DNA metabarcoding suggests adaptive seasonal variation of individual trophic traits in a critically endangered fish.

Dietary studies are critical for understanding foraging strategies and have important applications in conservation and habitat management. We applied a robust metabarcoding protocol to characterize the diet of the critically endangered freshwater fish Zingel asper (the Rhone streber). We conducted modelling and simulation analyses to identify and characterize some of the drivers of individual trophic trait variation in this species. We found that population density and ontogeny had minor effects on the trophic niche of Z. asper. Instead, our results suggest that the majority of trophic niche variation was driven by seasonal variation in ecological opportunity. The total trophic niche width of Z. asper seasonally expanded to include a broader range of prey. Furthermore, null model simulations revealed that the increase of between-individual variation in autumn indicates that Z. asper become more opportunistic relative to summer and spring, rather than being associated with a seasonal specialization of individuals. Overall, our results suggest an adaptive variation of individual trophic traits in Z. asper: the species mainly consumes a few ephemeropteran taxa (Baetis fuscatus and Ecdyonurus) but seems to be capable of adapting its foraging strategy to maintain its body condition. This study illustrates how metabarcoding data obtained from faeces can be validated and combined with individual-based modelling and simulation approaches to explore inter- and intrapopulational individual trophic traits variation and to test hypotheses in the conventional analytic framework of trophic ecology.

VTAM: A robust pipeline for validating metabarcoding data using controls.

To obtain accurate estimates for biodiversity and ecological studies, metabarcoding studies should be carefully designed to minimize both false positive (FP) and false negative (FN) occurrences. Internal controls (mock samples and negative controls), replicates, and overlapping markers allow controlling metabarcoding errors but current metabarcoding software packages do not explicitly integrate these additional experimental data to optimize filtering. We have developed the metabarcoding analysis software VTAM, which uses explicitly these elements of the experimental design to find optimal parameter settings that minimize FP and FN occurrences. VTAM showed similar sensitivity, but a higher precision compared to two other pipelines using three datasets and two different markers (COI, 16S). The stringent filtering procedure implemented in VTAM aims to produce robust metabarcoding data to obtain accurate ecological estimates and represents an important step towards a non-arbitrary and standardized validation of metabarcoding data for conducting ecological studies. VTAM is implemented in Python and available from: https://github.com/aitgon/vtam. The VTAM benchmark code is available from: https://github.com/aitgon/vtam_benchmark.

Spineless and overlooked: DNA metabarcoding of autonomous reef monitoring structures reveals intra- and interspecific genetic diversity in Mediterranean invertebrates.

The ability to gather genetic information using DNA metabarcoding of bulk samples obtained directly from the environment is crucial to determine biodiversity baselines and understand population dynamics in the marine realm. While DNA metabarcoding is effective in evaluating biodiversity at community level, genetic patterns within species are often concealed in metabarcoding studies and overlooked for marine invertebrates. In the present study, we implement recently developed bioinformatics tools to investigate intraspecific genetic variability for invertebrate taxa in the Mediterranean Sea. Using metabarcoding samples from Autonomous Reef Monitoring Structures (ARMS) deployed in three locations, we present haplotypes and diversity estimates for 145 unique species. While overall genetic diversity was low, we identified several species with high diversity records and potential cryptic lineages. Further, we emphasize the spatial scale of genetic variability, which was observed from locations to individual sampling units (ARMS). We carried out a population genetic analysis of several important yet understudied species, which highlights the current knowledge gap concerning intraspecific genetic patterns for the target taxa in the Mediterranean basin. Our approach considerably enhances biodiversity monitoring of charismatic and understudied Mediterranean species, which can be incorporated into ARMS surveys.

'Back to the future': how archaeological remains can describe salmon adaptation to climate change.

