Between but Not Within-Species Variation in the Distribution of Fitness Effects.

New mutations provide the raw material for evolution and adaptation. The distribution of fitness effects (DFE) describes the spectrum of effects of new mutations that can occur along a genome, and is, therefore, of vital interest in evolutionary biology. Recent work has uncovered striking similarities in the DFE between closely related species, prompting us to ask whether there is variation in the DFE among populations of the same species, or among species with different degrees of divergence, that is whether there is variation in the DFE at different levels of evolution. Using exome capture data from six tree species sampled across Europe we characterized the DFE for multiple species, and for each species, multiple populations, and investigated the factors potentially influencing the DFE, such as demography, population divergence, and genetic background. We find statistical support for the presence of variation in the DFE at the species level, even among relatively closely related species. However, we find very little difference at the population level, suggesting that differences in the DFE are primarily driven by deep features of species biology, and those evolutionarily recent events, such as demographic changes and local adaptation, have little impact.

Taming the massive genome of Scots pine with PiSy50k, a new genotyping array for conifer research.

Pinus sylvestris (Scots pine) is the most widespread coniferous tree in the boreal forests of Eurasia, with major economic and ecological importance. However, its large and repetitive genome presents a challenge for conducting genome-wide analyses such as association studies, genetic mapping and genomic selection. We present a new 50K single-nucleotide polymorphism (SNP) genotyping array for Scots pine research, breeding and other applications. To select the SNP set, we first genotyped 480 Scots pine samples on a 407 540 SNP screening array and identified 47 712 high-quality SNPs for the final array (called 'PiSy50k'). Here, we provide details of the design and testing, as well as allele frequency estimates from the discovery panel, functional annotation, tissue-specific expression patterns and expression level information for the SNPs or corresponding genes, when available. We validated the performance of the PiSy50k array using samples from Finland and Scotland. Overall, 39 678 (83.2%) SNPs showed low error rates (mean = 0.9%). Relatedness estimates based on array genotypes were consistent with the expected pedigrees, and the level of Mendelian error was negligible. In addition, array genotypes successfully discriminate between Scots pine populations of Finnish and Scottish origins. The PiSy50k SNP array will be a valuable tool for a wide variety of future genetic studies and forestry applications.

Limited gene exchange between two sister species of leaf beetles within a hybrid zone in the Alps.

Analysing genomic variation within and between sister species is a first step towards understanding species boundaries. We focused on two sister species of cold-resistant leaf beetles, Gonioctena quinquepunctata and G. intermedia, whose ranges overlap in the Alps. A previous study of DNA sequence variation had revealed multiple instances of mitochondrial genome introgression in this region, suggesting recent hybridization between the two species. To evaluate the extent of gene exchange resulting from these hybridization events, we sampled individuals of both species inside and outside the hybrid zone and analysed genomic variation among them using RAD-seq markers. Individual levels of introgression in the nuclear genome were estimated first by defining species-specific SNPs (displaying a fixed difference between species) a priori and second by using model-based methods. Both types of analyses indicated little gene exchange, if any, between species at the level of the nuclear genome. Whereas the first method suggested slightly more gene flow, we argue that it has likely overestimated introgression in the phylogeographic context of this study. We conclude that strong intrinsic barriers prevent genetic exchange at the level of the nuclear genome between the two species. The apparent discrepancy observed between introgression occurring in the nuclear and mitochondrial genomes could be explained by selection acting in favour of the latter. Also, these results have consequences for the phylogeographic study of each species, since we can assume that genetic diversity in the overlapping portion of their ranges is not the product of introgression.

Widespread co-occurrence of two distantly related mitochondrial genomes in individuals of the leaf beetle Gonioctena intermedia.

