Abstract

The phylogeny of the marine ectoparasitic gastropod family Pyramidellidae and its systematic placement have been a matter of debate for over a century. We employed nuclear (18S rDNA + 28S rDNA) and mitochondrial (16S rDNA + COI) gene fragments from 53 gastropod species (including 9 Pyramidellidae – 3 Turbonillinae and 6 Odostomiinae) in order to infer a phylogenetic hypothesis for Pyramidellidae. A Bayesian analysis of the combined data set of the four genes revealed monophyletic Pyramidellidae. The pyramidellid subgroups Turbonillinae and Odostomiinae were also recovered monophyletic. Tree reconstruction revealed Pyramidellidae deeply nested within Pulmonata implying a pulmonate origin of these ectoparasitic marine snails. However, the sister group of Pyramidellidae could not unambiguously be inferred due to lacking statistical support.

INTRODUCTION

The Pyramidellidae are marine ectoparasitic species (with a few exceptions) on a variety of hosts (e.g. polychaete worms or molluscs), partly exhibiting negative effects on cultured oysters (Crassostrea) or giant clams (Tridacna) (Schander, 1997). Pyramidellids pierce the tissue of their hosts with a buccal stylet and suck out body fluids using the muscular action of their buccal pump (Anke, 1948; Wise, 1996).

All pyramidellids possess a heterostrophic apex, a pair of more or less triangular tentacles, subepithelial median eyes, two salivary glands and an eversible proboscis (Schander, 1997).

According to the latest classification of Bouchet & Rocroi (2005), the Pyramidellidae are subdivided into four subfamilies, namely Odostomiinae, Pyramidellinae, Turbonillinae and Syrnolinae. The family comprises more than 5,000 named species, belonging to 350 genera and subgenera (Schander, van Aartsen & Corgan, 1999a).

The phylogenetic position of the Pyramidellidae within the Gastropoda has been debated for decades. This controversy was caused in part by the lack of information about the morphology of this taxon, but also due to changing views about gastropod phylogeny (Wise, 1996). Based on morphological characters, older studies placed Pyramidellidae within the ‘Prosobranchia’ (e.g. Golikov & Starobogatov, 1975; Gosliner, 1981; Robertson, 1985), due to possession of a spirally coiled calcareous shell into which the entire body is retractable, a foot with an operculum, a long proboscis and an anteriorly orientated mantle cavity. More recent studies have placed them in the Opisthobranchia, due to possession of a pallial kidney, subepithelial eyes on the median side of the tentacles, an ovotestis and a heterostrophic protoconch (e.g. Fretter & Graham, 1949; von Salvini-Plawen, 1980). Meanwhile, the traditional system of the Gastropoda with its three subclasses ‘Prosobranchia’, Opisthobranchia and Pulmonata was replaced by the concept of the Heterobranchia, introduced by Haszprunar (1985, 1988). This taxon comprises the ‘Lower Heterobranchia’ and the Euthyneura (including the Opisthobranchia and Pulmonata). Since then, the Pyramidellidae have been assigned to the paraphyletic ‘Lower Heterobranchia’ together with the Architectonicoidea, Omalogyroidea, Rissoelloidea and Valvatoidea, because they show several plesiomorphic heterobranch characters (Haszprunar, 1985, 1988; Healy, 1988, 1993; Ponder & Waren, 1988) (for a short review of the current state of pyramidellid phylogeny see also Wise, 1996).

However, molecular studies suggest a different scenario and support inclusion of the Pyramidellidae in the Euthyneura (Grande et al., 2004; Grande, Templado & Zardoya, 2008; Klussmann-Kolb et al., 2008; Dinapoli & Klussmann-Kolb, 2010). Grande et al. (2004, 2008) found them associated with Onchidella celtica (Onchidioidea), while Klussmann-Kolb et al. (2008) showed a sister-group relationship to Amphiboloidea, and Dinapoli & Klussmann-Kolb (2010) a close relationship to Glacidorboidea and Amphiboloidea.