A strategy for species to survive climate change will be to change adaptively their way of life. Understanding rapid adaptation to climate change is therefore a priority for current research. In this issue, Turrero et al. (2012) use an original approach to unravel life history trait responses to climate change in two fish species (Salmo trutta and S. salar). Going against the flow, the authors adopt the strategy of going back to the future by investigating the responses of fish to the warming periods that followed the Last Glacial Period (approximately 30-20,000 years BP). To do this, they analysed Salmo vertebrae from well-dated archaeological sites in northern Spain in order to uncover key life history traits, which they then compared to those of contemporary specimens. They found that, as the climate got warmer, Salmo species tended to reduce the time spent in growing areas and reached spawning areas at a younger age; this tendency began approximately 15,000 years BP and accelerated in contemporary periods. The implication is a lower age at maturity and a lower reproductive success, which they tentatively related to recent declines in population growth rate. This innovative study demonstrates how changes in life history traits are linked both to the population growth rate and to the evolutionary rate under climatic constraints, which may serve as a basis for future conservation research.

QDD: a user-friendly program to select microsatellite markers and design primers from large sequencing projects.

SUMMARY: QDD is an open access program providing a user-friendly tool for microsatellite detection and primer design from large sets of DNA sequences. The program is designed to deal with all steps of treatment of raw sequences obtained from pyrosequencing of enriched DNA libraries, but it is also applicable to data obtained through other sequencing methods, using FASTA files as input. The following tasks are completed by QDD: tag sorting, adapter/vector removal, elimination of redundant sequences, detection of possible genomic multicopies (duplicated loci or transposable elements), stringent selection of target microsatellites and customizable primer design. It can treat up to one million sequences of a few hundred base pairs in the tag-sorting step, and up to 50,000 sequences in a single input file for the steps involving estimation of sequence similarity. AVAILABILITY: QDD is freely available under the GPL licence for Windows and Linux from the following web site: http://www.univ-provence.fr/gsite/Local/egee/dir/meglecz/QDD.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Human ancient and extant mtDNA from the Gambier Islands (French polynesia): evidence for an early Melanesian maternal contribution and new perspectives into the settlement of easternmost Polynesia.

Molecular anthropology has been widely used to infer the origin and processes of the colonization of Polynesia. However, there are still a lack of representative geographical studies of Eastern Polynesia and unchallenged genetic data about ancient Polynesian people. The absence of both of these elements prevents an accurate description of the demographic processes of internal dispersion within the Polynesian triangle. This study provides a twofold analysis of ancient and modern mtDNA in the eastern part of French Polynesia: the Gambier Islands. The paleogenetic analyses conducted on burials of the Temoe Atoll (14(th) -17(th) centuries) represent the first fully authenticated ancient human sequences from Polynesia. The identification of the "Melanesian" Q1 mtDNA lineage in ancient human remains substantiates the Near Oceanic contribution to the early gene pool of this region. Modern samples originate from Mangareva Island. Genealogical investigations enable us to reliably identify the conservation of the Melanesian component in Easternmost Polynesia, despite recent European colonization. Finally, the identification of rare mutations in sequences belonging to haplogroup B4a1a1a provides new perspectives to the debate on the internal peopling of the Polynesian region. Altogether, the results laid out in our study put the emphasis on the necessity of controlled sampling when discussing the internal settlement of Polynesia.

Representativeness of microsatellite distributions in genomes, as revealed by 454 GS-FLX titanium pyrosequencing.

BACKGROUND: Microsatellites are markers of choice in population genetics and genomics, as they provide useful insight into patterns and processes as diverse as genome evolutionary dynamics and demographic processes. The acquisition of microsatellites through multiplex-enriched libraries and 454 GS-FLX Titanium pyrosequencing is a promising new tool for the isolation of new markers in unknown genomes. This approach can also be used to evaluate the extent to which microsatellite-enriched libraries are representative of the genome from which they were isolated. In this study, we deciphered potential discrepancies in microsatellite content recovery for two reference genomes (Apis mellifera and Danio rerio), selected on the basis of their extreme heterogeneity in terms of the proportions and distributions of microsatellites on chromosomes. RESULTS: The A. mellifera genome, in particular, was found to be highly heterogeneous, due to extremely high rates of recombination, with hotspots, but the only bias consistently introduced into pyrosequenced multiplex-enriched libraries concerned sequence length, with the overrepresentation of sequences 160 to 320 bp in length. Other deviations from expected proportions or distributions of motifs on chromosomes were observed, but the significance and intensity of these deviations was mostly limited. Furthermore, no consistent adverse competition between multiplexed probes was observed during the motif enrichment phase. CONCLUSIONS: This approach therefore appears to be a promising strategy for improving the development of microsatellites, as it introduces no major bias in terms of the proportions and distribution of microsatellites.

