Thierry B. Hoareau

An efficient method to find potentially universal population genetic markers, applied to metazoans

BackgroundDespite the impressive growth of sequence databases, the limited availability of nuclear markers that are sufficiently polymorphic for population genetics and phylogeography and applicable across various phyla restricts many potential studies, particularly in non-model organisms. Numerous introns have invariant positions among kingdoms, providing a potential source for such markers. Unfortunately, most of the few known EPIC (Exon Primed Intron Crossing) loci are restricted to vertebrates or belong to multigenic families.ResultsIn order to develop markers with broad applicability, we designed a bioinformatic approach aimed at avoiding multigenic families while identifying intron positions conserved across metazoan phyla. We developed a program facilitating the identification of EPIC loci which allowed slight variation in intron position. From the Homolens databases we selected 29 gene families which contained 52 promising introns for which we designed 93 primer pairs. PCR tests were performed on several ascidians, echinoderms, bivalves and cnidarians. On average, 24 different introns per genus were amplified in bilaterians. Remarkably, five of the introns successfully amplified in all of the metazoan genera tested (a dozen genera, including cnidarians). The influence of several factors on amplification success was investigated. Success rate was not related to the phylogenetic relatedness of a taxon to the groups that most influenced primer design, showing that these EPIC markers are extremely conserved in animals.ConclusionsOur new method now makes it possible to (i) rapidly isolate a set of EPIC markers for any phylum, even outside the animal kingdom, and thus, (ii) compare genetic diversity at potentially homologous polymorphic loci between divergent taxa.

High‐throughput microsatellite marker development in two sparid species and verification of their transferability in the family Sparidae

Recently, 454 sequencing has emerged as a popular method for isolating microsatellites owing to cost‐effectiveness and time saving. In this study, repeat‐enriched libraries from two southern African endemic sparids (Pachymetopon blochii and Lithognathus lithognathus) were 454 GS‐FLX sequenced. From these, 7370 sequences containing repeats (SCRs) were identified. A brief survey of 23 studies showed a significant difference between the number of SCRs when enrichment was performed first before 454 sequencing. We designed primers for 302 unique fragments containing more than five repeat units and suitable flanking regions. A fraction (<11%) of these loci were characterized with 18 polymorphic microsatellite loci (nine in each of the focal species) being described. Sanger sequencing of alleles confirmed that size variation was because of differences in the number of tandem repeats. However, a case of homoplasy and sequencing errors in the 454 sequencing were identified. These newly developed and four previously isolated loci were successfully used to identify polymorphic markers in nine other economically important species, representative of sparid diversity. The combination of newly developed markers with data from previous sparid cross‐species studies showed a significant negative correlation between genetic divergence to focal species and microsatellite transferability. The high level of transferability we described (48% amplification success and 32% polymorphism) suggests that the 302 microsatellite loci identified represent an excellent resource for future studies on sparids. Microsatellite marker development should commonly include tests of transferability to reduce costs and increase feasibility of population genetics studies in nonmodel organisms.

Late Glacial Demographic Expansion Motivates a Clock Overhaul for Population Genetics

The molecular clock hypothesis is fundamental in evolutionary biology as by assuming constancy of the molecular rate it provides a timeframe for evolution. However, increasing evidence shows time dependence of inferred molecular rates with inflated values obtained using recent calibrations. As recent demographic calibrations are virtually non-existent in most species, older phylogenetic calibration points (>1 Ma) are commonly used, which overestimate demographic parameters. To obtain more reliable rates of molecular evolution for population studies, I propose the calibration of demographic transition (CDT) method, which uses the timing of climatic changes over the late glacial warming period to calibrate expansions in various species. Simulation approaches and empirical data sets from a diversity of species (from mollusk to humans) confirm that, when compared with other genealogy-based calibration methods, the CDT provides a robust and broadly applicable clock for population genetics. The resulting CDT rates of molecular evolution also confirm rate heterogeneity over time and among taxa. Comparisons of expansion dates with ecological evidence confirm the inaccuracy of phylogenetically derived divergence rates when dating population-level events. The CDT method opens opportunities for addressing issues such as demographic responses to past climate change and the origin of rate heterogeneity related to taxa, genes, time, and genetic information content.

