Genomics of natural history collections for understanding evolution in the wild.

A long-standing question in biology is how organisms change through time and space in response to their environment. This knowledge is of particular relevance to predicting how organisms might respond to future environmental changes caused by human-induced global change. Usually researchers make inferences about past events based on an understanding of current static genetic patterns, but these are limited in their capacity to inform on underlying past processes. Natural history collections (NHCs) represent a unique and critical source of information to provide temporally deep and spatially broad time-series of samples. By using NHC samples, researchers can directly observe genetic changes over time and space and link those changes with specific ecological/evolutionary events. Until recently, such genetic studies were hindered by the intrinsic challenges of NHC samples (i.e. low yield of highly fragmented DNA). However, recent methodological and technological developments have revolutionized the possibilities in the novel field of NHC genomics. In this Special Feature, we compile a range of studies spanning from methodological aspects to particular case studies which demonstrate the enormous potential of NHC samples for accessing large genomic data sets from the past to advance our knowledge on how populations and species respond to global change at multiple spatial-temporal scales. We also highlight possible limitations, recommendations and a few opportunities for future researchers aiming to study NHC genomics.

In the presence of population structure: From genomics to candidate genes underlying local adaptation.

Understanding the genomic signatures, genes, and traits underlying local adaptation of organisms to heterogeneous environments is of central importance to the field evolutionary biology. To identify loci underlying local adaptation, models that combine allelic and environmental variation while controlling for the effects of population structure have emerged as the method of choice. Despite being evaluated in simulation studies, there has not been a thorough investigation of empirical evidence supporting local adaptation across these alleles. To evaluate these methods, we use 875 Arabidopsis thaliana Eurasian accessions and two mixed models (GEMMA and LFMM) to identify candidate SNPs underlying local adaptation to climate. Subsequently, to assess evidence of local adaptation and function among significant SNPs, we examine allele frequency differentiation and recent selection across Eurasian populations, in addition to their distribution along quantitative trait loci (QTL) explaining fitness variation between Italy and Sweden populations and cis-regulatory/nonsynonymous sites showing significant selective constraint. Our results indicate that significant LFMM/GEMMA SNPs show low allele frequency differentiation and linkage disequilibrium across locally adapted Italy and Sweden populations, in addition to a poor association with fitness QTL peaks (highest logarithm of odds score). Furthermore, when examining derived allele frequencies across the Eurasian range, we find that these SNPs are enriched in low-frequency variants that show very large climatic differentiation but low levels of linkage disequilibrium. These results suggest that their enrichment along putative functional sites most likely represents deleterious variation that is independent of local adaptation. Among all the genomic signatures examined, only SNPs showing high absolute allele frequency differentiation (AFD) and linkage disequilibrium (LD) between Italy and Sweden populations showed a strong association with fitness QTL peaks and were enriched along selectively constrained cis-regulatory/nonsynonymous sites. Using these SNPs, we find strong evidence linking flowering time, freezing tolerance, and the abscisic-acid pathway to local adaptation.

Combining population genomics and fitness QTLs to identify the genetics of local adaptation in Arabidopsis thaliana.

Evidence for adaptation to different climates in the model species Arabidopsis thaliana is seen in reciprocal transplant experiments, but the genetic basis of this adaptation remains poorly understood. Field-based quantitative trait locus (QTL) studies provide direct but low-resolution evidence for the genetic basis of local adaptation. Using high-resolution population genomic approaches, we examine local adaptation along previously identified genetic trade-off (GT) and conditionally neutral (CN) QTLs for fitness between locally adapted Italian and Swedish A. thaliana populations [Ågren J, et al. (2013) Proc Natl Acad Sci USA 110:21077-21082]. We find that genomic regions enriched in high FST SNPs colocalize with GT QTL peaks. Many of these high FST regions also colocalize with regions enriched for SNPs significantly correlated to climate in Eurasia and evidence of recent selective sweeps in Sweden. Examining unfolded site frequency spectra across genes containing high FST SNPs suggests GTs may be due to more recent adaptation in Sweden than Italy. Finally, we collapse a list of thousands of genes spanning GT QTLs to 42 genes that likely underlie the observed GTs and explore potential biological processes driving these trade-offs, from protein phosphorylation, to seed dormancy and longevity. Our analyses link population genomic analyses and field-based QTL studies of local adaptation, and emphasize that GTs play an important role in the process of local adaptation.

