Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea)

Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea)

Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Molecular Phylogenetics and Evolution jo...

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Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev

Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea) Thomas Wilke a,⇑, Martin Haase b, Robert Hershler c, Hsiu-Ping Liu d, Bernhard Misof e, Winston Ponder f a

Justus Liebig University Giessen, Department of Animal Ecology and Systematics, Heinrich-Buff-Ring 26-32 (IFZ), D-35392 Giessen, Germany Vogelwarte Hiddensee, Zoological Institute and Museum, University of Greifswald, Soldmannstr. 23, D-17489 Greifswald, Germany c Department of Invertebrate Zoology, Smithsonian Institution, P.O. Box 37012, Washington, DC 20013-7012, USA d Department of Biology, Metropolitan State College of Denver, P.O. Box 173362, Denver, CO 80217-3362, USA e Molecular Biodiversity Research, Zoologisches Forschungsinstitut und Museum A. Koenig, Adenauerallee 160, D-53113 Bonn, Germany f Australian Museum, 6 College Street, Sydney, 2010 NSW, Australia b

a r t i c l e

i n f o

Article history: Received 4 May 2012 Revised 18 October 2012 Accepted 29 October 2012 Available online xxxx Keywords: Gastropoda Rissooidea Hydrobiidae Systematics Structural alignment Phylogenetic signal Long-branch attraction

a b s t r a c t Although phylogenetic studies are increasingly utilizing multi-locus datasets, a review of GenBank data for the Gastropoda indicates a strong bias towards a few short gene fragments (most commonly COI, LSU rRNA, and SSU rRNA). This is particularly the case for the Rissooidea, one of the largest and most taxonomically difficult gastropod superfamilies. Here we analyze fragments of these three genes from 90 species to determine whether they can well resolve higher relationships within this superfamily, whether structurally aligned sequence datasets increase phylogenetic signal, and whether the inclusion of highly variable regions introduces noise. We also used the resulting phylogenetic data in combination with morphological/anatomical evidence to re-evaluate the taxonomic status of ‘hydrobioid’ family-level groups. Our results indicate that all three of the alignment strategies that were used resulted in phylogenies having similar signal levels. However, there was a slight advantage to using structural alignment for inferring family-level relationships. Moreover, the set of ‘standard’ gastropod genes supported recognition of many previously recognized families and provides new insight into the systematics of several problematic groups. However, some family-group taxa were unresolved and the relationships among families were also poorly supported, suggesting a need for more extensive sampling and inclusion of additional genes. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Although early molecular phylogenetic studies were based on single amino acid or nucleotide fragments, recent studies typically use multi-locus datasets, often including both mitochondrial and nuclear genes. Nonetheless, the selection of genes having appropriate resolution for a particular phylogenetic study is still a challenging task (e.g., Whelan et al., 2001; Townsend and Lopez-Giraldez, 2010). Furthermore, some genes lack the conservative regions that can be used to design primers that successfully amplify DNA in most or all members of a given taxon. Thus, it is not surprising that those genes for which ‘universal’ primers are available are heavily employed in phylogenetic studies. Given that most of these ‘universal’ primers were designed when DNA sequencing was still very time consuming and expensive, they typically amplify only relatively short fragments (sometimes only 25% of the length of the respective gene).

⇑ Corresponding author. Fax: +49 641 99 35709. E-mail address: [email protected] (T. Wilke).

The most commonly used mitochondrial genes in phylogenetic studies of gastropods are cytochrome c oxidase subunit I (COI) and large subunit rRNA (LSU rRNA or 16S rRNA). The most frequently used nuclear gene is the small subunit rRNA (SSU rRNA or 18S rRNA) gene. Out of 256,468 non-genome nucleotide sequences available in NCBI GenBank for the Gastropoda (as of 12 April 2012), 31,751 (12.3%) are COI, 14,726 (5.7%) are LSU rRNA, and 3047 (1.2%) are SSU rRNA. The majority of these sequences were generated utilizing available ‘universal’ primers such as the Folmer et al. (1994) primers for the COI gene (typically amplifying only 658 [excluding primer sequences] out of up to 1604 bp), the Palumbi et al. (1991) primers for LSU rRNA (typically amplifying 500–600 out of up to 1514 bp), and the Holland et al. (1991) primers for SSU rRNA (typically amplifying 400–600 out of up to 2239 bp). In some gastropod groups such as the superfamily Rissooidea, this bias is even more pronounced. Of 6974 individual rissooidean sequences in GenBank, 3626 (52.0%), 882 (12.6%), and 264 (3.8%) are from COI, LSU rRNA, and SSU rRNA, respectively. The majority of these were generated using the ‘universal’ primers (or variants of them) mentioned above, resulting in predominance of short fragments: >99% of the COI, LSU rRNA, and SSU rRNA rissooidean

1055-7903/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2012.10.025

Please cite this article in press as: Wilke, T., et al. Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.025

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sequences cover less than 50% of the respective total gene length (Fig. 1). The strong bias towards short gene fragments has important consequences for future phylogenetic studies of gastropods in general and the Rissooidea in particular. Even though researchers are increasingly using full-length sequences in their studies, they often have to rely on supplementary sequences taken from GenBank for comparison with and/or to augment either dataset. This is one of the reasons why many researchers have attempted to extract as much phylogenetic information as possible from (often short) fragments by applying, for example, sophisticated models of sequence evolution, codon models for protein-coding genes, or by using secondary structure information. The latter approach provides useful phylogenetic information (see, for example, Winnepenninckx et al., 1996; Lydeard et al., 2000, 2002a) and also enables an optimal alignment of sequences (e.g., Higgs, 2000). Although secondary structure alignment has become routine in phylogenetic studies, it is often unclear whether this method actually improves phylogenetic signal. Ribosomal DNAs and other nonprotein coding genes having a distinct secondary structure may contain a series of highly variable and rapidly evolving regions (i.e., loop regions) that significantly differ among taxa. Long autapomorphic insertions may add noise to the dataset and are prone to long-branch attraction, that is, the grouping of at least two long branches for reasons other than common ancestry (e.g., Felsenstein, 1978; Hendy and Penny, 1989; Huelsenbeck and Hillis, 1993; Bergsten, 2005). In order to avoid or reduce these problems some researchers have excluded highly variable regions a priori from their datasets. Although this is not considered to be a major problem when using long fragments, many researchers prefer not to further reduce the size of already short gene fragments. Interestingly, there is no general consensus as to whether information from highly variable regions reduces or increases phylogenetic signal at higher taxonomic levels. It is also uncertain as to whether secondary structure alignment will always outperform sophisticated probabilistic alignment algorithms such as PRANK (Löytynoja and Goldman, 2005), or highly conservative alignment strategies such as the heads-or-tails (HoT) alignment (Landan and Graur, 2007; but see Wise, 2010). There have been several studies of the relative performance of different alignment algorithms based on both model (e.g., Golubchik et al., 2007; Löytynoja and Goldman, 2008; Liu et al., 2009) and actual data sets (e.g., Whiting et al., 2006; Ahrens and Vogler, 2008). However, the results of these investigations are somewhat conflicting, indicating that performance may be dataset and gene specific.

Therefore, an optimized alignment strategy may enable a more robust delineation of phylogenetic relationships within the superfamily Rissooidea Grey, 1847, in general, and the position of the notoriously difficult ‘family’ Hydrobiidae Troschel, 1857, in particular. The Hydrobiidae was long considered to be one of the largest gastropod families with more than 400 recent genera assigned. Kabat and Hershler’s (1993) compendium of the Hydrobiidae s.l. (sometimes also referred to as ‘hydrobioids’, that is, hydrobiid-like taxa in terms of anatomical/morphological features; Davis, 1979) contained 75 family-level names with 725 generic names. Wilke et al.’s (2000a) study, which was based on three gene fragments (COI, LSU rRNA, and SSU rRNA), resolved the Hydrobiidae as distinct from the Amnicolidae and Pomatiopsidae. Wilke et al. (2001) subsequently used a larger dataset from two gene fragments (COI and SSU rRNA) to further delineate the Hydrobiidae s.s. as a monophyletic, primarily amphi-North Atlantic (Europe, western Asia, northern Africa, eastern North America) group. However, other family level groups included in that study were unresolved and/or poorly supported. Szarowska (2006) added several taxa (mostly belonging to the Hydrobiidae s.s.) to Wilke et al.’s (2001) dataset and largely confirmed the findings of the former study. Ponder et al. (2008) published the most comprehensive phylogeny of the Hydrobiidae s.l. to date; this study analyzed three genes (COI, LSU rRNA, and SSU rRNA) from more than 40 genus-level taxa. Their analysis delineated several well-supported family-level clades, but also demonstrated a need for a broader geographical sampling as most of the Australasian taxa studied were found to be highly divergent relative to the Hydrobiidae s.s. and thus may merit recognition as a separate family (see also Zielske et al., 2011). Given the partly conflicting classifications of the Hydrobiidae s.l., the limited geographical coverage of previous studies, and uncertainties about how alignment strategies for short DNA fragments affect phylogenetic signal, herein we analyze a large number of hydrobioid taxa from many parts of the world to determine: – whether structurally aligned datasets of rissooidean sequences and information from highly variable regions can increase phylogenetic signal, and – whether the most commonly used short DNA fragments in gastropods well resolve higher relationships within the Hydrobiidae s.l. We also use our results together with available morphological/ anatomical evidence to re-evaluate the taxonomic status of pertinent rissooidean family-level groups, and discuss strategies for future phylogenetic analyses of the Rissooidea. 2. Materials and methods 2.1. Materials We sampled representatives of 90 genus-level rissooidean taxa, including 24 members of the family Hydrobiidae (sensu Wilke et al., 2001). Representatives of the caenogastropod superfamilies Cypraeoidea, Calyptraeoidea, Cingulopsoidea, Littorinoidea, Eulimoidea, and the (informal group) Ptenoglossa were used as outgroup taxa (superfamily assignment according to Bouchet and Rocroi, 2005). Collection information, DNA voucher numbers, GenBank accession numbers and names of collectors are in Appendix A; morphological/anatomical features of pertinent family-level taxa are summarized in Appendix B. 2.2. DNA isolation, PCR and sequencing

Fig. 1. Proportion of size classes for sequences available at GenBank for three gene fragments in the superfamily Rissooidea (as of 12 April 2012).

