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RECONSTRUCTION OF THE PHYLOGENY OF THE

OPISTHOBRANCHIA (MOLLUSCA: GASTROPODA) BY MEANS

OF 18S AND 28S rRNA GENE SEQUENCES

VERENA VONNEMANN1, MICHAEL SCHRODL2, ANNETTE KLUSSMANN-KOLB3

AND HEIKE WAGELE1

1Lehrstuhl fur Spezielle Zoologie, Ruhr-Universitat Bochum, 44780 Bochum, Germany;2

Zoologische Staatssammlung Munchen, 81247 Munchen, Germany;3Zoologisches Institut der J. W. Goethe Universitat, 60054 Frankfurt am Main, Germany

(Received 20 November 2003; accepted 25 August 2004)

ABSTRACT

The complete 18S (SSU) rRNA gene and the partial 28S (LSU) rRNA gene of 49 taxa, including

representatives of the Opisthobranchia, Pulmonata and Allogastropoda, were sequenced to infer the

phylogeny of the Opisthobranchia and their major subordinate taxa Nudibranchia, Pleurobranchoidea,

Tylodinoidea, Sacoglossa, Cephalaspidea s.l. and Anaspidea. For the first time the enigmatic taxon

Acochlidiacea was included. Several tests were applied to the datasets to investigate relative evolutionary

rates of the taxa, saturation of substitution, base composition and the amount of phylogenetic

information. Maximum parsimony, maximum likelihood and distance methods of tree construction were

used. The opisthobranch subgroups, including the Acochlidiacea, were confirmed as monophyletic in

most analyses, whereas monophyly vs paraphyly of Opisthobranchia could not be resolved. All treesshowed a sister-group relationship between Nudibranchia and Pleurobranchoidea (Nudipleura). In most

trees, sister-group relationships between Cephalaspideas.s.and Anaspidea, and between Nudipleura and

Acteonoidea (Pupa solidula,Hydatina physis), were supported. The latter sister-group relationship supports

an affinity that has not been mentioned for nearly 40 years (Ghiselin, 1966, Malacologia,3: 327378). It

was rediscovered in the molecular analyses by Grande et al. (2004, Molecular Biology and Evolution, 21:

303313) and represents a valid and interesting opisthobranch clade.

INTRODUCTION

The Opisthobranchia are an assemblage of morphologically verydiverse snails and slugs occurring in marine habitats all over theworld. Their appearance ranges from inconspicuous caenogas-

tropod-like animals burrowing in sand to extravagantly colouredand highly derived swimming organisms. Their smallest repre-sentatives (Microhedylidae, Acochlidiacea) measure little morethan 1mm (Arnaud, Poizat & Salvini-Plawen, 1986), whereasthe largest species (Aplysia vaccariaWinkler, 1955, Anaspidea) canreach nearly 1m in length (Behrens, 1980). The earliest fossilopisthobranchs are from the Lower Carboniferous (ca 310Ma),resembling members of the extant Scaphandridae and Retusidae(Cephalaspidea; Gosliner, 1981). The number of species ofOpisthobranchia is of the order of 45005000 (Wa gele,unpublished) and is very different among the opisthobranchsubgroups (e.g. Tylodinoidea about 15 species, Nudibranchianearly 3000 species).

Despite numerous publications dealing with the Opisthobran-chia, their phylogeny is mainly unresolved. This is due to the lack

of comprehensive morphological investigations and the contra-dictory results produced by molecular studies (Thollesson, 1999;Dayrat et al., 2001;Wa gele, Vonnemann & Wa gele, 2003). Thepresent study is the first extensive phylogenetic examination ofopisthobranch 28S rRNA gene sequences and also the firstconsidering the domains D1, D2 and D3. Dayrat et al. (2001)investigated the D1 and D2 domains of 31 euthyneuran speciesand found that some groups were well supported, but resolutionwas poor for other major clades. In order to obtain an increase of

phylogenetic signal, additional characters (domain D3) havebeen included.

The aims of the present paper are to reconstruct opisthobranchphylogeny and to assess the suitability of the 28S rRNA gene forphylogenetic questions in comparison with the well-studied 18SrRNA gene.

MATERIALS AND METHODS

Biological material

Forty-three species of opisthobranchs and six outgroup species(Pulmonata and Allogastropoda) were collected at various sitesall over the world (Table 1). Within the scope of the present studythe partial 28S rRNA gene of 48 taxa and the complete 18SrRNA gene of 41 taxa were sequenced, and the remainingsequences were taken from GenBank.

DNA extraction, amplification and sequencing

Genomic DNA was extracted from alcohol-preserved specimensby means of the Blood and Tissue Kit (Qiagen), guided by theenclosed protocol. The amplification of the 18S rRNA generegion by PCR (Saiki et al., 1988) was performed with primersdeveloped by Trisha Spears (personal communication): forward(18A1), 50-CCT A(CT)C TGG TTG ATC CTG CCA GT-30

and reverse (1800), 50-GAT CCT TCC GCA GGT TCA CCTACG-30. The PCR was carried out in the thermal cycler Progene(Techne) under the following conditions: 95 8C for 4 min,followed by 38 cycles of 30 s at 94 8C, 30 s at 52.58C, 2.5min at72 8C and a final extension at 72 8C for 10 min. The domains D1D3 of the 28S rRNA gene were amplified with forward primerC10 (Dayrat et al., 2001): 50-ACC CGC TGA ATT TAA GCA

Correspondence:V.Vonnemann;e-mail: [email protected]

Journal of Molluscan Studies (2005) 71: 113125 doi: 10.1093/mollus/eyi014

q The Author 2005. Published by Oxford University Studies on behalf of The Malacological Society of London, all rights reserved.

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Table 1.List of specimens examined in this study, sampling localities, accession numbers and lengths of complete 18S and partial 28Sr DNA genes.

