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.

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.

An AFLP clock for absolute dating of shallow-time evolutionary history--too good to be true?

A major drawback of Amplified Fragment Length Polymorphisms (AFLP) as genetic makers for phylogeographic studies is their lack of a temporal dimension. In a recent publication in Molecular Ecology, Kropf et al. (2009) proposed a molecular clock for AFLP. In this comment we evaluate the proposed approach both theoretically and empirically. A linear increase with time is a prerequisite to use a genetic distance as molecular clock. Testing the relationship between genetic distance and time in the data of Kropf et al. (2009) for linearity revealed that the relationship was in fact not linear for their pooled data, as well as for one of the three species analyzed. Also, the relationship was not linear in two new species, where divergence times could be inferred from macrofossils. When applying the proposed molecular clock to data from eight species, dates obtained were plausible in some cases, but very improbable in others. The suggested genetic distance was also influenced by intrapopulation genetic diversity, leading to a potential bias. In the future, investigations of AFLP mutation rates combined with phylogeographic modelling may contribute to adding a time scale to the understanding of AFLP data.

Combined analysis of nuclear and mitochondrial markers provide new insight into the genetic structure of North European Picea abies.

Norway spruce of northern Europe expanded at the end of the last glacial out of one refugium in Russia. To provide a detailed insight into how the genetic structure in the northern European lineage of this species has been shaped by postglacial migration, recurrent pollen flow and marginality, we here compare variation at seven highly variable nuclear microsatellite loci in 37 populations (1715 trees) with mitochondrial DNA variation. Microsatellite diversity was high (H(E)=0.640) and genetic differentiation was low (F(ST)=0.029). The microsatellite structure supported a mitochondrial DNA (mtDNA)-based hypothesis of two migration routes out of a single Russian refugium; one northwestern over Finland to northern Scandinavia, and one southwestern across the Baltic Sea into southern Scandinavia. Microsatellite diversity was maintained along the southwestern migration routes, whereas a significant decrease was observed towards the north. In contrast, the mtDNA diversity suggested higher amounts of historical gene flow towards the north than along the southwestern migration route. This suggests that the loss of nuclear diversity after postglacial colonization has been efficiently replenished by pollen-mediated gene flow in the south. Towards the north, smaller effective population size because of more limited seed and pollen production may have caused decreased nuclear diversity and increased inbreeding, reflecting the ecological marginality of the species in the north.

Genetic structuring in three closely related circumpolar plant species: AFLP versus microsatellite markers and high-arctic versus arctic-alpine distributions.

Genetic structuring in response to the glacial cycles has been investigated for many plant species, but exclusively high-arctic ones have not been studied. Such extremely cold-adapted species have probably experienced range reductions under the present climate. Here we compare three predominantly selfing species of Draba with different distributions and hardiness (D. subcapitata, high-arctic; D. nivalis, arctic to arctic-alpine; D. fladnizensis, arctic-alpine) for genetic structuring on the basis of two different types of molecular markers (10 microsatellite loci and 160 amplified fragment length polymorphisms (AFLPs)). The degree of genetic structuring within these species is of particular interest because it has been shown that they contain many cryptic biological species. The high-arctic D. subcapitata had less phylogeographic structure, less diversity and fewer private alleles than the other two species, suggesting that long-distance dispersal may occur more frequently in the high arctic, that hardy plants may have higher probability for establishment after dispersal under high-arctic conditions and that high-arctic species may have experienced a bottleneck during the present interglacial. In contrast, D. fladnizensis and D. nivalis showed distinct phylogeographic structure and more diversity, suggesting separate long-term refugia in Eurasia and North America/Beringia. The AFLP markers revealed more phylogeographic structuring than the microsatellites, possibly because of the higher number of loci surveyed and/or because structure at very large geographic scales is blurred by high mutation rate leading to homoplasy at microsatellite loci. The number of genetic groups detected was in any case insignificant compared with the numerous cryptic biological species known within these species, supporting rapid development of sterility barriers.

Nuclear vs. plastid data: complex Pleistocene history of a circumpolar key species.

To fully understand the contemporary genetic structure of plants, both nuclear and plastid markers are needed. Three chloroplast DNA (cpDNA) lineages, which probably diverged before the major Pleistocene glaciations, have been identified in the circumpolar/circumboreal Vaccinium uliginosum. Here we investigate its nuclear DNA variation using nuclear ribosomal internal transcribed spacer (ITS) sequences, DNA ploidy level measurements and amplified fragment length polymorphisms (AFLPs). We also extend the cpDNA dataset. Two ITS lineages, corresponding to diploids and tetraploids, respectively, were identified. However, both main sequence types apparently occurred in most individual plants but showed ploidy-biased homogenization and possibly reflect paralogy predating the origin of V. uliginosum. The ploidy levels were largely consistent with the cpDNA lineages, suggesting that the initial cpDNA divergence followed early polyploidizations. Five main AFLP groups were identified, consistent with recent glacial refugia in Beringia, western Siberia, the southern European mountains and areas south/east of the Scandinavian and Laurentide ice sheets. Except from the southern European mountains, there has been extensive expansion from all refugia, resulting in several contact zones. Surprisingly, the presumably older ploidy and cpDNA patterns were partly inconsistent with the main AFLP groups and more consistent with AFLP subgroups. A likely major driver causing the inconsistencies is recent nuclear gene flow via unreduced pollen from diploids to tetraploids. This may prevent cytoplasmic introgression and result in overlayed patterns formed by processes dominating at different time scales. The data also suggest more recent polyploidizations, as well as several chloroplast capture events, further complicating this scenario. This study highlights the importance of combining different marker systems to unravel intraspecific histories.