Quantifying the individual impact of artificial barriers in freshwaters: A standardized and absolute genetic index of fragmentation.

Fragmentation by artificial barriers is an important threat to freshwater biodiversity. Mitigating the negative aftermaths of fragmentation is of crucial importance, and it is now essential for environmental managers to benefit from a precise estimate of the individual impact of weirs and dams on river connectivity. Although the indirect monitoring of fragmentation using molecular data constitutes a promising approach, it is plagued with several constraints preventing a standardized quantification of barrier effects. Indeed, observed levels of genetic differentiation GD depend on both the age of the obstacle and the effective size of the populations it separates, making comparisons of the actual barrier effect of different obstacles difficult. Here, we developed a standardized genetic index of fragmentation (F INDEX), allowing an absolute and independent assessment of the individual effects of obstacles on connectivity. The F INDEX is the standardized ratio between the observed GD between pairs of populations located on either side of an obstacle and the GD expected if this obstacle completely prevented gene flow. The expected GD is calculated from simulations taking into account two parameters: the number of generations since barrier creation and the expected heterozygosity of the populations, a proxy for effective population size. Using both simulated and empirical datasets, we explored the validity and the limits of the F INDEX. We demonstrated that it allows quantifying effects of fragmentation only from a few generations after barrier creation and provides valid comparisons among obstacles of different ages and populations (or species) of different effective sizes. The F INDEX requires a minimum amount of fieldwork and genotypic data and solves some of the difficulties inherent to the study of artificial fragmentation in rivers and potentially in other ecosystems. This makes the F INDEX promising to support the management of freshwater species affected by barriers, notably for planning and evaluating restoration programs.

A river runs through it: The causes, consequences, and management of intraspecific diversity in river networks.

Rivers are fascinating ecosystems in which the eco-evolutionary dynamics of organisms are constrained by particular features, and biologists have developed a wealth of knowledge about freshwater biodiversity patterns. Over the last 10 years, our group used a holistic approach to contribute to this knowledge by focusing on the causes and consequences of intraspecific diversity in rivers. We conducted empirical works on temperate permanent rivers from southern France, and we broadened the scope of our findings using experiments, meta-analyses, and simulations. We demonstrated that intraspecific (genetic) diversity follows a spatial pattern (downstream increase in diversity) that is repeatable across taxa (from plants to vertebrates) and river systems. This pattern can result from interactive processes that we teased apart using appropriate simulation approaches. We further experimentally showed that intraspecific diversity matters for the functioning of river ecosystems. It indeed affects not only community dynamics, but also key ecosystem functions such as litter degradation. This means that losing intraspecific diversity in rivers can yield major ecological effects. Our work on the impact of multiple human stressors on intraspecific diversity revealed that-in the studied river systems-stocking of domestic (fish) strains strongly and consistently alters natural spatial patterns of diversity. It also highlighted the need for specific analytical tools to tease apart spurious from actual relationships in the wild. Finally, we developed original conservation strategies at the basin scale based on the systematic conservation planning framework that appeared pertinent for preserving intraspecific diversity in rivers. We identified several important research avenues that should further facilitate our understanding of patterns of local adaptation in rivers, the identification of processes sustaining intraspecific biodiversity-ecosystem function relationships, and the setting of reliable conservation plans.

Inter- and extra-Indian admixture and genetic diversity in reunion island revealed by analysis of mitochondrial DNA.

Reunion Island is a French territory located in the western Indian Ocean. The genetic pattern of the Reunionese population has been shaped by contributions from highly contrasting regions of the world. Over the last 350 years, several migration waves and cultural and socio-economic factors have led to the emergence of six main ethnic groups in Reunion. India is one of the principal regions that contributed to the setting up of the Reunionese population. Diversity, demographic and admixture analyses were performed on mtDNA variation of the Reunionese of Indian ancestry, including the Malbar and Zarab ethnic groups, in order to question their history. Using a phylogeographical approach, we generated and analysed quantitative data on the contribution of the Indian parental populations. Furthermore, we showed that the settlement of Reunion Island by Indians did not involve a founder effect, except in the very beginning of the Reunionese settlement (at the end of the 17(th) century). The accuracy of our results was optimised by a re-evaluation of the classification of the Southern Asian mtDNA haplogroups. Finally, by comparing our results to a previous study dealing with the Reunionese population, we highlighted how ethno-historical data are critical for reconstructing the complex history of multiethnic populations.

