凉山州龙肘山锈红杜鹃与薄叶马银花根部真菌分子检测

【目的】为检验直接分子检测法用于揭示杜鹃花属(Rhododendron)植物根部真菌(Root-associated fungi,RAF)组成的有效性。【方法】采用不依赖于培养的分子检测技术直接从锈红杜鹃(R.bureavii)与薄叶马银花(R.leptothrium)的发根(Hair root)提取DNA,用真菌特异性引物扩增r DNA-ITS区、经克隆后测序,对获得的ITS序列进行分析;通过收集NCBI中与本研究的RAF相似性97%以上的所有序列对应的真菌来源(土壤或根系的身份)数据,分析真菌的生态学特性,并用FUNGuild软件提供的方法划分真菌的生态类型。【结果】从两种杜鹃花根部共检测到15种真菌,其中担子菌门(Basdiomycota)真菌3种,子囊菌门(Ascomycota)真菌12种。柔膜菌目(Helotiales)真菌在两种杜鹃花RAF群落中占据优势,并且在两种杜鹃花根系中均检测到该类真菌。此外,两种杜鹃花根部有多种生态类型的RAF共存,包括曾被频繁报道的杜鹃花类菌根菌Oidiodendron sp.和Rhizoscyphus sp.、内生真菌Phialocephala fortinii、共生一致病过渡型真菌Pezoloma ericae、外生菌根共生菌Meliniomyces sp.,以及腐生型真菌Myceana sp.、Lachnum virgineum、Herpotrichia sp.。【结论】直接分子检测法从两种杜鹃花属植物根部检测到的真菌谱系多样性较高、生态类型复杂,这一方法能较为全面地反映杜鹃花属植物RAF多样性。     

中国中部太白山不同海拔杜鹃兰根部内生真菌多样性

本研究以太白山自然保护区蒿坪站杜鹃兰Cremastra appendiculata为材料,采用菌丝团和根组织分离法进行真菌分离,并用ITS序列分子鉴定;用变性梯度凝胶电泳（denaturing gradient gel electrophoresis,DGGE）分析根部内生真菌多样性,研究海拔和根际土理化性质对真菌多样性的影响。分离到79株19种真菌,均为子囊菌门Ascomycota,隶属于座囊菌纲Dothideomycetes 1目2科2种和粪壳菌纲Sordariomycetes 7目10科17种,其中12种兰科菌根真菌。DGGE检测到80种真菌,其中兰科菌根真菌交链链格孢、锈腐土赤壳和木霉属真菌可通过组织分离法得到,担子菌门Basidiomycota 3种真菌只在DGGE检测到。随着海拔升高,根际土硝态氮和速效磷下降,导致真菌丰富度和香侬维纳指数降低,辛普森指数升高。本研究为发掘此地区杜鹃兰菌根真菌资源进行杜鹃兰人工培育奠定基础。     

黔中地区马尾松林下杜鹃根部内生真菌群落组成及其生态功能

探索黔中马尾松(Pinus massoniana)林下杜鹃(Rhododendron simsii)根内生真菌物种多样性和生态功能。采集黔中乌当(WD)、孟关(MG)、龙里(LL) 3个地区马尾松林下杜鹃的发根,提取真菌DNA进行高通量测序。分析真菌Alpha多样性,借助FUNGuild注释平台解析真菌的生态功能类别,探索真菌群落中的核心微生物组,并结合网络图展示菌群之间的关联性。结果,3个地区的杜鹃根部内生真菌多样性非常丰富,WD地区杜鹃内生真菌多样性和丰富度最高。试验共获得有效序列425799条,817个可操作分类单元(OTU),分属于6门19纲52目103科154属,主要隶属于子囊菌门(81.2%)、接合菌门(5.8%)和担子菌门(4.7%);优势纲、目、科分别为:散囊菌纲(43.0%)、散囊菌目(39.2%)、发菌科(39.2%);在属的水平上,青霉属(38.60%)占比最高,其次是木霉属(7.20%)、拟盘多毛孢属(6.10%)。根部的真菌拥有多种生态功能群,如未定义腐生菌(194 OTU)、植物病原菌(20 OTU)、土壤腐生菌(18 OTU)、外生菌(14 OTU)、地衣共生真菌(10 OTU)、杜鹃类菌根真菌(5 OTU)、木腐生菌(5 OTU)、丛枝菌根(4 OTU)、内生菌(2 OTU)、动物病原菌(8 OTU),以及多种混合营养型类群21类,102个Undefined种类在FUNGuild数据库中没有参考信息。根部真菌可以形成生态位共享模式,而且不同功能群之间存在耦合性,核心基因组与关键物种以真菌组形成的生态功能团表现。     

