Thalassas, 27 (2): 121-154 An International Journal of Marine Sciences

Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

KATRIN GÖBBELER(1,*) & ANNETTE KLUSSMANN-KOLB(1)

Key words: character evolution, BayesTraits, molecular systematics, diet, herbivory, Euthyneura

ABSTRACT Based on the presented data monophyly of Opisthobranchia is clearly rejected. However, The Opisthobranchia comprise a group of highly monophyly of the Euthyneura (comprising specialized gastropods with uncertain systematic Opisthobranchia and Pulmonata) is supported. affinities. Moreover, monophyly of the whole clade Furthermore, monophyly of most subgroups is has been repeatedly questioned. The present study revealed. The Runcinacea are found as a separate presents the currently most extensive analyses on clade clustering apart from the remaining opisthobranch phylogeny including 58 species from Cephalaspidea, the taxon in which they have been all major subgroups. A combination of four gene formerly classified. Furthermore, Aplysiomorpha markers as well as diverse molecular systematic and Pteropoda are recovered as a monophyletic analytical approaches are applied in order to shed new clade, while the Aplysiomorpha are found to be light on the evolution of this taxon. Special emphasis paraphyletic due to the position of a single taxon. is given to the reconstruction of ancestral diet In addition, Cylindrobullida are revealed as part of preferences since extant Opisthobranchia feed on a the sacoglossan subclade Oxynoacea denying their variety of different food items and the development of current separate status. The enigmatic Acochlidiacea dietary specialization is supposed to be important for are revealed as sister group to Eupulmonata. the evolution of these enigmatic marine gastropods. Ancestral diet preferences are reconstructed for monophyletic Euthyneura and all main subclades since (1) Institute for Ecology, Evolution and Diversity, Goethe- polyphyly of Opisthobranchia impeded reconstruction University Frankfurt, Siesmayerstrasse 70, 60054 Frankfurt am Main, Germany for this clade. Herbivory is found as the most likely e-mail: [email protected] ancestral diet of Euthyneura while carnivory probably *Current address: Department of Integrative Biology, University of Colorado Denver, P.O. Box 173364, Denver, CO evolved several times independently in different 80217, USA clades.

121 Katrin Göbbeler & Annette Klussmann-Kolb

Figure 1: Bayesian inference phylogram of the phylogenetic analyses (concatenated alignment of 18S rDNA, 28S rDNA, 16S rDNA and CO1), 50% majority rule consensus tree. Posterior probabilities are provided at the nodes; only support values above 0.5 are given. Taxonomic classifications (following Bouchet and Rocroi, 2005) are indicated and shaded on the right side (“lower Heterobranchia” = white; Opisthobranchia = light grey; Pulmontata = dark grey). The branch leading to the nudipleuran Dexiarchia was shortened by about 50% to allow better visibility.

122 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

INTRODUCTION Monophyly of the Opisthobranchia has been challenged by several authors due to the lack The Opisthobranchia are a group of gastro- of proper synapomorphies (Salvini-Plawen pods comprising morphologically diversified spe- and Steiner, 1996; Ponder and Lindberg, 1997) cies which are distributed globally in all marine caused by “rampant parallelism” (Gosliner and habitats. They are composed of about 6000 spe- Ghiselin, 1984) in this taxon. Moreover, previous cies (Wägele et al., 2008) currently divided into phylogenetic analyses often failed to reveal nine main clades: Cephalaspidea, Thecosomata, monophyly of this taxon both in morphology-based Gymnosomata, Aplysiomorpha, Acochlidiacea, studies (Dayrat and Tillier, 2002; Wägele and Sacoglossa, Cylindrobullida, Umbraculida and Klussmann-Kolb, 2005) and molecular systematic Nudipleura (Bouchet and Rocroi, 2005). Monophyly investigations (Thollesson, 1999; Dayrat et of these clades has been well supported in molecular al., 2001; Grande et al., 2004a, b; Klussmann- systematic studies (Wägele et al., 2003; Grande et Kolb et al., 2008; Dinapoli and Klussmann- al., 2004a, b; Vonnemann et al., 2005; Klussmann- Kolb, 2010; Jörger et al., 2010). In a recent Kolb and Dinapoli, 2006; Klussmann-Kolb et al., study focusing on phylogeny and systematics of 2008; Dinapoli and Klussmann-Kolb, 2010; Jörger Acochlidiacea, Jörger et. al (2010) propose the new et al., 2010). Furthermore, the pelagic Thecosomata clade Euopisthobranchia uniting Umbraculida, and Gymnosomata have been revealed as sister Aplysiomorpha, Cephalaspidea and Pteropoda. groups forming the Pteropoda (Klussmann-Kolb and This clade presents a “monophyletic remainder Dinapoli, 2006). of the (non-monophyletic) “Opisthobranchia” as traditionally defined” (Jörger et al., 2010, p. 7). The main evolutionary trend in Opisthobranchia is reduction or even loss of the shell (Grande et al., Opisthobranchia are supposed to form a clade 2004a) accompanied by development of diverse with pulmonate gastropods called Euthyneura defensive strategies (Wägele and Klussmann-Kolb, (Spengel, 1881). Monophyly of this clade has been 2005). Radiation of opisthobranchs has lead to detected in morphological (Ponder and Lindberg, parallelism and convergence of morphological 1997; Dayrat and Tillier, 2002) as well as molecular characters (Gosliner and Ghiselin, 1984; Gosliner, systematic studies (Thollesson, 1999; Wade and 1985, 1991) hampering morphology based Mordan, 2000; Knudsen et al., 2006) while other classification. Thus, phylogenetic hypotheses on molecular systematic studies reveal paraphyly of Opisthobranchia vary based on morphological Euthyneura (Dayrat et al., 2001; Grande et al., considerations (Schmekel, 1985; Bieler, 1992; 2004b; Klussmann-Kolb et al., 2008; Dinapoli Salvini-Plawen and Steiner, 1996; Dayrat and Tillier, and Klussmann-Kolb, 2010; Jörger et al., 2010). 2002; Mikkelsen, 2002; Wägele and Klussmann- Nevertheless, Thollesson (1999) as well as Jörger Kolb, 2005). Moreover, molecular phylogenetic et al. (2010) claimed the constant inclusion of both analyses reveal contradictory classifications as well pulmonate and opisthobranch taxa in phylogenetic (Thollesson, 1999; Dayrat et al., 2001; Grande et al., studies due to their possibly common origin. Thus, 2004a, b; Vonnemann et al., 2005; Klussmann-Kolb erroneous monophyly of either clade based on and Dinapoli, 2006; Klussmann-Kolb et al., 2008; incomplete taxon sampling can be avoided. We Dinapoli and Klussmann-Kolb, 2010; Jörger et al., follow this request in the present investigation by 2010) mainly due to differences in taxon sampling, incorporating both opisthobranch and pulmonate employed marker genes and outgroup determination. species in our analyses. Additionally, several “lower Thus, a common solution and a robust phylogeny of heterobranch” taxa are included to test monophyly Opisthobranchia are still lacking. of Euthyneura.

123 Katrin Göbbeler & Annette Klussmann-Kolb Figure 2: Taxonomic classification to (according Bouchet and Taxonomic 2005) Rocroi, indicated by braces. Neighbour-net Neighbour-net graph of the split decomposition analysis (concatenated alignment of 18S rDNA, 28S rDNA, 16S rDNA and CO1).

124 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

A highly specialized and possibly crucial feature of ancestral character states applying a Bayesian for evolution in Opisthobranchia is represented by their approach. For this purpose, we conducted a thorough diverse diet (Thompson, 1976; Rudman and Willan, literature search on diet of Opisthobranchia to 1998; Mikkelsen, 2002; Wägele, 2004). Many potential enable reconstruction of ancestral preferences. key characters for opisthobranch evolution are related This reconstruction is based on the currently most to diet and supposed to trigger exploration of new food comprehensive molecular systematic study on sources (Wägele, 2004). Some opisthobranch clades opisthobranch phylogeny covering all main subclades feed on algae (Aplysiomorpha, Sacoglossa); others are in order to account for their interrelationships and specialized on Porifera (Umbraculida), while diverse provide the framework for reconstruction of character carnivorous (Nudipleura) or even carnivorous or evolution. herbivorous food items (Cephalaspidea) are preferred in other groups. Some opisthobranch species exhibit MATERIALS AND METHODS remarkable adaptations to a special source of food, e.g. Nudibranchia living in mutualistic symbiosis Taxon sampling with photosynthetic dinoflagellates and sharing metabolites (Burghardt et al., 2005) or Sacoglossa The current study comprises a total of 86 taxa incorporating chloroplasts of their algal food in their with 58 Opisthobranchia covering all subclades. own digestive gland and using metabolites (Rumpho Additionally 18 Pulmonata (including all main et al., 2000; Händeler et al., 2009). The evolution subclades) and nine “lower Heterobranchia” (with of these highly specialized features is largely a special focus on the questionable opisthobranch unknown. Malaquias et al. (2009a) reconstructed the clade Acteonoidea) complement the taxon sampling. ancestral diet of Cephalaspidea comprising diversely The caenogastropod Littorina littorea was defined as specialized species, but up to now the ancestral diet outgroup taxon. of the last common ancestor of the Opisthobranchia remains a matter of debate. Haszprunar (1985) Sequences were primarily taken from GenBank, claimed that carnivory is the plesiomorphic supplemented by some newly generated sequences condition due to diet preferences of the possibly of crucial taxa. Specimens were collected worldwide basal opisthobranch taxon Architectibranchia. On by hand, snorkeling, or scuba diving and preserved the contrary, Mikkelsen (1996, 2002) argued that in 80-100% ethanol. Origin of all taxa and accession herbivory was the ancestral state. Vermeij and numbers of utilized sequences are summarized in Lindberg (2000, p. 423) suggested that “feeding on Table 1. sessile invertebrates may be the plesiomorphic mode of feeding from which herbivory arose in various DNA extraction, PCR and sequencing gastropod clades” (including Opisthobranchia). However, specific dietary preferences are likely Genomic DNA was extracted from muscle tissue the result of a complex interplay of phylogeny, prey via the DNeasy Tissue Kit (Qiagen, Hilden, Germany) structure and habitat (Mikkelsen, 1996). Thus, it according to the animal tissues/spin-column protocol. is important to reconstruct the ancestral state in order to gain new insights into the evolution of this We amplified two nuclear (complete 18S rDNA specialized character complex. and partial 28S rDNA) and two mitochondrial (partial 16S rDNA and CO1) gene fragments, which were We attempt to close this gap of knowledge by sequenced in both directions. Primer sequences and making use of the software BayesTraits (Pagel et al., PCR-protocols are given in Göbbeler and Klussmann- 2004) which is a powerful tool for the reconstruction Kolb (2010).

125 Katrin Göbbeler & Annette Klussmann-Kolb

Figure 3: Cladogram of the Baysian inference analysis (concatenated alignment of 18S rDNA, 28S rDNA, 16S rDNA and CO1; 50% majority rule consensus tree). Dietary coding of species indicated as colour coded (carnivory = red, herbivory = green, unselective = yellow) squares before species names. Results of reconstruction of character evolution mapped onto the tree coded as pie charts displaying color coded (carnivory = red, herbivory = green, unselective = yellow) fractions of the diverse dietary types (inferred from posterior probabilities) . Taxonomic classification (following Bouchet and Rocroi, 2005) provided at the right side, major clades are shaded..

126 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

PCR-products were purified from an agarose gel The Approximately Unbiased (AU) test using the QIAquick Gel Extraction Kit from Qiagen (Shimodaira, 2002) was employed to test an (Hilden, Germany). Sequencing was performed using alternative tree topology enforcing monophyly of a CEQ 2000 Beckmann Coulter capillary sequencer the Opisthobranchia. Likelihoods were calculated for at the scientific research lab in Frankfurt/Main. each nucleotide position in PAUP 4.0b10 (Swofford, 2002) for the constrained and unconstrained topology Sequence alignment and subsequently compared in CONSEL version 0.1 (Shimodaira and Hasegawa, 2001) in order to obtain Mafft version 6 (Katoh et al., 2005) was used p-values. for alignment of sequences under the linsi-option displaying one of the most accurate multiple sequence We investigated rate heterogeneity in the alignment methods. The results were analysed with sequences with a relative rate test using the software Aliscore (Misof and Misof, 2009) to filter ambiguous k2WuLi (Wu and Li, 1985). or randomly similar sites in the alignment which were subsequently deleted from the alignment. The Phylogenetic analyses maximal number of possible pairs was compared and gaps were treated as ambiguous characters. The best fitting model of sequence evolution for The single codon positions of the CO1-sequences each gene partition (single codon positions of CO1 were analysed separately. Details about alignment separately) was determined via MrModeltest 2.2 length and Aliscore results with excluded nucleotide (Nylander, 2004) based on the Akaike information positions are summarized in Table 2. criterion (AIC) prior to phylogenetic analyses. Details about the determined models are provided Statistical tests in Table 2.

