Molecular Phylogenetics and Evolution 55 (2010) 996–1007

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Molecular Phylogenetics and Evolution

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Out of Antarctica? – New insights into the phylogeny and biogeographyof the Pleurobranchomorpha (Mollusca, Gastropoda)

Katrin Göbbeler *, Annette Klussmann-KolbInstitute for Ecology, Evolution and Diversity, Biosciences, Goethe-University, Frankfurt am Main, Germany

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 September 2009Revised 18 November 2009Accepted 30 November 2009Available online 6 December 2009

Keywords:Ancestral-area analysesHistorical biogeographyMolecular clockOpisthobranchiaPhylogenyPhylogeographyPleurobranchomorpha

1055-7903/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ympev.2009.11.027

* Corresponding author. Address: Institute for EcolGoethe-University, Siesmayerstrasse 70, 60054 Frank+49 69 79824820.

E-mail addresses: goebbeler@bio.uni-frankfurt.dekolb@bio.uni-frankfurt.de (A. Klussmann-Kolb).

The aim of the current study was to gain new insights into the phylogeny, biogeography and evolution ofthe opisthobranch clade Pleurobranchomorpha. We focused on testing the hypothesis of an Antarctic ori-gin of this clade.

The combination of four gene markers (18S rDNA, 28S rDNA, 16S rDNA and CO1) was used to infer aphylogenetic hypothesis of the Pleurobranchomorpha employing Maximum likelihood and Bayesianinference methods. Four methodologically distinct approaches were applied to reconstruct the historicalbiogeography and dating of the tree was performed via relaxed molecular clock analysis.

Phylogenetic analyses supported the monophyly of the Pleurobranchomorpha and their sister grouprelationship to the Nudibranchia. Monophyly of the main subgroups Pleurobranchaeinae and Pleurobran-chinae could not be revealed. Reconstruction of the ancestral area of the Pleurobranchomorpha yieldeddifferent possibilities in the diverse analyses. However, the Pleurobranchinae most probably derived froman Antarctic origin. Estimation of divergence times revealed a long credible interval for the Pleurobranch-omorpha, whereas the Pleurobranchinae diverged in Early Oligocene and underwent rapid radiation dur-ing Oligocene and Early Miocene.

Divergence of the Pleurobranchinae into the Antarctic Tomthompsonia and the remaining species inEarly Oligocene coincides with two major geological events; namely the onset of glaciation in Antarcticaand the opening of the Drake Passage with following formation of the Antarctic Circumpolar Current(ACC). These sudden and dramatic changes in climate probably led to subsequent migration of the lastcommon ancestor of the remaining Pleurobranchinae into warmer regions, while the ACC may haveaccounted for larval dispersal to the Eastern Atlantic.

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1. Introduction

The Pleurobranchomorpha Schmekel (1985) or side-gilled seaslugs represent a clade of worldwide distributed marine gastro-pods. The species display a quite homogenous morphology anddefinitions of Pleurobranchomorpha include several diagnostic fea-tures like: internal, rectangular shell; presence of pedal gland;median buccal gland; internal, tubular vas deferens and protusiblepenis (Willan, 1987).

Traditionally, the Pleurobranchomorpha were considered to bethe sister group of the Umbraculida together forming the cladeNotaspidea (Burn, 1962; Willan, 1987; Tsubokawa and Miyazaki,1993). This assumption was challenged by Schmekel (1985), who

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ogy, Evolution and Diversity,furt am Main, Germany. Fax:

(K. Göbbeler), klussmann-

revealed the possible apomorphies to be plesiomorphic thus reject-ing the monophyly of this clade. Wägele and Willan (2000) recov-ered a sister group relationship of Pleurobranchomorpha andNudibranchia in a morphology-based cladistic analysis proposingthe new clade Nudipleura (apomorphies: possession of a bloodgland; androdiaulic reproductive system; absence (through loss)of osphradium). This sister group relationship was also revealedin further morphological (Wägele and Klussmann-Kolb, 2005;Martynov and Schrödl, 2008) as well as molecular analyses (Wol-lscheid-Lengeling et al., 2001; Vonnemann et al., 2005; Grandeet al., 2004a,b; Klussmann-Kolb et al., 2008).

The phylogenetic interrelationships of the pleurobranchomor-phan subclades are less well investigated. Hitherto only one phe-netic study (Willan, 1987), a study applying two-dimensionalelectrophoresis as a systematic method (Tsubokawa and Miyazaki,1993) and two poorly-resolved morphology-based cladistic analy-ses (Cervera et al., 2000; Martynov and Schrödl, 2008) are avail-able. Molecular analyses including more than a fewpleurobranchomorphan species are lacking until now. Followingthe latest classification of Bouchet and Rocroi (2005) the subclade

K. Göbbeler, A. Klussmann-Kolb / Molecular Phylogenetics and Evolution 55 (2010) 996–1007 997

Pleurobranchomorpha consists of a single superfamily (Pleurobr-anchoidea) with a single family: the Pleurobranchidae. This familyis divided into two subfamilies, the Pleurobranchaeinae compris-ing three valid genera: Pleurobranchaea with several described spe-cies distributed all over the world; as well as Euselenops andPleurobranchella each consisting of only a single species. The pres-ent study comprises two species of Pleurobranchaea and Euselenopsluniceps, while the Indo-West Pacific deep-sea species Pleuro-branchella nicobarica was not available for analyses.

The second subfamily Pleurobranchinae is divided into threetribes: Pleurobranchini, Berthellini and Bathyberthellini. The Pleu-robranchini consist of the genus Pleurobranchus representing themost species rich pleurobranchomorphan genus with four speciesincluded in the current study. The Berthellini comprise the generaBerthella, Berthellina, Pleurehdera and Tomthompsonia. Six species ofthe second largest pleurobranchomorphan genus Berthella are partof our study, as well as two species of Berthellina and the only spe-cies of Tomthompsonia. The tropical Indo-West Pacific species Pleu-rehdera haraldi was not available for this investigation. TheBathyberthellini proposed by Garcia et al. (1996) were supposedto comprise the genera Bathyberthella, Parabathyberthella andPolictenidia. However, in a recent investigation Martynov andSchrödl (2008) claimed that Parabathyberthella and Polictenidiashould be synonymized with Bathyberthella. Furthermore, they de-scribed the new monotypic genus Boreoberthella yielding a total oftwo valid genera represented by Bathyberthella antarctica in thepresent investigation due to unavailability of the newly describedarctic Boreoberthella.

