Contributions to Zoology, 86 (1) – 2017Isabel T Hyman; Irantzu de la Iglesia Lamborena; Frank Köhler: Molecular phylogenetics and systematic revision of the south-eastern Australian Helicarionidae (Gastropoda, Stylommatophora)

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Results

Comparative morphology

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All species described in this paper share some features of the reproductive system, which distinguish them from other Australian helicarionids: They all have a very short or absent vagina, the epiphallus lacks a caecum, and the epiphallic flagellum produces a spermatophore which has a spiralling shape and branched spines (with the exception of Cucullarion, which has just a single unbranched spine near the junction of the capsule and tail-pipe). Correspondingly, the flagellum has internal cryptae matching the shapes of the branches. Within this so delimited group the reproductive system is quite conserved, with the majority of differences occurring in the penial complex. However, the genera also exhibit variation in the shape of the albumen gland, the degree of folding of the spermoviduct, the presence or absence of a short vagina, and the length and shape of the bursa copulatrix. Some of these differences may relate to shell reduction. In particular, the folding of the spermoviduct is absent or minimal in Brevisentis and Mysticarion, the groups with the least shell reduction, and much more advanced in the remaining genera. Similarly, the albumen gland is an elongate triangle in Brevisentis (similar to the shape seen in other helicarionid snails), a less elongate triangle in Mysticarion, Peloparion and Ubiquitarion, and a modified, rounded shape in Parmavitrina. The bursa copulatrix is longest (relative to the length of the spermoviduct) in Brevisentis and shortest in Parmavitrina.

Brevisentis is the only snail genus included in this study. It is characterized by its glossy, discoidal to depressedly globose shell of 4.6-5.3 whorls with a rounded whorl profile. Unique aspects of its reproductive system include a penis with a verge and internally sculptured with a combination of longitudinal ridges and pustules or anastomosing ridges, and a tightly coiled spermatophore with short branching spines covering the entire tail-pipe. Within Brevisentis, the three species can be differentiated by shell shape (Fig. 1A): B. kaputarensis is significantly smaller than both B. jacksoniensis and B. atratus (P < 0.0001 for both SH and SD), and while the latter two species are similar to one another in size, B. atratus is flatter than B. jacksoniensis, which has a slightly raised spire (H/D ratio: P < 0.0001). Brevisentis kaputarensis is similar in shell shape to B. atratus, but has a flatter shell than B. jacksoniensis (H/D ratio: P < 0.0001).

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Fig. 1. Comparative analyses of shell shapes by plotting shell height (H) against shell diameter (D). A. Brevisentis, B. Mysticarion, C. Parmavitrina, D. Peloparion and Ubiquitarion.

The three congeners can also be differentiated by the length and shape of the penis (shorter, proximally swollen in B. jacksoniensis; longer, with internal narrowing in B. atratus; short, tubular in B. kaputarensis), by the internal sculpture of the penis (longitudinal pilasters becoming pustulose in B. jacksoniensis and B. atratus, anastomosing ridges in B. kaputarensis), and by the length and shape of the penial verge. The length of the penial verge is variable in B. kaputarensis and B. jacksoniensis but is always short in B. atratus.

The arboreal semislugs belonging to Mysticarion are united by their golden, globose shell of 3.0-4.5 whorls and generally pale body colour (with the exception of M. obscurior sp. nov., which has a more heavily pigmented body). In all species, the penis is relatively long and the epiphallus short. The penis interior contains longitudinal pilasters and a penial verge. The flagellum produces a spermatophore with branched spines present on the base of the capsule and continuing on the tail-pipe. The four species of Mysticarion can be differentiated by shell shape (visible on an H/D scatterplot; Fig. 1B) and body colour. Mysticarion obscurior sp. nov. could not be included in statistical tests due to insufficient material. M. porrectus is larger than its congeners, and is flatter and less globose than the other species, differing significantly in H/D ratio from both M. insuetus (P < 0.0001) and M. hyalinus (P < 0.0001). Mysticarion hyalinus is only slightly smaller than M. porrectus, with only a weakly significant difference in shell diameter (P < 0.05). Mysticarion obscurior sp. nov. is slightly smaller again, and is flatter than either M. hyalinus or M. porrectus; and M. insuetus is the smallest species, differing significantly in shell diameter from both M. porrectus and M. hyalinus (P < 0.001), with a relatively high spire. Anatomical differences include the flagellum and spermatophore shape (particularly the number of branches on the spermatophore and, correspondingly, the number of cryptae in the flagellum), the relative length of the penis and epiphallus, and the internal sculpture in the penis and size and shape of the penial verge.

