Minimally destructive hDNA extraction method for retrospective genetics of pinned historical Lepidoptera specimens.

The millions of specimens stored in entomological collections provide a unique opportunity to study historical insect diversity. Current technologies allow to sequence entire genomes of historical specimens and estimate past genetic diversity of present-day endangered species, advancing our understanding of anthropogenic impact on genetic diversity and enabling the implementation of conservation strategies. A limiting challenge is the extraction of historical DNA (hDNA) of adequate quality for sequencing platforms. We tested four hDNA extraction protocols on five body parts of pinned false heath fritillary butterflies, Melitaea diamina, aiming to minimise specimen damage, preserve their scientific value to the collections, and maximise DNA quality and yield for whole-genome re-sequencing. We developed a very effective approach that successfully recovers hDNA appropriate for short-read sequencing from a single leg of pinned specimens using silica-based DNA extraction columns and an extraction buffer that includes SDS, Tris, Proteinase K, EDTA, NaCl, PTB, and DTT. We observed substantial variation in the ratio of nuclear to mitochondrial DNA in extractions from different tissues, indicating that optimal tissue choice depends on project aims and anticipated downstream analyses. We found that sufficient DNA for whole genome re-sequencing can reliably be extracted from a single leg, opening the possibility to monitor changes in genetic diversity maintaining the scientific value of specimens while supporting current and future conservation strategies.

Detecting directional epistasis and dominance from cross-line analyses in alpine populations of Arabidopsis thaliana.

The contribution of non-additive genetic effects to the genetic architecture of fitness and to the evolutionary potential of populations has been a topic of theoretical and empirical interest for a long time. Yet, the empirical study of these effects in natural populations remains scarce, perhaps because measuring dominance and epistasis relies heavily on experimental line crosses. In this study, we explored the contribution of dominance and epistasis in natural alpine populations of Arabidopsis thaliana for 2 fitness traits, the dry biomass and the estimated number of siliques, measured in a greenhouse. We found that, on average, crosses between inbred lines of A. thaliana led to mid-parent heterosis for dry biomass but outbreeding depression for an estimated number of siliques. While heterosis for dry biomass was due to dominance, we found that outbreeding depression for an estimated number of siliques could be attributed to the breakdown of beneficial epistatic interactions. We simulated and discussed the implication of these results for the adaptive potential of the studied populations, as well as the use of line-cross analyses to detect non-additive genetic effects.

UV RESISTANCE LOCUS 8-Mediated UV-B Response Is Required Alongside CRYPTOCHROME 1 for Plant Survival in Sunlight under Field Conditions.

As sessile, photoautotrophic organisms, plants are subjected to fluctuating sunlight that includes potentially detrimental ultraviolet-B (UV-B) radiation. Experiments under controlled conditions have shown that the UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) controls acclimation and tolerance to UV-B in Arabidopsis thaliana; however, its long-term impact on plant fitness under naturally fluctuating environments remain poorly understood. Here, we quantified the survival and reproduction of different Arabidopsis mutant genotypes under diverse field and laboratory conditions. We found that uvr8 mutants produced more fruits than wild type when grown in growth chambers under artificial low-UV-B conditions but not under natural field conditions, indicating a fitness cost in the absence of UV-B stress. Importantly, independent double mutants of UVR8 and the blue light photoreceptor gene CRYPTOCHROME 1 (CRY1) in two genetic backgrounds showed a drastic reduction in fitness in the field. Experiments with UV-B attenuation in the field and with supplemental UV-B in growth chambers demonstrated that UV-B caused the cry1 uvr8 conditional lethal phenotype. Using RNA-seq data of field-grown single and double mutants, we explicitly identified genes showing significant statistical interaction of UVR8 and CRY1 mutations in the presence of UV-B in the field. They were enriched in Gene Ontology categories related to oxidative stress, photoprotection and DNA damage repair in addition to UV-B response. Our study demonstrates the functional importance of the UVR8-mediated response across life stages in natura, which is partially redundant with that of cry1. Moreover, these data provide an integral picture of gene expression associated with plant responses under field conditions.

Climate-induced range shifts drive adaptive response via spatio-temporal sieving of alleles.

