Left-Right Reversal Recurrently Evolved Regardless of Diaphanous-Related Formin Gene Duplication or Loss in Snails.

Bilateria exhibit whole-body handedness in internal structure. This left-right polarity is evolutionarily conserved with virtually no reversed extant lineage, except in molluscan Gastropoda. Phylogenetically independent snail groups contain both clockwise-coiled (dextral) and counterclockwise-coiled (sinistral) taxa that are reversed from each other in bilateral handedness as well as in coiling direction. Within freshwater Hygrophila, Lymnaea with derived dextrality have diaphanous related formin (diaph) gene duplicates, while basal sinistral groups possess one diaph gene. In terrestrial Stylommatophora, dextral Bradybaena also have diaph duplicates. Defective maternal expression of one of those duplicates gives rise to sinistral hatchlings in Lymnaea and handedness-mixed broods in Bradybaena, through polarity change in spiral cleavage of embryos. These findings led to the hypothesis that diaph duplication was crucial for the evolution of dextrality by reversal. The present study discovered that diaph duplication independently occurred four times and its duplicate became lost twice in gastropods. The dextrality of Bradybaena represents the ancestral handedness conserved across gastropods, unlike the derived dextrality of Lymnaea. Sinistral lineages recurrently evolved by reversal regardless of whether diaph had been duplicated. Amongst the seven formin gene subfamilies, diaph has most thoroughly been conserved across eukaryotes of the 14 metazoan phyla and choanoflagellate. Severe embryonic mortalities resulting from insufficient expression of the duplicate in both of Bradybaena and Lymnaea also support that diaph duplicates bare general roles for cytoskeletal dynamics other than controlling spiralian handedness. Our study rules out the possibility that diaph duplication or loss played a primary role for reversal evolution.

The genus Erhaia (Gastropoda, Truncatelloidea, Amnicolidae), with a new species from Bhutan.

The distribution of the five Erhaia (Gastropoda, Truncatelloidea, Amnicolidae) species that are diagnosed by both morphological and molecular data is combined with several records of less completely diagnosed nominal Erhaia species. The resulting distribution pattern is summarized in a map and is discussed herein. Erhaianorbui sp. nov. is described from Bhutan on the basis of shell morphology and two mitochondrial DNA barcoding markers. A molecular phylogeny is presented for the five Erhaia species for which molecular data are available, three of which form a separate clade and are from Bhutan.

The genera Erhaia and Tricula (Gastropoda, Rissooidea, Amnicolidae and Pomatiopsidae) in Bhutan and elsewhere in the eastern Himalaya.

Shells of the Rissooidea species that are known from Bhutan are characterized. Tricula montana is reported from that country for the first time. Two Erhaia species from Bhutan are described as new to science, viz. E. jannei sp. nov., and E. pelkiae sp. nov., The holotypes of the Erhaia species that were described from Nepal are figured with photographs for the first time and compared with the congeneric taxa from Bhutan and India. Erhaia nainitalensis is considered a senior synonym of E. chandeshwariensis. An identification key is presented for the Erhaia species of the Himalayan foothills.

Erhaia Davis & Kuo (Gastropoda, Rissooidea, Amnicolidae) also in Bhutan.

The occurrence of at least one species of Erhaia in Bhutan, viz. Erhaia wangchukisp. n., is confirmed by DNA sequencing. A second unnamed species from Bhutan, that might be congeneric, is known from only a single shell. According to the molecular analysis, E. wangchuki is most closely related to a still undescribed Erhaia species from China. These two species together with E. jianouensis and Akiyoshia kobayashii, both also from China, form a well supported clade. Awaiting additional molecular data, the apparent inconsistency regarding Erhaia versus Akiyoshia is not dealt with here. The extant true sister species of E. wangchuki could be among the four SE Himalayan species from Bhutan and Nepal that are classified with Erhaia on the basis of conchological data only.

Granopupa in Iran, monophyly, and the fossil Granariinae (Gastropoda, Pulmonata, Chondrinidae).