Several statistical tests were conducted on our Bayesian inference analysis was performed via data after exclusion of ambiguous sites and prior to MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001) phylogenetic analyses to estimate data quality and using separate models of evolution for each gene survey the results. partition. Two separate runs of four chains each (one cold, three heated) of a Metropolis – coupled The Incongruence Length Difference (ILD) Markov chain Monte Carlo algorithm operated for test (Farris et al., 1995) was used to examine the 2,000,000 generations. Likelihoods converged slowly, significance of incongruence in our combined dataset. thus the first 15,000 trees were ignored as burn-in for This test is implemented in PAUP 4.0b10 (Swofford, construction of the 50% majority rule consensus tree. 2002) as Partition Homogeneity test and was used to Posterior probabilities were calculated for each node, check if the single gene markers provide a congruent a value of 0.95 and higher being considered as good phylogenetic signal and can thus be concatenated statistical support. and analysed as a single dataset. We conducted 100 replicates of a heuristic search under the Maximum The result of this phylogenetic analysis was Parsimony criterion. reassessed by a split-decomposition analysis on the concatenated alignment using SplitsTree 4.9.1 Substitution saturation of the single datasets (Huson, 1998; Huson and Bryant, 2006). Split graphs was evaluated via the test by Xia et al. (2003) as show networks of phylogenetic relationships revealing implemented in the software package DAMBE (Xia conflicts in data sets. We constructed a neighbor-net and Xie, 2001). graph based on uncorrected p-distances.

127 Katrin Göbbeler & Annette Klussmann-Kolb

Table 1. Information on taxon sampling and Genbank accession numbers. * = sequences generated

forTable the current1: Information study. on taxon sampling and Genbank accession numbers. * = sequences generated for the current study

Genbank Accession Numbers Taxon Family/Subfamily Locality 18S 28S 16S CO1

CAENOGASTROPODA

Littorina littorea Littorinidae Genbank X91970 AJ488672 DQ093481 DQ093525

LOWER HETEROBRANCHIA

Orbitestella sp. Orbitestellidae Genbank EF489352 EF489377 EF489333 EF489397

Cima sp. Cimidae Genbank FJ917206 FJ917228 FJ917260 FJ917279

Rissoella rissoaformis Rissoellidae Genbank FJ917214 FJ917226 FJ917252 FJ917271

ACTEONOIDEA

Acteon tornatilis Acteonidae Genbank GQ845182 GQ845177 GQ845190 GQ845172 GQ845183

Pupa nitidula Acteonidae Genbank GQ845185 GQ845179 GQ845192 GQ845173

Rictaxis punctocaelatus Acteonidae Genbank GQ845186 EF489370 GQ845193 EF489393

Hydatina physis Aplustridae Genbank AY427515 AY427480 EF489320 GQ845174

Micromelo undata Aplustridae Genbank GQ845188 GQ845181 GQ845195 GQ845176

Bullina lineata Bullinidae Genbank GQ845189 ‐ GQ845196 AY296847

OPISTHOBRANCHIA

CEPHALASPIDEA

BULLOIDEA

Bulla striata Bullidae Genbank DQ923472 DQ986683 DQ986632 DQ986566

DIAPHANOIDEA

Diaphana sp. Diaphanidae Genbank DQ923455 EF489373 EF489325 EF489394

Toledonia globosa Diaphanidae Genbank EF489350 EF489375 EF489327 EF489395

HAMINOEOIDEA

Haminoea hydatis Haminoeidae Genbank AY427504 AY427468 EF489323 DQ238004

Atys cylindricus Haminoeidae Genbank DQ923458 DQ927228 ‐ DQ974671

Smaragdinella sp. Smaragdinellidae Genbank AJ224789 DQ927242 AF249257 AF249806

PHILINOIDEA

Scaphander lignarius Cylichnidae Genbank EF489348 EF489372 EF489324 DQ974663

Philine aperta Philinidae Genbank DQ093438 DQ279988 DQ093482 AY345016

Odontoglaja sp. Aglajidae Genbank DQ923450 DQ927218 ‐ DQ974655

128 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

Philinopsis pilsbryi Aglajidae Genbank AY427509 AY427474 AM421840 AM421888

Sagaminopteron Gastropteridae Genbank AY427513 AY427478 AM421815 AM421856 psychedelicum

Philinoglossa praelongata Philinoglossidae Genbank AY427510 AY427475 ‐ ‐

Retusa sp. Retusidae Genbank AY427511 AY427476 ‐ DQ974679

Pyrunculus sp. Retusidae Genbank DQ923465 DQ927237 ‐ DQ974678

RUNCINACEA

Runcina africana Runcinidae Genbank DQ923473 DQ927240 ‐ DQ974680

Ilbia ilbi Ilbiidae Australia, NSW GU213047* GU213052* GU213043* GU213057*

APLYSIOMORPHA

AKEROIDEA

Akera bullata Akeridae Genbank AY427502 AY427466 AF156127 AF156143

APLYSIOIDEA

Aplysia californica Aplysiidae Genbank AY039804 AY026366 AF192295 AF077759

Dolabrifera dolabrifera Aplysiidae Genbank DQ237960 DQ237973 AF156133 AF156149

Bursatella leachii Aplysiidae Genbank DQ237961 DQ237975 AF156130 AF156146

Dolabella auricularia Aplysiidae Genbank AY427503 AY427467 AF156132 AF156148

Stylocheilus longicauda Aplysiidae Genbank DQ237963 DQ237978 AF156140 AF156156

Petalifera petalifera Aplysiidae Genbank DQ237962 DQ237977 ‐ AY345020

PTEROPODA

THECOSOMATA

Cavolinia uncinnata Cavoliniidae Genbank DQ237964 DQ237983 ‐ DQ237997

Hyalocylis striata Cavoliniidae Genbank DQ237966 DQ237985 ‐ DQ237999

Clio pyramidata Cavoliniidae Genbank DQ237967 DQ237986 ‐ DQ238000

Cuvierina columnella Cavoliniidae Genbank DQ237965 DQ237984 ‐ DQ237998

GYMNOSOMATA

Pneumoderma atlantica Pneumodermatidae Genbank DQ237970 DQ237989 ‐ DQ238003

Spongiobranchaea Pneumodermatidae Genbank DQ237969 DQ237988 ‐ DQ238002 australis

UMBRACULIDA

Umbraculum umbraculum Umbraculidae Genbank AY165753 AY427457 EF489322 DQ256200

Umbraculum sp. Umbraculidae GU213048* GU213053* GU213044* GU213058* Mediterranean Sea

Tylodina perversa Tylodinidae France, GU213049* GU213054* GU213045* GU213059* Mediterranean Sea

129 Katrin Göbbeler & Annette Klussmann-Kolb

Tylodina fungina Tylodinidae Panama, Caribbean GU213050* GU213055* GU213046* GU213060* Sea ACOCHLIDIACEA

Unela glandulifera Microhedylidae Croatia AY427517 AY427482 EF489328 GU213061*

Pontohedyle milatchevitchi Microhedylidae Genbank AY427519 AY427484 EF489329 ‐

SACOGLOSSA

OXYNOACEA

Oxynoe antillarum Oxynoidae Genbank FJ917441 FJ917466 FJ917425 FJ917483

Lobiger viridis Oxynoidae Genbank GU213051* GU213056* EU140894 ‐

PLACOBRANCHACEA

Elysia viridis Placobranchidae Genbank AY427499 AY427462 EU140863 DQ471211

Placobranchus ocellatus Placobranchidae Genbank AY427497 AY427459 DQ480205 DQ471270

Bosellia mimetica Boselliidae Genbank AY427498 AY427460 DQ480203 DQ471214

Limapontia nigra Limapontiidae Genbank AJ224920 AY427465 ‐ ‐

CYLINDROBULLIDA

Cylindrobulla beauii Cylindrobullidae Genbank EF489347 EF489371 EF489321 ‐

NUDIPLEURA

NUDIBRANCHIA

DEXIARCHIA

Armina lovenii Arminidae Genbank AF249196 ‐ AF249243 AF249781

Flabellina verrucosa Flabellinidae Genbank AF249198 ‐ AF249245 AF249790

Eubranchus exiguus Eubranchidae Genbank AJ224787 ‐ AF249246 AF249792

Dendronotus dalli Dendronotidae Genbank AY165757 AY427450 AF249252 AF249800

ANTHOBRANCHIA

Bathydoris clavigera Bathydorididae Genbank AY165754 AY427444 AF249222 AF249808

Hypselodoris infucata Chromodorididae Genbank FJ917442 FJ917467 FJ917426 FJ917484

Chromodoris krohni Chromodorididae Genbank AJ224774 AY427445 AF249239 AF249805

Hoplodoris nodulosa Discodorididae Genbank FJ917443 FJ917469 FJ917428 FJ917486

Austrodoris kerguelenensis Dorididae Genbank AJ224771 ‐ EU823269 EU823218

Goniodoris nodosa Goniodorididae Genbank AJ224783 AY014157 AF249226 AJ223264

Acanthodoris pilosa Onchidorididae Genbank AJ224770 ‐ AJ225177 AJ223254

Limacia clavigera Polyceridae Genbank AJ224778 ‐ EF142952 AJ223268

PLEUROBRANCHOMORPHA

130 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

Tomthompsonia Pleurobranchinae Genbank AY427492 AY427452 EF489330 DQ237992 antarctica

Pleurobranchus peroni Pleurobranchinae Genbank AY427494 AY427455 EF489331 DQ237993

Berthellina citrina Pleurobranchinae Genbank FJ917448 FJ917476 FJ917436 FJ917494

Pleurobranchaea meckeli Pleurobranchaeinae Genbank FJ917449 FJ917481 FJ917439 FJ917499