Species Family Collecting locality Accession numbers*

Length of

complete 18S

Length of partial

28S (D1D3)

NUDIBRANCHIA

Bathydoris clavigera Bathydorididae Antarctica AY165754 2007 1383

Thiele, 1912 AY427444Chromodoris krohni Chromodorididae Spain, Atlantic Ocean AJ224774 1826 1064

(Verany, 1846) AY427445

Hypselodoris villafranca Chromodorididae Spain, Atlantic Ocean AJ224780 1832 1071

(Risso, 1818) AY427446

Dendrodoris fumata Dendrodorididae Great Barrier Reef, Australia AF249216 1842 1062

(Ruppell & Leuckart, 1830) AY427447

Cuthona nana Tergipedidae Helgoland, Germany AY165760 1881 1027

(Alder & Hancock, 1842) AY427448

Flabellina babai Flabellinidae Spain, Mediterranean Sea AY165768 1870 1045

(Schmekel, 1972) AY427449

Dendronotus dalli Dendronotidae USA, Atlantic Ocean AY165757 1947 1063

Bergh, 1879 AY427450

PLEUROBRANCHOIDEA

Euselenops luniceps Pleurobranchidae Great Barrier Reef, Australia AF249218 2047 1136(Cuvier, 1817) AY427451

Tomthompsonia antarctica Pleurobranchidae Antarctica AY427492 2105 1156

(Thiele, 1912) AY427452

Bathyberthella antarctica Pleurobranchidae Weddel Sea, Antarctica AF249219 2024 1123

Willan & Bertsch, 1987 AY427453

Berthellina citrina Pleurobranchidae Elba, Italy AY427493 2028 1090


Pleurobranchus peroni Pleurobranchidae Wollongong, NSW, Australia, AY427494 2022 1120

Cuvier, 1804 AY427455

Berthella stellata Pleurobranchidae Elba, Italy AY427495 2001 1166

(Risso, 1826) AY427456

TYLODINOIDEA

Umbraculum mediterraneum Umbraculidae Meteor Bank, Atlantic Ocean AY165753 1786 1069

(Lamarck, 1829) AY427457

Tylodina perversa Tylodinidae Spain, Mediterranean Sea AY427496 1796 1067

(Gmelin, 1791) AY427458

SACOGLOSSA

Placobranchus ocellatus Plakobranchidae Magnetic Island, QLD, Australia AY427497 1790 1053

van Hasselt, 1824 AY427459

Bosellia mimetica Boselliidae Elba, Italy AY427498 1793 1053

Trinchese, 1891 AY427460

Thuridilla bayeri Elysiidae Great Barrier Reef, Australia AF249220 1786 1053

Marcus, 1965 AY427461

Elysia viridis Elysiidae Mediterranean Sea AY427499 1789 1052

(Montagu, 1804) AY427462

Cyerce nigricans Caliphyllidae Eagle Island, QLD, Australia AY427500 1790 1063

(Pease, 1866) AY427463

Calliopaea bellula Limapontiidae Elba, Italy AY427501 1787 1044

dOrbigny, 1837 AY427464

Limapontia capitata Limapontiidae North Sea AJ224920 1786 1047

(O.F. Muller, 1773) AY427465

ANASPIDEA

Akera bullata Akeridae Kattegat, Denmark AY427502 1789 1047

Muller, 1776 AY427466

Aplysia californica Aplysiidae Genbank AY039804 1791 1049

Cooper, 1863 AY026366

Dolabella auricularia Aplysiidae Okinawa, Japan AY427503 1785 1042

(Lightfoot, 1786) AY427467

V. VONNEMANN ET AL.V. VONNEMANN ET AL.

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Table 1.(continued)

Species Family Collecting locality Accession numbers*

Length of

complete 18S

Length of partial

28S (D1D3)

CEPHALASPIDEA s.s.

Haminoea hydatis Haminoeidae Elba, Italy AY427504 1790 1037

(Linne, 1758) AY427468Phanerophthalmus smaragdinus Haminoeidae Lizard Island, QLD, Australia AY427505 1787 1036


Atys semistriatus Haminoeidae Dingo Beach, QLD, Australia AY427506 1787 1034

Pease, 1860 AY427470

Odontoglaja guamensis Aglajidae Orpheus Island, QLD, Australia AY427507 1789 1039

Rudman, 1978 AY427471

Navanax inermis

(Cooper, 1863)

A glaj id ae La P az, Gu lf of Cal if ornia, Me xico AY 42 75 08

AY427472

1790 1034

Chelidonura inornata Aglajidae Lizard Island, QLD, Australia AY165752 1796 1039

Baba, 1949 AY427473

Philinopsis pilsbryi Aglajidae Magnetic Island, QLD, Australia AY427509 1788 1034

(Eliot, 1900) AY427474

Philinoglossa praelongata Philinoglossidae Rovinj, Croatia AY427510 1784 1041

Salvini-Plawen, 1973 AY427475cf. Retusa sp. Retusidae Elba, Italy AY427511 1788 1035

AY427476

Bulla cf. striata Bullidae Elba, Italy AY427512 1787 1036

Bruguiere, 1792 AY427477

Sagaminopteron psychedelicum

Carlson & Hoff, 1974

Gastropteridae Magnetic Island, QLD, Australia AY427513

AY427478

1792 1042

Runcina adriatica Runcinidae Elba, Italy AY427514 1783 1044

Thompson, 1980 AY427479

ACTEONOIDEA

Hydatina physis Hydatinidae Hastings Point, NSW, Australia AY427515 1804 1087

(Linne, 1758) AY427480

Pupa solidula Acteonidae Dingo Beach, QLD, Australia AY427516 1842 1065

(Linne, 1758) AY427481

ACOCHLIDIACEAUnela glandulifera Microhedylidae Rovinj, Croatia AY427517 1787 1045

