An Integrated Taxonomic Study Of Primula Merrilliana Species Complex

Species is a separately evolving lineage in morphology,genetics,and niche.An accurate understanding of species diversity is essential for studies across a wide range of biological subdisciplines.However,delimiting species remains a challenge in evolutionary radiations,particularly in those herbaceous plants associated with micro-endemic,naturally fragmented distribution systems,where genotypic and phenotypic traits likely evolved discordantly,so that species identification is much more difficult.The Primula merrilliana complex,which is endemic to hilly and mountain areas in southern Anhui and northwest Zhejiang has high horticultural using value,used to be treated as one species but several clues suggested it may be composed of multiple species.Here we used multiple lines of evidence to test how many independently evolving lineages exist within the P.merrilliana complex to provide empirical research cases for the integrative taxonomy.The main research contents and results are as follows:Based on intensive field investigations in recent years,48 populations within the P.merrilliana complex were found.These populations covered the whole distribution area of the complex,of which 38 populations were distyled,and 10 populations were homostyled(6 long homostyled and 4 short homostyled).Firstly,the phylogenetic relationship of the populations was constructed and the operational taxonomic units(OTUs)were identified using ABGD,GMYC,and BP&P methods based on three sequence segments(ITS,trn L-F,and trn S-G).In addition,a total of 965 individuals from 36 populations representing the distributions and floral variations in each clade were sampled for detecting the population genetic differentiation degree among optimal OTUs and floral types based on SSR.Results showed that the complex could be divided into 4 OTUs(I–IV)which corresponded to the four clades recognized by the gene tree.There were 3 long homostyled populations and 8 distyled populations in OTU I located in the northwest corner of the distribution(Qiupu River Basin).OTU II included not only long homostyled and distyled populations but short homostyled populations,which had a wide distribution range(located in Anhui province and Fuyang city,Zhejiang province).OTU III and OTU IV were all distyled and restricted to a very narrow region(about 200 km~2)in the border area of Huangshan district and Yi county in the north of Huangshan Mountain.Average estimates of genetic diversity of disytled populations were generally high and significantly higher than that of homostyled populations within the optimal OTU and they have showed certain genetic differentiation.The morphological characteristics of 22 populations(14 distyled and 8 homostyled)representing 4 optimal OTUs were observed and analyzed.The principal component analysis of morphological variation was conducted based on three morphological data matrixes(total morphological traits,reproductive traits,and vegetative traits).R package mclust version 5.0 was used for cluster analyses.One-way ANOVA was used to test the statistical significance of morphological differences among recognized species using SPSS.Results showed that based on all 20 morphological traits datasets,there was strong support for three morphological clusters(?BIC<2.02)with 7.59%of individuals being misclassified.Under this scenario,all homotyled populations were grouped together and distyled populations of OTU I were separated from the other distyled populations.If only12 reproductive traits were taken into account,a two-cluster model was well supported(?BIC<5.41),i.e.homostyled group and distyled group,with only 2.17%of individuals being misclassified.When only using 7 vegetative traits,there were also two clusters.OTU I could be separated from the other OTUs(?BIC<6.32)but with relatively high misclassified individuals(13.02%).The artificial crossing of the above representative populations(except for CG and LHT due to the relatively small population size),including crosses within populations,among populations within,and among optimal OTUs,were conducted.Their fruit set,the size of seed,and seed viability were measured to examine the strength of postmating isolation among OTUs and between homostyled and distyled populations.Results showed that OTUs I and other OTUs have accumulated complete reproductive isolation.Within OTU II,the two homostyled populations in Zhejiang(DSC and BDS)were completely reproductive isolated from the other populations in Anhui.OTUs III and IV have formed complete reproductive isolation with other populations in the complex,but there was no obvious reproductive isolation between themselves.Niche differentiation among optimal OTUs,morphological species and biological species was explored based on 19 environmental factors and geographic data.Results showed that the two long-homostyled populations(BDS and DSC)in Zhejiang province occupied different niche from other population in the complex.While there was no significant niche differentiation among the populations in Anhui province.Our results indicated that P.merrilliana complex can be recognized four separately evolving metapopulation lineages:Primula qiupuensis J.W.Shao,P.merrilliana Schltr,P.zhexiensis X.He&J.W.Shao sp.nov.,P.wannanensis J.W.Shao,sp.nov..The extensive variation of breeding system,especially the floral morph transition from distyly(crossing)to homostyly(selfing)multiple times possibly promoted the rapid speciation within such a small geographic scale.This study case demonstrated that the genetic highly divergent but morphology indistinguishable possibly exhibited in herbs with fragment distributions,which led to the difficulties in species delimitation.Thus we highlight the importance of incorporating other characters,such as postzygotic reproductive isolation and geographic data,with commonly used molecular and morphological traits to infer species boundaries through an integrative taxonomic approach in such systems.