Indisputable Chondrinidae, Granariinae species, characterized by shell shape and apertural dentition, are known from Eocene deposits to the Recent. The generic classification of the extant species is based on conchological, anatomical and molecular data that are available now for most of the known species, including 'Granaria' persica as a representative of the once problematic group of so-called eastern Granaria species. According to molecular and anatomical characters, these eastern species have to be classified with Granopupa granum in Granopupa. Graniberia gen. n. is introduced for Granaria braunii on the basis of molecular and conchological data. For the pre-Pleistocene species, two generic names are equally well available now, viz. Granopupa and Granaria. Shell characters only do not enable a decision here. For the sake of nomenclatorial stability we propose to use Granaria for these species. Because both molecular and anatomical data most likely will never be known for the fossils, it will remain unclear whether the combined extant and extinct Granaria species form a monophyletic group.

Phylogeny of the land snail family Clausiliidae (Gastropoda: Pulmonata).

Clausiliidae is one of the most speciose and best-studied families of land snails. The family contributes to land snail diversity on a global scale, with three main centres of diversity: (1) western Eurasia (six subfamilies recognized), (2) East Asia (two subfamilies recognized) and (3) the neotropics (one subfamily recognized i.e. Neniinae). Despite a wealth of shell-morphological and anatomical studies, a well-supported phylogeny is lacking for the family. To provide a phylogenetic framework and reevaluate morphological and biogeographic observations on the family, we compiled a dataset consisting of partial 28S rRNA, histone H3 and histone H4 nucleotide sequences covering all clausiliid subfamilies, and 23 out of 25 tribes. Our analyses (MrBayes, BEAST, PhyML) divide the family into seven highly supported clades, which were retrieved by at least two of the three markers used, and which are more or less geographically confined. Three of these clades coincide with subfamilies recognized in the current classification (Alopiinae, Garnieriinae, Laminiferinae). The monophyly of four of the remaining six hitherto accepted subfamilies is not supported, with the New World subfamily Neniinae divided across two clades. All shell-morphological characters used in classical clausiliid classification were homoplasious at the subfamily level, with the exception of the type of shell aperture formation. In contrast to previous interpretations, our results suggest that the so-called 'apostrophic' aperture found in the neotropical clausiliids, and in a European (Laminiferinae) and a SE Asian (Garnieriinae) subfamily, is in fact the plesiomorphic condition among extant Clausiliidae. The widespread and fragmented geographic distribution of this type of aperture may therefore be considered relictual. Based on an inferred Late Cretaceous or Early Cenozoic European origin of the clade of extant Clausiliidae, the ancestor(s) of the neotropical Clausiliidae must have colonized the New World after the Atlantic Ocean had opened. A taxonomic revision is proposed.

Chiral speciation in terrestrial pulmonate snails.

On the basis of data in the literature, the percentages of dextral versus sinistral species of snails have been calculated for western Europe, Turkey, North America (north of Mexico), and Japan. When the family of Clausiliidae is represented, about a quarter of all snail species may be sinistral, whereas less than one per cent of the species may be sinistral where that family does not occur. The number of single-gene speciation events on the basis of chirality, resulting in the origin of mirror image species, is not closely linked to the percentage of sinistral versus dextral species in a particular region. Turkey is nevertheless exceptional by both a high percentage of sinistral species and a high number of speciation events resulting in mirror image species. Shell morphology and genetic background may influence the ease of chirality-linked speciation, whereas sinistrality may additionally be selected against by internal selection. For the Clausiliidae, the fossil record and the recent fauna suggest that successful reversals in coiling direction occurred with a frequency of once every three to four million years.

The complete mitogenome of Cylindrus obtusus (Helicidae, Ariantinae) using Illumina next generation sequencing.

