2018, Vol. 38文章信息
- 臧威, 刘春月, 谢广发, 沈赤, 刘新展, 邹彗君, 汤访评, 冯富娟, 孙剑秋, 白逢彦.
- ZANG Wei, LIU Chunyue, XIE Guangfa, SHEN Chi, LIU Xinzhan, ZOU Huijun, TANG Fangping, FENG Fujuan, SUN Jianqiu, BAI Fengyan.
- 浙江雁荡山山脉叶栖酵母菌资源与物种多样性
- Resources and species diversity of the phyllosphere yeasts from the Yandang Mountains in Zhejiang Province
- 生态学报. 2018, 38(11): 3920-3930
- Acta Ecologica Sinica. 2018, 38(11): 3920-3930
- http://dx.doi.org/10.5846/stxb201705040827
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文章历史
- 收稿日期: 2017-05-04
- 网络出版日期: 2018-03-02
2. 东北林业大学生命科学学院, 哈尔滨 150040;
3. 浙江古越龙山绍兴酒股份有限公司, 绍兴 312000;
4. 中国科学院微生物研究所真菌学国家重点实验室, 北京 100101
2. Life Science College, Northeast Forestry University, Harbin 150040, China;
3. Zhejiang Guyuelongshan Shaoxing Rice Wine Co., Ltd., Shaoxing 312000, China;
4. State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
酵母菌只是形态学术语而不是分类学名词, 是一类主要以芽殖或裂殖进行无性繁殖、有性繁殖阶段不形成子实体的单细胞真菌[1]。在植物、土壤、淡水、海水等生境中广泛分布着各类酵母菌, 与人类生活关系密切[2]。基于酵母菌在工业、农业、医学、食品等行业和基础研究、生态功能等领域的重要作用[1], 考察和分离各种生境中存在的酵母菌资源是生物技术发展的重要基础[3], 所以特定生境内酵母菌类群的分布情况一直受到广泛关注。对于酒类酿造[4-7]、传统食品发酵[8-10]、水环境[11]、土壤[12]、植物表面[3, 13-15]、人体[16]和患处[17-18]、极端环境[19]及其他生境[20]中酵母菌资源的研究成果也充分说明, 自然界中还有大量的酵母菌种类亟待发现。
雁荡山山脉位于浙江省东南部, 绵延数百公里, 按照地理位置可以分为北雁荡山、中雁荡山、南雁荡山、西雁荡山(泽雅)、东雁荡山(洞头半屏山), 气候温和湿润, 适合酵母菌栖息繁殖, 但是迄今对该地区野生酵母菌资源研究未见报道。我们采用经典的酵母菌分离培养方法和现代酵母菌分子鉴定技术, 对雁荡山山脉广阔区域内的叶栖酵母菌资源进行考察, 旨在弄清该地区叶栖酵母菌群落的多样性、组成和结构, 发现特殊酵母菌类群以进一步丰富我国酵母菌资源库。
1 材料与方法 1.1 采样地点研究范围集中在浙江省东南部雁荡山地区, 包括北雁荡山、中雁荡山、南雁荡山、西雁荡山(泽雅)、东雁荡山(洞头半屏山), 该地区属亚热带海洋性气候, 雨量充沛、气候温暖, 年平均气温13.5℃, 年平均降雨量1935.6 mm, 年平均无霜期269 d。
1.2 采样方法在2015—2016年间, 自雁荡山山脉采集树木半枯叶片暂存于无菌自封袋内, 尽快运回实验室进行叶栖酵母菌的分离和纯化。采样过程中, 对宿主植物信息、周围环境、纬度和经度等进行详细记载。
1.3 酵母菌分离参照Nakase和Takashima方法[21]进行。将采集的半枯叶片, 用凡士林粘贴在PDA平板的皿盖内侧, 在20℃下正置恒温培养7 d, 期间每天更换新鲜培养基。更换下的PDA平板在20℃下倒置培养至渐见菌落形成, 挑取形态不同的酵母菌菌落进行平板划线, 即可获得叶栖酵母菌的纯培养。
1.4 酵母菌DNA提取挑取少量在PDA斜面上生长旺盛的幼龄酵母菌菌体进行DNA微量提取, 参照Makimura方法[22]完成。
1.5 酵母菌26S rDNA D1/D2区PCR扩增参考Kurtzman和Robnett方法[23], 采用引物NL4(5′-GGTCCGTGTTTCAAGACGG-3′)和NL1(5′-GCATATCAATAAGCGGAGGAAAAG-3′)对菌株26S rDNA D1/D2进行扩增, 按照反应条件94℃ 1 min, 52℃ 1 min, 72℃ 1 min扩增30个循环。本研究中引物NL4和NL1的合成由生工生物工程(上海)股份有限公司完成, 北京六合华大基因科技有限公司对扩增产物进行测序。
