Scots pine - panmixia and the elusive signal of genetic adaptation.

Scots pine is the foundation species of diverse forested ecosystems across Eurasia and displays remarkable ecological breadth, occurring in environments ranging from temperate rainforests to arid tundra margins. Such expansive distributions can be favored by various demographic and adaptive processes and the interactions between them. To understand the impact of neutral and selective forces on genetic structure in Scots pine, we conducted range-wide population genetic analyses on 2321 trees from 202 populations using genotyping-by-sequencing, reconstructed the recent demography of the species and examined signals of genetic adaptation. We found a high and uniform genetic diversity across the entire range (global FST 0.048), no increased genetic load in expanding populations and minor impact of the last glacial maximum on historical population sizes. Genetic-environmental associations identified only a handful of single-nucleotide polymorphisms significantly linked to environmental gradients. The results suggest that extensive gene flow is predominantly responsible for the observed genetic patterns in Scots pine. The apparent missing signal of genetic adaptation is likely attributed to the intricate genetic architecture controlling adaptation to multi-dimensional environments. The panmixia metapopulation of Scots pine offers a good study system for further exploration into how genetic adaptation and plasticity evolve under gene flow and changing environment.

Sequence Capture of Mitochondrial Genome with PCR-Generated Baits Provides New Insights into the Biogeography of the Genus Abies Mill.

Mitochondrial DNA (mtDNA), being maternally inherited in plants of the family Pinaceae, is an important source of phylogeographic information. However, its use is hindered by a low mutation rate and frequent structure rearrangements. In the present study, we tested the method of genomic libraries enrichment with mtDNA via the sequence capture method yielding mtDNA data which were further used to reconstruct the phylogenetic tree of the genus Abies. The baits for hybrid capture were obtained by long-range PCR using primers designed on the basis of the assembly of Abies sibirica Ledeb. mitochondrial genome. Mitochondrial genomes of Picea sitchensis (Bong.) Carr., Larix sibirica Ledeb., and Keteleeria davidiana (Bertrand) Beissn. were used as an outgroup. The resulting phylogenetic tree consists of two sister branches, including the Eurasian and American species, respectively, with some exceptions. The subclade of A. sachalinensis (F. Schmidt) Mast. and A. veitchii Lindl. (Japan and Sakhalin islands) occupies a basal position in the branch of American firs, probably due to the complex history of fir migrations from North America to Eurasia. The tree has high support for majority of clades. For species represented by more than one sample an intraspecific variability was found which is suitable to design mtDNA markers for phylogeographic and population studies.

Genetic structure of a widespread alpine shrub Rhododendron aureum (Ericaceae) across East Asia.

The vast territory of East Asia, including southwestern Beringia, is considered to have been almost ice free during the Pleistocene. Cold-resistant flora may have persisted in this region expanding or contracting its range during the climate cooling. Only a few plant genera have been studied with a sampling area across their entire geographic range in East Asia; therefore, the understanding of the biogeographic history of alpine flora in this region remains limited. In the present study, genetic variation and population structure in 21 populations of the alpine shrub Rhododendron aureum across its range in East Asia were assessed using 18 microsatellite loci. Phylogenetic analyses revealed three main genetic groups: Siberia, Northeast, and North Pacific. According to the geographical pattern of genetic diversity, the North Pacific group includes populations from Kamchatka, south of Russian Far East, and territories close to central Japan. This group is the most diverse and likely diverged earlier than the Siberia and Northeast groups. Ecological niche modeling predicts range expansion of this species during the period of cooling and, together with demographic history, suggests that the divergence between the three main genetic groups predated the Last Glacial Maximum. Similar to other cold-resistant species such as Larix sibirica and Juniperus communis, the pattern of genetic diversity of R. aureum supports the survival of the species at high latitudes during the Pleistocene with limited contribution of the southern populations to expansion of the species range to the Northeast region and Siberia.

From America to Eurasia: a multigenomes history of the genus Abies.

The origin of conifer genera, the main components of mountain temperate and boreal forests, was deemed to arise in the Mesozoic, although paleontological records and molecular data point to a recent diversification, presumably related to Neogene cooling. The geographical area(s) where the modern lines of conifers emerged remains uncertain, as is the sequence of events leading to their present distribution. To gain further insights into the biogeography of firs (Abies), we conducted phylogenetic analyses of chloroplast, mitochondrial and nuclear markers. The species tree, generated from ten single-copy nuclear genes, yielded probably the best phylogenetic hypothesis available for Abies. The tree obtained from five regions of chloroplast DNA largely corresponded to the nuclear species tree. Ancestral area reconstructions based on fossil calibrated chloroplast DNA and nuclear DNA trees pointed to repeated intercontinental migrations. The mitochondrial DNA haplotype tree, however, disagreed with nuclear and chloroplast DNA trees. It consisted of two clusters: one included mainly American haplotypes, while the other was composed of only Eurasian haplotypes. Presumably, this conflict is due to inter-continental migrations and introgressive hybridization, accompanied by the capture of the mitotypes from aboriginal species by the invading firs. Given that several species inhabiting Northeastern Asia carry American mitotypes and mutations typical for the American cluster, whereas no Asian mitotypes were detected within the American species, we hypothesize that Abies migrated from America to Eurasia, but not in the opposite direction. The direction and age of intercontinental migrations in firs are congruent with other conifers, such as spruces and pines of subsection Strobus, suggesting that these events had the same cause.

