Zoltán T. Nagy

Multiple colonization of Madagascar and Socotra by colubrid snakes: evidence from nuclear and mitochondrial gene phylogenies.

Colubrid snakes form a speciose group of unclarified phylogeny. Their almost cosmopolitan distribution could be interpreted as a product of plate–tectonic vicariance. We used sequences of the nuclear c–mos, the mitochondrial cytochrome b and the 16S rRNA genes in 41 taxa to elucidate the relationships between the endemic colubrid genera found in Madagascar and in the Socotra archipelago. The well–resolved trees indicate multiple origins of both the Malagasy and the Socotran taxa. The Malagasy genus Mimophis was nested within the Psammophiinae, and the Socotran Hemerophis was closely related to Old World representatives of the former genus Coluber. The remaining 14 genera of Malagasy colubrids formed a monophyletic sister group of the Socotran Ditypophis (together forming the Pseudoxyrhophiinae). Molecular–clock estimates place the divergence of Malagasy and Socotran colubrids from their non–insular sister groups into a time–frame between the Eocene and Miocene. Over–seas rafting is the most likely hypothesis for the origin of at least the Malagasy taxa. The discovery of a large monophyletic clade of colubrids endemic to Madagascar indicates a need for taxonomic changes. The relationship of this radiation to the Socotran Ditypophis highlights the potential of the Indian Ocean islands to act as an evolutionary reservoir for lineages that have become extinct in Africa and Asia.

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.

A hands-on overview of tissue preservation methods for molecular genetic analyses

DNA studies have overwhelming importance in biological science. The aim of this paper is to present a compact and hands-on summary of widely available tissue preservation methods by listing dry, fluid/buffered and freezing techniques. Thereby, practical aspects, advantages and disadvantages, safety and feasibility issues of each method are discussed and compared.

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.

Cold Code: the global initiative to DNA barcode amphibians and nonavian reptiles

DNA barcoding facilitates the identification of species and the estimation of biodiversity by using nucleotide sequences, usually from the mitochondrial genome. Most studies accomplish this task by using the gene encoding cytochrome oxidase subunit I (COI; Entrez COX1). Within this barcoding framework, many taxonomic initiatives exist, such as those specializing in fishes, birds, mammals, and fungi. Other efforts center on regions, such as the Arctic, or on other topics, such as health. DNA barcoding initiatives exist for all groups of vertebrates except for amphibians and nonavian reptiles. We announce the formation of Cold Code, the international initiative to DNA barcode all species of these ‘cold‐blooded’ vertebrates. The project has a Steering Committee, Coordinators, and a home page. To facilitate Cold Code, the Kunming Institute of Zoology, Chinese Academy of Sciences will sequence COI for the first 10 specimens of a species at no cost to the steward of the tissues.

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.

Island Evolution and Systematic Revision of Comoran Snakes: Why and When Subspecies Still Make Sense

Species delimitation and species concepts have been a matter of debate among biodiversity researchers in the last decades, resulting in integrative taxonomy approaches and the use of modern species concepts, such as the phylogenetic, evolutionary or general lineage species concepts. The discussion of subspecies status and concepts has been addressed much less extensively, with some researchers completely refraining from recognizing subspecies. However, allopatric insular populations that are particularly differentiated have traditionally been assigned subspecies status. We studied the molecular phylogeny and morphology of endemic Comoran tree snakes of the genus Lycodryas. Taking an integrative taxonomic approach, we used the concept of independent lines of evidence to discriminate between evidence for specific and subspecific status. Molecular (mtDNA) and morphological data provided sufficient evidence to support four different taxa within Comoran Lycodryas. In a revision of this group, we propose two species, each with two subspecies. We present a discussion of the strong sexual dichromatism unique to Comoran Lycodryas within the genus and related genera that may be explained by sexual selection in combination with the absence of major predators. Then, we discuss the effects of insular evolution and the “island rule” on morphological traits in Comoran Lycodryas and in Liophidium mayottensis, another snake endemic to the Comoros. The absence of larger-bodied snakes may have promoted an increase in body size and the number of dorsal scale rows in these species. Finally, we discuss the subspecies concept, its applications and its significance for integrative taxonomy and for limiting taxonomic inflation. We emphasize that taxon descriptions should be based on an integrative approach using several lines of evidence, preferably in combination with statements on the underlying species concepts or operational criteria, to increase the objectivity and comparability of descriptions.

Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding

Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well‐supported clusters. Intraspecific Kimura 2‐parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra‐ and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species.