PULMONATA

SIPHONARIOIDEA

Siphonaria capensis Siphonariidae Genbank EF489335 EF489354 EF489301 EF489379

Siphonaria concinna Siphonariidae Genbank EF489334 EF489353 EF489300 EF489378

AMPHIBOLOIDEA

Phallomedusa solida Amphibolidae Genbank DQ093440 DQ279991 DQ093484 DQ093528

Amphibola crenata Amphibolidae Genbank EF489337 EF489356 EF489304 ‐

HYGROPHILA

Chilina sp. Chilinidae Genbank EF489338 EF489357 EF489305 EF489382

Lymnaea stagnalis Lymnaeidae Genbank AY427525 AY427490 EF489314 AY227369

Acroloxus lacustris Acroloxidae Genbank AY282592 EF489364 EF489311 AY282581

Latia neritoides Latiidae Genbank EF489339 EF489359 EF489307 EF489384

Bulinus tropicus Bulinidae Genbank AY282594 EF489366 EF489313 AY282583

EUPULMONATA

OTINOIDEA

Otina ovata Otinidae Genbank EF489344 EF489363 EF489310 EF489389

Smeagol phillipensis Smeagolidae Genbank FJ917210 FJ917229 FJ917263 FJ917283

ELLOBIOIDEA

Ophicardelus ornatus Ellobiidae Genbank DQ093442 DQ279994 DQ093486 DQ093530

SYSTELLOMMATOPHORA

Onchidella floridana Onchidiidae Genbank AY427521 AY427486 EF489317 EF489392

Onchidium verrucosum Onchidiidae Genbank AY427522 AY427487 EF489316 EF489391

STYLOMMATOPHORA

Arion silvaticus Arionidae Genbank AY145365 AY145392 EU541969 AF513018

Arianta arbustorum Helicidae Genbank AY546383 AY014136 AY546343 EF398269

Helix aspersa Helicidae Genbank X91976 AY014128 EU912832 AY546283

Deroceras reticulatum Limacidae Genbank AY145373 FJ917241 FJ917266 FJ917286

131 Katrin Göbbeler & Annette Klussmann-Kolb

Reconstruction of character evolution 16S-sequences were included to avoid the loss of phylogenetic signal at lower taxonomic levels. Character evolution of diet preferences was reconstructed using a Bayesian approach implemented Results of the relative rate test showed that in the software package BayesTraits (PC Version 1.0/ evolutionary rates differ among investigated taxa and Pagel et al., 2004) and based on the phylogenetic tree genetic markers. The major differences indicated by derived from Bayesian inference analysis. Reversible- the highest z-scores (up to 13.3) were found in the jump Markov chain Monte Carlo (MCMC) methods 18S-sequences of the nudibranch taxa Eubranchus were used to derive posterior probabilities of values of exiguus, Dendronotus dalli, Armina lovenii and traits at ancestral nodes of phylogenies. MultiState was Flabellina verrucosa. Regarding the 28S sequences selected as model of evolution and the rate deviation the highest z-scores between 5.0 and 7.7 were revealed was set to 12. A hyperprior approach was employed for the nudipleuran taxa Dendronotus dalli and with an exponential prior seeded from an uniform Pleurobranchaea meckeli. 16S and CO1-sequences on the interval 0 to 30. Thus, acceptance rates in the generally yielded lower z-scores with maximal values preferred range of 20 to 40% were achieved. A total of slightly more than 3.0 for the 16S-sequences of 5,000,000 iterations were run for each analysis of Orbitestella sp., Rissoella rissoaformis, the with the first 50,000 samples discarded as burn-in. sacoglossan Oxynoe antillarum, Lobiger viridis and Since posterior probabilities for ancestral states of the Elysia viridis as well as Pontohedyle milatchevitchi single runs partly varied, we calculated the arithmetic and Onchidium verrucosum and for the first codon mean of all samples for reconstruction of the ancestral position of CO1-sequences of Dendronotus dalli and condition. Additionally, a Bayes factor test was Hyalocylis striata as well as the second codon position conducted to test if there is support for one state over of the pteropodan Hyalocylis striata, Pneumoderma another as suggested in the BayesTraits manual for atlantica and Spongiobranchaea australis, and the the ancestral diet of Euthyneura. Diet preferences pulmonate Helix aspersa, Onchidella floridana were taken from the literature and classified as and Deroceras reticulatum. In the concatenated carnivorous, herbivorous or unselective. Information alignment the influence of the 18S sequences is strong on diet and coding for individual taxa and literature yielding z-scores of above 10.0 for the nudibranch sources are provided in Table 3. taxa Eubranchus exiguus, Dendronotus dalli, Armina lovenii and Flabellina verrucosa. RESULTS Phylogenetic analyses Statistical tests Bayesian inference analysis yielded a well The Incongruence Length Difference test (ILD) resolved tree topology with robust statistical support yielded a p-value of 0.01 implying that concatenation of terminal branches as well as deeper nodes. The of the four single gene fragments significantly phylogram of the Bayesian analysis with posterior improves the phylogenetic signal. probabilities given at the nodes is shown in Fig. 1.

Evaluation of substitution saturation revealed The Euthyneura comprising Opisthobranchia little saturation in the 16S-alignment and substantial and Pulmonata are retrieved as monophyletic in saturation in the 3rd codon position of CO1, although our analyses. In contrast to this, monophyly of the only 22 bp of this position were left after filtering Opisthobranchia is clearly rejected by our analyses with Aliscore. Thus the 3rd codon position of CO1 mainly due to the position of the Sacoglossa and the was excluded from further analyses, while the Acochlidiacea clustering well supported within the

132 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

(also polyphyletic) pulmonate taxa included in the apart from the remaining Cephalaspidea. The analyses. We additionally conducted an Approximately latter receive maximum statistical support with the Unbiased (AU) test to reevaluate this result. This test Diaphanidae found in a basal position and a further yielded a p-value of 0.997 for the unconstrained supported division into two main clades. The first topology implying polyphyly of the Opisthobranchia, clade comprises Bulla striata and the Retusidae, while a p-value of 0.003 was revealed for enforced while the second is composed of the two superfamilies monophyly of the Opisthobranchia. The latter value is Haminoeoidea and Philinoidea. The latter clade is below the significance value of 0.050 thus monophyly found to be paraphyletic due to the position of the of the Opisthobranchia is definitely rejected based on Retusidae who are traditionally assigned to this our dataset. superfamily.

The “lower heterobranch” outgroup taxa cluster The Aplysiomorpha and Pteropoda form a well well supported basal to all other taxa included in supported subclade within this main clade being sister our analyses. The Acteonoidea are revealed as sister taxon to the Runcinacea. However, statistical support group of the Rissoelloidea, together representing for this sister group relationship is non-existent and the sister group of Euthyneura. With regards to therefore this grouping should be considered with Euthyneura three main clades are recovered which are care. Akera bullata is found as the most basal offshoot supported by maximal posterior probability values. rendering the Aplysiomorpha paraphyletic since the remaining Aplysiomorpha included in our study form The first offshoot of the three euthyneuran clades the sister group of the Pteropoda. The Pteropoda is the Nudipleura. Monophyly of the Nudipleura as well themselves are divided into the two main subclades as of the two main subclades Pleurobranchomorpha Gymnosomata and Thecosomata. and Nudibranchia is supported by maximum statistical support values. The Nudibranchia are The third main clade revealed in this study furthermore divided into monophyletic Dexiarchia comprises pulmonate taxa and the opisthobranch and Anthobranchia. Sacoglossa and Acochlidiacea (Panpulmonata). Monophyly of the main subclades (Siphonarioidea, The two other clades form sister groups. One Hygrophila, Amphiboloidea and Eupulmonata) clade comprises the opisthobranch subclades is supported, but the Pulmonata are rendered Umbraculida, Cephalaspidea, Aplysiomorpha and paraphyletic due to the inclusion of Sacoglossa Pteropoda (called Euopisthobranchia by Jörger et and Acochlidiacea. The whole clade is divided al., 2010), while the other clade is composed of the into three main subclades without statistically pulmonate taxa and the opisthobranch subclades supported interrelationships. These subclades are the Sacoglossa and Acochlidiacea (termed Panpulmonata Siphonarioidea found monophyletic as the most basal by Jörger et al., 2010). The interrelationships of the offshoot, the Sacoglossa and a subclade comprising opisthobranch subclades in the Euopisthobranchia are Hygrophila, Amphiboloidea, Acochlidiacea and poorly resolved. Eupulmonata. The Sacoglossa are found to be monophyletic and display a well supported division Monophyly of the Umbraculida and its two into two main monophyletic subclades; on the one families Umbraculidae and Tylodinidae is strongly hand the Plakobranchacea and on the other hand the supported while their position basal to all other Oxynoacea clustering with the cylindrobullid taxon. taxa in this clade lacks statistical support. The Cephalaspidea are rendered paraphyletic due to the The monophyletic Hygrophila are found as the most unresolved position of the Runcinacea clustering basal offshoot of the third subclade, without statistical

133 Katrin Göbbeler & Annette Klussmann-Kolb

Table 2. Information on sequence alignments of the single markers and models of sequence evolution for Baysian analyses (pinv/I = proportion of invariable sites; α/G = gamma distribution shape parameter; GTR = Table 2: GeneralInformation Time Reversible).on sequence alignments of the single markers and models of sequence evolution for Baysian analyses (pinv/I = proportion of invariable sites; α/G = gamma distribution shape parameter; GTR = General Time Reversible).

Number of Length of alignment (after removal Excluded nucleotide positions Model of sequence Gene region taxa of ambiguous positions) (by Aliscore) evolution

18S rDNA 86 1783 43‐48, 113‐117, 160‐163, 179‐192, 230‐ GTR+I+G

320, 366‐370, 718‐720, 774‐829, 841‐ pinv = 0.3796

864, 871‐908, 929‐1250, 1264‐1270, α = 0.4720

1367‐1375, 1705‐1723, 1993‐2213,

2221‐2229, 2350‐2356, 2372‐2374,

2562‐2590, 2598‐2602, 2663

28S rDNA 79 954 96‐100,141‐150, 216‐218, 460‐480, 487‐ GTR+I+G

502, 511‐567, 574‐586, 640‐647, 664‐ pinv = 0.2752

672, 686‐750, 764‐809, 836‐845, 856‐ α = 0.6188

865, 875‐877, 904‐907, 937‐959, 966‐

988, 1150‐1413, 1540‐1557

16S rDNA 72 269 3‐22, 30‐35, 47‐64, 142‐152, 155‐159, GTR+I+G

170‐186, 196‐199, 215‐226, 258‐415, pinv = 0.2750

434‐552, 587‐612, 644‐652, 662‐675, α = 0.5667

690‐698

CO1 (1st position) 80 197 22‐24, 92‐94 GTR+I+G

pinv = 0.2731

α = 0.6195

CO1 (2nd position) 80 203 ‐ GTR+G

pinv= 0.3048

α = 0.7436 support, so that the interrelationships of Hygrophila, Most opisthobranch and pulmonate subclades Amphiboloidea and a clade of Acochlidiacea and receive split support in our network analysis; however Eupulmonata cannot be finally resolved. However, no split support could be detected for relationships weak statistical support is given for a sister group among the different subclades. relationship of Acochlidiacea and Eupulmonata. Within the latter grouping good support is received for In the outgroup and “lower heterobranch” taxa a sister group relationship of Otinoidea/Ellobioidea good split support is revealed for the Acteonoidea with Systellommatophora together representing the with some conflict regarding Acteon tornatilis which sister group of the Stylommatophora. shares split support with the Acteonoidea as well as with some nudibranch taxa. The remaining “lower The split network analysis (Fig. 2) confirms heterobranch” taxa cluster apart from the Acteonoidea conflict in the dataset. Additionally, it becomes without split support for these taxa. obvious that evolutionary rates and thus distances between taxa differ since lengths of edges vary Considerable split support is found for the between different clades. Nudipleura which exhibit very long parallel edges

134 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

indicating high evolutionary rates compared to Reconstruction of character evolution other taxa included in this analysis. Extensively long edges are present for the nudibranch subclade Our molecular phylogenetic analyses unambi- Dexiarchia, shorter ones lead to the Anthobranchia guously revealed polyphyly of Opisthobranchia, thus and Pleurobranchomorpha. reconstruction of ancestral diet preferences was per- formed for monophyletic Euthyneura (comprising Furthermore, considerable split support is detec- Opisthobranchia and Pulmonata) as well as its sister ted for each of the following taxa: Umbraculida, group consisting of Acteonoidea and Rissoelloidea. Aplysiomorpha and Cephalaspidea (without the taxon Furthermore, we traced dietary evolution for all main Runcinacea which itself receives good split support). lineages detected in our molecular systematic studies and all relevant opisthobranch and pulmonate clades. No split support could be detected for Sacoglossa The results are given as pie charts displaying different in our analysis; however the main subclades fractions (calculated as posterior probabilities) of the Plakobranchacea and Oxynoacea plus Cylindrobulla reconstructed dietary types and mapped onto the beauii receive considerable split support. phylogenetic tree in Fig. 3. Details about the posterior probabilities for all investigated clades are provided Additionally, no split support is found for the Pteropoda in Table 4. as their main subclades Thecosomata and Gymnosomata are separated by the acochlid Pontohedyle milatchevitchi The reconstruction of dietary evolution of the sharing split support with the Gymnosomata which Euthyneura strongly suggests that the last common themselves also receive good split support. ancestor was herbivorous. The posterior probability value for the herbivorous state is 0.85 compared The most surprising results are revealed for with 0.14 for a carnivorous state and 0.01 for an the Acochlidiacea which are not only lacking split unselective diet. The Bayes Factor test yielded a support but even cluster far apart in our network value of ~4.0 preferring the herbivorous state over the analysis. As already mentioned P. milatchevitchi carnivorous one and of ~8 preferring herbivory over shares split support with the Gymnosomata while the unselective state. Thus, there is support for an Unela glandulifera shares split support with both herbivorous state at this node. Eupulmonata and Hygrophila. The last common ancestor of Euthyneura along Regarding the pulmonate taxa good split support with the Acteonoidea/Rissoelloidea clade was is found for the Stylommatophora. Conflicting possibly also an herbivore (posterior probability: signals are found considering the interrelationships of 0.77, carnivore: 0.21, unselective: 0.02). In contrast Eupulmonata, since on the one hand split support is to this, ancestral Nudipleura representing the first given for a clade composed of Otinoidea/Ellobioidea offshoot of the Euthyneura most likely switched to and Systellommatophora and on the other hand carnivory, while the last common ancestor of all other for Otinoidea/Ellobioidea and Stylommatophora. Euthyneura included in this investigation was probably Generally, it becomes obvious that all pulmonate a herbivore (posterior probability: 0.89, carnivore: subclades are difficult to separate in this network 0.10, unselective: 0.01). The same holds true for the analyses probably because their evolutionary rates are two main subclades of the latter clade composed so much lower than those of the other clades included. of the opisthobranch Umbraculida, Cephalaspidea, Thus, Amphiboloidea is the only other pulmonate Aplysiomorpha and Pteropoda (Euopisthobranchia) subclade with detectable split support although the on the one hand and the pulmonate taxa along other taxa cluster in the expected clades as well. with the opisthobranch Sacoglossa and Acochlidiacea