Phylogenetic investigations within the Pyramidellidae are rare. Only two cladistic analyses, based on morphological data, have been published by Wise (1996) and Schander, Hori & Lundberg (1999b) and both treated only small groups within the family. The only molecular analysis to have examined interrelationships within the Pyramidellidae is that of Schander et al. (2003). They investigated the monophyly of the genera Odostomia and Turbonilla based on 16S rDNA gene sequences, and suggested that this marker should be combined with other data (e.g. molecular, morphological and developmental) to clarify more fully the evolutionary relationships.

The primary aims of our investigation were to verify the monophyly of the pyramidellid gastropods and to clarify their phylogenetic position within the Heterobranchia by applying molecular-systematic techniques. The data set comprised four markers (nuclear 28S rDNA + 18S rDNA and mitochondrial 16S rDNA + COI). A total of nine Pyramidellidae taxa belonging to Odostomiinae and Turbonillinae were included in the analysis. Representatives of the Onchidioidea, Amphiboloidea and Glacidorboidea were added in order to investigate the sister-group relationship of the Pyramidellidae.

MATERIAL AND METHODS

Taxon sampling

A total of 53 gastropod species (including 9 Pyramidellidae: 3 Turbonillinae and 6 Odostomiinae) were included (Table 1). Living specimens were preserved and stored in 100% ethanol. Most of the Pyramidellidae were collected intertidally.

Table 1.

Taxon sampling: collecting sites, accession numbers; sequences obtained in this study are marked with an asterisk.