The lack of a robust phylogeny of the Pleurobranchomorpha upto now hampered reconstruction of historical biogeography of thisclade. The current hypothesis on nudipleuran evolution along withrecent hypotheses on the origin of Antarctic marine benthic com-munities (Thatje et al., 2005) suggests a cold-water related origin,especially in the deep sea around the Antarctic shelf or shelteredshelf areas (Wägele et al., 2008). Thus, radiation of the major sub-groups Pleurobranchomorpha and Nudibranchia supposedly hap-pened around Antarctica about 40 Million years ago (Schrödl,2003; Martynov and Schrödl, 2008). Subsequently, radiation couldhave led to invasions into temperate and tropical seas as well asinto the deep sea along the northward flowing currents of the Ant-arctic bottom water (Wägele et al., 2008). The molecular phylog-eny of the Pleurobranchomorpha presented in the current studyoffers the opportunity to test this hypothesis on ancestral area dis-tribution of this clade.

In addition to reconstructing the historical biogeography, weconducted a molecular clock analysis in order to date the phyloge-netic tree of the Pleurobranchomorpha. This enables an assignmentof radiation events to certain geological time frames. This is espe-cially important for the Pleurobranchomorpha since the fossil re-cord of this clade is very poor. Members of the genus Berthellahave been dated back to the late Oligocene of France – about 24–26 Million years ago (Mya) (Valdés and Lozouet, 2000) which ledother authors to the conclusion that the Pleurobranchomorphaare probably about 30 My old (Wägele et al., 2008). However, theirfossil record is probably restricted due to their small and delicateshells, which might not be well preserved (Valdés, 2004). Addition-ally, a fossil record for the shell-less subfamily Pleurobranchaeinaeis completely missing.

The aim of the present study is to shed new light upon the evo-lution of a so far only morphologically examined clade of opistho-branch molluscs and investigate its origin. We present thecurrently most comprehensive molecular study of pleurobran-chomorphan phylogeny, which is used to assess their historicalbiogeography. In order to correlate biogeographical events to geo-logical incidents a relaxed molecular clock analysis was conductedto estimate node ages.

2. Materials and methods

2.1. Taxon sampling

17 species of Pleurobranchomorpha covering seven out of tendescribed genera were included in the current investigation. Thesingle species of the genera Pleurobranchella, Pleurehdera and therecently established Boreoberthella were not available for analyses.The taxon sampling was completed by representatives of all majoropisthobranch subgroups as well as two ‘‘lower Heterobranchia”and the Caenogastropod Littorina littorea as outgroup taxon yield-ing a total of 36 taxa.

Specimens were collected worldwide by hand, snorkeling, or scu-ba diving and preserved in 80–100% ethanol. Published sequencesfrom Genbank were utilized for some species. Origin of all taxa andaccession numbers of utilized sequences are summarized in Table 1.

2.2. DNA extraction, PCR and sequencing

Genomic DNA extraction from muscle tissue was performed byuse of the DNeasy Tissue Kit (Qiagen, Hilden, Germany) followingthe animal tissues/spin-column protocol.

Four gene fragments, including two nuclear (complete 18SrDNA and partial 28S rDNA) and two mitochondrial markers(partial 16S rDNA and CO1) were amplified. The primer sequenceswere obtained from former studies by Folmer et al., 1994; Simonet al., 1994; Wollscheid and Wägele, 1999; Dayrat et al., 2001and Vonnemann et al., 2005. All fragments were sequenced in bothdirections. See Appendix A1 in Supplementary data for details ofprimers and PCR-protocols.

PCR-products were purified from an agarose gel using the QIA-quick Gel Extraction Kit from Qiagen (Hilden, Germany). Subse-quent cycle sequencing was conducted with the CEQ DTCS QuickStart Kit (Krefeld, Germany). The final sequences were obtainedusing a CEQ 2000 Beckmann Coulter capillary sequencer at the sci-entific research lab in Frankfurt/Main or through SRD Scientific Re-search and Development (Bad Homburg, Germany).

2.3. Sequence alignment

Sequence alignments were performed using MUSCLE 3.6 (Ed-gar, 2004) under the default parameters. The results were cor-rected manually in BioEdit Version 7.0.9 (Hall, 1999). Severalinserts and hypervariable base positions were excluded fromthe alignments of 18S rDNA, 28S rDNA and 16S rDNA prior tophylogenetic reconstruction. Additionally, third codon positionsof the CO1-sequences were removed from the alignment dueto substantial substitution saturation. Details about alignmentlength and excluded positions are listed in Appendix A2 in Sup-plementary data.

2.4. Statistical tests

We performed several statistical tests on our data in order toestimate data quality and survey the results.

The incongruence length difference test (ILD) as described byFarris et al. (1995) evaluates the significance of incongruence ina combined dataset. This test is implemented in PAUP 4.0b10(Swofford, 2002) as Partition Homogeneity test and was usedto investigate if the datasets of the single gene markers providea congruent phylogenetic signal and therefore can be concate-nated and analysed as a single dataset. A heuristic search underthe Maximum parsimony criterion was conducted for 100 replicates.

The degree of substitution saturation was evaluated using thetest by Xia et al. (2003) as implemented in the software packageDAMBE (Xia and Xie, 2001).

Table 1Taxon sampling information. Taxon names and taxonomic classification according to Bouchet and Rocroi (2005), localities and Genbank accession numbers provided.

Taxon Family/Subfamily Locality Genbank accession numbers

18S 28S 16S CO1

CAENOGASTROPODALittorina littorea Littorinidae Genbank X91970 AJ488672 DQ093481 DQ093525

LOWER HETEROBRANCHIAOrbitestella sp. Orbitestellidae Genbank EF489352 EF489377 EF489333 EF489397Pupa solidula Acteonidae Genbank AY427516 AY427481 EF489319 DQ238006

OPISTHOBRANCHIACephalaspideaHaminoea hydatis Haminoeidae Genbank AY427504 AY427468 EF489323 DQ238004AplysiomorphaAkera bullata Akeridae Genbank AY427502 AY427466 AF156127 AF156143UmbraculidaUmbraculum umbraculum Umbraculidae Genbank AY165753 AY427457 EF489322 DQ256200Tylodina perversa Tylodinidae France, Mediterranean Sea AY427496 AY427458 FJ917424* AF249809ThecosomataHyalocylis striata Cavoliniidae Genbank DQ237966 DQ237985 – DQ237999SacoglossaOxynoe antillarum Oxynoidae Venezuela, Caribbean Sea FJ917441* FJ917466* FJ917425* FJ917483*

NudipleuraNudibranchia

Bathydoris clavigera Bathydorididae Genbank AY165754 AY427444 AF249222 AF249808Chromodoris krohni Chromodorididae Genbank AJ224774 AY427445 AF249239 AF249805Hypselodoris infucata Chromodorididae Australia, NSW FJ917442* FJ917467* FJ917426* FJ917484*

Rostanga calumus Discodorididae Australia, NSW FJ917451*

FJ917452*

FJ917453*

FJ917468* FJ917427* FJ917485*

Hoplodoris nodulosa Discodorididae Australia, NSW FJ917443* FJ917469* FJ917428* FJ917486*

