Evolution, plasticity and evolving plasticity of phenology in the tree species Alnus glutinosa.

Both traits and the plasticity of these traits are subject to evolutionary change and therefore affect the long-term persistence of populations and their role in local communities. We subjected clones from 12 different populations of Alnus glutinosa, located along a latitudinal gradient, to two different temperature treatments, to disentangle the distribution of genetic variation in timing of bud burst and bud burst plasticity within and among genotypes, populations, and regions. We calculated heritability and evolvability estimates for bud burst and bud burst plasticity and assessed the influence of divergent selection relative to neutral drift. We observed higher levels of heritability and evolvability for bud burst than for its plasticity, whereas the total phenological heritability and evolvability (i.e. combining timing of bud burst and bud burst plasticity) suggest substantial evolutionary potential with respect to phenology. Earlier bud burst was observed for the low-latitudinal populations than for the populations from higher latitudes, whereas the high-latitudinal populations did not show the expected delayed bud burst. This countergradient variation can be due to evolution towards increased phenological plasticity at higher latitudes. However, because we found little evidence for adaptive differences in phenological plasticity across the latitudinal gradient, we suggest differential frost tolerance as the most likely explanation for the observed phenological patterns in A. glutinosa.

Neutral and adaptive genomic signatures of rapid poleward range expansion.

Many species are expanding their range polewards, and this has been associated with rapid phenotypic change. Yet, it is unclear to what extent this reflects rapid genetic adaptation or neutral processes associated with range expansion, or selection linked to the new thermal conditions encountered. To disentangle these alternatives, we studied the genomic signature of range expansion in the damselfly Coenagrion scitulum using 4950 newly developed genomic SNPs and linked this to the rapidly evolved phenotypic differences between core and (newly established) edge populations. Most edge populations were genetically clearly differentiated from the core populations and all were differentiated from each other indicating independent range expansion events. In addition, evidence for genetic drift in the edge populations, and strong evidence for adaptive genetic variation in association with the range expansion was detected. We identified one SNP under consistent selection in four of the five edge populations and showed that the allele increasing in frequency is associated with increased flight performance. This indicates collateral, non-neutral evolutionary changes in independent edge populations driven by the range expansion process. We also detected a genomic signature of adaptation to the newly encountered thermal regimes, reflecting a pattern of countergradient variation. The latter signature was identified at a single SNP as well as in a set of covarying SNPs using a polygenic multilocus approach to detect selection. Overall, this study highlights how a strategic geographic sampling design and the integration of genomic, phenotypic and environmental data can identify and disentangle the neutral and adaptive processes that are simultaneously operating during range expansions.

The population genomic signature of environmental selection in the widespread insect-pollinated tree species Frangula alnus at different geographical scales.

The evaluation of the molecular signatures of selection in species lacking an available closely related reference genome remains challenging, yet it may provide valuable fundamental insights into the capacity of populations to respond to environmental cues. We screened 25 native populations of the tree species Frangula alnus subsp. alnus (Rhamnaceae), covering three different geographical scales, for 183 annotated single-nucleotide polymorphisms (SNPs). Standard population genomic outlier screens were combined with individual-based and multivariate landscape genomic approaches to examine the strength of selection relative to neutral processes in shaping genomic variation, and to identify the main environmental agents driving selection. Our results demonstrate a more distinct signature of selection with increasing geographical distance, as indicated by the proportion of SNPs (i) showing exceptional patterns of genetic diversity and differentiation (outliers) and (ii) associated with climate. Both temperature and precipitation have an important role as selective agents in shaping adaptive genomic differentiation in F. alnus subsp. alnus, although their relative importance differed among spatial scales. At the 'intermediate' and 'regional' scales, where limited genetic clustering and high population diversity were observed, some indications of natural selection may suggest a major role for gene flow in safeguarding adaptability. High genetic diversity at loci under selection in particular, indicated considerable adaptive potential, which may nevertheless be compromised by the combined effects of climate change and habitat fragmentation.

Ecological and evolutionary drivers of range size in Coenagrion damselflies.

Geographic range size is a key ecological and evolutionary characteristic of a species, yet the causal basis of variation in range size among species remains largely unresolved. One major reason for this is that several ecological and evolutionary traits may jointly shape species' differences in range size. We here present an integrated study of the contribution of ecological (dispersal capacity, body size and latitudinal position) and macroevolutionary (species' age) traits in shaping variation in species' range size in Coenagrion damselflies. We reconstructed the phylogenetic tree of this genus to account for evolutionary history when assessing the contribution of the ecological traits and to evaluate the role of the macroevolutionary trait (species' age). The genus invaded the Nearctic twice independently from the Palearctic, yet this was not associated with the evolution of larger range sizes or dispersal capacity. Body size and species' age did not explain variation in range size. There is higher flight ability (as measured by wing aspect ratio) at higher latitudes. Species with a larger wing aspect ratio had a larger range size, also after correcting for phylogeny, suggesting a role for dispersal capacity in shaping the species' ranges. More northern species had a larger species' range, consistent with Rapoport's rule, possibly related to niche width. Our results underscore the importance of integrating macroecology and macroevolution when explaining range size variation among species.

