Lessons from Timor: Shells are poor taxonomic indicators in Asperitas land snails (Stylommatophora,Dyakiidae)

The limacoid land snail Asperitas Gude, 1911 encompasses several vividly coloured, yet poorly known species that are distributed throughout the Lesser Sunda Islands, Sulawesi, and the Moluccas. We examined the variation in shell morphology, including the use of geometric morphometrics, and reproductive anatomy of snails from Timor and several adjacent islands. These studies revealed that none of the taxa described from Timor and considered to be endemic to this island could be reliably distinguished from any of the others. In addition to the systematic ambiguity of morphological characteristics, we uncovered rather low amounts of genetic differentiation in the mitochondrial marker cytochrome c oxidase subunit I that was not consistent with the current taxonomy. Based on these observations, we conclude that there is just a single Asperitas species on Timor that exhibits notable variability in shell characters and body colour. This variability is ascribed to the combined effects of polymorphic colour patterns with locally varying selection in different habitats and along altitudinal gradients. Individuals from the adjacent islands of Flores, Sumbawa, Solor and Romang, which are deemed to represent distinct species-level taxa, exhibit similar amounts of variation in shell and body colour. They exhibit a consistent reproductive anatomy and overall negligible amounts of mitochondrial differentiation from the populations on Timor. Patterns in shell variation do not lend support to previously held ideas that different Sunda Islands harbour distinct radiations of polytypic Asperitas species. By contrast, we suggest that the taxonomic diversity of Asperitas has been overestimated for the over-reliance on shell features and that many presently accepted taxa are likely mere synonyms of fewer and more widespread species.     

Evidence of a reproductive barrier between two forms of the marine periwinkle Littorina fabalis (Gastropoda)

Studies of allozyme variation may reveal unexpected patterns of genetic variation which challenge earlier conclusions of species delimitations based on morphological data. However, allozyme variation alone may not be sufficient to resolve this kind of problem. For example, populations of the marine intertidal snail Littorina fabalis (=Littorina mariae) from wave exposed parts end from protected parts of the same shores are distinguished by different alleles of arginine kinase (Ark) while indifferent, or very nearly so, in another 29 loci. Intermediate populations have large deficiencies of exposed/sheltered heterozygote classes of Ark and we have earlier suggested habitat-related selection in this locus as the explanation. In this study we estimated growth rate of individual snails of different Ark-genotypes in three different habitats (exposed, sheltered and intermediate). In all habitats the snails homozygous for alleles of ‘exposed’ type grew faster and matured at a larger size than did snails homozygous for alleles of ‘sheltered’ types. This relationship was indirectly confirmed in three additional sites of intermediate exposure where exposed AA-genotypes dominated among large (>8 mm) snails while the sheltered genotypes dominated among small (<5 mm) snails of truly sympatric samples. We furthermore found small differences in allele frequencies of two other loci (Pgi and Pgm-2) and in shell colour frequencies, comparing sympatric snails of exposed and sheltered Ark-homozygotes. Although we found no signs of habitat-related selection among snails of different Ark-genotype, or selection against heterozygotes, we cannot reject selection in Ark, as our experiments only covered one island, one season and grown-up snails. The coupling between allozyme and phenotypic characters in strictly sympatric samples of snails suggests the presence of two gene pools. Perhaps the large and small forms of L. fabalis represent very closely related cryptic taxa. However, introgression between them seems a possible explanation for the striking similarities in the vast majority of morphological and allozyme characters.     

