Studies Of Phylogeny And Phylogeography Of Smilax China Complex In East Asia

Smilax china complex is a typical polyploid complex, which occurs widely in East Asia. It has diverse ploidy level and variable morphology among different populations, especially between the widely distributed S. china and its relatives, which makes it difficult for accurate taxonomic identification, and some species (such as S. trinervula and S. biflora) remain taxonomically debatable. Based on previous research, the population ploidy and their geographical distribution in S. china complex were further studied using root tip chromosome counting and flow cytometry. Phylogenetic reconstruction of S. china complex and its relatives was conducted based on molecular data of cpDNA, ITS and single-copy nuclear gene agt\. Integrative taxonomy method was employed for the species re-delimitation of the diploids of S. china complex. The polyploid populations of S. china and two other species (S. trinervula and S. davidiana) which may be involved in its hybridization or polyploidization were studied using molecular systematic and population genetics methods. Based on molecular data and the geological and paleoclimatologic history of eastern Asia, the origin of S. china complex and the polyploidization history of S. china were discussed. The main results are as follows:1. Field investigation and supplementary sampling of Smilax china complexThe field work on the habitat investigation of the S. china complex started in 2009, and the sampling for molecular and chromosome analyses were conducted simultaneously. The investigation localities scattered throughout the distribution range of S. china complex except for Taiwan island and the Korean Peninsula. During the field work,12 populations were sampled for DNA matieral and 26 living individuals were transplanted into the HJC botanical garden.2. The ploidy level of Smilax china complex25 populations of 6 species were studied using chromosome tabletting and flow cytometrey, the result indicated that S. china complex has diverse ploidy levels including diploid, tetraploid, hexaploid and even aneuploid. The chromosome number of some populations were reported for the first time:S. china_SNJ/ZZ/YX 2n=32, S. trinervula_TT/ZH 2n=64 (4X), S. trinervula_PA/YS 2n=32, S. biflora_WJ2n=32 (2X), S. biflora_AM 2n=60. S. biflora was discovered to comprises two different ploidy levels, diploid and aneuploid. Combining with morphological and molecular evidences, the Amami population of S.biflora (2n= 60) was proved to be an undescribed new species. S. trinervula was found to comprises two different ploidy levels, diploid and tetraploid, and the result also indicated that populations south of the Yangtze River (westward to Guizhou) were diploid, and populations north of the Yangtze River (such as Dabie Mountains) and those from Japan were tetraploid.3. The phytogeny and species composition of Smilax china complexBased on 4 cpDNA fragments (matK, rbcL, trnS-trnG, psbA-trnH), nrITS and single copy nuclear gene agtl,80 individuals of 47 populations belonging to 21 species were studied with molecular systematic and divergence time estimation, the result indicated that S. china complex is a monophyletic group, while S. ferox, S. polycolea, S. lebrunii and S. glaucochina were excluded from the species complex. Together with the integrative taxonomy method, S. china complex was confirmed to include S. china L., S. china var. kuru Sakag. ex Yamam., S. amamiana ZS. Sun et P. Li, sp. nov., S. biflora Siebold ex Miq., S. davidiana A. DC, S. microdonta ZS. Sun & CX. Fu, sp. nov., S. nantoensis T. Koyama and S. trinervula Miq.4. Phylogeography of Smilax china4 cpDNA fragments (matK, rbcL, trnS-trnG, psbA-trnH) and nrITS were employed for the phylogeographical study of S. china (25 populations), S. trinervula (7 populations) and S. davidiana (6 populations). Based on molecular systematics, phylogeography and the fossil data, the evolutionary history of S. china complex was reconstructed. The result indicated that:1) S. china complex originated in the early Miocene (18.07 Ma); 2) In middle Miocene, S. china complex differentiated into 2 lineages:CJK (China-Japan-Korea) lineage and Chi (South and Southwest China), and the formation of East Asian monsoon in Miocene have important effects on the differentiation of the two lineages; 3) In late Miocene to Pliocene, the aridation of the middle and lower reaches of Yangtze River may promoted the differentiation of some diploid species in Chi lineage; 4) The emergence of the East China Sea landbridge promoted the migration of S. trinervula and S. china CJK lineage between China and Japan/Korea during the Quaternary ice age, and the corridor effect of East China Sea landbridge is not the same for different species; 5) The polyploid populations of S. china complex originated many times in different places, and the polyploidization of some lineages may occurred as early as the early Pliocene; 6) S. davidiana and S. trinervula were involved in the polyploidization of S. china. This result confirmed that hybridization played an important role in the formation of the hexaploid of S. china complex, and the hexaploids in eastern China may be hybrids between the local tetraploid and S. trinervula.5. Speciation patterns in Smilax china complexBased on the phylogenetic and phylogeographic studies, the geological history and the climate change of East Asia were deduced to have tremendous impacts on the speciation process of the S. china complex. There are at least 3 different speciation patterns existed in S. china complex, such as peripatric speciation, stasipatric speciation and hybridization & polyploidization. Two insular species, S. biflora and S. nantoensis, were derived from S. trinervula (Japan) and S. davidiana (mainland China), respectively, which agrees with the peripatric speciation pattern. S. amamiana has an unique chromosome number (2n=60), which is likely to promote the isolation of this species from the other species in & china complex, ie a process of stasipatric speciation. This study also indicated that hybridization & polyploidization is one of the most important speciation patterns in S. china complex.S. biflora and 5. amamiana, two insular species which are endemic to the Ryukyu Islands were proved to be originated from different genetic lineages, and their similarity on morphological characters (dwarf habit, small leaves, degradated tendrils, 1-2 flowered inflorescence, etc.) was deduced to be the result of parallel evolution due to the adaptation to the island habitat.6. Taxonomy treatment of the Smilax china complexBased on morphological, chromosomal, molecular and distibution data, the diploids of S. china complex were re-delimitated using integrative taxonomy method, and this work resolved the taxonomic status of some controversial taxa in S. china complex. The presented study discovered two undescribed new species:S. amamiana ZS. Sun & P. Li, sp. nov. and S.microdonta ZS. Sun & CX. Fu, sp. nov.. Based on the comparison of the new species and the other diploid species in S. china complex, detailed descriptions for the new species are given.The taxonomy arrangement of S. china complex was treated as follows:Smilax L., Sp. P1. 2:1028.1753.subgen. China P.Li & C.X.Fusect. ChinaT.Koyama(1) Smilax amamiana Z.S. Sun et P. Li sp. nov.(2) Smilax biflora Siebold ex Miq.(3) Smilax china L. S. china var. kuru Sakag. ex Yamam.(4) Smilax davidiana A.DC.(5) Smilax microdonta Z.S. Sun & C.X. Fu, sp. nov.(6) Smilax nantoensis T.Koyama(7) Smilax trinervula Miq.