DNA metabarcoding diet analysis for species with parapatric vs sympatric distribution: a case study on subterranean rodents.

Closely related sympatric species commonly develop different ecological strategies to avoid competition. Ctenomys minutus and C. flamarioni are subterranean rodents parapatrically distributed in the southern Brazilian coastal plain, showing a narrow sympatric zone. To gain understanding on food preferences and possible competition for food resources, we evaluated their diet composition performing DNA metabarcoding analyzes of 67 C. minutus and 100 C. flamarioni scat samples, collected along the species geographical ranges. Thirteen plant families, mainly represented by Poaceae, Araliaceae, Asteraceae and Fabaceae, were identified in the diet of C. minutus. For C. flamarioni, 10 families were recovered, with a predominance of Poaceae, Araliaceae and Asteraceae. A significant correlation between diet composition and geographical distance was detected in C. minutus, whereas the diet of C. flamarioni was quite homogeneous throughout its geographical distribution. No significant differences were observed between males and females of each species. However, differences in diet composition between species were evident according to multivariate analysis. Our results suggest some level of diet partitioning between C. flamarioni and C. minutus in the sympatric region. While the first species is more specialized on few plant items, the second showed a more varied and heterogeneous diet pattern among individuals. These differences might have been developed to avoid competition in the region of co-occurrence. Resource availability in the environment also seems to influence food choices. Our data indicate that C. minutus and C. flamarioni are generalist species, but that some preference for Poaceae, Asteraceae and Araliaceae families can be suggested for both rodents.

Multiplexing PCR allows the identification of within-species genetic diversity in ancient eDNA.

Sedimentary ancient DNA (sedaDNA) has rarely been used to obtain population-level data due to either a lack of taxonomic resolution for the molecular method used, limitations in the reference material or inefficient methods. Here, we present the potential of multiplexing different PCR primers to retrieve population-level genetic data from sedaDNA samples. Vaccinium uliginosum (Ericaceae) is a widespread species with a circumpolar distribution and three lineages in present-day populations. We searched 18 plastid genomes for intraspecific variable regions and developed 61 primer sets to target these. Initial multiplex PCR testing resulted in a final set of 38 primer sets. These primer sets were used to analyse 20 lake sedaDNA samples (11,200 cal. yr BP to present) from five different localities in northern Norway, the Alps and the Polar Urals. All known V. uliginosum lineages in these regions and all primer sets could be recovered from the sedaDNA data. For each sample on average 28.1 primer sets, representing 34.15 sequence variants, were recovered. All sediment samples were dominated by a single lineage, except three Alpine samples which had co-occurrence of two different lineages. Furthermore, lineage turnover was observed in the Alps and northern Norway, suggesting that present-day phylogeographical studies may overlook past genetic patterns. Multiplexing primer is a promising tool for generating population-level genetic information from sedaDNA. The relatively simple method, combined with high sensitivity, provides a scalable method which will allow researchers to track populations through time and space using environmental DNA.

DNA from soil mirrors plant taxonomic and growth form diversity.

Ecosystems across the globe are threatened by climate change and human activities. New rapid survey approaches for monitoring biodiversity would greatly advance assessment and understanding of these threats. Taking advantage of next-generation DNA sequencing, we tested an approach we call metabarcoding: high-throughput and simultaneous taxa identification based on a very short (usually <100 base pairs) but informative DNA fragment. Short DNA fragments allow the use of degraded DNA from environmental samples. All analyses included amplification using plant-specific versatile primers, sequencing and estimation of taxonomic diversity. We tested in three steps whether degraded DNA from dead material in soil has the potential of efficiently assessing biodiversity in different biomes. First, soil DNA from eight boreal plant communities located in two different vegetation types (meadow and heath) was amplified. Plant diversity detected from boreal soil was highly consistent with plant taxonomic and growth form diversity estimated from conventional above-ground surveys. Second, we assessed DNA persistence using samples from formerly cultivated soils in temperate environments. We found that the number of crop DNA sequences retrieved strongly varied with years since last cultivation, and crop sequences were absent from nearby, uncultivated plots. Third, we assessed the universal applicability of DNA metabarcoding using soil samples from tropical environments: a large proportion of species and families from the study site were efficiently recovered. The results open unprecedented opportunities for large-scale DNA-based biodiversity studies across a range of taxonomic groups using standardized metabarcoding approaches.