Complete mitochondrial sequences for haplogroups M23 and M46: insights into the Asian ancestry of the Malagasy population.

Through the sequencing of the complete mitochondrial genome of three individuals of Malagasy ancestry, we completed the characterization of the island southeastern Asian specific M46 haplogroup. We assumed that the association of the np 3588 and np 16278 polymorphisms were M46 specific. In addition, we characterized a novel basal M subhaplogroup: M23. This clade can be defined by one coding region transition at np 10295 and one control region transition at np 16263. Our data suggest the arrival of South Asian migrants before the start of the 15th century and highlights the fact that future studies dealing with the settlement of Madagascar should consider at least three potential source populations (Africa, Indonesia, and India).

One-locus-several-primers: A strategy to improve the taxonomic and haplotypic coverage in diet metabarcoding studies.

In diet metabarcoding analyses, insufficient taxonomic coverage of PCR primer sets generates false negatives that may dramatically distort biodiversity estimates. In this paper, we investigated the taxonomic coverage and complementarity of three cytochrome c oxidase subunit I gene (COI) primer sets based on in silico analyses and we conducted an in vivo evaluation using fecal and spider web samples from different invertivores, environments, and geographic locations. Our results underline the lack of predictability of both the coverage and complementarity of individual primer sets: (a) sharp discrepancies exist observed between in silico and in vivo analyses (to the detriment of in silico analyses); (b) both coverage and complementarity depend greatly on the predator and on the taxonomic level at which preys are considered; (c) primer sets' complementarity is the greatest at fine taxonomic levels (molecular operational taxonomic units [MOTUs] and variants). We then formalized the "one-locus-several-primer-sets" (OLSP) strategy, that is, the use of several primer sets that target the same locus (here the first part of the COI gene) and the same group of taxa (here invertebrates). The proximal aim of the OLSP strategy is to minimize false negatives by increasing total coverage through multiple primer sets. We illustrate that the OLSP strategy is especially relevant from this perspective since distinct variants within the same MOTUs were not equally detected across all primer sets. Furthermore, the OLSP strategy produces largely overlapping and comparable sequences, which cannot be achieved when targeting different loci. This facilitates the use of haplotypic diversity information contained within metabarcoding datasets, for example, for phylogeography and finer analyses of prey-predator interactions.

A from-benchtop-to-desktop workflow for validating HTS data and for taxonomic identification in diet metabarcoding studies.

The main objective of this work was to develop and validate a robust and reliable "from-benchtop-to-desktop" metabarcoding workflow to investigate the diet of invertebrate-eaters. We applied our workflow to faecal DNA samples of an invertebrate-eating fish species. A fragment of the cytochrome c oxidase I (COI) gene was amplified by combining two minibarcoding primer sets to maximize the taxonomic coverage. Amplicons were sequenced by an Illumina MiSeq platform. We developed a filtering approach based on a series of nonarbitrary thresholds established from control samples and from molecular replicates to address the elimination of cross-contamination, PCR/sequencing errors and mistagging artefacts. This resulted in a conservative and informative metabarcoding data set. We developed a taxonomic assignment procedure that combines different approaches and that allowed the identification of ~75% of invertebrate COI variants to the species level. Moreover, based on the diversity of the variants, we introduced a semiquantitative statistic in our diet study, the minimum number of individuals, which is based on the number of distinct variants in each sample. The metabarcoding approach described in this article may guide future diet studies that aim to produce robust data sets associated with a fine and accurate identification of prey items.

mtDNA polymorphisms in five French groups: importance of regional sampling.

