1、Research paper 研究论文 22 January 2023,42(1):252-262 菌物学报 Mycosystema ISSN1672-6472 CN11-5180/Q Doi:10.13346/j.mycosystema.220437 资助项目:国家自然科学基金(32170022,31493010,30970020,31760004);中国生态环境部生物多样性调查评估项目(2019HJ2096001006)This work was supported by the National Natural Science Foundation of China(32170022,3
2、1493010,30970020,31760004)and the Biodiversity Survey and Assessment Project of the Ministry of Ecology and Environment,China(2019HJ2096001006).*Corresponding author.E-mail:Received:2022-10-24;Accepted:2022-12-20 菌物学报 Copyright 2023 Institute of Microbiology,CAS.All rights reserved.|Http:/journals-T
3、el:+86-10-64807521 252 王向华 中国科学院昆明植物研究所副研究员。从事大型真菌系统分类学、生物地理学和群体遗传学研究。李玉乳菇(新种):欧洲槭乳菇在亚洲的近缘种 曹书琴1,王向华1*,秦位强2,吴芳3,刘铁志4,图力古尔5,李双龙6,陈作红7,刘冬梅8 1 中国科学院昆明植物研究所 东亚植物多样性与生物地理学重点实验室 云南省真菌多样性与绿色发展重点实验室,云南 昆明 650201 2 吉首大学张家界校区,湖南 张家界 427000 3 湖南省食用菌研究所,湖南 长沙 410013 4 赤峰学院化学与生命科学学院,内蒙古 赤峰 024000 5 吉林农业大学菌物研究所,吉
4、林 长春 130118 6 恩施土家族苗族自治州林业科学研究院,湖北 恩施 445000 7 湖南师范大学生命科学学院,湖南 长沙 410081 8 中国环境科学研究院,北京 100012 摘 要:槭乳菇 Lactarius acerrimus,乳菇中仅有的具有双孢担子的物种,曾经在中国被多次报道。我们在研究了该种的馆藏凭证标本后并未证实它在中国的分布,而是在中国发现了它的近缘种李玉乳菇 L.liyuanus(新种)。李玉乳菇的典型特征为:菌盖黄褐色具环纹、边缘具短毛,菌柄具窝斑,乳汁白色、不变色、具辣味,孢子具尖而高的条脊。它的担子为 4 孢,孢子较小具高而尖的条脊,这两点与槭乳菇明显不同。
5、文中提供了该种的条形码 ITS 序列、其他多基因序列和详细的形态描述,同时给出了对国内馆藏槭乳菇标本的重新鉴定结果。关键词:壳斗科;ITS;真菌区系;物种识别 Research paper 22 January 2023,42(1):252-262 Mycosystema ISSN1672-6472 CN11-5180/Q 菌物学报 253 引用本文 曹书琴,王向华,秦位强,吴芳,刘铁志,图力古尔,李双龙,陈作红,刘冬梅,2023.李玉乳菇(新种):欧洲槭乳菇在亚洲的近缘种.菌物学报,42(1):252-262 Cao SQ,Wang XH,Qin WQ,Wu F,Liu TZ,Bau Tol
6、gor,Li SL,Chen ZH,Liu DM,2023.New species Lactarius liyuanus in East Asia:a close relative of European L.acerrimus.Mycosystema,42(1):252-262 New species Lactarius liyuanus in East Asia:a close relative of European L.acerrimus CAO Shuqin1,WANG Xianghua1*,QIN Weiqiang2,WU Fang3,LIU Tiezhi4,BAU Tolgor5
7、,LI Shuanglong6,CHEN Zuohong7,LIU Dongmei8 1 CAS Key Laboratory for Plant Diversity and Biogeography of East Asia,Yunnan Key Laboratory for Fungal Diversity and Green Development,Kunming Institute of Botany,Chinese Academy of Sciences,Kunming 650201,Yunnan,China 2 Zhangjiajie Campus,Jishou Universit
8、y,Zhangjiajie 427000,Hunan,China 3 Hunan Edible Fungal Research Institute,Changsha 410013,Hunan,China 4 College of Chemistry and Life Sciences,Chifeng University,Chifeng 024000,Inner Mongolia,China 5 Institute of Mycology,Jilin Agricultural University,Changchun 130118,Jilin,China 6 Enshi Tujia and M
9、iao Autonomous Prefecture Academy of Forestry,Enshi 445000,Hubei,China 7 College of Life Sciences,Hunan Normal University,Changsha 410081,Hunan,China 8 Chinese Research Academy of Environmental Sciences,Beijing 100012,China Abstract:Lactarius acerrimus,the only milk cap with 2-spored basidia known,h
10、as been reported from China for several times.Examination on the voucher specimens,however,did not approve the presence of this species in China.Instead,we found a new species L.liyuanus,a close relative of L.acerrimus.It is recognized by the yellowish brown zonate pileus with hairy margin,scrobicul
11、ate stipe,white and unchanging latex and spores with high and acute ridges.It is easily distinguished from L.acerrimus by the 4-spored basidia and smaller spores with higher ridges.DNA barcode ITS sequences,additional multi-gene data and detailed morphological description are presented.Re-identifica
12、tion of the Chinese voucher specimens of L.acerrimus is given.Keywords:Fagaceae;ITS;mycota;species recognition INTRODUCTION In milk caps(Lactarius and Lactifluus),having 4-spored basidia is a rule for all species except for Lactarius acerrimus which strictly has 2-spored basidia producing the bigges