Molecular tools and analytical approaches for the characterization of farm animal genetic diversity.

Genetic studies of livestock populations focus on questions of domestication, within- and among-breed diversity, breed history and adaptive variation. In this review, we describe the use of different molecular markers and methods for data analysis used to address these questions. There is a clear trend towards the use of single nucleotide polymorphisms and whole-genome sequence information, the application of Bayesian or Approximate Bayesian analysis and the use of adaptive next to neutral diversity to support decisions on conservation.

New insights on lake sediment DNA from the catchment: importance of taphonomic and analytical issues on the record quality.

Over the last decade, an increasing number of studies have used lake sediment DNA to trace past landscape changes, agricultural activities or human presence. However, the processes responsible for lake sediment formation and sediment properties might affect DNA records via taphonomic and analytical processes. It is crucial to understand these processes to ensure reliable interpretations for "palaeo" studies. Here, we combined plant and mammal DNA metabarcoding analyses with sedimentological and geochemical analyses from three lake-catchment systems that are characterised by different erosion dynamics. The new insights derived from this approach elucidate and assess issues relating to DNA sources and transfer processes. The sources of eroded materials strongly affect the "catchment-DNA" concentration in the sediments. For instance, erosion of upper organic and organo-mineral soil horizons provides a higher amount of plant DNA in lake sediments than deep horizons, bare soils or glacial flours. Moreover, high erosion rates, along with a well-developed hydrographic network, are proposed as factors positively affecting the representation of the catchment flora. The development of open and agricultural landscapes, which favour the erosion, could thus bias the reconstructed landscape trajectory but help the record of these human activities. Regarding domestic animals, pastoral practices and animal behaviour might affect their DNA record because they control the type of source of DNA ("point" vs. "diffuse").

Post-glacial history of the dominant alpine sedge Carex curvula in the European Alpine System inferred from nuclear and chloroplast markers.

The alpine sedge Carex curvula ssp. curvula is a clonal, dominant graminoid found in the European Alps, the Carpathians, the Pyrenees and in some of the Balkan Mountains. It is a late-successional species of acidophilous alpine meadows that occurs on sites that were covered by ice during the last glacial maximum (LGM). By applying the amplified fragment length polymorphism (AFLP) fingerprinting and chloroplast DNA (cpDNA) sequencing, we attempted to identify the recolonization routes followed by the species after the last ice retreat. We relied on the genetic diversity of 37 populations covering the entire distributional range of the species. As a wind-pollinated species, C. curvula is characterized by a low level of population genetic differentiation. Nuclear and chloroplast data both support the hypothesis of a long-term separation of Eastern (Balkans and Carpathians) and Western (Alps and Pyrenees) lineages. In the Alps, a continuum of genetic depauperation from the east to the west may be related to a recolonization wave originating in the eastern-most parts of the chain, where the main glacial refugium was likely located. The Pyrenean populations are nested within the western Alps group and show a low level of genetic diversity, probably due to recent long-distance colonization. In contrast to the Alps, we found no phylogeographical structure in the Carpathians. The combination of reduced ice extension during the Würm period and the presence of large areas of siliceous substrate at suitable elevation suggest that in contrast to populations in the Alps, the species in the Carpathians underwent a local vertical migration rather than extinction and recolonization over long distance.

Ancient DNA analysis reveals divergence of the cave bear, Ursus spelaeus, and brown bear, Ursus arctos, lineages.

