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.

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.

Ancient DNA, lipid biomarkers and palaeoecological evidence reveals construction and life on early medieval lake settlements.

Direct evidence of ancient human occupation is typically established through archaeological excavation. Excavations are costly and destructive, and practically impossible in some lake and wetland environments. We present here an alternative approach, providing direct evidence from lake sediments using DNA metabarcoding, steroid lipid biomarkers (bile acids) and from traditional environmental analyses. Applied to an early Medieval Celtic settlement in Ireland (a crannog) this approach provides a site chronology and direct evidence of human occupation, crops, animal farming and on-site slaughtering. This is the first independently-dated, continuous molecular archive of human activity from an archeological site, demonstrating a link between animal husbandry, food resources, island use. These sites are under threat but are impossible to preserve in-situ so this approach can be used, with or without excavation, to produce a robust and full site chronology and provide direct evidence of occupation, the use of plants and animals, and activities such as butchery.

Persistence of arctic-alpine flora during 24,000 years of environmental change in the Polar Urals.

Plants adapted to extreme conditions can be at high risk from climate change; arctic-alpine plants, in particular, could "run out of space" as they are out-competed by expansion of woody vegetation. Mountain regions could potentially provide safe sites for arctic-alpine plants in a warmer climate, but empirical evidence is fragmentary. Here we present a 24,000-year record of species persistence based on sedimentary ancient DNA (sedaDNA) from Lake Bolshoye Shchuchye (Polar Urals). We provide robust evidence of long-term persistence of arctic-alpine plants through large-magnitude climate changes but document a decline in their diversity during a past expansion of woody vegetation. Nevertheless, most of the plants that were present during the last glacial interval, including all of the arctic-alpines, are still found in the region today. This underlines the conservation significance of mountain landscapes via their provision of a range of habitats that confer resilience to climate change, particularly for arctic-alpine taxa.

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.

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.

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.

Impact of ice ages on circumpolar molecular diversity: insights from an ecological key species.

We address the impact of the ice age cycles on intraspecific cpDNA diversity, for the first time on the full circumboreal-circumarctic scale. The bird-dispersed bog bilberry (or arctic blueberry, Vaccinium uliginosum) is a key component of northern ecosystems and is here used to assess diversity in previously glaciated vs. unglaciated areas and the importance of Beringia as a refugium and source for interglacial expansion. Eighteen chloroplast DNA haplotypes were observed in and among 122 populations, grouping into three main lineages which probably diverged before, and thus were affected more or less independently by, all major glaciations. The boreal 'Amphi-Atlantic lineage' included one haplotype occurring throughout northern Europe and one occurring in eastern North America, suggesting expansion from at least two bottlenecked, glacial refugium populations. The boreal 'Beringian lineage' included seven haplotypes restricted to Beringia and the Pacific coast of USA. The 'Arctic-Alpine lineage' included nine haplotypes, one of them fully circumpolar. This lineage was unexpectedly diverse, also in previously glaciated areas, suggesting that it thrived on the vast tundras during the ice ages and recolonized deglaciated terrain over long distances. Its largest area of persistence during glaciations was probably situated in the north, stretching from Beringia and far into Eurasia, and it probably also survived the last glaciation in southern mountain ranges. Although Beringia apparently was important for the initial divergence and expansion of V. uliginosum as well as for continuous survival of both the Beringian and Arctic-Alpine lineages during all ice ages, this region played a minor role as a source for later interglacial expansions.

The use of chloroplast DNA to resolve plant phylogenies: noncoding versus rbcL sequences.

Direct sequencing of polymerase chain reaction products is now an expanding area of plant systematics and evolution. Within angiosperms the rbcL gene has been widely sequenced and used for inferring plant phylogenies at higher taxonomic levels. Unfortunately rbcL does not usually contain enough information to resolve relationships between closely related genera, such as Hordeum, Triticum, and Aegilops. One solution to this problem could be to analyze noncoding regions of chloroplast DNA, which are supposed to evolve more rapidly than coding regions. Here we present pairwise comparisons among dicots and monocots for rbcL and two noncoding sequences of cpDNA (the trnL (UAA) intron and the intergenic spacer between the trnL (UAA) 3' exon and the trnF (GAA) gene). It appears that these regions evolve faster (more than three times faster, on average) than rbcL, as previously reported, and that the trnL intron evolves at a rate that is the same as that of the intergenic spacer. By the analysis of these regions, the genera Hordeum, Triticum, and Aegilops clearly could be distinguished. A phylogeny using trnL (UAA) intron sequences is also inferred for some species of the genus Gentiana L., clearly illustrating the phylogenetic utility of these zones at the generic level. The advantages and the disadvantages of the use of these regions to resolve plant phylogenies are discussed, as well as the desirability of a preliminary study before every large-scale analysis.