135 Katrin Göbbeler & Annette Klussmann-Kolb

(Panpulmonata) on the other hand. Both subclades al., 2010) or the Acteonoidea (Dayrat et al., 2001; received high posterior probabilities for the Klussmann-Kolb et al., 2008). Jörger et al. (2010) herbivorous state (0.79 and 0.98, respectively). claim inclusion of (formerly lower heterobranch) taxa Pyramidellidae and Glacidorboidea into Euthyneura Regarding the opisthobranch clades ancestral thus regaining monophyly of the clade. diet preferences differ. The last common ancestors of Nudipleura and Umbraculida probably were Monophyly of Opisthobranchia was challenged carnivorous, while the common ancestors of before since there are very few common apomorphic Cephalaspidea, Aplysiomorpha and Sacoglossa most features for this clade (Salvini-Plawen and probably were herbivores. The last common ancestor Steiner, 1996) and phylogenetic analyses based on of the Pteropoda probably also was a carnivore, but morphological or molecular data repeatedly yielded herbivorous as well as unselective diet also received paraphyly or even polyphyly of this clade (Thollesson, unexpectedly high posterior probabilities (carnivore: 1999; Dayrat et al., 2001; Dayrat and Tillier, 2002; 0.62, herbivore: 0.12, unselective: 0.26). Grande et al., 2004a, b; Wägele and Klussmann-Kolb, 2005; Klussmann-Kolb et al., 2008; Dinapoli and In summary, the results of the present study Klussmann-Kolb, 2010; Jörger et al., 2010). This result strongly support herbivorous diet as ancestral for is supported by our analyses comprising the most all Euthyneura. Carnivorous diet evolved at least extensive taxon sampling of all mentioned studies. five times independently according to our results (in Monophyly of Opisthobranchia is rejected in all our Nudipleura, Umbraculida, Pteropoda (Gymnosomata) analyses and based on several levels of evidence and twice in the Cephalaspidea). Furthermore, a (statistical tests, phylogenetic reconstruction, split generalization to unselective diet occurred randomly network analysis). across most clades. Monophyly of Pulmonata is generally accepted DISCUSSION based on morphological data (Tillier, 1984; Haszprunar, 1985; Nordsieck, 1992; Dayrat and Phylogeny of the Euthyneura Tillier, 2002). On the contrary, molecular data were not able to recover monophyly of this clade (Tillier The results of the present study confirm et al., 1996; Grande et al., 2004b, 2008; Knudsen monophyly of the Euthyneura while monophyly of et al., 2006; Klussmann-Kolb et al., 2008; Dinapoli the Opisthobranchia is strongly rejected. Hitherto, and Klussmann-Kolb, 2010; Jörger et al., 2010). The monophyly of Euthyneura was mainly based on results of the present study also support paraphyly of morphological analyses (Ponder and Lindberg, 1997; the Pulmonata. This is mainly due to the position of Dayrat and Tillier, 2002; Wägele and Klussmann- the supposedly opisthobranch taxon Acochlidiacea. Kolb, 2005) since members of this clade reveal Jörger et al. (2010) have proposed Acochlidiacea to be common features of the nervous (Haszprunar, 1985) closely related to Eupulmonata. This is confirmed by and reproductive system (Gosliner, 1981). Molecular our results. The position of the Sacoglossa which also systematic studies seldom revealed monophyly cluster in between the pulmonate taxa is unresolved. (Thollesson, 1999; Wade and Mordan, 2000; Knudsen They might represent the first offshoot of the whole et al., 2006); paraphyly was revealed more often, clade rendering Pulmonata (including Acochlidiacea) however mostly due to the position of clades with monophyletic. Inclusion of both Sacoglossa and uncertain systematic affinity like the Pyramidellidae Acochlidiacea into Pulmonata would support the (Grande et al., 2004b; Klussmann-Kolb et al., 2008; Panpulmonata-concept proposed by Jörger et al. Dinapoli and Klussmann-Kolb, 2010; Jörger et (2010).

136 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

The phylogenetic affinities of the Acteonoidea This early off split might also be the reason for have been a matter of debate for a long time. They were the high evolutionary rates present in Nudipleura either regarded as opisthobranchs mostly inhabiting a compared to all other taxa in this analysis resulting basal position (Gosliner, 1981, 1994; Ponder and in long branches in the phylogenetic tree and network Lindberg, 1997; Burn and Thompson, 1998; Dayrat analyses. If the Nudipleura split off early there and Tillier, 2002; Grande et al., 2004a, b; Vonnemann was plenty of time for accumulation of nucleotide et al., 2005; Klussmann-Kolb et al., 2008) or excluded changes. Unfortunately, this assumption cannot be into the “lower Heterobranchia” (Mikkelsen, 1996, underlined by the fossil record since the Nudibranchia 2002; Thollesson, 1999; Dayrat et al., 2001; Bouchet lack any fossils because of their missing shells and and Rocroi, 2005; Wägele and Klussmann-Kolb, the oldest known pleurobranchomorph fossil is only 2005; Dinapoli and Klussmann-Kolb, 2010; Jörger et dated back to about 26 Million years ago (Valdes al., 2010). The present study recovers the Acteonoidea and Lozouet, 2000). This suggests that this clade as sister group of the Rissoelloidea who are assigned is rather young, but, due to their small and delicate to the “lower Heterobranchia” (Bouchet and Rocroi, shells the fossil record of the Pleurobranchomorpha is 2005). Together both clades represent the sister likely incomplete (Valdes, 2004). Otherwise, the high group of the Euthyneura underlining the “lower evolutionary rates might hamper proper phylogenetic heterobranch” position of Acteonoidea outside but reconstruction and contribute to a basal placement of close to the Opisthobranchia. a derived clade. However, according to the results of the present study it seems reasonable to suggest that The first offshoot of the Euthyneura is represented the last common ancestor of Nudipleura separated by the opisthobranch clade Nudipleura. The Nudipleura early in evolution of euthyneuran gastropods and were established in 2000 by Wägele and Willan and that Nudipleura represent a single offshoot without a are composed of the sister taxa Nudibranchia and specific sister taxon. Pleurobranchomorpha. Monophyly of the Nudipleura as well as of Nudibranchia and Pleurobranchomorpha A clade composed of Umbraculida, Cephalaspidea, is supported by maximum statistical support values Aplysiomorpha and Pteropoda is the only monophyletic and considerable split support and is in accordance clade uniting several opisthobranch taxa; all other with former studies (Vonnemann et al., 2005; Wägele clades (Nudipleura, Sacoglossa and Acochlidiacea) and Klussmann-Kolb, 2005; Klussmann-Kolb et al., cluster separately. The clade of Umbraculida, 2008; Dinapoli and Klussmann-Kolb, 2010; Göbbeler Cephalaspidea, Aplysiomorpha and Pteropoda and Klussmann-Kolb, 2010; Jörger et al. 2010). has also been found in other molecular systematic analyses (Klussmann-Kolb et al., 2008; Dinapoli and However, the basal position of this morphologically Klussmann-Kolb, 2010; Göbbeler and Klussmann- derived clade is somewhat surprising although it has Kolb, 2010; Jörger et al., 2010) while it could not been revealed in most molecular systematic studies. be revealed in morphological analyses (Wägele and Nudipleura either cluster as sister group to the possibly Klussmann-Kolb, 2005) since common characters “lower heterobranch” clade Acteonoidea (Grande et are missing (Klussmann-Kolb and Dinapoli, 2006). al., 2004a, b; Vonnemann et al., 2005; Klussmann- However, Jörger et al. (2010) proposed to erect the Kolb et al., 2008) or represent the single first offshoot taxon Euopisthobranchia for these opisthobranchs and of Euthyneura like in the present study (Dinapoli proposed the presence of a gizzard as the unifying and Klussmann-Kolb, 2010; Jörger et al., 2010). synapomorphy. These results suggest that the last common ancestor of the Nudipleura evolved early as sister taxon to The Cephalaspidea are rendered paraphyletic in the last common ancestor of all other Euthyneura. our analyses due to the exclusion of the Runcinacea

137 Katrin Göbbeler & Annette Klussmann-Kolb

Table 3. Information on dietary of investigated species coded as carnivorous, herbivorous or

unspecific.Table Literature 3: Information sources on dietary for dietaryof investigated information species coded provided. as carnivorous, ‐ = unknown. herbivorous or unspecific. Literature sources for dietary information provided. - = unknown

Carnivorous Herbivorous Unselective Reference

CAENOGASTROPODA

Littorina littorea algae Lubchenco, 1978

LOWER HETEROBRANCHIA

Orbitestella sp. unicellular plants, Ponder and de Keyzer, detritus 1998 Cima sp. ‐

Rissoella rissoaformis Bacillariophyceae, Fretter, 1948 algal filaments, detritus ACTEONOIDEA

Acteon tornatilis Polychaeta Yonow, 1989

Pupa nitidula Polychaeta Rudman, 1972a

Hydatina physis Polychaeta Rudman, 1972b

Micromelo undata Polychaeta Burn and Thompson, 1998 Bullina lineata Polychaeta Taylor, 1986

OPISTHOBRANCHIA

CEPHALASPIDEA

BULLOIDEA

Bulla striata algae Malaquias et al., 2009a

DIAPHANOIDEA

Diaphana sp. ‐

Toledonia globosa ‐

HAMINOEOIDEA

Haminoea hydatis algae Malaquias et al., 2009a

Atys cylindricus algae Helbling, 1779

Smaragdinella sp. algae Rudman, 1972c

PHILINOIDEA

Scaphander lignarius Foraminifera, Hurst, 1965 Polychaeta, Bivalvia, Gastropoda, Crustacea, Echinodermata

138 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

Philine aperta Bivalvia Hansen, 1991

Odontoglaja sp. Polychaeta, Bivalvia Rudman, 1978

Philinopsis pilsbryi Opisthobranchia Rudman, 1972d

Sagaminopteron Porifera Becerro et al., 2006 psychedelicum Philinoglossa praelongata ‐

Retusa sp. Foraminifera, small Burn and Thompson, Mollusca 1998 Pyrunculus sp. Foraminifera, small Burn and Thompson, Mollusca 1998 RUNCINACEA

Runcina africana algae Rudman, 1971

Ilbia ilbi algae Rudman, 1971

APLYSIOMORPHA

AKEROIDEA

Akera bullata algae Hayward et al., 1990

APLYSIOIDEA

Aplysia californica algae Carefoot ,1981

Dolabrifera dolabrifera algae Willan, 1998a

Bursatella leachii algae Paige, 1988

Dolabella auricularia algae Pennings et al., 1993

Stylocheilus longicauda algae, Cyanobacteria Nagle et al., 1998

Petalifera petalifera algae Willan, 1998a

PTEROPODA

THECOSOMATA

Cavolinia uncinnata Bacillariophyceae, Newman, 1998 Dinoflagellata, Foraminifera, Radiolaria, zooplankton, phytoplankton Hyalocylis striata Bacillariophyceae, Boltovskoy, 1975 Dinoflagellata, Foraminifera, Radiolaria, zooplankton, phytoplankton Clio pyramidata Bacillariophyceae, Newman, 1998 Dinoflagellata, Foraminifera, Radiolaria,

139 Katrin Göbbeler & Annette Klussmann-Kolb

zooplankton, phytoplankton Cuvierina columnella Bacillariophyceae, Newman, 1998 Dinoflagellata, Foraminifera, Radiolaria, zooplankton, phytoplankton GYMNOSOMATA

Pneumoderma atlantica zooplankton Pafort‐van Iersel, 1985

Spongiobranchaea australis carnivorous Richter, 1977

UMBRACULIDA

Umbraculum umbraculum Porifera Willan, 1984

Umbraculum sp. Porifera Willan, 1984

Tylodina perversa Porifera Cyanobacteria Becerro et al., 2003 (nutritional value unclear, coded as “unknown”) Tylodina fungina Porifera Gabb, 1865

ACOCHLIDIACEA

Unela glandulifera microorganisms Burn, 1998a

Pontohedyle milatchevitchi microorganisms Burn, 1998a

SACOGLOSSA

OXYNOACEA

Oxynoe antillarum algae Morch, 1863

Lobiger viridis algae Morch, 1863

PLACOBRANCHACEA

Elysia viridis algae Thompson, 1976

Placobranchus ocellatus algae Burn, 1998b

Bosellia mimetica algae Marcus, 1978

Limapontia nigra algae Thompson, 1976

CYLINDROBULLIDA

Cylindrobulla beauii algae Burn, 1998b

NUDIPLEURA

NUDIBRANCHIA

DEXIARCHIA

140 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

Armina lovenii Cnidaria Thompson, 1988

Flabellina verrucosa Cnidaria Thompson and Brown, 1984 Eubranchus exiguus Cnidaria Thompson, 1988