Molecular Resources from Transcriptomes in the Brassicaceae Family.

The rapidly falling costs and the increasing availability of large DNA sequence data sets facilitate the fast and affordable mining of large molecular markers data sets for comprehensive evolutionary studies. The Brassicaceae (mustards) are an important species-rich family in the plant kingdom with taxa distributed worldwide and a complex evolutionary history. We performed Simple Sequence Repeats (SSRs) mining using de novo assembled transcriptomes from 19 species across the Brassicaceae in order to study SSR evolution and provide comprehensive sets of molecular markers for genetic studies within the family. Moreover, we selected the genus Cochlearia to test the transferability and polymorphism of these markers among species. Additionally, we annotated Cochlearia pyrenaica transcriptome in order to identify the position of each of the mined SSRs. While we introduce a new set of tools that will further enable evolutionary studies across the Brassicaceae, we also discuss some broader aspects of SSR evolution. Overall, we developed 2012 ready-to-use SSR markers with their respective primers in 19 Brassicaceae species and a high quality annotated transcriptome for C. pyrenaica. As indicated by our transferability test with the genus Cochlearia these SSRs are transferable to species within the genus increasing exponentially the number of targeted species. Also, our polymorphism results showed substantial levels of variability for these markers. Finally, despite its complex evolutionary history, SSR evolution across the Brassicaceae family is highly conserved and we found no deviation from patterns reported in other Angiosperms.

Is the ballan wrasse (Labrus bergylta) two species? Genetic analysis reveals within-species divergence associated with plain and spotted morphotype frequencies.

The ballan wrasse (Labrus bergylta) is a marine fish belonging to the family Labridae characterized by 2 main morphotypes that occur in sympatry: spotty and plain. Previous studies have revealed differences in their life-history traits, such as growth and maturation; however, the genetic relationship between forms is presently unknown. Using 20 recently developed microsatellite markers, we conducted a genetic analysis of 41 and 48 spotty and plain ballan wrasse collected in Galicia (northwest Spain). The 2 morphotypes displayed highly significant genetic differences to each other (FST = 0.018, P < 0.0001). A similar degree of genetic differentiation (FST = 0.025, P < 0.0001) was shown using the STRUCTURE clustering approach with no priors at K = 2. In this case, the frequency of spotty and plain morphotypes was significantly different (χ(2) = 9.46, P = 0.002). It is concluded that there is significant genetic heterogeneity within this species, which appears to be highly associated with the spotty and plain forms, but not completely explained by them. Given the previously demonstrated biological differences between morphotypes, and the present genetic analyses, we speculate about the convenience of a taxonomic re-evaluation of this species.

Phylogeography of a widespread species: pre-glacial vicariance, refugia, occasional blocking straits and long-distance migrations.

Phylogeographic studies give us the opportunity to reconstruct the historical migrations of species and link them with climatic and geographic variation. They are, therefore, a key tool to understanding the relationships among biology, geology and history. One of the most interesting biogeographical areas of the world is the Mediterranean region. However, in this area, the description of concordant phylogeographic patterns is quite scarce, which limits the understanding of evolutionary patterns related to climate. Species with one-dimensional distribution ranges, such as the strawberry tree (Arbutus unedo), are particularly useful to unravel these patterns. Here, we describe its phylogeographic structure and check for concordance with patterns seen in other Mediterranean plants: longitudinal/latitudinal clines of diversity, evidence for glacial refugia and the role of sea straits in dispersal. We also identify the most likely source for the disjunct Irish population. With this aim, we sequenced four chloroplast non-coding fragments of A. unedo from 23 populations covering its whole distribution. We determined the genetic diversity, population structure, haplotype genealogy and time to the most recent common ancestor. The genealogy revealed two clades that separated during the last 700 ky but before the last glacial maximum. One clade occupies Atlantic Iberia and North Africa, while the other occurs in the Western Mediterranean. The Eastern Mediterranean is inhabited by newer haplotypes derived from both clades, while the Irish population is closely related to Iberian demes. The straits of Sicily and Gibraltar partially restricted the gene flow. We concluded that a vicariance event during the Late Quaternary in the western end of the species' range followed by eastward migration seems a likely explanation for the observed phylogeographic pattern. The role of straits indicates an occasional communication between Europe and North Africa, suggesting that the latter was a novel refugia. The East-West genetic split in Iberia is consistent with the refugia-within-refugia model. Finally, the strawberry tree possibly reached Ireland from Iberia instead of throughout the maritime fringe of France as previously thought.

