Gontran Sonet

Next-Generation Museomics Disentangles One of the Largest Primate Radiations

Guenons (tribe Cercopithecini) are one of the most diverse groups of primates. They occupy all of sub-Saharan Africa and show great variation in ecology, behavior, and morphology. This variation led to the description of over 60 species and subspecies. Here, using next-generation DNA sequencing (NGS) in combination with targeted DNA capture, we sequenced 92 mitochondrial genomes from museum-preserved specimens as old as 117 years. We infer evolutionary relationships and estimate divergence times of almost all guenon taxa based on mitochondrial genome sequences. Using this phylogenetic framework, we infer divergence dates and reconstruct ancestral geographic ranges. We conclude that the extraordinary radiation of guenons has been a complex process driven by, among other factors, localized fluctuations of African forest cover. We find incongruences between phylogenetic trees reconstructed from mitochondrial and nuclear DNA sequences, which can be explained by either incomplete lineage sorting or hybridization. Furthermore, having produced the largest mitochondrial DNA data set from museum specimens, we document how NGS technologies can “unlock” museum collections, thereby helping to unravel the tree-of-life. [Museum collection; next-generation DNA sequencing; primate radiation; speciation; target capture.]

First Large-Scale DNA Barcoding Assessment of Reptiles in the Biodiversity Hotspot of Madagascar, Based on Newly Designed COI Primers

Background DNA barcoding of non-avian reptiles based on the cytochrome oxidase subunit I (COI) gene is still in a very early stage, mainly due to technical problems. Using a newly developed set of reptile-specific primers for COI we present the first comprehensive study targeting the entire reptile fauna of the fourth-largest island in the world, the biodiversity hotspot of Madagascar. Methodology/Principal Findings Representatives of the majority of Madagascan non-avian reptile species (including Squamata and Testudines) were sampled and successfully DNA barcoded. The new primer pair achieved a constantly high success rate (72.7–100%) for most squamates. More than 250 species of reptiles (out of the 393 described ones; representing around 64% of the known diversity of species) were barcoded. The average interspecific genetic distance within families ranged from a low of 13.4% in the Boidae to a high of 29.8% in the Gekkonidae. Using the average genetic divergence between sister species as a threshold, 41–48 new candidate (undescribed) species were identified. Simulations were used to evaluate the performance of DNA barcoding as a function of completeness of taxon sampling and fragment length. Compared with available multi-gene phylogenies, DNA barcoding correctly assigned most samples to species, genus and family with high confidence and the analysis of fewer taxa resulted in an increased number of well supported lineages. Shorter marker-lengths generally decreased the number of well supported nodes, but even mini-barcodes of 100 bp correctly assigned many samples to genus and family. Conclusions/Significance The new protocols might help to promote DNA barcoding of reptiles and the established library of reference DNA barcodes will facilitate the molecular identification of Madagascan reptiles. Our results might be useful to easily recognize undescribed diversity (i.e. novel taxa), to resolve taxonomic problems, and to monitor the international pet trade without specialized expert knowledge.

Genetic and Morphological Divergences in the Cosmopolitan Deep-Sea Amphipod Eurythenes gryllus Reveal a Diverse Abyss and a Bipolar Species

Eurythenes gryllus is one of the most widespread amphipod species, occurring in every ocean with a depth range covering the bathyal, abyssal and hadal zones. Previous studies, however, indicated the existence of several genetically and morphologically divergent lineages, questioning the assumption of its cosmopolitan and eurybathic distribution. For the first time, its genetic diversity was explored at the global scale (Arctic, Atlantic, Pacific and Southern oceans) by analyzing nuclear (28S rDNA) and mitochondrial (COI, 16S rDNA) sequence data using various species delimitation methods in a phylogeographic context. Nine putative species-level clades were identified within E. gryllus. A clear distinction was observed between samples collected at bathyal versus abyssal depths, with a genetic break occurring around 3,000 m. Two bathyal and two abyssal lineages showed a widespread distribution, while five other abyssal lineages each seemed to be restricted to a single ocean basin. The observed higher diversity in the abyss compared to the bathyal zone stands in contrast to the depth-differentiation hypothesis. Our results indicate that, despite the more uniform environment of the abyss and its presumed lack of obvious isolating barriers, abyssal populations might be more likely to show population differentiation and undergo speciation events than previously assumed. Potential factors influencing species’ origins and distributions, such as hydrostatic pressure, are discussed. In addition, morphological findings coincided with the molecular clades. Of all specimens available for examination, those of the bipolar bathyal clade seemed the most similar to the ‘true’ E. gryllus. We present the first molecular evidence for a bipolar distribution in a macro-benthic deep-sea organism.