Taxon Family Locality 18S complete 28S partial 16S partial COI complete ID DNA Master 
Lower Heterobranchia 
Acteonoidea 
Pupa nitidula Acteonidae GenBank GQ845185 GQ845179 GQ845192 GQ845173 – 
Hydatina physis Acteonidae GenBank AY427515 AY427480 EF489320 GQ845174 – 
Pyramidelloidea 
 Odostomiinae 
  Boonea seminuda Pyramidellidae GenBank AY145367 AY145395 AF355163 – – 
  Chrysallida sp. Pyramidellidae Jervis Bay, Australia GU331935* GU331925* GU331945* – AB17141029 
  Hinemoa sp. Pyramidellidae Jervis Bay, Australia GU331936* GU331926* GU331946* GU331955* AB17141116 
  Miralda sp. Pyramidellidae Jervis Bay, Australia GU331937* GU331927* GU331947* GU331956* AB17141105 
  Odostomia plicata Pyramidellidae Roscoff, France GU331938* GU331928* GU331948* GU331957* AB17141106 
  Pyrgisculus sp. Pyramidellidae Dunwich, Australia GU331939* GU331929* GU331949* GU331958* AB17141107 
 Turbonillinae        
  Cingulina sp. Pyramidellidae Jervis Bay, Australia GU331940* GU331930* GU331950* GU331959* AB17141119 
  Eulimella ventricosa Pyramidellidae GenBank FJ917213 FJ917235 FJ917255 FJ917274 – 
  Turbonilla elegantissima Pyramidellidae Roscoff, France GU331941* GU331931* GU331951* GU331960* AB17141095 
Glacidorboidea        
Glacidorbis rusticus Glacidorbidae GenBank FJ917211 FJ917227 FJ917264 FJ917284 – 
Opisthobranchia 
Umbraculoidea 
Umbraculum umbraculum Tylodinidae GenBank AY427499 AY427457 EF489322 DQ256200 – 
Tylodina perversa Tylodinidae GenBank AY427496 AY427458 FJ917264 AF249809 – 
Aplysiomorpha 
Aplysia californica Aplysiidae GenBank AY039804 AY026366 AF192295 AF077759 – 
Dolabella auricularia Aplysiidae GenBank AY427503 AY427467 AF156132 AF156148 – 
Dolabrifera dolabrifera Aplysiidae GenBank DQ237960 DQ237973 AF156133 AF156149 – 
Bursatella leachii Aplysiidae GenBank DQ237961 DQ237975 AF156130 AF156146 – 
Stylocheilus longicauda Aplysiidae GenBank DQ237963 DQ237978 AF156140 AF156156 – 
Cephalaspidea        
Toledonia globosa Diaphanidae GenBank EF489350 EF489375 EF489327 EF489395 – 
Haminoea hydatis Haminoeidae GenBank AY427504 AY427468 EF489323 DQ238004 – 
Philine aperta Philinidae GenBank DQ093438 DQ279988 DQ093482 AY345016 – 
Bulla striata Bullidae GenBank DQ923472 DQ986683 DQ986632 DQ986566 – 
Sacoglossa        
Oxyinoe antillarum Oxynoidae GenBank FJ917441 FJ917466 FJ917425 FJ917483 – 
Lobiger viridis Oxynoidae GenBank GU213051 GU213056 EU140863 – – 
Elysia viridis Placobranchidae GenBank AY427499 AY427462 AJ223398 DQ237994 – 
Placobranchus ocellatus Placobranchidae GenBank AY427497 AY427459 DQ480205 DQ471270 – 
Cyerce nigricans Polybranchiidae GenBank AY427500 AY427463 EU140843 DQ237995 – 
Bosellia mimetica Boselliidae GenBank AY427498 AY427460 DQ480203 DQ471214 – 
Pulmonata 
Amphiboloidea 
Phallomedusa solida Amphibolidae GenBank DQ093440 DQ279991 DQ093484 DQ093528 – 
Salinator sp. Amphibolidae Dunwich, Australia GU331942* GU331932* GU331952* GU331961* – 
Amphibola crenata Amphibolidae GenBank EF489350 EF489356 EF489304 – – 
Hygrophila 
Chilina sp. Chilinidae GenBank EF489338 EF489357 EF489305 EF489382 – 
Acroloxus lacustris Acroloxidae GenBank AY282592 EF489364 EF489311 AY282581 – 
Lymnaea stagnalis Lymnaeidae GenBank AY427525 AY427490 EF489314 AY227369 – 
Latia neritoides Latiidae GenBank EF489339 EF489359 EF489307 EF489384 – 
Bulinus tropicus Bulinidae GenBank AY282594 EF489366 EF489313 AY282583 – 
Planorbis planorbis Planorbidae GenBank EF012192 EF489369 EF489315 EF012175 – 
Physella acuta Planorbidae GenBank AY282600 EF489368 AY651241 AY282589 – 
Siphonarioidea 
Siphonaria capensis Siphonariidae GenBank EF489335 EF489354 EF489301 EF489379 – 
Siphonaria alternata Siphonariidae GenBank AY427523 AY427488 EF489299 – – 
Siphonaria concinna Siphonariidae GenBank EF489334 EF489353 EF486300 EF489378 – 
Eupulmonata 
 Stylommatophora 
  Arion silvaticus Arioninae GenBank AY145365 AY145392 AY947380 AY987918 – 
  Arianta arbustorum Helicidae GenBank AY546383 AY014136 AY546343 AY546263 – 
  Helix aspersa Helicidae GenBank X91976 AY014128 EU912832 AY546283 – 
  Helicella obvia Helicidae Mainz, Germany GU331943* GU331933* GU331953* GU331962* – 
  Deroceras reticulatum Agriolimacidae GenBank AY145373 FJ917241 FJ917266 FJ917286 – 
  Cochlicopa lubrica Cochlicopidae Ober-Olm, Germany GU331944* GU331934* GU331954* GU331963* – 
 Ellobioidea 
  Ophicardelus ornatus Ellobiidae GenBank DQ093442 DQ279994 DQ093486 DQ093486 – 
 Otinoidea 
  Otina ovata Otinidae GenBank EF489344 EF489363 EF489310 EF489389 – 
  Smeagol phillipensis Smeagolidae GenBank FJ917210 FJ917229 FJ917263 FJ917283 – 
 Onchidioidea 
  Onchidella floridana Onchididae GenBank AY427522 AY427487 EF489316 EF489391 – 
  Onchidium verruculatum Onchididae GenBank AY427521 AY427486 EF489317 EF489392 – 