Dendronotus dalli Cnidaria Bergh, 1879

ANTHOBRANCHIA

Bathydoris clavigera omnivorous, e.g. Wägele, 1989 Foraminifera, Porifera, Cnidaria, Echinodermata, Polychata, Mollusca Hypselodoris infucata Porifera Fontana, 1993

Chromodoris krohni Porifera Rudman and Bergquist, 2007 Hoplodoris nodulosa ‐

Austrodoris kerguelenensis Porifera Wägele, 1989

Goniodoris nodosa Bryozoa, Ascidiacea Thompson, 1988

Acanthodoris pilosa Bryozoa Müller, 1788

Limacia clavigera Bryozoa Müller, 1776

PLEUROBRANCHOMORPHA

Tomthompsonia antarctica (non‐selective) Hain et al., 1993 benthic deposit, e.g. Bacillariophyceae, Radiolaria, Porifera, Bryozoans Pleurobranchus peroni Ascidiacea Cuvier, 1804

Berthellina citrina Porifera Willan, 1984

Pleurobranchaea meckeli Cnidaria, Porifera, Cattaneo‐Vietti et al., Polychata, Mollusca 1993

PULMONATA

SIPHONARIOIDEA

Siphonaria capensis algae Maneveldt, 2006

Siphonaria concinna algae Gray, 1997

AMPHIBOLOIDEA

Phallomedusa solida detritus, Schacko, 1878 Bacillariophyceae Amphibola crenata detritus Stanisic, 1998a

HYGROPHILA

Chilina sp. microalgae Brace, 1983

141 Katrin Göbbeler & Annette Klussmann-Kolb

Lymnaea stagnalis detritus, carrion Sterry, 1997

Acroloxus lacustris Bacillariophyceae, Dillon, 2000 algae, rotted plants Latia neritoides Bacillariophyceae, Meyer‐Rochow and detritus Moore, 1988

Bulinus tropicus detritus, green algae, Madsen, 1992 Bacillariophyceae EUPULMONATA

OTINOIDEA

Otina ovata ‐

Smeagol phillipensis ‐

ELLOBIOIDEA

Ophicardelus ornatus algae Ross et al., 2009

SYSTELLOMMATOPHORA

Onchidella floridana algae Stanisic, 1998b

Onchidium verrucosum algae Stanisic, 1998b

STYLOMMATOPHORA

Arion silvaticus deadwood, detritus Kerney and Cameron, 1979 Arianta arbustorum plants Hägele, 2001

Helix aspersa plants Iglesias, 1999

Deroceras reticulatum plants Schley and Bees, 2003

which are commonly assigned to this taxon (Burn and Malaquias et al. (2009b) is the sister taxon to Runcina Thompson, 1998; Bouchet and Rocroi, 2005) and have africana of the Runcinidae rendering the Runcinacea been revealed as sister group to all Cephalaspidea monophyletic. Another controversial cephalaspidean (Grande et al., 2004a, b; Vonnemann et al., 2005). family is the Diaphanidae which has been excluded Malaquias et al. (2009b) performed extensive analyses from the Cephalaspidea and assigned to different on cephalaspidean phylogeny recovering exclusion of clades several times (e.g. Haszprunar, 1985; Salvini- Runcinacea from the Cephalaspidea in all analytical Plawen and Steiner, 1996; Jensen, 1996a). According attempts. Thus, he proposed (p. 36) that “Runcinacea to our results the Diaphanidae are definitely part of should be reinstated as a distinct taxonomic category the Cephalaspidea since they share split support and of equivalent rank to Cephalaspidea s.s.”; a postulation receive maximum statistical support values in tree supported by the present study. Additionally, the reconstruction. They are recovered basal to all other current study is the first to include more than one cephalaspidean taxa included in the present study genus of Runcinacea. Ilbia ilbi as member of the which is in congruence with the results of Malaquias Ilbiidae which were classified as incertae sedis by et al. (2009b).

142 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

The only statistically supported grouping in this represent the first offshoot in this clade while clade of Euopisthobranchia unites Aplysiomorpha Sacoglossa form the sister group to the third subclade. and Pteropoda. In this clade the aplysiomorph However, these interrelationships did not receive Akera bullata is found as the most basal offshoot statistical support. Similar though more or less which is consistent with former studies (Medina unresolved interrelationships were also revealed in and Walsh, 2000; Vonnemann et al., 2005; Wägele a study by Dinapoli and Klussmann-Kolb (2010) and Klussmann-Kolb, 2005) while the remaining while Klussmann-Kolb et al. (2008) and Jörger et Aplysiomorpha form the sister group to the Pteropoda al. (2010) recovered a sister group relationship of rendering the Aplysiomorpha paraphyletic. In contrast Siphonarioidea and Sacoglossa together representing to this, the split network analysis reveals good support the sister group to the third subclade. Phylogenetic for monophyletic Aplysiomorpha and monophyly of affinities of the Sacoglossa have also been a this clade has not been doubted in most phylogenetic matter of debate from morphological perspectives. analyses before (Thollesson, 1999; Medina and Walsh, Sacoglossa could hardly be linked to any other extant 2000; Dayrat and Tillier, 2002; Grande et al., 2004a, opisthobranch taxon (besides Cylindrobullida), since b; Vonnemann et al., 2005; Wägele and Klussmann- any apparent synapomorphy might be explained as Kolb, 2005; Klussmann-Kolb et al., 2008). Hence, this parallel evolution (Jensen, 1996b). result needs to be considered with caution and needs further investigation involving different markers and According to the present study the Sacoglossa possibly more taxa. incorporate Cylindrobulla beauii. This taxon was supposed to be part of a separate monogeneric The common ancestry of Aplysiomorpha and “group” called Cylindrobullida before (Bouchet and Pteropoda as found in the present study has already Rocroi, 2005) which was regarded as the sister group been revealed in a molecular systematic study of of the Sacoglossa (Jensen, 1996b). C. beauii clusters Dayrat et al. (2001) and was also recovered in along with the oxynoacean taxa included in this an extensive study on pteropodan phylogeny study receiving both maximum statistical support in (Klussmann-Kolb and Dinapoli, 2006). Furthermore, tree reconstruction and considerable split support in monophyly of Pteropoda comprising Gymnosomata network analysis. The latter has also been found in a and Thecosomata as sister taxa has been revealed molecular systematic study on Sacoglossan phylogeny in molecular systematic studies before (Klussmann- (Händeler and Wägele, 2006) as well as in the Kolb and Dinapoli, 2006; Klussmann-Kolb et al., most recent broad euthyneuran study performed by 2008, Jörger et al., 2010) and is confirmed in the Jörger et al. (2010). Furthermore, Cylindrobullida was current study. Morphological studies and traditional supposed to belong to the Oxynoacea in morphology- classifications by Thiele (1931), Hoffmann (1939) and based analyses (Mikkelsen, 1996, 2002). Thus, we Odhner (1939) also support monophyly of Pteropoda. suggest that the Cylindrobullida do not form a separate clade but possibly represent a subclade of sacoglossan The third main clade revealed in our analyses Oxynoacea. Furthermore, the divison of Sacoglossa in is composed of the Pulmonata along with the two main subclades (Oxynoacea and Plakobranchacea) opisthobranch clades Sacoglossa, Cylindrobullida recovered in the present study is in accordance with and Acochlidiacea. Jörger et al. (2010) proposed the former classifications (Jensen, 1996b; Bouchet and new clade Panpulmonata to unite these taxa. This Rocroi, 2005; Händeler and Wägele, 2006; Händeler clade is further divided into three main subclades: et al., 2009) and receives high statistical support Siphonarioidea, Sacoglossa and a statistically in tree reconstruction. However, split support for supported clade of Hygrophila, Amphiboloidea, Sacoglossa is missing and only present for the two Acochlidiacea and Eupulmonata. Siphonarioidea subclades.

143 Katrin Göbbeler & Annette Klussmann-Kolb

Table 4. Detailed results of the BayesTraits analyses. Arithmetic means of posterior probabilities (rounded to two decimal places) of the different diet preferences of last common ancestor of the Table 4: diverseDetailed clades results are of theprovided. BayesTraits The analyses. highest Arithmetic values formeans each of posterior clade are probabilities highlighted (rounded in bold. to two decimal places) of the different diet preferences of last common ancestor of the diverse clades are provided. The highest values for each clade are highlighted in bold.

Clade Carnivorous Herbivorous Unselective

Acteonoidea/Euthyneura 0.21 0.77 0.02

Euthyneura 0.14 0.85 0.01

Euthyneura without Nudipleura 0.10 0.89 0.01 Umbraculida/Cephalaspidea/Aplysiomorpha/Pteropoda 0.21 0.79 0 (Euopisthobranchia) Pulmonata/Sacoglossa/Acochlidiacea (Panpulmonata) 0 0.98 0.02

Acteonoidea/Rissoelloidea 0.82 0.10 0.08

Acteonoidea 1 0 0

Nudipleura 0.85 0.08 0.07

Umbraculida 0.99 0.01 0

Cephalaspidea without Runcinacea 0.24 0.73 0.03

Cephalaspidea/Aplysiomorpha/Pteropoda 0.09 0.90 0.01

Runcinacea/Aplysiomorpha/Pteropoda 0 0.99 0.01

Aplysiomorpha/Pteropoda 0 1 0

Aplysioidea 0 1 0

Aplysioidea/Pteropoda 0.01 0.98 0.01

Pteropoda 0.62 0.12 0.26

Sacoglossa 0 0.99 0.01

Sacoglossa/Hygrophila /Amphiboloidea/Acochlidiacea/Eupulmonata 0 0.89 0.11

Hygrophila 0 0.74 0.26

Hygrophila/Amphiboloidea/Acochlidiacea/Eupulmonata 0 0.67 0.33

Amphiboloidea/Acochlidiacea/Eupulmonata 0 0.68 0.32

Acochlidiacea/Eupulmonata 0.01 0.82 0.17

Eupulmonata 0 0.99 0.01

The subclade composed of Hygrophila, affinity to pulmonate taxa has repeatedly been Amphiboloidea, Acochlidiacea and Eupulmonata proposed before (Vonnemann et al., 2005; Klussmann- receives good statistical support. However, Kolb et al., 2008). In contrast to this, morphological interrelationships of these groups remain partly investigations reveal Acochlidiacea as sister group unresolved. Monophyly of the single subclades is to part of (polyphyletic) Cephalaspidea within other strongly supported in phylogenetic reconstruction. opisthobranch clades (Wägele and Klussmann- Furthermore, a sister group relationship of the Kolb, 2005) or regard them as sister group of the Acochlidiacea and the Eupulmonata receives a Sacoglossa (Gosliner and Ghiselin, 1984). However, posterior probabilities value of 0.94 which is close within these different clades Acochlidiacea group to the significance level of 0.95. This relationship with other mesopsammic taxa suggesting convergent supports the findings by Jörger et al. (2010) and an adaptions to this special habitat might mask the

