Neogastropod (Mollusca, Gastropoda) phylogeny: A step forward with mitogenomes.

The Neogastropoda (Mollusca, Gastropoda) encompass more than 15,000 described species of marine predators, including several model organisms in toxinology, embryology and physiology. However, their phylogenetic relationships remain mostly unresolved and their classification unstable. We took advantage of the many mitogenomes published in GenBank to produce a new molecular phylogeny of the neogastropods. We completed the taxon sampling by using an in-house bioinformatic pipeline to retrieve mitochondrial genes from 13 transcriptomes, corresponding to five families not represented in GenBank, for a final dataset of 113 taxa. Because mitogenomic data are prone to reconstruction artefacts, eight different evolutionary models were applied to reconstruct phylogenetic trees with IQTREE, RAxML and MrBayes. If the over-parametrization of some models produced trees with aberrant internal long branches, the global topology of the trees remained stable over models and softwares, and several relationships were revealed or found supported here for the first time. However, even if our dataset encompasses 60% of the valid families of neogastropods, some key taxa are missing and should be added in the future before proposing a revision of the classification of the neogastropods. Our study also demonstrates that even complex models struggle to satisfactorily handle the evolutionary history of mitogenomes, still leading to long-branch attractions in phylogenetic trees. Other approaches, such as reduced-genome strategies, must be envisaged to fully resolve the neogastropod phylogeny.

Delimiting species of marine gastropods (Turridae, Conoidea) using RAD sequencing in an integrative taxonomy framework.

Species delimitation in poorly known and diverse taxa is usually performed based on monolocus, DNA-barcoding-like approaches, while multilocus data are often used to test alternative species hypotheses in well-studied groups. We combined both approaches to delimit species in the Xenuroturris/Iotyrris complex, a group of venomous marine gastropods from the Indo-Pacific. First, COI sequences were analysed using three methods of species delimitation to propose primary species hypotheses. Second, RAD sequencing data were also obtained and a maximum-likelihood phylogenetic tree produced. We tested the impact of the level of missing data on the robustness of the phylogenetic tree obtained with the RAD-seq data. Alternative species partitions revealed with the COI data set were also tested using the RAD-seq data and the Bayes factor species delimitation method. The congruence between the species hypotheses proposed with the mitochondrial nuclear data sets, together with the morphological variability of the shell and the radula and the distribution pattern, was used to turn the primary species hypotheses into secondary species hypotheses. Allopatric primary species hypotheses defined with the COI gene were interpreted to correspond to intraspecific structure. Most of the species are found sympatrically in the Philippines, and only one is confidently identified as a new species and described as Iotyrris conotaxis n. sp. The results obtained demonstrate the efficiency of the combined monolocus/multilocus approach to delimit species.

Exon-Capture-Based Phylogeny and Diversification of the Venomous Gastropods (Neogastropoda, Conoidea).

Transcriptome-based exon capture methods provide an approach to recover several hundred markers from genomic DNA, allowing for robust phylogenetic estimation at deep timescales. We applied this method to a highly diverse group of venomous marine snails, Conoidea, for which published phylogenetic trees remain mostly unresolved for the deeper nodes. We targeted 850 protein coding genes (678,322 bp) in ca. 120 samples, spanning all (except one) known families of Conoidea and a broad selection of non-Conoidea neogastropods. The capture was successful for most samples, although capture efficiency decreased when DNA libraries were of insufficient quality and/or quantity (dried samples or low starting DNA concentration) and when targeting the most divergent lineages. An average of 75.4% of proteins was recovered, and the resulting tree, reconstructed using both supermatrix (IQ-tree) and supertree (Astral-II, combined with the Weighted Statistical Binning method) approaches, are almost fully supported. A reconstructed fossil-calibrated tree dates the origin of Conoidea to the Lower Cretaceous. We provide descriptions for two new families. The phylogeny revealed in this study provides a robust framework to reinterpret changes in Conoidea anatomy through time. Finally, we used the phylogeny to test the impact of the venom gland and radular type on diversification rates. Our analyses revealed that repeated losses of the venom gland had no effect on diversification rates, while families with a breadth of radula types showed increases in diversification rates, thus suggesting that trophic ecology may have an impact on the evolution of Conoidea.

Development of microsatellite primers in the protected species Viola elatior (Violaceae) using next-generation sequencing.