Dendrodoris fumata Dendrodorididae Australia, NSW FJ917444* FJ917470* – AF249799Goniodoris nodosa Goniodorididae Genbank AJ224783 AY014157 AF249226 AJ223264Armina lovenii Arminidae Genbank AF249196 – AF249243 AF249781Flabellina babai Flabellinidae Genbank AY165768 AY427449 – –Cuthona nana Tergipedidae Genbank AY165760 AY427448 – –

PleurobranchomorphaBathyberthella antarctica Pleurobranchinae Antarctica AF249219 AY427453 FJ917429* FJ917487*

Tomthompsonia antarctica Pleurobranchinae Genbank AY427492 AY427452 EF489330 DQ237992Berthella stellata Pleurobranchinae Italy AY427495 AY427456 FJ917430* FJ917488*

Berthella martensi Pleurobranchinae Australia, WA FJ917456*

FJ917457*FJ917471* FJ917431* FJ917489*

Berthella californica Pleurobranchinae Canada, Pacific FJ917445* FJ917472* FJ917432* FJ917490*

Berthella medietas Pleurobranchinae Australia, VIC FJ917454*

FJ917455*FJ917473* FJ917433* FJ917491*

Berthella platei Pleurobranchinae Chile FJ917446* FJ917474* FJ917434* FJ917492*

Berthella plumula Pleurobranchinae France, Atlantic FJ917447* FJ917475* FJ917435* FJ917493*

Berthellina citrina Pleurobranchinae Australia, NSW FJ917448* FJ917476* FJ917436* FJ917494*

Berthellina edwardsii Pleurobranchinae Ghana FJ917458*

FJ917459*FJ917477* – FJ917495*

Pleurobranchus peroni Pleurobranchinae Genbank AY427494 AY427455 EF489331 DQ237993Pleurobranchus membranaceus Pleurobranchinae France, Mediterranean Sea FJ917460*

FJ917461FJ917478* FJ917437* FJ917496*

Pleurobranchus hilli Pleurobranchinae Australia, WA FJ917462*

FJ917463*FJ917479* FJ917438* FJ917497*

Pleurobranchus reticulatus Pleurobranchinae Guinea FJ917464*

FJ917465*FJ917480* – FJ917498*

Euselenops luniceps Pleurobranchaeinae Genbank AF249218 AY427451 – –Pleurobranchaea meckeli Pleurobranchaeinae Spain, Mediterranean Sea FJ917449* FJ917481* FJ917439* FJ917499*

Pleurobranchaea californica Pleurobranchaeinae USA, California FJ917450* FJ917482* FJ917440* –

�, sequences generated in current study; –, missing sequences.

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The Approximately Unbiased (AU) test (Shimodaira, 2002) wasused to evaluate alternative tree topologies enforcing monophylyof the Pleurobranchaeinae or Berthellini, respectively. The likeli-hood at each nucleotide position was calculated for both alterna-tive topologies as well as for the unconstrained topology usingPAUP 4.0b10 (Swofford, 2002). The obtained likelihoods were usedto compute p-values in CONSEL version 0.1 (Shimodaira and Hase-gawa, 2001).

A relative rate test was conducted on the single gene markers aswell as on the concatenated alignment using the software k2WuLi(Wu and Li, 1985) to investigate rate heterogeneity in thesequences.

2.5. Phylogenetic analyses

2.5.1. Bayesian InferenceMrModeltest 2.2 (Nylander, 2004) based on the Akaike informa-

tion criterion (AIC) was applied for determination of the best fittingmodel of sequence evolution for all five gene partitions (single co-don positions of CO1 separately) prior to phylogenetic analyses. Fordetails about the models determined see Appendix A2 in Supple-mentary data.

Bayesian inference was conducted with MrBayes 3.1.2 (Huel-senbeck and Ronquist, 2001) using separate models of evolutionfor each of the five gene partitions. Two separate runs of four

K. Göbbeler, A. Klussmann-Kolb / Molecular Phylogenetics and Evolution 55 (2010) 996–1007 999

chains (one cold, three heated each) of a Metropolis coupled Mar-kov chain Monte Carlo algorithm operated for 1,000,000 genera-tions. Likelihoods converged slowly, thus the first 4000 sampledtrees were ignored as burn-in for construction of the 50% majorityrule consensus tree. Posterior probabilities were calculated foreach node in the consensus tree, a value of 0.95 and higher beingconsidered as good statistical support.

2.5.2. Maximum likelihoodMaximum likelihood analyses were performed using RAxML

7.0.3 (Stamatakis, 2006). This program uses a GTR model based ap-proach under the gamma model of rate heterogeneity. Modelparameters are estimated by RAxML for all data partitions. Preli-minary analyses to estimate the best settings for the final analysesyielded the best likelihood values for the default setting. This wasused to compute 200 best-known likelihood trees. Bootstrappingwith 1000 replicates was performed and the results were plottedonto the best-known likelihood tree. Bootstrap values above 75were considered as good statistical support.

2.6. Historical biogeography reconstructions

We used four different approaches to estimate the ancestralarea distribution of the Pleurobranchomorpha. Among these weretwo Maximum parsimony-based methods (WAAA and DIVA), aMaximum likelihood approach (lagrange) and a Bayesian analysis(BayesTraits). All reconstructions of historical biogeography wereperformed using the phylogenetic tree obtained in the Bayesiananalysis.

2.6.1. AreasRecent geographical distributions were used to assess the bio-

geographical history of the Pleurobranchomorpha. The biogeo-graphical regions were coded according to Frey and Vermeij(2008) as follows: Indo-West Pacific (IWP), temperate South-WestPacific (SP), Eastern Pacific (EP), Western Atlantic (WA) and EasternAtlantic (EA). Additionally, we coded the Antarctic region (A). Eachspecies was assigned to one or more areas based on distributioninformation. Detailed information about the distribution codingis available in Appendix A3 in Supplementary data.

2.6.2. Weighted ancestral-area analysisWeighted ancestral-area analysis (WAAA; Hausdorf, 1998) is a

cladistic method for estimating ancestral areas using reversibleparsimony in combination with a weighting scheme whichweights steps in positionally plesiomorphic branches higher thansteps in apomorphic branches. WAAA yields probability indices(PIs), indicating the relative probability that a certain area was partof the ancestral area for a particular node of the tree. Area optimi-zations were performed following Drovetski (2003), calculating theweighted gain steps (GSW) with the accelerated transformationoptimization and the weighted loss steps (LSW) with the delayedtransformation optimization. This procedure ensures the earliestpossible gain and latest possible loss for each area, which mini-mizes PIs for areas with rich species diversity (Drovetski, 2003).