Quaternary climate fluctuations drove many species to shift their geographic ranges, in turn shaping their genetic structures. Recently, it has been argued that adaptation may have accompanied species range shifts via the "sieving" of genotypes during colonisation and establishment. However, this has not been directly demonstrated, and knowledge remains limited on how different evolutionary forces, which are typically investigated separately, interacted to jointly mediate species responses to past climatic change. Here, through whole-genome re-sequencing of over 1200 individuals of the carnation Dianthus sylvestris coupled with integrated population genomic and gene-environment models, we reconstruct the past neutral and adaptive landscape of this species as it was shaped by the Quaternary glacial cycles. We show that adaptive responses emerged concomitantly with the post-glacial range shifts and expansions of this species in the last 20 thousand years. This was due to the heterogenous sieving of adaptive alleles across space and time, as populations expanded out of restrictive glacial refugia into the broader and more heterogeneous range of habitats available in the present-day inter-glacial. Our findings reveal a tightly-linked interplay of migration and adaptation under past climate-induced range shifts, which we show is key to understanding the spatial patterns of adaptive variation we see in species today.

A target capture approach for phylogenomic analyses at multiple evolutionary timescales in rosewoods (Dalbergia spp.) and the legume family (Fabaceae).

Understanding the genetic changes associated with the evolution of biological diversity is of fundamental interest to molecular ecologists. The assessment of genetic variation at hundreds or thousands of unlinked genetic loci forms a sound basis to address questions ranging from micro- to macroevolutionary timescales, and is now possible thanks to advances in sequencing technology. Major difficulties are associated with (i) the lack of genomic resources for many taxa, especially from tropical biodiversity hotspots; (ii) scaling the numbers of individuals analysed and loci sequenced; and (iii) building tools for reproducible bioinformatic analyses of such data sets. To address these challenges, we developed target capture probes for genomic studies of the highly diverse, pantropically distributed and economically significant rosewoods (Dalbergia spp.), explored the performance of an overlapping probe set for target capture across the legume family (Fabaceae), and built the general purpose bioinformatic pipeline CaptureAl. Phylogenomic analyses of Malagasy Dalbergia species yielded highly resolved and well supported hypotheses of evolutionary relationships. Population genomic analyses identified differences between closely related species and revealed the existence of a potentially new species, suggesting that the diversity of Malagasy Dalbergia species has been underestimated. Analyses at the family level corroborated previous findings by the recovery of monophyletic subfamilies and many well-known clades, as well as high levels of gene tree discordance, especially near the root of the family. The new genomic and bioinformatic resources, including the Fabaceae1005 and Dalbergia2396 probe sets, will hopefully advance systematics and ecological genetics research in legumes, and promote conservation of the highly diverse and endangered Dalbergia rosewoods.

Capacity of soil bacteria to reach the phyllosphere and convergence of floral communities despite soil microbiota variation.

Leaves and flowers are colonized by diverse bacteria that impact plant fitness and evolution. Although the structure of these microbial communities is becoming well-characterized, various aspects of their environmental origin and selection by plants remain uncertain, such as the relative proportion of soilborne bacteria in phyllosphere communities. Here, to address this issue and to provide experimental support for bacteria being filtered by flowers, we conducted common-garden experiments outside and under gnotobiotic conditions. We grew Arabidopsis thaliana in a soil substitute and added two microbial communities from natural soils. We estimated that at least 25% of the phyllosphere bacteria collected from the plants grown in the open environment were also detected in the controlled conditions, in which bacteria could reach leaves and flowers only from the soil. These taxa represented more than 40% of the communities based on amplicon sequencing. Unsupervised hierarchical clustering approaches supported the convergence of all floral microbiota, and 24 of the 28 bacteria responsible for this pattern belonged to the Burkholderiaceae family, which includes known plant pathogens and plant growth-promoting members. We anticipate that our study will foster future investigations regarding the routes used by soil microbes to reach leaves and flowers, the ubiquity of the environmental filtering of Burkholderiaceae across plant species and environments, and the potential functional effects of the accumulation of these bacteria in the reproductive organs of flowering plants.

Introgression is widespread in the radiation of carnivorous Nepenthes pitcher plants.

