Studies On The Lineage Divergence And Genetic Diversity Of Neolitsea Sericea (Lauraceae), An Endangered Species Endemic To East Asian Land-bridge Islands

Neolitsea sericea (Lauraceae) is an economically important tree species because of its characteristic aroma and timber uses. However, N. sericea is treated as an endangered/vulnerable species in the China Species Red List because of its limited resources in China. N. sericea, endemic to East Asian land-bridge islands, is a component species of broad-leaved evergreen forests. This dioecious, insect-pollinated tree with bird-dispersed seeds is distributed from Taiwan (Orchid Island), through the Ryukyus, to northeastern Japan, but also occurs disjuctly on offshore islands of East China (Zhoushan Archipelago) and South Korea. This species thus provides an ideal model system to examine Late Tertiary/Quaternary phylogeography in relation to configurations of land bridges and sea level changes, and specifically for the the East China Sea (ECS) region.In this study, we performed high-throughput transcriptome sequencing of N. sericea leaves using the Illumina HiSeqTM 2000 sequencing platform, and generated large transcript sequences for functional characterization and development of expressed sequence tag-simple sequence repeats (EST-SSRs) markers. A total of 61 populations from throughout the species’ range were genotyped using 10 EST-SSRs, nrITS and 5 chloroplast (cp) DNA sequence markers to assess current patterns of genetic diversity, population structure and the lineage divergence, supplemented by phylogenetic dating, ancestral area reconstructions and ecological niche modelling (ENM) of the species distributions during the Last Glacial Maximum (LGM) and at present. The main results are as follows:(1) Characterization of transcriptome and development of EST-SSR makersUsing the TRINITY assembly program, a total of 68,624 unigenes (mean length 733 bp) were obtained from about 54.7 million reads, and 41,130 unigenes of all the assembled unigenes showed similarity to public databases. From 68,624 unigenes, 13,213 EST-SSRs were identified. From the 13,213 EST-SSRs,1,191 primer pairs were designed for marker mining. After selecting 131 of these pairs at random for further validation,10 polymorphic pairs (3 outliers and 7 neutral loci) were identified as polymorphic SSR loci. We applied the 10 EST-SSRs to 50 populations to infer the genetic diversity and structure in N. sericea.(2) Genetic diversity and structure analyses based on EST-SSRsWe selected 50 populations to perform the current genetic diversity and structure analyses. We detected a negative correlation (r=-0.0906; P<0.05) between genetic diversity and the latitude. The STRUCTURE analyses identified two distinct lineages located in areas north and south of the’Tokara Gap’. The northern populations of N. sericea were further divided into two genetic subclusters, i.e. Zhoushan-Korea-Kyushu and Shikoku-Honshu. Both sublineages were detected in the southern regions of the main Japanese islands, but only Shikoku-Honshu sublineag was found in the northern regions of the main Japanese islands. Estimates of migration rate using BAYEASS showed that there was also relatively high gene flow from Zhoushan-Korea-Kyushu to Shikoku-Honshu sublineages.(3) Genetic diversity and structure analyses based on nrITS and cpDNA sequence polymorphismsBased on nrITS and cpDNA sequence markers, we selected 61 populations of N. sericea to infer current patterns of genetic diversity and structure. Two polymorphic sites yielded three ribotypes. The distribution of the three ribotypes revealed two distinct lineages across’Tokara Gap’. Based on the topology of ITS ribotype phylogeny, the BBM analysis of ancestral distribution areas supported a probably ancient distribution of N. sericea in Taiwan and Ryukyu islands, which subsequently migrated northward to Zhoushan islands, main Japanese islands and Korea. Based on the 17 polymorphic sites of four chloroplast DNA regions (psbA-trnH, trnL-trnF, psbC-trnS, rps16) surveyed across the 61 populations, a total of 20 haplotypes were identified. Except for H10, the areas north and south of the ’Tokara Gap’ harboured two sets of chlorotypes. A negative correlation (r=-0.05; P<0.05) between genetic diversity and the latitude was found. At the regional level, the areas south of the ’Tokara Gap’ had, on average, higher within-population genetic diversity when compared with the areas north of the’Tokara Gap’.(4) Past and future ecological niche models and demographic inferenceThe past (Last inter-glacial, Last Glacial Maximum), present and future distributions based on ecological niche modelling were constructed. During Last Glacial Maximum (LGM), N. sericea experienced massive habitat losses in North Japan but increases in Southeast China, East China Sea landbridge islands and South Japan. The distribution model for the future (2080) predicts that climate change will result in a 60% reduction of this species’potential range.Taken together, N. sericea in Zhoushan islands, main Japanese islands and Korea most likely originated from Taiwan and Ryukyu islands. The opening of Tokara Gap would have allowed allopatric populations of N. sericea on either side of this sea-barrier to diverge. Based on the IMa analyses of the combined nSSR/cpDNA data set, we dated the divergence of these northern and southern lineages to about 0.3 Ma (95% HPD:0.07-1.1 Ma). During inter-/postglacials of the Late Pleistocene, higher sea levels in the East China Sea (’ECS’) would have promoted range fragmentation and led to the regional disjuncts of N. sericea to disjunct regions.In summary, we conclude that the studies of historical phylogeograpgy and the genetic structure of N. sericea based on a multidisciplinary approach combining molecular phylogeographic, ENM, and phylogenetic approaches will contribute to a better understanding of the temporal origins and biogeographical processes underlying the formation of disjunct distribution patterns of N. sericea in East Asia. In addition, the genetic data reported is of fundamental importance to the development of management strategies for N. sericea.