Witold Wachowiak

High genetic diversity at the extreme range edge: nucleotide variation at nuclear loci in Scots pine (Pinus sylvestris L.) in Scotland

Nucleotide polymorphism at 12 nuclear loci was studied in Scots pine populations across an environmental gradient in Scotland, to evaluate the impacts of demographic history and selection on genetic diversity. At eight loci, diversity patterns were compared between Scottish and continental European populations. At these loci, a similar level of diversity (θsil=∼0.01) was found in Scottish vs mainland European populations, contrary to expectations for recent colonization, however, less rapid decay of linkage disequilibrium was observed in the former (ρ=0.0086±0.0009, ρ=0.0245±0.0022, respectively). Scottish populations also showed a deficit of rare nucleotide variants (multi-locus Tajimas D=0.316 vs D=−0.379) and differed significantly from mainland populations in allelic frequency and/or haplotype structure at several loci. Within Scotland, western populations showed slightly reduced nucleotide diversity (πtot=0.0068) compared with those from the south and east (0.0079 and 0.0083, respectively) and about three times higher recombination to diversity ratio (ρ/θ=0.71 vs 0.15 and 0.18, respectively). By comparison with results from coalescent simulations, the observed allelic frequency spectrum in the western populations was compatible with a relatively recent bottleneck (0.00175 × 4Ne generations) that reduced the population to about 2% of the present size. However, heterogeneity in the allelic frequency distribution among geographical regions in Scotland suggests that subsequent admixture of populations with different demographic histories may also have played a role.

Substantial heritable variation for susceptibility to Dothistroma septosporum within populations of native British Scots pine (Pinus sylvestris).

The threat from pests and pathogens to native and commercially planted forest trees is unprecedented and expected to increase under climate change. The degree to which forests respond to threats from pathogens depends on their adaptive capacity, which is determined largely by genetically controlled variation in susceptibility of the individual trees within them and the heritability and evolvability of this trait. The most significant current threat to the economically and ecologically important species Scots pine (Pinus sylvestris) is dothistroma needle blight (DNB), caused by the foliar pathogen Dothistroma septosporum. A progeny‐population trial of 4‐year‐old Scots pine trees, comprising six populations from native Caledonian pinewoods each with three to five families in seven blocks, was artificially inoculated using a single isolate of D. septosporum. Susceptibility to D. septosporum, assessed as the percentage of non‐green needles, was measured regularly over a period of 61 days following inoculation, during which plants were maintained in conditions ideal for DNB development (warm; high humidity; high leaf wetness). There were significant differences in susceptibility to D. septosporum among families indicating that variation in this trait is heritable, with high estimates of narrow‐sense heritability (0.38–0.75) and evolvability (genetic coefficient of variation, 23.47). It is concluded that native Scots pine populations contain sufficient genetic diversity to evolve lower susceptibility to D. septosporum through natural selection in response to increased prevalence of this pathogen.

High genetic similarity between Polish and North European Scots pine (Pinus sylvestris L.) populations at nuclear gene loci

Nucleotide polymorphisms in a set of 32 nuclear genes were studied in 19 mountain, peatbog and lowland populations of Scots pine representing known phenotypic races and populations of presumably relict character for the species in Poland. At 29 genes, the pattern of genetic variation was compared to 11 reference populations from Northern, Western and Southern Europe. Similar levels of nucleotide polymorphism and excess of low-frequency mutations were observed in Polish populations (πtot = 0.0055, D = −0.308) and as compared to the reference samples (πtot = 0.0054, D = −0.170). Bayesian assignment and conventional frequency-based statistics indicate that Polish populations share the same genetic background at the analysed nuclear gene markers. However, the populations showed a much closer genetic relationship with North European samples than other regional groups of populations. Across the very uniform genetic background of the populations, we identified several genes with outlier patterns of haplotype, polymorphism frequency variation and departures from compound neutrality tests. Our data indicate that the Central and North European parts of the Scots pine distribution seem particularly suitable for association genetic studies to link phenotypic and genetic variation at a large geographical scale.