The molecular work for the current study was done in three different laboratories, using three different protocols. For DNA

Please cite this article in press as: Wilke, T., et al. Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.025

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isolation, the methods described in Wilke et al. (2006), Liu et al. (2001), and Haase (2008) were used in the Wilke, Liu, and Haase labs, respectively. However, PCR of our three gene fragments (COI, LSU rRNA, and SSU rRNA) was done using the same primer combinations. These are primers LCO1490 and HCO2198 (Folmer et al., 1994) for the COI gene, 16Sar-L and 16Sbr-H (Palumbi et al., 1991) for the LSU rRNA gene, and the universal metazoan primers of Holland et al. (1991) for the SSU rRNA gene. Bidirectional sequencing was done either using a LI-COR DNA sequencer Long ReadIR 4200 (Wilke lab), an Amersham Biosciences MegaBACE 500 DNA analysis System (Haase lab) or a Life Technologies ABI 310 sequencer (Liu lab). 2.3. Sequence alignment The protein-coding mitochondrial COI sequences, which typically lack insertions and deletions (indels) in the Rissooidea (Wilke et al., 2001), were unambiguously aligned in BioEdit 7.0.9 (Hall, 1999) and trimmed to a total length of 638 bp. The only exception in our dataset is the family Iravadiidae, which is characterized by a gap of three bp (one amino acid). For the alignment of the two rRNA fragments, three different strategies were used. 2.3.1. Fully automated alignment (AA) based on the probabilistic PRANK algorithm Partial raw LSU and SSU rRNA sequences were aligned in the PRANKSTER graphical interface of the multiple sequence alignment program PRANK with empirical base frequencies, the Tamura–Nei substitution model and default settings for gap penalties. Ambiguously aligned sites (with a posterior probability of belonging to a certain structure state of 60.8) were excluded from subsequent analyses. Alignment of the SSU rRNA dataset (raw sequence length 486– 605 bp) resulted in a set of aligned sequences with a total length of 1123 bp. After removing ambiguous positions, 633 bp were retained for subsequent analyses. For the LSU rRNA dataset (raw sequence length 446–494 bp), the aligned dataset had a length of 749 bp with 386 of which were used for subsequent analyses. Together with the 638 bp long COI fragment, the combined dataset of three gene fragments had a total length of 1657 bp. 2.3.2. Heads-or-tails (HoT) alignment based on the Clustal X algorithm HoT alignment was done in three steps. First, the partial raw LSU and SSU rRNA sequences were aligned using the default settings in Clustal X (Thompson et al., 1997). In a second step, the original raw sequences were transformed in BioEdit into reverse-complement sequences, aligned as above and retransformed using the reverse complement function. In the final step, the forward and reverse-complement aligned datasets were combined and re-aligned in Clustal X. All positions that differed in the forward and reverse-complement aligned sequences were then excluded from subsequent analyses. The aligned SSU rRNA dataset had a length of total 695 bp, of which 174 ambiguous positions had to be removed, leaving 521 bp. For the LSU rRNA dataset, from a total of 548 bp, 124 bp had to be removed leaving 424 bp for subsequent analyses. Together with the COI fragment, the total HoT dataset thus had a length of 1583 bp. 2.3.3. Structural alignment (SA) using RNAsalsa The partial LSU and SSU rRNA sequences were aligned incorporating secondary structure information using RNAsalsa (Stocsits et al., 2009). For LSU rRNA we used the structure of Loligo bleekeri (GenBank accession number AB029616) from the Comparative RNA Web Site and Project (www.rna.ccbb.utexas.edu) and for

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SSU rRNA that of Littorina obtusata (X94274) from The European Ribosomal RNA Database incorporated in SILVA (http://www.arbsilva.de/). Prior to running RNAsalsa, the structure in dot-bracketnotation together with the underlying sequence were aligned with the remaining sequences in the profile alignment mode of Clustal X. Positions pairing within our fragments were identified using a PERL script written by one of us (BM). Positions that had their complements outside the fragment were re-coded as loops. Positions with random similarities were identified using Aliscore (Misof and Misof, 2009) and excluded from subsequent analyses. We defined a total of five partitions: COI, loop LSU rRNA, stem LSU rRNA, loop SSU rRNA, and stem SSU rRNA. The alignment of the SSU rRNA dataset yielded 671 aligned positions, of which 134 were removed, thus resulting in a total length of 537 bp. For the LSU rRNA dataset, 19 positions had to be removed out of 564 bp, leaving 545 bp for phylogenetic analyses (structural alignments in dot-bracket-notation for the LSU and SSU rRNAs genes are provided as electronic Supplementary material). Combined with the COI fragment, the SA dataset consisted of a total of 1720 bp. 2.4. Phylogenetic analyses Prior to the phylogenetic analyses of our three datasets (AA, HoT and SA), we ran MrModeltest v2.3 (Nylander, 2004) to infer the best fit model of sequence evolution for each dataset and each gene fraction under the Akaike Information Criterion (Akaike, 1974) and with correction for small sample sizes (Nylander, 2004). MrModeltest suggested for all three datasets the same model for the same gene fraction (i.e., GTR + I + G for the LSU and SSU rRNA fractions and GTR + G for the COI fraction). For the SA dataset, we additionally applied doublet and four by four nucleotide models for stem and loop regions, respectively. Bayesian phylogenetic reconstructions were conducted in MrBayes 3.1.2 (Huelsenbeck et al., 2001; Ronquist and Huelsenbeck, 2003) with two independent runs and gaps being treated as missing data. During the runs, every 100th tree was sampled and parameter convergence was monitored in Tracer 1.5. Individual analyses were terminated when final average standard deviations of split frequencies in MrBayes reached values of near or <0.01 and/or all effective sample sizes (ESS) calculated in Tracer reached values >350. Final split frequency values were 0.0079 after 30 million generations for the SA dataset. For the HoT dataset, these values never fell below 0.02 and for the AA dataset, they never became smaller than 0.012 during several runs of 50 million generations. However, the combined trees from the two runs of each analysis showed both high ESS values (all >2000) and a smooth frequency plot, indicating that the sampled trees very well represented the posterior distribution. For all datasets, we then computed consensus trees (‘half-’and ‘allcompat’) with the first 10% of generations ignored as burn in. 2.5. Test for substitution saturation The degree of saturation was tested exemplarily in the SA dataset for all five data partitions (i.e., COI, loop SSU rRNA, stem SSU rRNA, loop LSU rRNA, and stem LSU rRNA) using the entropy-based method of Xia et al. (2003) as implemented in DAMBE 5.2.9 (Xia and Lemey, 2009). Input values for the gamma shape parameter and invariant sites were obtained by Tracer from the respective parameter files. 2.6. Phylogenetic performance Simulation studies have shown that (i) the mean of all Bayesian posterior probability (BPP) values is a good proxy for the overall accuracy of a phylogenetic tree and that (ii) the probability that an individual clade is valid strongly correlates with its respective

Please cite this article in press as: Wilke, T., et al. Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.025

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Fig. 2. Bayesian tree of rissooidean taxa based on a structurally aligned datasets of three gene fragments. BPPs are provided at the nodes. The scale bar indicates the expected number of substitutions per site. The two primary outgroups, Cypraea tigris and Crepidula adunca, were removed from the tree a posteriori. Black bars indicate family-level assignments (with rissooidean families shown in bold); white bars indicate subfamily assignments within the Hydrobiidae s.s. For reasons of clarity, long branches were shortened (% values above respective branches indicate the fraction to which branches were reduced).

BPP value (Hall and Salipante, 2007a, also see Hall and Salipante, 2007b). Therefore, we here used mean BPP values from the ‘allcompat’ consensus trees to compare the overall phylogenetic

performance of our three analyses and individual BPP values to assess the probability that a higher level taxon (i.e., families) is valid.

Please cite this article in press as: Wilke, T., et al. Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.025

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2.7. Bayesian relative rates test (BRRT) For assessing the differential effects of alignment strategies on rate heterogeneity among lineages potentially causing long-branch attraction, we used the Bayesian relative rates test (BRRT) implemented in Cadence v1.08b (Wilcox et al., 2004). The test samples the posterior probability distribution of branch lengths obtained during a Bayesian tree search and estimates the distance from the most recent common ancestor (MRCA) to each terminal taxon. If the confidence interval of an estimated branch length of a given taxon does not overlap with the confidence intervals for the other taxa, there is significant rate heterogeneity. Our tests were based on the last 1000 trees sampled in the first runs of each analysis in MrBayes. Results were visualized using a script written in R 2.15 (Däumer et al., 2012). 3. Results 3.1. Test for substitution saturation The test, performed exemplarily for the SA dataset, showed little saturation for all data partitions under the assumption of a symmetrical tree as indicated by Iss values (index of substitution saturation) being significantly smaller than the respective Iss.c values (critical values) (COI: Iss = 0.492, Iss.c = 0.709; loop LSU rRNA: Iss = 0.263, Iss.c = 0.709; stem LSU rRNA: Iss = 0.173, Iss.c = 0.740; loop SSU rRNA: Iss = 0.115, Iss.c = 0.683; and stem SSU rRNA: Iss = 0.038, Iss.c = 0.556). Under the unlikely assumption of an extremely asymmetrical tree, Iss.c values are inherently lower (COI: 0.381; loop LSU rRNA: 0.381; stem LSU rRNA: 0.470; loop SSU rRNA: 0.358; and stem SSU rRNA: 0.364). Accordingly, in such a tree, the COI dataset would be saturated. 3.2. Bayesian inference of the Hydrobiidae s.l. The Bayesian tree based on structural alignment (Fig. 2) was composed of two outgroup taxa, a basal Barleeia lineage and a large clade containing the Rissooidea and other taxa, which render the former paraphyletic. However, the non-rissooidean taxa included in this clade (Littorinidae, Eatoniellidae, Eulimidae) grouped with the family Rissoidae (BPP = 0.93) and were well separated from most ‘hydrobioid’ taxa. Basal relationships were generally poorly resolved, resulting in several polytomies in the ‘halfcompat’ consensus tree. Previously recognized family level taxa generally received high BPP support, particularly the Hydrobiidae s.s., which not only had a BPP support of 1.0, but also was characterized by a relatively long branch leading to its most recent common ancestor (MRCA), indicating a high number of synapomorphies. The Bayesian tree based on the HoT alignment (Appendix C) was composed of the two outgroups, a basal lineages consisting of the assimineid Solenomphala scalaris, and a large clade containing all of the other taxa. The latter was poorly resolved and contained a large basal polytomy consisting of rissooidean and non-rissooidean taxa. However, most of the non-rissooidean taxa in this clade grouped with the family Rissoidae (BPP = 0.57), well separated from most ‘hydrobioid’ taxa. As in the SA analysis, relationships among families received poor BPP support, while most family level taxa were well supported. This was also the case for the Hydrobiidae s.s. (BPP = 1.0), although the branch leading to its MRCA was comparably short. The phylogenetic tree generated from the automatically aligned dataset (Appendix C) consisted of the two outgroups and a large basal polytomy. Most of the non-rissooidean taxa grouped with the Rissoidae. Basal relationships were again weakly supported

whereas, as in the trees based on SA and HoT alignments, most of the family level taxa were well supported, including the Hydrobiidae s.s. (BPP = 1.00). 3.3. Phylogenetic performance The ‘allcompat’ phylogenetic trees of 95 taxa consisted of 92 nodes each (actual trees not shown here). The means of all BPP values, here used as proxy for the overall accuracy of the respective phylogenetic tree, were 0.80 (SD ± 0.24) for the SA dataset, 0.78 (SD ± 0.25) for the HoT dataset, and 0.77 (SD ± 0.27) for the AA dataset. Given the highly skewed distribution of the BPP values in our trees, we also calculated the respective medians, which were 0.94, 0.88, and 0.89 for the SA, HoT, and AA datasets, respectively. A comparison of BPP supporting nominal rissooidean families indicated that both mean and medium values were considerably higher than average values over the whole phylogeny (Table 1). Note that for those families that were not resolved as monophyletic in all three analyses, we either omitted the taxon that rendered the family paraphyletic (i.e., Solenomphala scalaris within the family Assimineidae) or instead treated distinct subgroups, if applicable (i.e., Pomatiopsidae s.s. and ‘Tomichiinae’ for taxa previously assigned to the family Pomatiopsidae). The majority of family-level taxa received BPP support of 1.00 across the different analyses, with average values being highest in the structurally aligned dataset. However, a Kruskal–Wallis rank sum test indicated that family-level support values do not differ significantly among datasets (v2 = 3.874, df = 2, p = 0.144). 3.4. Bayesian relative rates test The results of the BRRTs, which show the effects of alignment strategies on rates of heterogeneity among lineages, are in Fig. 3. All three analyses yielded a relatively homogeneous band of taxa having small to moderate rates of heterogeneity. This is contrasted by a relatively small number of outliers. The latter taxa, having high rates of heterogeneity, include Pseudomerelina sp., Fairbankia australis, and Clenchiella sp. Solenomphala scalaris (HoT and AA datasets) and Barleeia oldroydi (SA dataset) also had deviant rates. Although the individual datasets have different outliers, there are no major differences recognizable in overall rates of heterogeneity.

