Speciation history of three closely related pines Pinus mugo (T.), P. uliginosa (N.) and P. sylvestris (L.)

Nucleotide polymorphisms at genomic regions including 17 nuclear loci, two chloroplast and one mitochondrial DNA fragments were used to study the speciation history of three pine species: dwarf mountain pine (Pinus mugo), peat‐bog pine (P. uliginosa) and Scots pine (P. sylvestris). We set out to investigate three specific speciation scenarios: (I) P. uliginosa is a homoploid hybrid between the other two, (II) the species have evolved without gene flow after divergence and (III) there has been substantial gene flow between the species since their divergence. Overall, the genetic data suggest that P. mugo and P. uliginosa share the same gene pool (average net divergence of 0.0001) and that the phenotypic differences (e.g. growth form) are most likely due to very limited areas of the genome. P. mugo and P. uliginosa are more diverged from P. sylvestris than from each other (average net divergence of 0.0027 and 0.0026, respectively). The nucleotide patterns can best be explained by the divergence with migration speciation scenario, although the hybrid speciation scenario with small genomic contribution from P. sylvestris cannot be completely ruled out. We suggest that the large amount of shared polymorphisms between the pine taxa and the lack of monophyly at all loci studied between P. sylvestris and P. mugo–P. uliginosa can largely be explained by relatively recent speciation history and large effective population sizes but also by interspecific gene flow. These closely related pine taxa form an excellent system for searching for loci involved in adaptive variation as they are differentiated in phenotype and ecology but have very similar genetic background.     

Hybridisation processes in sympatric populations of pines Pinus sylvestris L., P. mugo Turra and P. uliginosa Neumann

Natural hybridisation was postulated between the closely related pine species Pinus sylvestris and the P. mugo complex, however no clear evidence on propagation of mature hybrids in nature has been documented so far. To test the hybridisation hypothesis we applied chloroplast DNA (cpDNA) markers and isozymes in the analyses of 300 individuals representing the variety of morphological forms in the sympatric populations of P. sylvestris, P. mugo and P. uliginosa at the peat bog complex in the Sudety Mts., Poland. Additionally, the haplotypes of paternally inherited cpDNA of 149 open pollinated progeny derived from seeds were compared to the haplotypes of parental trees to access the intensity and direction of contemporary hybridisation. The morphologically highly variable polycormic (multi-stemmed) hybrids between P. mugo and P. uliginosa were identified. The second group of hybrids was found among the monocormic (single-stemmed) P. sylvestris-like individuals carrying the cpDNA from P. mugo complex. Hybrids of P. sylvestris as a pollen donor and P. mugo or P. uliginosa as a mother were not found, either in the group of examined trees, or among the open pollinated progeny. The results indicate that numerous hybrids can exist in the sympatric population of the species studied and that gene flow can successfully proceed from P. mugo complex to P. sylvestris. Hybridisation and ecological selection seems to play a significant role in diversification and evolution of the investigated species.     

Allozyme investigations on the genetic differentiation between closely related pines —Pinus sylvestris, P. mugo, P. uncinata, andP. uliginosa (Pinaceae)

In eight natural European populations of four closely related taxa of pines (Pinus sylvestris, P. mugo, P. uncinata, andP. uliginosa) starch-gel electrophoreses revealed altogether 58 alleles at 15 loci from nine enzyme systems. With Nei's genetic distance (D) the largest mean genetic distance (D = 0.171) was found betweenP. sylvestris andP. mugo, a distance corresponding to that between other closely related pine species. Mean genetic distances between the other taxa were less than half that value and characteristic for subspecies or varieties rather than for species. On the basis of our results we suggest that both,P. uliginosa andP. uncinata, could be the result of the ancient hybridization betweenP. sylvestris andP. mugo.     

Contribution to the taxonomy of Pinus uncinata (Pinaceae) based on cone characters

Eight natural populations of Pinus uncinata Ramond from the Spanish Pyrenees, five of P. mugo Turra from the Carpathians, the Sudethians and Abruzzian Apennines, three of P. sylvestris L. from the Central-European Lowland and the Iberian Peninsula and one of P. uliginosa Neumann from SW Poland have been compared biometrically in 16 cone characters. P. uncinata appears the most distant from all other taxa, differing by having the largest and most asymmetric cones and the thickest apophyses with the most prominent umbo. In cone characters, P. mugo is more similar to P. sylvestris than to P. uncinata. Nevertheless, P. mugo and P. sylvestris remain distant from each other in a number of morphological characters examined. P. uliginosa has cones intermediate between the other three taxa and is moreover very variable. The locality of P. uliginosa in the Silesian Lowland may be of a relict character and conserve genes from P. uncinata.     

