Molecular Systematics Research Of The Genus Farnicus (Grassaceae: Bamboo Subfamily)

Reproductive characteristics are most important in plant taxonomy.The majority of the woody bamboo species were described frequently without knowledge of the flowers because they have relatively long blooming intervals and vegetative features are more useful for identification in these infrequent flowering plants.Because the vegetative characters used for identification are likely to be subtle and variable in most cases,it is difficult to say how much they reflect the true evolutionary history of the organisms and it has not been proven either the vegetative-feature-based infrageneric taxonomy or the relationships between genera are cladistically sound.In other words,it is not clear whether diversification and evolution within the bamboo genera was based on the variation of reproductive characteristics,of vegetative characteristics,or both.The genus Fargesia Franchet is a good system to address this issue because it is one of the largest genera in woody and alpine bamboos,whose members have various morphological characteristics,wide distributions.Meanwhile,a vegetative feature-based infrageneric category has been established for this genus.Fargesia belongs to Tribe Arundinariinae,Bambusoideae,Poaceae or the temperate linage of woody bamboos.The species of this genus are widely distributed in medium altitude mountains from southwest China to east Himalaya.They are the main food of gaint panda,and play an important role in the local eco-system.After Fargesia was established,there had been a controversy of its classification and range of the genus.Some authors argued that Fargesia should be merged to other genera while others suggested the some species of other genera should be included into Fargesia or exclusion of some species to insure its monophyly.Yi(1988a,1996)once classified Fargesia into 2 sections and 6 series based on vegetative characteristics.However,only a few molecular systematic studies have been conducted to delimitate the range of Fargesia,and to infer specific composition and infrageneric relationships.In this study,thirty-seven species of Fargesia from Yi's(1988a,1996)infrageneric category,including the species transferred into Drepanostachyum,Himalayacalamus or Thamnocalamus by other authors were selected as ingroups,nine species from Yushania as the most closely related genus,two species from Thamnocalamus in the same subtribe Thamnocalaminae and two species from Pleioblastus in the same tribe Arundinarieae were selected as outgroups.Measurement and analysis of DNA sequence data analysis,together with morphological characters,were used to solve the range of Fargesia,specific composition and infrageneric relationships,and to look at the role of vegetative organs and reproductive organs in the Fargesia species diversity as well.The results are as follow:1.Measurement and analysis of 45 morphological characters of 37 Fargesia species showed that the species of different sections and series that Yi(1988a,1996)established were intersected,which does not support the sections and series that Yi established.2.Maximum parsimony,maximum likelihood and Bayesian analyses were conducted using separate and combined data sets of three cpDNA(matK,psbA-trnH and trnL-F),nrITS and rpb2 sequences,respectively.In all the phylogenetic trees,Pleioblastus amarus(Keng)Keng f.and P.gramineus(Bean)Nakai formed the basal clade;F.crassinoda Yi(=Th.spathiflorus(Trinius)Munro var.crassinodus(Yi)Stapleton)and F.damuniu Yi together with two Thamnocalamus species formed the Thamnocalamus clade;four species,once were transferred to Drepanostachyum or Himalayacalamus,including F.ampullaris Yi(=D.ampullare(Yi)Stapleton),F.semiorbiculata Yi(=D.semiorbiculatum(Yi)Stapleton),F.gyirongensis Yi(=H.falconeri(Munro)Keng f.)and F.collaris Yi(=H.collaris(Yi)Ohrnberger)formed the third clade Drepanostachyum clade;the remaining species of Fargesia and all species of Yushania formed a Fargesia + Yushania clade,except for the cpDNA trees,the Drepanostachyum and Fargesia + Yushania clades were not well resolved.On the MP trees obtained based on the the combined cpDNA,nrITS and rpb2 sequence data,the Fargesia + Yushania clade was first identified as a strongly statistically supported Clade A and B(MPB = 100,MLB = 96,PP = 0.99).The monophyly analysis showed that Clade A and B were monophyletic groups.Species in Clade A were subdivided into 4 subclades.Clade B was subdivided into subclades B1 and B2 with high statistical support(MPB = 100,MLB = 60,PP = 0.90)as well as in other trees based on the combined DNA sequence data.Yushania species did not form a monophyletic group but collapsed with Fargesia species in subclade B2.The comparison of the morphological characters,anthesis and distribution pattern showed that in the Fargesia + Yushania clade,the inflorescences are on top of leafy branches,and in Clade A,the inflorescences are compressed,unilateral and subtended by a series of leaf sheaths which expand as spatbes,of which the uppermost is generally longer than the inflorescence;the pistil has three stigmas.The species in Clade A species bloom mainly in spring in March to May,and occupy the northern part of the distribution of the Fargesia + Yushania clade.In Clade B,the species have open inflorescences,subtended by more or less expanded but nor spathe-like leaf sheaths that are always shorter than the inflorescence,or part of the inflorescences on a culm are unilateral,the pistil of most species