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

Cross-amplification and multiplexing of cpSSRs and nSSRs in two closely related pine species (Pinus sylvestris L. and P. mugo Turra)

Background: Simple sequence repeats (SSRs) are widespread molecular markers commonly used in population genetic studies. Nowadays, next-generation sequencing (NGS) methods allow identifying thousands of SSRs in one sequencing run, which greatly facilitates isolation and development of new SSRs. However, their usefulness as molecular markers still must be tested empirically on a number of populations to select SSRs with best parameters for future population genetic research. An alternative approach, cheaper and faster than isolation and characterization of new SSRs, involves cross-amplification of SSRs in closely related species. Aims: Our goal was to develop multiplex PCR protocols that will be useful in population genetic studies of Scots pine (Pinus sylvestris L.) and dwarf mountain pine (P. mugo Turra), and possibly other pine species. Methods: We tested 14 chloroplast (cpSSRs) and 22 nuclear (nSSRs) microsatellite markers originally designed for Japanese black pine (P. thunbergii Parl.), P. sylvestris and loblolly pine (P. taeda L.) in four populations of P. sylvestris and P. mugo across different locations in Europe. We designed six multiplex PCRs, which were subsequently screened for their ability to provide repeatable and high quality amplification products using capillary electrophoresis. Results: The transfer rate in our study was similar in both pine species, and it was very high for cpSSRs (93% and 86% for P. sylvestris and P. mugo, respectively) and moderate for nSSRs (59% for both species). We managed to design five well-performing multiplex reactions out of six initially tested. Most of the tested loci were polymorphic. Moreover, the allelic patterns detected at some cpSSRs were species-specific. Conclusions: We provide a set of five multiplexes which can be used in genetic studies of both P. sylvestris and P. mugo. Chloroplast marker PCP30277 is a good candidate for a cheap species diagnostic marker suitable for tracking interspecific gene flow between hybridizing species of P. sylvestris and P. mugo

Genetic variation of Scots pine (Pinus sylvestris L.) in Eurasia: impact of postglacial recolonization and human-mediated gene transfer

Key message: The range-wide level of genetic variation of Scots pine (Pinus sylvestris L.) is geographically structured. High admixture and low genetic structure of populations in Central Europe and Fennoscandia suggest past recolonization from multiple sources and the influence of human-mediated gene transfer. Gene pools of marginal and isolated stands require active conservation. Some areas of Scots pine distribution need further genetic studies. Context: Scots pine (Pinus sylvestris L.) seems to be a species of low conservation priority because it has a very wide Eurasian distribution and plays a leading role in many forest tree breeding programs. Nevertheless, considering its economic value, long breeding history, range fragmentation, and increased mortality, which is also projected in the future, it requires a more detailed description of its genetic resources. Aims: Our goal was to compare patterns of genetic variation found in biparentally inherited nuclear DNA with previous research carried out with mitochondrial and chloroplast DNA due to their different modes of transmission. Methods: We analyzed the genetic variation and relationships of 60 populations across the distribution of Scots pine in Eurasia (1262 individuals) using a set of nuclear DNA markers. Results: We confirmed the high genetic variation and low genetic differentiation of Scots pine spanning large geographical areas. Nevertheless, there was a clear division between European and Asian gene pools. The genetic variation of Asian populations was lower than in Europe. Spain, Turkey, and the Apennines constituted separate gene pools, the latter showing the lowest values of all genetic variation parameters. The analyses showed that most populations experienced genetic bottlenecks in the distant past. Ongoing admixture was found in Fennoscandia. Conclusions: Our results suggest a much simpler recolonization history of the Asian than European part of the Scots pine distribution, with migration from limited sources and possible founder effects. Eastern European stands seem to have descended from the Urals refugium. It appears that Central Europe and Fennoscandia share at least one glacial refugium in the Balkans and migrants from higher latitudes, as well as from south-eastern regions. The low genetic structure between Central Europe and Fennoscandia, along with their high genetic admixture, may result at least partially from past human activities related to the transfer of germplasm in the nineteenth and early twentieth centuries. In light of ongoing climate changes and projected range shifts of Scots pine, conservation strategies are especially needed for marginal and isolated stands of this species. Genetic research should also be complemented in parts of the species distribution that have thus far been poorly studied. © 2023, Institut National de Recherche en Agriculture, Alimentation et Environnement (INRAE)

Genetic Consequences of Hybridization in Relict Isolated Trees Pinus sylvestris and the Pinus mugo Complex

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