Mitochondrial genome heteroplasmy-the presence of more than one genomic variant in individuals-is considered only occasional in animals, and most often involves molecules differing only by a few recent mutations. Thanks to new sequencing technologies, a large number of DNA fragments from a single individual can now be sequenced and visualized separately, allowing new insights into intra-individual mitochondrial genome variation. Here, we report evidence from both (i) massive parallel sequencing (MPS) of genomic extracts and (ii) Sanger sequencing of PCR products, for the widespread co-occurrence of two distantly related (greater than 1% nucleotide divergence, excluding the control region) mitochondrial genomes in individuals of a natural population of the leaf beetle Gonioctena intermedia Sanger sequencing of PCR products using universal primers previously failed to identify heteroplasmy in this population. Its occurrence was detected with MPS data and may have important implications for evolutionary studies. It suggests the need to re-evaluate, using MPS techniques, the proportion of animal species displaying heteroplasmy.

Comparing phylogeographic hypotheses by simulating DNA sequences under a spatially explicit model of coalescence.

Computer simulations of genetic data are increasingly used to investigate the impact of complex historical scenarios on patterns of genetic variation. Yet, in most empirical studies, relatively large portions of species ranges are often treated as panmictic populations, ignoring the underlying spatial context. In some cases, however, a more accurate spatial model is required. We use a spatially explicit model of coalescence (easily constructed by overlaying a two-dimensional grid on maps displaying an estimate of past and current species ranges) to evaluate the potential of several summary statistics to differentiate three typical phylogeographic scenarios. We first explore the variation of each summary statistic within the boundaries of each phylogeographic scenario, and identify those that appear most promising for a comparison of historical scenarios and/or to infer historical parameters. We then combine a selected set of summary statistics in a single chi-square statistic and evaluate whether it can be used to differentiate past geographic fragmentation or range expansion from a simple scenario of isolation by distance. We also investigate the benefits of using a spatially explicit model by comparing its performance to alternative models that are less spatially explicit (lower geographic resolution). The results identify conditions in which each summary statistic is useful to infer the evolution of a species range, and allow us to validate our spatially explicit model of coalescence and our procedure to compare simulated and observed sequence data. We also provide a detailed description of the spatially explicit model of coalescence used, which is currently lacking.

Clinal variation in the temperature and photoperiodic control of reproductive diapause in Drosophila montana females.

Insect adaptation to climatic conditions at different latitudes has required changes in life-history traits linked with survival and reproduction. Several species, including Drosophila montana, show robust latitudinal variation in the critical day length (CDL), below which more than half of the emerging females enter reproductive diapause at a given temperature. Here we used a novel approach to find out whether D. montana also shows latitudinal variation in the critical temperature (CTemp), above which the photoperiodic regulation of diapause is disturbed so that the females develop ovaries in daylengths that are far below their CDL. We estimated CTemp for 53 strains from different latitudes on 3 continents after measuring their diapause proportions at a range of temperatures in 12 h daylength (for 29 of the strains also in continuous darkness). In 12 h daylength, CTemp increased towards high latitudes alongside an increase in CDL, and in 3 high-latitude strains diapause proportion exceeded 50% in all temperatures. In continuous darkness, the diapause proportion was above 50% in the lowest temperature(s) in only 9 strains, all of which came from high latitudes. In the second part of the study, we measured changes in CTemp and CDL in a selection experiment favouring reproduction in short daylength (photoperiodic selection) and by exercising selection for females that reproduce in LD12:12 at low temperature (photoperiodic and temperature selection). In both experiments selection induced parallel changes in CDL and CTemp, confirming correlations seen between these traits along latitudinal clines. Overall, our findings suggest that selection towards strong photoperiodic diapause and long CDL at high latitudes has decreased the dependency of D. montana diapause on environmental temperature. Accordingly, the prevalence and timing of the diapause of D. montana is likely to be less vulnerable to climate warming in high- than low-latitude populations.

Plasticity in Photoperiodism: Drosophila montana Females Have a Life-Long Ability to Switch From Reproduction to Diapause.

