| Reproductive isolation,the result of accumulation and interaction of multiple isolating barriers,has been thought to be crucial for maintaining species boundaries and integrity.These reproductive barriers at different stages are generally classified as pre-,post-pollination or postzygotic barriers.Previous studies suggest that species pairs with specialized pollination systems may have strong prepollination isolation and weak or no postpollination isolation.However,for species pairs with generalized pollination systems,prepollination isolation is relatively weaker as sharing pollinators may involve interspecific pollen transfer in which strong postpollination isolation becomes essential to prevent hybrids.However,reproductive isolation may act as the by-product of speciation and the strength of reproductive isolation among species could be determined by genetic distance.While nectarrewarding orchids tend to involve relatively generalized pollination systems,nectarless orchids tend to be specialized and involve deceptive pollination.Here we investigated 19 Dendrobium species to explore whether patterns of reproductive isolation relate to the pollination systems.Dendrobium species are epiphytic orchids with economic,medicinal and ornamental values,mainly distributed in Southeast Asia,where several species usually co-flower on the same host plant.Previous studies focusing on the reproductive biology report that deceptive pollination systems commonly occur in Dendrobium species.However,reproductive isolation mechanisms among Dendrobium species have not been systematically studied.We investigated 19 Dendrobium species in an orchid breeding station in Malipo,Yunnan Province and observed pollinator visits and conducted thousands of hand pollination experiments for three years,to quantify isolation barriers at six stages during sexual reproduction.Main results are below:1.To investigate whether flowers of Dendrobium species produce nectar,we randomly bagged inflorescences of the 19 species at bud stage and measured nectar volume and sugar concentration when flowers opened.Results show that 10 Dendrobium species produce little nectar(0.45-2.78 μL),with sugar concentration varying from 8.14%to 30.75%,while the other nine species produce no nectar.By reconstructing the phylogenetic tree,we conclude that nectar transition occurs within Dendrobium,from nectar-rewarding species to nectarless species,and from nectarless species to nectar-rewarding species.2.To estimate the strength of geographic isolation,we compared the overlap in distribution of 15 Dendrobium species.Location information of populations of each species was from records in the Global Biodiversity Information Facility(GBIF).We calculated the strength of geographic isolation,absolute and relative contributions to total reproductive isolation(RI).Results show that geographic isolation(RIgeography:0.5-1.0)is strong in most of 46 species pairs.Three-quarters of RI values are over or equal to 0.80.Geographic isolation has the highest absolute and relative contributions,indicating that geographic separation acting as the original factor plays a crucial role in isolating these species.3.To estimate the strength of phenological isolation,we recorded the flowering periods of 15 Dendrobium species in the field in 201 8 generally.We counted overlaps of flowering periods between species and calculated the strength of phenological isolation,absolute and relative contributions to total RI.Results show that RIphenology of 46 species pairs varies from 0 to 0.788.Two-thirds of RI values are less than or equal to 0.5.Phenology isolation has smaller absolute and relative contributions.4.To explore postpollination isolation mechanisms between species,we conducted 46 unidirectional species pairs in total under interspecific pollination treatment and quantified strengths of seven isolation barriers:fruit set,fruit size,fruit weight,seed set,embryo size,seed germination rate,F1 height.Results show that 24 of the 46 species pairs are completely isolated at fruit-set stage and most occur among distantly related species.To a certain extent,for most of species pairs,reproductive isolations of the remaining isolating barriers contribute to total isolation.However,no isolations of these postzygotic barriers occur among certain species pairs.Of the seven postpollination isolating barriers,fruit set(RIfruit set:-0.038-1.0)has the highest absolute and relative contributions to total isolation.5.We quantified the strength of prepollination(geography,phenology)and postpollination isolation barriers(including seven barriers).Results show that the mean absolute contribution of prepollination isolation of 46 species pairs(RIprepollination:0.563-1.0)to total isolation is greater(0.90069),while that of postpollination isolation(RIpostpollination:0-0.437)is smaller(0.09336).Prepollination isolations between species are strong,however,postpollination isolation barriers are indispensable.6.We reconstructed a phylogenetic tree of 15 Dendrobium species and conducted analyses of bivariate correlation between pairwise genetic distance and strength of each isolation barrier.Results show that strength of geographic isolation is negatively correlated with genetic distance,and strengths of reproductive isolation at fruit set stage and seed set stage are positively correlated with pairwise genetic distance.No significant correlations are observed between genetic distance and strengths of other six isolation barriers.The results indicate that reproductive isolation between closely related species is primarily maintained by geographic isolation.Nevertheless,reproductive isolation between sympatric distant species could be maintained by isolation at fruit set and seed set stages.We analyzed patterns of reproductive isolation between eight nectar-producing and seven nectarless species.Our results show that prepollination isolation(geographic isolation)still contributes most to total isolation for species pairs with different pollination systems,indicating that patterns of reproductive isolation in this genus are not affected by pollination systems.Strengths of postzygotic isolation among species are mainly determined by pairwise genetic distance. |
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