Introgression and hybridization are important processes in plant evolution, but they are difficult to study from a phylogenetic perspective, because they conflict with the bifurcating evolutionary history typically depicted in phylogenetic models. The role of hybridization in plant evolution is best documented in the form of allo-polyploidizations. In contrast, homoploid hybridization and introgression are less explored, although they may be crucial in adaptive radiations. Here we employ genome-wide data (ddRAD-seq, transcriptomes) to investigate the evolutionary history of Nepenthes, a radiation of c. 160 species of iconic carnivorous plants mainly from tropical Asia. Our data indicates that the main radiation is only c. 5 million years old, and confirms previous bifurcating phylogenies. However, due to a greatly expanded number of loci, we were able test for the first time the long-standing hypotheses of introgression and historical hybridization. The genus presents one very clear case of organellar capture between two distantly related but sympatric groups. Furthermore, all Nepenthes species show introgression signals in their nuclear genomes, as uncovered by a general survey of ABBA-BABA-like statistics. The ancestor of the rapid main radiation shows ancestry from two deeply diverged lineages, as indicated by phylogenetic network analyses. All major clades of the main radiation show further introgression both within and between each other, as suggested by admixture graphs. Our study supports the hypothesis that rapid adaptive radiations are hotspots of introgression in the tree of life, and highlights the need to consider non-treelike processes in evolutionary studies of Nepenthes in particular.

Identifying loci under selection via explicit demographic models.

Adaptive genetic variation is a function of both selective and neutral forces. To accurately identify adaptive loci, it is thus critical to account for demographic history. Theory suggests that signatures of selection can be inferred using the coalescent, following the premise that genealogies of selected loci deviate from neutral expectations. Here, we build on this theory to develop an analytical framework to identify loci under selection via explicit demographic models (LSD). Under this framework, signatures of selection are inferred through deviations in demographic parameters, rather than through summary statistics directly, and demographic history is accounted for explicitly. Leveraging the property of demographic models to incorporate directionality, we show that LSD can provide information on the environment in which selection acts on a population. This can prove useful in elucidating the selective processes underlying local adaptation, by characterizing genetic trade-offs and extending the concepts of antagonistic pleiotropy and conditional neutrality from ecological theory to practical application in genomic data. We implement LSD via approximate Bayesian computation and demonstrate, via simulations, that LSD (a) has high power to identify selected loci across a large range of demographic-selection regimes, (b) outperforms commonly applied genome-scan methods under complex demographies and (c) accurately infers the directionality of selection for identified candidates. Using the same simulations, we further characterize the behaviour of isolation-with-migration models conducive to the study of local adaptation under regimes of selection. Finally, we demonstrate an application of LSD by detecting loci and characterizing genetic trade-offs underlying flower colour in Antirrhinum majus.

Dioecy Is Associated with High Genetic Diversity and Adaptation Rates in the Plant Genus Silene.

About 15,000 angiosperm species (∼6%) have separate sexes, a phenomenon known as dioecy. Why dioecious taxa are so rare is still an open question. Early work reported lower species richness in dioecious compared with nondioecious sister clades, raising the hypothesis that dioecy may be an evolutionary dead-end. This hypothesis has been recently challenged by macroevolutionary analyses that detected no or even positive effect of dioecy on diversification. However, the possible genetic consequences of dioecy at the population level, which could drive the long-term fate of dioecious lineages, have not been tested so far. Here, we used a population genomics approach in the Silene genus to look for possible effects of dioecy, especially for potential evidence of evolutionary handicaps of dioecy underlying the dead-end hypothesis. We collected individual-based RNA-seq data from several populations in 13 closely related species with different sexual systems: seven dioecious, three hermaphroditic, and three gynodioecious species. We show that dioecy is associated with increased genetic diversity, as well as higher selection efficacy both against deleterious mutations and for beneficial mutations. The results hold after controlling for phylogenetic inertia, differences in species census population sizes and geographic ranges. We conclude that dioecious Silene species neither show signs of increased mutational load nor genetic evidence for extinction risk. We discuss these observations in the light of the possible demographic differences between dioecious and self-compatible hermaphroditic species and how this could be related to alternatives to the dead-end hypothesis to explain the rarity of dioecy.