Table 1 BPP support of rissooidean family-level taxa in Bayesian phylogenies generated based on three different alignment strategies. Only monophyletic groups (containing two or more taxa) are considered.

a

Taxon

SA alignment

HoT alignment

AA alignment

Amnicolidae Assimineidaea ‘Beddomeia group’ Bithyniidae Bythinellidae Iravadiidae Cochliopidae Emmericiidae ‘Geomelaniinae’ Hydrobiidae Lithoglyphidae Pomatiopsidae s.s. Rissoidae Stenothyridae Tateidae ‘Tomichiinae’ Truncatellidae s.s.

1.00 0.84 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.83 1.00

1.00 0.83 0.96 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.99 0.61 1.00 1.00 0.91 0.84 0.76

1.00 0.94 0.95 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.92 0.99 1.00 0.89 0.39 0.51

Mean (±SD) Median

0.98 (±0.05) 1.00

0.94 (±0.11) 1.00

0.92 (±0.18) 1.00

Without Solenomphala scalaris.

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However, the tree based on AA alignments had the most pronounced outliers (Fig. 3). 4. Discussion 4.1. Structurally aligned dataset of rissooidean sequences: phylogenetic signal, noise and long-branch attraction We applied different alignment strategies and algorithms for excluding ambiguously aligned sites in this study. All of the resulting tree topologies and associated support values were closely similar. However, in terms of absolute values, the structural alignment outperformed both the automated PRANK and HoT alignments. Although the difference is relatively small in terms of overall BPP support, the higher support for family-level taxa was pronounced. It is difficult to tell whether this increase in phylogenetic signal was a result of fewer homoplasies in the SA dataset, the additional phylogenetic information gained by differentially coding stem and loop regions, or a combination of both. Interestingly, in the structural models analyzed (see electronic Supplementary material), we did not find distinct structural synapomorphies similar to those delineated by Lydeard et al. (2002b) for higher-level molluscan taxa. Instead, synapomorphies within families were typically restricted to point mutations distributed across the two rRNAs investigated. Most family-groups were well supported by all three datasets. However, the higher mean support values based on the SA dataset are mostly due to strong support for a few families such as the Tateidae, Truncatellidae and Pomatiopsidae s.s. The SA alignment may thus increase the reliability of family-level assignments by utilizing additional structural information and therefore reducing the possibility of inferring false monophyletic groups. However, even slightly sub-optimal support values for family-level taxa (i.e., 0.95 6 x < 1.00), which are typically considered to be reliable in Bayesian analyses, may indicate problematic family-level assignments. Overall, the SA dataset had the longest total sequence length and the smallest number of highly variable sites preemptively excluded from subsequent phylogenetic analyses. This, in turn, indicates that highly variable regions did not significantly increase the mean noise level in the dataset. Moreover, these regions also do not appear to have increased the problem of rate heterogeneity among lineages (which would potentially cause long-branch attraction). In fact, the same number of outlier taxa (i.e., 4) had highly increased branch lengths in all three analyses, with the highest overall dispersion being evident in the AA dataset (Fig. 3). In summary, our results show that, when utilizing the three standard gene fragments for phylogenetic studies of rissooidean gastropods, all three alignment strategies yielded trees having similar phylogenetic signal. Structural alignment appears to perform better in resolving family-level groups, although this difference was not statistically significant. We therefore recommend using structural alignment in combination with a statistical method for identifying random signal for future family-level phylogenetic analyses of rissooidean snails. We also suggest conservative treatment of BPP support values. 4.2. Performance of short gene fragments Our findings indicate that the short DNA fragments most commonly used in gastropod phylogenetics well resolve family-level taxa within the Hydrobiidae s.l. However, higher relationships were poorly resolved. Although this may be partly due to our sampling design, which was optimized to infer family-level relationships, it is nonetheless surprising that even family-group sister relationships were poorly resolved. The short lengths of the frag-

Fig. 3. BRRT distributions of branch lengths obtained during tree searches based on AA alignment (bottom), HoT alignment (middle), and SA alignment (top). Black dots indicate individual data points, vertical lines the mean and white dots the 95% confidence interval for each taxon. Data points for non-rissooidean taxa were removed a posteriori. Only selected taxa are labeled.

ments used in this study may have contributed to this problem owing to the limited number of synapomorphies. We note that Dayrat et al. (2011), for example, analyzed phylogenetic relationships

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within basommatophoran gastropods using the same three gene fragments (although they used a considerably longer SSU rRNA fragment) and also had difficulty resolving deeper nodes. However, the use of short sequence lengths and/or the limited number of genes may not fully explain the poor resolution of higher taxa. Additional factors contributing to the limited resolution of higher relationships may include (i) sampling design (e.g., Dayrat et al., 2011), (ii) substitution saturation, which becomes more severe with increasing phylogenetic depth for a given gene (e.g. Dwivedi and Gadagkar, 2009), and (iii) structural homoplasies (e.g. Kjer et al., 2009). The latter particularly affects phylogenetic inference of higher taxa as structural similarities decrease with phylogenetic distance (e.g., Brown et al., 2009; Stocsits et al., 2009). However, the problem of resolving higher relationships extends beyond the number of genes and fragment lengths used per se. The performance of individual genes employed also needs to be considered. The ‘standard’ fragments used herein appear to have quite different levels of performance based on our analyses of saturation and random signal in the aligned datasets. All three genes performed moderately or very well at the family level. However, in higher taxa COI and possibly LSU rRNA are affected by saturation. Therefore much of the resolution at this level comes from the SSU rRNA gene, which, in turn, has numerous ‘noisy’ regions that had to be excluded from phylogenetic analyses, thus further decreasing the overall performance of the combined data set at higher taxonomic levels. In summary, the combination of ‘standard’ gastropod genes is very useful for phylogenetic studies targeting family-groups or lower rissooidean taxa. Moreover, given the possibility that some of the previously sequenced taxa cannot be recollected, the available genetic data are extremely valuable. However, given that deeper relationships within the Rissooidea cannot be well resolved with these genes, possibly due to an increased level of homoplasy, there is also a need for additional analyses based on more and/or longer gene fragments.

have never been considered to belong to the Hydrobiidae s.l. Moreover, the ‘Beddomeia group’, which is composed of several Australian genera, was previously included in the Hydrobiidae (e.g., Ponder et al., 1993) but resolved as an evolutionarily distinct lineage in our analyses. Additional studies are needed to confirm its scope and content. The pertinent family-level taxa that we recognize based on our analyses are listed below, together with brief anatomical/morphological diagnoses and taxonomic remarks. A more inclusive list of morphological and anatomical characters for each of the families is provided in Appendix B. Unless otherwise indicated, sister group relationships of these families are uncertain.

4.3. Family-level groups within the Hydrobiidae s.l. and the status of the Hydrobiidae s.s.

4.3.2. Family Assimineidae H. Adams & A. Adams, 1856 Clade support: The Assimineidae was rendered polyphyletic in all three analyses by placement of Solenomphala scalaris outside of the clade formed by other family members. However, even when this species was excluded, the remaining assimineids formed a poorly supported clade having BPPs of 0.84–0.94. The two South African taxa included in our study are anatomically distinct from other assimineids (H. Fukuda and W.F. Ponder, unpublished data) and given that we only sampled two other members of this very large and diverse family, our findings must be treated as preliminary. Distribution: Widely distributed in tropical and temperate areas. Diagnosis: The shell is conical to near planispiral; the head-foot has short to absent cephalic tentacles, a short, broad snout and omniphoric grooves. The marginal radular teeth are broad distally, and the oviduct gland is thickened ventrally (i.e., lacking a ventral channel) but there is no separate spermathecal duct. The penis is often simple but some glands and lobes can occur. Remarks: This family was recently reviewed by Fukuda and Ponder (2003) who recognized three informal groups, one of which is the terrestrial subfamily Omphalotropinae. The taxonomic status and phylogenetic relationships of this large family are in need of further investigation which is currently being undertaken by Dr. F. Fukuda and his co-workers.

The higher relationships within the Rissooidea are poorly known, in part because extensive morphological homoplasy in this group (e.g., Davis, 1979; Hershler and Ponder, 1998; also see Appendix B). Only a few of the 75 nominal family-level names that have been previously proposed for the Hydrobiidae s.l. are supported by synapomorphies. However, given that homoplasy in genetic data may also be a problem and some of the family-level clades inferred in the present study are supported by only a few genetic synapomorphies (indicated by short branches in the phylogenetic trees), conclusive delineation of family-group taxa may not always be possible. Inasmuch as our study was primarily designed to resolve the relationships of ‘hydrobioid’ gastropods, that is, the Hydrobiidae s.l., definitive conclusions regarding the taxonomic status of several of the other rissooidean families that we sampled cannot be made at this time. Some of these families (e.g., Barleeidae) are little sampled and thus their possible monophyly remains uncertain. Others, such as Emmericidae, have not been fully characterized morphologically and thus require additional study. One family is monotypic (Hydrococcidae) and another one is rather small (Falsicingulidae). For these reasons the following rissooidean families are not further discussed herein: Hydrococcidae Thiele, 1928; Falsicingulidae Slavoshevskaya, 1975; and Iravadiidae Thiele, 1928. We also do not treat the Rissoidae Gray, 1847 and Barleeiidae Gray, 1847, which

4.3.1. Family Amnicolidae Tryon, 1863 Clade support: Strong (BPP = 1.00 in all analyses) with a large number of synapomorphies. Distribution: Palearctic, Nearctic, and northern Asia. Diagnosis: Shell near planispiral to conical, umbilicate. Pallial roof often pigmented with a small number of dark bands. Penis bifurcate, having an internal gland which sometimes extends into the haemocoel; females having a spermathecal duct. Differs from the Cochliopidae, which also has a spermathecal duct, in that the duct to the albumen gland issues from the common duct (of the oviduct and spermathecal duct) instead of the oviduct. Differs from most other hydrobioid families in having a spermathecal duct. Differs from the Pomatiopsidae and Stenothyridae (which also have a spermathecal duct) in having internal penial glands. Some amnicolids have a dorsal keel on the egg capsule that is absent in other hydrobioid families. Remarks: Our analyses resolved two subfamilies with strong support (BPP = 1.00), the Amnicolinae Tryon, 1863 and the Baicaliinae P. Fischer, 1885 (also see Wilke, 2004; Szarowska and Wilke, 2004). The nominal subfamilies Emmericiinae Brusina, 1870 and Bythinellinae Kobelt, 1878, however, do not belong to this family (see below).