PREMISE OF THE STUDY: Viola elatior (Violaceae) is a Eurasian perennial plant species in which French populations are threatened by anthropogenic pressures. Microsatellite primers were developed to investigate its genetic structure and diversity. METHODS AND RESULTS: Eight microsatellite markers were isolated using next-generation sequencing. Loci were amplified and screened for 138 individuals in 17 populations from France. Two of the eight polymorphic loci presented no variability across populations. The total number of alleles per locus varied from two to four. Observed heterozygosity ranged from 0.051 to 1.000. All primers amplified successfully in the closely related species V. pumila. CONCLUSIONS: This set of microsatellites offers a valuable tool for assessing population genetic diversity of the species to improve its conservation and base management efforts. High observed heterozygosity values probably reflect the particular mating system of the species and suggest an important tendency to clonality.

Genetic variation and population structure in the endangered Hermann's tortoise: the roles of geography and human-mediated processes.

The Hermann's tortoise (Testudo hermanni) is an endangered land tortoise distributed in disjoint populations across Mediterranean Europe. We investigated its genetic variation by typing 1 mitochondrial locus and 9 nuclear microsatellites in approximately 300 individuals from 22 localities. Our goal was to understand the relative impact of natural and human-mediated processes in shaping the genetic structure and to identify the genetic priorities for the conservation of this species. We found that 1) all geographic areas are highly differentiated, mainly as a function of their distance but with a clear genetic discontinuity (F st values larger than 0.4) between the Eastern and the Western subspecies; 2) the contact zone between subspecies is located farthest to the west than previously believed, and it probably coincides with the delta of the largest Italian river; 3) extinction events due to climatic conditions in the Upper Palaeolithic and subsequent human-mediated translocations in the Neolithic possibly explain the unexpected similarity among Spain, Sicily, and Corsica. For conservation purposes, the large majority of genetic pools appears native although hybridization among subspecies, related to extensive 20th century trade of tortoises across Europe, is observed in Spain and some Italian samples. Most populations do not seem at immediate risk of low genetic variation, except the French population, which has very low nuclear genetic diversity (heterozygosity = 0.25) and where 50 out of 51 sampled animals shared the same mitochondrial sequence. In general, restocking and reintroduction plans should carefully consider the genetic background of the individuals.

Using next-generation sequencing to analyse the diet of a highly endangered land snail (Powelliphanta augusta) feeding on endemic earthworms.

Predation is often difficult to observe or quantify for species that are rare, very small, aquatic or nocturnal. The assessment of such species' diet can be conducted using molecular methods that target prey DNA remaining in predators' guts and faeces. These techniques do not require high taxonomic expertise, are applicable to soft-bodied prey and allow for identification at the species level. However, for generalist predators, the presence of mixed prey DNA in guts and faeces can be a major impediment as it requires development of specific primers for each potential prey species for standard (Sanger) sequencing. Therefore, next generation sequencing methods have recently been applied to such situations. In this study, we used 454-pyrosequencing to analyse the diet of Powelliphantaaugusta, a carnivorous landsnail endemic to New Zealand and critically endangered after most of its natural habitat has been lost to opencast mining. This species was suspected to feed mainly on earthworms. Although earthworm tissue was not detectable in snail faeces, earthworm DNA was still present in sufficient quantity to conduct molecular analyses. Based on faecal samples collected from 46 landsnails, our analysis provided a complete map of the earthworm-based diet of P. augusta. Predated species appear to be earthworms that live in the leaf litter or earthworms that come to the soil surface at night to feed on the leaf litter. This indicates that P. augusta may not be selective and probably predates any earthworm encountered in the leaf litter. These findings are crucial for selecting future translocation areas for this highly endangered species. The molecular diet analysis protocol used here is particularly appropriate to study the diet of generalist predators that feed on liquid or soft-bodied prey. Because it is non-harmful and non-disturbing for the studied animals, it is also applicable to any species of conservation interest.

Mixing plants from different origins to restore a declining population: ecological outcomes and local perceptions 10 years later.

