Efforts to detect loci under selection in plants have mostly focussed on single species. However, assuming that intraspecific divergence may lead to speciation, comparisons of genetic variation within and among recently diverged taxa can help to locate such genes. In this study, coalescent and outlier detection methods were used to assess nucleotide polymorphism and divergence at 79 nuclear gene fragments (1212 SNPs) in 16 populations (153 individuals) of the closely related, but phenotypically and ecologically distinct, pine taxa Pinus mugo , P. uliginosa and P. uncinata across their European distributions. Simultaneously, mitochondrial DNA markers, which are maternally inherited in pines and distributed by seeds at short geographic distance, were used to assess genetic relationships of the focal populations and taxa. The majority of nuclear loci showed homogenous patterns of variation between the taxa due to a high number of shared SNPs and haplotypes, similar levels of polymorphism, and low net divergence. However, against this common genetic background and an overall low population structure within taxa at mitochondrial markers, we identified several genes showing signatures of selection, accompanied by significant intra- and interspecific divergence. Our results indicate that loci involved in species divergence may be involved in intraspecific local adaptation. Electronic supplementary material The online version of this article (10.1007/s11295-018-1296-3) contains supplementary material, which is available to authorized users.

In species where females store sperm, males may try to influence paternity by the strategic placement of sperm within the female's sperm storage organ. Sperm may be mixed or layered in storage organs, and this can influence sperm use beyond a 'fair raffle'. In some insects, sperm from different matings is packaged into discrete packets (spermatodoses), which retain their integrity in the female's sperm storage organ (spermatheca), but little is known about how these may influence patterns of sperm use under natural mating conditions in wild populations. We examined the effect of the size and position of spermatodoses within the spermatheca and number of competing ejaculates on sperm use in female dark bushcrickets (Pholidoptera griseoaptera) that had mated under unmanipulated field conditions. Females were collected near the end of the mating season, and seven hypervariable microsatellite loci were used to assign paternity of eggs laid in the laboratory. Females contained a median of three spermatodoses (range 1-6), and only six of the 36 females contained more than one spermatodose of the same genotype. Both the size and relative placement of the spermatodoses within the spermatheca had a significant effect on paternity, with a bias against smaller spermatodoses and those further from the single entrance/exit of the spermatheca. A higher number of competing males reduced the chances of siring offspring for each male. Hence, both spermatodose size and relative placement in the spermatheca influence paternity success.

The dwarf mountain pine (Pinus mugo) and the Pyrenean pine (P. uncinata) constitute a pair of closely related coniferous taxa of poorly resolved evolutionary history and affinity, which inhabit numerous stands scattered over subalpine environments of European mountain ranges. The aim of the study was to investigate their phylogeography and mutual relationships, shedding new light on their taxonomy and the past of the alpine flora. Previous evolutionary reconstructions of the mountain pines relied mainly on bi‐parentally or paternally inherited markers that quickly homogenize between populations, showing rather shallow and recent differentiation of gene pools. Therefore, to contrast these pictures, we analyzed diversity and differentiation within a large set of new mitochondrial loci, inherited in maternal line and distributed by seeds at short geographical distances. Samples of the taxa were taken from 27 natural populations representing their range‐wide distributions—17 populations of P. mugo and 10 of P. uncinata. All markers appeared polymorphic, providing a total of 31 multilocus haplotypes. Two of the loci proved to be species‐diagnostic and nearly fixed between analyzed samples. Distribution of mitotypes indicate that allopatric populations of the taxa constitute separate mitochondrial haplogroups, and the two mountain pines have independent evolutionary history. However, introgression of P. mugo mitotypes by P. uncinata specimens revealed in the species contact zone in Western Alps shows that their speciation is not fully completed.

Estimates of genetic differentiation at intra- and interspecific level are often hindered by the lack of suitable molecular markers. Low phylogeographic resolution limits development of appropriate conservation strategies especially in case of endangered forest tree species with small and disjunct distribution. In this study, we assessed fine-scale genetic structure of relict and endangered peat bog pine ( Pinus uliginosa ) and two other closely related European pine species ( Pinus mugo and Pinus uncinata ) using a set of 15 newly developed maternally inherited and seed-mediated mitochondrial DNA ( mt DNA) markers and two previously known polymorphic mt DNA regions ( nad1 , nad7 ). Three main groups, corresponding in general to three investigated species were revealed in the haplotype network analysis. However, only P. uncinata was clearly distinct at all levels of analysis, whereas great genetic similarity and haplotype sharing was observed between P. uliginosa and P. mugo . Strong phylogeographic structure was found in P. uliginosa that showed high differentiation at relatively short geographical distance among populations and the existence of mitochondrial lineages of different evolutionary history. Hybridization with other pine species has likely contributed to genetic differentiation of P. uliginosa as indicated by contemporary distribution of mt DNA haplotypes. The research emphasizes the importance of accurate assessments of genetic structure of endangered species with complex evolutionary history for development of efficient conservation strategies.