4.3.3. Family Bithyniidae Gray, 1857 Clade support: Strong (BPP = 1.00 in all analyses) with a large number of synapomorphies.

Please cite this article in press as: Wilke, T., et al. Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.025

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Distribution: Palearctic/Oriental, possibly also Neotropic and African. Australia and Oceania. Diagnosis: Shell conical to ovate with variable sculpture on proto- and teleoconch; operculum calcareous with concentric growth lines; ctenidium with long filaments and food groove on floor of mantle cavity; female genitalia with distal seminal receptacle and anterior bursa copulatrix lying against capsule gland; penis with long, narrow tubular gland reaching into haemocoel where it is usually folded. 4.3.4. Family Bythinellidae Kobelt, 1878 Clade support: Strong (BPP = 1.00 in all analyses) with a large number of synapomorphies. Distribution: Western Palearctic. Diagnosis: Shell conical to globular, apex often blunt; protoconch with fine spiral threads; female genital system with distal seminal receptacle and elongate bursa copulatrix lying against albumen gland; capsule gland with closed sperm duct; penis with long tubular gland reaching into haemocoel. Remarks: Although our sampling of this group was limited, it nonetheless appears to be distinct from the Amnicolidae and thus we treat it as a separate family as previously suggested by Radoman (1976) (also see Szarowska and Wilke, 2004; Ponder et al., 2008). 4.3.5. Family Cochliopidae Tryon, 1866 Clade support: Strong (BPP = 1.00 in all analyses) with a large number of synapomorphies. Distribution: Primarily Neotropic, western Palearctic and eastern Nearctic. Diagnosis: Shell variable, ranging from planispiral to turriform, often strongly sculptured. Female having spermathecal duct, albumen gland often highly reduced, capsule gland often forming a thin walled brood pouch. Penis often lobate and/or ornamented with complex glands. A highly morphologically variable family that can be distinguished from most of the other ‘hydrobioid’ families by the combination of the presence of a female spermathecal duct, and the absence of omniphoric grooves. Distinctive features found in many cochliopid genera include transverse ciliary tracts on the left cephalic tentacle, an ovoviviparous reproductive mode, presence of apocrine glands or glandular papillae on the penis, and posteriorly folded albumen gland. Differentiated above from the Amnicolidae, which also has a spermathecal duct. Remarks: The cochliopid genera were reviewed by Hershler and Thompson (1992). 4.3.6. Family Emmericiidae Brusina, 1870 Clade support: Strong (BPP = 1.00 in all analyses) with a large number of synapomorphies. Distribution: Western Palearctic. Diagnosis: Shell ovate-conic or conical, sometimes sculptured with a well-developed spiral keel. Females having a ventrally closed albumen gland and lacking a spermathecal duct. Penis trifurcate, having two internal glands, one extending into the haemocoel. Differs from most hydrobiid families in having internal penial glands. Differs from the Amnicolidae and Bythinellidae (which also have these glands) in lacking a spermathecal duct; differs from the Bithyniidae (which also have these glands) in having a horny (not calcareous) operculum and lacking basal cusps on the central radula teeth. Remarks: Although only two representatives of this group were included in our study, our analyses nonetheless suggest that the nominal subfamily Emmericiinae does not belong to the Amnicolidae (see above) and that the Fontigentinae is not synonymous with the former. The Emmericiidae is recognized herein as a

separate family as previously suggested by Ponder et al. (2008) and others. 4.3.7. Family Hydrobiidae Stimpson, 1865 Clade support: Strong (BPP = 1.00 in all analyses) with a large number of synapomorphies. Distribution: Western Palearctic, eastern Nearctic, northern Neotropic, South Africa. Diagnosis: A highly morphologically and anatomically variable family characterized by the closed ventral wall of the female capsule gland. We are not aware of any uniquely shared morphological characters defining this group. Many hydrobiids have a pigmented coiled oviduct and/or glandular fields on the penis, which are not seen in other hydrobioid families. Remarks: The following hydrobiid subfamilies were highly supported (BPP = 1.00) in our analyses: Hydrobiinae Stimpson, 1865; Pseudamnicolinae Radoman, 1977; Nymphophilinae D.W. Taylor, 1966; Pyrgulinae Brusina, 1882 and Islamiinae Radoman, 1973. The Belgrandiinae de Stefani, 1877 does not appear to be monophyletic, thus the nominal subfamilies Belgrandiellinae Radoman, 1983 and/or Horatiinae D. W. Taylor, 1966 potentially remain valid. Mercuria similis was not positioned in any of the above subfamilies in the trees and thus may have to be assigned to its own subfamily. The Clenchiellinae D.W. Taylor, 1966 and Tateinae Thiele, 1925, which were treated hydrobiid subfamilies by Bouchet and Rocroi (2005), do not belong to the Hydrobiidae. 4.3.8. Family Lithoglyphidae Tryon, 1866 Clade support: Strong (BPPs 0.99–1.00) with a large number of synapomorphies. Distribution: Palearctic/Nearctic. Diagnosis: A morphologically cohesive group characterized by the closed ventral wall of the female capsule gland and a blade-like penis lacking large appendages and specialized glands. We do not know of any unique characters defining this group. Remarks: The two nominal subfamilies of the Lithoglyphidae, the Lithoglyphinae Tryon, 1866 (=Fluminicolinae Clessin, 1880) and the Benedictiinae Clessin, 1880, were rendered paraphyletic by the placement of western North American Fluminicola in all three analyses. Therefore, subfamilial relationships need further clarification. Furthermore, South American Potamolithus, which was previously classified in the Lithoglyphidae (Davis and da Silva, 1984), appears to belong to the family Tateidae (see below). 4.3.9. Family Moitessieriidae Bourguignat, 1863 Clade support: The three putative members of this family studied were part of a polytomy in the SA tree and thus their relationships were unresolved. However, the Moitessieriidae was delineated as monophyletic in the AA (BPP = 0.77) and HoT (BPP = 0.94) analyses. Distribution: Palearctic. Diagnosis: Shell conical to turriform; teleoconch often spirally sculptured; horny operculum occasionally with peg; intestine coiling loosely around style sac; one distal seminal receptacle that can be lost; bursa copulatrix large, behind albumen gland; penis sometimes with non-glandular lobe. Remarks: The phylogenetic relationships and taxonomic status of this little studied group require further study. 4.3.10. Family Pomatiopsidae Stimpson, 1865 Clade support: The Pomatiopsidae s.l. was not resolved as a monophyletic group in any of the trees. However, when Tomichia

Please cite this article in press as: Wilke, T., et al. Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.025

T. Wilke et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx

(Africa) and Coxiella (Australia) were excluded, the remaining taxa formed a monophyletic group having BPPs of 0.61–1.00. The latter two taxa formed a weakly supported clade (BPPs 0.39–0.83). Distribution: The Pomatiopsidae s.s. is Nearctic, eastern Palearctic and Oriental, the ‘Tomichiinae’ is southern Ethiopian and Australasian (see Remarks). Diagnosis: Shells are variable in shape and size, a spermathecal duct is present, and there are multiple cusps on the bases of the central teeth and, in some (Pomatiopsinae), omniphoric grooves on the head-foot. The penis is simple, often having an ejaculatory duct. Remarks: Bouchet and Rocroi (2005) recognized two nominal subfamilies based on Davis (1979) and subsequent investigations: the Pomatiopsinae Stimpson, 1865 (=Tomichiinae Wenz 1938; =Coxiellidae Iredale, 1943; =Oncomelaniidae Salisbury & Edwards, 1961; =Cecininae Starobogatov, 1983) and Triculinae Annandale, 1924. Although our sampling was limited, the resulting analyses nonetheless suggest that neither of these subfamilies is monophyletic (also see Kameda and Kato, 2011). Furthermore, Coxiella and Tomichia appear to form a monophyletic group distinct from the Pomatiopsidae s.s. The Tomichiinae (=Coxiellidae) is an available name for this clade which may merit recognition as a separate family. However, given the mixed support provided by our analyses, we recommend that additional studies be directed toward resolving the taxonomic status of the Coxiella–Tomichia clade. 4.3.11. Family Stenothyridae Tryon, 1866 Clade support: Strong (BPP = 1.00 in all analyses), with a large number of synapomorphies. Distribution: Palearctic, Oriental, Australia. Diagnosis: The shell has a circular aperture that is usually contracted in the adult. The operculum bears two flange-like projections on the inner surface and the foot has a metapodial tentacle. A spermathecal groove is present and the penis lacks glands but sometimes has a stylet. Remarks: This small estuarine and freshwater family is very distinct morphologically (see Appendix B). 4.3.12. Family Tateidae Thiele, 1925 Clade support: Strong (BPP = 1.00) in the SA analysis; weaker support in the AA (BPP = 0.89) and HoT (BPP = 0.91) analyses. However, the tateid clade was supported by a large number of synapomorphies in all of the trees. Sister-group relationships were not resolved in the AA and HoT analyses; the SA analysis suggests that Ascorhis tasmanica (Australia) may be the sister group to this family, although it is anatomically distinct (Ponder and Clark, 1988). Distribution: Australasian/Oceanic and possibly Neotropic (see remarks). Diagnosis: The shell is variable in shape, but usually pupiform to conical, horny operculum of many species with pegs or white smear on inner side; stomach of many Australasian and Oceanian taxa with fan-shaped caecum whose presence often coincides with a membranous junction of flank and face on the inner marginal teeth; female genitalia simple, usually with one distal seminal receptacle and a bursa copulatrix; ventral channel occasionally separated to form a vestibule; penis usually without appendages.

9

Remarks: We recognize Tateidae as having family rank (also see Ponder et al., 2008 and Zielske et al., 2011). The genus Potamolithus (South America), which was previously considered to be a lithoglyphid (Davis and da Silva, 1984; also see Wilke et al., 2001) was resolved as a member of this clade in all of our analyses.

