The draft genome sequence of the grove snail Cepaea nemoralis.

Studies on the shell color and banding polymorphism of the grove snail Cepaea nemoralis and the sister taxon Cepaea hortensis have provided compelling evidence for the fundamental role of natural selection in promoting and maintaining intraspecific variation. More recently, Cepaea has been the focus of citizen science projects on shell color evolution in relation to climate change and urbanization. C. nemoralis is particularly useful for studies on the genetics of shell polymorphism and the evolution of "supergenes," as well as evo-devo studies of shell biomineralization, because it is relatively easily maintained in captivity. However, an absence of genomic resources for C. nemoralis has generally hindered detailed genetic and molecular investigations. We therefore generated ∼23× coverage long-read data for the ∼3.5 Gb genome, and produced a draft assembly composed of 28,537 contigs with the N50 length of 333 kb. Genome completeness, estimated by BUSCO using the metazoa dataset, was 91%. Repetitive regions cover over 77% of the genome. A total of 43,519 protein-coding genes were predicted in the assembled genome, and 97.3% of these were functionally annotated from either sequence homology or protein signature searches. This first assembled and annotated genome sequence for a helicoid snail, a large group that includes edible species, agricultural pests, and parasite hosts, will be a core resource for identifying the loci that determine the shell polymorphism, as well as in a wide range of analyses in evolutionary and developmental biology, and snail biology in general.

Morphological and phylogenetic diversity of Waminoa and similar flatworms (Acoelomorpha) in the western Pacific Ocean.

The genus Waminoa currently contains two described species, which each contains two types of endosymbiotic algae. Waminoa individuals are basically brown in body color, derived from these algal symbionts, and their body shape has been described as "discoid to obcordate". They have been found as associates of various anthozoans (Cnidaria) in the Indo-Pacific Ocean and the Red Sea. In order to reveal the diversity of the genus Waminoa and their hosts, we conducted phylogenetic and morphological analyses on acoelomate flatworms specimens collected from Japan, Palau and Indonesia. At least 18 Waminoa morphotypes were found on at least 20 anthozoan host species, and two specimens were found on species of two sea stars. Overall, there were two main body shapes of specimens; obcordate, as seen in W. litus and W. brickneri, and the other molar-like with an elongated body. These two body shapes each represented a separate clade in 18S rDNA and mitochondrial cytochrome c oxidase subunit 1 (COI) phylogenetic trees, with W. brickneri included in the obcordate subclade. Automatic Barcode Gap Discovery (ABGD) analyses on COI sequences of our specimens revealed the presence of at least five operational taxonomic units (OTUs). These five OTUs consisted of one large group of all obcordate animals, three OTUs consisting of one specimen each within the molar-like clade, and one large group of the remaining molar-like specimens. Both clades contain numerous morphotypes and were associated with a variety of hosts. Finally, based on genetic distances, the molar-like specimens are considered as an unnamed genus group separate from Waminoa, which needs to be clarified in future studies.

Anchored hybrid enrichment generated nuclear, plastid and mitochondrial markers resolve the Lepanthes horrida (Orchidaceae: Pleurothallidinae) species complex.

Phylogenetic relationships in species complexes and lineages derived from rapid diversifications are often challenging to resolve using morphology or standard DNA barcoding markers. The hyper-diverse genus Lepanthes from Neotropical cloud forest includes over 1200 species and many recent, explosive diversifications that have resulted in poorly supported nodes and morphological convergence across clades. Here, we assess the performance of 446 nuclear-plastid-mitochondrial markers derived from an anchored hybrid enrichment approach (AHE) coupled with coalescence- and species network-based inferences to resolve phylogenetic relationships and improve species recognition in the Lepanthes horrida species group. In addition to using orchid-specific probes to increase enrichment efficiency, we improved gene tree resolution by extending standard angiosperm targets into adjacent exons. We found high topological discordance among individual gene trees, suggesting that hybridization/polyploidy may have promoted speciation in the lineage via formation of new hybrid taxa. In addition, we identified ten loci with the highest phylogenetic informativeness values from these genomes. Most previous phylogenetic sampling in the Pleurothallidinae relies on two regions (ITS and matK), therefore, the evaluation of other markers such as those shown here may be useful in future phylogenetic studies in the orchid family. Coalescent-based species tree estimation methods resolved the phylogenetic relationships of the L. horrida species group. The resolution of the phylogenetic estimations was improved with the inclusion of extended anchor targets. This approach produced longer loci with higher discriminative power. These analyses also disclosed two undescribed species, L. amicitiae and L. genetoapophantica, formally described here, which are also supported by morphology. Our study demonstrates the utility of combined genomic evidence to disentangle phylogenetic relationships at very shallow levels of the tree of life, and in clades showing convergent trait evolution. With a fully resolved phylogeny, is it possible to disentangle traits evolving in parallel or convergently across these orchid lineages such as flower color and size from diagnostic traits such as the shape and orientation of the lobes of the petals and lip.

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.

Using DNA-barcoding to make the necrobiont beetle family Cholevidae accessible for forensic entomology.

The beetle family Cholevidae (Coleoptera: Staphylinoidea), sometimes viewed as the subfamily Cholevinae of the Leiodidae, consists of some 1700 species worldwide. With the exception of specialized cave-dwelling species and species living in bird and mammal nests and burrows, the species are generalized soil-dwellers that, at least in temperate regions, are mostly found on vertebrate cadavers. Although they have been regularly reported from human corpses, and offer potential because of many species' peak activity in the cold season, they have not been a focus of forensic entomologists so far. This is probably due to their small size and the difficulty in identifying the adults and their larvae. In this paper, we show that DNA-barcoding can help make this group of necrobiont beetles available as a tool for forensic research. We collected 86 specimens of 20 species of the genera Catops, Fissocatops, Apocatops, Choleva, Nargus, Ptomaphagus, and Sciodrepoides from the Netherlands and France and show that a broad "barcoding gap" allows almost all species to be easily and unambiguously identified by the sequence of the "barcoding gene" cytochrome c oxidase I (COI). This opens up the possibility of adding Cholevidae to the set of insect taxa routinely used in forensic entomology.

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.