ616794

 

Anteaeolidiella cacaotica (Stimpson, 1855)

Eastern Australia

14 Feb 10

MNCN/ADN: 51922

JX087528

JX087455

JX087590

 

Baeolidia moebii Bergh, 1888

Philippines

16 Apr 08

CASIZ 177602

HQ616770

HQ616733

HQ616799

 

Berghia coerulescens (Laurillard, 1830)

Croatia

03 Dec 04

ZSM Mol 20041584

JQ997049

JQ996845

JQ996946

 

Bulbaeolidia alba (Risbec, 1928)

Philippines

21 May 09

CASIZ 180387

JQ997012

JQ996805

JQ996904

 

“Cerberilla” annulata (Quoy and Gaimard, 1832)

Marshall Is.

24 Jul 00

CASIZ 182227

JQ996866

JQ996967

 

“Cerberilla” bernadettae Tardy, 1965

Spain (EA)

06 Apr l08

MNCN/ADN: 51957

JX087555

JX087489

JX087625

 

“Cerberilla” cf. affinis (Quoy and Gaimard, 1832)

Philippines

16 May 09

CASIZ 180421

JQ996863

JQ996964

 

Limenandra nodosa Haefelfinger and Stamm, 1958

Balearic Is. (Spain, MED)

Sep 2007

MNCN/ADN 24.923

HQ616768

HQ616731

HQ616797

 

Spurilla neapolitana (delle Chiaje, 1841)

Italy

17 Mar 09

MNCN/ADN: 51976

JX087584

JX087521

JX087659

Babakinidae Roller, 1973

Babakina anadoni (Ortea, 1979)

Brazil

Feb 06

MNRJ 10893

HQ616746

HQ616709

HQ616775

 

Babakina indopacifica Gosliner,

González-Duarte and Cervera, 2007

Philippines (GB)

20 Mar 08

HM162754

HM162678

HM162587

Facelinidae Bergh, 1889

Burnaia helicochorda Miller, 1987

Victoria, Australia

26 Oct 07

NMV F152957

KT200150

  

Victoria, Australia

13 Feb 08

NMV F155816

KT200148

KT200147

KT200149 

 

Cratena peregrina Gmelin, 1791

Senegal

30 May 05

MNCN 15.05/53691

HQ616752

HQ616715

HQ616781

 

Dondice banyulensis Portmann and Sandmeier, 1960

Spain (EA)

26 May 09

MNCN 15.05/53693

HQ616740

HQ616804

  

Spain (MED, GB)

AF249782

 

Facelina annulicornis (Chamisso and Eysenhardt, 1821)

Azores Is. (Portugal)

11 Jun 02

CASIZ 186793

JQ997076

JQ996881

JQ996986

 

Favorinus branchialis (Rathke, 1806)

Spain (EA)

26 Jun 07

MNCN 15.05/53695

HQ616761

HQ616724

HQ616790

 

Favorinus elenalexiarum García and Troncoso, 2001

Costa Rica (EP, GB)

17 Apr 07

HM162755

HM162679

HM162588

 

Godiva quadricolor (Barnard, 1927)

South Africa (EA, GB)

09 Jan 08

HM162692

HM162602

HM162508

 

Hermosita hakunamatata (Ortea, Caballer and Espinosa, 2003)

Mexico

17 Feb 06

CASIZ 174088

KP143674

KP143670

KP143678

 

Moridilla brockii Bergh, 1888

Philippines

29 Apri 11

CASIZ 186245

JQ997083

JQ996888

JQ996994 

 

Noumeaella isa Marcus and Marcus, 1970

Philippines

01 May 11

CASIZ 186249

JQ997084

JQ996889

JQ996995 

 

Phidiana lynceus Bergh, 1867

Cuba

21 Jul 08

MNCN/ADN: 51995

JX087562

JX087497

JX087633

 

Phyllodesmium horridum (Macnae, 1954)

South Africa (EA, GB)

03 Jan 08

HM162757

HM162681

HM162590

 

Pleurolidia juliae Burn, 1966

Philippines

05 May 05

CASIZ 186217

JQ997094

JQ996899

JQ997007

 

