The species of Oxytropis DC. of section Gloeocephala Bunge (Fabaceae) from Northeast Asia: genetic diversity and relationships based on sequencing of the intergenic spacers of cpDNA and ITS nrDNA.

Phylogenetic relationships within Oxytropis DC. sect. Gloeocephala Bunge from Northeast Asia were studied using plastid intergenic spacers (psbA-trnH + trnL-trnF + trnS-trnG) and ITS nrDNA. Populations of O. anadyrensis Vass., O. borealis DC., O. middendorffii Trautv., O. trautvetteri Meinsh., and O. vasskovskyi Jurtz. were monomorphic or characterised by a low level of chloroplast genetic diversity (h varied from 0.143 to 0.692, and π from 0.0001 to 0.0005). Presumably, the low genetic diversity was a result of the severe bottlenecks during Pleistocene glaciation-interglacial cycles. Twenty chlorotypes were identified; species studied had no shared chlorotypes. Chlorotypes of O. anadyrensis, O. borealis, and O. middendorffii formed two lineages each, while the chlorotypes of O. trautvetteri and O. vasskovskyi formed one separate lineage each in the phylogenetic network. There were specific diagnostic markers of cpDNA in each lineage, excluding O. vasskovskyi. The presence of a species-specific diagnostic marker in O. trautvetteri and specific markers in two lineages of O. anadyrensis support circumscribing these taxa as independent species. Regarding ITS nrDNA polymorphism, five ribotypes were detected. The differences revealed in plastid and nuclear genomes of Oxytropis sect. Gloeocephala confirmed that the Asian sector of Megaberingia was the main centre of diversification of arctic legumes.

Rhizobial Microsymbionts of Kamchatka Oxytropis Species Possess Genes of the Type III and VI Secretion Systems, Which Can Affect the Development of Symbiosis.

A collection of rhizobial strains isolated from root nodules of the narrowly endemic legume species Oxytropis erecta, O. anadyrensis, O. kamtschatica, and O. pumilio originating from the Kamchatka Peninsula (Russian Federation) was obtained. Analysis of the 16S ribosomal RNA gene sequence showed a significant diversity of isolates belonging to families Rhizobiaceae (genus Rhizobium), Phyllobacteriaceae (genera Mesorhizobium, Phyllobacterium), and Bradyrhizobiaceae (genera Bosea, Tardiphaga). A plant nodulation assay showed that only strains belonging to genus Mesorhizobium could form nitrogen-fixing nodules on Oxytropis plants. The strains M. loti 582 and M. huakuii 583, in addition to symbiotic clusters, possessed genes of the type III and type VI secretion systems (T3SS and T6SS, respectively), which can influence the host specificity of strains. These strains formed nodules of two types (elongated and rounded) on O. kamtschatica roots. We suggest this phenomenon may result from Nod factor-dependent and -independent nodulation strategies. The obtained strains are of interest for further study of the T3SS and T6SS gene function and their role in the development of rhizobium-legume symbiosis. The prospects of using rhizobia having both gene systems related to symbiotic and nonsymbiotic nodulation strategies to enhance the efficiency of plant-microbe interactions by expanding the host specificity and increasing nodulation efficiency are discussed.

Late Pleistocene origin of the entire circumarctic range of the arctic-alpine plant Kalmia procumbens.

The circumarctic ranges of arctic-alpine plants are thought to have been established in the late Pliocene/early Pleistocene, when the modern arctic tundra was formed in response to climate cooling. Previous findings of range-wide genetic structure in arctic-alpine plants have been thought to support this hypothesis, but few studies have explicitly addressed the temporal framework of the genetic structure. Here, we estimated the demographic history of the genetic structure in the circumarctic Kalmia procumbens using sequences of multiple nuclear loci and examined whether its genetic structure reflects prolonged isolation throughout the Pleistocene. Both Bayesian clustering and phylogenetic analyses revealed genetic distinction between alpine and arctic regions, whereas detailed groupings were somewhat discordant between the analyses. By assuming a population grouping based on the phylogenetic analyses, which likely reflects a deeper intraspecific divergence, we conducted model-based analyses and demonstrated that the intraspecific genetic divergence in K. procumbens likely originated during the last glacial period. Thus, there is no need to postulate range separation throughout the Pleistocene to explain the current genetic structure in this species. This study demonstrates that range-wide genetic structure in arctic-alpine plants does not necessarily result from the late Pliocene/early Pleistocene origin of their circumarctic ranges and emphasizes the importance of a temporal framework of the current genetic structure for understanding the biogeographic history of the arctic flora.