Universal primers for amplification of three non-coding regions of chloroplast DNA.

Six primers for the amplification of three non-coding regions of chloroplast DNA via the polymerase chain reaction (PCR) have been designed. In order to find out whether these primers were universal, we used them in an attempt to amplify DNA from various plant species. The primers worked for most species tested including algae, bryophytes, pteridophytes, gymnosperms and angiosperms. The fact that they amplify chloroplast DNA non-coding regions over a wide taxonomic range means that these primers may be used to study the population biology (in supplying markers) and evolution (inter- and probably intraspecific phylogenies) of plants.

Genetic structure of Rhododendron ferrugineum at a wide range of spatial scales.

Rhododendron ferrugineum L. (Ericaceae) is a subalpine shrub found throughout the Pyrenees and Alps at elevations of 1600-2200 m. We examined relationships between genetic and geographic distance, using 115 dominant amplified fragment length polymorphism (AFLP) markers to assess genetic structure over a wide range of spatial scales. We sampled 17 sites with distances of 4 km to more than 1000 km between them. At these scales we detected no association between geographic distance and genetic distance between populations. This suggests that genetic drift and gene flow are not in equilibrium for these populations. This pattern could have resulted from recent and rapid postglacial colonization, from more recent human disturbance, or as a function of frequent and random "natural" long-distance colonization. At two of our sites we used transects (two horizontal and two vertical with respect to slope at each site) to sample at distances ranging from 10 m to more than 5000 m. At this scale we observed a positive relationship between genetic and spatial distance along two vertical transects, one at each site. We hypothesize that isolation-by-distance at this smaller scale is a function of restricted gene flow via seed dispersal.

Phylogenetic use of noncoding regions in the genus Gentiana L.: chloroplast trnL (UAA) intron versus nuclear ribosomal internal transcribed spacer sequences.

Sequence divergence was estimated within noncoding sequences of both chloroplast DNA (cpDNA) trnL (UAA) intron and nuclear ribosomal DNA (nrDNA) internal transcribed spacer sequences (ITS1 and ITS2) for 10 species of the genus Gentiana L. (Gentianaceae). Comparisons of evolutionary rates among these sequences (cpDNA versus nrDNA, ITS1 versus ITS2) were performed. It appears that sequence divergence is on average two to three times higher in ITSs than in the trnL intron sequences and higher in ITS1 than in ITS2. Both the cpDNA intron and ITSs of nrDNA give concordant phylogenetic trees. However, the ITS-based phylogeny displays higher bootstrap values. At the intrageneric level, at least in Gentiana, ITSs (especially ITS2) sequences seem to be more appropriate in the assessment of plant phylogenies. Nevertheless, the cpDNA trnL intron seems to be preferable at the intergeneric level.

The evolutionary history of the two karyotypic groups of the common shrew, Sorex araneus, in Poland.

Genetic variability within and among two karyotypic groups and five chromosome races of the common shrew (Sorex araneus) in Poland was assayed by sequencing a 1023 bp part of the cytochrome b gene (mtDNA) from 28 individuals. Thirty-four variable positions defined 21 distinct haplotypes with a maximum sequence divergence of 0.88%. No significant differentiation in the cytochrome b gene between Western and Eastern Karyotypic groups was found. Haplotype diversity estimates within the races and groups sampled were high (h = 0.800-0.928), while nucleotide diversity estimates were low (pi = 0.0034-0.0053). The distribution of pairwise nucleotide differences fits well with expectations of a "sudden expansion" model. High haplotype diversity was accompanied by relatively high expected heterozygosity (H(E)) values in nuclear genes (calculated over 47 enzyme loci: H(E) = 0.031 - 0.049), giving no evidence for a recent bottleneck after the process of post-Pleistocene recolonization of Poland by the shrews. Thus, for S. araneus chromosome races in Poland, the data on the cytochrome b gene variability support the hypothesis assuming the Robertsonian fusions having spread into an ancestral acrocentric distribution.

Multiple maternal origins and weak phylogeographic structure in domestic goats.