Protaeolidiella atra Baba, 1955

Japan

17 Feb 04

NSMT-Mo 78853

KP143676

KP143672

KP143680

  

Japan

18 Feb 04

NSMT-Mo 78852

KP143675

KP143671

KP143679

 

Pruvotfolia longicirrha (Eliot, 1906)

Cape Verde

Mar 10

MNCN 15.05/53703

HQ616760

HQ616723

HQ616789

 

Pruvotfolia pselliotes (Labbé, 1923)

France (EA)

05 Sep 04

MNCN 15.05/53705

HQ616762

HQ616725

HQ616791

 

Sakuraeolis enosimensis (Baba, 1930)

California (GB)

13 Dec 07

HM162758

HM162682

HM162591

Fionidae Alder and Hancock, 1855

Fiona pinnata (Eschscholtz, 1831)

Morocco (EA)

22 Dec 10

MNCN/ADN: 51997

JX087558

JX087492

JX087628

Flabellinidae Bergh, 1881

Flabellina affinis (Gmelin, 1791)

Balearic Is. (Spain, MED)

14 Jul 07

MNCN 15.05/53696

HQ616753

HQ616716

HQ616782

 

Flabellina babai Schmekel, 1972

Chafarinas Is. (MED)

25 Feb 07

MNCN 15.05/53698

HQ616754

HQ616717

HQ616783

 

Flabellina baetica García-Gómez, 1984 

Spain (EA)

14 Jan 05

MNCN 15.05/53699

HQ616755

HQ616718

HQ616784

 

Flabellina ischitana Hirano and Thompson, 1990

Morocco (EA)

07 Mar 08

MNCN 15.05/53700

HQ616756

HQ616719

HQ616785

 

Flabellina pedata (Montagu, 1815)

Spain (MED)

13 Oct 07

MNCN 15.05/53702

HQ616758

HQ616721

HQ616787

Piseinotecidae Edmunds, 1970

Piseinotecus gabinieri (Vicente, 1975)

Spain (MED)

13 Oct 07

MNCN/ADN: 52000

JX087561

JX087495

JX087631

 

Piseinotecus gaditanus Cervera, García-Gómez and García, 1987

Spain (EA)

20 Jun 07

MNCN 15.05/53704

HQ616759

HQ616722

HQ616788

 

Piseinotecus sp.

Philippines (GB)

22 Apr 08

HM162694

HM162604

HM162510

ATL Atlantic Ocean, EA eastern Atlantic Ocean, EP eastern Pacific, GB GenBank, MED Mediterranean

Sequence alignment and phylogenetic analyses

Sequences were assembled and edited with Geneious Pro version 4.7.6 (Drummond et al. 2009), aligned in MAFFT (Katoh et al. 2009), and further checked using MacClade version 4.06 (Maddison and Maddison 2005). The most variable regions from the 16S rRNA alignment were removed using the default settings in Gblocks (Talavera and Castresana 2007). Excluding “indel-rich” regions, the tree was in general very similar when including the variable regions. Therefore, final analyses were performed including all bases. Sequences of COI, 16S, and H3 were trimmed to 657, 445, and 327 base pairs, respectively.

The best-fit evolutionary model (GTR + I + G for the three genes) was determined in MrModeltest version 2.3 (Nylander 2004), using the Akaike information criterion (Akaike 1974). MrBayes version 3.1.2 (Ronquist and Huelsenbeck 2003) was used for Bayesian inference analysis and to estimate posterior probabilities (PP) for node support with two runs of 10,000,000 generations each. Convergence was checked in TRACER version 1.5 (Drummond and Rambaut 2007) with a burn-in of 25 %. Maximum likelihood (ML) analyses were performed using the software RAxML version 7.0.4 (Stamatakis 2006), and node support was assessed with nonparametric bootstrapping (BS) with 5000 replicates, random starting trees, and parameters estimated from each data set under the model selected for the original data set. Only nodes supported by BS ≥70 (Hillis and Bull 1993) and PP ≥0.95 (Alfaro et al. 2003) are discussed.

