菱科（Trapaceae）的冰期避难所和物种分化研究

Quaternary glaciation has a strong influence on most of the existing creatures’ geographic distribution and genetic structure. Glacial Refugia and the colonization and differentiation of population in postglacial epoch are the focus of current studies. However, existing relevant research on aquatic plants is fragmental. Trapaceae is a representative research target. Fossil evidence shows that Trapaceae originates from Laurasia, and that a large number of the species have died out in the Pleistocene. The current existing Trapa are unevenly distributed throughout Eurasia and Africa, whose distribution centers are China and the Far East in Russia. Here, the Heilongjiang and Tumen River valley, and the middle and lower reaches of Yangtze River, are two morphology variation centers. This study plans to collect samples from at least 85% species (110 populations) of Trapa, sequencing the entire chloroplast genome of all species，develop 4-6 polymorphic cpDNA fragments and 16 pairs of nucleus microsatellites molecular markers to mark the samples. Consequently, we expect to reveal phylogenetic relationships between species and spatial genetic structures of the population, rebuild phylogeography history, additionally, we will speculate on Glacial Refugia and postglacial recolonization models of Trapaceae and compare with terrestrial plants.We will combine phylogenetic relationships with morphology characters, clarify the morphological classification boundaries between Trapa species. Ultimately, this study is focused on finding greater aquatic glacial refuge in order to explore the influence of Quaternary glaciation on aquatic plant’s geographic distribution patterns.

第四纪冰期对现存大多数生物的地理分布格局和遗传结构有重要影响，冰期避难所及冰期后种群的扩张与分化等成为研究热点。然而，关于水生植物的相关研究仅有支离破碎的信息。菱科是有代表性的研究对象，化石证据表明其起源于古劳亚大陆，更新世大量灭绝。现存单型属（Trapa）不均衡分布于欧亚大陆和非洲，分布中心在中国和俄罗斯远东，其中黑龙江及图门江流域和长江中下游是二个形态变异中心。本研究拟在整个分布区采集菱属物种数超过85%以上（110个群体）实验材料，完成所有种叶绿体全基因组测序，自主开发具多态性4-6个cpDNA片段和16对核SSR引物标记实验材料，揭示种间谱系进化关系和群体空间遗传结构，重建谱系地理历史，推测菱属冰期避难所及冰期后种群迁移模式，比较与陆生植物有何不同；将谱系进化关系与形态特性相结合，澄清菱属种间的形态分类界线。旨在发现水生植物的冰期避难所，探讨第四纪冰期对水生植物地理分布格局影响。

本项目完成调查了中国、俄罗斯和日本共61不同地点菱属植物资源调查，采集了每个地点所分布的所有菱属材料（包括标本、叶片和成熟的果实）。总个体数（叶片材料）为2454份，110个种群。还采集到了1500多个成熟的菱角。通过菱角、植株等形态特性鉴定结果为20个种、7个变种和2个待定种。自主开发了21对具有多态性的微卫星引物，对所有2454份样本进行荧光标记。通过高通量测序方法完成81个样本（包括20个物种、3个变种和1个待定种）叶绿体全基因组拼接，用于叶绿体全基因组系统进化关系分析。同时还拼接出这81个样本的ITS序列，用于系统进化关系分析。还采集了10个种新鲜活体叶片，做了流式细胞分析，鉴定染色体倍性。发现菱属有二倍体和异源杂交起源的四倍体二种倍性存在，四倍体有格菱（T. pseudoincisa）、丘角菱（T. japonica）、弓角菱（T. arcuata）和兴凯菱（T. khankensis）4个物种和四角矮菱（T. natnas var. pumila）、南昌格菱（T. pseudoincisa var. nanchangensis）二个变种和一个待定种；四倍体物种仅分布于东亚；菱属起源中心在东亚的长江流域、黑龙江和图们江流域和日本；黑龙江流域是最北部的冰期避难所；不同流域之间的基因流屏障对菱属的系统进化有重要意义；菱角的角数、果冠的大小及菱角本身的突起等均可作为形态分类性状。