2.6.3. Dispersal–vicariance analysisMaximum parsimony reconstruction of ancestral distributions

was performed using dispersal–vicariance analysis as imple-mented in DIVA 1.1 (Ronquist, 1996). This event-based approachreconstructs ancestral distributions without any prior assumptionsabout the area relationships (Ronquist, 1996). Optimal ancestraldistributions are inferred by minimizing dispersal and extinctionevents, vicariance being the default mode of speciation (Ronquist,1997).

The default settings were used for DIVA, since they proved to besuitable for our data in initial trials. The input file was generatedusing MacClade 4.0 (Maddison and Maddison, 2000) by importinga binary character matrix and the phylogenetic tree.

2.6.4. Bayesian estimation of ancestral character statesA Bayesian approach as implemented in the BayesTraits (PC ver-

sion 1.0) software package (Pagel et al., 2004) was used to recon-struct ancestral character states regarding area distribution forselected nodes in the phylogeny. BayesTraits uses reversible-jumpMarkov chain Monte Carlo (MCMC) methods to derive posteriorprobabilities and the values of traits at ancestral nodes of phylog-enies. MultiState was selected as model of evolution and MCMC asmethod of analysis. Rate deviation was set to 35. A hyperprior ap-proach was employed with an exponential prior seeded from a uni-form on the interval 0–10. Thus, acceptance rates in the preferredrange of 20–40% were achieved. A total of 1,010,000 iterationswere run for each analysis with the first 10,000 samples discardedas burn-in. Since posterior probabilities for ancestral areas of thesingle runs partly varied, we calculated the arithmetic mean ofall samples for reconstruction of the ancestral distribution.

2.6.5. Likelihood-based analysis of geographic range evolutionThe software Lagrange was used for a likelihood-based ap-

proach of historical biogeography. A beta-version of the software,along with a configurator for compiling an input file for the analy-sis, is available from http://www.reelab.net/lagrange/configurator/index.

This method is based on a stochastic, continuous-time modelfor geographic range evolution by dispersal, extinction and clado-genesis (DEC-model: Ree et al., 2005; Ree and Smith, 2008). Thismodel specifies current transition rates between ranges along phy-logenetic branches and uses these to infer likelihoods of ancestralrange inheritance scenarios at branching events (Ree and Smith,2008).

Ranges were not constrained in the adjacency matrix since dis-persal is principally possible between all areas. The maximumrange size was restricted to three, which displays the maximumrange size of recent species. No combination of ranges was ex-cluded from the analyses. Dispersal constraints were defined in asingle matrix for a time period up to 40 Mya. Parameters wereset to 100 for directly adjacent areas, areas with moderate openwater bodies between them were set to 50, 25 was chosen for areaswith large open water bodies in between and areas not adjacent toeach other were set to a value of 5.

2.7. Molecular dating

The procedure of molecular clock dating is disputed (Graur andMartin, 2004; Heads, 2005; Pulquerio and Nichols, 2007). However,this approach may provide insights into the origins and diversifica-tion of organisms with poor or lacking fossil record (Welch andBromham, 2005), as is the case for the Pleurobranchomorpha.

BEAST v1.4.8 (Drummond and Rambaut, 2007) was used to esti-mate divergence times using the relaxed phylogenetics method ofDrummond et al. (2006). BEAST uses a Bayesian relaxed molecularclock while incorporating tree uncertainty in the MCMC process toinfer divergence times. A Yule process-speciation prior and anuncorrected log-normal model of rate variation were implementedin each analysis (Drummond et al., 2006).

Taxon sets were defined on the caenogastropod outgroup Litto-rina littorea, the Opisthobranchia including Pupa solidula and thePleurobranchinae including Euselenops luniceps, which had beenplaced there in former phylogenetic analyses. The latter two taxonsets were chosen, because of the availability of relatively certainfossil data for these groups (Tracey et al., 1993; Valdés and Lozouet,

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2000). Therefore, node constraints were assigned to these twotaxon sets with a normal prior distribution. Normal mean andstandard deviation were set to 210 and 6 for the Opisthobranchia +Pupa solidula as well as 31.5 and 0.9 for the Pleurobranchinae +Euselenops luniceps, respectively.

After an initial period of fine-tuning the operators, ten separateMCMC analyses were run for 10,000,000 generations (burn-in 10%)with parameters sampled every 1000 steps to ensure effectivesample size (ESS) values above 200 for most parameters. Indepen-dent runs were combined in LogCombiner v1.4.8 implemented inthe software package BEAST v1.4.8. ESS values of each parameterwere measured with Tracer v1.4.1 (Rambaut and Drummond,2008).

Tree topologies were assessed using TreeAnnotator v1.4.8implemented in the software package BEAST v1.4.8 to generate aconsensus tree out of all sampled trees and FigTree v1.2.2 (Ram-baut, 2009) was used to show the consensus tree along with nodeages and node bars.

3. Results

3.1. Statistical tests

The incongruence length difference (ILD) test revealed that thecombination of the four gene partitions improves the phylogeneticsignal with a p-value of 0.01. Thus concatenation of the singlegenes is reasonable.

Evaluation of substitution saturation showed little saturation inthe 16S-alignment and substantial saturation in the 3rd codon po-sition of CO1. The latter was therefore removed from further anal-yses. 16S-sequences were retained in the analyses to avoid the lossof phylogenetic signal at lower taxonomic levels.

The relative rate test revealed that evolutionary rates differamong the investigated taxa and genetic markers. The highest z-scores indicating the major differences were found in the 18S rDNAand 28S rDNA sequences of the dexiarchian taxa Armina lovenii,Flabellina babai and Cuthona nana. 16S rDNA and CO1-sequencesgenerally yielded much lower z-scores. Regarding the Pleuro-branchomorpha the highest z-scores were found in the 18S rDNAsequences of Berthella stellata, Berthella martensi and Berthella plu-mula and the 28S rDNA sequences of Berthella stellata, Berthella plu-mula and Pleurobranchaea meckeli. In the concatenated alignmentz-scores were maximal for the dexiarchian taxa and Berthellastellata.

3.2. Phylogenetic analyses

The different phylogenetic approaches yielded mostly congru-ent results concerning phylogenetic relationships and the treetopology of the Pleurobranchomorpha was identical in both analy-ses. Good posterior probabilities were achieved for most nodes.The resulting phylogram of the Bayesian analysis is shown inFig. 1. Posterior probabilities and Maximum likelihood bootstrapsupport values are indicated at the nodes.

Our results reject the existence of the Notaspidea, a clade com-prising Umbraculida and Pleurobranchomorpha as sister groups.Instead our data confirm a sister group relationship of Nudibran-chia and Pleurobranchomorpha, forming the so called Nudipleura.Meanwhile the Umbraculida are detected monophyletic in theBayesian analysis as the sister group of a clade composed ofCephalaspidea, Aplysiomorpha and Pteropoda.