《菌物学报》“菌根真菌专刊”征稿通知

<正>真菌与植物关系密切、共同发展、协同进化,在自然界长期演化过程中,真菌与植物逐渐建立了共生关系。业已证实,菌根真菌与植物根系建立的共生体是生物圈内典型的互惠共生关系。菌根真菌几乎涉及真菌界所有门类。其中,丛枝菌根真菌(AMF)属于专性活体营养的共生真菌,已分离鉴定近300种,隶属球囊菌门1纲4目11科25属;外生菌根真菌隶属担子菌门、子囊菌门、接合菌门和无性态真菌,全球约5 000~6 000种,     

中国种子植物内生真菌资源及菌植协同进化

综述了中国种子植物内生真菌资源研究概况,比较了裸子植物和被子植物内生真菌种类,它们都具有肉座菌目(Hypocreales),粪壳菌目(Sordariales),散囊菌目(Eurotiales),毛霉目(Mucorales)及不产孢类(Myceliasterilia)内生真菌。裸子植物内生真菌涉及52个属,既包括高等的子囊菌和担子菌,也包括低等的卵菌(Oomycetes)和接合菌(Zygomycetes)类。被子植物涉及60个属,主要为高等的子囊菌(Ascomycetes)和担子菌(Basidiomycetes),低等的卵菌和接合菌报道很少。双子叶植物涉及40个属,单子叶植物内生真菌涉及30个属,两类被子植物所报道的内生真菌只有11个属相同。裸子植物与双子叶植物内生真菌相似程度较高,都具有炭角菌目(Xylariales)、格孢腔菌目(Pleosporales)、柔膜菌目(Helotiales)和白粉菌目(Erysiphales),刺盘孢菌属(Colletotrichum)、拟茎点霉属(Phomopsis)、枝孢霉属(Cladosporium)、地霉属(Geotrichum)等内真菌,共20个属相同。各类种子植物具有自己独特的一些内生真菌。还对植物与其内生真菌的协同进化关系进行了分析。     

白及内生真菌多样性研究

白及( Bletilla striata)是兰科地生型多年生植物,也是我国传统中药材之一。利用菌根技术进行白及的保护和人工栽培,需要获得白及可培养的内生真菌。该研究以广西野生的白及根和叶为材料,采用分离培养法分离内生真菌,并结合真菌形态特征,及其核糖体的转录间隔区( ITS)序列分析,确定内生真菌的分类地位。结果表明：从2株白及植物90块组织中分离获得37株内生真菌,鉴定为15个分类单元,由9个属组成,分属于2门4纲7目8科,包括锤舌菌纲( Leotiomycetes)、座囊菌纲( Dothideomycetes)和粪壳菌纲( Sordariomy-cetes),伞菌纲( Agaricomycetes)。从根中分离获得内生真菌12种,蜡壳菌属为优势属;从叶中分离获得内生真菌3种,刺盘孢属为优势属;刺盘孢菌属( Colletotrichum)和蜡壳菌属( Sebacina)真菌的相对多度值均达到20％;4株担子菌均分布于根中,叶组织中未有分布。根组织中内生真菌的多样性指数(H＝1.863)高于叶组织(1.098)。该研究结果及其所分离培养的担子菌类真菌,为更好地利用菌根技术进行白及等兰科植物资源的保护与可持续利用奠定了基础。     