Novel colour polymorphisms in a hybrid zone of Mandarina (Gastropoda: Pulmonata)

A hybrid zone of the land snails Mandarina mandarina and Mandarina chichijimana in the oceanic Bonin Islands was studied morphologically and genetically to show the potential of hybridization as a source of morphological novelties. These species are clearly distinguishable on the basis of allozymes, colour polymorphisms, shell form and genital morphology, but exhibit a hybrid zone from the northeast to southwest of Chichijima island. There is a cline in the frequency of the colour patterns characteristic of each of the species, and specimens with intermediate colour pattern on the shells appear in the hybrid zone. Not only specimens with colour patterns that are overlapping of the patterns of the two species, but also specimens with unique colour patterns appear in the hybrid populations. These unique colour patterns are not found in the pure populations of both species or other Mandarina species in Chichijima Islands, and it is suggested that these are produced by the hybridization. Because of the appearance of many types of unique colour patterns, variability of the colour polymorphism in the hybrid populations are remarkably higher than that in the pure populations. This result suggests that the novel morphology is produced by the hybridization between species with distinctive morphology. This reveals the importance of hybridization as a source of morphological variation, diversity and evolutionary novelty.     

Statistical discrimination of sex in Melanoides tuberculata (Gastropoda: Thiaridae)

Systematists may rely on morphometric differences among samples of specimens for the recognition of living and fossil species, even though morphometric differentiation may be caused by non-genetic factors, such as ecophenotypy, differential growth rates and taphonomic mixing. When genetic differences between sexes or among closely related species are expressed as differences in the morphology of the individual or population, potentially valuable information becomes available to the systematist for a variety of genetic and ecological investigations. We have studied the morphology of the freshwater snail Melanoides tuberculata (Muller, 1774) in Israel, where males occur in what would otherwise be normally parthenogenetic (all female) populations. In modern M. tuberculata, sex may be determined by observation of gonadal tissue; in fossil specimens, any classification according to sex must be accomplished using only preservable features of the mineralized shell. Previous research confirmed that in large samples, mean shell shape of male and female snails differed significantly, but the degree of difference was too small to identify the sex of any individual specimen. We apply a three stage process that results with a high degree of accuracy in the discrimination of individual M. tuberculata specimens by sex on the basis of continuous morphological characters: (1) measurement of many aspects of shell morphology of individuals of known sex, and stepwise discrimination to discover which of the variables, if any, contribute to the morphometric differentiation of males from females (one time only, for the species); (2) use of these selected variables in a clustering procedure to make a preliminary assignment of each specimen to sex; (3) use of cluster assignments in a discrimination procedure to optimally predict sex. For species that exhibit morphometric differences between two groups, and for which continuous morphometric variation precludes the a priori recognition of discrete clusters, this sequential procedure may be of broad applicability. These objective methods may be applied to the discrimination within any set of specimens for which the hypothesis of two, and only two, constituent groups may be entertained.     

Extreme,continuous variation in an island snail: local diversification and association of shell form with the current environment

On Rosemary Island, a small continental island (11 km²) in the Dampier Archipelago, Western Australia, snails of the genus Rhagada have extremely diverse morphologies. Their shells vary remarkably in size and shape, with the latter ranging from globose to keeled‐flat, spanning the range of variation in the entire genus. Based primarily on variation in shell morphology, five distinct species are currently recognized. However, a study of 103 populations has revealed continuity of shell form within a very closely‐related group. A phylogenetic analysis of specimens from Rosemary Island, and other islands in the Dampier Archipelago, indicates that much of the morphological variation has evolved on the island, from within a monophyletic group. Within the island, snails with distinct shell morphologies could not be distinguished based on variation in mitochondrial DNA or their reproductive anatomy. The shell variation is geographically structured over a very fine scale, with clines linking the extreme forms over distances less than 200 m. Although there is no evidence that the different forms have evolved in isolation or as a consequence of drift, there is a strong association between keeled‐flat shells and rocky habitats, suggesting that shell shape may be of adaptive significance. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 756–769.     