1.6 序列分析将测得酵母菌26S rDNA D1/D2序列在GenBank核酸序列数据库中进行同源序列搜索(BLAST search), 比较实验菌株与已知菌株序列的相似程度。采用Clustal X软件比对相应模式菌株序列与实验菌株26S rDNA D1/D2序列的碱基差异[24]。通过MEGA5软件和Neighbor-Joining方法, 构建分子系统树以明确相关酵母菌的分类地位[25-26]。根据系统树上标注的序列号(accession numbers), 可以从GeneBank下载相关菌株26S rDNA D1/D2参考序列。
1.7 数据分析相对频率(Relative frequency, RF)是指某雁荡山地区分离到的某种叶栖酵母菌占该地区叶栖酵母菌总菌株数的百分数, 若≥1%则属于常见种类。
根据以下Shannon-Weiner公式计算多样性指数(Diversity index, H′), 式中k是指雁荡山某地区分离到的常见叶栖酵母菌种类的总数, Pi是指某种叶栖酵母菌的菌株数量占该地区叶栖酵母菌全部菌株数的百分数。
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丰度(Richness, R), 是指某雁荡山地区分离到的常见叶栖酵母菌种类数。
相似性系数(Similarity coefficients, Cs), 根据Sorenson系数公式进行计算, Cs=2j/(a+b), 式中j是指雁荡山山脉两个地区之间都可以分离到的常见叶栖酵母菌种类数, a是指雁荡山山脉一个地区可以分离到的常见叶栖酵母菌种类数, b是指雁荡山山脉另一个地区可以分离到的常见叶栖酵母菌种类数。
采用bootstrap指数获得期望物种丰富度(S), 在如下计算公式中So代表雁荡山某地区分离到的常见叶栖酵母菌种类数, Pk代表某种叶栖酵母菌的菌株数占该地区叶栖酵母菌全部菌株数的百分数, m是指全部样本数量。借助EstimateS软件完成分析和计算, 通过Excel绘制雁荡山山脉叶栖酵母菌的物种累积曲线(Species accumulation curves)。
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从北雁荡山、中雁荡山、南雁荡山、西雁荡山和东雁荡山分别采集到树木半枯叶片标本30、32、30、31和30份, 分离获得酵母菌144、209、257、337株和171株。参照1118株酵母菌26S rDNA D1/D2序列在GenBank核酸序列数据库中同源序列的搜索(BLAST search)结果, 采用Clustal X软件将相应模式菌株与实验菌株26S rDNA D1/D2序列进行比较分析并选择典型菌株绘制分子系统树, 其中1072个菌株与已知模式菌株26S rDNA D1/D2序列相似性在99%以上, 被鉴定为37属56种(图 1)。
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| 图 1 基于26S rDNA D1/D2序列构建雁荡山山脉典型酵母菌株与近缘种间的系统进化树 Fig. 1 The phylogenetic tree based on 26S rDNA D1/D2 sequences, showing the relationships among representative strains and their close relatives 编号中E、S、W、N和M分别表示该菌株分离自东雁荡山、南雁荡山、西雁荡山、北雁荡山和中雁荡山 |
在雁荡山山脉分离得到1118个叶栖酵母菌株中, 有46个菌株26S rDNA D1/D2序列较为特殊, 与最近缘的已知种类26S rDNA D1/D2序列均存在1%以上的碱基差异, 根据目前国际酵母菌分类标准[27-29]疑似属于新的酵母菌类群。其中, 分离自北雁荡山共有23株、中雁荡山7株、南雁荡山10株、西雁荡山2株和东雁荡山4株。通过Clustal X软件比较并与相应模式菌株比对, 包括子囊菌酵母4属5种和担子菌酵母9属14种(表 1)。
| 疑似新种 Proposed new species |
菌株 Strains |
最近缘模式种(序列号)与相似性/% Closest relatives (accession number) and sequence identity |
宿主植物 Hosts |
纬度和经度 Latitude and longitude |
地点 Sites |
|
| 子囊菌酵母Ascomycetous taxa | ||||||
| Collophora sp. | M2212 | C. paarla (GQ154611) |