(Kowalewsky, 1901) AY427482

Parahedyle cryptophthalma Microhedylidae Elba, Italy AY427518 1787 1045

(Westheide & Wawra, 1974) AY427483

Pontohedyle milatchevitchi Microhedylidae Elba, Italy AY427519 1788 1043


Hedylopsis spiculifera Hedylopsidae Rovinj, Croatia AY427520 1786 1035


PULMONATA

Onchidella floridana Onchidiidae Flatts Village, Bermuda AY427521 1790 1049

(Dall, 1885) AY427486

Onchidium cf. verruculatum Onchidiidae Cockle Bay, QLD, Australia AY427522 1791 1050

(Cuvier, 1830) AY427487

Siphonaria alternata Siphonariidae Flatts Village, Bermuda AY427523 1786 1046

(Say, 1826) AY427488

Cepaea hortensis Helicidae Grietherbusch, Rhine, Germany AY427524 1788 1058

(OF Muller 1774) AY427489

Lymnaea stagnalis Lymnaeidae Grietherbusch, Rhine, Germany AY427525 1800 1042

(Linnaeus, 1758) AY427490

ALLOGASTROPODA

Odostomia sp. Pyramidellidae Banyuls sur Mer, France,

Mediterranean Sea

AY427526

AY427491

1791 1044

*Accession number for complete 18S rRNA gene sequence above, accession number for partial 28S rRNA gene below.

Specimens have been deposited at either the Zoologische Staatssammlung Munchen (ZSM) or at the Lehrstuhl Spezielle Zoologie, Ruhr-Universita t Bochum.

PHYLOGENY OF OPISTHOBRANCHIAPHYLOGENY OF OPISTHOBRANCHIA

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T-30 and reverse primer D3: 50-GAC GAT CGA TTT GCA CGTCA-30 under the same conditions as the 18S rRNA gene. EachPCR reaction mix (50 ml) contained 5ml of 10 PCR buffer(Qiagen), 10ml of Q-Solution (Qiagen), 5ml of dNTP-mix(2 mM per dNTP), 0.5ml of each primer (18A1 and 1800 or C1and D3), 0.3 ml of Taq polymerase (Qiagen), 0.25 to 3.0 ml ofgenomic DNA and 21.5525.7 ml H2O.

After purification of the PCR products by means of theQIAquick PCR purification kit (Qiagen), the 18S and 28S rRNA

genes were sequenced directly by the chain-termination method(Sanger, Miklen & Coulson, 1977) using the Thermo-sequenasefluorescent-labelled primer cycle sequencing kit (Amersham) onthe automated sequencers 4000 and 4000IR2 (Licor). Bothstrands of the 18S and 28S rRNA genes were sequenced using theprimers from the PCR and several internal primers (Table 2).

Sequence alignment

Sequences were aligned with CLUSTAL X (Thompson et al.,1997) using the default parameters (pairwise parameters: gapopening 10, gap extension 0.1; alignment parameters: gap

opening 10, gap extension 0.2), but subsequent manual correc-tion was necessary. This was based on different and partlysubjective criteria: elimination of obvious errors like displacedsequence parts, optimization of the homologies among groupsthat are well supported morphologically and maximization ofcorrespondences by insertion of gaps. This manual treatment wasexecuted by means of the program GENEDOC (Nicholas &Nicholas, 1997).

Phylogenetic signal

The software PHYSID (Phylogenetic Signal Detection, Wa gele& Ro dding, 1998) allows ana prioriestimation of the informationcontent of aligned sequences without reference to a tree topologyor to a model of sequence evolution. The method consists of asearch for splits in an alignment supported by putativeapomorphies. If the most frequent partitions are compatible astrong phylogenetic signal is assumed (Wa gele, 1996;Wa gele &Ro dding, 1998). An a posteriori evaluation can follow bycomparing the splits with the dendrograms obtained bymaximum parsimony, maximum likelihood or distance methodsand examinating the fit of the splits on the trees.

Another (a posteriori) approach for evaluating the phylogeneticsignal of the 18S and 28S rRNA gene alignments is the

permutation-tail probability (PTP) test (Archie, 1989). Thistest is based on comparison of the length of the most parsimonioustree with the lengths of trees obtained after randomly permutat-ing the character state assignments within each character. Thepermutation-tail probability is then defined as the estimate of theproportion of times that a tree can be found that is as shortor shorter than the original tree (Faith & Cranston, 1991). ThePTP test was conducted by means of PAUP 4.0 beta 3a(Swofford, 1999).

Statistical tests and phylogenetic reconstruction

Base composition and saturation of substitution were investigatedby means of the software PAUP 4.0 beta 3a (Swofford, 1999) andrates of evolution by means of the software k2WuLi ( Wu & Li,1985).

Alltree-building methods were conducted using PAUP 4.0beta3a (Swofford, 1999). Parameters of maximum-parsimony analyseswere: ACCTRAN, gaps counted as fifth base; heuristic searchoptions: stepwise addition random, branch-swapping option TBR. The option gaps are missing data was not used, sinceelongations in sequences are separate characters, and should becoded as such. The parameters for distance and maximum-likelihood methods were determined with likelihood ratio testsusing Modeltest Version 3.06 (Posada & Crandall, 1998).

For testing incongruence in combined analyses, the incon-gruence length difference (ILD) test (Farris et al., 1994) wasapplied. With this test, it can be inferred whether combining thedata improves phylogenetic accuracy (P. 0.01) or whetheraccuracy of the combined data suffers relative to the individualpartitions (P, 0.001,Cunningham, 1997).

RESULTS

Sequence alignment

The alignment of the 18S rRNA gene (28S rRNA gene)sequences comprises 2211 (1149) positions, of which 1124 (441)positions are constant, 293 (149) are variable but parsimony-uninformative and 794 (559) are parsimony-informative.

Parts with uncertainties of homology were removed from thealignments (unpublished data of secondary structure analyses ofsenior author). In the 18S rRNA gene the Pleurobranchoideapossess long non-alignable insertions (positions 865 1004 in

Table 2. Primers used for sequencing the complete 18S and partial 28SrRNA genes.