The cave bear, Ursus spelaeus, represents one of the most frequently found paleontological remains from the Pleistocene in Europe. The species has always been confined to Europe and was contemporary with the brown bear, Ursus arctos. Relationships between the cave bear and the two lineages of brown bears defined in Europe, as well as the origins of the two species, remain controversial, mainly due to the wide morphological diversity of the fossil remains, which makes interpretation difficult [1, 2]. Sequence analysis of ancient DNA is a useful tool for resolving such problems because it provides an independent source of data [3]. We previously amplified a short DNA fragment of the mitochondrial DNA control region (mt control region) of a 40,000-year-old Ursus spelaeus sample [4]. In this paper, we describe the DNA analysis of two mtDNA regions, the control region and the cytochrome b gene. Control region sequences were obtained from ten samples of cave bears ranging from 130,000 to 20,000 years BP, and one particularly well-conserved sample gave a complete cyt b sequence. Our data demonstrate that cave bears split largely before the lineages of brown bears around 1.2 million years ago. Given its abundance, its wide distribution in space and time, and its large morphological diversity, the cave bear is a promising model for direct observation of the evolution of sequences throughout time, extinction periods, and the differentiation of populations shaped by climatic fluctuations during the Pleistocene.

High-throughput microsatellite genotyping in ecology: improved accuracy, efficiency, standardization and success with low-quantity and degraded DNA.

Microsatellite markers have played a major role in ecological, evolutionary and conservation research during the past 20 years. However, technical constrains related to the use of capillary electrophoresis and a recent technological revolution that has impacted other marker types have brought to question the continued use of microsatellites for certain applications. We present a study for improving microsatellite genotyping in ecology using high-throughput sequencing (HTS). This approach entails selection of short markers suitable for HTS, sequencing PCR-amplified microsatellites on an Illumina platform and bioinformatic treatment of the sequence data to obtain multilocus genotypes. It takes advantage of the fact that HTS gives direct access to microsatellite sequences, allowing unambiguous allele identification and enabling automation of the genotyping process through bioinformatics. In addition, the massive parallel sequencing abilities expand the information content of single experimental runs far beyond capillary electrophoresis. We illustrated the method by genotyping brown bear samples amplified with a multiplex PCR of 13 new microsatellite markers and a sex marker. HTS of microsatellites provided accurate individual identification and parentage assignment and resulted in a significant improvement of genotyping success (84%) of faecal degraded DNA and costs reduction compared to capillary electrophoresis. The HTS approach holds vast potential for improving success, accuracy, efficiency and standardization of microsatellite genotyping in ecological and conservation applications, especially those that rely on profiling of low-quantity/quality DNA and on the construction of genetic databases. We discuss and give perspectives for the implementation of the method in the light of the challenges encountered in wildlife studies.

High performance computation of landscape genomic models including local indicators of spatial association.

With the increasing availability of both molecular and topo-climatic data, the main challenges facing landscape genomics - that is the combination of landscape ecology with population genomics - include processing large numbers of models and distinguishing between selection and demographic processes (e.g. population structure). Several methods address the latter, either by estimating a null model of population history or by simultaneously inferring environmental and demographic effects. Here we present samßada, an approach designed to study signatures of local adaptation, with special emphasis on high performance computing of large-scale genetic and environmental data sets. samßada identifies candidate loci using genotype-environment associations while also incorporating multivariate analyses to assess the effect of many environmental predictor variables. This enables the inclusion of explanatory variables representing population structure into the models to lower the occurrences of spurious genotype-environment associations. In addition, samßada calculates local indicators of spatial association for candidate loci to provide information on whether similar genotypes tend to cluster in space, which constitutes a useful indication of the possible kinship between individuals. To test the usefulness of this approach, we carried out a simulation study and analysed a data set from Ugandan cattle to detect signatures of local adaptation with samßada, bayenv, lfmm and an FST outlier method (FDIST approach in arlequin) and compare their results. samßada - an open source software for Windows, Linux and Mac OS X available at http://lasig.epfl.ch/sambada - outperforms other approaches and better suits whole-genome sequence data processing.

Heterogeneity of three molecular data partition phylogenies of mints related to M. x piperita (Mentha; Lamiaceae).