Populations of the Large-flowered Sandwort (Arenaria grandiflora L.) in the Fontainebleau forest (France) have declined rapidly during the last century. Despite the initiation of a protection program in 1991, less than twenty individuals remained by the late 1990s. The low fitness of these last plants, which is likely associated with genetic disorders and inbreeding depression, highlighted the need for the introduction of non-local genetic material to increase genetic diversity and thus restore Fontainebleau populations. Consequently, A. grandiflora was introduced at three distant sites in the Fontainebleau forest in 1999. Each of these populations was composed of an identical mix of individuals of both local and non-local origin that were obtained through in vitro multiplication. After establishment, the population status (number of individuals, diameter of the plants, and number of flowers) of the introduced populations was monitored. At present, two populations (one of which is much larger than the other) persist, while the third one became extinct in 2004. Analyses of the ecological parameters of the introduction sites indicated that differences in soil pH and moisture might have contributed to the differences in population dynamics. This introduction plan and its outcome attracted interest of local community, with those who supported the plan and regarded its 10-year result as a biological success (i.e., persistent populations were created), but also those who expressed reservations or disapproval of the plan and its outcome. To understand this controversy, a sociological study involving 27 semi-structured interviews was carried out. From these interviews emerged three areas of controversy: alteration of the identity of the plant, alteration of the identity of its territory, and the biological and ethical consequences of the techniques used for the experimental conservation.

Population genetic structure and colonisation history of the tool-using New Caledonian crow.

New Caledonian crows exhibit considerable variation in tool making between populations. Here, we present the first study of the species' genetic structure over its geographical distribution. We collected feathers from crows on mainland Grande Terre, the inshore island of Toupéti, and the nearby island of Maré where it is believed birds were introduced after European colonisation. We used nine microsatellite markers to establish the genotypes of 136 crows from these islands and classical population genetic tools as well as Approximate Bayesian Computations to explore the distribution of genetic diversity. We found that New Caledonian crows most likely separate into three main distinct clusters: Grande Terre, Toupéti and Maré. Furthermore, Toupéti and Maré crows represent a subset of the genetic diversity observed on Grande Terre, confirming their mainland origin. The genetic data are compatible with a colonisation of Maré taking place after European colonisation around 1900. Importantly, we observed (1) moderate, but significant, genetic differentiation across Grande Terre, and (2) that the degree of differentiation between populations on the mainland increases with geographic distance. These data indicate that despite individual crows' potential ability to disperse over large distances, most gene flow occurs over short distances. The temporal and spatial patterns described provide a basis for further hypothesis testing and investigation of the geographical variation observed in the tool skills of these crows.

Fast, cost-effective development of species-specific microsatellite markers by genomic sequencing.

Microsatellites are the genetic markers of choice for many population genetic studies, but must be isolated de novo using recombinant approaches where prior genetic data are lacking. Here we utilized high-throughput genomic sequencing technology to produce millions of base pairs of short fragment reads, which were screened with bioinformatics toolsets to identify primers that amplify polymorphic microsatellite loci. Using this approach we isolated 13 polymorphic microsatellites for the blue duck (Hymenolaimus malacorhynchos), a species for which limited genetic data were available. Our genomic approach eliminates recombinant genetic steps, significantly reducing the time and cost requirements of marker development compared with traditional approaches. While this application of genomic sequencing may seem obvious to many, this study is, to the best of our knowledge, the first attempt to describe the use of genomic sequencing for the development of microsatellite markers in a non-model organism or indeed any organism.

Establishing causes of eradication failure based on genetics: case study of ship rat eradication in Ste. Anne archipelago.

Determining the causes of a failed eradication of a pest species is important because it enables an argued adjustment of the methodologies used and the improvement of the protocols for future attempts. We examined how molecular monitoring can help distinguish between the two main reasons for an eradication failure (i.e., survival of some individuals vs. recolonization after eradication). We investigated genetic variation in seven microsatellite loci in ship rat (Rattus rattus) populations from four islets off the Martinique coast (French Caribbean). In 1999 an eradication attempt was conducted on the four islets. Three years later rats were observed again on two of them. We compared the genetic signatures of the populations before and after the eradication attempt. On one of the islands, the new rat population was likely a subset of the pre-eradication population. A weak genetic differentiation was found between them, with almost no new alleles observed in the new population and moderate F(ST) values (0.15). Moreover, assignment procedures clustered the two populations together. In contrast, on the other islet, many new alleles were observed after the eradication attempt, resulting in an increase in genetic diversity (from 2.57 to 3.57 mean number of alleles per locus) and strong F(ST) values (0.39). Moreover, genetic clustering clearly separated the two samples (i.e., before and after the eradication attempt) in two different populations. Thus, to achieve long-term eradication on these islets, it seems necessary to redevelop the eradication procedure to avoid individuals surviving and to prevent reinvasion, probably from the mainland, by installing permanent trapping and poisoning devices and conducting regular monitoring. We strongly encourage wildlife managers conducting eradication campaigns to integrate molecular biological tools in their protocols, which can be done easily for most common invasive species.