Unravelling the effects of contemporary and historical range expansion on the distribution of genetic diversity in the damselfly Coenagrion scitulum.

Although genetic diversity provides the basic substrate for evolution, there are a limited number of studies that assess the impact of recent climate change on intraspecific genetic variation. This study aims to unravel the degree to which historical and contemporary factors shape genetic diversity and structure across a large part of the range of the range-expanding damselfly Coenagrion scitulum (Rambur, 1842). A total of 525 individuals from 31 populations were genotyped at nine microsatellites, and a subset was sequenced at two mitochondrial genes. We inferred the importance of geography, environmental factors, and recent range expansion on genetic diversity and structure. Genetic diversity decreased going westwards, suggesting a signature of historical post-glacial expansion from east to west and the presence of eastern refugia. Although genetic differentiation decreased going northwards, it increased in the northern edge populations, suggesting a role of contemporary range expansion on the genetic make-up of populations. The phylogeographical context was proven to be essential in understanding and identifying the genetic signatures of local contemporary processes. Within this framework, our results highlight that recent range expansion of a good disperser can decrease genetic diversity and increase genetic differentiation which should be considered when devising suitable conservation strategies.

Local and regional founder effects in lake zooplankton persist after thousands of years despite high dispersal potential.

We reconstructed the genetic structure of a planktonic crustacean Daphnia longispina living in high mountain lakes and ponds in the Pyrenees to investigate whether it was shaped by persistent founder effects originating shortly after the last glacial maximum or by ongoing dispersal and effective migration (gene flow). We found that the genetic structure can largely be explained by a single colonization event following gradual deglaciation of the Pyrenees ~10 000-15 000 years ago. Nuclear genetic diversity declined steeply from southeast to northwest, suggestive of serial colonization of available habitats with advancing deglaciation. The spatial genetic structure suggests that founder effects were major determinants of the present-day diversity, both at the catchment level and at the level of individual water bodies, further supporting extremely low effective migration rates. This study reveals a prime example of a founder effect that is both long lasting and maintained at small spatial scales. Our data suggest a process of isolation by colonization as a result of strong priority effects and monopolization. We found evidence for the spread of haplotypes with Pyrenean ancestry across the Palaearctic over distances up to 5500 km, although the local genetic structure after colonization was hardly influenced by contemporary dispersal. Finally, our data also suggest that mitochondrial mutation rates in the studied populations were seven times higher than typically assumed. Overall, we show that founder effects can persist for centuries even at small spatial scales at which the potential for dispersal is high.

Rapid range expansion increases genetic differentiation while causing limited reduction in genetic diversity in a damselfly.

Many ectothermic species are currently expanding their geographic range due to global warming. This can modify the population genetic diversity and structure of these species because of genetic drift during the colonization of new areas. Although the genetic signatures of historical range expansions have been investigated in an array of species, the genetic consequences of natural, contemporary range expansions have received little attention, with the only studies available focusing on range expansions along a narrow front. We investigate the genetic consequences of a natural range expansion in the Mediterranean damselfly Coenagrion scitulum, which is currently rapidly expanding along a broad front in different directions. We assessed genetic diversity and genetic structure using 12 microsatellite markers in five centrally located populations and five recently established populations at the edge of the geographic distribution. Our results suggest that, although a marginal significant decrease in the allelic richness was found in the edge populations, genetic diversity has been preserved during the range expansion of this species. Nevertheless, edge populations were genetically more differentiated compared with core populations, suggesting genetic drift during the range expansion. The smaller effective population sizes of the edge populations compared with central populations also suggest a contribution of genetic drift after colonization. We argue and document that range expansion along multiple axes of a broad expansion front generates little reduction in genetic diversity, yet stronger differentiation of the edge populations.

SNP discovery using Paired-End RAD-tag sequencing on pooled genomic DNA of Sisymbrium austriacum (Brassicaceae).

Single nucleotide polymorphisms SNPs are rapidly replacing anonymous markers in population genomic studies, but their use in non model organisms is hampered by the scarcity of cost-effective approaches to uncover genome-wide variation in a comprehensive subset of individuals. The screening of one or only a few individuals induces ascertainment bias. To discover SNPs for a population genomic study of the Pyrenean rocket (Sisymbrium austriacum subsp. chrysanthum), we undertook a pooled RAD-PE (Restriction site Associated DNA Paired-End sequencing) approach. RAD tags were generated from the PstI-digested pooled genomic DNA of 12 individuals sampled across the species distribution range and paired-end sequenced using Illumina technology to produce ~24.5 Mb of sequences, covering ~7% of the specie's genome. Sequences were assembled into ~76 000 contigs with a mean length of 323 bp (N(50)  = 357 bp, sequencing depth = 24x). In all, >15 000 SNPs were called, of which 47% were annotated in putative genic regions based on homology with the Arabidopsis thaliana genome. Gene ontology (GO) slim categorization demonstrated that the identified SNPs covered extant genic variation well. The validation of 300 SNPs on a larger set of individuals using a KASPar assay underpinned the utility of pooled RAD-PE as an inexpensive genome-wide SNP discovery technique (success rate: 87%). In addition to SNPs, we discovered >600 putative SSR markers.