Volatiles as Chemosystematic Markers for Distinguishing Closely Related Species within the Pinus mugo Complex

Headspace solid‐phase microextraction (HS‐SPME) coupled to GC/MS analysis was used to identify the constituents of pine‐needle volatiles differentiating three closely‐related pine species within the Pinus mugo complex, i.e., P. uncinata Ramond ex DC., P. uliginosa G.E.Neumann ex Wimm ., and P. mugo Turra . Moreover, chemosystematic markers were proposed for the three analyzed pine species. The major constituents of the pine‐needle volatiles were α‐pinene (28.4%) and bornyl acetate (10.8%) for P. uncinata, δ‐car‐3‐ene (21.5%) and α‐pinene (16.1%) for P. uliginosa, and α‐pinene (20%) and δ‐car‐3‐ene (18.1%) for P. mugo. This study is the first report on the application of the composition of pine‐needle volatiles for the reliable identification of closely‐related pine species within the Pinus mugo complex.     

Genetic Variation and Differentiation of Peat-Bog and Dry-Meadow Populations of the Dwarf Mountain Pine Pinus mugo Turra in the Highlands of the Ukrainian Carpathians

Based on electrophoretic analysis of 21 isozyme loci controlling 10 enzyme systems, the intra- and interpopulation variation was studied in two peat-bog and three dry-meadow populations of the dwarf mountain pine Pinus mugoTurra from the highlands of the Ukrainian Carpathians. In the studied samples (a total of 164 trees), on average 62% of the studied genes were polymorphic; the mean heterozygosity was 21.3%. The dry-meadow populations differed from the peat-bog populations by allele and genotype diversity and by heterozygosity although the indices characterizing population heterogeneity (F _st and G _st) were small (0.027 and 0.032, respectively). Nei's genetic distances between the populations ranged of 0.011 to 0.032 with the mean of 0.018.     

Geographical patterns of nucleotide diversity and population differentiation in three closely related European pine species in the Pinus mugo complex

Nucleotide polymorphism at 12 nuclear loci and two mitochondrial gene fragments was studied in three closely related pine species from the Pinus mugo complex in populations across the species distributional range in Europe. Despite large differences in the census sizes of the populations, high and similar levels of nucleotide diversity (θ_sil = ～0.013–0.017) were found at nuclear loci in the three pine species. More rapid decay of overall linkage disequilibrium (LD) and recombination to diversity ratio (ρ/θ) was observed across the species distributional range in P. mugo (ρ = 0.0369 ± 0.0028; ρ/θ = ～2.2) than in P. uncinata (ρ = 0.0054 ± 0.0011; ρ/θ = ～0.4) and P. uliginosa (ρ = 0.0051 ± 0.0010, ρ/θ = ～0.4). However, regional groups of P. mugo showed similar levels of LD and ρ/θ ratio to the other species. An excess of rare nucleotide variants was found in P. mugo at four loci, but, overall, the allelic frequency spectrum in the three species did not deviate significantly from neutrality (multilocus Tajima's D = ?0.681, D = ?0.118 and D = ?0.266, P > 0.05, respectively). Bayesian clustering methods showed no clear correspondence of clusters to species or geographical regions. Some differences between populations and species were found in a hierarchical analysis of molecular variance (AMOVA) and in the distribution of the mitochondrial DNA haplotypes, suggesting rather limited gene flow between the taxa and ongoing divergence. As all three pine taxa have similar genetic backgrounds, they form an excellent system for searching for loci involved in adaptive variation and speciation. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 225–238.     

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.     

Nuclear microsatellite markers reveal the low genetic structure of <Emphasis Type="Italic">Pinus mugo</Emphasis> 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.     

Population history of European mountain pines Pinus mugo and Pinus uncinata revealed by mitochondrial DNA markers

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.     