Phylogenetic reconstructions with molecular tools are now widely used, thanks to advances in PCR and sequencing technologies. The choice of the molecular target still remains a problem because too few comparative data are available. This is particularly true for hybrid taxa, where differential introgression of genome parts leads to incongruity between data sets. We have studied the potential of three data partitions to reconstruct the phylogeny of mints related to M. x piperita. These included nuclear DNA (ITS), chloroplast DNA (non-coding regions trnL intron, intergenic spacers trnL-trnF, and psbA-trnH), and AFLP and ISSR, markers. The taxonomic sampling was composed of hybrids, diploid and polyploid genomes. Since the genealogy of cultivated mint hybrids is known, they represent a model group to compare the usefulness of various molecular markers for phylogeny inference. Incongruities between ITS, chloroplast DNA, and AFLP-ISSR phylogenetic trees were recorded, although DNA fingerprinting data were congruent with morphological classification. Evidence of chloroplast capture events was obtained for M. x piperita. Direct sequencing of ITS led to biased results because of the existence of pseudogenes. Sequencing of cloned ITS further failed to provide evidence of the existence of the two parental copy types for M. x piperita, a sterile hybrid that has had no opportunity for concerted evolution of ITS copies. AFLP-ISSR data clustered M. x piperita with the parent that had the largest genome. This study sheds light on differential of introgression of different genome regions in mint hybrids.

Family-assisted inference of the genetic architecture of major histocompatibility complex variation.

With their direct link to individual fitness, genes of the major histocompatibility complex (MHC) are a popular system to study the evolution of adaptive genetic diversity. However, owing to the highly dynamic evolution of the MHC region, the isolation, characterization and genotyping of MHC genes remain a major challenge. While high-throughput sequencing technologies now provide unprecedented resolution of the high allelic diversity observed at the MHC, in many species, it remains unclear (i) how alleles are distributed among MHC loci, (ii) whether MHC loci are linked or segregate independently and (iii) how much copy number variation (CNV) can be observed for MHC genes in natural populations. Here, we show that the study of allele segregation patterns within families can provide significant insights in this context. We sequenced two MHC class I (MHC-I) loci in 1267 European barn owls (Tyto alba), including 590 offspring from 130 families using Illumina MiSeq technology. Coupled with a high per-individual sequencing coverage (~3000×), the study of allele segregation patterns within families provided information on three aspects of the architecture of MHC-I variation in barn owls: (i) extensive sharing of alleles among loci, (ii) strong linkage of MHC-I loci indicating tandem architecture and (iii) the presence of CNV in the barn owl MHC-I. We conclude that the additional information that can be gained from high-coverage amplicon sequencing by investigating allele segregation patterns in families not only helps improving the accuracy of MHC genotyping, but also contributes towards enhanced analyses in the context of MHC evolutionary ecology.

Mitochondrial DNA polymorphism, phylogeography, and conservation genetics of the brown bear Ursus arctos in Europe.

Some small European populations of the brown bear (Ursus arctos) are threatened by the risk of extinction in the near future. The reinforcement of these populations with bears from other regions might provide a solution to their future survival. However, before any population transfer, the different conservation units must be identified. The phylogeographic approach has been advocated for this purpose. The different European populations were assayed for mitochondrial (mt) DNA polymorphism. A remarkable degree of concordance was found between the geographic distribution and the mtDNA haplotypes. Two clearly distinct lineages differing by more than 7% in mtDNA control region sequences were found and, furthermore, the western lineage appears to be organized into two clades which correspond to two different ancestral refugia. The potential conservation units can be deduced from these results, and a management policy can consequently be inferred. This study clearly demonstrates the relevance of the molecular phylogeographic approach to the identification of conservation units.

Phylogeographical evidence of gene flow among common crossbill (Loxia curvirostra, aves, fringillidae) populations at the continental level

Common Crossbill subspecies have been described according to morphological traits, vocalizations and geographical distribution. In this study, we have tried to determine whether the subspecies correspond to clear-cut mitochondrial DNA lineages, by sequencing 717 bp of the control region from individuals taken at several sampling locations in North America and the Western Palaearctic. We find 22 haplotypes from the 37 sampled individuals with a mean divergence of 0.0118 +/- 0.0069 (mean +/- SD). We find a mixing of the mitochondrial haplotypes at the continental level among the different types or subspecies previously described. Morphological differentiation (in bill size and shape essentially) shows the possibility of rapid local adaptation to fluctuating resources (coniferous seeds), without necessarily promoting the development of reproductive barriers between morphs.

Genetic diversity and assignment tests among seven French cattle breeds based on microsatellite DNA analysis.