Species limits in molecular phylogenies: a cautionary tale from Australian land snails (Camaenidae: Amplirhagada Iredale, 1933)

By complementing two independent systematic studies published recently on the Western Australian land snail Amplirhagada, we compare levels of morphological variation in shells and genitalia with those in the mitochondrial markers cytochrome c oxidase (COI) and 16S to evaluate the utility of mtDNA markers for delimiting species. We found that penial morphology and mitochondrial divergence are generally highly consistent in delimiting species, while shells have little overall taxonomic utility in these snails. In addition to this qualitative correspondence, there is almost no overlap between intraspecific and interspecific genetic distances in COI, with the highest intraspecific and lowest interspecific distance being 6%. This value is twice the general level suggested as a DNA barcode threshold by some authors and higher than the best average found in stylommatophoran land snails. Although in Amplirhagada land snails DNA barcoding may provide meaningful information as a first‐pass approach towards species delimitation, we argue that this is due only to specific evolutionary circumstances that facilitated a long‐termed separate evolution of mitochondrial lineages along spatial patterns. However, because in general the amounts of morphological and mitochondrial differentiation of species depend on their evolutionary history and age, the mode of speciation, distributional patterns and ecological adaptations, and absence or presence of mechanisms that prevent gene flow across species limits, the applicability of DNA barcoding has to be confirmed by morphological studies for each single group anew. Based on evidence from both molecular and morphological markers, we describe six new species from the Bonaparte Archipelago and revise the taxonomy of a further two. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 165 , 337–362.     

Redescription of the western Balkan species Xerocampylaea waldemari and its phylogenetic relationships to other Urticicolini (Gastropoda: Hygromiidae)

The proper identification of species has significant implications for conservation and general knowledge of ecosystem variety. It is especially important for biodiversity hotspots and former Pleistocene refugia, such as the Balkans. In this paper, we evaluate the taxonomic status of the endemic Balkan species Fruticicola waldemari A. J. Wagner, 1912, using both recently collected material and museum specimens. Phylogenetic analyses based on two mitochondrial markers, 16S rRNA and cytochrome c oxidase subunit I, show that this species is closely related to the representatives of Xerocampylaea, including a conchologically similar and partly sympatric species, X. erjaveci. Analyses of shell and genital morphology as well as mitochondrial DNA sequences indicate that X. waldemari and X. erjaveci are separate but closely related species. These species differ in shell morphology (size, umbilicus diameter, microsculpture) as well as genital morphology. Nevertheless, a further study with more comprehensive sampling of both taxa is required to fully understand the complex pattern of genetic and morphological variation observed.     

Mitochondrial phylogeny of extant Hawaiian tree snails (Achatinellinae)

Hawaiian tree snails in the endemic subfamily Achatinellinae display a staggering variety of shell colors and banding patterns. Despite numerous attempts to classify this morphological variation, a conclusive phylogeny has not been proposed. To improve conservation efforts, we sought to better understand the species identities and phylogenetic relationships among the extant species of Achatinella and Partulina using partial mitochondrial 16S ribosomal DNA sequences. The reconstructed phylogeny showed a high degree of support for more recent branches, but gave little support to deeper nodes. The most confident branches challenge previous systematic arrangements of these snails, grouping species that previously had been placed into different subgenera. High levels of sequence divergence within some species may reflect the long-term isolation of subpopulations. Rapid rates of sequence divergence may have saturated base substitutions and contributed to the lack of resolution of higher-order relationships. We did not find support for the monophyly of the Achatinella species, nor thus for a single colonization of Oahu from Maui Nui.     