Hybridization in contact zone between temperate European pine species

Hybridization studies are important to advance our understanding of the interspecific gene flow and its evolutionary consequences in closely related species. Hybridization and admixture patterns were assessed in a contact zone and reference populations of European pine species using sequence data from 26 nuclear genes and a species-diagnostic cpDNA marker. Reference populations formed three distinct genetic clusters comprising Pinus sylvestris, Pinus mugo/Pinus uliginosa, and Pinus uncinata. Evidence of population structure was found only in P. uliginosa. Based on phenotypic characteristics and molecular data, we identified five groups of individuals in the contact zone in Poland, comprising forms of the parental species and intermediates that were most probably the result of interspecific crosses. A combination of nuclear gene sequence data and a diagnostic organelle marker were used to show that hybridization is frequent in the contact zone and results in hybrid trees with distinct phenotypic identity. The influence of selection in maintaining hybrid phenotypes in environments unsuited to parental species was inferred from nucleotide polymorphism data. A lack of admixture in reference populations suggests that hybridization has not occurred during post-glacial migration and so the contact zone represents a distinct, active example of ongoing evolution. Pine populations in this zone will be a valuable system for studying the genetic basis of hybrid advantage in environmental conditions untypical of pure parental species.

Different patterns of genetic structure of relict and isolated populations of endangered peat-bog pine (Pinus uliginosa Neumann)

Recent changes in environmental conditions in populations of peat-bog pine (Pinus uliginosa Neumann) caused rapid decline or even extinction of the species in several stands in Central Europe. Conservation strategies forP. uliginosa require information about the evolutionary history and genetic structure of its populations. Using isozymes we assessed the genetic structure ofP. uliginosa from four isolated stands in Poland and compared the results to genetic structures of other closely related pine species including eight populations ofPinus mugo, ten ofPinus sylvestris and one ofPinus uncinata. The level of genetic variability ofP. uliginosa measured by the mean number of alleles per locus and average heterozygosity was similar to others related toP. uliginosa taxa from the reference group but it differs among populations. High genetic similarity was found between two populations ofP. uliginosa from Low Silesian Pinewood. The populations were genetically distinct as compared to other populations includinglocus classicus of the species from the peat bog at Batorów Reserve. Very low genetic distance (Dn = 0.002) and small genetic differentiation (GST = 0.003) were found betweenP. uliginosa andP. mugo in the sympatric populations of the species from Zieleniec peat bog suggesting the ongoing natural hybridisation and genetic contamination of peat-bog pine from this area. Some evidence for skew in allele frequency distribution potentially due to recent bottleneck was found in population from Low Silesian Pinewood. The analysed open pollinated progeny derived from twoP. uliginosa stands from Low Silesian Pine-wood showed the excess of homozygotes as compared to the maternal trees indicating high level of inbreeding (F = 0.105,F = 0.081). The results are discussed in the context of evolution ofP. uliginosa populations, taxonomic relationships between the analysed species and conservation strategies for active protection of peat-bog pine.

Contrasting patterns of genetic variation in core and peripheral populations of highly outcrossing and wind pollinated forest tree species

This study of nuclear microsatellite loci demonstrates genetic relationships between twenty four Scots pine populations from Europe and Asia Minor. The analysed populations were assigned to several groups that corresponded to the geographical regions of their occurrence. Significant differentiation over short geographical distances was observed between isolated populations within the Iberian and Anatolian Peninsulas (Asia Minor), which contrasted with the absence of genetic differentiation between distant populations, for instance in central and northern Europe. Despite the signs of isolation found in peripheral stands, a similar level of genetic variation and no evidence of a recent bottleneck was found across the populations.