Domestic animals have played a key role in human history. Despite their importance, however, the origins of most domestic species remain poorly understood. We assessed the phylogenetic history and population structure of domestic goats by sequencing a hypervariable segment (481 bp) of the mtDNA control region from 406 goats representing 88 breeds distributed across the Old World. Phylogeographic analysis revealed three highly divergent goat lineages (estimated divergence >200,000 years ago), with one lineage occurring only in eastern and southern Asia. A remarkably similar pattern exists in cattle, sheep, and pigs. These results, combined with recent archaeological findings, suggest that goats and other farm animals have multiple maternal origins with a possible center of origin in Asia, as well as in the Fertile Crescent. The pattern of goat mtDNA diversity suggests that all three lineages have undergone population expansions, but that the expansion was relatively recent for two of the lineages (including the Asian lineage). Goat populations are surprisingly less genetically structured than cattle populations. In goats only approximately 10% of the mtDNA variation is partitioned among continents. In cattle the amount is >/=50%. This weak structuring suggests extensive intercontinental transportation of goats and has intriguing implications about the importance of goats in historical human migrations and commerce.

Recent divergence between two morphologically differentiated subspecies of bluethroat (Aves: Muscicapidae: Luscinia svecica) inferred from mitochondrial DNA sequence variation.

We assessed the mitochondrial DNA sequence divergence of a 718 bp fragment of the control region and 1007 bp of the cytochrome b gene between two allopatric morphologically different subspecies of bluethroat (Luscinia svecica). None of the 17 total haplotypes was shared between L. s. namnetum and L. s. svecica. However, the mean distances between subspecies were very low for both fragments (0.00168 +/- 0.00001 (mean +/- SE) for the control region; 0.00306 +/- 0.00016 for the cytochrome b gene). Only one substitution made the two subspecies genetically differentiated, highlighting their recent divergence. Interestingly, the control region was not more variable than the cytochrome b gene.

Genetic introgression as a potential to widen a species' niche: insights from alpine Carex curvula.

Understanding what causes the decreasing abundance of species at the margins of their distributions along environmental gradients has drawn considerable interest, especially because of the recent need to predict shifts in species distribution patterns in response to climatic changes. Here, we address the ecological range limit problem by focusing on the sedge, Carex curvula, a dominant plant of high-elevation grasslands in Europe, for which two ecologically differentiated but crosscompatible taxa have been described in the Alps. Our study heuristically combines an extensive phytoecological survey of alpine plant communities to set the niche attributes of each taxon and a population genetic study to assess the multilocus genotypes of 177 individuals sampled in typical and marginal habitats. We found that ecological variation strongly correlates with genetic differentiation. Our data strongly suggest that ecologically marginal populations of each taxon are mainly composed of individuals with genotypes resulting from introgressive hybridization. Conversely, no hybrids were found in typical habitats, even though the two taxa were close enough to crossbreed. Thus, our results indicate that genotype integrity is maintained in optimal habitats, whereas introgressed individuals are favored in marginal habitats. We conclude that gene flow between closely related taxa might be an important, although underestimated, mechanism shaping species distribution along gradients.

Molecular phylogeny and evolution of Sorex shrews (Soricidae: insectivora) inferred from mitochondrial DNA sequence data.

Shrews of the genus Sorex are characterized by a Holarctic distribution, and relationships among extant taxa have never been fully resolved. Phylogenies have been proposed based on morphological, karyological, and biochemical comparisons, but these analyses often produced controversial and contradictory results. Phylogenetic analyses of partial mitochondrial cytochrome b gene sequences (1011 bp) were used to examine the relationships among 27 Sorex species. The molecular data suggest that Sorex comprises two major monophyletic lineages, one restricted mostly to the New World and one with a primarily Palearctic distribution. Furthermore, several sister-species relationships are revealed by the analysis. Based on the split between the Soricinae and Crocidurinae subfamilies, we used a 95% confidence interval for both the calibration of a molecular clock and the subsequent calculation of major diversification events within the genus Sorex. Our analysis does not support an unambiguous acceleration of the molecular clock in shrews, the estimated rate being similar to other estimates of mammalian mitochondrial clocks. In addition, the data presented here indicate that estimates from the fossil record greatly underestimate divergence dates among Sorex taxa.

Amplification of oak DNA from ancient and modern wood.

A polymorphic noncoding region of chloroplast DNA (cpDNA) was successfully amplified by the polymerase chain reaction (PCR) from various oak wood samples, including recent and more ancient (about 600-years-old) samples from different oak species. Adaptation of DNA isolation and amplification protocols was necessary to obtain this result. Polymorphisms useful to distinguish species or geographical origin of these samples could be scored through sequencing. These polymorphisms include one substitution and two microsatellite-type polymorphisms, due to a variable number of A/T repeats. Identical results were obtained independently in two separate laboratories.