Molecular Phylogenetics And Biogeography Of The Worldwide Smilacaceae (Liliales)

Smilacaceae are unique (woody vine, climbing with tendrils, leaves with rich reticular venation), worldwide distributed, plant family in monocots. Although an over250years of classification history, due to the variability and complexity of their morphological traits, and the breadth of their distribution, classification revision and systematics of Smilacaceae have never been well resolved. Their interspecific phylogenetic relationship has been a unsolved issue in the order Liliales. In addition, the worldwide distribution pattern of Smilacaceae has also intrigued the attention and interest of many biologists. However, because of their worldwide distribution, and lacking of suitable phylogenetic framework and evolutionary time frame, no one has been able to conduct a comprehensive in-depth research to understand the evolutionary history and biogeography of Smilacaceae. With nearly20years of accumulation of plant materials and literature, in this study, we used modern methods of molecular biology and bioinformatics to reconstruct a molecular phylogeny and the biogeographic history of Smilacaceae. Our results provide a framework to solve some controversial problems in taxonomy, and help elucidate evolutionary relationships within the family. Moreover, the biogeography analysis provides insights into the origin and evolution of distribution ranges of Smilacaceae. The main findings are summarized as follows:1) Phylogeny and classification of Smilacaceae:Through world-wide sampling of species of Smilacaceae, we obtained135taxa of the family from6continents and35countries. These taxa covered all sections of Smilax and Heterosmilax, and major global distribution zones of the family. Representatives from Dioscoreales, Asparagales, and Liliales were used as outgroup. We generated sequences of ribosomal DNA ITS, the chloroplast DNA matK, rbcL, ndhA, ndhF gene and the rpll6intron regions, as well as single-copy nuclear genes At103and agt, a total of eight molecular fragments. Using the data, we reconstructed a well-revolved the molecular phylogenetic tree of Smilacaceae use maximum parsimony, maximum likelihood and Bayesian inference methods. The resulting phylogeny is well-resolved, showing five major lineages was recognized within Smilacaceae:the New World clade, Smilax aspera clade, the West China-Himalaya clade, the Old World prickles clade, and the Old World woody clade. It showed that except Sect. Nemexia, the previous morphology based classification of Smilax is not supported. Some taxonomic characteristics like multiple anthers, raceme inflorescence, and prickles were found to have multiple origins in this family. The result showed a conspicuous Old World-New World distribution pattern, strong geographic grouping of lineages. Based on the comprehensive phylogenetic results, we proposed that the status of Heterosmilax should be diminished, and the lineage should be recognized as a section status in Smilax. This change results in a monotypic Smilacaceae including only Smilax consisting of210species. We proposed the need of a revision of Smilax by combing molecular phylogenetic results, morphological characters and geographic distribution pattern.2) Character evolution in SmilacaceaeWe inferred the evolutionary trends of six morphological characteristics of Smilax by reconstructing the ancestral character state using the unordered parsimony model. The result showed the ancestor of Smilacaceae were woody vine, had single umbels without a prophylla. Through reconstruction of the evolutionary history of ancestral characters state of Smilacaceae, we found that the ancestral character states of Smilacaceae has been retained in the lineages of the New World and Old World. The reason for this may be related to the better adaptability of this morphological trait combination to various environments. Over60%of Smilacaceae species occur in the Sino-Japanese and Sin-Himalaya flora with exceptional morphological diversity, which suggests recent diversification of Smilacaceae in the region. This may due to the rapid uplift of Himalaya-Hengduan Mountain region, that resulted in geographic isolation, climatic changes that promoted differentiation.3) Historical biogeography of Smilacaceae Based on the phylogenetic framework of Smilacaceae reconstructed by eight DNA markers and comprehensive fossil records, we conducted divergence time estimation, ancestral area reconstruction, and diversification rate analyses of Smilacaceae. The results suggested the area of origin, migration and dispersal routes of Smilacaceae. The late Cretaceous period (-97Ma) in North America were suggested the time and origin center of the family. Species of Smilacaceae in the Eocene and Oligocene boundary might experience a mass extinction, and the divergence of the New World and Old World lineages were associated with dramatic climatic cooling at that time. The diversification events of the Old World lineage in Oligocene period (～38Ma-～26.7Ma) were likely linked to the collision between India and Euroasia. Through the CorSim analyses of diversification rate analyses of Smilacaceae, we found Smilacaceae experienced two rapid diversification periods occurred in the Early Miocene (～20Ma-～13Ma) and Pliocene (～5Ma-～2Ma), respectively. In addition, we also detected the diversification rate disparity between the Old World and New World clades. More complex geological history and topography of Asia may explain the faster rate observed in Old World clade.Inter-continental disjunction of SmilacaceaeSpecies of Smilax in the Late Miocene-Pliocene period (～7.5. Ma-～4Ma) experienced multiple expansions, migration and dispersal events, leading to today’s global distribution pattern. Ten major transoceanic intercontinental disjunctions were detected in the family, including eastern Asia and North America disjunction, North America and Europe disjunction, long distance dispersal between South America and Hawaii, Southeast Asia and Australia, South Asia and Africa, and Southeast Asia between Oceanic islands. In addition to isolation and migration hypothesis, long-distances dispersal was also found to be a major factor in the formation of the pantropic distribution pattern of Smilacaceae. This study provided an important example for the pantropical disjunct phytogeography, and valuable information to the global phytogeographic map.