The Nudipleura and within the Nudibranchia and Pleurobranch-omorpha are recovered monophyletic with maximum statisticalsupport values. The Nudibranchia are divided into monophyletic

Anthobranchia and Dexiarchia, the latter exhibit extremely longbranches displaying their high substitution rates.

Based on morphological data the Pleurobranchomorpha and itssingle family Pleurobranchidae are traditionally subclassified intothe Pleurobranchaeinae and the Pleurobranchinae. We are not ableto recover this classification based on our molecular data due tothe position of Euselenops luniceps (lacking 16S rDNA and CO1-se-quences). This taxon is traditionally assigned to the Pleurobran-chaeinae, but clusters deep in the Pleurobranchinae in ouranalyses. Apart from the position of this one taxon a division ofthe Pleurobranchomorpha/Pleurobranchidae into two main sub-groups (the Pleurobranchaea spp. and the Pleurobranchinae withE. luniceps) is evident.

Within the more species rich clade Pleurobranchinae the Ant-arctic species Tomthompsonia antarctica is found as the most basaloffshoot, while the remaining taxa form two main clades. Oneclade consists of five Berthella species, the other comprises themonophyletic Pleurobranchini and a clade composed of Bathy-berthella antarctica, Euselenops luniceps, Berthella californica andthe Berthellina species as sister groups. Hence, Tomthompsonia doesnot belong to the Berthellini according to our results and the Ber-thellini as well as within the Berthella species are found to be para-phyletic. This is due to a sister group relationship of Berthellacalifornica to the two Berthellina species included in the analysesand no closer relationship to the other Berthella species forming amonophyletic group on their part. The monophyly of the Bathyber-thellini can not be assessed since only a single species is includedin our analyses. The exact position of Euselenops luniceps in theaforementioned clade remains unresolved due to low statisticalsupport values. It is worth mentioning that some species of Berth-ella (B. plumula, B. stellata and B. martensi) also reveal very longbranches due to high substitution rates.

3.3. Historical biogeography reconstructions

The results of the four different approaches to reconstruct thehistorical biogeography of the Pleurobranchomorpha are summa-rized in Table 2 and plotted on the phylogenetic tree in Fig. 2.

Weighted ancestral-area analysis favours three regions asancestral for the Pleurobranchomorpha: Eastern Pacific, EasternAtlantic and Antarctica. However, the Pleurobranchinae mostprobably originated from waters surrounding Antarctica.

Dispersal–vicariance analysis inferred the Eastern Atlantic andcombinations of Antarctica and Eastern Pacific as well as Antarc-tica, Eastern Pacific and Eastern Atlantic as ancestral areas for thePleurobranchomorpha. The origin of the Pleurobranchinae isreconstructed to be in Antarctica and the Eastern Atlantic.

The Bayesian estimation of ancestral area reconstruction showsambiguous results regarding the origin of Pleurobranchomorpha.Antarctica and Eastern Pacific are slightly favoured as ancestralareas, although an origin in Indo-West Pacific, Southern Pacificand Western Atlantic can not be ruled out. The Pleurobranchinaemost probably originated in Antarctica.

The Maximum likelihood approach implemented in lagrange fa-vours a pleurobranchomorphan origin in the Eastern Atlantic,while the ancestral area of the Pleurobranchinae most probablyis Antarctica.

3.4. Molecular dating

Apart from the questionable position of a single taxon (Goniod-oris nodosa) the ‘‘relaxed” molecular clock analyses via Beast v1.4.8(Fig. 3) yielded the same tree topology as the Bayesian analysis viaMrBayes 3.2.1 (Fig. 1). Posterior probability support values are max-imal for the main groupings. Regarding the Pleurobranchomorpha,

Fig. 1. Bayesian inference phylogram of the phylogenetic analyses, 50% majority rule consensus tree. Posterior probabilities of the Bayesian analysis and Bootstrap supportvalues of the Maximum likelihood analysis are provided at the nodes. Only support values above 0.5 and 50, respectively, are indicated. Pleurobranchomorphan taxa aremarked by color frames.

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only the position of Euselenops luniceps remains unsupported andthus unresolved.

Beast 95% HPD (highest posterior density) credible intervals arerelatively long for most nodes besides the Pleurobranchinae, wherethey are noticeably shorter. Due to these long credible intervalsestimation of divergence times for the Nudipleura and Nudibran-chia is limited. According to our analyses the split of Nudibranchiaand Pleurobranchomorpha most probably occurred in the Creta-ceous but can not be assigned to a more definite point in time.The Pleurobranchomorpha diverged into the Pleurobranchaea spp.and Pleurobranchinae (including Euselenops luniceps) about37 Mya with a long 95% HPD from 32 to 89 Mya.

Dating back the split-off of Pleurobranchaea spp. is also ham-pered by long credible intervals ranging from 3.6 to 53.9 Mya witha value of 4.7 Mya in the maximum clade credibility tree.

Estimated divergence times for the Pleurobranchinae indicate arapid radiation during the Oligocene and early Miocene. All mainlineages evolved in a period of about 10 to 15 Million years. After

the split-off of Tomthompsonia antarctica about 31 Mya (95%HPD: 30.2–33.7 Mya) the remaining Pleurobranchinae split intotwo main clades some 28.5 Mya (95% HPD: 25.9–32.2 Mya). TheBerthella clade rapidly split of into lineages leading to the differentspecies (approximately 26.1; 24.7 and 20.3 Mya). Radiation of thesecond clade took place nearly at the same time (21 and19.7 Mya), while the Pleurobranchini diverged into single speciesa little later (about 11.5, 9.9 and 3.7 Mya).

4. Discussion

4.1. Phylogeny of the Pleurobranchomorpha

The results of the present study confirm the existence andmonophyly of the Nudipleura Wägele and Willan, 2000 com-posed of Pleurobranchomorpha and Nudibranchia. Thus, a sistergroup relationship of Pleurobranchomorpha and Umbraculida as

Table 2Historical biogeography reconstruction of the Pleurobranchomorpha and the Pleurobranchinae – summary of the results of the different analytical approaches.