Mining microsatellite markers from public expressed sequence tags databases for the study of threatened plants.

BACKGROUND: Simple Sequence Repeats (SSRs) are widely used in population genetic studies but their classical development is costly and time-consuming. The ever-increasing available DNA datasets generated by high-throughput techniques offer an inexpensive alternative for SSRs discovery. Expressed Sequence Tags (ESTs) have been widely used as SSR source for plants of economic relevance but their application to non-model species is still modest. METHODS: Here, we explored the use of publicly available ESTs (GenBank at the National Center for Biotechnology Information-NCBI) for SSRs development in non-model plants, focusing on genera listed by the International Union for the Conservation of Nature (IUCN). We also search two model genera with fully annotated genomes for EST-SSRs, Arabidopsis and Oryza, and used them as controls for genome distribution analyses. Overall, we downloaded 16 031 555 sequences for 258 plant genera which were mined for SSRsand their primers with the help of QDD1. Genome distribution analyses in Oryza and Arabidopsis were done by blasting the sequences with SSR against the Oryza sativa and Arabidopsis thaliana reference genomes implemented in the Basal Local Alignment Tool (BLAST) of the NCBI website. Finally, we performed an empirical test to determine the performance of our EST-SSRs in a few individuals from four species of two eudicot genera, Trifolium and Centaurea. RESULTS: We explored a total of 14 498 726 EST sequences from the dbEST database (NCBI) in 257 plant genera from the IUCN Red List. We identify a very large number (17 102) of ready-to-test EST-SSRs in most plant genera (193) at no cost. Overall, dinucleotide and trinucleotide repeats were the prevalent types but the abundance of the various types of repeat differed between taxonomic groups. Control genomes revealed that trinucleotide repeats were mostly located in coding regions while dinucleotide repeats were largely associated with untranslated regions. Our results from the empirical test revealed considerable amplification success and transferability between congenerics. CONCLUSIONS: The present work represents the first large-scale study developing SSRs by utilizing publicly accessible EST databases in threatened plants. Here we provide a very large number of ready-to-test EST-SSR (17 102) for 193 genera. The cross-species transferability suggests that the number of possible target species would be large. Since trinucleotide repeats are abundant and mainly linked to exons they might be useful in evolutionary and conservation studies. Altogether, our study highly supports the use of EST databases as an extremely affordable and fast alternative for SSR developing in threatened plants.

Population Structure of a Widespread Species under Balancing Selection: The Case of Arbutus unedo L.

Arbutus unedo L. is an evergreen shrub with a circum-Mediterranean distribution that also reaches the Eurosiberian region in northern Iberia, Atlantic France, and a disjunct population in southern Ireland. Due to the variety of climatic conditions across its distribution range, the populations of A. unedo were expected to display local adaptation. Conversely, common garden experiments revealed that diverse genotypes from a range of provenances produce similar phenotypes through adaptive plasticity, suggesting the action of stabilizing selection across its climatically heterogeneous range. Nonetheless, since a uniform response might also result from extensive gene flow, we have inferred the population structure of A. unedo and assessed whether its extended and largely one-dimensional range influences gene flow with the help of AFLP genotypes for 491 individuals from 19 populations covering the whole range of the species. As we had anticipated, gene flow is restricted in A. unedo, providing further support to the hypothesis that stabilizing selection is the most likely explanation for the homogeneous phenotypes along the range. The Euro-Siberian populations were not particularly isolated from the Mediterranean. Instead, there was a distinct genetic divide between the populations around the Mediterranean Sea and those sampled along Atlantic coasts from northern Africa up to Ireland. This genetic structure suggests the action of historic rather than biogeographic factors as it seems consistent with a scenario of independent glacial refugia in the Atlantic and Mediterranean portions of the range of A. unedo. Genetic exchange was likewise restricted within each set of populations. Nevertheless, isolation-by-distance (IBD) was stronger, and F ST increased faster with distance, along the Atlantic, suggesting that gene flow might be larger among Mediterranean populations. Genetic diversity was significantly lower in NW Iberia and Ireland than in other populations whereas Ireland was more closely related to populations in NW Iberia than to a population in Atlantic France, suggesting a postglacial stepping-stone colonization of the Atlantic coast. Altogether, our results show that stabilizing selection is able to homogenize the phenotypic response even when population structure is strong, gene flow is constrained, and the phylogeographic past is complex.