With the current rate of biodiversity loss, conservation management practices require a comprehensive understanding of eco‐evolutionary relationships, history, and genetic structure of species. Assessments of genetic diversity are crucial, especially in rare, endemic, or threatened forest tree species with small and isolated populations, such as peat bog pine (Pinus uliginosa N.). Here, we used a novel approach, combining genetic diversity assessment, ecological niche modeling, and population demography inference to explore the complex history of a few remnant populations of this endangered pine. To asses the relative influence of isolation and fragmentation on genetic diversity in the taxonomic context, the patterns of genetic variation found in P. uliginosa were contrasted with those observed in its close relatives with much bigger distribution ranges and larger populations (Pinus sylvestris, Pinus mugo, and Pinus uncinata). We found a similar level of genetic diversity across the species at nuclear loci but contrasting patterns of variability distribution at chloroplast markers. We detected the signatures of an ancient genetic bottleneck dated at around 26 400 years ago, indicating a drastic reduction in the population size of P. uligionosa during the Last Glacial Maximum. In addition, we found substantial differentiation between current populations as a result of enhanced genetic drift during long‐lasting isolation. The research suggests potential conservation management strategies for peat bog pine and emphasizes the importance of using complementary approaches for their successful development.

Scots pine (Pinus sylvestris L.) and the taxa from the P. mugo complex can hybridize in the contact zones and produce fertile hybrids. A unique example of an early Holocene relict population of P. sylvestris and P. uliginosa (a taxon from the P. mugo complex) growing on the tops of Jurassic sandstone rocks is located in Błędne Skały (Sudetes). Phenotypically, there are trees resembling P. sylvestris, P. uliginosa and intermediate forms between them. We expected that some of P. sylvestris and/or P. uliginosa-like trees could be in fact cryptic hybrids resembling one of the parental phenotypes. To address this question, we examined randomly sampled individuals, using a set of plastid (cpDNA), nuclear (nDNA) and mitochondrial (mtDNA) markers as well as biometric characteristics of needles and cones. The results were compared to the same measurements of allopatric reference populations of the P. sylvestris and the P. mugo complex (Pinus mugo s.s, P. uncinata and P. uliginosa). We detected cpDNA barcodes of the P. mugo complex in most individuals with the P. sylvestris phenotype, while we did not detect cpDNA diagnostic of P. sylvestris within P. uliginosa-like trees. These results indicate the presence of cryptic hybrids of the P. sylvestris phenotype. We found only three typical P. sylvestris individuals that were clustered with the species reference populations based on needle and cone characteristics. Most trees showed intermediate characteristics between P. sylvestris and P. uliginosa-like trees, indicating intensive and probably long-lasting hybridization of the taxa at this area and subsequent gene erosion of parental species.

Mountain plants, challenged by vegetation time contractions and dynamic changes in environmental conditions, developed adaptations that help them to balance their growth, reproduction, survival, and regeneration. However, knowledge regarding the genetic basis of species adaptation to higher altitudes remain scarce for most plant species. Here, we attempted to identify such corresponding genomic regions of high evolutionary importance in two closely related European pines, Pinus mugo and P. uncinata, contrasting them with a reference lowland relative—P. sylvestris. We genotyped 438 samples at thousands of single nucleotide polymorphism (SNP) markers, tested their genetic differentiation and population structure followed by outlier detection and gene ontology annotations. Markers clearly differentiated the species and uncovered patterns of population structure in two of them. In P. uncinata three Pyrenean sites were grouped together, while two outlying populations constituted a separate cluster. In P. sylvestris, Spanish population appeared distinct from the remaining four European sites. Between mountain pines and the reference species, 35 candidate genes for altitude-dependent selection were identified, including such encoding proteins responsible for photosynthesis, photorespiration and cell redox homeostasis, regulation of transcription, and mRNA processing. In comparison between two mountain pines, 75 outlier SNPs were found in proteins involved mainly in the gene expression and metabolism.

We analysed data on nucleotide polymorphism at 78 nuclear genes to search for signatures of divergence and selection in populations from across the distribution of Pinus sylvestris in Europe. Data on variation at a large set of maternally inherited mitochondrial DNA markers were also analysed. Most nuclear loci showed homogeneous patterns of variation across populations from Spain, Central Europe, Nordic countries and Scotland. Mitochondrial markers did not reveal strong population structure but suggested admixture of samples carrying different mitochondrial lineages in certain parts of the distribution. At nuclear DNA loci, we found high nucleotide polymorphism, generally low population differentiation and weak signals of isolation by distance. Together with the mitochondrial DNA results, these patterns seem most likely to be associated with a process of colonization from multiple sources and efficient gene flow. The results are in line with demographic inference indicating establishment of Central European and Nordic populations before the Last Glacial Maximum and admixture of colonization fronts from mainland Europe in Scottish populations. Coalescent and outlier detection methods identified several genes showing molecular signatures of selection especially in response to temperature variation in ecologically and phenotypically divergent populations. The study provides a large set of markers for analysis of genetic diversity in populations of P. sylvestris, including candidate genes that are implicated in local adaptation and population divergence.