Nadir Alvarez

Restriction site-associated DNA sequencing, genotyping error estimation and de novo assembly optimization for population genetic inference

Restriction site‐associated DNA sequencing (RADseq) provides researchers with the ability to record genetic polymorphism across thousands of loci for nonmodel organisms, potentially revolutionizing the field of molecular ecology. However, as with other genotyping methods, RADseq is prone to a number of sources of error that may have consequential effects for population genetic inferences, and these have received only limited attention in terms of the estimation and reporting of genotyping error rates. Here we use individual sample replicates, under the expectation of identical genotypes, to quantify genotyping error in the absence of a reference genome. We then use sample replicates to (i) optimize de novo assembly parameters within the program Stacks, by minimizing error and maximizing the retrieval of informative loci; and (ii) quantify error rates for loci, alleles and single‐nucleotide polymorphisms. As an empirical example, we use a double‐digest RAD data set of a nonmodel plant species, Berberis alpina, collected from high‐altitude mountains in Mexico.

Broad-scale adaptive genetic variation in alpine plants is driven by temperature and precipitation

Identifying adaptive genetic variation is a challenging task, in particular in non‐model species for which genomic information is still limited or absent. Here, we studied distribution patterns of amplified fragment length polymorphisms (AFLPs) in response to environmental variation, in 13 alpine plant species consistently sampled across the entire European Alps. Multiple linear regressions were performed between AFLP allele frequencies per site as dependent variables and two categories of independent variables, namely Moran’s eigenvector map MEM variables (to account for spatial and unaccounted environmental variation, and historical demographic processes) and environmental variables. These associations allowed the identification of 153 loci of ecological relevance. Univariate regressions between allele frequency and each environmental factor further showed that loci of ecological relevance were mainly correlated with MEM variables. We found that precipitation and temperature were the best environmental predictors, whereas topographic factors were rarely involved in environmental associations. Climatic factors, subject to rapid variation as a result of the current global warming, are known to strongly influence the fate of alpine plants. Our study shows, for the first time for a large number of species, that the same environmental variables are drivers of plant adaptation at the scale of a whole biome, here the European Alps.

Forecasting changes in population genetic structure of alpine plants in response to global warming.

Species range shifts in response to climate and land use change are commonly forecasted with species distribution models based on species occurrence or abundance data. Although appealing, these models ignore the genetic structure of species, and the fact that different populations might respond in different ways because of adaptation to their environment. Here, we introduced ancestry distribution models, that is, statistical models of the spatial distribution of ancestry proportions, for forecasting intra‐specific changes based on genetic admixture instead of species occurrence data. Using multi‐locus genotypes and extensive geographic coverage of distribution data across the European Alps, we applied this approach to 20 alpine plant species considering a global increase in temperature from 0.25 to 4 °C. We forecasted the magnitudes of displacement of contact zones between plant populations potentially adapted to warmer environments and other populations. While a global trend of movement in a north‐east direction was predicted, the magnitude of displacement was species‐specific. For a temperature increase of 2 °C, contact zones were predicted to move by 92 km on average (minimum of 5 km, maximum of 212 km) and by 188 km for an increase of 4 °C (minimum of 11 km, maximum of 393 km). Intra‐specific turnover—measuring the extent of change in global population genetic structure—was generally found to be moderate for 2 °C of temperature warming. For 4 °C of warming, however, the models indicated substantial intra‐specific turnover for ten species. These results illustrate that, in spite of unavoidable simplifications, ancestry distribution models open new perspectives to forecast population genetic changes within species and complement more traditional distribution‐based approaches.

Genetic diversity in widespread species is not congruent with species richness in alpine plant communities

The Convention on Biological Diversity (CBD) aims at the conservation of all three levels of biodiversity, that is, ecosystems, species and genes. Genetic diversity represents evolutionary potential and is important for ecosystem functioning. Unfortunately, genetic diversity in natural populations is hardly considered in conservation strategies because it is difficult to measure and has been hypothesised to co-vary with species richness. This means that species richness is taken as a surrogate of genetic diversity in conservation planning, though their relationship has not been properly evaluated. We tested whether the genetic and species levels of biodiversity co-vary, using a large-scale and multi-species approach. We chose the high-mountain flora of the Alps and the Carpathians as study systems and demonstrate that species richness and genetic diversity are not correlated. Species richness thus cannot act as a surrogate for genetic diversity. Our results have important consequences for implementing the CBD when designing conservation strategies.

Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens.

In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes loci dropout. This should enable the application of hyRAD to analyses at broader evolutionary scales.

Gene flow among wild and domesticated almond species: insights from chloroplast and nuclear markers

Hybridization has played a central role in the evolutionary history of domesticated plants. Notably, several breeding programs relying on gene introgression from the wild compartment have been performed in fruit tree species within the genus Prunus but few studies investigated spontaneous gene flow among wild and domesticated Prunus species. Consequently, a comprehensive understanding of genetic relationships and levels of gene flow between domesticated and wild Prunus species is needed. Combining nuclear and chloroplastic microsatellites, we investigated the gene flow and hybridization among two key almond tree species, the cultivated Prunus dulcis and one of the most widespread wild relative Prunus orientalis in the Fertile Crescent. We detected high genetic diversity levels in both species along with substantial and symmetric gene flow between the domesticated P. dulcis and the wild P. orientalis. These results were discussed in light of the cultivated species diversity, by outlining the frequent spontaneous genetic contributions of wild species to the domesticated compartment. In addition, crop‐to‐wild gene flow suggests that ad hoc transgene containment strategies would be required if genetically modified cultivars were introduced in the northwestern Mediterranean.