4.3.13. Family Truncatellidae Gray, 1840 Clade support: The Truncatellidae s.l. was poorly supported in the SA analysis (BPP = 0.89) and depicted as polyphyletic in the HoT and AA analyses. However, the two nominal subfamilies Truncatellinae Gray, 1840 and Geomelaniinae Kobelt & Möllendorff, 1897 were delineated as clades; the latter received perfect support (BPP = 1.00 in all analyses) while the former was highly supported only in the SA analysis. Distribution: Nearctic, Neotropic, Palearctic. Diagnosis: The shell is elongate and usually decollate in adults, with axial sculpture common. The head-foot has omniphoric grooves, a short foot and a long snout that is involved in locomotion. The oviduct gland lacks a ventral channel and the penis is simple. Remarks: Given that the Truncatellidae s.l. received poor support and may even be polyphyletic, and the two nominal subfamilies are characterized by distinct anatomical synapomorphies, additional studies will be needed to determine whether the latter merit recognition as separate families. Note that the superfamily Rissooidea appears to be polyphyletic in all of the trees. There was also no support for a distinct ‘hydrobioid’ unit. In fact, the Hydrobiidae appear to be more closely related to rissoid and even littorinid taxa than to some of the other hydrobioid families. Given that the family Hydrobiidae is now clearly defined and differentiated from other families within the Rissooidea (based on molecular evidence), we discourage continued use of the terms Hydrobiidae s.s. and Hydrobiidae s.l. We also note that there is an obvious need to continue searching for morphological and/or anatomical evidence to support the monophyly of the Hydrobiidae and other families that are recognized herein primarily based on molecular data. We also see a need for further studies of family-level taxa that (a) were not included in our study (e.g., Ceacidae); (b) were poorly resolved and/or potentially deserve family rank (e.g., Ascorhis spp., Assimineidae, ’Beddomeia group’, ‘Fontigentinae’, ’Tomichiinae’); and (c) were potentially affected by long-branch attraction in our analyses (e.g., Iravadiidae, Eatoniellidae).

Acknowledgments This work was largely inspired by the extensive work on the Rissooidea by George M. Davis and his life-long commitment to resolving the evolutionary history of this challenging but fascinating superfamily. The study was, in part, supported by grants of the German Science Foundation (WI 1902/8-1, 2 to TW and HA 4752/2-1 to MH). Our analyses could not have been done without the kind help of many colleagues who provided samples or assistance in determining the species studied (see Appendix A).

Please cite this article in press as: Wilke, T., et al. Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.025

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T. Wilke et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx

Appendix A Classification, locality information and GenBank accession numbers for the taxa studied. See Discussion for details of classification. Classification Calyptraeoidea Crepidulidae

Cingulopsoidea Eatoniellidae

Cypraeoidea Cypraeidae

Eulimoidea Eulimidae

Littorinoidea Littorinidae

Rissooidea Amnicolidae

Assimineidae

Species

Locality

GenBank accession # COI/LSU rRNA/ SSU rRNA

Crepidula adunca Sowerby, 1825

USA, Washington, Friday Harbor (COI, LSU rRNA), Canada, Vancouver (SSU rRNA)

AF546047 (Collin, 2003) AF545987 (Collin, 2003) X94277 (Winnepenninckx et al., 1998)

Eatoniella atropurpurea (Frauenfeld, 1876)

Australia, New South Wales, Balmoral, Edwards Beach; coll.: W.F. Ponder, 21 August 1998; USNM 892216

JX970598 JX970523 JX970556

Cypraea tigris Linnaeus, 1758

Indian Ocean (COI, LSU rRNA), Japan, Minabe (SSU rRNA)

AY161722 (Meyer, 2003) AY161489 (Meyer, 2003) AF055654 (Harasewych et al., 1998)

Balcis eburnea A. Adams, 1861

Information not provided in original publication

AF120636 (Giribet and Wheeler, 2002) DQ280051 (Giribet et al., 2006) AF120519 (Giribet and Wheeler, 2002)

Littorina obtusata (Linnaeus, 1758)

United Kingdom, Isles of Scilly (COI, SSU rRNA); United Kingdom, Pembroke Dock (LSU rRNA)

AJ622947 (Williams and Reid, 2004) U46812 (Williams and Reid, 2004) AJ488715 (Williams and Reid, 2004)

Amnicola limosa (Say, 1817)

USA, Michigan, Washtenaw County, Blind Lake (42.38°N, 84.02°W); coll.: J. Burch

AF213348 (Wilke et al., 2000a) AF212903 (Wilke et al., 2000a) AF212916 (Wilke et al., 2000a)

Antroselates spiralis Hubricht, 1963

USA, Indiana, Harrison Co., Harrison Cave Spring; coll.: J.J. Lewis, 29 July 1995; USNM 883970

AF354758 (Liu et al., 2001) JX970524 JX970557

Baicalia costata W. Dybowski, 1875

Russia, Lake Baikal, northern slope of Chornaye Canyon; coll.: T. Backeljau, 18 June 1991; USNM 854741

JX970599 JX970525 JX970558

Erhaia jianouensis (Liu & Zhang, 1979)

China, Fujian, Nanping, Tianxi (26.9925°N, 118.3874°E); coll.: G.M. Davis

AF367652 (Wilke et al., 2001) EU573984 (Ponder et al., 2008) AF367688 (Wilke et al., 2001)

Maackia bythiniopsis (Lindholm, 1909)

Russia, Lake Baikal, Listvyanka; coll.: T. Sitnikova; 7 May 2000

HQ623173 HQ623157 HQ623165

Marstoniopsis insubrica (Küster, 1853)

Germany, Rostock, Warnow River near the feeder of the Rostock waterworks (54.09°N, 12.12°E); coll.: M.L. Zettler

AF322408 (Wilke and Falniowski, 2001) AY341257 (Wilke, 2004) AF367676 (Wilke et al., 2001)

Moria kikuchii (Habe, 1961)

Japan, Tagawa, Hikosan Soeda-machi, Hikosan Shrine; coll.: G.M. Davis

AF213350 (Wilke et al., 2000a) AF212905 (Wilke et al., 2000a) AF212918 (Wilke et al., 2000a)

Assiminea grayana Fleming, 1828

Germany, Vareler Watt, ca. 1 km N mouth of Jade River; coll. J. Hartmann, 24 February 2004

HQ623170 HQ623153 HQ623162

‘Assiminea’ sp. 1

South Africa, Knysna Lagoon near Ashmead Channel (34.0592°S, 23.0664°E); coll.: R. Barnes

– JX970526 JX970559

‘Assiminea’ sp. 2

South Africa, Knysna Lagoon, Thesens Island (34.0472°S, 22.0472°E); leg.: B. Allanson, 26 January 2000, det.: T. Wilke

JX970600 JX970527 JX970560

Please cite this article in press as: Wilke, T., et al. Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.025

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Appendix A (continued) Classification

Barleeiidae

‘Beddomeia group’

Bithyniidae

Bythinellidae

Iravadiidae

Cochliopidae

Species

Locality

GenBank accession # COI/LSU rRNA/ SSU rRNA

Solenomphala scalaris (Heude, 1882)

China, Sichuan Province, Tianggongmiao Township, Huangba Village, Eryang River (30.58758°N, 117.38607°E); coll.: G.M. Davis

JX970601 – JX970561

Barleeia oldroydi (P. Bartsch, 1920)

USA, California, Monterey County, Pacific Grove; coll.: D.R. Lindberg; USNM 892576

JX970602 – JX970562

Beddomeia paludinella (Reeve, 1857)

Australia, Tasmania, tributary of Thirteen Mile Creek; coll.: S.A. Clark, 9 February 1995

JX970603 JX970528 JX970563

Phrantela marginata (Petterd, 1889)

Australia, Tasmania, tributary of Thirteen Mile Creek; coll.: S.A. Clark, 9 February 1995

JX970604 JX970529 JX970564

Victodrobia sp.

Australia, Victoria, tributary of Aceron River near Burton; coll.: J.H. Waterhouse & S.A. Clark, 8 December 1988

– JX970530 JX970565

Bithynia tentaculata (Linnaeus, 1758)

USA, New York, Dutchess County, Hudson River, Cruger Island; coll.: R. Hershler & D.L. Strayer, 8 August 1995; USNM 883969

JX970605 JX970531 JX970566

Parafossarulus cf. manchouricus (Bourguignat, 1860)

China, Anhui Province, Zhujia Township, Ming Sheng Village, Qiupu Stream (30.67347°N, 117.45493°E); coll.: G.M. Davis

HQ623174 HQ623158 HQ623166

Bythinella austriaca (Frauenfeld, 1856)

Austria, Steiermark, National Park ‘Kalkalpen’ (47.77°N, 14.38°E); coll.: M. Haase

AF213349 (Wilke et al., 2000a) AF212904 (Wilke et al., 2000a) AF212917 (Wilke et al., 2000a)

Bythinella pannonica (Frauenfeld, 1865)

Slovakia, Slovensky Kras, Hrhov Spring (48.616°N, 20.769°E); coll.: A. Falniowski & M. Szarowska, USNM 1010151

AY222650 (Szarowska and Wilke, 2004) AY222660 (Szarowska and Wilke, 2004) EU573994 (Ponder et al., 2008)

Clenchiella sp.

Australia, Queensland, Townsville, Three Mile Creek; coll.: W.F. Ponder, 24 February 1997; USNM 854876

JX970606 – JX970567

Fairbankia australis Hedley, 1901

Australia, Queensland, Cockle Bay, Magnetic Island; coll.: W.F. Ponder, 27 February 1997; USNM 892108

JX970607 JX970532 JX970568

Pseudomerelina sp.

Australia, Queensland, Townsville, Three Mile Creek; coll.: W.F. Ponder, 22 February 1997; USNM 892106

JX970608 JX970533 JX970569

Heleobops carrikeri Davis & McKee, 1989

USA, Maryland, Dorchester Co., Little Choptank River at the end of Ragged Point Road (38.5388°N, 76.2729°W); coll.: G.M. Davis USA, Maryland, Dorchester Co., Town Point at the end of Town Point Road (38.5418°N, 76.2080°W); coll.: G.M. Davis

AF213347 (Wilke et al., 2000a) AF212902 (Wilke et al., 2000a) AF212915 (Wilke et al., 2000a) AF367645 (Wilke et al., 2001) EU573990 (Ponder et al., 2008) AF367678 (Wilke et al., 2001)

Mexipyrgus carranzae Taylor, 1966

Mexico, Coahuila, Mojarral West Laguna, Cuatro Cienegas basin; coll.: D.A. Hendrickson; USNM 854813

AF129325 (Hershler et al., 1999) JX970534 JX970570

Semisalsa stagnorum (Gmelin, 1791)

The Netherlands, Kaaskenswaters, Zierikzee (51.65582°N, 3.93580°E); coll.: T. Wilke

JQ973024 (Kroll et al., 2012) JX970535 JX970571

Spurwinkia salsa (Pilsbry, 1905)

USA, Maryland, Dorchester Co., Town Point at the end of Town Point Road (38.5418°N, 76.2080°W); coll.: G.M. Davis

AF367633 (Wilke et al., 2001) EU573991 (Ponder et al., 2008) AF367663 (Wilke et al., 2001)

Onobops jacksoni (Bartsch, 1953)

(continued on next page)

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Appendix A (continued) Classification

Species

Locality

GenBank accession # COI/LSU rRNA/ SSU rRNA

Emmericia expansilabris Bourguignat, 1880

Croatia, Izvor River, freshwater spring (type locality); coll.: A. Falniowski

Emmericia patula (Brumati, 1838)

Italy, Trieste-Trst, San Giovanni-Stivan, Timavo Springs; coll.: S. Turk & B. Sket, 2 July 1996; USNM 854802

– EU573985 (Ponder et al., 2008) EU573995 (Ponder et al., 2008) – JX970536 JX970572

Falsicingulidae

Falsicingula athera Bartsch, 1936

Japan, Hokkaido Prefecture, Etorofu Island; coll.: H. Fukuda

HQ623172 HQ623155 HQ623164

‘Fontigentinae’

Fontigens nickliniana (I. Lea, 1838)