BACKGROUND: This study describes how the complete mitogenome of a terrestrial snail, Cylindrus obtusus (Draparnaud, 1805) was sequenced without PCRs from a collection specimen that had been in 70% ethanol for 8 years. The mitogenome was obtained with Illumina GAIIx shot gun sequencing. Although the used specimen was collected relatively recently and kept in a DNA-friendly preservative (not formalin as frequently used with old museum specimens), we believe that the exclusion of PCRs as facilitated by NGS (Next Generation Sequencing) removes a great obstacle in DNA sequencing of collection specimens. A brief comparison is made between our Illumina GAIIx approach and a similar study that made use of the Roche 454-FLX platform. RESULTS: The mtDNA sequence of C. obtusus is 14,610 bases in length (about 0.5 kb larger than other stylommatophoran mitogenomes reported hitherto) and contains the 37 genes (13 protein coding genes, two rRNAs and 22 tRNAs) typical for metazoans. Except for a swap between the position of tRNA-Pro and tRNA-Ala, the gene arrangement of C. obtusus is identical to that reported for Cepaea nemoralis. The 'aberrant' rearrangement of tRNA-Thr and COIII compared to that of other Sigmurethra (and the majority of gastropods), is not unique for C. nemoralis (subfamily Helicinae), but is also shown to occur in C. obtusus (subfamily Ariantinae) and might be a synapomorphy for the family Helicidae. CONCLUSIONS: Natural history collections potentially harbor a wealth of information for the field of evolutionary genetics, but it can be difficult to amplify DNA from such specimens (due to DNA degradation for instance). Because NGS techniques do not rely on primer-directed amplification (PCR) and allow DNA to be fragmented (DNA gets sheared during library preparation), NGS could be a valuable tool for retrieving DNA sequence data from such specimens. A comparison between Illumina GAIIx and the Roche 454 platform suggests that the former might be more suited for de novo sequencing of mitogenomes.

Reappraisal of the "Molecular phylogeny of Western Palaearctic Helicidae s.l. (Gastropoda: Stylommatophora)": when poor science meets GenBank.

A paper on the 'Molecular phylogeny of Western Palaearctic Helicidae s.l.', published by Steinke et al. (2004) in this journal, is critically analysed. Several obvious errors are corrected and methodological weaknesses are revealed. BLAST searches on the sequences published in that paper and now in GenBank, showed high percentages of similarity of the alleged species with taxa that are considered only distantly related in the literature. Inspection of the so-called voucher specimens showed that some shells were misidentified, whereas others contained dirt or were bleached, indicating that these had been collected empty. Obviously the sequences published for those species could not have originated from those specimens, which cannot be considered vouchers therefore, even if they are from the same locality. In other instances, spurious sequences were published for correctly identified voucher specimens. For several species for which we collected specimens ourselves, the COI or the 16S sequence, or both, clearly differed from the results published by Steinke et al. The consequences of our results for the molecular data on helicid gastropods and their classification are listed.

Internal selection against the evolution of left-right reversal.

Among metazoan species, left-right reversals in primary asymmetry have rarely gone to fixation. This suggests that a general mechanism suppresses the evolution of polarity reversal. Most metazoans appear externally symmetric and reproduce by external fertilization or copulation with genitalia located in the midline. Thus, reversal should generate little exogenous disadvantage when interacting with the external environment or in mating with the common wild-type. Accordingly, an endogenously caused fitness reduction may be responsible for the general absence of reversed species. However, how this selection operates is little understood. Phenotypic changes associated with reversal are usually inseparable from zygotic pleiotropy. By exploiting hermaphroditism and the maternal inheritance of left-right polarity, we generated dextral and sinistral snails that share the same zygotic genotype. Before hatching, these sinistrals developed lethal morphological anomalies more frequently than dextrals. Their shell shape at maturity differed from the mirror image of the dextral shell. These interchiral differences demonstrate pleiotropy in maternal effects of the polarity or linked genes. Variation in interchiral differences between parental crosses suggests the presence of epistatic variation in relative performance of sinistrals. Our results show that internal selection operates against polarity reversal, and we suggest that this is due to changes in blastomere configuration.

The development and evolution of left-right asymmetry in invertebrates: lessons from Drosophila and snails.