Multispecies genetic structure and hybridization in the Betula genus across Eurasia.

Boreal and cool temperate forests are the major land cover of northern Eurasia, and information about continental-scale genetic structure and past demographic history of forest species is important from an evolutionary perspective and has conservation implications. However, although many population genetic studies of forest tree species have been conducted in Europe or Eastern Asia, continental-scale genetic structure and past demographic history remain poorly known. Here, we focus on the birch genus Betula, which is commonly distributed in boreal and cool temperate forests, and examine 129 populations of two tetraploid and four diploid species collected from Iceland to Japan. All individuals were genotyped at seven to 18 nuclear simple sequence repeats (nSSRs). Pairwise FST' among the six species ranged from 0.285 to 0.903, and genetic differentiation among them was clear. structure analysis suggested that Betula pubescens is an allotetraploid and one of the parental species was Betula pendula. In both species pairs of B. pendula and B. plathyphylla, and B. pubescens and B. ermanii, genetic diversity was highest in central Siberia. A hybrid zone was detected around Lake Baikal for eastern and western species pairs regardless of ploidy level. Approximate Bayesian computation suggested that the divergence of B. pendula and B. platyphylla occurred around the beginning of the last ice age (36 300 years BP, 95% CI: 15 330-92 700) and hybridization between them was inferred to have occurred after the last glacial maximum (1614 years BP, 95% CI: 561-4710), with B. pendula providing a higher contribution to hybrids.

The extent and meaning of hybridization and introgression between Siberian spruce (Picea obovata) and Norway spruce (Picea abies): cryptic refugia as stepping stones to the west?

Boreal species were repeatedly exposed to ice ages and went through cycles of contraction and expansion while sister species alternated periods of contact and isolation. The resulting genetic structure is consequently complex, and demographic inferences are intrinsically challenging. The range of Norway spruce (Picea abies) and Siberian spruce (Picea obovata) covers most of northern Eurasia; yet their geographical limits and histories remain poorly understood. To delineate the hybrid zone between the two species and reconstruct their joint demographic history, we analysed variation at nuclear SSR and mitochondrial DNA in 102 and 88 populations, respectively. The dynamics of the hybrid zone was analysed with approximate Bayesian computation (ABC) followed by posterior predictive structure plot reconstruction and the presence of barriers across the range tested with estimated effective migration surfaces. To estimate the divergence time between the two species, nuclear sequences from two well-separated populations of each species were analysed with ABC. Two main barriers divide the range of the two species: one corresponds to the hybrid zone between them, and the other separates the southern and northern domains of Norway spruce. The hybrid zone is centred on the Urals, but the genetic impact of Siberian spruce extends further west. The joint distribution of mitochondrial and nuclear variation indicates an introgression of mitochondrial DNA from Norway spruce into Siberian spruce. Overall, our data reveal a demographic history where the two species interacted frequently and where migrants originating from the Urals and the West Siberian Plain recolonized northern Russia and Scandinavia using scattered refugial populations of Norway spruce as stepping stones towards the west.

Clinal variation at phenology-related genes in spruce: parallel evolution in FTL2 and Gigantea?

Parallel clines in different species, or in different geographical regions of the same species, are an important source of information on the genetic basis of local adaptation. We recently detected latitudinal clines in SNPs frequencies and gene expression of candidate genes for growth cessation in Scandinavian populations of Norway spruce (Picea abies). Here we test whether the same clines are also present in Siberian spruce (P. obovata), a close relative of Norway spruce with a different Quaternary history. We sequenced nine candidate genes and 27 control loci and genotyped 14 SSR loci in six populations of P. obovata located along the Yenisei river from latitude 56°N to latitude 67°N. In contrast to Scandinavian Norway spruce that both departs from the standard neutral model (SNM) and shows a clear population structure, Siberian spruce populations along the Yenisei do not depart from the SNM and are genetically unstructured. Nonetheless, as in Norway spruce, growth cessation is significantly clinal. Polymorphisms in photoperiodic (FTL2) and circadian clock (Gigantea, GI, PRR3) genes also show significant clinal variation and/or evidence of local selection. In GI, one of the variants is the same as in Norway spruce. Finally, a strong cline in gene expression is observed for FTL2, but not for GI. These results, together with recent physiological studies, confirm the key role played by FTL2 and circadian clock genes in the control of growth cessation in spruce species and suggest the presence of parallel adaptation in these two species.

Southern montane populations did not contribute to the recolonization of West Siberian Plain by Siberian larch (Larix sibirica): a range-wide analysis of cytoplasmic markers.