Genetic structure and evolution of Alpine polyploid complexes: Ranunculus kuepferi (Ranunculaceae) as a case study

The alpine white‐flowered buttercup, Ranunculus kuepferi Greuter & Burdet, is a polyploid complex with diploids endemic to the southwestern Alps and polyploids – which have been previously described as apomictic – widespread throughout European mountains. Due to the polymorphic status of both its ploidy level and its reproductive mode, R. kuepferi represents a key species for understanding the evolution of polyploid lineages in alpine habitats. To disentangle the phylogeography of this polyploid taxon, we used cpDNA sequences and AFLP (amplified fragment length polymorphism) markers in 33 populations of R. kuepferi representative of its ploidy level and distribution area. Polyploid individuals were shown to be the result of at least two polyploidization events that may have taken place in the southwestern Alps. From this region, one single main migration of tetraploids colonized the entire Alpine range, the Apennines and Corsica. Genetic recombination among tetraploids was also observed, revealing the facultative nature of the apomictic reproductive mode in R. kuepferi polyploids. Our study shows the contrasting role played by diploid lineages mostly restricted to persistent refugia and by tetraploids, whose dispersal abilities have permitted their range extension all over the previously glaciated Alpine area and throughout neighbouring mountain massifs.

Origin and expansion of the allotetraploid Aegilops geniculata, a wild relative of wheat

*This study reconstructs the phylogeography of Aegilops geniculata, an allotetraploid relative of wheat, to discuss the impact of past climate changes and recent human activities (e.g. the early expansion of agriculture) on the genetic diversity of ruderal plant species. *We combined chloroplast DNA (cpDNA) sequencing, analysed using statistical parsimony network, with nonhierarchical K-means clustering of amplified fragment length polymorphism (AFLP) genotyping, to unravel patterns of genetic structure across the native range of Ae. geniculata. The AFLP dataset was further explored by measurement of the regional genetic diversity and the detection of isolation by distance patterns. *Both cpDNA and AFLP suggest an eastern Mediterranean origin of Ae. geniculata. Two lineages have spread independently over northern and southern Mediterranean areas. Northern populations show low genetic diversity but strong phylogeographical structure among the main peninsulas, indicating a major influence of glacial cycles. By contrast, low genetic structuring and a high genetic diversity are detected in southern Mediterranean populations. Finally, we highlight human-mediated dispersal resulting in substantial introgression between resident and migrant populations. *We have shown that the evolutionary trajectories of ruderal plants can be similar to those of wild species, but are interfered by human activities, promoting range expansions through increased long-distance dispersal and the creation of suitable habitats.

Ancient and recent evolutionary history of the bruchid beetle, Acanthoscelides obtectus Say, a cosmopolitan pest of beans

Acanthoscelides obtectus Say is a bruchid species of Neotropical origin, and is specialized on beans of the Phaseolus vulgaris L. group. Since the domestication and diffusion of beans, A. obtectus has become cosmopolitan through human‐mediated migrations and is now a major pest in bean granaries. Using phylogeographic methods applied to mitochondrial DNA (mtDNA) and nuclear microsatellite molecular markers, we show that the origin of this species is probably further south than Mesoamerica, as commonly thought. Our results also indicate that A. obtectus and its Mesoamerican sister species Acanthoscelides obvelatus, two morphologically close species differing principally in voltinism, speciated in allopatry: A. obtectus (multivoltine) arising in Andean America and A. obvelatus (univoltine) in Mesoamerica. In contrast to Mesoamerica where beans fruit once yearly, wild beans in Andean America fruit year‐round, especially in regions showing little or no seasonality. In such habitats where resources are continuously present, multivoltinism is adaptive. According to existing hypotheses, multivoltinism in A. obtectus is a new adaptation that evolved after bean domestication. Our data suggest the alternative hypothesis that multivoltinism is an older trait, adapted to exploit the year‐round fruiting of wild beans in relatively aseasonal habitats, and allowed A. obtectus to become a pest in bean granaries. This trait also permitted this species to disperse through human‐mediated migrations associated with diffusion of domesticated beans. We also show that diversity of Old World A. obtectus populations can be quite well explained by a single colonization event about 500 bp. Human‐mediated migrations appear not to be rare, as our results indicate a second more recent migration event from Andean America to Mexico.

Parallels in the evolution of the two largest New and Old World seed-beetle genera (Coleoptera, Bruchidae)

This study provides the first phylogenetic analysis of a large sample of the two largest genera of seed‐beetles, Acanthoscelides Schilsky and Bruchidius Schilsky, which mostly feed on legumes (Fabaceae). The goal of this study was to investigate evolutionary patterns in relation to biogeography and host‐plant associations. We used three mitochondrial molecular markers and parsimony and Bayesian inference methods to reconstruct the phylogeny of 76 species. In addition, we critically reviewed host‐plant records in the literature for these two bruchid genera. Our results demonstrated the existence of two major clades, one New World and one largely Old World, which generally correspond to the two genera. Yet, current classification of several species is erroneous, so that both genera as currently defined are paraphyletic. We highlighted a strong trend toward specialization (with high taxonomic conservatism in host‐plant use) exhibited by the two studied genera. However, we showed the existence of several host shifts during the evolution of this group of bruchids. Our phylogenetic hypotheses and our evaluation of host‐plant associations both suggest that the two genera have undergone parallel evolution, as they have independently colonized similar host plants in their respective areas of distribution. Our estimation of divergence times indicated a more ancient origin for bruchids than that suggested by the fossil records. Interestingly, the suggested timing of diversification is consistent with the hypothesis of a radiation that could have occurred contemporaneously with the diversification of their legume hosts.