Photoperiodic reproductive diapause is an essential part of female life cycle in several insect species living on high latitudes, where overwintering in reproductive stage involves high risks for survival and progeny production. The sensitive period (SP), during which photoperiodic cues can trigger the switch from direct development to diapause, can last from a few hours or days after emergence to the entire life span of females. Moreover, in some species, sexually mature females can enter post-reproductive diapause as a response to decreasing day length and/or temperature. We studied the duration of SP for diapause induction and the females' ability to enter post-reproductive diapause at short day lengths in Drosophila montana strains from different latitudes in Europe, North America, and Japan. Our study shows that the females of this species have a life-long SP and that they retain an ability to switch between reproduction and diapause as a response to back-and-forth changes in day length for at least 3 months. D. montana strains from different latitudes showed high variation in females' ability to enter post-reproductive diapause; females of the southern strains generally requiring longer time and/or lower temperature to enter this stage than those of the northern strains. Moreover, the proportion of females that switched to post-reproductive diapause in 3 weeks in short day conditions at 16 °C showed positive correlation with the critical day length (CDL) for diapause induction and the latitudinal and continental origin of the strains. Life-long SP increases females' flexibility to respond to short-term changes in environmental conditions and enables reproducing females to switch to post-reproductive diapause when the days get shorter and colder toward the autumn. This ability can play a major role in species phenology and should be taken into account in theoretical and empirical studies on insect adaptation to seasonal variation.

Nanda-Hamner Curves Show Huge Latitudinal Variation but No Circadian Components in Drosophila Montana Photoperiodism.

Insect species with a wide distribution offer a great opportunity to trace latitudinal variation in the photoperiodic regulation of traits important in reproduction and stress tolerances. We measured this variation in the photoperiodic time-measuring system underlying reproductive diapause in Drosophila montana, using a Nanda-Hamner (NH) protocol. None of the study strains showed diel rhythmicity in female diapause proportions under a constant day length (12 h) and varying night lengths in photoperiods ranging from 16 to 84 h at 16°C. In the northernmost strains (above 55°N), nearly all females entered diapause under all photoperiods and about half of them even in continuous darkness, while the females of the southern strains showed high diapause proportions only in the circadian 24 h photoperiod. Significant correlation between the strains' mean diapause proportions in ≥ 24 h photoperiods and critical day length (CDL; half of the females enter diapause) suggests at least partial causal connection between the traits. Interestingly, females of the northern strains entered diapause even in ≤ 24 h photoperiods, where the night length was shorter than their critical night length (24 h - CDL), but where the females experienced a higher number of Light:Dark cycles than in 24 h photoperiods. NH experiments, performed on the control and selection lines in our previous selection experiment, and completed here, gave similar results and confirmed that selection for shorter, southern-type CDL decreases female diapausing rate in non-circadian photoperiods. Overall, our study shows that D. montana females measure night length quantitatively, that the photoperiodic counter may play a prominent but slightly different role in extra short and extra long photoperiods and that northern strains show high stability against perturbations in the photoperiod length and in the presence of LD cycles. These features are best explained by the quantitative versions of the damped external coincidence model.

Juvenile hormone pathway in honey bee larvae: A source of possible signal molecules for the reproductive behavior of Varroa destructor.

The parasitic mite Varroa destructor devastates honey bee (Apis mellifera) colonies around the world. Entering a brood cell shortly before capping, the Varroa mother feeds on the honey bee larvae. The hormones 20-hydroxyecdysone (20E) and juvenile hormone (JH), acquired from the host, have been considered to play a key role in initiating Varroa's reproductive cycle. This study focuses on differential expression of the genes involved in the biosynthesis of JH and ecdysone at six time points during the first 30 hr after cell capping in both drone and worker larvae of A. mellifera. This time frame, covering the conclusion of the honey bee brood cell invasion and the start of Varroa's ovogenesis, is critical to the successful initiation of a reproductive cycle. Our findings support a later activation of the ecdysteroid cascade in honey bee drones compared to worker larvae, which could account for the increased egg production of Varroa in A. mellifera drone cells. The JH pathway was generally downregulated confirming its activity is antagonistic to the ecdysteroid pathway during the larva development. Nevertheless, the genes involved in JH synthesis revealed an increased expression in drones. The upregulation of jhamt gene involved in methyl farnesoate (MF) synthesis came into attention since the MF is not only a precursor of JH but it is also an insect pheromone in its own right as well as JH-like hormone in Acari. This could indicate a possible kairomone effect of MF for attracting the mites into the drone brood cells, along with its potential involvement in ovogenesis after the cell capping, stimulating Varroa's initiation of egg laying.