内蒙古地区白桦外生菌根形态类型及分子鉴定

以内蒙古不同地区白桦外生菌根为材料,采用形态解剖学方法和分子生物学手段对与白桦共生的外生菌根真菌多样性进行全面的调查,并经对所测序列与GenBank和Database比对。结果显示:在内蒙古地区与白桦共生的外生菌根真菌共13种,其中担子菌7种,子囊菌4种,分别来自于丝膜菌属、丝盖伞属、蜡壳耳属、毛革菌属、滑菇属和空团菌属、块菌属、地怀菌属。其中,菌根类型T₈和T₁₁未能提出其总DNA,根据其外生菌根形态类型并参照Agerer体视显微镜菌根图谱和Haug菌根图谱进行比较,分类鉴定为荷顿氏疣柄牛肝菌和白桦外生菌根真菌一种。结果表明,内蒙古地区白桦外生菌根真菌多样性相对较高,且与利用地上子实体鉴定的外生菌根真菌种类有一定的区别。     

四川黄龙沟优势兰科植物菌根真菌多样性及其季节变化

在自然条件下,兰科菌根真菌对兰花的种子萌发和植株生长都是必不可少的。为了解高原兰科植物菌根真菌的多样性状况及其季节性变化规律,选取了四川黄龙沟的两种生境中生长的8种优势兰科植物,分别于植株的萌芽期(4月份)、生长期(7月份)和果期(9月份)采集营养根进行菌根真菌的多样性研究。其中,黄花杓兰(Cypripedium flavum)、少花鹤顶兰(Phaiusdelavayi)、二叶匍茎兰(Galearis diantha)和广布小蝶兰(Ponerorchis chusua)分布在开阔生境;筒距兰(Tipularia szechuanica)、小花舌唇兰(Platanthera minutiflora)、珊瑚兰(Corallorhiza trifida)和尖唇鸟巢兰(Neottia acuminate)则分布在密林生境。通过对分离所得的50个菌株进行形态观察和ITS序列测定相结合的鉴定,共获得菌根真菌41种。对担子菌和子囊菌分别进行的系统发育树构建结果显示,子囊菌为优势种类(35种),以柔膜菌目(Helotiales)、炭角菌目(Xylariales)和肉座菌目(Hypocreales)内的种类为主,担子菌则以胶膜菌(Tulasnellaceaesp.)为主。在8种兰科植物中,二叶匐茎兰表现出极高的专一性,其菌根真菌均属于Hypocrea。其余兰科植物的菌根真菌分别属于不同的科,专一性相对较低。物种丰富度和Simpson多样性指数分析结果表明,密林生境的兰科植物的菌根真菌多样性在各生长季节基本高于开阔生境。此外,两种生境的优势兰科植物的菌根真菌物种多样性随生长季节转变所呈现的变化规律是相似的:萌发期和生长期的多样性均较高,峰值出现在生长期,到果期时则大幅下降。这与高原兰科植物的生长特性及营养供求规律基本相符。     

菌根真菌与施肥对杜鹃苗养分吸收的影响

以2年生杜鹃(Rhododendron simsii)苗为研究对象,采用J1(杜鹃花类菌根真菌混合接菌)、J2(杜鹃花类菌根真菌和马尾松外生菌混合接菌)、J3(不接菌,对照)等3种接菌组合与不同氮磷钾施肥组合研究菌根真菌与氮磷钾肥对杜鹃苗生长及养分吸收的影响。结果表明,杜鹃幼苗的生长指标和氮磷钾含量总体表现为接菌处理优于不接菌处理,J2处理下施用尿素1.1 g·株-1、钙镁磷1 g·株-1、氯化钾0.7 g·株-1对杜鹃苗生长及氮磷钾含量积累的促进作用最为明显。数据表明,J2处理促进N向叶积累,促进K向茎叶积累,促进P向根茎积累;J1处理促进N和P向根茎积累,促进K向茎叶积累。     

