Phylogeny And Biogeography Of Scrophularia And Phylogeography Of S. Incisa Complex

1. Phylogenetic and biogeographic study of global ScrophulariaScrophualria Linn, is a typical genus with Eurasia-North America disjunction, comprising the herbal sect. Scrophularia and semi-shrubby sect. Caninae. The whole genus include more than 200 species. In spite of 250 years of taxonomic history, the variations of morphological characters of Scrophularia, especially the leaf and flower are complex. Therefore, the taxonomy and phylogeny of Scrophularia have remained unresolved. Moreover, though Scrophularia is distributed widely, previous studies have been limited to a particular area (like Cental Asia, or North America or Europe). The phylogeny and biogeography of global Scrophualria has not been studied yet. Therefore, the origin, diversification, migration, and evolution time frame of Scrophularia remain to be interesting questions. Based on over several years’ materials collection and GenBank downloaded data, our reasearch involved 91 individuals from 76 taxa, covering all the distribution areas and representatives of all taxonomical sections. We sequcenced 4 cpDNA fragements and 1 nrlTS, and with combined 5 loci reconstructed a robust phylogenetic tree of Scrophularia. Through the phylogenetic framework, biogeographic analyses, and temporal diversification of Scrophularia on a global basis, we try to answer the following questions:the origin and evolution history of scrophualria, the phylogenetic position of East Asia and North America Scrophualria, and whether the genus migrated from East Asia to North America. The main results are listed below:1) Phylogeny of Scrophularia and taxonomy We sequenced four cpDNA fragments of trnL-trnF, trnQ-rps16, psbA-trnH, trnG-trnS and ITS4-ITSleu and reconstruct the molecular phylogenetic tree of 71 world-wide distributed ingroup species through ML (Maximum Likelihood), MP (Maximum Parsimony) and BI (Bayesian Inference) analyses. We obtained a highly supported Scrophularia phylogenetic tree for the first time. The tree suggested that global Scrophualria consist of 5 clades:the early diverged Eurasia widely distributed S. umbrosa clade, Mediterranean distributed herbal Scrophularia clade, Central Asia distributed semi-shruby Caninae clade, North America distributed herbal Scrophularia clade and East Asia distributed herbal Scrophularia clade. The tree indicated that three species from Japan and Korea (S. musashiensis, S. duplicato-serrata, S. takesimensis) are embedded inside North America clade, which does not support the hypothesis of Scrophularia in North America was originated from East Asia. We speculated that there was a dispersal event from North America to Japan and Korea by bering land bridge at the end of Miocene (-7.5 Ma), resulting in the East Asia-North America disjunction. Central Asia distributed semi-shruby Caninae is a robust supported monophyly and embedded in the sect. Scrophularia, namely the herbal sect. Scrophularia is a paraphyletic group.2) Biogeographic history of Scrophulaira To estimated the divergence time of Scrophularia, we selected 6 calibration points, including 5 confidently assigned fossils and an estimated crown node of Lamiales from previous study, Our results show that the divergence time between Scrophulria and Teedia is 27.77 Ma. Employing this time, we calibrated the lineage of Scrophularia using beast analysis and reconstructed the ancestral area by RASP. The result showed that the common ancestor of Scrophualn’a is originated in Mediterranean at c.18.9 Ma during the mid-Miocene, and subsequently migrated eastward into Central Asia and East Asia; migrated westward into North America through the Atlantic land bridge, resulting in the Eurasian-North America disjunction distribution pattern. Our results suggested that North America clade contains 3 subclades and there was a dispersal event from Eastern North America to Antilles Islands, followed by rapid radiation and divergence. At the same time there was also a migration event from North America to East Asia through the berign land bridge, followed by migration from Japan-Korean Peninsula back to North America. For the first time, we suggested that East Asia Scrophularia is consisted of 3 subclades which diverged at c.5 Ma:1) E1, Tibet clade; 