144 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

phylogenetic signal (Schrödl and Neusser, 2010). features for the evolution of this clade (Thompson, Monophyly of Acochlidiacea is strongly supported 1976; Rudman and Willan, 1998; Mikkelsen, 2002; in our tree reconstruction analysis and has also Wägele, 2004). Major evolutionary radiations of been revealed in former studies (Vonnemann et opisthobranchs are connected to habitat and diet al., 2005; Wägele and Klussmann-Kolb, 2005; (Rudman and Willan, 1998; Wägele, 2004). The Klussmann-Kolb et al., 2008). Surprisingly both occupation of diverse feeding niches enabled by included taxa of Acochlidiacea cluster far apart in development of particular morphological structures our network analyses. Unela glandulifera clusters for dietary specialization is even considered as among eupulmonate taxa resembling placement the “driving force” of opisthobranch evolution of the whole clade in tree reconstruction, while (Thompson, 1976; Mikkelsen, 2002). Pontohedyle milatchevitchi shares spilt support with Gymnosomata. Nevertheless, Acochlidiacea might Nevertheless up to now, assessment of their be monophyletic and share split support which is trophic relationships was mainly based on scattered not revealed in the graphical output of the split data (Malaquias et al., 2009a). We conducted a network analyses showing a two dimensional picture thorough literature search to compile information on of relationships of diverse taxa. Furthermore, the dietary sources of a great variety of species in order to Acochlidiacea have never been associated with the reconstruct the ancestral diet preferences of this clade. Pteropoda, so that we would regard the latter result Opisthobranch gastropods reveal highly specialized as erroneous and still consider the Acochlidiacea (as feeding habits which may be classified into different well as the Pteropoda) as monophyletic. categories regarding their size, feeding mode or type of food. This study focuses on reconstructing The Eupulmonata consist of Stylommatophora, dietary preferences based on a division in herbivorous Systellommatophora, Ellobioidea and Otinoidea versus carnivorous sources, additionally coding for (Bouchet and Rocroi, 2005). Several molecular unselective food items. The reconstruction is based systematic studies support their monophyly (Tillier et on a robust molecular phylogenetic hypothesis for the al., 1996; Wade and Mordan, 2000; Klussmann-Kolb respective taxa. et al., 2008; Dinapoli and Klussmann-Kolb, 2010) including the present one. Moreover, monophyly Our molecular systematic analyses unambiguously of Stylommatophora is revealed in the present revealed polyphyly of Opisthobranchia; therefore, investigation which is in accordance with several we decided to reconstruct the evolution of diet for other studies (e.g. Tillier et al., 1996; Wade and monophyletic Euthyneura and all main subclades Mordan, 2000; Dayrat and Tillier, 2002; Grande et al., separately. 2004b, 2008; Klussmann-Kolb et al., 2008; Dinapoli and Klussmann-Kolb, 2010). The Stylommatophora The ancestral gastropod in general is envisaged as form the sister-group of a clade composed of unselective (Vermeij and Lindberg, 2000). Carnivory Systellommatophora, Ellobioidea and Otinoidea, as well as herbivory might have arisen out of this which is congruent with the results of Dinapoli and unspecific grazing by progressively selecting plant or Klussmann-Kolb (in 2010) as well. animal components of the food (Fretter et al., 1998). In Evolution of diet contrast to this, our results strongly support herbivory as the ancestral state implying that carnivory evolved Ancestral diet preferences of Opisthobranchia several times independently. Furthermore, the ancestral have been a matter of debate because the evolution of Euthyneuran was possibly not an unselective grazer. special nutrition strategies as well as the specialization Inclusion of the dubious opisthobranch subclade on particular food items were discerned to be crucial Acteonoidea and its sister group Rissoelloidea does

145 Katrin Göbbeler & Annette Klussmann-Kolb

not alter the overall tendency towards an herbivorous major marine animal phyla, except Echinodermata last common ancestor. At first sight, this result (Willan, 1998b). This further specialization probably might seem to contradict parsimonious principles contributes to the species richness especially in the because Acteonoidea, Nudipleura, and Umbraculida Nudibranchia subclade (~3000 species; Wägele and (three basal clades of Euthyneura) are carnivores. Klussmann-Kolb, 2005). Several different diet-related Thus, it would seem to be more parsimonious to specializations have occurred in Nudibranchia. suggest carnivory as the ancestral diet preference. The nudibranch subclade Aeolidoidea (member Vermeij and Lindberg (2000) claim that the ancestral of Dexiarchia) is able to store cnidocysts of their gastropod was an unselective grazer by counting cnidarian prey and use them for their own defence evolutionary transitions from microphagy-carnivory (Kälker and Schmekel, 1976). This yielded to an to herbivory and vice versa assuming the smaller increase in food sources, since cnidarian colonies number to represent the more parsimonious pattern of could be fully explored and resulted in diversification transition. We applied their method to our phylogeny to more than 500 species in this subclade (Wägele, and counted evolutionary transitions from one state 2004). Other taxa of Aeolidoidea live in mutualistic to the other two in all possible directions only symbiosis with photosynthetic dinoflagellates and considering major clades. These analyses confirm make use of their metabolites (Burghardt et al. herbivory as the most parsimonious ancestral feeding 2005). This specialization may also account for mode, because the fewest evolutionary steps are the higher number of species in the respective taxa necessary for transition from herbivory to carnivory (Wägele, 2004). The Chromodorididae (a taxon as well as microphagy based on our phylogenetic of the nudibranch Anthobranchia) display a clear hypothesis. A possible explanation for our results is pattern of food specificity at both genus and species that carnivorous species occur more clustered than level (Rudman and Bergquist, 2007). They further herbivores which are widespread all over clades. If incorporate distasteful and anti-feedant secondary species revealing the same preferences are grouped metabolites of their prey into their skin or even into together, it takes only one evolutionary step to special organs (MDF-mantle dermal formations) for switch from one state to another; whereas, multiple their own protection (Wägele, 2004; Wägele et al., transitions are necessary, if species occur separated 2006; Rudman and Bergquist, 2007). The evolution from each other. Thus, carnivory is not the most of these special mantle glands is regarded as the key possible ancestral state, although carnivorous clades factor for extensive radiation of this clade, since the are predominantly found at the base of the tree. Based storage in special organs might have led to feeding on on our phylogeny, herbivory is clearly preferred as the even more toxic sponges enhancing food resources ancestral mode of feeding for Euthyneura. (Wägele, 2004).

The last common ancestors of the Acteonoidea/ In the future, it would be interesting to evaluate Rissoelloidea clade as well as of Nudipleura if radiation of Nudipleura coincided with radiation (representing the first offshoots in our study) of some of their food items implying that Nudipleura independently switched to a carnivorous life style. immediately adapted to existence of new sources. At While the Acteonoidea specialized on polychaetes the moment, such an approach is hampered by the fact as a definite food item, the species-rich Nudipleura that the age of the Nudipleura is unknown due to rare further specialized on a variety of different and fossil record and large credible intervals (~260-110 seldom selected food sources of animal origin Mya / Dinapoli and Klussmann-Kolb, 2010; ~175-75 like Porifera, Cnidaria, Bryozoa and Ascidiacea Mya / Göbbeler and Klussmann-Kolb, 2010, ~260-115 which are partly unpleasant or toxic for most other Mya / Jörger et al. 2010) in the only molecular clock predators. In fact nudibranch diet encompasses all analyses incorporating this taxon.

146 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

The last common ancestor of Umbraculida, The Aplysiomorpha and Pteropoda form a clade Cephalaspidea, Aplysiomorpha, and Pteropoda with herbivory as strongly supported ancestral diet, (Euopisthobranchia) possibly was herbivorous however ancestral Pteropoda expanded their food although the (statistically not significant) first sources. Carnivory is here favoured as the ancestral offshoot is represented by the definitely carnivorous diet, but herbivory and unselective sources cannot be Umbraculida. excluded. Thus, the last common ancestor of the whole clade possibly grazed on algae like extant aplysiomorph The Cephalaspidea, Aplysiomorpha, and species and change to pelagic life style in Pteropoda Pteropoda are a morphologically well defined was followed by exploration of new food sources. It is taxon sharing an apomorphic feature related to diet: possibly not effective to be restricted to a certain food the muscular oesophageal gizzard with gizzard source if living in the pelagial with limited abilities of plates (Klussmann-Kolb and Dinapoli, 2006). The self navigation. Otherwise, the Thecosomata possess a possession of the gizzard with plates is probably gizzard which is probably related to herbivorous diet related to herbivory displaying the plesiomorphic preferences (Klussmann-Kolb and Dinapoli, 2006). condition within this clade (Wägele, 2004; Thus, herbivory might be the ancestral condition for Klussmann-Kolb and Dinapoli, 2006), an assumption all Pteropoda and Gymnosomata changed to carnivory supported by the present study. The structure of the accompanied by loss of the gizzard. gizzard differs among the taxa, it consists of at least ten larger plates in the Aplysiomorpha, whereas the The Aplysiomorpha also exhibit special Runcinacea and Thecosomata possess four plates adaptations to their prey. Some taxa produce a purple and a further reduction to three plates has occurred “ink” out of pigments ingested with the algae they in the Bulloidea and other Cephalaspidea. Wägele feed upon used to deter predators (Willan, 1998a). (2004) assumed that evolution of three large plates Most species sequester secondary metabolites of allowed a higher diversification through exploration algae in their digestive gland; however if these are of different kinds of food causing higher diversity in used for defensive behavior (Wägele et al., 2006) or the bulloid Cephalaspideans (~260 species) than in the not (Pennings, 1994) is disputed. Aplysiomorpha (~75 species). The last common ancestor of Pulmonata, The last common ancestor of the Cephalaspidea Sacoglossa and Acochlidiacea probably also was (without Runcinacea) was possibly also an herbivore. an herbivore. However, there seems to be a trend to Cephalaspideans feed on a variety of different unselective feeding especially in freshwater species. food items and carnivory has possibly evolved at least twice during evolution of this clade which The Sacoglossa are highly specialized algal is in accordance with a former character tracing feeders and the evolution of a special tooth for analysis in Cephalaspidea (Malaquias et al., 2009a). cutting algal cell walls is regarded as essential Both herbivorous (Bulloidea) and carnivorous for their radiation (Wägele, 2004; Händeler et al., (Philinoidea) subclades display rich species diversity 2009). Moreover, some sacoglossans are able to store (about 260 and 500 species respectively/Wägele, chloroplasts of their food items and make use of the 2004) underlining evolutionary success of both produced metabolites (Rumpho et al., 2000), which groups specializing in different diets. Both feeding is a possible further key character for evolution of strategies benefit from the evolution of a highly this taxon (Wägele, 2004; Händeler et al., 2009). Our specialized gizzard with three plates displaying the results suggest that the last common ancestor of the key character for higher diversity in Cephalaspidea Sacoglossa already fed upon algae and developed the (Wägele, 2004). specialized features enabling radiation of the clade.

147 Katrin Göbbeler & Annette Klussmann-Kolb

In summary, our study demonstrates that the characters and their evolution in Euthyneura is diverse dietary strategies of the opisthobranch necessary to fully understand the evolution of this subclades most probably evolved from an herbivorous taxon. Moreover, disentangling the interrelationships ancestral state and were established in the different of the well defined subclades of Euthyneura needs lineages. This contradicts previous assumptions that further research. For this purpose new marker genes carnivory was the plesiomorphic feeding preference are irreplaceable since resolution is too low with the in Opisthobranchia (Haszprunar, 1985). Furthermore, conventional ones. the results of the present study indicate that interactions of prey structure, habitat, and anatomy Acknowledgements might not have had a strong influence on phylogeny of the whole clade, but were probably accounting for The German Academic Exchange Service the diversification within the subclades as already (DAAD) financially supported a collecting trip to claimed for the Cephalaspidea (Malaquias et al., Australia for the first author. Permission for collecting 2009a). was given by the NSW Department of Primary Industries (permit number p07/0058). Conclusions Georg Mayer (Melbourne), John Healy (Brisbane) and Angela Dinapoli (Frankfurt) provided help during Our phylogenetic analyses yield some intriguing the field trip to Australia. insights into the phylogeny of euthyneuran gastropods. DNA samples or specimens were provided by Heike Wägele and Katharina Händeler (Bonn), The Opisthobranchia are undoubtedly rendered Michael Schrödl and Enrico Schwabe (Munich) as polyphyletic. The single monophyletic lineages of well as Patrick Krug (Los Angeles). Nudipleura, Sacoglossa and Acochlidiacea evolved Heike Wägele (Bonn) and Michael Schrödl more or less independently, while the Umbraculida, (Munich) made valuable comments on an earlier Cephalaspidea, Aplysiomorpha and Pteropoda version of this manuscript. Jan Müller (Frankfurt) (Euopisthobranchia) form a well supported clade. assisted in literature research of dietary preferences. The second author is supported by the Biodiversity Herbivory as most likely ancestral diet of all and Climate Research Centre (BiK-F), Frankfurt/Main. Euthyneura is clearly supported by our analyses implying that carnivory evolved several times REFERENCES independently in the different subclades. While the exploration of new diets coincide with adaptive Becerro MA, Turon X, Uriz MJ, Templado J (2003). Can radiation in some clades, like carnivory in Nudipleura a sponge feeder be a herbivore? Tylodina perversa or different herbivorous as well as carnivorous food (Gastropoda) feeding on Aplysina aerophoba items in Cephalaspidea, species numbers of other (Demospongiae). Biological Journal of the Linnean clades, like the Umbraculida which also changed to Society, 78: 429-438. carnivorous diet, remained limited. Becerro MA, Starmer JA, Paul VJ (2006). Chemical defense of cryptic and aposematic gasteroptid molluscs feeding Thus, it seems possible that dietary preferences on their host sponge Dysidea granulosa. Journal of along with exploration of new diet resources might Chemical Ecology, 32: 1491-1500. have supported the evolutionary success of different Bergh LSR (1879). On the nudibranchiate gasteropod lineages. However, diet is clearly only one of probably Mollusca of the North Pacific Ocean with special many features responsible for the success of a certain reference to those of Alaska. Scientific Results clade and a closer look at other possibly important Exploration Alaska 1: 127-188.