95.85 | Daphniphyllum oldhami | 28°6′56″120°52′28″ | 中雁荡 |
| M2421 | Rubus chingii | 28°6′44″120°52′18″ | ||||
| Coniosporium sp. | N0112 | C. apollinis (NG042587) |
90.94 | Cyclobalanopsis sp. | 28°21′42″121°4′10″ | 北雁荡 |
| N1412 | Cunninghamia lanceolata | 28°22′8″121°5′37″ | ||||
| N1211 | 90.65 | Shrub sp. | 28°22′12″121°5′43″ | |||
| M2511 | 91.13 | Lindera reflexa | 28°6′42″120°52′18″ | 中雁荡 | ||
| Elsinoe sp. | N0511 | E. eucalypticola (DQ923530) |
92.87 | Aceraceae sp. | 28°21′42″121°4′10″ | 北雁荡 |
| N2911 | Cunninghamia lanceolata | 28°22′5″121°5′21″ | ||||
| Taphrina sp.1 | N0111 | T. padi (AF492048) |
98.18 | Cyclobalanopsis sp. | 28°21′42″121°4′10″ | 北雁荡 |
| N0512 | Aceraceae sp. | 28°21′42″121°4′10″ | ||||
| N0513 | ||||||
| N1421 | Cunninghamia lanceolata | 28°22′8″121°5′37″ | ||||
| N1431 | ||||||
| N1432 | ||||||
| N1811 | Ilex sp. | 28°22′6″121°5′31″ | ||||
| N1812 | ||||||
| M3111 | Cinnamomum camphora | 28°6′30″120°52′13″ | 中雁荡 | |||
| M1411 | Symplocos stellaris | 28°6′48″120°52′13″ | ||||
| M1412 | ||||||
| Taphrina sp.2 | N0721 | T. padi (AF492048) |
97.95 | Cryptomeria fortunei | 28°21′48″121°3′57″ | 北雁荡 |
| N0722 | ||||||
| 担子菌酵母Basidiomycetous taxa | ||||||
| Bulleribasidium sp.1 | E1541 | B. foliicola (KY106250) |
98.06 | Rhus chinensis | 27°49′52″121°7′11″ | 东雁荡 |
| Bulleribasidium sp.2 | W2742 | B. setariae (AB119463) |
97.08 | Rhododendron mariesii | 28°2′13″120°22′18″ | 西雁荡 |
| Carlosrosaea sp. | N3011 | C. vrieseae (KP691955) |
96.79 | Ligustrum sp. | 28°22′5″121°5′21″ | 北雁荡 |
| Chrysozyma sp.1 | S0711 | C. fushanensis (AB176591) |
98.06 | Loropetalum chinensis | 27°35′53″120°16′18″ | 南雁荡 |
| S0731 | ||||||
| Chrysozyma sp.2 | S0712 | C. griseoflava (KY106920) |
97.35 | Loropetalum chinensis | 27°35′53″120°16′18″ | 南雁荡 |
| Colacogloea sp.1 | S1011 | C. falcate (KY106942) |
96.18 | Cibotium barometz | 27°35′58″120°16′22″ | 南雁荡 |
| Colacogloea sp.2 | N0921 | C. foliorum (KY106943) |
96.15 | Cryptomeria fortunei | 28°21′47″121°3′59″ | 北雁荡 |
| Colacogloea sp.3 | N2121 | C. eucalyptica (EU075184) |
97.11 | Schima superba | 28°22′2″121°5′26″ | 北雁荡 |