have two stigmas.The species in Clade B species bloom mainly from April to August(rarely to September)and occupy the southern part of the distribution of the Fargesia + Yushania clade.The conclusions are as follow:1)Yi's(1088a,1996)Fagesia is polyphyletic,some species once were thought to belong to Fargesia are not the species of this genus,F.crassinoda(=Th.spathiflorus var.crassinoduns)and F.damuniu are Thamnocalamus species.F.ampullari.s(=D.ampullare),F.semiorbiculata(=D.semiorbiculatum),F.gyirongensis(=H.falconeri)and F.collaris(=H.collaris)are Drepanostachyum species.2)DNA sequence analysis shows that Yushania species are inserted into a branch of Fargesia and this indicated that the Yushania does not exist,and Yushania species should be classified as Fargesia.3)The range of Fargesia should include most species of Fargesia and the entire species of Yushania.F.damuniu,F.crassinoda,F.semiorbiculata,F.collaris,F.ampullaris and F.gyirongensis were excluded,while F.fractiflexa Yi and F.membranacea Yi should be included in Fargesia.The main phenotypic character of Fargesia are:rhizomes pachymorph,with short or long neck,the inflorescences are subtended by a series of leaf sheaths expanded more or less as spathes,or the open inflorescences are subtended by more or less but never spathe-like expanded leaf sheaths.4)For the infrageneric relationship of Fargesia,Fargesia species were divided into two clades,Clade A(F.spathacea Clade)and Clade B(F.communis Clade).In Clade A,the inflorescences are compressed,unilateral and subtended by a series of persistent and delicate leaf sheaths which expand as spathes;the pistil has three stigmas.In Clade B,the open inflorescences are somewhat subtended but never spathe-like expanded leaf sheaths;the pistil has two or rarely two to three stigmas.In Clade A,the sheath scar almost equals the nodal ridge in species of subclade A1,while more prominent and ridged in that of subclade A2-4.In Clade B,the culm buds are semiorbiculate and unappressed in subclade B1,while in subclade B2,all species have long ovoid and appressed culm bude.Apart from subclade B1,relationship among species in each sub-clade are uncertain,complex was formed in each subclade.5)The morphological characteristics of the A and B clades dapted to its geographic environment.In Fargesia,species in Clade A are mainly distributed in north part of the Fargesia distribution area,and those in Clade B in south part.In Clade A,the compressed,unilateral inflorescences subtended by spathe-like leaf sheathes might be the adaption to the cold and dry weather in north.In Clade B,the open inflorescences and long rhizomes neck might be the adaption to the warm and moist weather in south.Because of thefeatural differentiation,distribution area of bamboo species in Clade B,especially the species which originally belonged to Yushania,was enlarged.6)The analysis showed itlikely that the reproductive organs play an important role in the evolution and adaptation of Fargesia species divided into A and B clades,and the vegetative characteristics play an important role in the evolution within A and B clades.3.To verify the reliability of the species defined on the vegetation characteristics,the wide distribution species F.decurvata Lu and F.dracocephala Yi were selected.Ten populations of F.decurvata and F.dracocephala were investigated in their entire distribution.The result shows that there is no difference between the floral organs of F.decurvata and F.dracocephala,five major characters exhibited great variation not only at population level,but at individual level within a population,even the culm level within an individual and in different parts of the same culm.Cluster analyses based on 29 vegetative morphology characteristics showed that 10 populations of F.decurvata and F.dracocephala were not divided into two species,but they were well separated with outgroup.MP and NJ trees based on ITS sequences showed the same results with the cluster analysis on morphological characters.The results indicated the following conclusions:1)The publishment of F.dracocephala was the result of discontinuous sampling of F.decurvata,and F.dracocephala should be treated as the synonym of F.decurvata.The artificial isolation of character(s)lead to the result that a virtual species be defined as different ones.In the bamboo species definition,it is necessary to study as many collections as possible from whole area where a taxon may occur,and select suitable features for the definition.2)It's also shows that some Fargesia species that classification based on the vegetative morphology characteristics were unreasonable and these species may need the evidence of reproductive organs and the molecular systematics study.4.This article offered additional descriptions about reproductive organs characteristics of F.canaliculata Yi,F.qinlingensis Yi et J.X.Shao,F.funiushanensis Yi,and F.subflexuo.sa Yi and Drepanostachyum semiorbiculatum(Yi)Stapleton(=Fargesia semiorbiculata Yi).By verifying the reproductive organs,it was confirmed that the combination of D.semiorbiculatum is reasonable.In conclusion,the molecular phylogenetic studies and the reproductive characteristics analyses suggest that the classification of Fargesia based on the vegetative morphology characteristics were unreasonable and the feature of reproductive organs play an important role in Fargesia species evolution and species definition.Therefore,the current bamboo plant classified based on vegetative features may need further confirmation.