13、t spores among all milk caps(9 15812 m,Hesler&Smith 1979;Moser 1983;Heilmann-Clausen et al.1998;Basso 1999;Knudsen&Vesterholt 2008),and grows in deciduous woods(especially with oaks and hornbeams).The irregular cap and anastomosing gills are good field characters.Originally described from Bayern,Ger
14、many,L.acerrimus is mostly found in warm environment in Europe and uncommon in Nordic countries(Heilmann-Clausen et al.1998;Knudsen&Vesterholt 2008).Lohwag(1937),for the first time reported L.acerrimus in China,based on a specimen collected from“Nguluk”,a village near“Lidjiang”(Lijiang)in northweste
15、rn Yunnan.Zang(1996)listed four specimens collected from Lijiang under L.acerrimus.Yuan&Sun(1995),Zhuang(2005)and Chen&Zhang(2020)added Sichuan,Shaanxi,Gansu and Hunan to the known distribution of L.acerrimus in China.In the“Redlist of Chinas Biodiversity Macrofungi”,CAO Shuqin et al./New species La
16、ctarius liyuanus in East Asia:a close relative of European L.acerrimus 研究论文 菌物学报 254 L.acerrimus is ranked as“Least Concern”.Our more than twenty years intensive field investigation in northwestern Yunnan,however never found milk caps that perfectly fit L.acerrimus in morphology.Instead,at quite man
17、y places in northern,central and southern China,we collected quite many specimens that could be confused with L.acerrimus in the field but have 4-spored basidia and smaller spores.BLASTn of ITS sequences of these specimens also suggests a close relationship with L.acerrimus.Analysis on the morpholog
18、y and DNA sequence data of these specimens and re-examination on Chinese voucher specimens under L.acerrimus excluded the presence of L.acerrimus in China and discovered a new species as its counterpart in Asia.1 MATERIALS AND METHODS 1.1 Morphology Morphological study basically followed Wang(2017).
19、For the holotype,40 spores were measured.Voucher specimens are deposited in the Cryptogamic Herbarium,Kunming Institute of Botany,Chinese Academy of Sciences(HKAS section,KUN),Mycological Herbarium of Hunan Normal University,China(MHHNU)and Mycological Herbarium of Chifeng University,China(CFSZ).1.2
20、 DNA extraction,PCR amplification,and Gene cloning DNA extraction and PCR amplification followed Wang(2017).When PCR of the whole ITS region failed,primer pairs ITS1F(or ITS5)+5.8S and ITS3+ITS4 were used to amplify the ITS1 and ITS2 regions separately.Following the recommendation for publishing new
21、 fungal species version 3.0(Aime et al.2021),we sequenced the following secondary barcode loci for some of the specimens:LSU,ef1a,rpb1 and rpb2.General primers published by Liu et al.(1999),Moncalvo et al.(2000),Matheny et al.(2007)and Morehouse et al.(2003)were used to amplify rpb2,LSU,rpb1 and ef1
22、a respectively.When PCR using these general primers failed,we used the following primers:for LSU,LR3(5-CCGTG TTTCAAGACGGG-3,reverse)and LR3R(5-GT CTTGAAACACGGACC-3,forward);for rpb2,rpb2-MF(5-GGTGGCTYAAYCARGGYAC-3,forward)and rpb2-MR(5-TTNGCCARATGYT GCCAYTTGA-3,reverse);for ef1a,983F,1567R and an in
23、ternal forward primer ef1a-MF(5-ACA ARATGGACACNACCAAGG-3);for rpb1,two Russulaceae-specific pimers Af-Rus(5-GARTG CCCWGGKCATTTYGG-3,forward),Cr-Rus(5-CYGCAATRTCRTTGTCCATGTA-3,reverse)and two internal primers rpb1-MF(5-CCRTCG TYTGGAVYCAYTGCAA-3,forward)and rpb1-MR(5-TCRGCCTCACARAYCATCTTAC-3,reverse).