| Derxomyces sp. | S0372 | D. simaoensis (KY107627) |
97.28 | Sloanea sinensis | 27°36′6″120°16′14″ | 南雁荡 |
| S1412 | Elaeocarpus decipiens | 27°35′57″120°16′22″ | ||||
| Fellozyma sp. | N3051 | F. inositophila (KY107699) |
93.19 | Ligustrum sp. | 28°22′5″121°5′21″ | 北雁荡 |
| S0952 | 93.01 | Phoebe chekiangensis | 27°35′56″120°16′20″ | 南雁荡 | ||
| Kockovaella sp. | S0541 | K. barringtoniae (AB292854) |
98.04 | Camellia oleifera | 27°36′4″120°16′11″ | 南雁荡 |
| Kondoa sp.1 | M151 | K. myxariophila (AF189904) |
96.10 | Rosa laevigata | ？ | 中雁荡 |
| E1511 | Rhus chinensis | 27°49′52″121°7′11″ | 东雁荡 | |||
| E2111 | Hovenia acerba | 27°50′5″121°7′13″ | ||||
| E2711 | Ficus erecta | 27°50′8″121°7′16″ | ||||
| Kondoa sp.2 | S1322 | K. myxariophila (AF189904) |
97.37 | Syzygium jambos | 27°35′58″120°12′23″ | 南雁荡 |
| S1512 | Euonymus carnosus | 27°35′57″120°16′22″ | ||||
| Saitozyma sp. | W0951 | S. flavus (AF075497) |
91.83 | Adina rubella | 28°2′11″120°22′20″ | 西雁荡 |
| N0931 | Cryptomeria fortunei | 28°21′47″121°3′59″ | 北雁荡 | |||
| N0941 | ||||||
| N0961 | ||||||
| N0991 | ||||||
从雁荡山山脉分离到的1118株叶栖酵母菌被鉴定为37属56种已知种类和13属19种疑似新种(图 1和表 1)。其中, 在东雁荡山有12种常见类群, 占该地区叶栖酵母菌的95.4%；在南雁荡山有14种常见类群, 占该地区叶栖酵母菌的86.8%；在西雁荡山有9种常见类群, 占该地区叶栖酵母菌的94.4%；在北雁荡山有14种常见类群, 占该地区叶栖酵母菌的93.3%；中雁荡山分离到13种常见种类, 占该地区叶栖酵母菌的94%(表 2)。雁荡山山脉常见的叶栖酵母菌种类中, 只有Derxomyces mrakii和Derxomyces pseudoschimicola两种酵母菌在东雁荡山、西雁荡山、南雁荡山、北雁荡山、中雁荡山都可以分离到；除北雁荡山的优势酵母为Derxomyces pseudoschimicola之外, 其他地区分离到的叶栖酵母菌中Derxomyces mrakii均表现出明显的分布优势；特别是在东雁荡山, Derxomyces mrakii在该地区常见叶栖酵母菌中的相对频率可以达到55% (表 2)。
| 分类单元 Taxon |
东雁荡 East |
南雁荡 South |
西雁荡 West |
北雁荡 North |
中雁荡 Middle |
| Bannoa hahajimensis | 11.7 | ||||
| Bannoa ogasawarensis | 6.4 | 3.1 | 17.4 | 1.4 | |
| Bullera alba | 1.4 | ||||
| Bulleribasidium foliicola | 1.2 | ||||
| Bulleribasidium variabile | 3.5 | ||||
| Coniochaeta velutina | 1.4 | ||||
| Coniosporium sp. | 2.1 | ||||
| Cryptococcus sp. | 1.9 | ||||
| Derxomyces bambusicola | 1.4 | ||||
| Derxomyces boekhoutii | 3.3 | ||||
| Derxomyces mrakii | 55.0 | 43.6 | 36.8 | 13.9 | 41.6 |