2) E2, Central-Northwestern-Southwestern China clade; 3) E3, East China clade (including the widely distributed S. ningpoensis complex), respectively. We found two origin of Scrophularia in Korean and Japan:some species come from China by short distance migration and others come from North America by long distance migration. The ancestral area of Scmphularia in Mediterranean region included 3 subclades and the dispersal event also happened from Iberian Peninsula and South Europe to Macaronesia. Our results strongly supported that the Mediterranean distributed herbal Scmphularia clade and Central Asia distributed semi-shruby Caninae clade were sister groups, for the sect. Caninae was obviously the adaptive evolutionary clade at the end of Miocene (-7 Ma), in order to adapt to the arid environment of Central Asia caused by uplift of QTP.2. Phylogeographic research of 5.incisa complex circular the QTPPhylogenetic study suggested that there is a well-support monophyly complex consisting of three relative species from QTP and adjacent regions. The three species are S. kiriloviana from Xinjiang, S. inicsa from Qinghai-Gansu-Inner Mongolia and S. dentata from Tibet. They have similar morphological characters and habitat, and are ideal type materials to study the speciation mechanism of this hotpot.Our research covering 14 populations of S. kiriloviana from Xinjiang and one from Central Asia,11 populations of S. inicsa from Qinghai,3 from Gansu, and one from Inner Mongolia,3 populations of S. dentata from Tibet, applied three kinds of markers, including cpDNA, nrSSR and nrITS to study on the phylogeography of S. incisa complex. The main results are listed below:1) Phylogeographic of S. inicsa complex based on cpDNA For the cp sequences, we got 30 haplotypes, grouping into two clades, Cenral Asia Clade and China Clade. The China Clade was further grouped into three subcaldes, Xinjiang subclade, Qinghai-Gansu-Inner Mongolia subclade, Tibet subclade, respectively. Diverence time estimation of cpDNA haplotypes showed that their ancestral is originated at 4.5Ma during early Pliocene and the divergence time of China Clade was 3.28 Ma. This time node is consistent with the last uplift of QTP:3.5-1.6 Ma and the aritify of Tarim:from 3.7 Ma to present. We speculated that the vicariance caused by the uplift of QTP led to allopathic speciation of S. incisa complex. The ancestral area reonstruction analyses of RASP showed that the three species originated in Central Asia and subsequently migrated eastward through Xinjiang to Qinghai, Gansu, Ningxia, and Mongolia Plateau, also through the Himalayan to Tibet.2) Genetic structure analysis of S. incisa complex based on nrSSR. The result suggested that when K=3, populations of the S. incisa complex were divided into 3 gene pools:red gene pool of Central Asia-Xinjiang-Inner Mongolia, green gene pool of Qinghai-Gansu, blue gene pool of Tibet, respectively. This result is consistent with cpDNA dataset and the differences are that in this data set Central Asia, Xinjiang and Inner Mongolia group together. We speculated that the genetic structure could have arisen by the gene flow between Central Asia and Xinjiang and incomplete lineage sorting between Xinjiang and Inner Mongolia. The haplotype analysis of ITS is also generally consistent with SSR, indicating the early divergence between the haplotypes from Central Asia, Xinjiang and Inner Mongolia. The differences are that Western Qinahai-Gansu shared the red haplotype of Xinjiang clade and Tibet shared the green haplotype of Qinghai clade. Incomplete lineage sorting between Xinjiang and Qinghai could be an explanation for this scenario. We also speculated that there is an asymmetric hybridization event between Qinghai and Tibet.3. Taxonomic revision suggestionCombining molecular phylogenetic, morphologic and geographic evidences, we suggestd that Scrophularia is composed of five sections, namely sect. Caninae, sect. Scrophularia, sect. Americana, sect. China, and sect. Umbrosa. S, incisa complex distributed in Central Asia is S. incisa subsp. incisa and S. incisa subsp. kiriloviana, respectively. S. dentata from Tibet is a good species, while taxa from Inner Mongolia and Gansu-Qinghai are S. incisa subsp. yuandong and S. incisa subsp. qinghai. And we also suggested that S. koraiensis as the synonym of S. kakudensis.