Incongruence between molecular phylogeny and morphological classification in amphipod crustaceans: a case study of Antarctic lysianassoids

In Antarctic waters, the superfamily Lysianassoidea is one of the most important amphipod groups both in terms of species number and abundance. Dominant members of this superfamily are species of the orchomenid complex, found throughout the Southern Ocean. This study presents the first molecular phylogenetic analysis based on a representative subset of the Antarctic species belonging to different orchomenid genera and hence provides a framework for a systematic revision of these taxa. The current classification of the orchomenid genera is mainly based on mouthpart morphology. The validity of these morphological characters was assessed by resolving phylogenetic relationships using nuclear 28S rRNA and mitochondrial cytochrome oxidase subunit I sequences. The molecular data rejected most of the previously proposed taxonomic subdivisions within this complex. The genera Abyssorchomene and Orchomenella as well as the subgenus Orchomenopsis appeared to be non-monophyletic. This implies that the supposed diagnostic characters are likely a result of convergent evolution. Further, our results indicated the necessity of a revision of the family-level systematics.

DNA Barcoding Amphibians and Reptiles

Only a few major research programs are currently targeting COI barcoding of amphibians and reptiles (including chelonians and crocodiles), two major groups of tetrapods. Amphibian and reptile species are typically old, strongly divergent, and contain deep conspecific lineages which might lead to problems in species assignment with incomplete reference databases. As far as known, there is no single pair of COI primers that will guarantee a sufficient rate of success across all amphibian and reptile taxa, or within major subclades of amphibians and reptiles, which means that the PCR amplification strategy needs to be adjusted depending on the specific research question. In general, many more amphibian and reptile taxa have been sequenced for 16S rDNA, which for some purposes may be a suitable complementary marker, at least until a more comprehensive COI reference database becomes available. DNA barcoding has successfully been used to identify amphibian larval stages (tadpoles) in species-rich tropical assemblages. Tissue sampling, DNA extraction, and amplification of COI is straightforward in amphibians and reptiles. Single primer pairs are likely to have a failure rate between 5 and 50% if taxa of a wide taxonomic range are targeted; in such cases the use of primer cocktails or subsequent hierarchical usage of different primer pairs is necessary. If the target group is taxonomically limited, many studies have followed a strategy of designing specific primers which then allow an easy and reliable amplification of all samples.

Exploring Species Level Taxonomy and Species Delimitation Methods in the Facultatively Self-Fertilizing Land Snail Genus Rumina (Gastropoda: Pulmonata)

Delimiting species in facultatively selfing taxa is a challenging problem of which the terrestrial pulmonate snail genus Rumina is a good example. These snails have a mixed breeding system and show a high degree of shell and color variation. Three nominal species (R. decollata, R. saharica and R. paivae) and two color morphs within R. decollata (dark and light) are currently recognized. The present study aims at evaluating to what extent these entities reflect evolutionary diverging taxonomic units, rather than fixed polymorphisms due to sustained selfing. Therefore, a phylogenetic analysis of nuclear (ITS1, ITS2) and mitochondrial DNA (COI, CytB, 12S rDNA, 16S rDNA) sequences was performed. Putative species in Rumina, inferred from the mitochondrial DNA phylogeny, were compared with those proposed on the basis of the COI gene by (1) DNA barcoding gap analysis, (2) Automatic Barcode Gap Discovery, (3) the species delimitation plug-in of the Geneious software, (4) the Genealogical Sorting Index, and (5) the General Mixed Yule Coalescent model. It is shown that these methods produce a variety of different species hypotheses and as such one may wonder to what extent species delimitation methods are really useful. With respect to Rumina, the data suggest at least seven species, one corresponding to R. saharica and six that are currently grouped under the name R. decollata. The species-level status of R. paivae is rejected.