Name Sequence (50 3 0) Reference

18A1seq CTG GTT GAT CCT GCC

AGT CAT ATG C

According to PCR primer

400F ACG GGT AAC GGG GAA

TCA GGG

Wollscheid, Dreyer & Englisch

470F CAG CAG GCA CGC AAA

TTA CCC


700F GTC TGG TGC CAG CAG

CCG CG


1155F CTG AAA CTT AAA GGA

ATT GAC GG


1600F CGT CCC TGC CCT TTG

TAC ACA CC


1800seq GAT CCT TCC GCA GGT

TCA CCT ACG


1500R CAT CTA GGG CAT CAC

AGA CC


1155R CCG TCA ATT CCT TTA

AGT TTC AG


700R CGC GGC TGC TGG CAC

CAG AC


400R CCC TGA TTC CCC GTT

ACC CGT


28SC1 ACC CGC TGA ATT TAA

GCA T


28SC2F (C20)* GAA AAG AAC TTT GAA

GAG AGA GT

Dayratet al. 2001, modified

28 SD2 F CCC GTC TTG AA A CA C

GGA CCA AGG

Vonnemannet al., present study

28SD3 GAC GAT CGA TTT GCA

CGT CA


28 SD2 R CCT TGG TCC GTG TTT

CAA GAC GGG

Vonnemannet al., present study

28SC2R (C2)* ACT CTC TCT TCA AAG

TTC TTT TC

Dayratet al., 2001, modified

* Names of unmodified primers in brackets (Dayrat et al., 2001). Molecular Phylogeny Working group, Department of Spezielle Zoologie,

Ruhr-Universitat Bochum.


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structure E23_3) in region V4 (Wuyts, van de Peer &De Wachter, 2001). In structure 43 (region V7: positions18921974) the sequence lengths of Nudibranchia, andespecially ofBathydoris clavigera, are much higher than of theother opisthobranch taxa and show a high divergence. These tworegions were excluded from the analyses. In the 28S rRNA gene,length variation and extensive sequence divergence occur in thedomains D2 and D3. Therefore, the alignment positions 491539(D2), 667729 (D2) a nd 11131398 (D3) were not considered

in the phylogenetic analyses.

Phylogenetic signal

The application of PHYSID (Wa gele & Ro dding, 1998) revealsthat the signal-to-noise relationship is adverse in both alignments.Most opisthobranch subgroups are supported by as many orfewer putative apomorphies as nonsense groups, i.e. thephylogenetic signals for these clades are not higher thanbackground noise. The most frequent partition separates Cuthonaand Flabellina (Nudibranchia) from the remaining taxa, sup-ported by 217/102 (18S dataset/28S dataset) putative apomor-phies. Concerning the major opisthobranch clades thePleurobranchoidea show the best signal (70/42 putative apo-morphies), followed by Nudibranchia (50/34), Nudipleura (31/24), Acteonoidea (31/21), Sacoglossa (10/14), Cephalaspideas.s.(12/4), Tylodinoidea (6/10), Anaspidea Cephalaspidea s.s.(7/4), Anaspidea (2/8) and Acochlidiacea (1/1).

The permutation-tail probability (PTP) test on the other handreveals significant phylogenetic information in the 18S as well asin the 28S dataset. After generation of 100 random data sets,computed tree lengths were compared with the MP trees (50%majority rule consensus;Figs 1, 2) of the original data. In the 18SrRNA gene dataset, the shortest tree length of the unpermutateddata is 2903 and the shortest random set length is 5996, resultingin the minimum possible PTP of 0.01. In the 28S the PTP is also0.01 (actual length 3413, shortest random set length 5678).

Statistical tests

Execution of the chi-squared test shows a significant hetero-

geneous base composition in the 18S (P 0) as well as the 28S(P 0.02) rRNA gene data, caused primarily by the sequences ofthe Nudibranchia. After exclusion of this taxon, Pvalues of 0.89(18S) and 0.92 (28S) are obtained. Additional exclusion of thePleurobranchoidea leads to a homogeneous composition of bases(18S, P 1.0; 28S, P 0.99). Regarding the AT and GC-content of the 18S rRNA gene sequences a progressive increase ofAT is observed from Cladobranchia (Nudibranchia, 38.4%) toAnthobranchia (Nudibranchia, 42.4%), Pleurobranchoidea(43.7%), Acteonoidea (46.8%) and the remaining taxa(48.1%). This trend is not as distinct in the 28S rRNA gene(Cladobranchia, 31.7%; Anthobranchia, 32.5%; Pleurobran-choidea, 34.2%; Acteonoidea, 32.9%; others, 36.8%). Investi-gation of the nucleotide substitutions by plotting the P-distancesagainst the absolute number of transitions and transversions (not

shown) indicated that the 18S and 28S rRNA gene sequences arenot saturated.

The relative-rate test (Sarich & Wilson, 1973) reveals thatevolutionary rates are significantly different in both genes. TheCladobranchia (Nudibranchia) show the highest rates in bothgenes, with a maximum Z-score of 15.9 between Dendronotus dalliand Siphonaria alternata (Pulmonata) in the 18S rRNA gene and10.9 between Dendronotus dalli and Dolabella auricularia(Anaspidea) in the 28S rRNA gene. The Anthobranchia(Nudibranchia) show higher rates than the other taxa exceptCladobranchia (18S, 5.910.1; 28S, 2.67.4), and the Pleuro-branchoidea evolve faster than the remaining taxa exceptNudibranchia (18S, 5.09.8; 28S, 1.77.0). Within the

Pleurobranchoidea the very high rates of Berthella stellata arestriking (18S, 7.314.7; 28S, 2.39.3). Its Z-scores are muchhigher than the values of the other Pleurobranchoidea andeven higher than those of the Anthobranchia. Among the othergroups the substitution rates are comparatively homogeneous(18S, 05.0; 28S, 04.7), except for Pupa solidula and Hydatinaphysis, which are distinguished by slightly higher Z-scores in the18S gene (0.15.9)