Genetic variability and relationships among six native French cattle breeds (Abondance, Tarentaise, Villard de Lans, Montbéliarde, Limousin, and Charolais) and one foreign breed (Holstein) were investigated using 23 microsatellite markers. These breeds were also compared with four Swiss breeds genotyped in a previously published study. Interestingly, the French alpine breeds have smaller population sizes but showed higher genetic variability than the larger Holstein breed. Neighbor-joining trees and PCA (principal components analysis) showed that alpine breeds tend to cluster together. Abondance and Tarentaise breeds were closely related, whereas the Holstein was highly differentiated from all breeds analyzed. Two different assignment tests for determining the breed of origin of individuals were compared: "direct" and "exclusion-simulation" approaches. The exclusion-simulation significance test correctly assigns fewer individuals than the direct approach but provides a confidence level (e.g., P < 0.01) for each individual being assigned. Accurate assignment with high statistical confidence is required for animal traceability. Unfortunately, the accuracy of assignment greatly decreases as the threshold level of confidence of assignment increases (e.g., from P < 0.05 to P < 0.001). Assignment accuracy also greatly declines as the level of population differentiation decreases below the level often found between related breeds (e.g., F(ST) < 0.1).

DNA metabarcoding multiplexing and validation of data accuracy for diet assessment: application to omnivorous diet.

Ecological understanding of the role of consumer-resource interactions in natural food webs is limited by the difficulty of accurately and efficiently determining the complex variety of food types animals have eaten in the field. We developed a method based on DNA metabarcoding multiplexing and next-generation sequencing to uncover different taxonomic groups of organisms from complex diet samples. We validated this approach on 91 faeces of a large omnivorous mammal, the brown bear, using DNA metabarcoding markers targeting the plant, vertebrate and invertebrate components of the diet. We included internal controls in the experiments and performed PCR replication for accuracy validation in postsequencing data analysis. Using our multiplexing strategy, we significantly simplified the experimental procedure and accurately and concurrently identified different prey DNA corresponding to the targeted taxonomic groups, with ≥ 60% of taxa of all diet components identified to genus/species level. The systematic application of internal controls and replication was a useful and simple way to evaluate the performance of our experimental procedure, standardize the selection of sequence filtering parameters for each marker data and validate the accuracy of the results. Our general approach can be adapted to the analysis of dietary samples of various predator species in different ecosystems, for a number of conservation and ecological applications entailing large-scale population level diet assessment through cost-effective screening of multiple DNA metabarcodes, and the detection of fine dietary variation among samples or individuals and of rare food items.

Using next-generation sequencing for molecular reconstruction of past Arctic vegetation and climate.

Palaeoenvironments and former climates are typically inferred from pollen and macrofossil records. This approach is time-consuming and suffers from low taxonomic resolution and biased taxon sampling. Here, we test an alternative DNA-based approach utilizing the P6 loop in the chloroplast trnL (UAA) intron; a short (13-158 bp) and variable region with highly conserved flanking sequences. For taxonomic reference, a whole trnL intron sequence database was constructed from recently collected material of 842 species, representing all widespread and/or ecologically important taxa of the species-poor arctic flora. The P6 loop alone allowed identification of all families, most genera (>75%) and one-third of the species, thus providing much higher taxonomic resolution than pollen records. The suitability of the P6 loop for analysis of samples containing degraded ancient DNA from a mixture of species is demonstrated by high-throughput parallel pyrosequencing of permafrost-preserved DNA and reconstruction of two plant communities from the last glacial period. Our approach opens new possibilities for DNA-based assessment of ancient as well as modern biodiversity of many groups of organisms using environmental samples.

Are cattle, sheep, and goats endangered species?