Genetic diversity and population structure in contemporary house sparrow populations along an urbanization gradient.

House sparrow (Passer domesticus) populations have suffered major declines in urban as well as rural areas, while remaining relatively stable in suburban ones. Yet, to date no exhaustive attempt has been made to examine how, and to what extent, spatial variation in population demography is reflected in genetic population structuring along contemporary urbanization gradients. Here we use putatively neutral microsatellite loci to study if and how genetic variation can be partitioned in a hierarchical way among different urbanization classes. Principal coordinate analyses did not support the hypothesis that urban/suburban and rural populations comprise two distinct genetic clusters. Comparison of FST values at different hierarchical scales revealed drift as an important force of population differentiation. Redundancy analyses revealed that genetic structure was strongly affected by both spatial variation and level of urbanization. The results shown here can be used as baseline information for future genetic monitoring programmes and provide additional insights into contemporary house sparrow dynamics along urbanization gradients.

Temperature-related natural selection in a wind-pollinated tree across regional and continental scales.

Adaptive genetic variation is a key factor in evolutionary biology, but the detection of signatures of natural selection remains challenging in nonmodel organisms. We used a genome scan approach to detect signals of natural selection in the Black alder (Alnus glutinosa), a widespread wind-pollinated tree. Gene flow through pollen dispersal is believed to be high in this species, and we therefore expected to find a clear response to natural selection. In combination with two different landscape genetic approaches, we determined which environmental variables were most associated with the inferred selection. This analysis was performed on a regional scale (northern Belgium) and on a continental scale (Europe). Because climate-related differences are much more pronounced at the continental scale, we expected to find more selection-sensitive genetic markers across Europe than across northern Belgium. At both spatial scales, a substantial number of genetic loci were considered outliers, with respect to neutral expectations, and were therefore identified as selective. Based on results from our combined approach, four putative selective loci (or 2.5%) were recovered with high statistical support. Although these loci seemed to be associated with different environmental variables, they were mainly temperature-related. Our study demonstrates that the use of complementary methods in landscape genetics allows the discovery of selective loci which otherwise might stay hidden. In combination with a genome scan, the selective loci can be verified and the nature of the selection pressure can be identified.

Development and characterization of eight polymorphic microsatellite markers for Daphnia atkinsoni (Crustacea: Ctenodaphnia).

A microsatellite-enriched genomic library was developed for the water flea Daphnia atkinsoni Baird, 1859, a dominant species of intermittent wetlands in Europe. Eight polymorphic microsatellite markers were successfully optimized. Characterization of 77 individuals from Belgium and Spain showed moderate (in the former) to high (in the latter) levels of polymorphism with two to 11 alleles per locus and an observed heterozygosity ranging from 0 to 0.87. Some of these microsatellite markers were successfully amplified in three other Daphnia species (D. magna n = 4, D. similis n = 6; D. obtusa n = 6).

A cryptic invasion within an invasion and widespread introgression in the European water frog complex: consequences of uncontrolled commercial trade and weak international legislation.

In Western Europe, many pond owners introduce amphibians for ornamental purposes. Although indigenous amphibians are legally protected in most European countries, retailers are circumventing national and international legislation by selling exotic nonprotected sibling species. We investigated to what extent non-native species of the European water frog complex (genus Pelophylax) have become established in Belgium, using morphological, mitochondrial and nuclear genetic markers. A survey of 87 sampling sites showed the presence of non-native water frogs at 47 locations, mostly Marsh frogs (Pelophylax ridibundus). Surprisingly, at least 19% of all these locations also harboured individuals with mitochondrial haplotypes characteristic of Anatolian water frogs (Pelophylax cf. bedriagae). Nuclear genotyping indicated widespread hybridization and introgression between P. ridibundus and P. cf. bedriagae. In addition, water frogs of Turkish origin obtained through a licensed retailer, also contained P. ridibundus and P. cf. bedriagae, with identical haplotypes to the wild Belgian populations. Although P. ridibundus might have invaded Belgium by natural range expansion from neighbouring countries, our results suggest that its invasion was at least partly enhanced by commercial trade, with origins as far as the Middle East. Also the invasion and rapid spread of Anatolian lineages, masked by their high morphological similarity to P. ridibundus, is likely the result of unregulated commercial trade. We expect that Anatolian frogs will further invade the exotic as well as the native range of P. ridibundus and other Pelophylax species elsewhere in Western and Central Europe, with risks of large-scale hybridization and introgression.