Taxon Family Locality 18S complete 28S partial 16S partial COI complete ID DNA Master 
Lower Heterobranchia 
Acteonoidea 
Pupa nitidula Acteonidae GenBank GQ845185 GQ845179 GQ845192 GQ845173 – 
Hydatina physis Acteonidae GenBank AY427515 AY427480 EF489320 GQ845174 – 
Pyramidelloidea 
 Odostomiinae 
  Boonea seminuda Pyramidellidae GenBank AY145367 AY145395 AF355163 – – 
  Chrysallida sp. Pyramidellidae Jervis Bay, Australia GU331935* GU331925* GU331945* – AB17141029 
  Hinemoa sp. Pyramidellidae Jervis Bay, Australia GU331936* GU331926* GU331946* GU331955* AB17141116 
  Miralda sp. Pyramidellidae Jervis Bay, Australia GU331937* GU331927* GU331947* GU331956* AB17141105 
  Odostomia plicata Pyramidellidae Roscoff, France GU331938* GU331928* GU331948* GU331957* AB17141106 
  Pyrgisculus sp. Pyramidellidae Dunwich, Australia GU331939* GU331929* GU331949* GU331958* AB17141107 
 Turbonillinae        
  Cingulina sp. Pyramidellidae Jervis Bay, Australia GU331940* GU331930* GU331950* GU331959* AB17141119 
  Eulimella ventricosa Pyramidellidae GenBank FJ917213 FJ917235 FJ917255 FJ917274 – 
  Turbonilla elegantissima Pyramidellidae Roscoff, France GU331941* GU331931* GU331951* GU331960* AB17141095 
Glacidorboidea        
Glacidorbis rusticus Glacidorbidae GenBank FJ917211 FJ917227 FJ917264 FJ917284 – 
Opisthobranchia 
Umbraculoidea 
Umbraculum umbraculum Tylodinidae GenBank AY427499 AY427457 EF489322 DQ256200 – 
Tylodina perversa Tylodinidae GenBank AY427496 AY427458 FJ917264 AF249809 – 
Aplysiomorpha 
Aplysia californica Aplysiidae GenBank AY039804 AY026366 AF192295 AF077759 – 
Dolabella auricularia Aplysiidae GenBank AY427503 AY427467 AF156132 AF156148 – 
Dolabrifera dolabrifera Aplysiidae GenBank DQ237960 DQ237973 AF156133 AF156149 – 
Bursatella leachii Aplysiidae GenBank DQ237961 DQ237975 AF156130 AF156146 – 
Stylocheilus longicauda Aplysiidae GenBank DQ237963 DQ237978 AF156140 AF156156 – 
Cephalaspidea        
Toledonia globosa Diaphanidae GenBank EF489350 EF489375 EF489327 EF489395 – 
Haminoea hydatis Haminoeidae GenBank AY427504 AY427468 EF489323 DQ238004 – 
Philine aperta Philinidae GenBank DQ093438 DQ279988 DQ093482 AY345016 – 
Bulla striata Bullidae GenBank DQ923472 DQ986683 DQ986632 DQ986566 – 
Sacoglossa        
Oxyinoe antillarum Oxynoidae GenBank FJ917441 FJ917466 FJ917425 FJ917483 – 
Lobiger viridis Oxynoidae GenBank GU213051 GU213056 EU140863 – – 
Elysia viridis Placobranchidae GenBank AY427499 AY427462 AJ223398 DQ237994 – 
Placobranchus ocellatus Placobranchidae GenBank AY427497 AY427459 DQ480205 DQ471270 – 
Cyerce nigricans Polybranchiidae GenBank AY427500 AY427463 EU140843 DQ237995 – 
Bosellia mimetica Boselliidae GenBank AY427498 AY427460 DQ480203 DQ471214 – 
Pulmonata 
Amphiboloidea 
Phallomedusa solida Amphibolidae GenBank DQ093440 DQ279991 DQ093484 DQ093528 – 
Salinator sp. Amphibolidae Dunwich, Australia GU331942* GU331932* GU331952* GU331961* – 
Amphibola crenata Amphibolidae GenBank EF489350 EF489356 EF489304 – – 
Hygrophila 
Chilina sp. Chilinidae GenBank EF489338 EF489357 EF489305 EF489382 – 
Acroloxus lacustris Acroloxidae GenBank AY282592 EF489364 EF489311 AY282581 – 
Lymnaea stagnalis Lymnaeidae GenBank AY427525 AY427490 EF489314 AY227369 – 
Latia neritoides Latiidae GenBank EF489339 EF489359 EF489307 EF489384 – 
Bulinus tropicus Bulinidae GenBank AY282594 EF489366 EF489313 AY282583 – 
Planorbis planorbis Planorbidae GenBank EF012192 EF489369 EF489315 EF012175 – 
Physella acuta Planorbidae GenBank AY282600 EF489368 AY651241 AY282589 – 
Siphonarioidea 
Siphonaria capensis Siphonariidae GenBank EF489335 EF489354 EF489301 EF489379 – 
Siphonaria alternata Siphonariidae GenBank AY427523 AY427488 EF489299 – – 
Siphonaria concinna Siphonariidae GenBank EF489334 EF489353 EF486300 EF489378 – 
Eupulmonata 
 Stylommatophora 
  Arion silvaticus Arioninae GenBank AY145365 AY145392 AY947380 AY987918 – 
  Arianta arbustorum Helicidae GenBank AY546383 AY014136 AY546343 AY546263 – 
  Helix aspersa Helicidae GenBank X91976 AY014128 EU912832 AY546283 – 
  Helicella obvia Helicidae Mainz, Germany GU331943* GU331933* GU331953* GU331962* – 
  Deroceras reticulatum Agriolimacidae GenBank AY145373 FJ917241 FJ917266 FJ917286 – 
  Cochlicopa lubrica Cochlicopidae Ober-Olm, Germany GU331944* GU331934* GU331954* GU331963* – 
 Ellobioidea 
  Ophicardelus ornatus Ellobiidae GenBank DQ093442 DQ279994 DQ093486 DQ093486 – 
 Otinoidea 
  Otina ovata Otinidae GenBank EF489344 EF489363 EF489310 EF489389 – 
  Smeagol phillipensis Smeagolidae GenBank FJ917210 FJ917229 FJ917263 FJ917283 – 
 Onchidioidea 
  Onchidella floridana Onchididae GenBank AY427522 AY427487 EF489316 EF489391 – 
  Onchidium verruculatum Onchididae GenBank AY427521 AY427486 EF489317 EF489392 – 