Cryptic, genetically extremely divergent, polytypic, convergent, and polymorphic taxa in Madagascan Tropidophora (Gastropoda: Pomatiasidae)

Madagascar's magnificent and environmentally threatened endemic radiation of the land-snail genus Tropidophora Troschel has recently been classified into three subgenera, 95 species and 142 varieties, based on often subtle conchological variation among small samples; it seems best to ignore temporarily this confusing plethora of names until true biological species and their relationships are better understood. The author's field work in 1990 succeeded in obtaining live Tropidophora from 40 populations distributed throughout the island. Allozyme analysis (108 snails, 15 loci, 117 alleles) yielded a cladogram suggesting nine genetically coherent taxa (T. taxa A-I), each represented in the collections by 1–10 populations. Comparisons among shells (total 1634) and penes (total 31 from 20 populations representing eight of the nine taxa) revealed: (1) two conchologically indistinguishable taxa (H and I) fixed for alternative alleles at 13 of 15 loci, with only a very subtle difference in penes, and with mosaic and overlapping geographical distributions in the Northeast; (2) two extremely polytypic taxa (C in the Southwest, F in the Southeast) with parallel trends toward depressed, broadly umbilicate, heavily sculptured shells with apertural lips widely reflected at the umbilicus at inland, more arid sites, resulting in sympatric convergence; (3) one southeastern taxon (G) in which the penis apparently doubles in length but the shell does not change 6.0 km to the northwest, but in which the shell shifts dramatically in sculpture, colour, and lip reflection 0.5 km to the westnorthwest; and (4) generally such extreme intra- and inter-population variation in shell and male-genital characters as to render many of them dubious at best for systematics. Thus the Madagascan Tropidophora present a fascinatingly complex problem in evolutionary morphology/ecology, the solution of which will require even more extensive collecting, followed by molecular comparisons or detailed anatomical comparisons, or both. The total number of biological species is still likely to be quite large, despite irrelevance of much of the current taxonomy, because many smaller species remain to be discovered.     

Phylogeny and reclassification of the Caucasigenini radiation from the Caucasus region (Gastropoda,Hygromiidae)

The Caucasigenini is an endemic radiation of hygromiid land snails from the Caucasus region. A phylogenetic analysis of morphological characters of the genitalia and the shell showed that the morphological characters are insufficient for resolving the relationships within the Caucasigenini. Convergences of the few parsimony informative characters in other groups of the Hygromiidae demonstrate that these characters are not reliable indicators of phylogenetic relationships. Phylogenetic analyses of sequences of cox1, 16S rDNA, 5.8S rDNA, ITS2 and 28S rDNA revealed several well‐supported groups. The relationships among these groups could not be resolved. It is likely that these groups originated in a rapid radiation during the uplift of the Caucasus. Based on the molecular phylogeny, we propose a new classification of the species of the Caucasigenini and establish a new genus, Lazicana gen. n.     

Phylogeography and morphological variability in land snails: the Sicilian Marmorana (Pulmonata, Helicidae)

Land snails have long been recognized as suitable organisms for studying phenotypic differentiation and phylogeny in relation to geographical distribution. Morphological data (shell and anatomy biometry on different geographical scales) and partial sequences from mitochondrial genes (cytochrome oxidase subunit I , 16S rDNA) were used to test whether morphological patterns match phylogeny in a diversified group of Sicilian rock-dwelling land snails belonging to the genus Marmorana . The taxonomic implications of the three character sets (shell and anatomical biometry and molecular data) were also considered. The inferred phylogenetic relationships do not match morphological (shell and genitalia) patterns. This result may significantly modify the current taxonomy. Mitochondrial based reconstructions define several supported clades well correlated with geographic distribution and populations were found to be distributed parapatrically. The progressive decline in mitochondrial DNA sequence similarity over a distance of 250 km is consistent with a model of isolation by distance, a pattern previously recognized for other groups of land snails. For one clade of Marmorana , colonization along Mediterranean trade routes appears to be a possibility.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 809–823.     

Molecular insights into the taxonomy of Hypanis (Bivalvia, Cardiidae, Lymnocardiinae) in the Black Sea lagoons

In this study, we used data from morphology and three DNA markers to assess the taxonomic status of the putative bivalve species Hypanis colorata and Hypanis angusticostata in a Black Sea lagoon, the Razelm Lake in Romania. The morphological data (the shape of shell ribs and the multivariate analysis of morphometric variance of three variables constructed as the ratios between the main dimensions of the shell) confirmed that the two analyzed species are distinct morphological entities. Three molecular markers, one from the nuclear genome (18S rRNA) and two from the mitochondrial genome (16S rRNA and COI), showed extremely reduced sequence divergence (0?C0.1%) between the two putative species. Based on these results, we suggest that H. angusticostata and H. colorata are morphotypes of a single species.     