| Derxomyces pseudoschimicola | 1.2 | 5.4 | 6.2 | 37.5 | 9.1 |
| Derxomyces qinlingensis | 1.2 | 1.2 | |||
| Elsinoe sp. | 1.4 | ||||
| Erythrobasidium hasegawianum | 1.4 | ||||
| Fellozyma inositophila | 3.0 | ||||
| Golubevia pallescens | 1.4 | ||||
| Kockovaella mexicanus | 1.6 | ||||
| Kondoa sp.1 | 1.8 | ||||
| Leucosporidium intermedium | 2.7 | ||||
| Moesziomyces aphidis | 1.8 | ||||
| Oberwinklerozyma yarrowii | 1.2 | ||||
| Phyllozyma productus | 1.8 | ||||
| Rhodosporidiobolus ficis | 1.2 | ||||
| Rhodosporidiobolus odoratus | 2.3 | 1.4 | |||
| Ruinenia pyrrosiae | 2.3 | ||||
| Saitozyma sp. | 2.8 | ||||
| Sporobolomyces carnicolor | 2.7 | 1.9 | |||
| Sporobolomyces pararoseus | 4.2 | 6.7 | |||
| Sporobolomyces phaffii | 4.7 | 5.6 | 1.4 | ||
| Symmetrospora coprosmae | 1.8 | 1.4 | 4.3 | ||
| Symmetrospora gracilis | 1.4 | ||||
| Taphrina sp.1 | 5.6 | 1.4 | |||
| Taphrina sp.2 | 1.4 | ||||
| Tilletiopsis sp. | 1.9 | ||||
| Tilletiopsis washingtonensis | 5.8 | ||||
| Udeniomyces megalosporus | 1.9 | 6.5 | 3.3 | ||
| Udeniomyces pyricola | 15.2 | 29.1 | 18.7 | ||
| 合计 | 95.4 | 86.8 | 94.4 | 93.3 | 94.0 |
| 稀有种类; The rare taxa (RF﹤1%); 东雁荡山East Yandang mountain: Bulleribasidium foliicola, Bulleribasidium sp.1, Ceraceosorus africanus, Erythrobasidium sp., Hannaella sinensis, Microsporomyces magnisporus, Naganishia albida, Tilletiopsis sp. 南雁荡山South Yandang mountain: Bannoa hahajimensis, Chrysozyma sp.1, Chrysozyma sp.2, Colacogloea falcata, Colacogloea sp.1, Cystobasidiomycetes sp., Derxomyces boekhoutii, Derxomyces sp., Dioszegia zsoltii, Exobasidium symploci-japonicae, Fellozyma sp., Golubevia pallescens, Kockovaella sp., Kockovaella vietnamensis, Kondoa sp.2, Microsporomyces magnisporus, Microstroma phylloplanum, Nielozyma formosana, Phyllozyma corallina, Rhodotorula sp., Sporobolomyces koalae, Sporobolomyces pararoseus, Symmetrospora coprosmae, Takashimella formosensis. 西雁荡山West Yandang mountain: Bannoa ogasawarensis, Bulleribasidium sp.2, Cryptococcus sp., Derxomyces bambusicola, Dioszegia zsoltii, Hannaella surugaensis, Myriangium sp., Oberwinklerozyma yarrowii, Piskurozyma cylindricus, Rhodotorula sp., Saitozyma sp. 