Some of the pyramidellids species could not be identified to species level. For reference these were assigned voucher numbers (Table 1). Sequence data are deposited in GenBank. Photographs of the shells are available from the authors upon request. DNA is stored permanently at the Zoologische Staatssammlung München (ZSM) as part of DFG-funded project DNA Bank Network (www.dnabank-network.org).

DNA extraction, amplification and sequencing

DNA was isolated from foot tissue or the entire animal using the DNeasy Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions.

Approximately 1,800 bp of the nuclear 18S rRNA, 1,000 bp of the nuclear 28S rRNA (domains D1–D3), 450 bp of the mitochondrial 16S rRNA and 600 bp of the mitochondrial COI gene were amplified and sequenced.

The PCR technique was used to amplify defined gene fragments; see Supplementary material Table S1 for details of primers and PCR conditions. Thermal cycling was performed with a Primus 96 Advanced Gradient Thermal Cycler (Peqlab, Erlangen, Germany). Negative controls to check for contaminations (dH2O) were included in each reaction array.

Successful PCRs were cleaned and prepared for sequencing using the QIAquick Gel Extraction Kit (Qiagen). Corresponding bands were cut out from a 1.4% agarose gel and both sense and antisense strands were sequenced either on the ABN 3130 XL Applied Biosystems capillary sequencer at the SRD GmbH, Bad Homburg, or on the CEQ 2000 Beckmann Coulter capillary sequencer at the Institute for Ecology, Evolution and Diversity, Frankfurt/Main.

Sequence editing and alignment

BLAST searches (Altschul et al., 1990) were performed to compare the amplified sequences with all sequences stored in the GenBank database (www.ncbi.nlm.nih.gov/Genbank/index.html) in order to find out if the correct genes have been amplified.

The software Chromas lite 2.0.1 (www.technelysium.com.au/chromas_lite.html) was used to edit the sequence chromatograms of each amplified fragment by eye.