USA, Michigan, Kalamazoo County, Kalamazoo River basin; coll.: J. Legge, 30 October 1995; USNM 854740

JX970609 – JX970573

Hydrobiidae

‘Adrioinsulana’ conovula (Frauenfeld, 1863)

Croatia, Pag Island, Zubovici (44.52076°N, 14.97237°E); coll.: A. Falniowski

AF367628 (Wilke et al., 2001) EU573986 (Ponder et al., 2008) AF367656 (Wilke et al., 2001)

Adriohydrobia gagatinella (Küster, 1852)

Croatia, Krka River near Skradin (43.81712°N, 15.92353°E); leg.: A. Falniowski, det.: T. Wilke

AF317857 (Wilke and Falniowski, 2001) EU573987 (Ponder et al., 2008) AF367657 (Wilke et al., 2001)

Avenionia brevis berenguieri (Bourguignat, 1882)

France, Gard, spring of the fountain of St.Victor-La Coste (44.057°N, 4.636°E); coll.: M. Bodon

AF367638 (Wilke et al., 2001) – AF367670 (Wilke et al., 2001)

Belgrandia thermalis (Linnaeus, 1767)

Italy, Tuscany, Pisa, S. Giuliano Terme, thermal channel near S. Giuliano (43.751°N, 10.440°E); coll.: M. Bodon

AF367648 (Wilke et al., 2001) – AF367684 (Wilke et al., 2001)

Belgrandiella kusceri (Wagner, 1914)

Slovenia, spring of Rakek (type locality); coll.: A. Falniowski

JX970610 JX970537 JX970574

Cincinnatia winkleyi (Pilsbry, 1912)

USA, Maine, Cumberland County, Spurwink River (43.57°N, 70.25°W); coll.: G.M. Davis

AF118370 (Wilke and Davis, 2000) AF212901 (Wilke et al., 2000a) AF212914 (Wilke et al., 2000a)

Dianella thiesseana (Kobelt, 1878)

Greece, Lake Trichonida at Loutres Mirtias (38.56553°N, 21.61978°E); coll.: A. Falniowski

AY676127 (Szarowska et al., 2005) AY676121 (Szarowska et al., 2005) AY676125 (Szarowska et al., 2005)

Ecrobia ventrosa (Montagu, 1803)

United Kingdom, Norfolk, The Wash, Snettisham lagoon RSPB bird reserve (52.863°N, 0.460°E); coll.: B. James

AF118335 (Wilke and Davis, 2000) AF478402 (Wilke, 2003) AF367681 (Wilke et al., 2001)

Euxinipyrgula milachevitchi (Golikov and Starobogatov, 1966) Fissuria boui Boeters, 1981

Russia, Sea of Azov, Miusski Liman (47.27°N, 38.77°E); leg.: F. Riedel, det.: T. Wilke

EF379290 (Wilke et al., 2007) EF379306 (Wilke et al., 2007) EF379280 (Wilke et al., 2007) AF367654 (Wilke et al., 2001) – AF367690 (Wilke et al., 2001)

Emmericiidae

France, Alpes Maritimes, Peymeinade, spring near La Prouveresse (43.64279°N, 6.88735°E); coll.: M. Bodon

Graziana alpestris (Frauenfeld, 1863)

Italy, Liguria, Savona, Molino, spring at Porra River (44.219°N, 8.255°E); coll.: M. Bodon

AF367641 (Wilke et al., 2001) AY676123 (Szarowska et al., 2005) AF367673 (Wilke et al., 2001)

Hauffenia tellinii (Pollonera, 1898)

Italy, Friuli-Venetia Julia, Gorizia, Isonzo River near Sagrado, spring (45.8743°N, 13.4856°E); coll.: M. Bodon Croatia, spring of Vrana River, between Vrana and Radosinovci (43.92532°N, 15.58799°E); coll.: A. Falniowski

AF367640 (Wilke et al., 2001) EU573988 (Ponder et al., 2008) AF367672 (Wilke et al., 2001) AF367637 (Wilke et al., 2001)

France, Hérault, Etang du Prévost (43.513°N,

AF278808 (Wilke et al., 2000b)

Horatia klecakiana Bourguignat, 1887

Hydrobia acuta

AY222656 (Szarowska and Wilke, 2004) AF367669 (Wilke et al., 2001)

Please cite this article in press as: Wilke, T., et al. Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.025

13

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Appendix A (continued) Classification

Species

Locality

GenBank accession # COI/LSU rRNA/ SSU rRNA

(Draparnaud, 1805)

3.897°E); coll.: C. Casagranda

AY222659 (Szarowska and Wilke, 2004) AF367680 (Wilke et al., 2001)

‘Hydrobia’ knysnaensis (Krauss, 1848)

South Africa, Van Stadens Estuary (33.96°S, 25.22°W); coll.: P. Teske

JX970611 JX970538 JX970575

Islamia piristoma Bodon & Cianfanelli, 2001

Italy, Liguria, La Spezia, Arcola, spring at Magra River (44.1042°N, 9.9337°E); coll.: M. Bodon

AF367639 (Wilke et al., 2001) – AF367671 (Wilke et al., 2001)

Mercuria similis (Draparnaud, 1805)

Italy, Friuli-Venetia-Julia, Udine, Aquileia, Canale Panigai near Panigai (45.7415°N, 13.3408°E); coll.: M. Bodon

AF367646 (Wilke et al., 2001) AF478393 (Wilke, 2003) AF367682 (Wilke et al., 2001)

Notogillia wetherbyi (Dall, 1885)

USA, Florida, Marion County, Rainbow Springs; coll.: F.G. Thompson; UF 263135

AF52091 (Hershler et al., 2003b) JX970539 JX970576

Nymphophilus minckleyi Taylor, 1966

Mexico, Coahuila, Laguna Churince, Cuatro Cienegas basin; coll.: R. Hershler & J.J. Landye; USNM 863502

AF354771 (Liu et al., 2001) – JX970577

Orientalina callosa (Paulucci, 1881)

Italy, Abruzzo, Pescara, Caramanico Terme (42.1571°N, 14.0167°E); coll.: M. Bodon

Pauluccinella minima (Paulucci, 1881)

Italy, S. Egidio, Lago di Piediluco; coll.: M. Bodon

AF367649 (Wilke et al., 2001) – AF367685 (Wilke et al., 2001) JX970612 JX970540 JX970578

Pseudamnicola lucensis (Issel, 1866)

Italy, Tuscany, Lucca, Bagni di Lucca, Bagni Caldi, thermal spring (44.007°N, 10.585°E); coll.: M. Bodon

AF367651 (Wilke et al., 2001) AF478394 (Wilke, 2003) AF367687 (Wilke et al., 2001)

Pyrgula annulata (Linnaeus, 1767)

Italy, Brescia, Lake Garda, Desenzano del Garda (45.48°N, 10.55°E); coll.: M. Bodon

AY341258 (Wilke, 2004) AY676122 (Szarowska et al., 2005) AY676124 (Szarowska et al., 2005)

Sadleriana fluminensis (Küster, 1853)

Slovenia, Mocilnik near Vrhnika, main spring of Ljubljanica River (45.9516°N, 14.2954°E); coll.: A. Falniowski & M. Szarowska, 29 August 2001

Hydrococcidae

Hydrococcus brazieri (Tenison-Woods, 1876)

Australia, Victoria, Queens Cliff Yacht Club, Swan Bay, Port Phillip (38.23°S, 144.67°E); coll.: W.F. Ponder; AMNH C306250

AY273996 (Szarowska and Wilke, 2004) AY222657 (Szarowska and Wilke, 2004) EU573996 (Ponder et al., 2008) – JX970541 JX970579

Lithoglyphidae

Benedictia baicalensis (Gerstfeldt, 1859)

Russia, Lake Baikal, Listvenichnyi Bay (51.869314°N; 105.824544°E); leg.: Rusinek, det.: T. Sitnikova, 26 February 2006

HQ623171 HQ623154 HQ623163

Fluminicola coloradensis Morrison, 1940

USA, Utah, Wasatch County, Provo River; coll.: P. Hovingh, 10 August 1995; USNM 1123926

AF520931 (Hershler et al., 2003b) JX970542 JX970580

Lithoglyphus naticoides (C. Pfeiffer, 1828)

Poland, Narew River near Drozdowo (53.13853°N, 22.15180°E); coll.: A. Falniowski & M. Szarowska

AF367642 (Wilke et al., 2001) AY222654 (Szarowska and Wilke, 2004)

Bythiospeum cf. diaphanum (Michaud, 1831)

France, Gard, Lirac, Source de la Nizon (44.03°N, 4.68°E); coll.: M. Bodon

AF367674 (Wilke et al., 2001) AF367634 (Wilke et al., 2001) – AF367664 (Wilke et al., 2001)

Moitessieria cf. puteana Coutagne, 1883

France, Alpes Maritimes, Peymeinade, spring near La Prouveresse (43.64279°N, 6.88735°E); coll.: M. Bodon

AF367635 (Wilke et al., 2001) EU573992 (Ponder et al., 2008) AF367665 (Wilke et al., 2001)

Moitessieriidae

(continued on next page)

Please cite this article in press as: Wilke, T., et al. Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.025

14

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Appendix A (continued) Classification

Pomatiopsidae s.s.

Rissooidae

Stenothyridae

Tateidae

Species

Locality

GenBank accession # COI/LSU rRNA/ SSU rRNA

Sardopaladilhia plagigeyerica Manganelli, Bodon, Cianfanelli, Talenti & Giusti, 1998

Italy, Sardinia, Grotta di San Giovanni su Anzu (40.3203°N, 9.6158°E); leg.: C. Carletti & S. Bonni, 22 August 2000, det.: M. Bodon

HQ623176 HQ623160 HQ623168

Cecina cf. manchurica A. Adams, 1861

Russia, Kuril Islands, Shikotan Island, Otradnaya Bay (43.86695°N, 146.79172°E); coll.: T.A. Pearce, 12 August 1998

JX970613 – JX970581

Gammatricula chinensis Davis, Liu & Chen, 1990

China, Zhejiang Province, Kaiwa Co, Tong Cun Town, Bai Keng Village (29.0008°N, 118.2578°E); leg.: C.-E. Chen, det.: G.M. Davis

AF253067 (Davis et al., 1998) EU573993 (Ponder et al., 2008)

Lacunopsis sp.

China, Yunnan Province, Xishuangbanna, Mengla (21.51198°N, 101.54668°E); coll.: G.M. Davis

AF213343 (Wilke et al., 2000a) AF212897 (Wilke et al., 2000a) AF212910 (Wilke et al., 2000a)

Oncomelania h. hupensis Gredler, 1881

China, Anhui, Dalin (30.8780°N, 118.9143°E); coll.: G.M. Davis

AF254547 (Wilke et al., 2000c) DQ212859 (Wilke et al., 2006) AF367667 (Wilke et al., 2001)

Pomatiopsis lapidaria (Say, 1817)

USA, Michigan, Washtenaw County, Bridgewater Township, near Raisin River at Allen Road (42.0893°N, 83.9725°W); coll.: J. Burch, 8 October 1998

AF367636 (Wilke et al., 2001) AY676118 (Szarowska et al., 2005) AF367666 (Wilke et al., 2001)

Tricula sp.