Genomic signatures of convergent adaptation to Alpine environments in three Brassicaceae species.

It has long been discussed to what extent related species develop similar genetic mechanisms to adapt to similar environments. Most studies documenting such convergence have either used different lineages within species or surveyed only a limited portion of the genome. Here, we investigated whether similar or different sets of orthologous genes were involved in genetic adaptation of natural populations of three related plant species to similar environmental gradients in the Alps. We used whole-genome pooled population sequencing to study genome-wide SNP variation in 18 natural populations of three Brassicaceae (Arabis alpina, Arabidopsis halleri, and Cardamine resedifolia) from the Swiss Alps. We first de novo assembled draft reference genomes for all three species. We then ran population and landscape genomic analyses with ~3 million SNPs per species to look for shared genomic signatures of selection and adaptation in response to similar environmental gradients acting on these species. Genes with a signature of convergent adaptation were found at significantly higher numbers than expected by chance. The most closely related species pair showed the highest relative over-representation of shared adaptation signatures. Moreover, the identified genes of convergent adaptation were enriched for nonsynonymous mutations, suggesting functional relevance of these genes, even though many of the identified candidate genes have hitherto unknown or poorly described functions based on comparison with Arabidopsis thaliana. We conclude that adaptation to heterogeneous Alpine environments in related species is partly driven by convergent evolution, but that most of the genomic signatures of adaptation remain species-specific.

Identification of sex-linked markers in the sexually cryptic coco de mer: are males and females produced in equal proportions?

Lodoicea maldivica (coco de mer) is a long-lived dioecious palm in which male and female plants are visually indistinguishable when immature, only becoming sexually dimorphic as adults, which in natural forest can take as much as 50 years. Most adult populations in the Seychelles exhibit biased sex ratios, but it is unknown whether this is due to different proportions of male and female plants being produced or to differential mortality. In this study, we developed sex-linked markers in Lodoicea using ddRAD sequencing, enabling us to reliably determine the gender of immature individuals. We screened 589 immature individuals to explore sex ratios across life stages in Lodoicea. The two sex-specific markers resulted in the amplification of male-specific bands (Lm123977 at 405 bp and Lm435135 at 130 bp). Our study of four sub-populations of Lodoicea on the islands of Praslin and Curieuse revealed that the two sexes were produced in approximately equal numbers, with no significant deviation from a 1:1 ratio before the adult stage. We conclude that sex in Lodoicea is genetically determined, suggesting that Lodoicea has a chromosomal sex determination system in which males are the heterogametic sex (XY) and females are homogametic (XX). We discuss the potential causes for observed biased sex ratios in adult populations, and the implications of our results for the life history, ecology and conservation management of Lodoicea.

Adaptation to local climate in multi-trait space: evidence from silver fir (Abies alba Mill.) populations across a heterogeneous environment.

Heterogeneous environments, such as mountainous landscapes, create spatially varying selection pressure that potentially affects several traits simultaneously across different life stages, yet little is known about the general patterns and drivers of adaptation in such complex settings. We studied silver fir (Abies alba Mill.) populations across Switzerland and characterized its mountainous landscape using downscaled historical climate data. We sampled 387 trees from 19 populations and genotyped them at 374 single-nucleotide polymorphisms (SNPs) to estimate their demographic distances. Seedling morphology, growth and phenology traits were recorded in a common garden, and a proxy for water use efficiency was estimated for adult trees. We tested whether populations have more strongly diverged at quantitative traits than expected based on genetic drift alone in a multi-trait framework, and identified potential environmental drivers of selection. We found two main responses to selection: (i) populations from warmer and more thermally stable locations have evolved towards a taller stature, and (ii) the growth timing of populations evolved towards two extreme strategies, 'start early and grow slowly' or 'start late and grow fast', driven by precipitation seasonality. Populations following the 'start early and grow slowly' strategy had higher water use efficiency and came from inner Alpine valleys characterized by pronounced summer droughts. Our results suggest that contrasting adaptive life-history strategies exist in silver fir across different life stages (seedling to adult), and that some of the characterized populations may provide suitable seed sources for tree growth under future climatic conditions.