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.

Rare arctic-alpine plants of the European Alps have different immigration histories: the snow bed species Minuartia biflora and Ranunculus pygmaeus.

Minuartia biflora and Ranunculus pygmaeus are circumarctic plants with a few isolated occurrences in the European Alps. We analysed amplified fragment length polymorphism (AFLP) and chloroplast DNA sequence data to unravel the history of their immigration into the Alps and to provide data on their circumpolar phylogeography. In spite of the similar ecological requirements of the two species, they exhibit strikingly different immigration histories into the Alps. In M. biflora, the Alpine populations are most probably derived from source populations located between the Alpine and Scandinavian ice sheets, in accordance with the traditional biogeographic hypothesis. In contrast, the Alpine populations of R. pygmaeus cluster with those from the Tatra Mountains and the Taymyr region in northern Siberia, indicating that the distant Taymyr area served as source for the Alpine populations. Both species showed different levels of genetic diversity in formerly glaciated areas. In contrast to the considerable AFLP diversity observed in M. biflora, R. pygmaeus was virtually nonvariable over vast areas, with a single phenotype dominating all over the Alps and another, distantly related one dominating the North Atlantic area from Greenland over Svalbard to Scandinavia. The same pattern was observed in chloroplast DNA sequence data. Thus, postglacial colonization of R. pygmaeus was accompanied by extreme founder events.

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.

How to track and assess genotyping errors in population genetics studies.

Genotyping errors occur when the genotype determined after molecular analysis does not correspond to the real genotype of the individual under consideration. Virtually every genetic data set includes some erroneous genotypes, but genotyping errors remain a taboo subject in population genetics, even though they might greatly bias the final conclusions, especially for studies based on individual identification. Here, we consider four case studies representing a large variety of population genetics investigations differing in their sampling strategies (noninvasive or traditional), in the type of organism studied (plant or animal) and the molecular markers used [microsatellites or amplified fragment length polymorphisms (AFLPs)]. In these data sets, the estimated genotyping error rate ranges from 0.8% for microsatellite loci from bear tissues to 2.6% for AFLP loci from dwarf birch leaves. Main sources of errors were allelic dropouts for microsatellites and differences in peak intensities for AFLPs, but in both cases human factors were non-negligible error generators. Therefore, tracking genotyping errors and identifying their causes are necessary to clean up the data sets and validate the final results according to the precision required. In addition, we propose the outline of a protocol designed to limit and quantify genotyping errors at each step of the genotyping process. In particular, we recommend (i) several efficient precautions to prevent contaminations and technical artefacts; (ii) systematic use of blind samples and automation; (iii) experience and rigor for laboratory work and scoring; and (iv) systematic reporting of the error rate in population genetics studies.

Amplified fragment length polymorphism versus random amplified polymorphic DNA markers: clonal diversity in Saxifraga cernua.

Random amplified polymorphic DNA (RAPD) markers are sensitive to changes in reaction conditions and may express polymorphisms of nongenetic origin. Taxa with variable chromosome numbers are particularly challenging cases, as differences in DNA content may also influence marker reproducibility. We addressed these problems by comparing RAPD and amplified fragment length polymorphism (AFLP) analyses of clonal identity and relationships in a chromosomally variable arctic plant, the polyploid Saxifraga cernua, which has been thought to be monoclonal over large geographical distances. Fifty-seven plants from four Greenland populations were analysed using a conservative scoring approach. In total, 26 AFLP and 32 RAPD multilocus phenotypes (putative clones) were identified, of which 21 were identical and each of the remaining five AFLP clones was split into two to three very similar RAPD clones. This minor difference can be explained by sampling error and stochastic variation. The pattern observed in Greenland corroborates our previous results from Svalbard, suggesting that rare sexual events in S. cernua are sufficient to maintain high levels of clonal diversity even at small spatial scales. We conclude that although AFLP analysis is superior in terms of efficiency, RAPDs may still be used as reliable markers in small low-tech laboratories.

The Baltic Sea as a model system for studying postglacial colonization and ecological differentiation, exemplified by the red alga Ceramium tenuicorne.