China, Sichuan Province, Huang Ba (30.540408°N, 103.241238°E); coll.: G.M. Davis

AF253071 (Davis et al., 1998) AF212895 (Wilke et al., 2000a) AF411141 (Wilke et al., 2001)

Alvania novarensis Frauenfeld, 1867

Australia, New South Wales, Balmoral, Edwards Beach; coll.: W.F. Ponder, 21 August 1998; USNM 892218

– JX970543 JX970582

Rissoa labiosa (Montagu, 1803)

Croatia, canal at Ploce (43.04528°N, 17.43627°E); coll.: A Falniowski, 5 September 2001

AY676128 (Szarowska et al., 2005) AY676117 (Szarowska et al., 2005) AY676126 (Szarowska et al., 2005)

Setia turriculata Monterosato, 1884

Bulgaria, Nessebar, bay NW of dam to Nessebar Peninsula (42.660°N, 27.717°E); coll.: T. Wilke

AF253084 (Davis et al., 1998) AY222652 (Szarowska and Wilke, 2004) AF367655 (Wilke et al., 2001)

Stenothyra cf. glabra A. Adams, 1861

China, Anhui Province, Sun Bang Township, Daliu Nat. Village, Xigan Stream (30.8780°N, 118.9143°E); coll.: G.M. Davis, T. Wilke

HQ623177 HQ623161 HQ623169

Stenothyra sp.

Australia, Queensland, Magnetic Island, Cockle Bay; coll.: W.F. Ponder, 22 February 1997; USNM 906000

JX970614 JX970544 JX970583

Fluvidona orphana Miller, Ponder & Clark, 1999

Australia, New South Wales, Sea Acres reserve, Port Macquarie; coll. S.A. Clark, 1989

– JX970545 JX970584

Fluviopupa sp.

New Caledonia, La Coulée; coll.: P. Bouchet, 17 November 1992; AMS C

JX970615 JX970546 JX970585

Halopyrgus pupoides (Hutton, 1882)

New Zealand, South Island, Cable Bay Reserve; coll.: M. Haase; AMS C408337

Hemistomia winstonefi (Haase and Bouchet, 1998) Leptopyrgus melbourni Haase, 2008

New Caledonia, La Coulée; coll.: P. Bouchet, 17 November 1992; AMS C 476047 New Zealand, North Island, Awakino Gorge, W bank of SH3, 150 m S intersection with Awakau Road; coll.: M. Haase

JX970616 JX970547 JX970586 JX970617 JX970548 JX970587 AY631075 (Haase, 2005) AY634053 (Haase, 2005) JX970588

Obtusopyrgus alpinus Haase, 2008

New Zealand, South Island, S side of Broken River Ski Club Road, SE of Arthur’s Pass; coll.:

AY631088 (Haase, 2005) AY634066 (Haase, 2005)

AF367668 (Wilke et al., 2001)

Please cite this article in press as: Wilke, T., et al. Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.025

15

T. Wilke et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx

Appendix A (continued) Classification

‘Tomichiinae’

Truncatellidae

Species

GenBank accession # COI/LSU rRNA/ SSU rRNA

M. Haase

JX970589

Opacuincola delira Haase, 2008

New Zealand, South Island,, NE of Karamea, Oparara River valley, Crazy Paving Cave; leg.: M. Haase, C. Mosimann, D.J. Roscoe, det.: M. Haase

AY631090 (Haase, 2005) AY634068 (Haase, 2005) JX970590

Potamolithus ribeirensis Pilsbry, 1911

Brazil, Sao Paulo, Iporanga River, Iporanga City; coll.: M. Bichuette & C. Santos; USNM 892038

JX970618 JX970549 JX970591

Potamopyrgus antipodarum (Gray, 1843)

Great Britain, London, West India Dock (51.5033°N, 0.011°W); leg.: T. Worsfold, det.: J. Dyson

EU573983 (Ponder et al., 2008) EU573989 (Ponder et al., 2008) EU573997 (Ponder et al., 2008)

Tatea huonensis (TenisonWoods, 1876)

Australia, New South Wales, Manly Lagoon; coll.: W.F. Ponder

JX970619 JX970550 JX970592

Coxiella striata (Reeve, 1842)

Australia, Lake Victoria, Point Lonsdale (38.28°S, 144.60°E); coll.: W.F. Ponder; AMNH c. 306253

– JX970551 JX970593

Tomichia ventricosa (Reeve, 1842)

South Africa, West Cape, between Struisbaai & Elim (34.6690°S, 19.8980°E); coll.: D. Herbert, 12 February 2000; AMS C433801

– JX970552 JX970594

Geomelania inornata Chitty, 1853

Jamaica, Trelawny Perish, Cocknit County, 2 km N of Quickstep (18.2580°N, 77.7075°W); coll.: G. Rosenberg

AF367629 (Wilke et al., 2001) HQ623156 AF367659 (Wilke et al., 2001)

Geomelania minor C.B. Adams, 1849

Jamaica, Manchester Parish, Auchtembeddie, Cracken Run, S of Troy (8.2203°N, 77.6228°W); coll D.G. Robinson & I.V. Muratov, 12 February 1997 Jamaica, Trelawny Perish, N of Falmouth (18.4910°N, 77.6577°W); coll.: G. Rosenberg & I.V. Muratov

JX970620 JX970553 JX970595

Truncatella scalaris (Michaud, 1830)

Jamaica, Trelawny Perish, N of Falmouth (18.4968°N, 77.6630°W); coll.: G. Rosenberg & I.V. Muratov, 12 September 1996

JX970621 JX970554 JX970596

Ascorhis tasmanica Martens, 1858

Australia, NSW, Carrel Bay; coll.: W.F. Ponder

AF129330 (Hershler et al., 1999) JX970555 JX970597

Truncatella pulchella (Pfeiffer, 1839)

?

Locality

AF253085 (Davis et al., 1998) AY222653 (Szarowska and Wilke, 2004) AF367658 (Wilke et al., 2001)

Please cite this article in press as: Wilke, T., et al. Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.025

Absent

Oval

Horny

Paucispiral or multispiral

Eccentric

Apertural varix

Operculum Shape

Structure

Coiling

Nucleus position

Long tapering

Normal

Absent

Absent Absent

Absent Present

Absent

Posterior pallial tentacle Absent Absent

Absent

?

Uniform or Variable unpigmented

Metapodial tentacle Omniphoric groove

Absent or present (longitudinal band)

Cephalic tentacle pigment

Absent

Parallel-sided (?)

Cephalic tentacle shape

Moderately long and wide Short to absent

Cochliopidae

Horny

Oval

Absent

Pyriform

Faint, wrinkled spiral threads

Absent Absent

Pyriform

Tapered

Normal

?

Central or eccentric Absent

Absent Absent

Absent Absent

Absent

?

?

?

Normal

Absent

?

Horny

?

Paucispiral or multispiral

Near-circular to oval Horny

Lithoglyphidae

Normal

Variable

Smooth or wrinkled

Horny

Absent Absent

Absent

Absent

Sometimes with longitudinal band

Tapered (?)

Normal

Absent

Absent Absent

Absent

Absent

Eccentric

Paucispiral

Horny

Oval

Often present

Pyriform

?

Normal

Absent or present Pyriform Absent

?

Valvatiform, globular to turriform

Tateidae

Elongate conic

Truncatellidae

Absent

Absent or rarely present

Long, used in locomotion

Calcareous plate absent or present

Eccentric

Paucispiral

Horny

Oval

Often present

Pyriform

Smooth or with spirals and/or axial ribs

Absent Absent

Absent

Usually absent, sometimes present

Absent Present

Absent

Absent

Uniform to unpigmented

Tapered or parallel sided Short to long and narrow

Normal

Without or with peg(s); sometimes with white smear

Eccentric

Paucispiral

Horny

Oval

Rarely present

Pyriform

Smooth or pits or wrinkles or spiral threads

Absent, but several Usually longitudinal pigment rings may pigment if present, be present sometimes anterior black band

Long and narrow

Long and mobile

Absent Usually present Present Absent (Pomatiopsinae) or absent (triculinae)

Absent

Absent

Normal to long, sometimes used in locomotion Short to moderately long and narrow Uniform or unpigmented

Eccentric to subcentric 2 projections

Paucispiral

Near circular to circular Horny

Absent

Circular

Smooth or Smooth weakly punctate (?)

With or Absent without peg in nucleus

Eccentric

Paucispiral

Horny

Oval

Absent

Normal

Smooth

Normal

Elongate conic to Conic to ovate neritiform conic

Stenothyridae

Last whorl Normal, last whorl rarely Normal contracted detached Spiral pores or Smooth or with Smooth except for Smooth or keeled Axial ribs or threads or weak to (rarely) pits in spiral rows rarely smooth smooth strong spirals or or spiral ribs (rarely) axials

Absent

Conical to turriform

Moitessieriidae Pomatiopsidae

Usually without band, Absent rarely with (longitudinal) band

Tapered

Normal

Absent

Highly variable Eccentric

Highly variable Paucispiral

Horny

Highly variable Oval

Near circular to pyriform Absent

Spirals (?)

Smooth or with Usually smooth or spirals with spirals, rarely with collabral elements

Normal

Planispiral to Trochiform, conical, elongate-conic ovate-conic

Rarely present Often present Absent

Variable

With or without Sometimes pigment, very with variable transverse or longitudinal bands ? Usually present, sometime absent Absent Absent

Parallel sided

Normal

Without peg

Smooth or with spiral keel

Normal

Conical, ovate-conic

Emmericiidae Hydrobiidae

Smooth, ? wrinkled, or with spirals or collabral elements

Globular to Planispiral to conical or blunt- elongate-conic conical Normal Rarely uncoiled Smooth Smooth or with spirals and/or collabral elements

Bythinellidae

Concentric, Paucispiral nucleus may be paucispiral Eccentric Eccentric

Calcareous

Oval

Eccentric or rarely central Absent; Without rarely peg pegs present

Ciliated rugae at base of right Absent cephalic tentacle

Normal

Head-foot Snout shape

White smear or pegs on inner Absent side; other projections

Oval to circular Horny, rarely with outer calcareous layer Paucispiral, rarely multispiral

Pyriform

Aperture shape

Protoconch sculpture

Smooth or spiral threads or axial ribs, sometimes reticulate Faint, wrinkled spiral threads, sometimes with axial ribs or reticulate Near circular Pyriform to pyriform Usually Rarely absent present

Usually absent, rarely with Smooth or transverse and/or spiral elements with weak to (rarely) strong spirals or (rarely) axials Usually absent, rarely with Usually transverse and/or spiral elements smooth, sometimes with fine pits or spirals

Normal

Conical

Assimineidae Bithyniidae

Trochiform, conical, or ovate-conic Conical to depressed trochiform Normal Normal

Amnicolidae

Teleoconch sculpture

Coiling

Shell Shape

Characters

Some morphological and anatomical characters of selected rissooidean families. For descriptions of the characters and their states see Hershler and Ponder (1998).