Evolutionary history of a widespread Indo-Pacific goby: The role of Pleistocene sea-level changes on demographic contraction/expansion dynamics

Compared to endemics, widespread species are of particular interest to retrace recent evolutionary history. These species have a large population size which provides a clearer genetic signature of past events. Moreover, their wide geographic range increases the potential occurrence of evolutionary events (expansion, divergence, etc.). Here, we used several coalescent-based methods to disentangle the evolutionary history of a widespread amphidromous goby (Sicyopterus lagocephalus), in the light of sea-level variations during the Pleistocene. Using 75 samples recovered from three biogeographic regions (Western Indian Ocean, Melanesia and Polynesia), we analysed a portion of the cytochromeb gene and confirmed three major haplogroups, each specific to a region. Furthermore, we found that: (1) the Melanesian haplogroup was the oldest while the two peripheral regions hosted daughter haplogroups; (2) two centrifugal colonisation events occurred from Melanesia to the periphery, each synchronised with periods of strong paleo-ENSO episodes; (3) the demographic contraction-expansion events were linked to Pleistocene sea-level changes; (4) Melanesia and Polynesia acted as efficient refuges during the Last Glacial Maximum. These results highlight the importance of studying widespread species to better understand the role of climate changes and paleo-oceanography on the evolution of biodiversity.

Secondary contact and asymmetrical gene flow in a cosmopolitan marine fish across the Benguela upwelling zone

The combination of oceanographic barriers and habitat heterogeneity are known to reduce connectivity and leave specific genetic signatures in the demographic history of marine species. However, barriers to gene flow in the marine environment are almost never impermeable which inevitably allows secondary contact to occur. In this study, eight sampling sites (five along the South African coastline, one each in Angola, Senegal and Portugal) were chosen to examine the population genetic structure and phylogeographic history of the cosmopolitan bluefish (Pomatomus saltatrix), distributed across a large South-east Atlantic upwelling zone. Molecular analyses were applied to mtDNA cytochrome b, intron AM2B1 and 15 microsatellite loci. We detected uncharacteristically high genetic differentiation (FST 0.15–0.20; P<0.001) between the fish sampled from South Africa and the other sites, strongly influenced by five outlier microsatellite loci located in conserved intergenic regions. In addition, differentiation among the remaining East Atlantic sites was detected, although mtDNA indicated past isolation with subsequent secondary contact between these East Atlantic populations. We further identified secondary contact, with unidirectional gene flow from South Africa to Angola. The directional contact is likely explained by a combination of the northward flowing offshore current and endogenous incompatibilities restricting integration of certain regions of the genome and limiting gene flow to the south. The results confirm that the dynamic system associated with the Benguela current upwelling zone influences species distributions and population processes in the South-east Atlantic.

Putative origins of the fungus Leptographium procerum.

Appropriate management of invasive fungi requires adequate understanding of their global diversities and movement histories. The fungus Leptographium procerum is associated with root-colonizing forest insects in pine forests throughout the world, and may have contributed to the aggressive behaviour of the red turpentine beetle (Dendroctonus valens) in the beetles invasive range in China. We used microsatellites and mating type loci to investigate the global diversity of L. procerum and the source population of L. procerum associated with D. valens in China. Clustering analyses supported the separation of the fungal data set into three genetically and geographically-distinct clusters: Europe, North America, and China. The fungus had the highest genetic diversity in Europe, followed by North America and China. Analyses using Approximate Bayesian Computation supported Europe as the most likely source of the North American and Chinese populations. Overall, the results suggested that Europe is the global centre of diversity of L. procerum. Furthermore, they suggested that L. procerum most likely arrived in China independently of D. valens and adopted this beetle as a vector after its introduction.

Evaluating the resolution power of new microsatellites for species identification and stock delimitation in the Cape hakes Merluccius paradoxus and Merluccius capensis (Teleostei: Merlucciidae)

The utility of 15 new and 17 previously published microsatellite markers was evaluated for species identification and stock delimitation in the deep-water hake Merluccius paradoxus and the shallow-water hake Merluccius capensis. A total of 14 microsatellites were polymorphic in M. paradoxus and 10 in M. capensis. Two markers could individually discriminate the species using Bayesian clustering methods and a statistical power analysis showed that the set of markers for each species is likely to detect subtle genetic differentiation (FST < 0·006) that will be valuable to delimit and characterize genetic stocks.

