Phylogenetic Reasearch On Lactarius In Yunnan Province

Lactarius commonly known as milky fungus,which is a member of Basidiomycetes,Hymenomycetes,Agaricales,Russulaceae.The largest characteristic of species is when they have been damaged the sporocarp of this genus will excreting milk-like fluids.As the most common fungi in Yunnan province the wild yield and species of Lactarius are also huge.Lactarius is a common trade fungus in the market,most spcies of Lactarius can be eaten,but some species are not edible.The correct understanding of species and their phylogenetic relationships is the basis of sustainable utilization for wildlife resources.However,there are many species of Lactarius,and the morphological characteristics of them are deeply influenced by environment.Therefore,the classical classification based on morphological features may not be accurate,so it is necessary for us to make in-depth research of it.Nowadays,molecular biology is the most reliable technique for fungal identification and phylogenetic analysis.Except as a testimony of traditional taxonomy this technology is also a supplement and revise of classical classification.In this study,41 samples from different regions of Yunnan Province were sequenced by clone sequencing method.The molecular identification of the samples was carried out by blast based on the sequencing results of two sequences.Molecular identification through ITS,LSU sequencing blast and Phylogenetic analysis on them though maximum likelihood and neighbor-joining.Phylogenetic trees will be established based on ITS and LSU sequences,including constructed respectively and co-constructed.The main conclusions are as follows:(1)The results of molecular identification showed that the samples SP15,SP27 and SP28 were L.corrugis subgroup,L.vellereus voucher,L.akahatsu,and the support rate of LSU sequences is highly.According to ITS identification results,SP18,SP19 and SP25 were renamed as L.rubrocorrugatus,L.kesiyae,L.hygrophoroides.Based the conclusion of ITS identification sample SP1,SP6 and SP37 are L.deliciosus,just as the results of morphological identification.SP3?SP4?SP5?SP7?SP17 are L.hatsudake.Based on morphological and molecular identification results SP24 and SP39 are L.volemus.The results of comprehensive morphological identification and ITS comparison showed that SP33,SP40 are L.hirtipes.According to the results of molecular identification.SP21 and SP41 similar species of L.Glaucescens.SP20 and SP36 are Lactifluus Glaucescens which has a highly ITS blast supportion.Some samples were found not belong to the fungi of the genus Lactarius.The ITS sequence showed that SP23 and SP35 Russula delica,which belongs to Russula;SP13 and SP32 are fungi of the mushroom,but they could't be named accurately;SP29 and SP30 could't be named too.Compare with the ITS fragment LSU sequence is more conservative,so it's not suitable to the identify Lactarius by using LSU sequence.In the Gene Bank of NCBI the ITS sequence data is more perfect than the LSU sequence data.On the other hand,variation sites of ITS sequences are also huge.In the phylogenetic tree which was builted based on ITS fragment has the same results with molecular identification,so it's suggested that ITS fragments as the main fragment for Lactarius phylogenetic analysis and study.(2)The result of Molecular systematics study:Different phylogenetic tree would be get if we creat it based on different single gene sequence.Based on the LSU sequence,we would get a phylogenetic tree which is really different from Molecular identification.Most of the developmental tree based on ITS sequence coincide with the identification results.And if we creating a phylogenetic tree withing these two sequences the conclusion was more reliable.In the phylogenetic tree which was established by ITS and LSU fragment shows that sample SP3-7,SP7,SP17(L.hatsudake);SP11 and SP25(L.hygrophoroides);SP12,SP22(Lactifluus cf.luteolus);SP33,SP40(L.hirtipes);SP20,SP36(Lactifluus glaucescens);SP21,SP41(Lactifluus aff.glaucescens)gather in the same branch,just as the resslt of morphological and molecular identification.According the tree except SP34?SP37 all sample of L.deliciosus were in the same branch and it also has a highly support rate.SP13 can't be re named accurately by using molecular identification method.It can only be determined that it is a member of Russulas.However,according to phylogenetic tree,it is speculated that SP13 is Russula delica,no matter which sequence is used,this sample is always combined with SP23(Russula delica)in the same branch.For L.deliciosus sample SP1?SP2?SP6 and L.hengduanensis(SP27,SP31)were always divided into the same branch,the support of this branch was higher than 70%.Although SP29,SP37,SP34 and SP28(SP34 and SP37 are L.deliciosus)belong to different species of Lactarius,but they are in the same group no matter which method we used to build phylogenetic trees.Morphological identification results of SP15 is L.volemus,but according to the molecular identification results SP15 is L.corrugis.In each of the different phylogenic trees,it could be combined with the SP24(L.volemus)each time,and he remaining two samples of L.volemus(SP38,SP39)were classified in one branch each time.There may be some homology among SP10(L.pilosus)SP27(L.vellereus),through the collection site of these two samples was different they always get together in the same group.The division of SP18 and SP30 is also the same.Based on different method SP14(L.angustizonatus)and SP19(L.kesiyae)would be divided in different group.Perhaps interspecific difference among SP15?SP30 and other samples is too great so no matter we use each methord to building a phylogenic trees they are always divided from other samples.