Comparative analysis of two sister Erythrophleum species (Leguminosae) reveal contrasting transcriptome-wide responses to early drought stress.

With the ongoing climate change, African rainforests are expected to experience severe drought events in the future. In Africa, the tropical genus Erythrophleum (Fabaceae) includes two forest sister timber tree species displaying contrasting geographical distributions. Erythrophleum ivorense is adapted to wet evergreen Guineo-Congolian forests, whereas E. suaveolens occurs in a wider range of climates, being found in moist dense forests but also in gallery forests under a relatively drier climate. This geographical distribution pattern suggests that the two species might cope differently to drought at the genomic level. Yet, the genetic basis of tolerance response to drought stress in both species is still uncharacterized. To bridge this gap, we performed an RNA-seq approach on seedlings from each species to monitor their transcriptional responses at different levels of drought stress (0, 2 and 6 weeks after stopping watering seedlings). Monitoring of wilting symptoms revealed that E. suaveolens displayed an earlier phenotypic response to drought stress than E. ivorense. At the transcriptomic level, results revealed 2020 (1204 down-regulated/816 up-regulated) and 1495 differentially expressed genes (DEGs) in response to drought stress from a total of 67,432 and 66,605 contigs assembled in E. ivorense and E. suaveolens, respectively. After identifying 30,374 orthologs between species, we found that only 7 of them were DEGs shared between species, while 587 and 458 were differentially expressed only in E. ivorense or E. suaveolens, respectively. GO and KEGG enrichment analysis revealed that the two species differ in terms of significantly regulated pathways as well as the number and expression profile of DEGs (Up/Down) associated with each pathway in the two stress stages. Our results suggested that the two studied species react differently to drought. E. suaveolens seems displaying a prompt response to drought at its early stage strengthened by the down-regulation of many DEGs encoding for signaling and metabolism-related pathways. A considerable up-regulation of these pathways was also found in E. ivorense at the late stage of drought, suggesting this species may be a late responder. Overall, our data may serve as basis for further understanding the genetic control of drought tolerance in tropical trees and favor the selection of crucial genes for genetically enhancing drought resistance.

Genetic evidence confirms polygamous mating system in a crustacean parasite with multiple hosts.

Mating systems are diverse in animals, notably in crustaceans, but can be inferred from a limited set of parameters. Baeza and Thiel (2007) proposed a model predicting mating systems of symbiotic crustaceans with three host characteristics and the risk of predation. These authors proposed five mating systems, ranging from monogamy to polygynandry (where multiple mating occurs for both genders). Using microsatellite loci, we tested the putatively mating system of the ectoparasite crab Dissodactylus primitivus. We determined the mating frequencies of males and females, parentage assignment (COLONY & GERUD software) as well as the contents of female spermathecae. Our results are globally consistent with the model of Baeza and Thiel and showed, together with previous aquarium experiments, that this ectoparasite evolved a polygamous mating system where males and females move between hosts for mate search. Parentage analyses revealed that polyandry is frequent and concerns more than 60% of clutches, with clutches being fertilized by up to 6 different fathers. Polygyny is supported by the detection of eight males having sired two different broods. We also detected a significant paternity skew in 92% of the multipaternal broods. Moreover, this skew is probably higher than the estimation from the brood because additional alleles were detected in most of spermathecae. This high skew could be explained by several factors as sperm competition or cryptic female choice. Our genetic data, combined with previous anatomic analyses, provide consistent arguments to suggest sperm precedence in D. primitivus.