Taxonomic Boundaries and Craniometric Variation in the Treeshrews (Scandentia,Tupaiidae) from the Palawan Faunal Region

The taxonomy of treeshrews (Order Scandentia) has long been complicated by ambiguous morphological species boundaries, and the treeshrews of the Palawan faunal region of the Philippines are no exception. Four named forms in the genus Tupaia Raffles, 1821, have been described from four island groups based on subtle qualitative morphological characters, and as many as three distinct species have been recognized. A recent molecular phylogenetic study of relationships among Tupaia species suggests that the two currently-recognized treeshrew species from the Palawan faunal region diverged very recently relative to other sister-species divergences within the genus and may not represent species-level taxonomic entities. Here we review the taxonomic and biogeographic histories of the Tupaia taxa from this region. We also collected craniodental data from 133 skulls of all four named forms, representing five island populations, and conducted univariate and multivariate analyses on these data. Our morphometric results are consistent with molecular results, further suggesting that there is insufficient evidence to recognize T. moellendorffi Matschie, 1898, as a separate species from T. palawanensis Thomas, 1894. Our analyses also revealed a craniodentally divergent population from the island of Balabac, which has never been considered a distinct subspecies (or species) from the population on Palawan. These results have conservation implications for the island populations in our analyses, but additional surveys and molecular evidence will be required to fully assess conservation priorities for the treeshrews of the Palawan faunal region.     

Divergence in the shell morphology of the land snail genus Aegista (Pulmonata: Bradybaenidae) under phylogenetic constraints

The role of natural selection in phenotypic evolution is central to evolutionary biology. Phenotypic evolution is affected by various factors other than adaptation, and recent focus has been placed on the effects of phylogenetic constraints and niche conservatism on phenotypic evolution. Here, we investigate the relationship between the shell morphology and habitat use of bradybaenid land snails of the genus Aegista and clarify the causes of the divergence in shell morphology among phylogenetically related species. The results of ancestral state reconstruction showed that arboreal species have evolved independently from ground‐dwelling species at least four times. A significant association was found between shell shape and habitat use, despite the existence of a certain degree of phylogenetic constraint between these traits. A principal component analysis showed that arboreal species tend to have a relatively high‐spired shell with a narrow umbilicus. By contrast, ground‐dwelling species have a low‐spired shell with a wide umbilicus. Although the latitude and elevation of the sampling locations showed no relationship with shell morphology, the geology of the sampling locations affected the shell size of arboreal species. The development of a well‐balanced shell shape is one effective method for reducing the cost of locomotion under the force of gravity in each life habitat, resulting in the divergence in shell morphology and the independent evolution of morphologically similar species among different lineages. The present study suggests that ecological divergence is probably the cause of shell morphology divergence in land snails. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 114 , 229–241.     

Divergent shell shape as an antipredator adaptation in tropical land snails

Although many land snails exhibit amazingly divergent shell shapes in the tropics, the functions of these remain obscure. Here we show that a modified aperture shape acts as an impediment specifically to predation by a snail-eating snake. Pareas iwasakii (Colubridae: Pareatinae) uses a unique method to feed on land snails: the snake extracts the soft body from the shell through the aperture by alternately retracting its mandibles. The snail Satsuma caliginosa (Camaenidae: Camaeninae) has apertural variation in regard to the presence of snail-eating snakes. Our experiments demonstrated that the distorted aperture mechanically impeded predation by this gape-limited predator, interrupting the mandibular movements. In contrast, congeneric snails with round apertures did not escape predation by snakes. The paleobiogeography of the focal area indicates that the subspecies Satsuma caliginosa picta, which does not have apertural modification, was derived from a defensive ancestor after the extinction of snail-eating snakes. Our study suggests a possibility that snail-eating snakes are responsible for divergent shell shapes in a variety of tropical land snails.     

