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