The sequences were aligned with the software MAFFT v. 6 (Katoh & Toh, 2008) using the E-INS-I option, which is suitable for sequences containing large unalignable regions such as loop regions of rRNA.

The alignment was checked for random similarity with the software Aliscore 0.2 (Misof & Misof, 2009). This newly developed method is based on Monte Carlo resampling within a sliding window and detects randomly similar sites (including ambiguously aligned positions and nonsignal sections) that might have negative effects on tree reconstruction. For the current analysis the following parameters were used: a sliding window with size w = 6; gaps were treated as ambiguous characters (-N option); and a maximum number of possible pairwise comparisons were applied. Noisy alignment positions identified with Aliscore were excluded prior to phylogenetic analyses (see Supplementary material Table S2).

Statistical tests

Base compositional heterogeneity within data can mislead phylogenetic reconstruction (Foster, 2004; Jermiin et al., 2004; Blanquart & Lartillot, 2006; Reumont et al., 2009), so a χ2 test was conducted using the software PAUP 4.0 Beta 10 (Swofford, 2002) to test for homogeneity of base frequencies across taxa.

Comparison of tree topologies based on single markers showed less resolution than the combined data and did not produce conflicting topologies. Therefore, a concatenated data set comprising all four markers was used for further analyses.

Phylogenetic analyses

A Bayesian analysis was performed with MrBayes v. 3.1.2 (Huelsenbeck & Ronquist, 2001). Likelihood parameters were estimated separately for each gene (and each codon position within coding sequences) using a character partition. Appropriate models for the analyses were selected after running MrModeltest v. 2.2 (Nylander, 2004) and using the Akaike information criterion. For information about evolutionary models see Supplementary material Table S3. The analysis was performed for 5,000,000 generations, with a sample frequency of 10 (additional analyses using sampling frequencies of 100 and 1,000 steps were also conducted, but all received trees showed the same topology). We examined the log-likelihood values of the analysis and discarded the first 50,000 generations as burnin, because the likelihood values had already reached a plateau. Support for nodes is expressed as posterior probabilities (PP).

RESULTS

Alignment

The software Aliscore determined various ambiguous alignment positions. These positions were excluded from further analyses (see Supplementary material Table S2). However, after examining the new alignments by eye it was decided to exclude all the remaining 23 third-codon positions of COI due to noisy and missing phylogenetic signal.

Statistical tests

The base composition within the alignments of all markers used (18S rRNA, 28S rRNA and 16S rRNA as well as the first two codon positions of COI) showed significant homogeneity (P ≥ 0.05).

An incongruence length difference test (ILD) test was performed to test for conflicting phylogenetic signal between the various genes (partition homogeneity test in PAUP). This test was not significant (P = 0.01; 10,000 replicates) but the individual gene trees showed low resolution and only a few nodes were well supported in the four individual-gene data sets and these were also retrieved in the concatenated analysis. Problems with the ILD test have been extensively documented and its limited power to detect incongruence shown (Darlu & Lecointre, 2002). Further analyses were conducted with the combined data set (3,377 bp).

Phylogenetic analyses

The reconstructed Bayesian 50% majority-rule consensus tree shows good resolution with high statistical support at almost all nodes (Fig. 1).

Figure 1.

Bayesian inference phylogram (50% majority-rule consensus tree) based on the concatenated sequences of 18S rRNA, 28S rRNA, 16S rRNA and COI (without third-codon positions); support values are PP.

Figure 1.

Bayesian inference phylogram (50% majority-rule consensus tree) based on the concatenated sequences of 18S rRNA, 28S rRNA, 16S rRNA and COI (without third-codon positions); support values are PP.

The Pyramidellidae are monophyletic with high statistical support (PP = 1.00). The same is true for the pyramidellid subfamilies Turbonillinae (PP = 1.00) and Odostomiinae (PP = 1.00). The Pyramidellidae, Glacidorboidea and Amphiboloidea together form a well-supported clade (PP = 1.00), sister to the Hygrophila with significant support (PP = 0.98). The Eupulmonata are monophyletic (including Onchidioidea, Ellobioidea, Otinoidea and Stylommatophora) with high support (PP = 1.00) and are sister to a clade comprising the Hygrophila, Amphiboloidea, Glacidorboidea and Pyramidellidae (PP = 0.98). The Sacoglossa (PP = 1.00) and the Siphonarioidea (PP = 1.00) are both monophyletic and appear as offshoots between the remaining Opisthobranchia and Pulmonata. Finally, the analysis recovered a sister-group relationship between Aplysiomorpha and Cephalaspidea (PP = 0.95), which formed the sister taxon to the Umbraculoidea (PP = 0.96).

