Phylogeography Of Iris Ensata (Iridaceae), A Disjunct Species In East Asia, And Role Of Pollinators On The Persistence Of Rear Edge Populations

Understanding the patterns of species’geographic distribution, their causes and their consequences are core issues in ecology and evolution biology. Many studies have proved that present abiotic factors could limit one species’ geographic distribution through affecting the species’ physiological and ecological functions. In past two decades, the studies of phylogeography have showed that climate fluctuations and the related geological events (e.g. uplifts of mountains and sea-level oscillations) during glacial periods had strongly shaped present geographic distribution of plants. Besides abiotic factors, the geographic distribution of one species could be influenced by biotic interactions (e.g., pollination) and the dispersal ability of species.Though glacials had no direct effect on the flora of East Asia, climatic fluctuations, sea-level oscillations and land-bridges configuration during glacial periods had dramatically effect on geographic distribution and population genetic structure of species. Meanwhile, the landscape features in East Asia also played an important role in shaping the genetic structure of species. The plants with disjunctive distribution in east of Eastern Asia provide ideal models to infer intraspecific divergence and the relation between divergence and environmental changes.The response of species’ geographic distribution to abiotic and/or biotic environment changes is likely to be determined by the response at range margins. Located at the low-latitude margins of species’ distribution range, rear edge populations were relicts as most populations of one species shifted northward during interglacial period. The rear edge populations are the hotspot of genetic diversity of species. However, little is known about the effects of biotic interaction on persistence of rear edge populations, which would help us fully understand and predict the response of rear edge populations or even a species to environment changes in future.Iris ensata (Iridaceae) is a species disjunctively distributed in Tianmu Mts. of East China, Northeast of China (NEC), Eastern Russian, Korea and Japan. Based on dataset of cpDNA sequences and nSSRs, we assessed genetic structure of I. ensata populations. Combined approximate Bayes computation (ABC) with ecological niche modelling, we inferred demographic dynamics. We also characterized breeding system and potential factors affecting sexual reproduction in rear edge populations. Main conclusions are as follows.1. Thirteen polymorphism primers were developed for I. ensata using the method of improved biotin-streptavidin capture, and nine of them were used to analyze of genetic structure. Based on nSSR dataset, PCoA, Barrier analysis and STRUCTURE clustering analyses supported that all populations were grouped into three clades, Tianmu Mts., Japan-South Korea, NEC. There were genetic mixtures of Japan-South Korea and NEC in Jeju Island. ABC analysis strongly supported the "secondary contact" scenario:Japan populations and ancestral populations of South Korea consequently diverged from NEC population, followed by secondary contact between populations of South Korea and Japan since 910 generations. Moderate genetic diversity (HE=0.618, HE=0.618) and high genetic divergence(FST=0.245, P<0.001) were detected at the species level. The genetic diversity level of South Korea populations was significant higher than those of other regions (P< 0.05). Remarkable genetic sub-structures were detected in Tianmu Mts., Japan and South Korea.2. Fifteen haplotypes were observed using joint analysis of five fragments including matK, trnK intron,psbJ-petA,petL-psbE and ndhA intron. Low nucleotide diversity (π=0.57×10-3), moderate haplotype diversity (Hd=0.753) and significant geographic structure (GST=0.834<NSt=0.927) (P<0.01) were revealed based on cpDNA dataset. TCS analysis revealed 15 haplotypes were clustered into three lineages, Tianmu Mts. (Lineage Ⅰ), Japan-Jeju Island (Lineage II), and South Korea-NEC-Eastern Russian (Lineage III). Incomplete lineage sorting was revealed in lineage III in the maximum likelihood tree and the Bayes tree. Neutral test and mismatch distribution analysis indicated populations of Japan and NEC had experienced expansion during the Late Pleistocene, while populations of Tianmu Mts. did not show significant expansion. Bayesian molecular clock analysis showed that the divergence of three lineages was dated from 0.45 Ma (95% HPD:0.76-0.20 Ma), which was accordant with the geologic events that the submergence of land-bridge across East China Sea (ECS) or the Korea Strait.3. Compared with distribution range at present, results of Ecological Niche Modeling showed that the potential distribution range in the LGM was hardly contracted southward, which was accordant with adaptation of I. ensata to cool conditions. During the LGM, the migration of I. ensata was not revealed across the ECS land bridge, while the contact between regions was weak across the Korea/Tsushima Strait land bridge.4. Lack of apomixes and spontaneous selfing, seed production of I. ensata was completely dependent on the asymmetrical specialized pollination system between this iris and its pollinators, Bombus trifasciatus in Tianmu Mts. Visitation rate of pollinators was stable in natural populations. Accordingly, pollen limitation was low (13.3%-40.3%) in natural populations. In contrast, pollination mutualism was collapsed in two ex-situ populations, which were located at low altitudes owning same or similar latitude with natural populations, and pollen limitation was high (83.1% -100%) due to low pollinator visitation or pollinator absence. Inbreeding depression extent was 0.409 and 0.159 evaluated with seed setting and germination, respectively.In conclusion,I. ensata was fragmented and contracted into three independent refugia along with the disappearance of land-bridges during mid- to late Pleistocene. During the LGM, the role of the ECS land bridge had not revealed, thus populations of Tianmu Mts. had experienced strict allopatric divergence. However, secondary contact could be facilitated to link populations of South Korea and Japan by the land bridge of the Korea/Tsushima Strait. As I. ensata had poor dispersal ability and high habitat preference, natural fragmentation had enhanced its intra-specific divergence and facilitated local adaptation. Pollination mutualism had played a predominant role in the persistence of I. ensata in rear edge populations. Intense inbreeding depression acted as the selective agent to maintaining its outcrossing mating system.