Maximum-parsimony analysis

A maximum-parsimony analysis of the complete 18S rRNA genewith Odostomia sp. (Pyramidellidae) as outgroup produced twoshortest topologies (length 2903 steps, CI 0.63, RI 0.78,RC 0.49). The strict consensus tree is shown inFigure 1. Allopisthobranch subgroups appear monophyletic, with the Nudi-pleura (Nudibranchia and Pleurobranchoidea), Acteonoidea(Pupa solidula and Hydatina physis) and Sacoglossa forming onemajor clade and Cephalaspidea s.s., Anaspidea, Tylodinoideaand Acochlidiacea forming the other. The Acteonoidea are thesister group of the Nudipleura and the members of the Pulmonataare basal in the tree. The bootstrap analysis (1000 replicates)reveals low support for the position of the major opisthobranchgroups. Monophyletic groups with high bootstrap support are,for example, Nudipleura (100%), Pleurobranchoidea (100%),Nudibranchia (90%), Acteonoidea (100%), Nudipleura plusActeonoidea (98%), Tylodinoidea (97%), Sacoglossa (100%)and Cephalaspidea s.s. except Runcina adriatica (100%). Thesister-group relationship of Cephalaspidea s.s. and Anaspidea issupported by a bootstrap value of only 56%; the sister-grouprelationship between these two groups and the Tylodinoidea byonly 59%. The positions of the Sacoglossa, Acochlidiacea andPulmonata are not resolved at all and the acochlidiacean speciesHedylopsis spiculiferadoes not form a monophyletic group with theother Acochlidiacea.

The result of the maximum-parsimony analysis of the 28SrRNA gene (Fig. 2; 16 shortest trees, length 3413 steps,CI 0.45, RI 0.63, RC 0.28) shows several correspon-dences with the results based on the 18S rRNA gene alignment.Subordinate taxa of the Opisthobranchia are monophyletic

except the Acochlidiacea (as above). The sister-group relation-ships between Nudipleura and Acteonoidea as well as betweenCephalaspidea s.s. and Anaspidea are confirmed, and a sister-group relationship between Sacoglossa and Tylodinoidea isproposed. The bootstrap tree does not differ significantly fromthe maximum-parsimony tree, but resolution is lower. Mono-phyletic groups with high bootstrap support are, for example,Nudibranchia (97%), Pleurobranchoidea (99%), Nudipleura(100%), Acteonoidea (100%), Cephalaspidea s.s. (88%),Anaspidea (100%), Sacoglossa (100%), Acochlidiacea withoutHedylopsis spiculifera (99%) and Tylodinoidea (91%). The sister-group relationship between Nudipleura and Acteonoidea has abootstrap value of only 54%, and that between Cephalaspideas.s. and Anaspidea has a bootstrap value of 76%.

The incongruence length-difference test (Farris et al., 1994)

shows aPvalue of 0.003 (1000 replicates), indicating a significantdegree of incongruence. Cunningham (1997) showed thatcombining genes generally improved phylogenetic accuracy,even in cases where incongruence was detected. Only whenP values were lower than 0.001 did the combined data sufferrelative to the individual partitions. Therefore, the 18S and 28SrRNA gene data sets have also been analysed combined.

The combined analysis of the two genes (Fig. 3) shows atopology (8 shortest trees, length 6352 steps, CI 0.53,RI 0.7, RC 0.37) that is better resolved than the 28S tree,but not as good as the 18S tree. The Pulmonata are presented asthe sister group of the Opisthobranchia, the remaining taxa formthree major clades, of which the relationships are unresolved.


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One clade consists of Nudipleura Acteonoidea Sacoglossa,the second of Cephalaspidea s.s. Anaspidea Tylodinoideaand the third of the Acochlidiacea. The bootstrap analysis showshigher resolution compared with the bootstrap trees of the singlegenes. All opisthobranch subgroups are supported by highbootstrap values, as well as Nudibranchia plus Pleurobranchoi-dea (100%), Nudipleura plus Acteonoidea (98%) and Cepha-laspideas.s. plus Anaspidea (91%).

Distance methods

For choosing the most appropriate model of sequenceevolution, hierarchical likelihood-ratio tests (Goldman, 1993)are conducted by means of the program Modeltest Version3.06 (Posada & Crandall, 1998). For both datasets the best-fitmodel is the Tamura-Nei model (Tamura & Nei, 1993) with

gamma distribution shape parameters of a 0.31 (18S) anda 0.52 (28S). The proportions of invariable sites are 0.27(18S) and 0.23 (28S). The phylogram of the 18S dataset is

shown in Figure 4, revealing that branch lengths areextremely short in all groups except Nudibranchia, Pleuro-branchoidea and Acteonoidea. The results of the distance treesand bootstrap values (18S and 28S gene) are presented inFigure 5A, B. For better clarity and easier comparison ofopisthobranch intrarelationships we prefer to present thedistance and maximum-likelihood trees as cladograms andnot as phylograms, and tree presentation is limited to therelationships of the major opisthobranch groups. If they arenot monophyletic, the excluded taxa are specified separately.In Figure 5A, B it can be seen that several taxa have varyingpositions within the cladograms (Sacoglossa, Anaspidea,Acochlidiacea, Pulmonata), and even the monophyly of

Figure 1.Strict consensus tree of two shortest maximum-parsimony trees based on complete 18S rRNA gene (length 2903 steps, CI 0.63, RI 0.78,RC 0.49). Bootstrap indices (50% majority rule) are given.