148 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

Bieler R (1992). Gastropod phylogeny and systematics. Dayrat BT, Tillier S (2002). Evolutionary relationships Annual Reviews of Ecology and Systematics, 23: 311- of euthyneuran gastropods (Mollusca): a cladistic 338. re-evaluation of morphological characters. Zoological Boltovskoy D (1975). Ecological aspects of zooplankton Journal of the Linnean Society, 135: 403-470. (Foraminifera, Pteropoda and Chaetognatha) of the Dillon RT jr ( 2000). The ecology of fresh water molluscs. southwestern Atlantic Ocean. Veliger, 18: 203-216. University Press, Cambridge. Bouchet P, Rocroi JP (2005). Classification and nomenclator Dinapoli A, Klussmann-Kolb A (2010). The long way of gastropod families. Malacologia, 47: 1-397. to diversity – phylogeny and evolution of the Brace RC (1983). Observations on the morphology and Heterobranchia (Mollusca: Gastropoda). Molecular behaviour of Chilina fluctuosa Gray (Chilinidae), with a Phylogenetics and Evolution, 55: 60-76. discussion on the early evolution of pulmonate gastropods. Farris JS, Källersjö M, Kluge AG, Bult C (1995). Testing Philosophical Transactions of the Royal Society of significance of incongruence. Cladistics, 10: 315-319. London, Series B-Biological Sciences, 300: 463-491. Fontana A (1993). Defensive allomones in three species Burghardt I, Evertsen J, Johnsen G, Wägele H (2005). Solar of Hypselodoris (Gastropoda: Nudibranchia) from the powered seaslugs - Mutualistic symbiosis of aeolid Cantabrian Sea. Journal of Chemical Ecology, 19: Nudibranchia (Mollusca, Gastropoda, Opisthobranchia) 339-356. with Symbiodinium. Symbiosis, 38: 227-250. Fretter V (1948). The structure and life history of some Burn R (1998a). Order Acochlidea. In: PL Beesley, GJB minute prosobranchs of rock pools: Skeneopsis Ross, A Wells, Eds, Mollusca: The Southern Synthesis, planorbis (Fabricius), Omalogyra atomus (Phillipi), CSIRO Publishing, Melbourne, 959-960. Risoella diaphana (Adler) and Rissoella opalina Burn R (1998b). Order Sacoglossa. In: PL Beesley, GJB (Jeffreys). Journal of the Marine Biological Association Ross, A Wells, Eds, Mollusca: The Southern Synthesis, of the UK, 27: 597-632. CSIRO Publishing, Melbourne, 961-974. Fretter V, Graham A, Ponder WF, Lindberg DR (1998). Burn R, Thompson TE (1998). Order Cephalaspidea. In Prosobranchs. Introduction. In: PL Beesley, GJB Ross, PL Beesley, GJB Ross, A Wells, Eds, Mollusca: The B Wilson, Eds. Mollusca. The Southern Synthesis. Southern Synthesis, CSIRO Publishing, Melbourne, SCIRO Publishing, Melbourne, 605-638. 943-959. Gabb WM (1865). Description of new species of marine Carefoot TH (1981). Nutrition of a coral rubble-inhabiting shells from the coast of California. Proceedings of the herbivore, the sea hare Aplysia. Proceedings of the Californian Academy of Science, 3: 182-190. Fourth International Coral Reef Symposium, Manila, Göbbeler K, Klussmann-Kolb A (2010). Out of Antarctica? 2: 665-669. - New insights into the phylogeny and biogeography Cattaneo-Vietti R, Burlando B, Senes L (1993). Life history of the Pleurobranchomorpha (Mollusca, Gastropoda). and diet of Pleurobranchaea meckelii (Opisthobranchia: Molecular Phylogenetics and Evolution, 55: 996-1007. Notaspidea). Journal of Molluscan Studies, 59: 309-313. Gosliner TM (1981). Origins and relationships of Cuvier GL (1804). Mémoire sur la Phyllidie et sur le primitive members of the Opisthobranchia (Mollusca: Pleurobranche, deux nouveaux genres de mollusques Gastropoda). Biological Journal of the Linnean Society, de l’ordre des gastéropodes, et voisins des patelles 16: 197-225. et des oscabrions, dont l’un est nu dont l’autre porte Gosliner TM, Ghiselin MT (1984). Parallel evolution in une coquille cachée. Annales du Muséum National opisthobranch gastropods and its implications for d’Histoire Naturelle Paris, 5: 266-276. phylogenetic methodology. Systematic Biology, 33: Dayrat B, Tillier A, Lecointre G, Tillier S (2001). New 255-247. clades of euthyneuran gastropods (Mollusca) from Gosliner TM (1985). Parallelism, parsimony and the testing 28S rRNA sequences. Molecular Phylogenetics and of phylogenetic hypotheses: the case of opisthobranch Evolution, 19: 225-235. gastropods. In: ES Vrba, Ed, Species and speciation.

149 Katrin Göbbeler & Annette Klussmann-Kolb

Transvaal Museum Monograph No. 4, Transvaal morphology of the velar structures. Marine Biology, Museum, Pretoria, 105-107. 111: 263-270. Gosliner TM (1991). Morphological parallelism in Haszprunar G (1985). The Heterobranchia - a new concept opisthobranch gastropods. Malacologia, 32: 313-327. of the phylogeny of the higher Gastropoda. Zeitschrift Gosliner TM (1994). Gastropoda: Opisthobranchia. In: für zoologische Systematik und Evolutionsforschung, FW Harrison, AJ Kohn, Eds, Microscopic Anatomy of 23: 15-37. Invertebrates, Vol. 5: Mollusca I, Wiley-Liss, Inc., New Hayward PJ, Wigham GD, Yonow N (1990). Mollusca I: York, 253-355. Polyplacophora, Scaphopoda, and Gastropoda. In: PJ Grande C, Templado J, Cervera JL, Zardoya R (2004a). Hayward, JS Ryland, Eds, The Marine Fauna of the Phylogenetic relationships among Opisthobranchia British Isles and North-West Europe, Clarendon Press, (Mollusca: Gastropoda) based on mitochondrial cox Oxford, 628-730. 1, trnV and rrL genes. Molecular Phylogenetics and Helbling GS (1779). Bulla cylindrica. In: Ignaz Edlen Evolution, 33: 378-388. von Born, Ed, Abhandlungen einer Privatgesellschaft Grande C, Templado J, Cervera JL, Zardoya R (2004b). in Böhmen zur Aufnahme der Mathematik, der Molecular Phylogeny of Euthyneura (Mollusca: väterlandischen Geschichte, und der Naturgeschichte, Gastropoda). Molecular Biology and Evolution, 21: Vol 4, 122. 303-313. Hoffmann H (1939). Opisthobranchia. In: HG Bronns, Ed, Grande C, Templado J, Zardoya R (2008). Evolution of Klassen und Ordnungen des Tierreichs, Bd 3, Abt 2, gastropod mitochondrial genome arrangements. BMC Buch 3, Akademische Verlagsgesellschaft, Leipzig. Evolutionary Biology, 8: 61. Huelsenbeck JP, Ronquist F (2001). MrBAYES; Bayesian Gray DR (1997). Temporal variation in foraging behaviour of inference for phylogeny. Bioinformatics, 17: 754-755. Patella granularis (Patellogastropoda) and Siphonaria Hurst A (1965). Studies on the structure and function of the concinna (Basommatophora) on a South African shore. feeding apparatus of Philine aperta with a comparative Journal of Molluscan Studies, 63: 121-130. consideration of some other opisthobranchs. Hägele B (2001). Determinants of seasonal feeding of Malacologia, 2: 281-347. the generalist snail Arianta arbustorum at six sites Huson DH (1998). SplitsTree: analyzing and visualizing dominated by Senecioneae. Oecologia, 128: 228-236. evolutionary data. Bioinformatics, 14: 68-73. Hain S, Wägele H, Willan RC (1993). Tomthompsonia Huson DH, Bryant D (2006). Application of phylogenetic spiroconchalis Wägele and Hain, 1991 (Opisthobranchia, networks in evolutionary studies. Molecular Biology Notaspidea): a junior synonym of Adeorbis antarcticus and Evolution, 23: 254-267. Thiele, 1912 (Prosobranchia: Truncatelloidea) with Iglesias J (1999). Field observations on feeding of the notes on diet and histology. Journal of Molluscan land snail Helix aspersa Müller. Journal of Molluscan Studies, 59: 366-370. Studies, 65: 411-423. Händeler K, Wägele H (2006). Preliminary study on Jensen KR (1996a). The Diaphanidae as a possible sister molecular phylogeny of Sacoglossa and a compilation group of the Sacoglossa (Gastropoda, Opisthobranchia). of their food organisms. Bonner Zoologische Beiträge, In: J Taylor, Ed, Origin and evolutionary radiation of 55: 231-254. the Mollusca, University Press, Oxford, 231-247. Händeler K, Grzymbowski YP, Krug PJ, Wägele H (2009). Jensen KR (1996b). Phylogenetic systematics and Functional chloroplasts in metazoan cells - a unique classification of the Sacoglossa (Mollusca, Gastropoda, evolutionary strategy in animal life. Frontiers in Opisthobranchia). Philosophical Transactions of the Zoology, 6: 28. Royal Society of London Series B-Biological Sciences, Hansen B (1991). Feeding behaviour in larvae of the 351: 91-122. opisthobranch Philine aperta. 2. Food size spectra and Jörger K, Stöger S, Kano Y, Fukuda H, Knebelsberger particle selectivity in relation to larval behaviour and T, Schrödl M (2010). On the origin of Acochlidia

150 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

and other enigmatic euthyneuran gastropods, with Maneveldt G (2006). The role of encrusting coralline algae implications for the systematics of Heterobranchia. in the diets of selected intertidal herbivores. Journal of BMC Evolutionary Biology, 10: 323. Applied Phycology, 18: 619-627. Kälker H, Schmekel L (1976). Bau und Funktion des Marcus E (1978). On a new species of Bosellia. Boletim de Cnidosacks der Aeolidoidea (Gastropoda, Zoologia da Universidade de Sao Paulo, 3: 1-6. Nudibranchia). Zoomorphology, 86: 41-60. Medina M, Walsh PJ (2000). Molecular systematic of Katoh K, Kuma K, Toh H, Miyata T (2005). MAFFT version the order Anaspidea based on mitochondrial DNA 5: improvement in accuracy of multiple sequence sequences (12S, 16S, and CO1). Molecular Phylogenetics alignment. Nucleic Acids Research, 33: 511-518. and Evolution, 15: 41-58. Kerney MP, Cameron RAD (1979). A field guide to the Meyer-Rochow VB, Moore S (1988). Biology of Latia land snails of Britain and north-west Europe. Collins, neritoides Gray 1850 (Gastropoda, Pulmonata, London. Basommatophora): the only light-producing freshwater Klussmann-Kolb A, Dinapoli A (2006). Systematic position snail in the world. Internationale Revue der gesamten of the pelagic Thecosomata and Gymnosomata within Hydrobiologie, 73: 21-42. Opisthobranchia (Mollusca, Gastropoda) - revival of Mikkelsen PM (1996). The evolutionary relationships of the Pteropoda. Journal of Zoological Systematics and Cephalaspidea S.L. (Gastropoda: Opisthobranchia): A Evolutionary Research, 44: 118-129. phylogenetic analysis. Malacologia, 37: 375-442. Klussmann-Kolb A, Dinapoli A, Kuhn K, Streit B, Mikkelsen PM (2002). Shelled opisthobranchs. Advances in Albrecht C (2008). From sea to land and beyond - New Marine Biology, 42: 69-132. insights into the evolution of euthyneuran Gastropoda Misof B, Misof K (2009). A Monte Carlo approach (Mollusca). BMC Evolutionary Biology, 8: 57. successfully identifies randomness in multiple sequence Knudsen B, Kohn AB, Nahir B, McFadden CS, Moroz L alignments: A more objective means of data exclusion. (2006). Complete DNA sequence of the mitochondrial Systematic Biology, 58: 21-34. genome of the sea-slug, Aplysia californica: Mörch OAL (1863). Révision des espèces du genre Conservation of the gene order in Euthyneura. Oxynoe Rafinesque et Lobiger Krohn. Journal de Molecular Phylogenetics and Evolution, 38: 459-469. Conchyliologie, 11: 43-48. Lubchenco J (1978). Plant species diversity in a marine Müller OF (1776). Zoologiae Danicae. Prodromus seu intertidal community: importance of herbivore animalium Daniae et Norvegiae ingenarum characteres, food preference and algal competitive abilities. The nomina, et synonyma imprimis popularium, p. I-XXXII, American Naturalist, 112: 23-39. 1-242. Madsen H (1992). A comparative study on the food locating Müller OF (1788). Zoologia Danica sev animalium Daniae ability of Helisoma duryi, Biomphalaria camerunensis et Norvegiae rariorum ac minus notorum descriptiones and Bulinus truncatus (Pulmonata, Planorbidae). et historia, Volume 2, 51-80. Journal of Applied Ecology, 29: 70-78. Nagle GT, Camacho FT, Paul VJ (1998). Dietary preferences Malaquias MAE, Berecibar E, Reid DG (2009a). of the opisthobranch mollusc Stylocheilus longicauda Reassessment of the trophic position of Bullidae from secondary metabolites produced by the tropical (Gastropoda: Cephalaspidea) and the importance of diet cyanobacterium Lyngbya majuscula. Marine Biology, in the evolution of cephalaspidean gastropods. Journal 132: 267-273. of Zoology, 277: 88-97. Newman L (1998). Order Thecosomata. In: PL Beesley, GJB Malaquias MAE, Mackenzie-Dodds J, Bouchet P, Gosliner Ross, A Wells, Eds, Mollusca: The Southern Synthesis, T, Reid DG (2009b). A molecular phylogeny of the CSIRO Publishing, Melbourne, 980-985. Cephalaspidea sensu lato (Gastropoda: Euthyneura): Nordsieck H (1992). Phylogeny and system of the Pulmonata. Architectibranchia redefined and Runcinacea Archiv für Molluskenkunde, 121: 31-52. reinstated. Zoologica Scripta, 38: 23-41. Nylander JAA (2004). MrModeltest v2. [Program distributed