24、For sequences with unphasable INDELs,cloning method described by Wang et al.(2015)was used.1.3 Sampling and phylogenetic analyses ITS region of 35 specimens were amplified.These specimens were collected from Hubei,Hunan,Inner Mongolia,Jilin,Sichuan and Yunnan in China.In addition,three specimens of
25、L.inquinatus(XHW3706),L.mediterraneensis(XHW5749)and L.yazooensis(YJH574)collected by authors were sequenced for genetic comparison with the new species.When sequences had heterozygous INDELs or ambiguous sites,PCR products were sequenced bidirectionally to make contigs of the amplified regions or v
26、erify the ambiguous sites.Flot et al.s(2006)method was used to determine the INDEL heterozygosity and phase the sequences into two copies,thus generating 44 sequences for the 35 specimens.The new ITS sequences generated by this study were assembled and edited by Sequencher 4.1.4(Gene Codes Corporati
27、on,Ann Arbor)and submitted to GenBank with accessions OP680929 OP680972 and OQ121683.For 20 of the 35 specimens,we obtained multi-locus data.GenBank accessions of these sequences are:OP938269 OP938288(LSU),OP994149,OP994153OP994172(ef1a),OP965651 OP965672(rpb2),OP994119 OP994142(rpb1).Besides the IT
28、S sequences above,three categories of ITS data were included in the phylogenetic analysis:(1)Matches with similarity 97%and coverage 90%by BLASTn in GenBank using our queries(with the 18S and 28S ends trimmed);(2)Sequences of species with morphological similarities,i.e.,L.acatlanensis,L.austrozonari
29、us,L.dilutisalmoneus,L.haugiae,L.Research paper 22 January 2023,42(1):252-262 Mycosystema ISSN1672-6472 CN11-5180/Q 菌物学报 255 Fig.1 Original tree generated by neighbor-joining(NJ)analysis of Lactarius liyuanus and its look-alikes based on ITS sequences,rooted with L.lignyotus and L.oomsisiensis.NJ bo
30、otstrap higher than 70%are indicated above or by the branches except for the 47%value for the big clade formed by the 35 specimens sequenced.Sample names are presented in the order of species name,voucher specimen,GenBank accession,and the geographical origin.For the 35 studied specimens,all were co
31、llected from China and the province names are given.CAO Shuqin et al./New species Lactarius liyuanus in East Asia:a close relative of European L.acerrimus 研究论文 菌物学报 256 indozonarius,L.mediterraneensis,L.psammicola,L.sinozonarius,L.wenquanensis,L.yazooensis,L.zonarioides,L.zonarius;(3)Published seque
32、nces of some Asian species in L.subsect.Zonarii to which the new species belongs morphologically,i.e.L.albidoarmeniacus,L.angustizonatus,L.inquinatus,L.pallidozonarius and L.pohangensis.Two species of L.subg.Plinthogalus,L.lignyotus and L.oomsisiensis were used as outgroups.Alignment was made by the
33、 online version of the multiple sequence alignment program MAFFT v7(Katoh&Toh 2008),using the L-INS-I strategy.It was manually adjusted in BioEdit v.7.1.3.0(Hall 1999).The dataset consisted of 82 sequences and 873 bases.No partitioning was set for the ITS dataset,considering relatively low variation
34、 among the species investigated.Due to the high similarity among sequences,Maximum Likelihood and Bayesian Inference methods were not able to give a resolved topology with satisfactory supports for the backbones and even some species(Verbeken et al.2014;Lee et al.2019;Wang et al.2021).Therefore we u
35、sed the Neighbor-Joining method implemented in MEGA X(Kumar et al.2018)to infer the phylogenetic relationships,using the following settings:bootstrap method(with 1 000 replicates)as test of phylogeny,p-distance as substitution model,transitions and transversions both included for substitutions,unifo
36、rm rates among sites,homogenous pattern among lineages and pairwise deletion for gaps treatment.A NJ bootstrap(NJ-BS)70%was considered as significant support for a node to be monophyletic.Genetic distance was calculated in MEGA X.2 RESULTS 2.1 ITS genealogy Among the 35 specimens sequenced,nine spec