Why is the molecular identification of the forensically important blowfly species Lucilia caesar and L. illustris (family Calliphoridae) so problematic

Species of the fly genus Lucilia are commonly used in forensic investigations to estimate the postmortem interval (PMI). Two close-related species Lucilia caesar and L. illustris are difficult to identify. Previous studies showed that the mitochondrial cytochrome c oxidase subunit I (COI) marker could be used to identify many Lucilia species. However, mixed results were obtained for L. caesar and L. illustris due to some European specimens showing identical haplotypes. Here, we investigated 58 new European male specimens of L. illustris and L. caesar whose morphological identifications were checked and for which COI fragments were sequenced. In addition, two other mitochondrial (cytochrome c oxidase subunit II and 16S) and two nuclear (internal transcribed spacer 2 and 28S ribosomal RNA) markers were obtained for a subset of these samples. For each marker, genetic divergence within each species was in the same range as between species, confirming the close relationship between both species. Moreover, for each of the gene fragments, both species shared at least one haplotype/genotype. Hence, none of the molecular markers tested could be used, alone or in combination, to discriminate between L. illustris and L. caesar.

Utility of GenBank and the Barcode of Life Data Systems (BOLD) for the identification of forensically important Diptera from Belgium and France.

Abstract Fly larvae living on dead corpses can be used to estimate post-mortem intervals. The identification of these flies is decisive in forensic casework and can be facilitated by using DNA barcodes provided that a representative and comprehensive reference library of DNA barcodes is available. We constructed a local (Belgium and France) reference library of 85 sequences of the COI DNA barcode fragment (mitochondrial cytochrome c oxidase subunit I gene), from 16 fly species of forensic interest (Calliphoridae, Muscidae, Fanniidae). This library was then used to evaluate the ability of two public libraries (GenBank and the Barcode of Life Data Systems – BOLD) to identify specimens from Belgian and French forensic cases. The public libraries indeed allow a correct identification of most specimens. Yet, some of the identifications remain ambiguous and some forensically important fly species are not, or insufficiently, represented in the reference libraries. Several search options offered by GenBank and BOLD can be used to further improve the identifications obtained from both libraries using DNA barcodes.

Species limits, interspecific hybridization and phylogeny in the cryptic land snail complex Pyramidula: The power of RADseq data

Restriction site-associated DNA sequencing (RADseq) was used to jointly assess phylogenetic relationships, interspecific hybridization and species delimitation in the cryptic, non-model land snail complex Pyramidula. A robust phylogeny was inferred using a matrix of concatenated sequences of almost 1,500,000bp long, containing >97,000 polymorphic sites. Maximum likelihood analyses fully resolved the phylogenetic relationships among species and drastically improved phylogenetic trees obtained from mtDNA and nDNA gene trees (COI, 16S rRNA, 5.8S rRNA, ITS2 and 28S rRNA sequence data). The best species delimitation scenario was selected on the basis of 875 unlinked single nucleotide polymorphisms, showing that nine Pyramidula species should be distinguished in Europe. Applying D-statistics provided no or weak evidence of interspecific hybridization among Pyramidula, except for some evidence of gene flow between two species.

Towards a revision of the Neotropical soldierless termites (Isoptera : Termitidae): redescription of the genus Anoplotermes and description of Longustitermes, gen. nov.*

Soldierless termites are well represented in the Neotropics where they constitute about one-third of the total termite species richness. However, despite their substantial diversity, they have been neglected by most taxonomists because they lack soldiers. Species identification therefore relies upon worker characters, in particular the anatomy of the digestive tract and the enteric valve armature. Here, in order to provide a solid basis for future taxonomic work on this group, we supply detailed descriptions of the type species of the genus Anoplotermes, A. pacificus, and of a few common and widespread species, which we retain in Anoplotermes: A. banksi, A. parvus and A. janus, sp. nov. We transfer Anoplotermes manni to a new genus, Longustitermes, and place six species into synonymy. This redefinition of the genus Anoplotermes is complemented by DNA sequences of four genetic markers to allow species identification by molecular techniques and future phylogenetic studies. This study represents a first step towards a complete revision of the neotropical Anoplotermes-group.