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some major taxa is not supported (Pulmonata by exclusion ofCepaea hortensis and/or Lymnaea stagnalis, Cephalaspidea s.s. by

exclusion of Runcina adriatica). Bootstrap values in the 18S treeare high for e.g. Cladobranchia (100%), Nudipleura (96%),Acteonoidea (100%), the latter plus Nudipleura (86%),Sacoglossa (92%), Cephalaspidea s.s. except Runcina adriatica(99%), Anaspidea (84%) and Tylodinoidea (99%). For 28S,they are high, for example, Anthobranchia (98%), Clado-branchia (100%), Nudibranchia (98%), Pleurobranchoidea(99%), Nudipleura (99%), Acteonoidea (100%), Anaspidea(99%), Cephalaspidea s.s. except Runcina adriatica (90%),Sacoglossa (100%) and Tylodinoidea (97%).

A distance analysis of the combined datasets is not carried outbecause the ILD test is presently only implemented withparsimony.

Maximum-likelihood analysis

The phylogenetic reconstructions by means of the maximum-likelihood method were also conducted with the settings obtainedfrom the likelihood-ratio tests (see above).

The trees based on 18S and 28S rRNA genes (Fig 5C, D) againshow differences. Monophyly is shown for the major opistho-branch groups except for the Acochlidiacea (because ofHedylopsisspiculifera). MoreoverRuncina adriaticais excluded again from theCephalaspideas.s. in the 18S tree. Bootstrap analyses could notbe carried out because of the computation time required. Thetwo datasets were not analysed simultaneously under themaximum-likelihood criterion (see above).

Comparing all four analyses (Fig. 5A D), the sister-taxarelationship of Nudipleura and Acteonoidea occurs three times,

Figure 2. Strict consensus tree of 16 shortest maximum-parsimony trees based on partial 28S rRNA gene (length 3413 steps, CI 0.45, RI 0.63,RC 0.28). Bootstrap indices (50% majority rule) are given.


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and the sister-taxa relationship of Cephalaspidea s.s. andAnaspidea twice.

DISCUSSION

To date, the phylogeny of the Opisthobranchia is unresolved,both in respect of the origin of the whole group as well as of theinterrelationships of its major clades. Two possible evolutionaryscenarios have been discussed: Opisthobranchia and Pulmonataevolved from a common ancestor and are therefore sister-groups(e.g.Gosliner, 1981, 1994; Gosliner & Ghiselin, 1984;Wade &Mordan, 2000;Mikkelsen, 2002;Wa geleet al., 2003); Pulmonataoriginated within the Opisthobranchia, rendering thelatter paraphyletic (e.g. Haszprunar, 1985; Tillier, Masselot,

Guerdoux & Tillier, 1994; Tillier, Masselot & Tillier, 1996;Ponder & Lindberg, 1997;Thollesson, 1999;Dayratet al., 2001).

A third phylogenetic hypothesis for the Euthyneura hasrecently been presented by Grande et al. (2003), based on theanalysis of eight mitochondrial genes. They recovered thepulmonates as a polyphyletic group with the Stylommatophoraas an early split and the basommatophoran pulmonateSiphonariaplaced within the opisthobranchs.

Because of the well-known rampant parallelism in opistho-branch gastropods, there are very few uniquely derivedattributes in the Opisthobranchia (Gosliner & Ghiselin, 1984).Mikkelsen (2002) mentioned the following synapomorphies:loss of the adult operculum, presence of parapodia (withthree reversals) and loss of the posterior stomach chamber.Many phylogenetic investigations concerning the various

Figure 3.Strict consensus tree of 8 shortest maximum-parsimony trees based on the combined dataset of 18S and 28S rRNA genes (length 6352 steps,CI 0.53, RI 0.7, RC 0.37). Bootstrap indices (50% majority rule) are given.


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opisthobranch subgroups have been carried out and haveproduced some good results. The Cephalaspidea s.l. arecommonly accepted as a paraphyletic group consisting of themonophyletic group Cephalaspideas.s.and a cluster of primitiveforms, the Acteonoidea (Acteonidae, Hydatinidae and Ring-iculidae). Apomorphies of Cephalaspidea s.s. are the ciliatedstrips flexed at the mantle margin and the presence of threegizzard plates (Mikkelsen, 1996). The Acteonoidea are eithera basal and probably paraphyletic opisthobranch group(e.g. Gosliner, 1994) or have even been excluded from theOpisthobranchia, belonging to the lower heterobranchs

(e.g. Mikkelsen, 1996). The lower heterobranchs compriseseveral families, for example Rissoellidae, Architectonicidae andPyramidellidae, that in the past have been variously aligned withprosobranchs or opisthobranchs.

The Anaspidea (including Akera) are well supported as amonophyletic group by several synapomorphies from thereproductive system, defensive glands, radula, gizzard andnervous system (Morton & Holme, 1955; Ghiselin, 1966;Mikkelsen, 1996; Medina & Walsh, 2000). The Sacoglossa,with reallocation of the genus Cylindrobulla, are confirmed asmonophyletic by, for example, the uniserial radula and the ascus

Figure 4.Distance phylogram based on the complete 18S rRNA gene. Model of sequence evolution: Tamura-Nei model (Tamura & Nei, 1993), gammadistribution shape parameter 0.31, proportions of invariable sites 0.27.


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(Mikkelsen, 1998, 2002). Monophyly of the Notaspidea(Pleurobranchoidea plus Tylodinoidea) has been rejected byseveral workers (Minichev & Starobogatov, 1978; Schmekel,

1985; Salvini-Plawen, 1991; Salvini-Plawen & Steiner, 1996;Wa gele & Willan, 2000). Characters presented as apomorphiesbyWillan (1987), for example a bipinnate gill on the right side ofthe body and longitudinally slit rhinophores, have since beenshown to be plesiomorphies. Monophyly of the Tylodinoidea issupported by a cuticularized thickened ring just inside the mouth(Willan, 1987) and the concentration of the nervous system withpaired cerebral, pleural-intestinal and pedal ganglia (Schmekel,1985). The Pleurobranchoidea are united by the internal,rectangular shell and the presence of a pedal gland (Willan,1987). Schmekel (1985) was the first to propose a sister-grouprelationship of Pleurobranchoidea and Nudibranchia, confirmedby Salvini-Plawen (1990, 1991) and Wa gele & Willan (2000).The latter authors named the new taxon Nudipleura forPleurobranchoidea plus Nudibranchia, characterized by the

possession of a blood gland, an androdiaulic reproductive systemand the loss of the osphradium.A polyphyletic origin has often been assumed for the

Nudibranchia (e.g. Minichev, 1970) but recent analysessupport their monophyly (Wa gele & Willan, 2000). Apomor-phies are, for example, the presence of a vacuolatedepithelium and a longitudinally orientated pericardial complex(Wa gele & Willan, 2000).