DNA barcoding of reef brittle stars (Ophiuroidea, Echinodermata) from the southwestern Indian Ocean evolutionary hot spot of biodiversity

Abstract In anticipation of the current biodiversity crisis, it has become critical to rapidly and accurately assess biodiversity. DNA barcoding has proved efficient in facilitating the discovery and description of thousands of species and also provides insight into the dynamics of biodiversity. Here, we sequenced a portion of the mitochondrial cytochrome c oxidase subunit I (COI) gene from all morphospecies of reef brittle stars collected during a large‐scale biodiversity survey in the southwestern Indian Ocean (SWIO). Three methods of species delineation (Automatic Barcode Gap Discovery, Generalized Mixed Yule Coalescent model, and Bayesian Poisson Tree Processes) showed concordant results and revealed 51 shallow reef species in the region. Mean intraspecific genetic distances (0.005–0.064) and mean interspecific genetic distances within genera (0.056–0.316) were concordant with previous echinoderm studies. This study revealed that brittle‐star biodiversity is underestimated by 20% within SWIO and by >40% when including specimens from the Pacific Ocean. Results are discussed in terms of endemism, diversification processes, and conservation implications for the Indo‐West Pacific marine biodiversity. We emphasize the need to further our knowledge on biodiversity of invertebrate groups in peripheral areas.

Diagnostic description and geographic distribution of four new cryptic species of the blue-spotted maskray species complex (Myliobatoidei: Dasyatidae; Neotrygon spp.) based on DNA sequences

Nine morphologically similar but genetically distinct lineages in the blue-spotted maskray species complex, previously Neotrygon kuhlii (Müller and Henle) qualify as cryptic species. Four of these lineages have been previously described as Neotrygon australiae Last, White and Séret, Neotrygon caeruleopunctata Last, White and Séret, Neotrygon orientale Last, White and Séret, and Neotrygon varidens (Garman), but the morphological characters used in the descriptions offered poor diagnoses and their geographic distributions were not delineated precisely. The objective of the present work is to complete the description of the cryptic species in the complex. Here, an additional four lineages are described as new species on the basis of their mitochondrial DNA sequences: Neotrygon bobwardi, whose distribution extends from the northern tip of Aceh to the western coast of Sumatera; Neotrygon malaccensis, sampled from the eastern part of the Andaman Sea and from the Malacca Strait; Neotrygon moluccensis, from the eastern half of the Banda Sea; and Neotrygon westpapuensis from the central portion of northern West Papua. The geographic distributions of N. australiae, N. coeruleopunctata, N. orientale, and N. varidens are updated. For each species, a diagnosis is provided in the form of a combination of private or partly-private nucleotides at 2–4 nucleotide sites along a 519-base pair fragment of the CO1 gene. We believe that the present taxonomic revision will provide information relevant to the sound management and conservation of cryptic species of the blue-spotted maskray in the Coral Triangle region.

Investigating the origin of vagrant dusky groupers, Epinephelus marginatus (Lowe, 1834), in coastal waters of Réunion Island.

Due to their geographic isolation, biotas of oceanic islands are likely influenced by episodic long distance dispersal events, but such observations are scarce. In June 2012, fishermen from Réunion Island caught an unknown specimen of grouper, identified as dusky grouper Epinephelus marginatus (Lowe, 1834). This was highly unexpected considering the large distance of its closest verified occurrence (South Africa, ∼2500km). To identify the origin of this specimen and the mechanisms driving this potential long distance colonization, we combined genetic analyses and hydrodynamic connectivity modeling approaches. Molecular markers and samples from various locations across the distribution range resulted in the identification of three putative source populations. The Réunion specimen clustered genetically with South Africa. The estimated spawning period in relation to the connectivity modeling of larvae showed no possible direct connection between South Africa and Réunion. However, connectivity was predicted through intermediate stepping stone populations likely located around the southern tip of Madagascar, where the occurrence of the species has yet to be verified. The results further highlight the potential role of the cyclone Bingiza (February 2011) in the connection between Madagascar and Réunion. This shows that cyclones may be an important driver in long distance colonization of oceanic islands.