Appendix B

16 T. Wilke et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx

Please cite this article in press as: Wilke, T., et al. Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.025

Assimineidae Bithyniidae

Terminal

Cusps on outer marginal teeth Cusps on both sides

Absent

Zero or one

One

Seminal receptacle position relative to renal oviduct Bursa copulatrix

Distal

Distal

Renal oviduct position relative to gonopericardial duct Seminal receptacle number

Cochliopidae

Large to very Elongate

Usually present

Distal

Distal

One

Distal

Coiled

Lobulated

Tight Straight

Absent

Elongate

Distal

One

Distal

Simple coil

Lobulated (few large lobes)

Tight Straight

Absent

Usually

Usually present, sometimes absent Distal

Distal

Usually lobulated, sometimes simple Coiled

Tight Usually straight, sometimes looped

Sometimes present

Present

Distal

One

Distal

Coiled

?

Tight Straight

Absent

Larger on inner Larger on Larger on marginal tooth inner marginal inner marginal

Cusps on inner side Cusps on both sides Rounded

Continuous

Similar in size

Cusps on outer side Cusps on both sides Rounded

Cusps on outer side Cusps on both sides Rounded

Continuous

Long

Outer wing longer

absent

Cusps on outer side Cusps on both sides Rounded

Continuous

Outer wing longer

1–4 pairs

Short

Cutting edge much shorter than wing Long

1–4 pairs

Cutting edge much shorter than wing Absent to long Continuous

Lithoglyphidae

Continuous

Short or long

Outer wing longer or much longer

0–6 pairs

?

?

Cutting edge much shorter than wing

1–3 pairs

Present

Distal

Usually present, rarely absent

Distal

Coiled

Usually lobulated

Tight Usually (always?) Straight

Absent or present

Distal if present

Simple coil

?

Present

Distal

Large

Distal

Coiled and posterior to renopericardial duct (different from other families) Proximal

Distinctly lobulated

Tight Straight

Small to large

Distal

Small to large,

Proximal

Usually one, One sometimes multiple or none

Distal

Coiled

Lobulated

Reduced to vestigial or absent

Truncatellidae

Pointed

Inner

Outer

?

Short

About equal to cutting edge

3–4 pairs

Large

One (Truncatellinae), absent (Geomelaniinae) Distal

Distal

Simple and short or with single coil

?

Tight Straight

(continued on next page)

Very variable

Distal

One or zero

Distal

Coiled

Lobulated

Tight Straight or looped

Absent

Larger on inner marginal Subequal or similar in size

Rounded

Cusps on both sides

Membranous, sometimes continuous Cusps on outer side

Short to long

Cutting edge much shorter than wing

1–4 pairs

Absent or long and Fan-shaped or absent narrow

Subequal

Pointed

Inner

Inner to near terminal Pointed to rounded

?

Short

About equal to a little longer than cutting edge

3–6 pairs

Outer

Subequal

Highly variable

Tateidae

Absent Absent Absent Well developed, Large or small, opposite Elongate oval oval, middle of gill anterior part or middle or posterior part of ctenidium (if present), less than twice as long as broad to more than three times longer than broad

Outer

?

Short

Slightly shorter to longer than cutting edge

2–5 pairs

Absent; Absent intestine with long loop around style sac Wide and loose Tight Straight or Straight looped

Similar in size

One; sometimes partly One or zero or totally embedded in albumen gland

Distal

Coiled

Lobulated

Tight Straight

Absent

Larger on inner Larger on inner marginal marginal

Stenothyridae

Well developed, Well developed rarely absent

Absent Absent Small, opposite Well developed anterior part of or small ctenidium (if present), less than twice as long than broad

Absent or reduced

Moitessieriidae Pomatiopsidae

Cusps on outer Cusps on outer side ? side Cusps on both Cusps on inner side or ? sides (?) on both sides Rounded Rounded or wide Rounded

Continuous

0 to ca. 4 (?) pairs Longer or much longer than cutting edge Highly variable

Absent Absent Highly variable Medium-sized or large, narrow, posteriorly positioned

Highly variable Well developed

Emmericiidae Hydrobiidae

Well developed Usually well ? developed, rarely reduced or absent Absent Absent Absent Large, more Usually small, ? than three times oval, sublonger than centrally broad, opposite positioned to posterior part of ctenidium (slightly behind middle)

Bythinellidae

1–6 pairs

Distal

Coiled

Coiled

Renal oviduct structure

Lobulated

Lobulated

Tight Straight or looped

Absent

Female reproductive system Ovary structure

Intestine coil around style sac Tight Intestine in pallial cavity Straight

Gastrointestinal tract Caecum

Outer marginal distal end Rounded Rounded to (narrow pointed, narrow, very broad rounded, broad) Cusps on marginal teeth Markedly larger on inner marginal Markedly (equal to subequal in size, larger on markedly larger on inner inner marginal) marginal

Terminal

Length of ‘‘neck’’ of lateral Short teeth Junction of flank and face of Continuous lateral teeth Cusps on inner marginal teeth Cusps on outer side

0 to several pairs Short to very short relative to cutting edge Moderate to long ?

Very reduced Well to absent developed, with long filaments Absent Absent Present Medium-sized, oval, sub-centrally Well Large, more positioned developed, than three oval times longer than broad, opposite to anterior part of ctenidium

Usually well developed, rarely reduced

Amnicolidae

Radula Number of basal denticles on 1–3 pairs central teeth Length of lateral teeth outer Outer wing longer flanks relative to cutting edge

Food groove Osphradium

Mantle cavity Ctenidium

Characters

Appendix B (continued)

T. Wilke et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx 17

Please cite this article in press as: Wilke, T., et al. Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.025

Amnicolidae

Tubular gland (in haemocoel) usually present, rarely absent

Usually absent, sometimes present Absent Absent Absent

Kuroda and Habe (1957), Fretter and Graham (1978), Davis et al. (1985), Hershler and Hubricht (1988), Hershler and Thompson (1988), Thompson and Hershler (1991), Davis and Kang (1995), Davis and Rao (1997), Hershler et al. (2003a)

Penis: internal glands

Penis: ejaculatory duct Penial stylet

References Fukuda and Ponder (2003, 2004, 2005, 2006), Fukuda et al. (2006)

Absent or present Sometimes present

Absent

Penis: non-glandular lobes

Nonapocrine glands sometimes present

Lobes

Absent

Lobes

Penis: apocrine or similar external glands

Male reproductive system Testis structure

No

Thin glandular wall to thick glandular wall Absent Terminal to subterminal

Ventral wall of pallial oviduct Closed (open, closed forming ventral channel, thick and glandular)

Spermathecal duct Present Position of female pallial Terminal genital opening relative to capsule gland Female receives sperm via No pericardium/kidney

Variable

large Posterior

Bythinellidae

Krull (1935), Lilly (1953), Chung (1984), Ponder (2003)

Absent Rarely present

Long tubular gland in penis and haemocoel

Absent

Absent

?

No

Absent Terminal

Ventral channel

Radoman (1983), Falniowski (1987), Szarowska (1996), Haase et al. (2007)

Absent Absent

Long tubular gland in penis and haemocoel

Absent

Absent

Lobes

No

Absent Terminal

Closed sperm duct

Lying Lying against against albumen gland capsule gland Capsule About equal gland larger than albumen gland

Assimineidae Bithyniidae

Relative sizes of albumen and Variable capsule glands

Bursa copulatrix position Sometimes partly embedded in relative to albumen gland albumen gland

Characters

Appendix B (continued)

Davis et al. (1982), Hershler and Thompson (1992)

Absent Absent or present

Usually simple lobes, sometimes consisting of a sac or compound lobes Papilla, apocrine, or several other types of glands sometimes present; glands sometimes stalked Sometimes present Absent

No

Present Terminal

Radoman (1983), Szarowska (2006)

No

Absent Terminal

Closed

Partly or completely embedded within, or on right side of albumen land Variable

Lithoglyphidae

Sometimes present Absent

Thompson (1968), Radoman (1983), Hershler (1994), Szarowska (2006)

Kozhov (1936), Krause (1949), Radoman (1983), Thompson (1984), Hershler and Frest (1996), Hershler (1999), Sitnikova (2000, 2001), Hershler et al. (2007)

Absent Rarely present

Absent

Absent

Narrow Absent glandular fields (ridges) or large, circular glandular units often present, sometimes borne on crests

Usually simple Compound lobes lobes, sometimes a sac or compound lobes

No

Absent Terminal or subterminal

Tubular gland (in haemocoel) and large gland in penis opening to separate lobe Absent Absent Absent Absent

Absent

Absent

?

No

Absent Terminal

Closed

Sometimes partly embedded in albumen gland Variable

Emmericiidae Hydrobiidae

present Usually lying Posterior against albumen gland Variable About equal (albumen gland sometimes folded posteriorly, highly reduced in ovoviviparous taxa) Closed Closed

Cochliopidae

Boeters and Gittenberger (1990), Bodon and Giusti (1991), Bernasconi (1994)

Absent Absent

Sometimes present Absent (?)

Absent

?

No

Absent Terminal

Ventral channel

?

Posterior

Davis (1967, 1968, 1979, 1981), Davis et al. (1976, 1983, 1984, 1986, 1992), Davis and Kang (1990)

Often present Absent or (rarely) present

Absent or (rarely) present Absent

Absent or (rarely) present

Lobes

In a few taxa

Present Terminal

Thick and glandular

Variable

Posterior

Moitessieriidae Pomatiopsidae

Kosuge (1969), Hoagland and Davis (1979), Davis et al. (1986, 1988), Fukuda (1994), Hosaka and Fukuda (1996), Tamaki et al. (2002)

?

Posterior

Truncatellidae

Mostly absent

Sometimes present

Absent

Lobes

No

Absent Terminal or at middle

Davis and da Silva (1984), Ponder and Clark (1990), Ponder et al. (1991), Haase and Bouchet (1998, 2006), Miller et al. (1999), Haase (2008), Clark (2009), Haase et al. (2010)

Fretter and Graham (1962), Kosuge (1966), Rosenberg (1996)

Absent Absent

Absent

Absent

Absent

Lobes

Via kidney in Truncatellinae only

Absent Terminal

Short Ventral channel, occasionally separated to (Truncatellinae), form vestibule long (Geomelaniinae)

Capsule gland usually larger than albumen gland

Usually posterior

Tateidae

Absent Absent Sometimes present Rarely present

Absent

Absent

Absent

Lobes

No

No ventral channel – unclear as to whether thin or thick glandular wall ventrally Present Terminal

Variable

rarely absent Posterior

Stenothyridae

18 T. Wilke et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx

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T. Wilke et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx

Appendix C Bayesian trees of rissooidean taxa based on heads-or-tails (I) and fully automated PRANK (II) alignments of three gene fragments (COI, LSU rRNA, and SSU rRNA). BPP are provided at the nodes. The scale bar indicates the expected number of substitutions per site

19

according to the model of sequence evolution applied. The two primary outgroups, Cypraea tigris and Crepidula adunca, were removed from the tree a posteriori. For reasons of clarity, long branches were shortened (% values above respective branches indicate the fraction to which branches were reduced).

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T. Wilke et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx

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Please cite this article in press as: Wilke, T., et al. Pushing short DNA fragments to the limit: Phylogenetic relationships of ‘hydrobioid’ gastropods (Caenogastropoda: Rissooidea). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.025