Scots pine growing on forested mires in Abernethy Forest,Strathspey, Scotland

Summary

A substantial proportion of the Abernethy Forest Reserve has Scots pine (Pinus sylvestris) growing on the surfaces of a variety of mires. The hydrology of the mires has been affected by drainage and peat cutting but this area is unusual in having had a long period of protection from grazing by domestic stock. There are three main types of pine populations found on these mires. Woodland bog comprises predominantly bog vegetation with abundant pine seedlings due to the heavy seed rain from the surrounding woodland. Only a few very small trees survive, which are stunted, heavily diseased and have very low seed production. Wooded bog also comprises predominately bog vegetation but there are scattered mature trees of a moderate height with an open canopy. The trees are fertile and can form uneven aged stands with regeneration. Bog woodland is a predominantly woodland vegetation with tall, dense tree cover on deep peat. The trees are well grown with a dense canopy. A few remnants of bog vegetation remain in the ground flora although most have been replaced by woodland bryophytes and shrubs. Each of these three types is described and their development is discussed.     

Geography determines genetic relationships between species of mountain pine (Pinus mugo complex) in western Europe

Aim Our aims were to test whether morphological species of mountain pines were genetically supported in the western part of the distribution range of the Pinus mugo species complex (Pinus mugo Turra sensu lato), to resolve genetically homogeneous clusters of populations, to determine historical demographic processes, and to assess the potential hybridization of mountain pines with Scots pine, Pinus sylvestris L. Location Populations were sampled in the Iberian System, the Pyrenees, the French Mont Ventoux, Vosges and Jura mountains, the German Black Forest and throughout the Alps. This corresponded to a range‐wide sampling for mountain pine sensu stricto (Pinus uncinata Ram.) and to a sampling of the western parts of the ranges of dwarf mountain pine (Pinus mugo Turra sensu stricto) and bog pine/peatbog pine [Pinus rotundata Link/Pinus × pseudopumilio (Willk.) Beck]. Methods In total, 786 individuals of P. mugo sensu lato from 29 natural populations, and 85 individuals of P. sylvestris from four natural populations were genotyped at three chloroplast microsatellites (cpSSRs). Populations were characterized for standard genetic diversity statistics and signs of demographic expansion. Genetic structure was explored using analysis of molecular variance, differentiation statistics and Bayesian analysis of population structure (BAPS). Results One hundred haplotypes were identified in P. mugo sensu lato. There was a stronger differentiation between geographical regions than between morphologically identified taxa (P. mugo sensu stricto, P. uncinata and P. rotundata/P. ×pseudopumilio). Overall genetic differentiation was weak (G_ST = 0.070) and displayed a clear phylogeographic structure [N_ST = 0.263, N_ST > N_ST (permuted), P < 0.001]. BAPS identified a Pyrenean and an Alpine gene pool, along with several smaller genetic clusters corresponding to peripheral populations. Main conclusions The core regions of the Pyrenees and Alps were probably recolonized, respectively by P. uncinata and P. uncinata/P. mugo sensu stricto, from multiple glacial refugia that were well connected by pollen flow within the mountain chains. Pinus rotundata/P. × pseudopumilio populations from the Black Forest, Vosges and Jura mountains were probably recolonized from various glacial populations that kept their genetic distinctiveness despite late glacial and early Holocene expansion. Marginal P. uncinata populations from the Iberian System are compatible with elevational shifts and long‐term isolation. The causes of haplotype sharing between P. mugo sensu lato and P. sylvestris require further research.     

Blaue,rote und gelbgrüne männlichePinus-Blüten

Differences in colours of male strobili, originally encountered in races ofPinus mugo, are found among other pine species too: Species of subsect.Cembrae flower bright red, whereas in subsect.Cembroides yellowgreen is dominant. Recently, blue and grey-blue male flowers have been discovered inP. engelmannii from Mexico,P. palustris, P. elliotti andP. densa from Florida. These flower colour differences apparently reflect some species relationships.

     

Molecular signatures of divergence and selection in closely related pine taxa

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.     

Cross-species amplification and characterization of microsatellite loci in Pinus mugo Turra

Pinus mugo (dwarf mountain pine) is an important component of European mountain ecosystems. However, little is known about the present genetic structure and population differentiation of this species at the DNA level, possibly due to a lack of nuclear microsatellite markers (SSR) developed for Pinus mugo. Therefore in this study we transferred microsatellite markers originally developed for Pinus sylvestris and Pinus taeda to Pinus mugo. This cross-species amplification approach is much faster and less expensive than isolation and characterization of new microsatellite markers. The transfer rates from the source species to Pinus mugo were moderately low (26%). There were no differences in microsatellite repeat motifs between the source species and Pinus mugo. Nuclear microsatellite markers successfully transferred to Pinus mugo can be applied to various genetic studies on this species, due to the high level of their polymorphism and high value of polymorphic information content.     

