Reproductive isolation is a fundamental require- ment for speciation and includes several sequential stages. Few studies have determined the relative contributions of pre- and post-zygotic reproductive isolation in plants, especially between relative species with clear differentiation in flower form. To investigate the mechanisms responsible for repro- ductive isolation in sympatric A4ussaenda pubescens var. alba and Mussaenda shikokiana （Rubiaceae） in Guangxi Province, China, we made observations of flowering phenology, patterns of insect visitation, and conducted pollination experiments, including artificial hybridization. The two species had over- lapping flowering times and were pollinated by overlapping pollinators; however, their relative importance differed significantly with M. pubescens visited more commonly by bees and M. shikokiana more frequently by butterflies. Using vegetative and floral characters and molecular evidence based on nuclear ribosomal internal and external transcribed spacer regions we detected seven naturally occurring hybrids among a sample of approximately 125 individuals. Hybrids were characterized by morphologies that most closely resembled their maternal parents based on chloroplast evidence. Studies of artificially synthesized and natural hybrids demonstrated that hybrid seed had very low germination rates and naturally occurring hybrids exhibited pollen sterility. Post-zygotic reproductive isolating mechanisms play a primary role in limiting gene exchange between co-occurring species and maintaining species integrity in areas of sympatry.
Reproductive isolation defines the biological species concept and plays a key role in the formation and maintenance of species. The relative contributions of different isolating stages has been suggested to be closely associated with phylogenetic relatedness. Few studies have focused on the relative contributions of pre- versus post-zygotic mechanisms, and even fewer have been conducted under strict phylogenetic frameworks. Pre-and post-zygotic reproductive isolation stages have been investigated in the sister species Mussaenda kwangtungensis and M. pubescens var. alba. The two species have partly overlapping distribu-tion ranges and flowering times, while the principal pollinators differed strikingly for them, demonstrating strong pre-zygotic isolations. Natural hybrids were detected by simple sequence repeat markers and their maternal parents were identified based on chloroplast gene sequen-ces. Five out of 81 individuals were suggested to be hybrids that fall into the categories F2, BC1, and BC2 by the NewHybrids analysis. Interspecific crossings resulted in significantly reduced fruit set and seed germination rates. Phylogenetic analysis revealed short Kimura-2-parameter distance between M. kwangtungensis and M. pubescens var. alba. These findings strongly supported the hypothesis that for species with a closer phylogenetic relationship, pre-zygotic isolation plays an important part in limiting gene exchange in sympatric areas.
Natural hybridization frequently occurs in plants and can facilitate gene flow between species, possibly resulting in species refusion. However, various reproductive barriers block the formation of hybrids and maintain species integrity. Here, we conducted a field survey to examine natural hybridization and reproductive isolation （RI） between sympatric populations of Primula secundiflora and P. poissonii using ten nuclear simple sequence repeat （SSR） loci. Although introgressive hybridization occurred, species boundaries between P. secundiflora and P. poissonii were maintained through nearly complete reproductive isolation. These interfertite species provide an excellent model for studying the RI mechanisms and evolutionary forces that maintain species boundaries.
Yanping XieXingfu ZhuYongpeng MaJianli ZhaoLi LiQingjun Li