Sex is determined by XY chromosomes across the radiation of dioecious Nepenthes pitcher plants.

Species with separate sexes (dioecy) are a minority among flowering plants, but dioecy has evolved multiple times independently in their history. The sex-determination system and sex-linked genomic regions are currently identified in a limited number of dioecious plants only. Here, we study the sex-determination system in a genus of dioecious plants that lack heteromorphic sex chromosomes and are not amenable to controlled breeding: Nepenthes pitcher plants. We genotyped wild populations of flowering males and females of three Nepenthes taxa using ddRAD-seq and sequenced a male inflorescence transcriptome. We developed a statistical tool (privacy rarefaction) to distinguish true sex specificity from stochastic noise in read coverage of sequencing data from wild populations and identified male-specific loci and XY-patterned single nucleotide polymorphsims (SNPs) in all three Nepenthes taxa, suggesting the presence of homomorphic XY sex chromosomes. The male-specific region of the Y chromosome showed little conservation among the three taxa, except for the essential pollen development gene DYT1 that was confirmed as male specific by PCR in additional Nepenthes taxa. Hence, dioecy and part of the male-specific region of the Nepenthes Y-chromosomes likely have a single evolutionary origin.

Insights into the genetic architecture of sexual dimorphism from an interspecific cross between two diverging Silene (Caryophyllaceae) species.

The evolution of sexual dimorphism in species with separate sexes is influenced by the resolution of sexual conflicts creating sex differences through genetic linkage or sex-biased expression. Plants with different degrees of sexual dimorphism are thus ideal to study the genetic basis of sexual dimorphism. In this study we explore the genetic architecture of sexual dimorphism between Silene latifolia and Silene dioica. These species have chromosomal sex determination and differ in the extent of sexual dimorphism. To test whether QTL for sexually dimorphic traits have accumulated on the sex chromosomes and to quantify their contribution to species differences, we create a linkage map and performed QTL analysis of life history, flower and vegetative traits using an unidirectional interspecific F2 hybrid cross. We found support for an accumulation of QTL on the sex chromosomes and that sex differences explained a large proportion of the variance between species, suggesting that both natural and sexual selection contributed to species divergence. Sexually dimorphic traits that also differed between species displayed transgressive segregation. We observed a reversal in sexual dimorphism in the F2 population, where males tended to be larger than females, indicating that sexual dimorphism is constrained within populations but not in recombinant hybrids. This study contributes to the understanding of the genetic basis of sexual dimorphism and its evolution in Silene.

Conservation genetics: Linking science with practice.

Conservation genetics is a well-established scientific field. However, limited information transfer between science and practice continues to hamper successful implementation of scientific knowledge in conservation practice and management. To mitigate this challenge, we have established a conservation genetics community, which entails an international exchange-and-skills platform related to genetic methods and approaches in conservation management. First, it allows for scientific exchange between researchers during annual conferences. Second, personal contact between conservation professionals and scientists is fostered by organising workshops and by popularising knowledge on conservation genetics methods and approaches in professional journals in national languages. Third, basic information on conservation genetics has been made accessible by publishing an easy-to-read handbook on conservation genetics for practitioners. Fourth, joint projects enabled practitioners and scientists to work closely together from the start of a project in order to establish a tight link between applied questions and scientific background. Fifth, standardised workflows simplifying the implementation of genetic tools in conservation management have been developed. By establishing common language and trust between scientists and practitioners, all these measures help conservation genetics to play a more prominent role in future conservation planning and management.

Publisher Correction: Low genetic variation is associated with low mutation rate in the giant duckweed.

The original HTML version of this Article had an incorrect Published online date of 20 March 2019; it should have been 18 March 2019. This has been corrected in the HTML version of the Article. The PDF version was correct from the time of publication.

Low genetic variation is associated with low mutation rate in the giant duckweed.

Mutation rate and effective population size (Ne) jointly determine intraspecific genetic diversity, but the role of mutation rate is often ignored. Here we investigate genetic diversity, spontaneous mutation rate and Ne in the giant duckweed (Spirodela polyrhiza). Despite its large census population size, whole-genome sequencing of 68 globally sampled individuals reveals extremely low intraspecific genetic diversity. Assessed under natural conditions, the genome-wide spontaneous mutation rate is at least seven times lower than estimates made for other multicellular eukaryotes, whereas Ne is large. These results demonstrate that low genetic diversity can be associated with large-Ne species, where selection can reduce mutation rates to very low levels. This study also highlights that accurate estimates of mutation rate can help to explain seemingly unexpected patterns of genome-wide variation.