Source of specimens and morphology

The specimens studied were borrowed from the Museum Victoria (Melbourne, Australia). Specimens were dissected by dorsal incision. Their internal features were examined and drawn under a stereoscopic microscope with the aid of a camera lucida. The buccal mass was removed and soaked in a 10 % sodium hydroxide solution to dissolve the connective and muscle tissue, leaving only the radula and jaws. Both, radula and jaws, were then rinsed in water, dried, and mounted for examination under a scanning electron microscopy (SEM).

Results

Phylogenetic analysis

The combined data set yielded a sequence alignment of 1429 positions. No saturation was observed across genes and codon positions, not even in the third position (not shown). The resulting combined tree provided better resolution than H3, COI, or 16S separately (not shown). Figure 1 shows the phylogenetic hypothesis based on the combined data set constructed by Bayesian inference. The topology of the ML tree was identical (not shown), although the bootstrap values were lower than the PP in deeper nodes.
Fig. 1

Phylogenetic hypothesis for B. helicochorda based on the combined data set (H3 + COI + 16S) inferred by Bayesian analysis (BI). Numbers above branches represent posterior probabilities from BI. Numbers below branches represent bootstrap values from ML. B. helicochorda specimens in bold. B Babakinidae, Fbl Flabellinidae, P Piseinotecidae, F Facelinidae, D Dendronotidae, Fi Fionidae

Burnaia helicochorda and the remaining species of Aeolidiidae included in this study clustered in two distinct and well-separated clades (PP = 1, BS = 100, and PP = 0.99, BS = 72, respectively). B. helicochorda were nested among different facelinid species (PP = 0.99, BS = 51): Pruvotfolia longicirrha (Eliot, 1906), P. pselliotes (Labbé, 1923), Phidiana lynceus Bergh, 1867, Moridilla brockii Bergh, 1888, Cratena peregrina (Gmelin, 1791), Facelina annulicornis (Chamisso and Eysenhardt, 1821), Sakuraeolis enosimensis (Baba, 1930), Favorinus branchialis (Rathke, 1806), Favorinus elenalexiarum Garcia and Troncoso, 2001, Protaeolidiella atra Baba, 1955 and Pleurolidia juliae Burn, 1966. However, the relationships between them were not resolved, and Facelinidae was not recovered as monophyletic.

Diagnosis of Burnaia following Miller (2001):

Body long, narrow, oral tentacles moderately long, tapered, rhinophores longish, lamellate almost to base; cerata cylindrical, long, mobile (diverticula loosely spiralled), arranged in horseshoe-shaped arches, in single rows; cleioproctic, anus in centre of second arch, renal pore abanal, immediately in front of arch, reproductive apertures at tip of rear limb of first arch; foot narrow, rounded at front end extending as short, blunt lobes; oral glands small, club-shaped; radular tooth a very low, wide arch, blade narrowish, denticles almost uniform; jaw masticatory process complexly denticulate.
  • Burnaia helicochorda (Miller 1987)

  • (Figure 2)
    Fig. 2

    Burnaia helicochorda (Miller 1987). a, b Frontal and dorsal view of the living animal, photograph by L. Altoff. c, d Scanning electron photographs (NMV F155816). c General view of the radula, scale bar 125 μm. d Radular teeth, scale bar 75 μm. e Complete view of the jaw, scale bar 0.5 mm. f Detailed view of the masticatory border, scale bar 125 μm

  • Aeolidia helicochorda Miller 1987: 391, figs. 1–5.

Type locality:

Goat Island Bay, New Zealand.

Type material:

The holotype was deposited at the Museum of New Zealand (M.87451) (Miller (1987).

Material examined

(NMV F155816), one specimen, adult, mature, partially dissected, 11 mm in length preserved, Victoria, Australia, collected by Leon Altoff, 13.ii.08; (NMV F152957), one specimen, adult, mature, 5 mm in length preserved, Victoria, Australia, collected by Leon Altoff, 26.x.07.