The unique nature of body handedness, which is distinct from the anteroposterior and dorsoventral polarities, has been attracting growing interest in diverse biological disciplines. Recent research progress on the left-right asymmetry of animal development has focused new attention on the mechanisms underlying the development and evolution of invertebrate handedness. This exploratory review of currently available information illuminates the prospective value of Drosophila and pulmonate snails for innovative new research aimed at elucidating these mechanisms. For example, findings in Drosophila and snails suggest that an actin filament-dependent mechanism may be evolutionarily conserved in protostomes. The polarity conservation of primary asymmetry across most metazoan phyla, which visceral handedness represents, indicates developmental constraint and purifying selection as possible but unexplored mechanisms. Comparative studies using Drosophila and snails, which have the great advantages of using genetic and evolutionary approaches, will accelerate our understanding of the mechanisms governing the conservation and diversity of animal handedness.

Whole-body enantiomorphy and maternal inheritance of chiral reversal in the pond snail Lymnaea stagnalis.

Sinistral and dextral snails have repeatedly evolved by left-right reversal of bilateral asymmetry as well as coiling direction. However, in most snail species, populations are fixed for either enantiomorph and laboratory breeding is difficult even if chiral variants are found. Thus, only few experimental models of chiral variation within species have been available to study the evolution of the primary asymmetry. We have established laboratory lines of enantiomorphs of the pond snail Lymnaea stagnalis starting from a wild population. Crossing experiments demonstrated that the primary asymmetry of L. stagnalis is determined by the maternal genotype at a single nuclear locus where the dextral allele is dominant to the sinistral allele. Field surveys revealed that the sinistral allele has persisted for at least 10 years, that is, about 10 generations. The frequency of the sinistral allele showed large fluctuations, reaching as frequent as 0.156 in estimate under the assumption of Hardy-Weinberg equilibrium. The frequency shifts suggest that selection against chiral reversal was not strong enough to counterbalance genetic drift in an ephemeral small pond. Because of the advantages as a model animal, enantiomorphs of L. stagnalis can be a unique system to study aspects of chirality in diverse biological disciplines.

Ancient DNA elucidates the controversy about the flightless island hens (Gallinula sp.) of Tristan da Cunha.

A persistent controversy surrounds the flightless island hen of Tristan da Cunha, Gallinula nesiotis. Some believe that it became extinct by the end of the 19th century. Others suppose that it still inhabits Tristan. There is no consensus about Gallinula comeri, the name introduced for the flightless moorhen from the nearby island of Gough. On the basis of DNA sequencing of both recently collected and historical material, we conclude that G. nesiotis and G. comeri are different taxa, that G. nesiotis indeed became extinct, and that G. comeri now inhabits both islands. This study confirms that among gallinules seemingly radical adaptations (such as the loss of flight) can readily evolve in parallel on different islands, while conspicuous changes in other morphological characters fail to occur.

Biogeography: molecular trails from hitch-hiking snails.

Darwin was fascinated by the transportation of land snails across great swathes of open ocean by birds--he even immersed snails in sea water to see how long they would survive. Here we follow a molecular phylogenetic trail that reveals the incredible transequatorial dispersal of the land snail Balea from Europe to the Azores and the Tristan da Cunha islands, and back again. This long-distance dispersal is unexpected for what are proverbially considered the most pedestrian of creatures.

Widespread polyphyly among Alopiinae snail genera: when phylogeny mirrors biogeography more closely than morphology.

Consider a group of species that is evenly divided by an easily identifiable complex morphological character. Most biologists would assume that this character should provide better phylogenetic information than, say, the spatial distribution of these species over a fairly continuous 500-km radius area. Paradoxically, this is not the case among terrestrial snail genera in the clausiliid subfamily Alopiinae. Phylogenetic analysis using the nuclear markers ITS1/ITS2 and mitochondrial markers COI/12S reveals widespread homoplasy in the clausilial apparatus (a complex aperture-closing mechanism), and concomitant extensive polyphyly among Carinigera, Isabellaria, and Sericata. In contrast, phylogenetic relationships as revealed by molecular data are closely congruent with biogeography at a relatively small scale. A combination of extremely low vagility and extremely high morphological convergence has conspired to produce this unexpected result. Implications as to the function of the clausilial apparatus are discussed.