The monophyly of the interstitial Acochlidiacea is supportedby their unique external organization. They possess a distinctvisceral hump separated from the rest of the body, in which thehead-foot complex can be partially or completely retracted(Schro dl, personal communication;Rankin, 1979).

The pelagic Gymnosomata and Thecosomata have not yetbeen studied phylogenetically and are not considered in thepresent paper.

Our knowledge about the phylogeny of the subgroups withinthe opisthobranchs is fragmentary because extensive morpho-logical analyses based on cladistic principles are lacking andmolecular analyses have produced contradictory or poorlyresolved trees (e.g. compare the molecular studies by Thollesson,1999,andDayratet al., 2001with the morphological analysis byWa gele & Willan, 2000). Mikkelsen (1996), in her thoroughmorphological analyses of Cephalaspidea s.l., found a sister-group relationship of Cephalaspideas.s.and Anaspidea, togetherrepresenting the sister-group of the Sacoglossa. In 2002 sheconducted a combined analysis of characters from four publishedphylogenies based on morphological characters (Willan, 1987;Mikkelsen, 1996; Jensen, 1996, 1997;Wa gele & Willan, 2000),although the resulting cladogram was not well resolved. Themost derived clade consisted of Tylodinoidea and Nudipleura,

forming a monophyletic group with Anaspidea and Cephalaspi-dea s.s. All groups together were the sister taxon of theSacoglossa. A sister-group relationship of Anaspidea andCephalaspideas.s. was also evident in molecular analyses of the16S rRNA gene in the study byThollesson (1999)and of the 18SrRNA gene, 16S rRNA gene and COI gene by Wa gele et al.(2003).

Practically nothing can be said about the position ofAcochlidiacea, Thecosomata and Gymnosomata, because onlyvery few data are available.Gosliner & Ghiselin (1984)presentedthe Acochlidiacea as the sister-group of the Sacoglossa basedon morphological characters. Dayrat et al. (2001) found amonophyletic group consisting of Thecosomata, Gymnosomata

Figure 5.Distance (branch lengths not considered) and maximum-likelihood trees (branch lengths not considered) based on complete 18S rRNA andpartial 28S rRNA genes. For better understanding and easier comparison, the terminal taxa are condensed into monophyletic major groups. Bootstrapindices (50% majority rule) are given. Models of sequence evolution were determined by means of Modeltest Version 3.06 ( Posada & Crandall, 1998).A.Distance tree based on the complete 18S rRNA gene. B.Distance tree based on the partial 28S rRNA gene. C.Maximum-likelihood tree based on thecomplete 18S rRNA gene.D. Maximum-likelihood tree based on the partial 28S rRNA gene.


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and Anaspidea, based on analyses of the 28S rRNA gene, whereasGymnosomata was not part of the Euthyneura but grouped withthe caenogastropods in the study ofThollesson (1999).

An important contribution to the solution of opisthobranchphylogeny was made by Grande et al. (2004)by means of theirmolecular study of the Euthyneura, based on eight mitochondrialgenes. They recovered the Sacoglossa as the most basal group anda well-supported clade consisting of Anaspidea, Cephalaspideaand Tylodinoidea. The Nudibranchia formed a paraphyletic

group, because the Pleurobranchoidea were located within them.Certainly the most striking affinity was the sister-grouprelationship between Acteonoidea and Nudibranchia plusPleurobranchoidea (discussed below).

The results of the present study, based on the 18S and 28SrRNA genes and reconstructed by means of maximum parsi-mony, maximum likelihood and distance methods, can besummarized as follows.

Opisthobranchia is mainly presented as a monophyletic groupif Pupa solidula and Hydatina physis are considered opistho-branchs. Opisthobranchia can be either para- or monophy-letic, because Pulmonata is arranged among Opisthobranchiain the maximum-likelihood and distance trees, or as a basalparaphyletic group in the maximum-parsimony analyses.

Monophyly of the major opisthobranch clades is supported inmost analyses, usually with high bootstrap values. However,Acochlidiacea forms a monophyletic group only in themaximum-parsimony (18S and combined tree) and distanceanalyses, and a high bootstrap value is found only in themaximum-parsimony tree of the combined dataset. Further-more Runcina adriaticais excluded from the Cephalaspidea s.s.in the maximum-likelihood and distance tree of the 18S rRNAgene. In addition to the topological support, the monophyly ofthe pleurobranchoidean taxa is corroborated by the corre-sponding insertion (structure E23_3) in region V4 of the 18SrRNA gene.

The arrangement of the major opisthobranch clades showsvariability caused by the lack of phylogenetic signal in bothalignments, with exceptions of the following three points.

The sister-group relationship of Nudibranchia and Pleuro-

branchoidea (Nudipleura) is confirmed in all analyses withhigh bootstrap values (96100%).

The monophyly of Cephalaspidea s.s. plus Anaspidea is alsoconfirmed (except in distance trees), although bootstrapsupport is lower than for Nudipleura (56 91%).