According to classical markers, France has been reported to be regionally heterogeneous. Here, we propose to test the homogeneity of the French mitochondrial gene pool by analysing D-Loop and coding regions polymorphisms in 210 individuals stemming from five regions. The data set obtained was also used to test the ability of mitochondrial DNA to detect well historically established admixtures (admixtures between British/Irish people and native Breton people in our case). For these purposes, the sampling procedure was subject to special care, concerning the individuals' geographical origin and maternal pedigree. The mtDNA analysis revealed some regional specificities in haplogroup distribution, which is discussed in terms of successive settlements of France. Statistical analyses were conducted to investigate mtDNA diversity and structure within and between British, Irish and French groups. They tended to show affinities between Morbihan region and Britain plus Ireland. Furthermore, genetic evidences were in line with the fact that Morbihan region results from an admixture event, agreeing with historical evidences of successive migrations from Britain and Ireland into Brittany. These results also tended to outline the fact that two geographically very adjacent samples (Morbihan and Finistère), sharing a cultural and linguistic area, can present a distinct genetic pattern. Although mtDNA analyses were able to identify a historically reported admixture event, we point out here the high influence of the sampling procedure and representativeness over the migrations hypothesis. We also underline the importance of regional sampling for studies on the spread and/or origin of specific European haplogroups (here U5a1a and U8).

Microsatellite primers in Parietaria judaica (Urticaceae) to assess genetic diversity and structure in urban landscapes.

PREMISE OF THE STUDY: Urbanization is one of the main factors contributing to loss of genetic diversity, as the resulting landscape fragmentation and habitat loss induce species isolation. However, studies of genetic structure and diversity in urbanized landscapes are still rare. We characterized microsatellite primers for Parietaria judaica to study this environment. • METHODS AND RESULTS: Eleven microsatellite loci from P. judaica, an urban plant, were isolated using shotgun pyrosequencing, and the simple sequence repeat (SSR) markers were screened in 20 individuals of P. judaica. The loci were tested on 166 individuals from three populations in different cities. The number of alleles ranged from two to 19, and expected and observed heterozygosity values ranged from 0.019 to 0.912 and 0.019 to 0.448, respectively. • CONCLUSIONS: The markers amplified well in the species and will be useful for examining genetic diversity and population genetic structure in this urban plant.

QDD version 3.1: a user-friendly computer program for microsatellite selection and primer design revisited: experimental validation of variables determining genotyping success rate.

Microsatellite marker development has been greatly simplified by the use of high-throughput sequencing followed by in silico microsatellite detection and primer design. However, the selection of markers designed by the existing pipelines depends either on arbitrary criteria, or older studies on PCR success. Based on wet laboratory experiments, we have identified the following factors that are most likely to influence genotyping success rate: alignment score between the primers and the amplicon; the distance between primers and microsatellites; the length of the PCR product; target region complexity and the number of reads underlying the sequence. The QDD pipeline has been modified to include these most pertinent factors in the output to help the selection of markers. Furthermore, new features are also included in the present version: (i) not only raw sequencing reads are accepted as input, but also contigs, allowing the analysis of assembled high-coverage data; (ii) input data can be both in fasta and fastq format to facilitate the use of Illumina and IonTorrent reads; (iii) A comparison to known transposable elements allows their detection; (iv) A contamination check can be carried out by BLASTing potential markers against the nucleotide (nt) database of NCBI; (v) QDD3 is now also available imbedded into a virtual machine making installation easier and operating system independent. It can be used both on command-line version as well as integrated into a Galaxy server, providing a user-friendly interface, as well as the possibility to utilize a large variety of NGS tools.

Combining genetic and demographic data for prioritizing conservation actions: insights from a threatened fish species.

Prioritizing and making efficient conservation plans for threatened populations requires information at both evolutionary and ecological timescales. Nevertheless, few studies integrate multidisciplinary approaches, mainly because of the difficulty for conservationists to assess simultaneously the evolutionary and ecological status of populations. Here, we sought to demonstrate how combining genetic and demographic analyses allows prioritizing and initiating conservation plans. To do so, we combined snapshot microsatellite data and a 30-year-long demographic survey on a threatened freshwater fish species (Parachondrostoma toxostoma) at the river basin scale. Our results revealed low levels of genetic diversity and weak effective population sizes (<63 individuals) in all populations. We further detected severe bottlenecks dating back to the last centuries (200-800 years ago), which may explain the differentiation of certain populations. The demographic survey revealed a general decrease in the spatial distribution and abundance of P. toxostoma over the last three decades. We conclude that demo-genetic approaches are essential for (1) identifying populations for which both evolutionary and ecological extinction risks are high; and (2) proposing conservation plans targeted toward these at risk populations, and accounting for the evolutionary history of populations. We suggest that demo-genetic approaches should be the norm in conservation practices. We combined genetic and demographic data from a threatened freshwater fish species (Parachondrostoma toxostoma) at the river basin scale for conservation purposes. Genetic diversity and effective population sizes are very low, probably due to the strong genetic bottlenecks detected in this study. The species spatial distribution and abundance also decreased during the last decades.