北雁荡山North Yandang mountain: Carlosrosaea sp., Colacogloea falcate, Colacogloea sp.2, Colacogloea sp.3, Derxomyces boekhoutii, Fellozyma sp., Meira nashicola, Moesziomyces aphidis, Papiliotrema aurea, Sporobolomyces koalae. 中雁荡山Middle Yandang mountain: Collophora sp., Coniosporium sp., Cryptococcus sp., Derxomyces boekhoutii, Dioszegia zsoltii, Gjaerumia minor, Jaminaea angkorensis, Kondoa sp.1, Oberwinklerozyma yarrowii, Papiliotrema aurea, Rhodotorula sp., Tilletiopsis sp. | |||||
多样性指数(H′)和丰度(R)可以反映该地区叶栖酵母菌的物种多样性程度, 在雁荡山山脉广阔地区分布的叶栖酵母菌具有丰富的多样性。从多样性指数(H′)来看, 北雁荡山﹥中雁荡山﹥南雁荡山﹥西雁荡山﹥东雁荡山；根据酵母菌的丰度值, 也可以看出分离自北雁荡山、中雁荡山和南雁荡山的叶栖酵母菌种类相对更多(表 3)。
| 地区 Area |
多样性指数 Diversity index (H′) |
丰度 Richness(R) |
地区 Area |
多样性指数 Diversity index (H′) |
丰度 Richness(R) |
|
| 东雁荡山East | 1.55 | 12 | 北雁荡山North | 1.84 | 14 | |
| 南雁荡山South | 1.66 | 14 | 中雁荡山Middle | 1.76 | 13 | |
| 西雁荡山West | 1.61 | 9 |
相似性系数可以比较雁荡山山脉两个地区之间叶栖酵母菌物种组成的相似性程度。根据表 4发现, 中雁荡山常见叶栖酵母菌组成与东、南、西、北雁荡山等周边地区的相似性系数较高, 达到0.4—0.52；而南雁荡山与西雁荡山、中雁荡山常见叶栖酵母菌组成最为相近, 相似性系数达到0.52的较高水平；东、西雁荡山和南、北雁荡山的叶栖酵母菌种类的相似程度则较低, 相似性系数分别为0.19和0.21。
| 地区Area | 南雁荡山South | 西雁荡山West | 北雁荡山North | 中雁荡山Middle |
| 东雁荡山East | 0.23 | 0.19 | 0.31 | 0.4 |
| 南雁荡山South | 0.52 | 0.21 | 0.52 | |
| 西雁荡山West | 0.17 | 0.45 | ||
| 北雁荡山North | 0.44 |
以东雁荡山、西雁荡山、南雁荡山、北雁荡山和中雁荡山都可以分离到的Derxomyces mrakii为代表, 对雁荡山山脉属于同一种类的叶栖酵母菌物种资源D1/D2序列进行分析和比较。结果发现, 分离自雁荡山山脉的广布种Derxomyces mrakii包括13种基因型, 根据每种基因型与模式菌株CBS8288的D1/D2序列比较结果及其在雁荡山不同地区分布的相对频率(表 5), 其中与模式菌株D1/D2序列完全相同的基因型01最常见, 在雁荡山各个地区可以占到Derxomyces mrakii菌株的87.4%—98.2%。
| 基因型 Genetype |
东雁荡山 East |
南雁荡山 South |
西雁荡山 West |
北雁荡山 North |
中雁荡山 Middle |
与模式菌株碱基差异数量 Sequence identities |
| 01 | 87.4 | 98.2 | 96 | 95 | 96.6 | 0 |
| 02 | 3.2 | 1.1 | 1 (T→C) | |||
| 03 | 1.1 | 2 (C→T, C→A) | ||||
| 04 | 1.1 | 1 (T→C) | ||||
| 05 | 1.1 | 1 (T→C) | ||||
| 06 | 0.8 | 1 (C→A) | ||||
| 07 | 1.1 | 1 (C→T) | ||||
| 08 | 0.8 | 1 (G→A) | ||||
| 09 | 0.8 | 1 (A→G) | ||||
| 10 | 1.8 | 0.8 | 1.1 | 1 (T→C) | ||
| 11 | 5 | 1 (T→C) | ||||
| 12 | 0.8 | 3 (A→T, C→G, A→G) | ||||
