Phylogeographical Study Of An Intercontinental Disjunct Smilax Aspera:a Basal Taxon Of Smilacaceae

Smilax aspera is one of the components of the evergreen broad-leaved sclerophyllous forest. This species originated from the warm and humid Paleo-Tethys evergreen forest. Smilax aspera is now mainly distributed in the Mediterranean region, East Africa, South Asia and the transition zone between India and the Himalaya region. Based on the previous multi-gene phylogenetic analysis of Smilacaceae, S. aspera was found to be sister to the remainder of the Old World clade with in the family diverging off in the Late Eocene. Previous studies of paleovegetation, paleoclimate and paleogeography have shown that the warm and humid Paleo-Tethys evergreen forest was widely distributed in the Paleo-Mediterranean region. However, the plate tectonics and temperature decrease during the mid-Miocene period caused a serial extinction of numerous palaeotropical taxa. Little is known about the phylogeographic history of these Paleo-Tethys relic taxa in South Asia, Africa and Mediterranean region during the climate change in the Late Tertiary. The aims of this study focuses on elucidating the phylogeographic patterns of S. aspera, to understand the origin, evolution and migration history of the species. This research will contribute a better understanding of how Paleo-Tethys evergreen flora responsed to environmental changes since the Late Tertiary.A total of38populations and57specimens were collected across the entire S. aspera distribution area. Three cpDNA loci (matK, trnC-ycf6and atp B-rbcL) and two nuclear loci (ITS and single-copy gene Agtl) were sequenced across all the samples. Morphological classification data were also considered. Phylogenetic analysis, population genetic statistics, molecular dating and the reconstruction of ancestral areas were used to clarify the biogeographic pattern of S. aspera and its evolutionary history. The main results are listed below:1) The genetic diversity and phylogeographic structure of Smilax asperaBased either on the cpDNA data set or combined sequences data of the three cpDNA regions and ITS, all phylogenetic trees strongly support the monophyly of S. aspera, meanwhile two major clades were recovered within it:East Africa-South Asia and Himalaya lineage and circum-Mediterranean lineage. Based on either the ITS or combined sequences data, two subclades were recovered in East Africa-South Asia and Himalaya lineage:East Africa sub-lineage and South Asia-Himalaya sub-lineage. Hierarchical AMOVA based on cpDNA in terms of continental distribution allocated66.48%variation among regions which is much higher than that among populations within region and within populations. The genetic polymorphism of cpDNA in S. aspera is much higher at species level (hT=0.906, πT×10-3=4.91) than that at population level (hs=0.152; πs×10-3=0.33), and the value of NST(0.911) is significantly larger than the value of GST(0.822)(U=7.6, P<0.01), These results indicated a distinct lineages geographical structure existed at species level.2) The formation of intercontinental disjunct distribution of Smilax asperaThe BEAST result based on cpDNA and fossil suggested that the widespread distributed S. aspera divergenced in the middle Miocene (13.13Ma), East Africa-South Asia and Himalaya lineage differentiated in late Miocene (6.11Ma), followed by the generation of East Africa sub-lineages and South Asia and Himalaya sub-lineages which differentiated from the late Miocene (Agtl:4.11Ma; combined data:2.56Ma; ITS:7.76Ma). The reconstruction of ancestral areas using Bayesian Binary MCMC method based on cpDNA showed that geographic isolation events had been occurred between5. aspera in circum-Mediterranean region and East Africa-South Asia and Himalaya region, and East Africa-South Asia and Himalaya lineage originated from east Africa. Moreover the Agt1data showed the South Asia-Himalaya sub-lineage is in a more evolutionary position in topology. By all of these, we indicated the South Asia-Himalaya sub-lineage derived from east Africa populations.During the middle Miocene, global temperatures became cooled, which caused subtropical elements retreated to refugial areas and resulted in the distribution range of S. aspera shift away along with the shrink of subtropical elements. As a result, the disjunct distribution between the circum-Mediterranean region and East Africa-South Asia and Himalaya region formed. The closure of the Tethys Sea induced the formation of the "Gomphoterium land bridge" during the Miocene, and this land bridge would have enabled migration of the S. aspera lineage from east Africa to Asia with the expanded tropical forests associated with the Middle Miocene thermal maximum. The cooler and drier climates during the Late Miocene to Pliocene may induced this vicarious events, and then a secondary disjunct distribution patterns formed between the East Africa and South Asia-Himalaya. The rapid differentiation in the South Asia lineage probably related with the uplift of Himalayas in Pliocene which induced geological and climate change in this region.3) Differentiation and dynamic history of circum-Mediterranean lineageThe chronogram based on combined sequences data and the genetic diversity patterns based on cpDNA revealed a strong degree of genetic differentiation between the eastern and western Mediterranean population, and the circum-Mediterranean lineage can be further divided into four geographic sub-lineages:Iberian sub-lineage and Apennine sub-lineage in the west Mediterranean, while Balkan sub-lineage and Minor Asia sub-lineage in the east Mediterranean. Both of Bayesian Binary MCMC analysis and S-DIVA analysis indicated that the west Mediterranean was the most likely ancestral area of S. aspera in Mediterranean region, with the crown age estimated at8.45Ma coincide with the Messinian salinity crisis occurred in the late Miocene (7.2-5.3Ma). So we infered that the demise of the tropical-subtropical climate was intensified by the Messinian salinity, which induced differentiation via vicariance between the eastern and western Mediterranean population. A second phase of differentiation occurred during the Pliocene to Pleistocene. This period was characterized by the repetition of the glacial-interglacial cycles, which resulted in cycles of fragmentation and re-expansion of S. aspera in the distribution area and finally four sub-lineages in the Mediterranean region were shaped. We found that the Strait of Gibraltar does not constitute a barrier to gene flow for S. aspera which is resulted from the seed spreading by different birds migration between Mediterranean mainlands and islands.4) The taxonomy meaning of Smilax aspera By the wild populations survey we found the habitat of circum-Mediterranean lineage and East Africa-South Asia and Himalaya lineage has been completely different, which suggested the different habitats of S. aspera have led to a significant differentiation at the molecular level. Based on branch classification analysis of morphological traits, we found a differentiation trend exits between these two lineages. Based on molecular evidence and morpholorgy evidence, the two lineages could be treated as two subspecies.