Differentiation in adaptive traits between neighbouring bog and mineral soil populations of Scots pine Pinus sylvestris

I used a reciprocal sowing experiment in the field to reveal differentiation in adaptive traits between two neighbouring northern populations of Scots pine Pinus sylvestris. 1 compared a peat bog population with a mineral soil population. Seedling survival was monitored during seven growing seasons and the plants were then harvested to obtain data on pine traits associated with growth and resource allocation, i.e. height, needle length, total dry weight, relative growth, proportion root and proportion needles. Seeds from the peat bog populations had lower germination capacity and were smaller than those from the mineral soil population. Despite their smaller size, the seeds from the bog population were superior for establishment of pines on the bog. On the mineral soil, the traits were strikingly similar in the two pine populations. In contrast, the traits were more variable and differentiated on the bog. Here, the native bog pines grew faster and had a larger proportion root than those originating from the population on the adjacent mineral soil. It is suggested that the differentiation between peat bog populations and mineral soil populations might represent a major direction of differentiation in northern Scots pine populations.     

Molecular and paleo-climatic data uncover the impact of an ancient bottleneck on the demographic history and contemporary genetic structure of endangered Pinus uliginosa

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.     

A critical evaluation of reproductive barriers between closely related species using DNA markers - a case study in Pinus

Former controlled crosses between twelve Pinus montana var. rostrata (Pinus mugo complex) and eight P. sylvestris clones revealed that only two P. sylvestris had efficiently fertilised P. montana. Two species-diagnostic chloroplast DNA markers were applied to verify the species purity of the parental clones. All maternal P. montana were unambiguously confirmed to belong to the P. mugo complex at both chloroplast DNA marker loci. Six P. sylvestris clones carried the `sylvestris' haplotypes. However, the same two P. sylvestris clones that had efficiently fertilised P. montana displayed the chloroplast haplotypes diagnostic to the P. mugo complex. The patterns of highly polymorphic cpDNA microsatellite markers in parents and offspring ruled out contamination by foreign pollen. We concluded that the two clones successful in the crosses represent fertile hybrids between the two species with P. mugo as the pollen donor. Consequently, DNA markers are proposed for verifying or falsifying the success of artificial fertilisation in general. The existence of crossing barriers between the two Pinus species, meaningful to the postulated natural hybridisation and the evolution of their populations in sympatric stands, was indicated and is newly discussed.     

Genetic variability of natural populations and man-made stands of common pine (Pinus sylvestris L.) in Kremenets Hills and Maloe Polese

Comparative analysis of 19 allozyme loci of Pinus sylvestris L was performed on three relic populations and six uneven-aged man-made stands to evaluate genetic variability of common pine in Kremenets Hills and Maloye Polesye. Low heterozygosity was characteristic of all natural populations and of four forest stands (H_o = 0.182–0.205), whereas in two forest stands the level of heterozygosity was noticeably higher (H_o = 0.245–0.300). Both allelic and genotypic variability of natural populations was reproducible in the man-made forest. However, the genetic distance between forest stands and natural populations was considerably higher than that among three natural populations (D_Nav = 0.014 and D_Nav = 0.003, respectively).     

Early phenology and growth trait variation in closely related European pine species

Closely related taxa occupying different environments are valuable systems for studying evolution. In this study, we examined differences in early phenology (bud set, bud burst) and early growth in a common garden trial of closely related pine species: Pinus sylvestris, P. mugo, and P. uncinata. Seeds for the trial were sourced from populations across the ranges of each species in Europe. Over first 4 years of development, clear differences were observed between species, while the most significant intraspecific differentiation was observed among plants from P. sylvestris populations from continental European locations. Trait differences within P. sylvestris were highly correlated with altitude and latitude of the site of origin. Meanwhile, P. mugo populations from the Carpathians had the earliest bud set and bud flush compared to other populations of the species. Overall, populations from the P. mugo complex from heterogeneous mountain environments and P. sylvestris from the Scottish Highlands showed the highest within‐population variation for the focal traits. Although the three species have been shown to be genetically highly similar, this study reveals large differences in key adaptive traits both among and within species.