Persistent history of the bird-dispersed arctic-alpine plant Vaccinium vitis-idaea L. (Ericaceae) in Japan.

Arctic-alpine plants have expanded and contracted their ranges in response to the Pleistocene climate oscillations. Today, many arctic-alpine plants have vast distributions in the circumarctic region as well as marginal, isolated occurrences in high mountains at lower latitudes. These marginal populations may represent relict, long-standing populations that have persisted for several cycles of cold and warm climate during the Pleistocene, or recent occurrences that either result from southward step-wise migration during the last glacial period or from recent long-distance dispersal. In light of these hypotheses, we investigated the biogeographic history of the marginal Japanese populations of the widespread arctic-alpine plant Vaccinium vitis-idaea (Ericaceae), which is bird-dispersed, potentially over long distances. We sequenced three nuclear loci and one plastid DNA region in 130 individuals from 65 localities covering its entire geographic range, with a focus on its marginal populations in Japan. We found a homogenous genetic pattern across its enormous range based on the loci analysed, in contrast to the geographically structured variation found in a previous study of amplified fragment length polymorphisms in this species. However, we found several unique haplotypes in the Japanese populations, excluding the possibility that these marginal populations result from recent southward migration. Thus, even though V. vitis-idaea is efficiently dispersed via berries, our study suggests that its isolated populations in Japan have persisted during several cycles of cold and warm climate during the Pleistocene.

Molecular evidence for ancient relicts of arctic-alpine plants in East Asia.

Following climate cooling at the end of the Tertiary, arctic-alpine plants attained most of their extant species diversity. Because East Asia was not heavily glaciated, the importance of this region as a location for the long-term persistence of these species and their subsequent endemism during the Pleistocene was proposed in early discussions of phytogeography. However, this hypothesis remains to be fully tested. Here, we address this hypothesis by elucidating the phylogenetic history of Phyllodoce (Ericaceae). A phylogenetic tree based on multiple nuclear loci revealed that Phyllodoce nipponica was not derived from widespread species such as the arctic-alpine Phyllodoce caerulea, but rather represented an independent lineage sister to the clade of widespread relatives. Molecular dating indicated a mid-Pleistocene divergence of P. nipponica. These findings exclude the hypothesis that P. nipponica was derived from an arctic-alpine species that extended its range southwards during recent glacial periods. Instead, our results support the hypothesis that P. nipponica is an ancestral species which persisted in the Japanese archipelago during the mid- and late Pleistocene. Our findings demonstrate support for the early proposal and shed light on the importance of the Japanese archipelago for the evolution and persistence of arctic-alpine species.

The allopolyploid Arabidopsis kamchatica originated from multiple individuals of Arabidopsis lyrata and Arabidopsis halleri.

Polyploidization, or genome duplication, has played a critical role in the diversification of animals, fungi and plants. Little is known about the population structure and multiple origins of polyploid species because of the difficulty in identifying multiple homeologous nuclear genes. The allotetraploid species Arabidopsis kamchatica is closely related to the model species Arabidopsis thaliana and is distributed in a broader climatic niche than its parental species. Here, we performed direct sequencing of homeologous pairs of the low-copy nuclear genes WER and CHS by designing homeolog-specific primers, and obtained also chloroplast and ribosomal internal transcribed spacer sequences. Phylogenetic analysis showed that 50 individuals covering the distribution range including North America are allopolyploids derived from Arabidopsis lyrata and Arabidopsis halleri. Three major clusters within A. kamchatica were detected using Bayesian clustering. One cluster has widespread distribution. The other two are restricted to the southern part of the distribution range including Japan, where the parent A. lyrata is not currently distributed. This suggests that the mountains in Central Honshu and surrounding areas in Japan served as refugia during glacial-interglacial cycles and retained this diversity. We also found that multiple haplotypes of nuclear and chloroplast sequences of A. kamchatica are identical to those of their parental species. This indicates that multiple diploid individuals contributed to the origin of A. kamchatica. The haplotypes of low-copy nuclear genes in Japan suggest independent polyploidization events rather than introgression. Our findings suggest that self-compatibility and gene silencing occurred independently in different origins.