Geographical distribution

Originally described from North of New Zealand (Miller 1987), this species has been also reported in eastern Australia (Rudman 2001; present study) and Tasmania (Grove 2015).

External morphology (Fig. 2a, b)

The body is elongate and narrow when fully extended, tapering gradually to the fairly short tail. The foot corners are rounded. The body colour is translucent white, but the dorsal surface may have orange pigmentation, which is denser from the area immediately posterior to the head to the posterior end of the body. The rhinophores, oral tentacles, and foot corners are also covered with orange. From the anterior base of the rhinophores to the edge of the head, there is longitudinal orange band that joins a U-shaped mark, which surrounds the edge of the head and runs forward to the base of the oral tentacles. The rhinophores, which are a bit shorter than the oral tentacles, are perfoliate, bearing up to 18 lamellae. A pair of black eyes is visible at the posterior side of the rhinophores base.

The cerata are arranged in arches, with gaps between the arches that decrease posteriorly. Each arch contains between two and 11 cerata. They extend from behind the rhinophores to the posterior end of the body. The ceratal length is variable, but all are slender, cylindrical, with a small swelling at the apical part. The cerata are translucent, but covered by orange pigmentation. The olive green ramifications of the digestive gland are visible through the ceratal wall and have several constrictions over their length. The distal 1/3 of the cerata is opaque greyish green. On the right side of the body, the gonopore is situated among the cerata of the anteriormost group. The anus is on the right side of the body, in a cleioproctic position, located within the second ceratal arch.

Anatomy (Fig. 2c–f)

The radula is uniseriate (11 × 0.1.0, NMV F155816). The teeth decrease in size towards the posterior region of the radula (Fig. 2c). The radular teeth are pectinate with 58–124, fine, elongate, and acutely pointed denticles (Fig. 2d). The jaws are translucent and oval (Fig. 2e). The masticatory border of the jaws is finely denticulate (Fig. 2f). The oral glands occur dorsolaterally to the buccal bulb. They are moderately short and increase in size towards the posterior end. Salivary glands were not observed.

The reproductive system was not examined so as not to damage the specimen, but was described in detail by Miller (1987, Fig. 5).

Discussion

The inclusion of B. helicochorda within the paraphyletic Facelinidae represents an intriguing and unexpected outcome. As Miller (1987, 2001) pointed out, in body shape B. helicochorda resembles Limenandra nodosa and L. fusiformis. In addition, the pectinate radula could be considered as the typical radula of the Aeolidiidae members (Carmona et al. 2014a, b, c). However, our molecular study separates B. helicochorda from Aeolidiidae and places it in a clade composed by facelinids. Carmona et al. (2013) and more recently Carmona et al. (2015) found out a similar situation with the species P. atra and P. juliae, formerly considered as primitive members of Aeolidiidae and now clearly situated within Facelinidae. This last outcome was supported by the feeding habits of these two species, since they prey on hydroids instead of sea anemones and other anthozoans that represent the characteristic prey of aeolidiids (Gosliner et al. 2008; Carmona et al. 2013). Nevertheless, we were unable to find information about the diet of B. helicochorda. Finally, in terms of morphology and internal anatomy, our study suggests re-evaluation of the apparent morphological synapomorphies that have traditionally united the aeolidid genera and species is needed. A more general and comprehensive revision of the Facelinidae and Aeolidida is also necessary given the fact that Facelinidae is not monophyletic (Carmona et al. 2013, 2015; present study).

Notes

Declarations

Acknowledgments

We are deeply grateful to R. Wilson and L. Altoff who helped to provide specimens and images, respectively, for this study. 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 is CEI·MAR journal publication 101.

Authors’ Affiliations

(1)
Departamento de Biología, Facultad de Ciencias del Mar y Ambientales, Campus de Excelencia Internacional del Mar (CEI·MAR), Universidad de Cádiz
(2)
Departamento de Biología, Edificio de Biología, Campus de Excelencia Internacional UAM+CSIC, Universidad Autónoma de Madrid
(3)
Department of Invertebrate Zoology, California Academy of Sciences

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Copyright

© Springer-Verlag Berlin Heidelberg and AWI 2015

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