DISCUSSION

This study is the first molecular phylogenetic analysis to include more than a small number of Pyramidellidae and to use a multilocus data set.

Previous molecular phylogenetic studies (e.g. Grande et al., 2004, 2008; Klussmann-Kolb et al., 2008; Dinapoli & Klussmann-Kolb, 2010) supported the inclusion of the Pyramidellidae within the Euthyneura, using a wide range of nonheterobranch taxa (e.g. Vetigastropoda and Caenogastropoda) as well as ‘Lower Heterobranchia’ (e.g. Rissoelloidea, Omalogyroidea and Architectonicoidea) for their analyses. However, statistical support for various clades within the Euthyneura, especially for subgroups of Pulmonata, was lacking in several of these studies (e.g. Klussmann-Kolb et al., 2008; Dinapoli & Klussmann-Kolb, 2010), mainly due to substitution saturation in the sequences. In order to avoid or reduce noisy phylogenetic signal, only taxa with a complete or nearly complete sequence set were included in the present analyses. Moreover, only taxa with similar substitution rates were used for the phylogenetic analysis. As in other studies (e.g. Wägele & Mayer, 2007; Wägele et al., 2009) we excluded taxa which showed long branches in previously published trees (e.g. Nudipleura) to improve phylogenetic signal.

For the following phylogenetic discussion only PP of ≥0.95 are considered statistically significant. Hence, relationships with lower support values will not be discussed.

Phylogenetic implications

Pyramidellidae are considered to be monophyletic on the basis of apomorphic features such as an alimentary tract with a buccal stylet (Wise, 1996). However, only a few molecular systematic investigations have so far included more than one representative of the group (Grande et al., 2004, 2008; Klussmann-Kolb et al., 2008). The phylogenetic study of Dinapoli & Klussmann-Kolb (2010) was based on a concatenated data set of four genes and included three Pyramidellidae, revealing them as a monophyletic group within Pulmonata. These findings have been examined in greater detail in the current study, which includes even more Pyramidellidae. Our results support the placement of Pyramidellidae within the Pulmonata.

Schander et al. (2003) investigated the monophyly of the pyramidellid genera Odostomia and Turbonilla, using the single mitochondrial 16S rRNA gene but the resulting topologies were weakly supported or unresolved. The present study (based on complete 18S rRNA, partial 28S rRNA and partial 16S rRNA as well as COI) supported monophyly of both Turbonillinae and Odostomiinae. Further multigene analyses including representatives of the other two subfamilies (Pyramidellinae and Syrnolinae) should be conducted. Although the pyramidellid taxon sampling of the present multilocus study is the most comprehensive to date, more taxa should be included in future analyses.

Schander et al. (2003) erected the new node-based informal name Liostomini for a clade of various genera within the Odostomiinae, all with an intorted protoconch. The Liostomini could not be detected in the present study. However, most of the taxa included by Schander et al. (2003) were missing (e.g. Liostomia, Brachystomia, Jordaniella, Spiralinella and Parthenina). It should be mentioned that in the study of Schander et al. (2003) the node support for the Liostomini was weak (maximum bootstrap 64%) using an all-taxon alignment. The same applies to the reduced Liostomini clade alignment which showed an unresolved topology. Statistically significant support for the monophyly of the Liostomini is therefore missing.

Two morphological cladistic analyses (Wise, 1996; Schander et al., 1999b) have been published to date. Owing to differences in taxon sampling, comparisons between these two studies and with our own are difficult. The odostomiine genera Sayella, Petitilla and Houbricka were not included in our study. Nevertheless, the clade Odostomiinae was supported in all three studies. This group is defined by the following synapomorphies: a single, prominent and acute fold on the upper half of the columella perpendicular to the columella axis, unnotched anterior mentum edge, mentum not bifurcate, presence of tentacular pads and salivary gland ducts within the walls of buccal pump (Schander et al., 1999b). The sampling within the clade Turbonillinae is too different to permit comparison among the three studies.