PATTERNS OF QUANTITATIVE VARIATION IN LEPIDOPTERAN WING MORPHOLOGY: THE CONVERGENT GROUPS HELICONIINAE AND ITHOMIINAE (PAPILIONOIDEA: NYMPHALIDAE)

Wing morphology has historically been a major focus in taxonomic and evolutionary studies of lepidopterans. However, general patterns of quantitative variation and diversification in wing sizes and shapes and the factors underlying them have been unexplored. A morphometric study of wing variation in the convergent heliconine and ithomine butterflies reveals remarkable similarities, both in their morphologies at a given size and in their patterns of allometry and variability. The groups differ primarily in the relative lengths of inner and outer forewing margins, with larger species being more similar across groups than smaller ones. Allometric size-scaling variation accounts for more than 90% of the total morphological variation in the two groups and thus seems to be the major determinant of wing shape. Forewings and hind wings are isometric in size (area) with respect to one another; however, wing shape within and among groups is significantly allometric, resulting in considerable shape differences between small and large species. A strong trend of increasing variability from anterior to posterior along the wings is consistent with hypotheses of aerodynamic constraint. Wings and bodies represent classical morphological “character suites” in that size and shape variation are more tightly correlated within suites than among them. Such complexes argue against the overriding importance of aerodynamic factors, such as wing load and muscle development, in constraining gross morphology.     

Molecular phylogeny, taxonomy and evolution of the land snail genus Iberus (Pulmonata: Helicidae)

Partial DNA sequences of two mitochondrial genes [cytochrome oxidase subunit I (COI) and 16S rRNA] from 59 specimens of Iberus were used to test the validity of the described morphospecies of this genus, and examine genetic divergences within and between main phylogenetic groups. Both gene fragments showed phylogenetic concordance. The COI gene was found to be faster evolving than the 16S gene and was fully protein-coding with no insertions or deletions. 16S rRNA was more informative than COI for resolving basal nodes. Both individual and combined analyses of the two gene fragments revealed five main phylogroups. These five groups are genetically unique lineages that are allopatrically distributed and considered to have full species status. Further subdivisions were also considered. Shell morphology was suitable for delimiting species boundaries, but several incongruences between morphology and mtDNA phylogeny were observed. These incongruences were considered consequence of hybridization between Iberus cobosi and Iberus marmoratus , and the result of shell shape polymorphism in Iberus rositai . According to spatial patterns of sequence divergence, life habits and shell morphology may be concluded that the keeled-flat shelled snails independently originated several times within Iberus and they could represent cases of similar shell adaptation to a karstic arid environment.     

The evolution of extreme shell shape variation in the land snail Ainohelix editha: a phylogeny and hybrid zone analysis

Ainohelix editha from Hokkaido, Japan, exhibit great geographical variation in their shell morphology. In particular, A. editha in two quite separate locations, Shimamaki and Samani, are striking because they are extremely flat and have a sharp keel, whereas at adjacent sites the shells are globular or depressed-globular. We used mitochondrial 16S rRNA and nuclear ITS-2 sequences to infer a phylogeny among 47 snails from 29 locations. Snails from the two keeled-flat populations clustered separately in the phylogeny, suggesting that this unusual shell form could have evolved independently. A morphological analysis of shells collected along a transect between keeled-flat and globular snail sites showed a cline for shell shape and the angle of the keel. Two different mtDNA lineages were found across the transect, with a cline for an ITS-2 single nucleotide polymorphism. Together, the results may suggest a lack of reproductive isolation between keeled-flat and globular snails, with possible introgression by hybridization.