| Natural hybridization is ubiquitous in plants and is an important way of speciation.Reproductive isolation is a key step in the process of hybrid speciation.Calanthe is a genus of the tribe Collabieae,Epidenroideae,Orchidaceae.The Chinese Calanthe species are mainly distributed in the south of the Yangtze River.We discovered a new species,C.sieboldopsis,in a valley in Jinggangshan,Ji’an,Jiangxi Province.The morphological characteristics of its flowers are between those of C.sieboldii and C.graciliflora,which are distributed in the same region.The flowering periods of the three species are overlaped,and we recorded pollen flow from C.sieboldii to C.graciliflora.Therefore,it is inferred that C.sieboldopsis may be a hybrid of C.sieboldii and C.graciliflora.This study aim to determine whether there is natural hybridization between C.sieboldii and C.graciliflora by integrating evidences from morphological characteristics,strength of prezygotic and postzygotic isolations,and genetic variations among the two species.Firstly,we compared the the morphological characteristics among the three Calanthe species.Secondly,to examine the strength of prezygotic isolation,we recorded the flowering phenology,observed pollinator behaviors,and conducted breeding system experiments.Thirdly,to examine the strength of postzygotic isolation,we assessed the fitness of hybrids reproducted from artificial pollination experiments.Finally,we sequenced the chloroplast genes(mat K,ycf1 and rps16-trn Q-UUG)and nuclear genes(ITS)of C.sieboldii,C.sieboldopsis and C.graciliflora,and determined potential hybridization events of the two Calanthe species.The main conclusions of this study are as follows:(1)Morphological characteristics.We found that the leaves,fruits,plant heights,and inflorescences of the three species were similar,but there were significant differences in the size of the flowers.Moreover,the 13 flower features of C.sieboldopsis are between those of C.sieboldii and C.graciliflora,including the length and width of dorsal sepal,the length and width of lateral sepals,the length and width of lip,the length and width of petals,the length and width of mid-lobe,the length and width of lateral lobe and spur length.The morphological characteristics suggest that there might be natural hybridization among the three Calanthe species.(2)Prezygotic isolation.The distribution areas and flowering phenology of the three Calanthe species are overlaped.The pollinators of C.sieboldii are three carpenter bees: Xylocopa chinensis,X.rufipes and X.appendiculata.The pollinators of C.graciliflora are Melea mangkamensis and Bombus flavescens.We did not observed the transfer of pollinaria of C.sieboldii to the column of C.graciliflora mediated by Xylocopa spp.We did recorded that Melea mangkamensis could transfer the pollinaria of C.graciliflora to the stigma of C.sieboldii.These indicate that the prepollination-prezygotic isolation is relatively weak in C.sieboldii and C.graciliflora.In addition,the pollinarium of C.sieboldii is significantly larger than the opening of the flower of C.graciliflora,thus the transfer of pollinaria from C.sieboldii to C.graciliflora are unlikely to happen under natural conditions.Therefore,there might be asymmetry natural hybridization between C.sieboldii and C.graciliflora.The overlapping distribution area and flowering period phenology and shared pollinators provided the possibility of natural hybridization between C.sieboldii and C.graciliflora.(3)Postzygotic isolation.The fitness study showed that the hybrids of C.sieboldii and C.graciliflora(P1♀ × P2 and P2♀ × P1♂)could produce viable and germinating seeds,indicating postzygotic isolation is relatively weak among the two Calanthe species;The progeny of the P1♀ × P2 have higher fitness than the progeny of the P2♀× P1 ♂,indicating that the two have different affinities;The progeny of the hybridization of C.sieboldopsis and C.sieboldii(Hy♀×P1♂)had higher fitness than the progeny of the cross with C.graciliflora(Hy♀×P2♂),indicating that the fitness of backcrossing is asymmetric;The postzygotic isolation between C.sieboldii and C.graciliflora is weak,which led to the easy occurrence of natural hybridization between the two.(4)Analysis of the chloroplast genomes.The three Calanthe species had little difference in the size of the chloroplast genomes,GC contents,and the boundaries of the IR region.The phylogeneny based on the whole chloroplast genomes of 9 Calanthe species and other 6 orchids showed that C.sieboldii,C.sieboldopsis,and C.graciliflora were clustered into a branch with a bootstrap value of 100%.However,C.sieboldii and C.sieboldopsis are very closely related with little variations between their chroloplast genomes.And C.graciliflora has certain differentiation from C.sieboldii and C.sieboldopsis.(5)Hapotypes of ITS and cp DNA regions.From the analysis results of the nuclear gene sequence(ITS)and the chloroplast gene sequence(mat K,rps16-trn Q-UUG,and ycf1),it can be seen that C.sieboldii and C.graciliflora have their own specific chloroplast haplotypes,and most individuals of C.sieboldopsis have the same haplotype of C.sieboldii.This indicated that C.sieboldii and C.graciliflora were the parents of C.sieboldopsis.And the natural hybridization of C.sieboldii and C.graciliflora had asymmetry. |