37、imens have ITS region with INDELs.Among the nine specimens,five have one INDEL and four have two INDELs.These INDELs are both in the ITS1 and ITS2 regions.In the ITS tree,these 35 samples formed a clade with NJ-BS 47%,not reaching 70%,the significant support value.Within the clade,there were one ter
38、minal clade lacking support(I)and three well-supported terminal clades(II IV).All samples from northern China(Inner Mongolia and Jilin)were in clade I.Besides the northern samples,clade I included samples from central(Hubei and Hunan)and southwestern China(Yunnan)as well.Clade II(NJ-BS 99%)had five
39、samples from Hunan(only two samples were shown in the tree).Samples of clade III(NJ-BS 99%)were all collected from central China(Hubei and Hunan)and those of clade IV(NJ-BS 100%)from eastern(Zhejiang,HKAS 126521,not shown in the tree),central(Hubei and Hunan)and southwestern China(Yunnan).The clades
40、 of these 35 specimens formed the closest relatives of European L.acerrimus with NJ-BS 89%.The successive relative of this big assemblage was L.mediterraneensis consisting of two European samples and two Asian samples(NJ-BS 88%).All other species included in the analysis had significant supports but
41、 all backbones lacked supports.2.2 Taxonomy Lactarius liyuanus X.H.Wang,S.Q.Cao&W.Q.Qin,sp.nov.Fig.2,Fig.3A C,Fig.4 MycoBank:MB 846877 Etymology:named after Prof.Yu LI,in honour of his great contribution to the study on Chinese edible mushrooms,on the occasion of his 80th birthday.Holotype:China:Hub
42、ei Prov.,Fang Co.,Yerengu Town,Xihaoping,31 Jul.2011,X.H.Wang,no.2936(HKAS 73496).Diagnosis:The species is characterized by the yellowish brown zonate pileus with hairy margin,scrobiculate stipe,white unchanging hot latex,macrocystidia embeded in hymenium and spores stripped with high ridges.It is d
43、istinguished from L.acerrimus by the 4-spored basidia and smaller spores with higher ridges.Basidiocarps big,stout.Pilei 42140 mm diam.,at first convex to applanate with a decurved and hairy margin,gradually becoming depressed to funnel-shaped,margin applanate to wavy,with short hairs or nearly glab
44、rous when mature,pale yellow to orange yellow(3A6 3A8),yellowish brown at maturity;surface zonate,viscid when Research paper 22 January 2023,42(1):252-262 Mycosystema ISSN1672-6472 CN11-5180/Q 菌物学报 257 Fig.2 Lactarius liyuanus(XHW2936,holotype).A:Basidiocarps.B:Basidiospores.C:Pleuromacrocystidia.D:
45、Cheilomacrocystidia.E:Pileipellis.wet,sometimes hygrophanous and striate at margin.Lamella 3 8 mm broad,straight to decurrent,crowded when young,medium crowded to subdistant when mature,sometimes forking near the stipe,rarely anastomosing,cream-colored,light yellow to light orange,yellowish-brown or
46、 orange-brown when mature.Stipe 20 3510 20 mm,central or eccentrical,cylindrical,equal or tapering downwards,concolorous with or paler than gills,hollow,often with pits.Context 48 mm thick,compact,white,pale cream to pale yellow,smell unpleasant,slightly sour,taste acrid.Latex copious,white,pale cre
47、am,unchanging,very rarely changing to pale yellow after exposed,very hot.Basidiospores(240/11/10)(7.0)7.5 8.59.5(10.5)(6.0)6.5 7.58.0(10.0)m Q=(1.07)1.09 1.28(1.36),Q=1.180.06 holotype:(40/1/1)(7.5)8.08.5 9.0(9.5)6.5 7.0 8.0 m,Q=(1.12)1.13 1.29(1.36),Q=1.200.05,ellipsoid,rarely subglobose;ornamentat
48、ion 1.0 2.0 m high,composed of actute ridges partially connected,not forming closed meshes,isolated warts and short ridges common,often with transparent dots between two short ridges;plage not or distally amyloid.Basidia 4-spored,40 70917.5 m,clavate.Pleuromacrocystidia common,3072 4 10 m,embedded i
49、n hymenium,not or slightly projecting beyond the basidia,narrowly fusiform to almost cylindrical with a tapering apex,apex often moniliform,with granular or agglomerated contents.Hymenial pseudocystidia rare to common,2 5 m broad,some with slightly inflated apex,with sparse contents,colourless.Lamel
50、la edge sterile,rarely fertile;cheilomacrocystidia abundant,25 594 8 m,narrowly fusiform,with granular or needle-like contents;marginal cells 8 254 10 m,cylindrical,CAO Shuqin et al./New species Lactarius liyuanus in East Asia:a close relative of European L.acerrimus 研究论文 菌物学报 258 Fig.3 Lactarius li