Divergence in Glucosinolate Profiles between High- and Low-Elevation Populations of Arabidopsis halleri Correspond to Variation in Field Herbivory and Herbivore Behavioral Preferences.

Variation in local herbivore pressure along elevation gradients is predicted to drive variation in plant defense traits. Yet, the extent of intraspecific variation in defense investment along elevation gradients, and its effects on both herbivore preference and performance, remain relatively unexplored. Using populations of Arabidopsis halleri (Brassicaceae) occurring at different elevations in the Alps, we tested for associations between elevation, herbivore damage in the field, and constitutive chemical defense traits (glucosinolates) assayed under common-garden conditions. Additionally, we examined the feeding preferences and performance of a specialist herbivore, the butterfly Pieris brassicae, on plants from different elevations in the Alps. Although we found no effect of elevation on the overall levels of constitutive glucosinolates in leaves, relative amounts of indole glucosinolates increased significantly with elevation and were negatively correlated with herbivore damage in the field. In oviposition preference assays, P. brassicae females laid fewer eggs on plants from high-elevation populations, although larval performance was similar on populations from different elevations. Taken together, these results support the prediction that species distributed along elevation gradients exhibit genetic variation in chemical defenses, which can have consequences for interactions with herbivores in the field.

Ecological divergence plays an important role in strong but complex reproductive isolation in campions (Silene).

New species arise through the evolution of reproductive barriers between formerly interbreeding lineages. Yet, comprehensive assessments of potential reproductive barriers, which are needed to make inferences on processes driving speciation, are only available for a limited number of systems. In this study, we estimated individual and cumulative strengths of seven prezygotic and six postzygotic reproductive barriers between the recently diverged taxa Silene dioica (L.) Clairv. and S. latifolia Poiret using both published and new data. A combination of multiple partial reproductive barriers resulted in near-complete reproductive isolation between S. dioica and S. latifolia, consistent with earlier estimates of gene flow between the taxa. Extrinsic barriers associated with adaptive ecological divergence were most important, while intrinsic postzygotic barriers had moderate individual strength but contributed only little to total reproductive isolation. These findings are in line with ecological divergence as driver of speciation. We further found extensive variation in extrinsic reproductive isolation, ranging from sites with very strong selection against migrants and hybrids to intermediate sites where substantial hybridization is possible. This situation may allow for, or even promote, heterogeneous genetic divergence.

Population genomic evidence for plant glacial survival in Scandinavia.

Quaternary glaciations have played a major role in shaping the genetic diversity and distribution of plant species. Strong palaeoecological and genetic evidence supports a postglacial recolonization of most plant species to northern Europe from southern, eastern and even western glacial refugia. Although highly controversial, the existence of small in situ glacial refugia in northern Europe has recently gained molecular support. We used genomic analyses to examine the phylogeography of a species that is critical in this debate. Carex scirpoidea Michx subsp. scirpoidea is a dioecious, amphi-Atlantic arctic-alpine sedge that is widely distributed in North America, but absent from most of Eurasia, apart from three extremely disjunct populations in Norway, all well within the limits of the Weichselian ice sheet. Range-wide population sampling and variation at 5,307 single nucleotide polymorphisms show that the three Norwegian populations comprise unique evolutionary lineages divergent from Greenland with high between-population divergence. The Norwegian populations have low within-population genetic diversity consistent with having experienced genetic bottlenecks in glacial refugia, and host private alleles that probably accumulated in long-term isolated populations. Demographic analyses support a single, pre-Weichselian colonization into Norway from East Greenland, and subsequent divergence of the three populations in separate refugia. Other refugial areas are identified in North-east Greenland, Minnesota/Michigan, Colorado and Alaska. Admixed populations in British Columbia and West Greenland indicate postglacial contact. Taken together, evidence from this study strongly indicates in situ glacial survival in Scandinavia.