Method Pleurobranchomorpha Pleurobranchinae

WAAA (probability indices) Eastern Pacific 0.51 Antarctica 1.2Eastern Atlantic 0.48 Eastern Atlantic 0.43Antarctica 0.45 Eastern Pacific 0.31Indo-West Pacific 0.30 Indo-West Pacific 0.30Southern Pacific 0.17 Western Atlantic 0.22Western Atlantic 0.14 Southern Pacific 0.20

DIVA Antarctica/Eastern PacificEastern AtlanticAntarctica/Eastern Pacific/Eastern Atlantic

Antarctica/Eastern Atlantic

BayesTraits Antarctica 0.23 Antarctica 0.30Eastern Pacific 0.22 Indo-West Pacific 0.19Indo-West Pacific 0.17 Eastern Pacific 0.17Southern Pacific 0.14 Southern Pacific 0.14Western Atlantic 0.14 Western Atlantic 0.13Eastern Atlantic 0.09 Eastern Atlantic 0.07

Lagrange (relative probability) Eastern Atlantic 0.48 Antarctica 0.21Antarctica/Eastern Atlantic/Indo-West Pacific

0.09

Indo-West Pacific 0.08

Fig. 2. Results of the four methodological approaches to reconstruct historical biogeography of the Pleurobranchomorpha plotted on a cladogram of the phylogeny of thePleurobranchomorpha resulting from Bayesian and Maximum likelihood analyses. Distribution coding of extant species indicated behind species names. Results of weightedancestral-area analyses are indicated above, DIVA results below branches. Output of the Bayesian approach is displayed as pie chart, lagrange findings as colored squares.

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‘‘Notaspidea” (Burn, 1962; Willan, 1987) is rejected rendering thelatter polyphyletic. This finding is in accordance with morphologi-cal (Wägele and Klussmann-Kolb, 2005; Martynov and Schrödl,2008) as well as molecular (Wollscheid-Lengeling et al., 2001;Grande et al., 2004a,b; Vonnemann et al., 2005; Klussmann-Kolbet al., 2008) systematic studies.

Regarding the phylogeny of the Pleurobranchomorpha the re-sults of current study are partly incongruent with the traditionalmorphology-based classification (Willan, 1987; Bouchet and Roc-roi, 2005); however, these results are preliminary since not all va-lid genera could be included in the analyses. The monophyly of thetwo main subgroups Pleurobranchinae and Pleurobranchaeinae

could not be supported due to the position of a single taxon. Thistaxon, Euselenops luniceps is assigned to the Pleurobranchaeinaeand clusters within this group in morphology-based analyses (Cer-vera et al., 2000; Martynov and Schrödl, 2008). However, our re-sults retrieve E. luniceps forming a clade with Bathyberthella,Berthellina and Berthella species in a rather derived position withinthe Pleurobranchinae. Although the exact position or the sistergroup of E. luniceps could not be unambiguously detected due tolow statistical support values the placement in the aforementionedclade was found well supported in the Bayesian analysis. Since 16SrDNA and CO1-sequences are missing for E. luniceps this resultmight be regarded as erroneous. However, the Pleurobranchaeinae

Fig. 3. Maximum clade credibility tree displaying results of the relaxed molecular clock analyses. Posterior probabilities and age estimations (in Million years) are provided atthe branches. Grey node bars indicate 95% HPD (highest posterior density) credible intervals. Geological time scale is based on the International Stratigraphic chart by theInternational Commission on Stratigraphy (2008). (L, Lower; P, Pliocene).

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have not been recovered monophyletic in single analyses of the18S rDNA and 28S rDNA datasets (data not shown). Furthermore,the monophyly of the Pleurobranchaeinae was also doubted forthis dataset in the Approximately Unbiased (AU) test. The AU testyielded p-values of 0.916 for the topology without constraints and0.131 for constrained monophyly of Pleurobranchaeinae. The latterp-value is much lower than the p-value of the non-constrainttopology, but the monophyly of Pleurobranchaeinae cannot be def-initely disclaimed as the value is not below the significance level of0.050. However, the only other molecular study comprising E. luni-ceps and five other Pleurobranchinae recovered E. luniceps in a de-rived position within the Pleurobranchinae rendering the latterparaphyletic (Vonnemann et al., 2005). The morphology-basedmonophyly of the Pleurobranchaeinae is drawn on different char-acters, including the absence of a shell, a papillate oral veil, sepa-rated rhinophores, a highly ramified medial buccal gland shape,polygonal jaw elements, and a serial receptaculum seminis (Mar-cus and Gosliner, 1984; Cervera et al., 2000; Martynov and Schrödl,2008). Although these features involve diverse organ systems, ourresults indicate that they might have evolved independently. How-ever, this explanation is not parsimonious. If we assume mono-phyly of the Pleurobranchaeinae it might as well be possible thatthe position of the Pleurobranchaea spp. basal to the other pleuro-branchomorphan taxa is erroneous. This placement might perhapsresult from long branch attraction to other basal taxa due to sharedrandom similarity. Thus, the position of Euselenops luniceps might

represent the placement of the whole group implying that Pleuro-branchaeinae arose from Pleurobranchinae rendering the latterparaphyletic.

To sum up, there is an obvious incongruence between molecu-lar- and morphology-based results. Continuative comprehensiveanalyses incorporating diverse genetic markers or ESTs and addi-tional taxa especially of the Pleurobranchaeinae as well as acces-sory morphological characters are needed to finally clarify thedubious phylogenetic position of E. luniceps.

According to our investigation the Antarctic species Tomthom-psonia antarctica appears as the most basal offshoot of the Pleuro-branchinae which is congruent with the results of Martynov andSchrödl (2008). This is especially important because of its impacton subsequent historical biogeography reconstructions.

The Berthellini and even the genus Berthella are rendered para-phyletic by our analyses. The AU test yielded p-values of 0.916 forthe topology without constraints and 0.030 for the topology withenforced monophyly for Berthellini, which is below the signifi-cance level of 0.050. Former morphology-based analyses werenot able to resolve relationships among Berthella species probablydue to the fact that they show a mixture of plesiomorphic and apo-morphic features (Cervera et al., 2000; Martynov and Schrödl,2008). The latter authors regard the genus Berthella as possiblynon-monophyletic representing a thus far unresolved evolutionarygrade rather than a clade (Martynov and Schrödl, 2008). Thisassumption is supported by the results of the present study.

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In contrast to this, the Pleurobranchini with its single genusPleurobranchus seem to represent a valid clade, since they arerecovered monophyletic in the present molecular systematic anal-yses as well as in former morphology-based analyses (Cerveraet al., 2000; Martynov and Schrödl, 2008). However, the positionof the Pleurobranchini within the Pleurobranchinae varies. Cerveraet al. (2000) found them in a basal position as the sister group to allremaining Pleurobranchinae whereas Martynov and Schrödl(2008) could not regain this basal position. The latter is consistentwith the results of the current study where the Pleurobranchini arefound in a rather derived position.

4.2. Out of Antarctica?

Extant Pleurobranchomorpha are distributed worldwide in allmarine habitats from the shallow intertidal zone to epipelagial re-gions and from tropical seas to the cold-waters surrounding Ant-arctica. The most species rich regions today are the Indo-WestPacific and the Eastern Atlantic including the Mediterranean.There, Pleurobranchomorpha occur mainly in shallow temperateand tropical waters.