| 13 | 6.3 | 2 (A→G, C→G) |
基于雁荡山山脉常见叶栖酵母菌在东雁荡山、西雁荡山、南雁荡山、北雁荡山和中雁荡山的分布数据(表 2), 采用EstimateS软件对该地区叶栖酵母菌多样性进行了分析和计算, 并绘制出雁荡山山脉叶栖酵母菌的物种累积曲线(图 2)。
|
| 图 2 雁荡山山脉叶栖酵母菌的物种累积曲线 Fig. 2 The species accumulation curves for the yeasts from the Yandang Mountain area 1：物种累积曲线；2、3：在95%置信区间内物种累积曲线的下限与上限95% |
根据物种累积曲线特征和EstimateS软件计算结果, 其Bootstrap值为46.95, 估计在本项研究中已经将雁荡山地区80.94%的叶栖酵母菌分离出来。
3 讨论雁荡山是我国华东地区的重要山脉之一, 幅员辽阔、资源丰富, 在生物多样性研究方面受到广泛重视[30]。该地区温度、湿度等环境因子适合野生酵母菌栖息, 系统、全面的研究雁荡山地区酵母菌资源情况具有一定代表性。在野生酵母菌研究方面, 植物叶表是人们比较关注的酵母菌栖息环境[31-32], 而且每年秋季的成熟植物叶片上酵母菌的种类和数量最为丰富[33]。现在一般认为, 同土壤等生境相比较, 叶表可能更容易分离到特殊种类的酵母菌。
目前国际上通行的酵母菌鉴定依据主要是26S rRNA基因D1/D2区域的序列组成[23, 27], 种内不同菌株之间D1/D2序列差异应该在1%以内, 如果株间该序列碱基差异在1%以上则基本可以判定为不同种类[28]。近年来酵母菌分类系统变化较大[34-36], 在进行雁荡山地区叶栖酵母菌的序列比对和系统发育分析时, 参照最新的酵母菌分类系统对鉴定结果进行了必要的修正。
分离自雁荡山山脉的1072株叶栖酵母菌, 经过26S rRNA基因D1/D2序列比对和系统发育分析, 属于56个已知类群(图 1)；另外还有46个菌株26S rDNA D1/D2序列与已知种类差异明显, 根据26S rRNA基因D1/D2序列比对和系统发育分析结果, 基本可以确定这些菌株代表 19个新类群(表 1)；尽管有些菌株, 还需要详细研究其ITS序列特征和生理生化特征、细胞形态特征、培养特征以进一步证明其新种地位, 但是疑似新种比较常见的事实也说明, 研究雁荡山地区叶栖酵母菌资源具有重要的理论意义。
在雁荡山地区有38种常见叶栖酵母菌类群(表 2), 包括Derxomyces属5种, Sporobolomyces属3种, Bannoa、Bulleribasidium、Rhodosporidiobolus、Symmetrospora、Taphrina、Tilletiopsi和Udeniomyces属各2种, Bullera、Coniochaeta、Coniosporium、Cryptococcus、Elsinoe、Erythrobasidium、Fellozyma、Golubevia、Kockovaella、Kondoa、Leucosporidium、Moesziomyces、Oberwinklerozyma、Phyllozyma、Ruinenia和Saitozyma属各1种, 其中Derxomyces mrakii在该地区分离到的叶栖酵母菌中具有明显分布优势。Hamamoto和Nakase[37]最早从新西兰的三种植物Leptospermum scoparium、Nothofagus fusca、Pseudowintera colonata上分离到Derxomyces mrakii培养物, 并将其鉴定为Bullera属新种, Wang和Bai[38]在建立Derxomyces属时将其重合组合在Derxomyces属中。在分析比较雁荡山地区Derxomyces mrakii的D1/D2序列时, 发现大量培养物与模式酵母的序列并非完全一致, 表现出一定程度的遗传多样性(表 5)。
雁荡山山脉叶栖酵母菌资源丰富(表 3), 根据不同地区常见叶栖酵母菌组成(表 2)和相似程度(表 4), 发现中雁荡山与东雁荡山、西雁荡山、南雁荡山和北雁荡山叶栖酵母菌相似性系数达到0.4—0.52, 表明中雁荡山与其他4个地区的叶栖酵母菌种类组成更加相似；而东、西雁荡山之间和南、北雁荡山之间叶栖酵母菌组成相似性系数分别为0.19和0.21, 酵母菌种类组成存在更大差异；可能地理上相近地区的酵母菌物种分布具有更大相似性, 这个推测可以供今后野生酵母菌资源考察工作参考。
在生物多样性研究领域, 物种累积曲线被广泛用于样本量充分性的判断以及物种丰富程度的估计[39]。根据图 2, 我们已经分离到雁荡山地区绝大多数的叶栖酵母菌种类, 研究结果可以真实反映出该地区叶栖酵母菌资源和物种多样性的存在状况。
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