Species Delimitation In The Subfamily Coryloideae Of Betulaceae In China

As the cornerstone for each biological branch,species delimitation is highly critical in modern taxonomy.The subfamily Coryloideae of the Betulaceae comprises four distinct genera,Corylus,Ostryopsis,Ostrya and Carpinus.In this study,we conducted the preliminary species delimitation for these genera based on the intergrative species concept and multiple lines of evidence from DNA sequence variations,morphological gaps at the population level and other lines of evidence.Main results were summarized as follows.1)We examined sequence variation of two maternally inherited chloroplast DNA fragments and AFLP polymorphisms of 32 populations for three species of Ostryopsis,O.davidiana,O.nobilis and O.intermedia respectively distributed in northern China and southeast Qinghai-Tibet Plateau.Each of two distinct cpDNA haplotype groups was fixed by O.davidiana or O.intermedia respectively while both were found in O.nobilis.All analyses of AFLP polymorphisms delimitated three species very well and suggested that O.intermedia is more closely related to O.davidiana than to O.nobilis.Bayesian clustering analysis(K = 2)further suggested that the genomic composition of O.intermedia showed a slightly mosaic origin from the other two species although mostly from O.davidiana.The origin of O.intermedia through hybrid speciation due to southward migration of O.davidiana and the following hybridization with O.nobilis may provide the most reasonable explanation for the inconsistence between phylogenetic relationships of three species and degrees of genetic diversity from cpDNA sequence or AFLP dataset.Flowering times and inter-/intra-specific pollination experiments of three Ostryopsis species in a common gardern by transplanting the northern O.davidiana to the southern sympatric site of the other twos suggested clear pre-pollination and post-pollination reproductive isolations between threes.Based on this and previous studies,these three species are welldelimitated and may represent ‘good species' supported by multiple lines of evidence from morphological and genetic variarions(ITS,SSR and AFLP),niche divergences and reproductive isolations.This case study further suggests DNA polymorphisms should be widely used for species delimitation.Evidence from morphological and population genetic variation should be basic method to conduct the species delimitation using the intergrative species concepts because of the universality and practicability.2)We collected materials for 110 individuals from 22 natural populations of five species of the genus Ostrya for species delimitation in China based on DNA sequence variations.We sequenced four chloroplast DNA fragments(trnH-psbA,trnL-trnF,rps16 and trnG)and the nuclear ITS region for all samples.Sequence variations of Ostrya from four cpDNA fragments identified three groups that showed no correspondence to any morphological delimitation because of the incomplete lineage sorting and/or possible inter-specific introgression in the history.However,phylogenetic analyses of ITS sequence variations discerned four species,O.japonica,O.rehderiana,O.trichocarpa and O.chinensis while O.yunnanensis nested within O.chinensis.Morphological clustering also discerned four species and showed the complete consistency with DNA sequence variations.Moreover,our phylogenetic analyses based on ITS sequence variations suggested that O.trichocarpa comprised an isolated lineage different from the other Eurasian ones.Based on these results,hop-hornbeams in China should be treated as four distinct species.Our results further highlight the importance of ITS sequence variations in delimitating and discerning the closely related species in plants.3)We used 1609 individuals from 181 populations of eight species(including two varieties)for species delimitations of the genus Corylus in China.We collected population data based on ITS sequence variations and 10 SSR loci,morphological variations at the population level and niche divergences between assumed species.Our analyses of ITS sequence variations firsly identified three well delimitated monophyletic groups corresponding to morphological delimitation of C.sieboldiana,C.ferox and C.fargesii.The fourth monophyletic group(better treated as one species complex)comprised C.yunnanensis,C.wulingensis,C.heterophylla var.heterophylla and C.heterophylla var.sutchuenensis.Two monophyletic groups were identified for sampled individuals of the Chinese hazel.The cloned ITS sequences of C.wangii comprised two distinct clades with respective relationships with twomonophyletic groups.Sequence variations of two cpDNA fragments identified four groups that showed no correspondence to morphological delimitation and to the ITS results,but are consistent with four geographical regions fron north to south.Our population genetic data based on SSR data and niche divences further suggested that the species complex(by ITS)should be divided into two tentative species: C.yunnanensis and C.heterophylla,and C.wulingensis should be reduced into C.heterophylla.C.wangii is not an isolated and evolving lineage and individuals ascribed to it are hybrids from between C.yunnanensis and C.ferox.SSR data,morphological clustering and geographical distributions supported that two separated species should be acknowledged within the Chinese hazel.In total,7 species can be recognized although further taxonomic revisions should be made afterwards.4)We firstly established two new species for the genus Carpinus(C.langaoensis and C.tibetana)in China based on ITS sequene variations,morphological variations at the population level and niche divergences.We then collected ITS sequences from445 inidivudals of 89 popluatlions of all 30 species in China.Our comprehensive analyses suggested 9 species can be well delimitated within the genus based on sequence variation of this nuclear DNA fragment and morphological variations and others are tentatively treated as three species complexes without clear species delimitation within each of them.