Interspecific gene flow and ecological selection in a pine (Pinus sp.) contact zone

Nucleotide polymorphisms in a set of nuclear genes were studied in a sympatric population of pines Pinus mugo and Pinus sylvestris that includes trees classified as pure species and polycormic (multi-stemmed) individuals of potentially hybrid origin. Patterns of genetic diversity were compared between those groups of samples and to the reference allopatric populations of the species in Europe. Polymorphisms at the gene loci clearly distinguished pure parental species as measured by conventional frequency-based statistics and Bayesian assignment of samples into separate genetic clusters. Most individuals classified based on phenotypic assessments as putative hybrids were genetically very similar to P. mugo showing no existing average net divergence and genetic assignment to the same genetic cluster. On the other hand, individuals of P. sylvestris showed homogenous genetic background to the reference populations of the species from Central and Northern Europe. Ten individuals of admixed genetic composition were found in all three groups of samples; however, the majority of hybrids except one individual were identified across the samples classified as P. mugo and polycormic pines. Those trees that contained a mixture of nuclear gene haplotypes observed in the reference populations of pure species and cpDNA from P. mugo, most likely represent the first generation of hybrids. Analysis of the allelic frequency spectra and compound neutrality tests identified deviations from neutrality at several genes. This contact zone seems suitable for selection of a mapping population both in hybrid and parental species for admixture mapping to effectively search for polymorphisms that may play role in species adaptive variation and speciation.

Utility of closely related taxa for genetic studies of adaptive variation and speciation: Current state and perspectives in plants with focus on forest tree species

Studies of adaptation and speciation have greatly benefited from rapid progress of DNA sequencing and genotyping technologies. Comparative genomics of closely related taxa has great potential to advance evolutionary research on genetic architecture of adaptive traits and reproductive isolation. Such studies that utilized closely related plant species and ecotypes have already provided some important insights into genomic regions and/or genes that are potentially involved in local adaptation and speciation. The choice of an appropriate species model for such research is crucial. The paper discusses current approaches used to reveal the patterns of intra‐ and interspecific divergence due to natural selection. Its outcomes in herbaceous plants and forest trees are briefly summarized and compared to reveal general regularities concerning evolutionary processes. We then highlight the importance of multispecies studies and discuss the utility of several related pine taxa as fine candidates for evolutionary inferences. Genetically similar but ecologically and phenotypically diverged taxa seem a promising study system to search for genomic patterns of speciation and adaptive variation.

Reconstructing the plant mitochondrial genome for marker discovery: a case study using Pinus

Whole‐genome‐shotgun (WGS) sequencing of total genomic DNA was used to recover ~1 Mbp of novel mitochondrial (mtDNA) sequence from Pinus sylvestris (L.) and three members of the closely related Pinus mugo species complex. DNA was extracted from megagametophyte tissue from six mother trees from locations across Europe, and 100‐bp paired‐end sequencing was performed on the Illumina HiSeq platform. Candidate mtDNA sequences were identified by their size and coverage characteristics, and by comparison with published plant mitochondrial genomes. Novel variants were identified, and primers targeting these loci were trialled on a set of 28 individuals from across Europe. In total, 31 SNP loci were successfully resequenced, characterizing 15 unique haplotypes. This approach offers a cost‐effective means of developing marker resources for mitochondrial genomes in other plant species where reference sequences are unavailable.

Nuclear microsatellite markers reveal the low genetic structure of Pinus mugo Turra (dwarf mountain pine) populations in Europe

Twenty-one populations (555 individuals) covering the entire native range of Pinus mugo Turra (dwarf mountain pine) were investigated for genetic variation scored at 13 nuclear microsatellite markers (nSSRs). The main objective of the present study was to determine the genetic structure across the present distribution of the species and locate populations of different genetic compositions. Most of the genetic variation was observed within the populations (95%). The assignment of populations based on Bayesian clustering methods revealed that the Sudeten populations of P. mugo form a separate genetic cluster. These stands have likely been established through the founder effects of Alpine migrants. The distribution and level of SSR polymorphisms, along with no evidence of isolation by distance or phylogeographic structure, indicate that the present populations of P. mugo have diverged relatively recently and originate from a larger glacial distribution of the species. One peripheral stand from Italy had the lowest values of most calculated genetic variation indices. This stand could therefore be more susceptible to genetic drift and a negative impact of predicted environmental changes. We discuss our findings with respect to previously published results on the genetic and morphological variation of P. mugo and with consideration for the conservation genetics of the species.