A multilocus species delimitation reveals a striking number of species of coralline algae forming Maerl in the OSPAR maritime area.

Maerl beds are sensitive biogenic habitats built by an accumulation of loose-lying, non-geniculate coralline algae. While these habitats are considered hot-spots of marine biodiversity, the number and distribution of maerl-forming species is uncertain because homoplasy and plasticity of morphological characters are common. As a result, species discrimination based on morphological features is notoriously challenging, making these coralline algae the ideal candidates for a DNA barcoding study. Here, mitochondrial (COI-5P DNA barcode fragment) and plastidial (psbA gene) sequence data were used in a two-step approach to delimit species in 224 collections of maerl sampled from Svalbard (78°96'N) to the Canary Islands (28°64'N) that represented 10 morphospecies from four genera and two families. First, the COI-5P dataset was analyzed with two methods based on distinct criteria (ABGD and GMYC) to delineate 16 primary species hypotheses (PSHs) arranged into four major lineages. Second, chloroplast (psbA) sequence data served to consolidate these PSHs into 13 secondary species hypotheses (SSHs) that showed biologically plausible ranges. Using several lines of evidence (e.g. morphological characters, known species distributions, sequences from type and topotype material), six SSHs were assigned to available species names that included the geographically widespread Phymatolithon calcareum, Lithothamnion corallioides, and L. glaciale; possible identities of other SSHs are discussed. Concordance between SSHs and morphospecies was minimal, highlighting the convenience of DNA barcoding for an accurate identification of maerl specimens. Our survey indicated that a majority of maerl forming species have small distribution ranges and revealed a gradual replacement of species with latitude.

Genetic guidelines for the conservation of the endangered polyploid Centaurea borjae (Asteraceae).

Appropriate management of species of conservation concern requires designing strategies that should include genetic information as small population size and restricted geographic range can reduce genetic variation. We used AFLPs to investigate genetic variation within and among populations of the endangered narrow endemic Centaurea borjae, and found no evidence for genetic impoverishment despite its <40 km range and potential for vegetative propagation. Genetic variation was comparable to other plants with similar life history (88 % occurring within populations) and potential clone mates were less frequent than expected. Nonetheless, populations separated by few hundred meters showed signs of significant genetic differentiation suggesting low gene flow between them. Our results suggested that the three geographically closer populations located at the center of the range might be treated as a single management unit, while the remaining ones could be considered independent units. We found evidence of fine-scale spatial genetic structure up to 80 m indicating that the collection of germplasm for ex-situ conservation should focus on individuals separated >80 m to maximize genetic variation.

Population structure and range expansion: the case of the invasive gastropod Cyclope neritea in northwest Iberian Peninsula.

Biotic invasions have a reputation for unpredictable behavior. Here, we report how slight changes in human activity responsible for the introduction and range expansion of a non-native mollusk have led to detectable differences in the genetics of the invasion. Cyclope neritea is a non-predatory gastropod introduced to 2 areas of the European Atlantic: the northwest Iberian Peninsula (NWIP) and the French Atlantic coast (FAC). Shellfish seabed farming is intense in both areas but focuses on different commercial species. Using mitochondrial gene sequences, the lower genetic diversity recorded along the NWIP suggests a more homogeneous range of source populations than in the FAC. Unlike FAC, genetic diversity and haplotype composition in the NWIP correlate with the date of first occurrence of C. neritea at each site rather than with geographical location. Although this pattern evokes the genetic signature expected under a serial-founder colonization model from a single initial enclave, a comparison with samples from potential source populations suggests that the NWIP probably experienced several independent reintroductions. The jump dispersal pattern of C. neritea in the NWIP, together with the observation that populations established in the same year are genetically undifferentiated, point to human transport as the most plausible explanation for the current range expansion. Despite evidence for human-mediated dispersal, C. neritea managed to develop a seemingly non-random genetic pattern in the NWIP. It is suggested that caution must be exerted when interpreting genetic patterns in invaders.