While many species were confined to southern latitudes during the last glaciations, there has lately been mounting evidence that some of the most cold-tolerant species were actually able to survive close to the ice sheets. The contribution of these higher latitude outposts to the main recolonization thrust remains, however, untested. In the present study, we use the first range-wide survey of genetic diversity at cytoplasmic markers in Siberian larch (Larix sibirica; four mitochondrial (mt) DNA loci and five chloroplast (cp) DNA SSR loci) to (i) assess the relative contributions of southern and central areas to the current L. sibirica distribution range; and (ii) date the last major population expansion in both L. sibirica and adjacent Larix species. The geographic distribution of cpDNA variation was uninformative, but that of mitotypes clearly indicates that the southernmost populations, located in Mongolia and the Tien-Shan and Sayan Mountain ranges, had a very limited contribution to the current populations of the central and northern parts of the range. It also suggests that the contribution of the high latitude cryptic refugia was geographically limited and that most of the current West Siberian Plain larch populations likely originated in the foothills of the Sayan Mountains. Interestingly, the main population expansion detected through Approximate Bayesian Computation (ABC) in all four larch species investigated here pre-dates the LGM, with a mode in a range of 220,000-1,340,000 years BP. Hence, L. sibirica, like other major conifer species of the boreal forest, was strongly affected by climatic events pre-dating the Last Glacial Maximum.

Cytoplasmic DNA variation and biogeography of Larix Mill. in northeast Asia.

Range-wide variation in 54 populations of Dahurian larch (Larix gmelinii) and related taxa in Northeast Asia was assessed with four mitochondrial PCR-RFLP and five chloroplast SSR markers. Eleven mitotypes and 115 chlorotypes were detected. The highest diversity was observed in the southern Russian Far East where hybrids of L. gmelinii, L. olgensis and L. kamtschatica are distributed. In contrast, only two mitotypes occurred in L. cajanderi and L. gmelinii. The Japanese larch (L. kaempferi) was found to be closely related to populations of L. kamtschatica inhabiting the Kuril Islands and South Sakhalin, populations from the northern part of Sakhalin being more closely related to continental species. In general, both mitochondrial (G(ST) = 0.786; N(ST) = 0.823) and chloroplast (G(ST) = 0.144; R(ST) = 0.432) markers showed a strong phylogeographical structure and evidence of isolation-by-distance. Yet both markers did not allow a clear delineation of species borders. In particular, and contrary to expectations, cpDNA was not significantly better than mtDNA at delineating species borders. This lack of concordance between morphological species and molecular markers could reflect extensive ancestral haplotype sharing and past and ongoing introgression. Finally the distribution of mtDNA and cpDNA variation suggests the presence of several refugia during Pleistocene glacial intervals. In particular, mtDNA and cpDNA reveal weak but visible differentiation between L. gmelinii and L. cajanderi, suggesting independent glacial histories of these species.

Conflicting phylogenies of Larix (Pinaceae) based on cytoplasmic and nuclear DNA.

The phylogeny of Larix species was studied using chloroplast DNA RFLPs and nuclear ITS sequences and AFLPs. The study resolved the sister relationships between the Eurasian and Asian species and the monophyletic origin of each major geographic group. It also provides strong evidence for an early splitting of American Larix from other species in the genus. The discrepancy between phylogenies based on chloroplast DNA and nuclear DNA, previously observed by Qian et al. [Can. J. For. Res. 25 (1995) 1197], is confirmed. Various explanations for the incongruence between chloroplast DNA and nuclear DNA are discussed, and a tentative reconstruction of Eurasian Larix phylogeography is proposed.

Nuclear and cytoplasmic variation within and between Eurasian Larix (Pinaceae) species.

The genetic variation in nuclear and cytoplasmic markers was investigated in 28 populations belonging to Eurasian Larix species (L. decidua, L. sibirica, L. gmelinii, L. olgensis, and L. kaempferi). Nuclear genetic variation was assessed at 214 AFLP loci, and both PCR-RFLP and four microsatellite loci were used to estimate variation of the chloroplast DNA. Variation of the mitochondrial genome was measured using RFLPs. Although population differentiation at both nuclear and chloroplast markers was much weaker than at mitochondrial DNA, it nonetheless corroborated the grouping observed with mitochondrial DNA. The AFLPs led to the same population grouping as mtDNA. Notably, the presence of two ancient western and eastern groups within L. sibirica was confirmed and possible postglacial routes inferred. The genetic composition of the northernmost L. sibirica population in our sample established that it is located at the confluence of the eastern and western recolonization routes. The joint use of the three markers also indicated that populations around Lake Baikal are hybrids between L. gmelinii and L. sibirica, with L. gmelinii primarily acting as the pollinator. Finally, AFLP-based estimates of nucleotide variation were an order of magnitude larger than the strikingly low estimates of nucleotide variation recently reported in Pinus sylvestris.