The large semislugs of the genus Parmavitrina have flattened shells of 2.5-4.0 whorls with a wide aperture and, in some species, a membraneous base. All species have a strongly keeled tail and a cephalic shield. The genital morphology defining this group consists of a penis with primarily longitudinal sculpture and sometimes with a penial verge, a short vagina and bursa copulatrix, distinct cryptae in the flagellum and adjacent epiphallus, and a spermatophore with branching spines present on the capsule and a short, smooth tail-pipe. Each species can be separated from its congeners by a combination of penis size and shape, presence and shape of a penial verge, and sculpture of the penis interior, and thickness of the penis and penial sheath. Some of the species can be distinguished based on shell shape (although we have insufficient measurement data for P. maculosa sp. nov. and P. flavocarinata sp. nov.). For example, P. megastoma and P. flavocarinata sp. nov. are smaller than the remaining taxa (P. megastoma differs significantly in SD from P. planila­bris, P. disposita and P. rubrica, P < 0.0001). Parmavitrina planilabris has a significantly higher H/D ratio than P. rubrica and P. disposita (in both cases P < 0.0001), indicating a taller shell. However, P. rubrica and P. disposita cannot be separated based on shell shape alone, with no significant differences between SH and H/D ratio and only a weak difference in SD (P < 0.01) (Fig. 1C).

Parmavitrina encompasses two morphologically distinct groups, each containing three similar species: the planilabris-group (P. planilabris, P. rubrica, P. disposita), and the megastoma-group (P. megastoma, P. maculosa sp. nov., P. flavocarinata sp. nov.). Species in the planilabris-group are larger with a higher whorl count (3.5-4.0), have a large penis, very short vagina and bursa copulatrix, and longer spines on the spermatophore. In contrast, the species in the mega­stoma-group are smaller, with shells of fewer than 3.0 whorls, have a slender penial complex, a longer vagina and bursa copulatrix, and short, highly branched spines on the spermatophore. However, the two groups are also unified by numerous morphological characters as outlined above.

Cucullarion, another group of moderately large semislugs, contains two species with very reduced shells that are covered by extensive mantle lobes. This group represents the most northerly representative of the southeastern Australian radiation, exhibiting the highest degree of shell reduction. The two species, C. parkini and C. albimaculosus, are unified by their reduced shell, retaining calcium only on the plate-like dorsal surface, and slender bodies. Due to the degenerate shell, only shell diameter and number of whorls were measured, and due to lack of sufficient material no statistical comparisons could be made. Both species have a highly folded spermoviduct and a moderately long bursa copulatrix with an elongate sac. The flagellum is long and slender with only a single internal crypt in the adjacent epiphallus, indicating a spermatophore with a single unbranched spine. The penial anatomy varies between the two species and features a combination of longitudinal pilasters and circular pilasters or pustules.

Two small semislugs so far placed together in the genus Peloparion are similar in shell shape and size and both have rounded, black-lined shell lappets. However, while they cannot be separated based on their shell shape (P > 0.2), Ubiquitarion iridis is significantly smaller (SH, SD: P < 0.0001) and they can be clearly distinguished on an H/D scatterplot (Fig. 1D). Their genital anatomy is also rather similar, with a medium length, tubular penis, a short vagina and a moderate length bursa copulatrix. However, the epiphallus, flagellum, spermatophore and penis interior differ considerably.

Molecular analyses

The final concatenated sequence dataset contained a total of 96 sequences of 16S, including 14 Genbank sequences, and 93 COI sequences, including 12 Genbank sequences. Some samples have been represented by either only the COI or 16S sequence while the lacking sequence fragment was coded as missing data. However, we had usually at least one complete set of sequences for each species identified by comparative morphology. The final 16S alignment had a total length of 1,123 aligned nucleotide positions. This alignment was used to calculate uncorrected p-distances between all sequences. Of these 1,123 alignment positions, 859 were retained after removing ambiguous alignment positions with Gblocks for subsequent phylogenetic analysis.

The time-reversible model with invariant sites and a gamma distribution of rates (GTR+I+G; Tavaré, 1986) has been found to be the best-fit model of sequence evolution for both the 16S and COI sequence datasets by means of both the Bayesian and Akaike Information Criterion. Xia’s et al. (2003) test indicated no or little saturation in both mitochondrial fragments (Iss < Iss.c with P < 0.001).

The best maximum likelihood tree (Fig. 2) was rooted by using the designated out-group sequences, which were selected based on the phylogenetic tree presented by Hyman et al. (2007). Among the in-group, the southeastern Australian helicarionid radiation, some but not all genera as currently circumscribed have been retrieved as monophyletic groups. While the monophyly of Brevisentis, and Cucullarion as presently delimited has been confirmed with high nodal support, the genera Mysticarion, Peloparion, Parmavitrina and Desidarion have each been found to be polyphyletic with generally high bootstrapping support.