For about 10 000 years, farmers have been managing cattle, sheep, and goats in a sustainable way, leading to animals that are well adapted to the local conditions. About 200 years ago, the situation started to change dramatically, with the rise of the concept of breed. All animals from the same breed began to be selected for the same phenotypic characteristics, and reproduction among breeds was seriously reduced. This corresponded to a strong fragmentation of the initial populations. A few decades ago, the selection pressures were increased again in order to further improve productivity, without enough emphasis on the preservation of the overall genetic diversity. The efficiency of modern selection methods successfully increased the production, but with a dramatic loss of genetic variability. Many industrial breeds now suffer from inbreeding, with effective population sizes falling below 50. With the development of these industrial breeds came economic pressure on farmers to abandon their traditional breeds, and many of these have recently become extinct as a result. This means that genetic resources in cattle, sheep, and goats are highly endangered, particularly in developed countries. It is therefore important to take measures that promote a sustainable management of these genetic resources; first, by in situ preservation of endangered breeds; second, by using selection programmes to restore the genetic diversity of industrial breeds; and finally, by protecting the wild relatives that might provide useful genetic resources.

Pleistocene colonization of afro-alpine 'sky islands' by the arctic-alpine Arabis alpina.

The afro-alpine region comprises the high mountains of Ethiopia and tropical East Africa, which represent biological 'sky islands' with high level of endemism. However, some primarily arctic-alpine plants also occur in the afro-alpine mountains. It has been suggested that these plants are Tertiary relicts, but a recent worldwide study of Arabis alpina suggests that this species colonized the region twice during the Pleistocene. Here we investigate the detailed colonization history of A. alpina in the afro-alpine region based on chloroplast DNA sequences from 11 mountain systems. The results confirm the twice-into-Africa scenario. The Asian lineage is confined to the mountains closest to the Arabian Peninsula, on opposite sides of the Rift Valley (Simen Mts and Gara Muleta in Ethiopia), suggesting long-distance dispersal of this lineage. The African lineage is divided into two phylogeographic groups with distinct geographic distribution. The observed pattern is consistent with isolation of the African lineage in at least two interglacial refugia, located on separated highlands, followed by range expansion in cooler period(s), when the afro-alpine habitat extended further down the mountains. Several long-distance dispersal events, also across the Rift Valley, are suggested by single haplotypes observed outside the area occupied by the phylogeographic groups they belonged to.

A spatial analysis method (SAM) to detect candidate loci for selection: towards a landscape genomics approach to adaptation.

The detection of adaptive loci in the genome is essential as it gives the possibility of understanding what proportion of a genome or which genes are being shaped by natural selection. Several statistical methods have been developed which make use of molecular data to reveal genomic regions under selection. In this paper, we propose an approach to address this issue from the environmental angle, in order to complement results obtained by population genetics. We introduce a new method to detect signatures of natural selection based on the application of spatial analysis, with the contribution of geographical information systems (GIS), environmental variables and molecular data. Multiple univariate logistic regressions were carried out to test for association between allelic frequencies at marker loci and environmental variables. This spatial analysis method (SAM) is similar to current population genomics approaches since it is designed to scan hundreds of markers to assess a putative association with hundreds of environmental variables. Here, by application to studies of pine weevils and breeds of sheep we demonstrate a strong correspondence between SAM results and those obtained using population genetics approaches. Statistical signals were found that associate loci with environmental parameters, and these loci behave atypically in comparison with the theoretical distribution for neutral loci. The contribution of this new tool is not only to permit the identification of loci under selection but also to establish hypotheses about ecological factors that could exert the selection pressure responsible. In the future, such an approach may accelerate the process of hunting for functional genes at the population level.

A new individual-based spatial approach for identifying genetic discontinuities in natural populations.

The population concept is central in evolutionary and conservation biology, but identifying the boundaries of natural populations is often challenging. Here, we present a new approach for assessing spatial genetic structure without the a priori assumptions on the locations of populations made by adopting an individual-centred approach. Our method is based on assignment tests applied in a moving window over an extensively sampled study area. For each individual, a spatially explicit probability surface is constructed, showing the estimated probability of finding its multilocus genotype across the landscape, and identifying putative migrants. Population boundaries are localized by estimating the mean slope of these probability surfaces over all individuals to identify areas with genetic discontinuities. The significance of the genetic discontinuities is assessed by permutation tests. This new approach has the potential to reveal cryptic population structure and to improve our ability to understand gene flow dynamics across landscapes. We illustrate our approach by simulations and by analysing two empirical datasets: microsatellite data of Ursus arctos in Scandinavia, and amplified fragment length polymorphism (AFLP) data of Rhododendron ferrugineum in the Alps.