A sister-group relationship of Nudipleura and Acteonoidea(28/21 putative apomorphies detected by PHYSID) isshown in all trees (except the maximum-likelihood tree ofthe 18S rRNA gene). This is remarkable because theActeonoidea are usually considered the most basal opistho-branchs or are even excluded from this taxon (Mikkelsen1996, 2002), whereas the Nudipleura are usually regardedas highly derived (e.g. Wa geleet al., 2003). At first sight thisrelationship therefore seems to be due to a long-branchproblem, because Pupa, Hydatina and especially the Nudi-

pleura shows higher evolutionary rates than the other taxa.On the other hand, it is possible that there are real sistertaxa, because there are some similar morphological char-acters. Ghiselin, who united Acoela (Nudipleura andTylodinoidea) with Hydatinidae and Acteonidae on thebasis of their reproductive system, mentioned that Eliot(1910) notes a number of [] similarities which imply thatthere is an affinity between the Acoela and the Hydatinidaeand Acteonidae (Ghiselin, 1966: 371). In contrast to thephylogeny proposed by Ghiselin (1966), Nudipleura andActeonoidea represent a very derived clade in the maxi-mum-parsimony and distance trees of the present study. Inthe maximum-likelihood analyses on the other hand they

partly appear as basal taxa. Although this is the secondstudy to show this sister-group relationship, the introductionof a name for the clade Nudipleura Acteonoidea ispostponed until further support is available.

In the present study Tylodinoidea does not form a cladewith Nudipleura but shows different positions in thedifferent analyses due to the lack of phylogenetic infor-mation in both genes. A relationship with Cephalaspideaand Anaspidea, as suggested by Grande et al. (2003), is

shown in the maximum-parsimony trees of the18S rRNA(Fig. 1), the combined dataset (Fig. 3) and the maximum-likelihood tree of the 18S rRNA gene (Fig. 5C).

The position of Sacoglossa is also variable. In thephylogenetic reconstructions of the 18S rRNA gene andthe combined datasets, this taxon shows an affinity withNudipleura and Acteonoidea, but in the trees based on the28S rRNA gene no such relationship can be recognized. Abasal position, as lin Grande et al. (2004), is not supportedat all.

Acochlidiacea is not recognized as a monophyletic group inseveral analyses, because Hedylopsis spiculifera, a member ofthe family Hedylopsidae, does not group with the threemembers of the Microhedylidae (Unela, Pontohedyle, Para-hedyle). Except for the 28S maximum-likelihood tree,

Acochlidiacea shows a more or less basal position, mostlyin close proximity to the Pulmonata. The amount of phylogenetic information in the 18S and

28S rRNA gene sequences at the family and genus levels israther different concerning the several opisthobranchsubgroups. In all analyses the phylogeny of Nudibranchiais in accordance with Wa gele & Willan (2000). Theposition of the pleurobranchoidean genera, on the otherhand, is extremely variable, dependent on which gene andmethod is used. The arrangement of Cephalaspidea s.s. iscomparatively consistent. The Aglajidae (Chelidonura, Odon-toglaja, Navanax, Philinopsis) is recovered as a monophyleticgroup in each analysis, and the Haminoeidae (Phaner-ophthalmus, Atys, Haminoea) in each except in the 18Sdistance tree. Runcina, if included, occupies the most basal

position within the Cephalaspidea s.s. and Retusa representsthe sister-group of the Haminoeidae except in the 18Sdistance tree where it groups within the Haminoeidae. Thepositions of Philinoglossa, Sagaminopteron and Bulla arevariable. The basal position of Runcina adriatica is compa-tible with morphological observations, because Runcinapossesses four gizzard plates, whereas all other membersof the Cephalaspidea s.s. have three (Mikkelsen, 1996),implying that four plates represent the plesiomorphiccharacter state. Concerning Sacoglossa, Limapontiidae(Limapontia, Calliopaea) is recovered in each tree and aclose affinity between Thuridilla, Elysia, Placobranchus andBosellia (Placobranchoidea) is always shown except in the28S distance tree. The position of Cyerce is variable.Microhedylidae, containing Unela, Parahedyle and Pontohe-dyle, is always monophyletic.

Nucleotide composition bias and rate heterogeneity, found inboth genes, are estimated to have no affect on the accuracy of thereconstructed phylogenies. The detected differences in the AT-content reflect phylogenetic relationships already established onthe basis of morphological investigations, i.e. Cladobranchia,Anthobranchia and Pleurobranchoidea (Wa gele & Willan,2000). The relative-rate test supports the same groups bydifferent classes of substitution rates (Cladobranchia .Anthobranchia . Pleurobranchoidea . remaining taxa). Theonly case where higher substitution rates can potentially misleadphylogeny estimation is that of the Acteonoidea, because within


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the remaining taxa the Acteonoidea show the highest evol-utionary rates.

The suitability of 18S and partial 28S rRNA genes forinvestigating the phylogeny of the Opisthobranchia must beassessed critically. Although monophyly of the individualsubgroups is supported, the relationships of several majorclades remain uncertain. The analyses with PHYSID reveal arelatively low content of phylogenetic information in bothgenes. This is probably due to the many highly conserved

positions and the loss of phylogenetic signal in the variablepositions by multiple hits. The content of phylogeneticinformation is higher in the 18S than in the (investigatedpart) of the 28S rRNA gene, probably attributed to thegreater sequence length of the 18S rRNA gene.

ACKNOWLEDGEMENTS

This project was supported by several grants from the GermanScience Foundation to the authors: Wa 618/5 (HW), Wa618/7 (HW, VV, MS) and KL 1303/1 (AKK). We wish toexpress our sincere thanks to some colleagues who providedmaterial for this study: J. Brodie (Townsville), B. Bolland(Okinawa), J. Bohn (Munich), P. Duran (Santiago de Chile),A. Fahrner (Munich), G. Haszprunar (Munich), N. Michiels(Mu nster), M. Raupach (Bochum) and G. Steiner (Vienna).Thanks also to two unknown reviewers and M. Thollesson forvaluable criticism.

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