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Fig. 2. Best maximum likelihood tree based on analysis of the concatenated data set of fragments of the mitochondrial genes 16S and COI. Ambiguous alignment sites in 16S removed by using Gblocks. Numbers on branches indicate nodal support based on 200 thorough bootstrap replicates for a total of 10 independent runs. Scale bar indicating 5% of modelled sequence divergence.

The analysis of the concatenated dataset revealed the sister pair of Helicarion spp. and ‘Peloparioniridis to form the most basal offshoot among the southeastern Australian radiation. Cucullarion was found to be the sister of all remaining in-group clades containing Parmavitrina+Desidarion, Brevisentis, Peloparion helenae, and Mysticarion, respectively (Fig. 2). However, there was usually only weak nodal support for the principal branching patterns in the phylogenetic tree. In order to explore this instability further, we have analysed the two sequence datasets separately. These analyses revealed that the two mitochondrial fragments supported vastly different topologies with respect to the relationships between the principal clades (Figs 3-4). In particular the COI tree is characterized by extremely low nodal support for the relationships among the principal clades. Moreover, the in-group was non-monophyletic as Helicarion clustered among the out-group taxa (Fig. 3). By contrast, in the 16S tree the monophyly of the in-group is well-supported while the relationships among its principal clades attracted weak nodal support only (Fig. 4).

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Fig. 3. Best maximum likelihood tree based on analysis of the fragment of the mitochondrial gene COI. Numbers on branches indicate nodal support based on 200 thorough bootstrap replicates for a total of 10 independent runs. Scale bar indicating 4% of modelled sequence divergence.

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Fig. 4. Best maximum likelihood tree based on analysis of the fragment of the mitochondrial gene 16S. Ambiguous alignment sites removed by using Gblocks. Numbers on branches indicate nodal support based on 200 thorough bootstrap replicates for a total of 10 independent runs. Scale bar indicating 4% of modelled sequence divergence.

By contrast to relationships among principal branches, the relationships among the tips of the tree are consistently well-supported and there has been little, if any, conflict between the topologies produced for different data sets. Some noteworthy findings are that among Brevisentis, specimens from Wollemi NP identified as B. atratus do not cluster with any of the three currently recognized species, and are thought to represent a yet undescribed species. However, because of the lack of reproductively mature specimens, we currently refrain from naming this species.

We also found that Mysticarion is monophyletic only if Fastosarion staffordorum is included. Sequences of this taxon group closely with M. hyalinus with very little genetic differentiation (Tables 1-2). The rather small amount of genetic differentiation confirms that M. porrectus, once suggested to be a species complex, is simply a widely distributed species. Specimens initially identified as M. insuetus occupied two highly distinct clades. This genetic differentiation is underpinned by subtle morphological differences that are in support of the taxonomic distinctiveness of these two previously unrecognized groups. Based on the specimens from the Hunter Valley, we describe a new species, M. obscurior sp. nov., below. Neither Desidarion nor Parmavitrina are monophyletic: Parmavitrina planilabris groups very closely with both D. rubricus and D. dispositus. The two Desidarion species also share a number of morphological characters with Parmavitrina; based on these similarities we treat both genus names as synonyms below. One population of D. rubricus from around Taree shows some genetic divergence. However, this is accompanied by insignificant anatomical differences. The two new species P. maculosa sp. nov. and P. flavocarinata sp. nov., recognized by their morphology, group closely with P. mega­stoma. The two species currently placed in Peloparion, P. helenae and P. iridis, do not group together; based on this and on considerable anatomical differences, we describe a new genus for P. iridis below.

The evolutionary divergence within and between species was estimated by calculating uncorrected pairwise distances across all sequences. We found that range of intraspecific distances in COI (0-0.04) and 16S (0-0.72) overlapped with the range of interspecific distances in both genes to some degree (COI: 0.029-0.157; 16S: 0.02-0.234) (Fig. 5, Tables 1-2). However, there has been no overlap between the average intraspecific distances per species (< 0.028, on average 0.011 in COI; < 0.022, on average 0.006 in 16S) and the average interspecific distance between any two congeners (0.036-0.095, on average 0.072 in COI; 0.029-0.114, on average 0.073 in 16S).

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Fig. 5. Comparison of intra- and interspecific genetic p-distances for the two mitochondrial fragments analysed. A. Frequency distributions of distances in COI. B. Frequency distributions of distances in 16S.

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Table 1. Average pairwise distances in 16S within and between species under pairwise removal of gaps.

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Table 2. Average pairwise distances in COI within and between species.