Only very few pleurobranchomorphan species live around Ant-arctica today. Four different genera, most of them monotypic, aredistinguished: Tomthompsonia Wägele and Hain, 1991, Bathyberth-ella Willan, 1983, Parabathyberthella Garcia et al., 1994 and Polict-enidia Garcia et al., 1996. However, Martynov and Schrödl (2008)synonymized the monotypic genera Parabathyberthella and Policte-nidia with the senior Bathyberthella. Thus, only two valid Antarcticgenera (the monotypic Tomthompsonia as well as Bathyberthellacomprising four species) remain which are both included in thepresent study. All of these genera belong to the Pleurobranchinae.Thus, no extant member of the Pleurobranchaeinae occurs aroundAntarctica. Schrödl (2003) and Wägele et al. (2008) proposed a sce-nario in which the early nudipleuran evolution took place aroundthe cooling Antarctica about 40 or 30 Mya, respectively. Thus, radi-ation of major subgroups like the Pleurobranchomorpha was re-lated to cold-waters, followed by invasions to temperate andtropical seas along the northward flowing currents of the Antarcticbottom water (Martynov and Schrödl, 2008; Wägele et al., 2008).

Unfortunately, the possible origin of the Nudipleura could notbe assessed by the present analytical approaches since the taxonsampling of the Nudibranchia with more than 3000 extant speciesis not sufficient for this purpose. However, the Antarctic speciesBathydoris clavigera was found as the most basal offshoot of thesubclade Anthobranchia in the present study. This finding has al-ready been discovered in former studies (Wägele and Willan,2000; Vonnemann et al., 2005; Wägele and Klussmann-Kolb,2005). In addition, the deep and polar water genus Doridoxa is sup-posed to be the first offshoot and thus most basal group of the sec-ond nudibranch subclade Dexiarchia (Schrödl et al., 2001). Since noDoriodoxa species was included in the present analyses we couldnot reassess this result. However, according to current research(Wägele and Willan, 2000; Schrödl et al., 2001; Vonnemannet al., 2005; Wägele and Klussmann-Kolb, 2005; Martynov andSchrödl, 2008) Antarctic and deep water species are the most basaloffshoots of all main nudipleuran subgroups besides the Pleuro-branchaeinae without any extant Antarctic species. However, thebasal offshoot of Pleurobranchaeinae according to Martynov andSchrödl (2008) is Pleurobranchella nicobarica a deep-sea speciesand thus presumably cold-water adapted. This leads us to the con-clusion that possibly all Nudipleura evolved from an Antarctic ori-gin supporting the assumption by Schrödl (2003) and Wägele et al.(2008). However, this hypothesis needs to be verified in futurestudies comprising a comprehensive nudibranch taxon sampling.

The age of the Nudipleura and the estimation of split-off timeare difficult regarding the present results as well since all age esti-

mations have long credible intervals. The unexpectedly high ageestimations of Nudibranchia and Nudipleura might be due to ex-tremely high evolutionary rates of the cladobranch taxa (Arminalovenii, Flabellina babai, Cuthona nana) compared to all other taxaincluded in this analysis which possibly constrains age estimation.

Regarding the Pleurobranchomorpha historical biogeographyreconstruction is hampered by the fact that only three membersof the Pleurobranchaeinae without any extant Antarctic speciesare included in the analyses. This is reflected in ambiguous resultsidentifying three possible ancestral areas for this clade: Antarctica,Eastern Atlantic and Eastern Pacific. However, this result does notexclude the hypothesis of an Antarctic origin. If the Pleurobranch-omorpha evolved from an Antarctic origin the ancestor of the Pleu-robranchaeidae probably migrated into warmer regions at timeswhen temperatures started decreasing around Antarctica afterthe Early Eocene Climate Optimum (50–52 Mya; Zachos et al.,2001). Since the world stayed largely ice-free during the MiddleEocene (Burgess et al., 2008) dispersal happened before onset ofglaciation. Radiation into the extant species must then have takenplace in warmer regions afterwards, while Antarctic ancestors be-came extinct.

Finally, an Antarctic origin is clearly supported for the Pleuro-branchinae comprising about 80% of the extant pleurobranchomor-phan species by the results of most historical biogeographyreconstruction methods in our study. The estimated time of divisionof this clade is between 30.2 and 33.7 Mya in Early Oligocene. At thistime Antarctica was neither connected to Australia (until Early Eo-cene; Lawver and Gahagan, 2003) nor to South America (until Mid-dle Eocene; Houle, 1999) and the water gap between Africa andAntarctica was extensive (Houle, 1999). The split-off time of thePleurobranchinae into the Antarctic Tomthompsonia and theremaining species coincides with two major geological events inthe Antarctic region. On the one hand, a significant drop of air andwater temperatures was recorded for the Southern Oceans and Ant-arctica at the end of Eocene (Zinsmeister, 1982; Ditchfield et al.,1994) followed by the onset and rapid expansion of Antarctic glaci-ation in the time period from the Late Eocene to Early Oligocene(Ditchfield et al., 1994; Zachos et al., 2001). This abrupt coolingevent dramatically altered circulation patterns and temperature re-gimes and thus caused a sudden change in shelf faunas (Zinsmeister,1982). The appearance of remarkably cooler conditions implied aperiod of environmental stress for Antarctic molluscan fauna result-ing in a decrease in diversity (Zinsmeister, 1982). We assume thatthese sudden and dramatic climate changes leading to difficult con-ditions under decreasing temperatures probably led to dispersal ofthe remaining Pleurobranchinae into warmer regions. Migrationout of Antarctica in glacial periods is regarded as a common patternfor different species (Brandt et al., 2007; Clarke, 2008).

On the other hand, division of the Pleurobranchinae coincideswith opening of the Drake Passage �31 Mya (Lawver and Gahagan,2003) resulting in a complete circum-Antarctic seaway. Moreover,the so called Antarctic Circumpolar Current (ACC) emerged as aseries of eastward-flowing jets about 30 Mya (Lawver and Gaha-gan, 2003; Livermore et al., 2005). As the strongest of these jetsthe Polar Front is a strong barrier to free north–south exchangeof water representing a distinctive biogeographical discontinuity(Crame, 1999; Clarke et al., 2005; Barnes et al., 2006). Strong cur-rents and deep passages of water can be effective barriers to dis-persal of planktonic larvae of marine animals occurring oncontinental shelves (Wilson et al., 2009) as well as to small plank-tonic animals like Krill (Patarnello et al., 1996). Nevertheless, ourresults indicate that the last common ancestor of the Pleurobran-chinae (excluding Antarctic Tomthompsonia) managed to dispersenorthwards most probably to Western Atlantic and Eastern Pacificcoasts of South America via the Drake Passage. Dispersal acrossthe Drake Passage has already been revealed for the Antarctic