All molecular studies that have considered the position of Pyramidellidae, including our own, have yielded the same result: they cluster deep within the Euthyneura, so that a basal position in the Heterobranchia is improbable (Grande et al., 2004, 2008; Klussmann-Kolb et al., 2008; Dinapoli & Klussmann-Kolb, 2010). However, the sister group of the Pyramidellidae is still ambiguous. Grande et al. (2004, 2008) showed a sister-group relationship with Onchidella celtica (Onchidioidea). Klussmann-Kolb et al. (2008) found them associated with Amphiboloidea and Dinapoli & Klussmann-Kolb (2010) supposed a close relationship with Glacidorboidea (Pulmonata) and Amphiboloidea (Pulmonata).

The current study revealed a well-supported clade comprising the Pyramidellidae, Glacidorboidea and Amphiboloidea. Nevertheless, statistical support for the clade including Glacidorboidea and Pyramidellidae is insignificant; it remains unclear whether the Glacidorboidea or Amphiboloidea are more closely related to the Pyramidellidae. Additional members of Glacidorboidea and Amphiboloidea should be included to attempt to clarify these relationships.

The monophyly and sister-group relationships of the Sacoglossa, Siphonarioidea, Hygrophila, Eupulmonata and Opisthobranchia have been discussed in detail in the study of Dinapoli & Klussmann-Kolb (2010), based on a more extensive data set. The results of the present study are similar.

Evolutionary implications

The Pyramidellidae are remarkable diverse, comprising more than 5,000 named species belonging to 350 genera and subgenera (Schander et al., 1999a). Dietary specialization has already been discussed by several authors in relation to the adaptive radiations of some marine gastropods, especially in the Opisthobranchia (e.g. Thompson, 1976; Mikkelsen, 1996, 2002; Wägele, 2004; Händeler & Wägele, 2007). It can be assumed that the switch to a carnivorous lifestyle (the potential sister taxa are herbivorous) opened for the Pyramidellidae a wide variety of new food sources (e.g. Bivalvia, Gastropoda and Polychaeta). The potential key adaptive character related to feeding is the piercing stylet associated with an acrembolic proboscis.

Pulmonate apomorphies such as the pneumostome and pulmonary vessels, presence of a procerebrum and dorsal bodies (Dayrat & Tillier, 2002) are missing in the Pyramidellidae. Hence, these characters have apparently been lost during evolution, but this awaits explanation. Comparative morphological studies are rendered difficult by the fact that pyramidellids show unique structural adaptations and miniaturization for their parasitic lifestyle. Further studies of this enigmatic taxon are necessary, including morphological investigations. Recent studies have utilized computer-based three-dimensional reconstruction to investigate the anatomy of tiny gastropods such as Acochlidiacea (Neusser et al., 2006) or Omalogyridae (Bäumler et al., 2008) and this may be a promising new approach that could shed light on the Pyramidellidae.

SUPPLEMENTARY MATERIAL

Supplementary material is available at Journal of Molluscan Studies online.

ACKNOWLEDGEMENTS

For providing us with pyramidellid material we are grateful to Michael Schrödl and Enrico Schwabe from the Zoologische Staatssammlung in München (ZSM) and Constantine Mifsud (Malta). Many thanks to John Healy (Brisbane), Georg Mayer (Melbourne) and Katrin Göbbeler (Frankfurt) for their support during a collecting trip in Australia. We are also grateful to Bruce Marshall (Wellington) and Richard Taylor (Leigh) for their support during a collecting trip in New Zealand. We also thank Claudia Nesselhauf (Frankfurt) for her help with DNA isolation and amplification. This project was funded by the German Science Foundation (KL 1303/4-1, 4-2), by the German Academic Exchange Service and by the Vereinigung von Freunden und Förderern der Goethe-Universität. The second author is also supported by the Biodiversity and Climate Research Centre (Bik-F).

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