From Late Miocene to Holocene: processes of differentiation within the Telestes genus (Actinopterygii: Cyprinidae).

Investigating processes and timing of differentiation of organisms is critical in the understanding of the evolutionary mechanisms involved in microevolution, speciation, and macroevolution that generated the extant biodiversity. From this perspective, the Telestes genus is of special interest: the Telestes species have a wide distribution range across Europe (from the Danubian district to Mediterranean districts) and have not been prone to translocation. Molecular data (mtDNA: 1,232 bp including the entire Cyt b gene; nuclear genome: 11 microsatellites) were gathered from 34 populations of the Telestes genus, almost encompassing the entire geographic range. Using several phylogenetic and molecular dating methods interpreted in conjunction with paleoclimatic and geomorphologic evidence, we investigated the processes and timing of differentiation of the Telestes lineages. The observed genetic structure and diversity were largely congruent between mtDNA and microsatellites. The Messinian Salinity Crisis (Late Miocene) seems to have played a major role in the speciation processes of the genus. Focusing on T. souffia, a species occurring in the Danube and Rhone drainages, we were able to point out several specific events from the Pleistocene to the Holocene that have likely driven the differentiation and the historical demography of this taxon. This study provides support for an evolutionary history of dispersal and vicariance with unprecedented resolution for any freshwater fish in this region.

High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries.

Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next-generation sequencing on 454 GS-FLX Titanium platforms. The procedure was calibrated on a model species (Apis mellifera) and validated on 13 other species from various taxonomic groups (animals, plants and fungi), including taxa for which severe difficulties were previously encountered using traditional methods. We obtained from 11,497 to 34,483 sequences depending on the species and the number of detected microsatellite loci ranged from 199 to 5791. We thus demonstrated that this procedure can be readily and successfully applied to a large variety of taxonomic groups, at much lower cost than would have been possible with traditional protocols. This method is expected to speed up the acquisition of high-quality genetic markers for nonmodel organisms.

Cross-species amplification of 41 microsatellites in European cyprinids: A tool for evolutionary, population genetics and hybridization studies.

BACKGROUND: Cyprinids display the most abundant and widespread species among the European freshwater Teleostei and are known to hybridize quite commonly. Nevertheless, a limited number of markers for conducting comparative differentiation, evolutionary and hybridization dynamics studies are available to date. FINDINGS: Five multiplex PCR sets were optimized in order to assay 41 cyprinid-specific polymorphic microsatellite loci (including 10 novel loci isolated from Chondrostoma nasus nasus, Chondrostoma toxostoma toxostoma and Leuciscus leuciscus) for 503 individuals (440 purebred specimens and 63 hybrids) from 15 European cyprinid species. The level of genetic diversity was assessed in Alburnus alburnus, Alburnoides bipunctatus, C. genei, C. n. nasus, C. soetta, C. t. toxostoma, L. idus, L. leuciscus, Pachychilon pictum, Rutilus rutilus, Squalius cephalus and Telestes souffia. The applicability of the markers was also tested on Abramis brama, Blicca bjoerkna and Scardinius erythrophtalmus specimens. Overall, between 24 and 37 of these markers revealed polymorphic for the investigated species and 23 markers amplified for all the 15 European cyprinid species. CONCLUSIONS: The developed set of markers demonstrated its performance in discriminating European cyprinid species. Furthermore, it allowed detecting and characterizing hybrid individuals. These microsatellites will therefore be useful to perform comparative evolutionary and population genetics studies dealing with European cyprinids, what is of particular interest in conservation issues and constitutes a tool of choice to conduct hybridization studies.