K. Göbbeler, A. Klussmann-Kolb / Molecular Phylogenetics and Evolution 55 (2010) 996–1007 1005

nudibranch Doris kergulensis (Wilson et al., 2009), species ofNemertea (Thornhill et al., 2008) and ostracod Crustaceans (Woodet al., 1999). Moreover, dispersal to Indo-West Pacific coasts ofAustralia via the South Tasman Rise and Tasmania seems possible.The regular occurrence of kelp rafts has been confirmed for thisregion (Smith, 2002). Large numbers of bivalves, bryozoans,spirobids and hydroids can survive transport on kelps apparentlyeven over several months (Helmuth et al., 1994) as might eggcapsules of Pleurobranchinae. Investigations on diverse taxa haveshown that the ACC and the Polar Front are no distinct dispersalbarriers: species of Bivalvia (Page and Linse, 2002), Bryozoa(Barnes and Griffiths, 2008), decapod and copepod Crustaceans(Gorny, 1999; Razouls et al., 2000), Echinodermata (Hunter andHalanych, 2008) and Notothenioid fish (Bargelloni et al., 2000)have been found in and out of Antarctica. In addition, the formationof the ACC may also have supported the dispersal of benthicorganisms (Beu et al., 1997; Razouls et al., 2000; Barnes andGriffiths, 2008) not only circum-Antarctically (Brandt et al., 1999)but also between various high latitude localities particularly fororganisms with planktonic larval dispersal (Crame, 1999) likesome Pleurobranchinae.

Since circum-Antarctic circulation tends to move northward asit is driven eastward by the wind, Lawver and Gahagan (2003) ex-pect the ACC to travel north in the Eastern Atlantic region. Thus,larvae or egg cases of Pleurobranchinae might perhaps have beendispersed from Antarctica to the Eastern Atlantic, where the spe-cies might have spread northwards along the African continent.Furthermore, the northward movement of deep water formed inWeddell Sea facilitates close faunal connections between SouthernOceans and other basins (Vinogradova, 1997; Brandt et al., 2007).Indeed abyssal faunas tend to have strong links to other oceansparticularly the Atlantic (Brandt et al., 2007). Antarctic bottomand intermediate waters flow towards the equator, the faster inter-mediate water can emerge at the surface (e.g., in West-southernAfrica) after only months (Barnes et al., 2006). Taking into accountthat intracapsular developmental times of opisthobranch gastro-pods from the Weddell Sea are extraordinary long (several months)and that juveniles are capable of drifting along with currents (Hainand Arnaud, 1992) dispersal might also have occurred this way.

Our data indicate that soon after dispersal out of Antarctica ra-pid radiation of the clade along with invasions into temperate andtropical areas began. No special migration pattern could be ob-served for the investigated taxa, all temperate and tropical marineareas were probably colonized in a relatively short amount of timeregarding the dispersal possibilities of slugs. This assumption is notjust supported by the results of the molecular clock analysis, butcan also be inferred from extremely short branch lengths in theBayesian and Maximum likelihood analyses.

Berthella species emerged first about 26 Mya, which nicely fitstheir fossil record (Valdés and Lozouet, 2000). Subsequently, thisclade rapidly split-off into the separate species, which indicatesthat some of the species (Berthella stellata, B. plumula, B. martensi)are more than 20 My old. Since age estimation for this clade mighthave been hampered by the high evolutionary rates of exactlythese three taxa this result has to be considered with caution.

Pleurobranchini evolved considerably later (about 11.5 Mya),their radiation was probably related to warm and shallow waters(Martynov and Schrödl, 2008). The same holds true for the remain-ing Berthella and Berthellina species, which evolved between 16 and7 Mya probably also in warm and shallow waters.

According to our results, Bathyberthella antarctica recolonizedthe Antarctic region about 20 Million years ago (Mya) in a periodof warmer climate from Late Oligocene to Middle Miocene (Zachoset al., 2001) that followed initial glaciation in Antarctica. This is inaccordance with the expectation that taxa immigrate into Antarc-tica during global warming and emigrate at cooler times (Clarke

and Crame, 1992; Barnes et al., 2006). This contradicts the assump-tion that Bathyberthella species may be relics of basal pleurobran-chomorphan radiation in ancient Antarctic waters (Martynov andSchrödl, 2008). Nevertheless, the scenario of recolonization of theAntarctic shelf seems reasonable and has been regarded as likelybefore (Wägele et al., 2008).

5. Conclusions

The present study provides first insights into the molecularphylogeny and biogeography of the Pleurobranchomorpha. Histor-ical biogeography reconstruction yielded ambiguous results. How-ever, an Antarctic origin seems reasonable implying that the lastcommon ancestor of the Pleurobranchaeidae left Antarctica in Mid-dle Eocene when the climate began to cool.

The results of the present study strongly suggest an Antarctic ori-gin of the Pleurobranchinae. The division of this clade into the Ant-arctic Tomthompsonia and the remaining species in Early Oligocenecoincides with two major geological events in the Southern Ocean– onset of glaciation in Antarctica and opening of the Drake Passagewith formation of the Antarctic Circumpolar Current (ACC). We sup-pose that these dramatic and sudden changes in climate conditionshave led to the migration of the last common ancestor of the Pleuro-branchinae (excluding Tomthompsonia) into warmer regions, whererapid radiation and dispersal into all temperate and tropical regionsfollowed. The ACC may have contributed to dispersal of larvae to theEastern Atlantic. Recolonization of Antarctica by Bathyberthella spe-cies probably occurred in a period of warmer climate in Early Mio-cene. Preferences for habitats providing at least temperateconditions can also be inferred from our analyses.

Acknowledgements

We are thankful to the German Academic Exchange Service(DAAD) for financial support of a field trip to Australia for the firstauthor. Permission for collecting was given by Department of Pri-mary Industries, Victoria (permit number RP921) and the NSWDepartment of Primary Industries (permit number P07/0058).Georg Mayer (Melbourne, Australia) provided chemicals and JohnHealy (Brisbane, Australia) assisted in export of the specimens. An-gela Dinapoli (Frankfurt, Germany) helped collecting thespecimens.

DNA samples and two sequences were supplied by VerenaVonnemann and Heike Wägele (Bonn, Germany). Furthermore,we are indebted to some colleagues who provided material: JuanLucas Cervera (Cadiz, Spain), Rhanor Gillette (Urbana, IL, USA), Lou-ise Page (Victoria, BC, Canada), Michael Schrödl and Enrico Schwa-be (ZSM, Munich, Germany) and Sid Staubach (Chicago, IL, USA).The first author participated in a biogeography workshop by RickRee (Chicago, IL, USA) and got helpful assistance with lagrange.

The second author is supported by the Biodiversity and ClimateResearch Centre (BiK-F), Frankfurt/Main.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ympev.2009.11.027.

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