151 Katrin Göbbeler & Annette Klussmann-Kolb

by the author.] Evolutionary Biology Centre, Uppsala Journal of Natural History, 5: 647-675. University. Rudman WB (1972a). Structure and functioning of the Odhner NHJ (1939). Opisthobranchiate Mollusca from the gut in the Bullomorpha (Opisthobranchia). Part 2. western and northern coasts of Norway. Kongelige Acteonidae. Journal of Natural History, 6: 311-324. Norske Videnskabers Selskab, 1: 1–93. Rudman WB (1972b). The anatomy of the opisthobranch Pafort-van Iersel GJS (1985). Contributions to pelagic genus Hydatina and the functioning of the mantle zoogeography of the mid North Atlantic Ocean, 1. cavity and alimentary canal. Zoological Journal of the Distribution of pteropods, heteropods and silver Linnean Society, 51: 121-139. hatchetfishes. In: GJS Pafort-van Iersel (Ed). A Rudman WB (1972c). The herbivorous opisthobranch genera contribution to pelagic zoogeography of the mid North Phanerophthalmus A. Adams and Smaragdinella A. Atlantic Ocean. University of Amsterdam. Adams. Journal of Molluscan Studies, 40: 189-210. Pagel M, Meade A, Barker D (2004). Baysian Estimation of Rudman WB (1972d). A comparative study of the genus ancestral character states on phylogenies. Systematic Philinopsis Pease, 1860 (Aglajidae, Opisthobranchia). Biology, 53: 673-684. Pacific Science, 26: 381-399. Paige JA (1988). Biology, metamorphosis and postlarval Rudman WB (1978). A new species and genus of the development of Bursatella leachii plei Rang Aglajidae and the evolution of the philinacean (Gastropoda: Opisthobranchia). Bulletin of Marine opisthobranch molluscs. Zoological Journal of the Science, 42: 65-75. Linnean Society, 62: 89-107. Pennings SC, Nadeau MT, Paul VJ (1993). Selectivity Rudman WB, Willan RC (1998). Opisthobranchia, and growth of the generalist herbivore Dolabella Introduction. In: PL Beesley, GJB Ross, A Wells, Eds, auricularia feeding upon complementary resources. Mollusca: The Southern Synthesis, CSIRO Publishing, Ecology, 74: 879-890. Melbourne, 915-942. Pennings SC (1994). Interspecific variation in chemical Rudman WB, Bergquist PR (2007). A review of feeding defenses in the sea hares (Opisthobranchia: Anaspidea). specificity in the sponge-feeding Chromodorididae Journal of Experimental Marine Biology and Ecology, (Nudibranchia: Mollusca). Molluscan Research, 27: 180: 203-219. 60-88. Ponder W F, Lindberg, DR (1997). Towards a phylogeny of Rumpho ME, Summer EJ, Manhart JR (2000). Solar- gastropod molluscs: an analysis using morphological powered sea slugs. Mollusc/algal chloroplast symbiosis. characters. Zoological Journal of the Linnean Society, Plant Physiology, 123: 29-38. 119: 83-265. Salvini-Plawen L, Steiner G (1996). Synapomorphies and Ponder WF, de Kreyzer RG (1998). Superfamily Valvatoidea. plesiomorphies in higher classification of Mollusca. In: In: PL Beesley, GJB Ross, A Wells, Eds Mollusca: The J Taylor, Ed, Origin and evolutionary radiation of the Southern Synthesis, CSIRO Publishing, Melbourne, Mollusca, University Press, Oxford, 29-51. 854-858. Schacko G (1878). Die Zungenbewaffnung der Richter G (1977). Jäger, Fallensteller und Sammler. Zur Gattung Amphibola. Jahrbücher der Deutschen Ernährung planktonischer Schnecken. Natur und Malakozoologischen Gesellschaft, 5: 1–9. Museum, 107: 221-234. Schley D, Bees MA (2003). Delay dynamics of the slug Ross P, Minchinton T, Ponder W (2009). The ecology of Deroceras reticulatum, an agricultural pest. Ecological molluscs in Australian salt marshes. In: N Saintilan, Modelling, 162: 177–198. P Adam, R Connelly, P Dale, P Laegdsgaard, Eds, Schmekel L (1985). Aspects of Evolution within the Australian saltmarsh ecology, CSIRO Publishing, Opisthobranchia. In: KM Wilbur, Ed, The Mollusca, Melbourne. Academic Press, London, 221-267. Rudman WB (1971). Structure and functioning of the gut in Schrödl M, Neusser TP (2010). Towards a phylogeny the Bullomorpha (Opisthobranchia). Part 1. Herbivores. and evolution of Acochlidia (Mollusca: Gastropoda:

152 Molecular phylogeny of the Euthyneura (Mollusca, Gastropoda) with special focus on Opisthobranchia as a framework for reconstruction of evolution of diet

Opisthobranchia). Zoological Journal of the Linnean (Mollusca: Gastropoda). Zoological Journal of the Society, 158: 124-154. Linnean Society, 82: 345-362. Shimodaira H, Hasegawa M (2001). CONSEL: for Tillier S, Masselot M, Tillier A 1996. Phylogenetic assessing the confidence of phylogenetic tree selection. relationships of the pulmonate gastropods from rRNA Bioinformatics, 17: 1246-1247. sequences, and tempo and age of the sytommatophoran Shimodaira H (2002). An approximately unbiased test of radiation. In: J Taylor, Ed, Origin and evolutionary phylogenetic tree selection. Systematic Biology, 51: radiation of the Mollusca, University Press, Oxford, 492-508. 267-284. Spengel J (1881). Die Geruchsorgane und das Nervensystem Valdes A, Lozouet P (2000). Opisthobranch Molluscs from der Mollusken. Zeitschrift für Wissenschaftliche the tertiary of the aquitaine basin (South-Western Zoologie, 35: 333-383. France), with descriptions of seven new species and a Stamatakis A (2006). RAxML-VI-HPC: maximum new genus. Palaeontology, 43: 457-479. likelihood-based phylogenetic analyses with thousands Valdes A (2004). Phylogeography and phyloecology of of taxa and mixed models. Bioinformatics, 22: 2688- dorid nudibranchs (Mollusca, Gastropoda). Biological 2690. Journal of the Linnean Society, 83: 551-559. Stanisic J (1998a). Order Basommatophora. In: PL Beesley, Vermeij GJ, Lindberg DR (2000). Delayed herbivory and the GJB Ross, A Wells, Eds, Mollusca: The Southern assembly of marine benthic ecosystems. Palaeobiology, Synthesis, CSIRO Publishing, Melbourne, 1067-1075. 26: 419-430. Stanisic J (1998b). Order Systellommatophora. In: PL Vonnemann V, Schrödl M, Klussmann-Kolb A, Wägele Beesley, GJB Ross, A Wells, Eds, Mollusca: The H (2005). Reconstruction of the phylogeny of the Southern Synthesis, CSIRO Publishing, Melbourne, Opisthobranchia (Mollusca: Gastropoda) by means 1060-1067. of 18S and 28S rRNA gene sequences. Journal of Sterry P (1997). Complete British Wildlife photo guide. Molluscan Studies, 71: 113-125. Harper Collins Publishers, Ltd, London. Wade CM, Mordan PB (2000). Evolution within the Swofford DL (2002). PAUP*. Phylogenetic analysis using gastropod molluscs; using the ribosomal RNA gene- parsimony (*and other methods). Version 4_10b. cluster as an indicator of phylogenetic relationships. Sunderland, MA, Sinauer Associates. Journal of Molluscan Studies, 66: 565-570. Taylor JD (1986). Diets of sand living predatory gastropods Wägele H (1989). Diet of some arctic nudibranchs at Piti Bay, Guam. Asian Marine Biology, 3: 47-58. (Gastropoda, Opisthobranchia, Nudibranchia). Marine Thiele J (1931). Handbuch der systematischen Weichtierkunde. Biology, 100: 439-441. II. Subclassis Opisthobranchia, Gustav Fischer Verlag, Wägele H, Willan RC (2000). Phylogeny of Nudibranchia. Jena. Zoological Journal of the Linnean Society, 130: 83-181. Thollesson M (1999). Phylogenetic analysis of Euthyneura Wägele H, Vonnemann V, Wägele JW (2003). Towards (Gastropoda) by means of the 16S rRNA gene: use of a phylogeny of the Opisthobranchia. In: C Lydeard, a fast gene for higher-level phylogenies. The Royal DR Lindberg, Eds, Molecular systematics and Society of London, 266: 75-83. phylogeography of Mollusks, Smithsonian Institution Thompson TE (1976). Biology of Opisthobranch Molluscs, Press, Washington DC, 185-228. Volume 1, Ray Society, London. Wägele H (2004). Potential key characters in Opisthobranchia Thompson TE, Brown GH (1984). Biology of Opisthobranch (Gastropoda, Mollusca) enhancing adaptive radiation. Molluscs, Volume 2, Ray Society, London. Organisms, Diversity and Evolution, 4: 175-188. Thompson TE (1988). Molluscs: Benthic Opisthobranchs Wägele H, Klussmann-Kolb A (2005). Opisthobranchia (Mollusca: Gastropoda). In: Synopsis of the British (Mollusca, Gastropoda) - more than just slimy slugs. Fauna, 8: 1-356. Shell reduction and its implications on defence and Tillier A (1984). Relationships of gymnomorph gastropods foraging. Frontiers in Zoology, 2: 3.

153 Katrin Göbbeler & Annette Klussmann-Kolb

Wägele H, Ballesteros M, Avila C (2006). Defensive glandular structures in opisthobranch molluscs - From histology to ecology. Oceanography and Marine Biology - an Annual Review, 44: 197-276. Wägele H, Klussmann-Kolb A, Vonnemann V, Medina M (2008). Heterobranchia I. The Opisthobranchia. In: WF Ponder, DR Lindberg, Eds, Phylogeny and evolution of the Mollusca, University of California Press, Berkeley, 385-408. Willan RC (1984). A review of diets in the Notaspidea (Mollusca: Opisthobranchia). Journal of the Malacological Society of Australia, 6: 125-142. Willan RC (1998a). Order Anaspidea. In: PL Beesley, GJB Ross, A Wells, Eds, Mollusca: The Southern Synthesis, CSIRO Publishing, Melbourne, 974-977. Willan RC (1998b). Order Nudibranchia. In: PL Beesley, GJB Ross, A Wells, Eds, Mollusca: The Southern Synthesis, CSIRO Publishing, Melbourne, 990. Wu CI, Li WH (1985). Evidence for higher rates of nucleotides substitution in rodents than in man. Proceedings of the National Academy of Sciences of the USA, 82: 1741-1745. Xia X, Xie Z (2001). DAMBE: data analysis in molecular biology and evolution. Journal of Heredity, 92: 371-373. Xia X, Xie Z, Salemi M, Chen L, Wang Y (2003). An index of substitution saturation and its application. Molecular Phylogenetics and Evolution, 26: 1-7. Yonow N (1989). Feeding observations on Acteon tornatilis (Linnaeus) (Opisthobranchia: Acteonidae). Journal of Molluscan Studies, 55: 97-102.

154