The Baltic Sea provides a unique model system for studying genetic effects of postglacial colonization and ecological differentiation, because all marine organisms must have immigrated after the opening of the Danish Straits 8000 years ago and responded to the development of the steep Skagerrak-Baltic salinity gradient. The red alga Ceramium tenuicorne shows conspicuous variation in growth and reproduction along this gradient. Herein we obtained reproductive data coupled with two types of molecular markers, one organellar (cox2-3 spacer sequences of mitochondrial DNA; mtDNA) and one mainly nuclear (random amplified polymorphic DNAs; RAPDs). Nine main populations were sampled in a nested spatial hierarchy including three salinity regions (Oslofjorden, Kattegat, and the Baltic Sea), and nine additional populations were sampled for the mtDNA analysis. Asexuality was frequent at low (Baltic) and medium (Kattegat) salinities but virtually absent at the highest salinity (Oslofjorden). Five mtDNA haplotypes were observed, of which two highly divergent ones were common. One was restricted to and fixed in Oslofjorden, and the other, which was closely related to the three rare haplotypes, was found from southernmost Norway via Kattegat into the Baltic. The RAPD data revealed, on the other hand, a continuous cline corresponding to the salinity gradient, with 27.4% divergence among salinity regions and most of the variation stored at the smallest spatial scale analysed (64.2%; within 1 m2 subpopulations). The combined data suggest colonization from a diverse Atlantic glacial gene pool followed by (1) lineage sorting of ancestral mtDNA polymorphisms and (2) strong differential selection among nuclear genotypes along the salinity gradient, including selection for nonrecombinant multiplication of those best fit to the marginal low-salinity habitats.

Molecules and morphology in concert. II. The Festuca brachyphylla complex (Poaceae) in Svalbard.

We used a combined molecular and morphological approach to unravel variation in the autogamous Festuca brachyphylla polyploid complex in the arctic archipelago of Svalbard. Forty populations were analyzed for random amplified polymorphic DNAs (RAPDs) and 46 morphological characters. Eighteen RAPD multilocus phenotypes were observed in the 86 plants analyzed, based on 30 polymorphic markers. Multivariate analyses of the RAPD data revealed four distinct groups of multilocus phenotypes; in contrast, the variation was more or less continuous in multivariate analyses of the morphological data. However, we identified several individual morphological characters that unambiguously discriminated among the four groups of RAPD multilocus phenotypes. Analysis of type material suggests that the four groups in Svalbard can be referred to Festuca baffinensis, F. brachyphylla, F. hyperborea, and F. edlundiae. This study shows that concerted analysis of molecules and morphology is a powerful tool in low-level taxonomy.

Molecules and morphology in concert: tests of some hypotheses in arctic Potentilla (Rosaceae).

We developed a combined molecular and morphological approach to unravel complex variation at low taxonomic levels, exemplified by some arctic members of Potentilla. Twenty-one populations from Svalbard were analyzed for random amplified polymorphic DNAs (RAPDs) and 64 morphological characters to test the hypotheses that (1) the P. nivea complex (section Niveae) consists of three taxa (P. chamissonis, P. insularis, and P. nivea), (2) three "eco-morphotypes" in P. pulchella (section Multifidae) should be considered different taxa, and (3) P. insularis originated as an intersectional hybrid (Niveae × Multifidae). Twenty-two RAPD multilocus phenotypes were observed in the 136 plants analyzed based on 35 markers. Three fairly distinct groups of RAPD phenotypes were identified in the P. nivea complex based on multivariate analyses and an analysis of molecular variance (AMOVA; 77.6% among-group variation). The variation within the P. nivea complex was more or less continuous in multivariate analyses of the morphological data. We identified, however, several individual morphological characters that separated unambiguously among the three groups of RAPD phenotypes, revealing that these groups correspond to the previously hypothesized taxa. Many identical RAPD multilocus phenotypes were observed in the "eco-morphotypes" of P. pulchella, suggesting that its conspicuous morphological variation is caused by plasticity or by genetic variation at a small number of loci. The hypothesis of the hybrid origin of P. insularis was not supported by the RAPD data. Overall, very little RAPD variation was observed within populations of the four taxa (2.1-16.7% in AMOVA analyses; average genotypic diversity, D, was 0.10-0.30). We conclude that detailed, concerted analysis of molecules and morphology is a powerful tool in low-level taxonomy.

Molecular evidence for polyploid origins in Saxifraga (Saxifragaceae): the narrow arctic endemic S. svalbardensis and its widespread allies.

The recently described polyploid Saxifraga svalbardensis is endemic to the arctic archipelago of Svalbard. We investigated relationships among four closely related species of Saxifraga in Svalbard and tested three previously proposed hypotheses for the origin of S. svalbardensis: (1) differentiation from the morphologically and chromosomally variable polyploid S. cernua; (2) hybridization between the diploid S. hyperborea and S. cernua; and (3) hybridization between the tetraploid S. rivularis and S. cernua. Fifteen populations were analyzed using random amplified polymorphic DNAs (RAPDs) and nucleotide sequences of the chloroplast gene matK and the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). RAPD and matK data suggest that S. svalbardensis has originated from a hybrid with S. rivularis as the maternal parent and S. cernua as the paternal parent, possibly a single time, whereas ITS data could not be used to discriminate among the hypotheses. The data also suggest that the diploid S. hyperborea is a progenitor of the tetraploid S. rivularis. The four populations examined of S. svalbardensis were virtually identical for RAPD and ITS markers, whereas S. cernua showed high levels of variation, suggesting that the latter polyploid either has formed recurrently or has undergone considerable differentiation since its origin.