亚热带森林菌根植物根系真菌群落结构对氮磷添加的响应

植物根系真菌对维系植物的营养吸收和健康具有重要作用。本研究分析了不同菌根类型植物根系中真菌的群落结构对外源氮(N)、磷(P)、氮和磷(NP)输入的响应。试验采集了无添加(对照)和N、P、NP添加处理下亚热带森林3种菌根类型(丛枝菌根、外生菌根、欧石楠菌根)9种植物的根系，运用高通量测序技术检测根系中真菌的多样性和群落组成。结果表明：9种植物根系真菌群落均主要由担子菌门和子囊菌门组成；P添加下子囊菌门的相对多度显著低于对照，而担子菌门的相对多度显著高于对照。欧石楠菌根植物根系中子囊菌门的相对多度显著高于丛枝菌根和外生菌根植物，而其担子菌门的相对多度显著低于丛枝菌根和外生菌根植物。与对照相比，P添加显著降低了植物根系中真菌的α多样性，改变了不同菌根类型植物根系中真菌的群落组成，而N添加和菌根类型的影响不明显。与对照和N添加相比，NP添加使全部植物根系中真菌群落变异程度更大，即群落整体更加趋异，而外生菌根植物根系中的真菌群落比丛枝菌根植物根系中的真菌群落变异更小，即群落更趋同。综上，P养分是影响亚热带森林土壤中树木根系真菌群落结构的关键因素。本研究有助于提升对全球环境变化下亚热带地区植物根系...     

Sebacinales are common mycorrhizal associates of Ericaceae

Previous reports of sequences of Sebacinales (basal Hymenomycetes) from ericoid mycorrhizas raised the question as to whether Sebacinales are common mycorrhizal associates of Ericaceae, which are usually considered to associate with ascomycetes. Here, we sampled 239 mycorrhizas from 36 ericoid mycorrhizal species across the world (Vaccinioideae and Ericoideae) and 361 mycorrhizas from four species of basal Ericaceae lineages (Arbutoideae and Monotropoideae) that do not form ericoid mycorrhizas, but ectendomycorrhizas. Sebacinales were detected using sebacinoid-specific primers for nuclear 28S ribosomal DNA, and some samples were investigated by transmission electron microscopy (TEM). Diverging Sebacinales sequences were recovered from 76 ericoid mycorrhizas, all belonging to Sebacinales clade B. Indeed, some intracellular hyphal coils had ultrastructural TEM features expected for Sebacinales, and occurred in living cells. Sebacinales belonging to clade A were found on 13 investigated roots of the basal Ericaceae, and TEM revealed typical ectendomycorrhizal structures. Basal Ericaceae lineages thus form ectendomycorrhizas with clade A Sebacinales, a clade that also harbours ectomycorrhizal fungi. This further supports the proposition that Ericaceae ectendomycorrhizas involve ectomycorrhizal fungal taxa. When ericoid mycorrhizas evolved secondarily in Ericaceae, a shift of mycobionts occurred to ascomycetes and clade B Sebacinales, hitherto not described as ericoid mycorrhizal fungi.     

New Neotropical Sebacinales Species from a Pakaraimaea dipterocarpacea Forest in the Guayana Region,Southern Venezuela: Structural Diversity and Phylogeography

Pakaraimaea dipterocarpacea, a member of the Dipterocarpaceae endemic in the Guayana region, is associated with a diverse community of ectomycorrhizal (ECM) fungi. Amongst the 41 ECM fungal species detected in a 400 m² P. dipterocarpacea ssp. nitida plot in Southern Venezuela, three species belonged to the Sebacinales. We tested whether ECM anatomotype characterization can be used as a feasible element in an integrative taxonomy in this diverse fungal group, where the relevance of fruitbody morphology for species delimitation seems limited. Using a combination of ECM morpho-anatomical characterizations and phylogenetic analyses based on nuclear ITS and LSU sequences, we report three new species. The main distinguishing features of Sebacina guayanensis are the yellowish cell walls together with conspicuous undifferentiated, uniform compact (type B) rhizomorphs. Staghorn-like hyphae are characteristic of S. tomentosa. The combination of clusters of thick-walled emanating hyphae, including hyphae similar to awl-shaped cystidia with basal dichotomous or trichotomous ramifications, and the presence of type B rhizomorphs were characteristic of a third, yet unnamed species. The three species belong to three different, possibly specifically tropical clades in Sebacinales Group A. The geographic distribution of phylogenetically related strains was wide, including a Dicymbe forest in Guyana and an Ecuadorian rainforest with Coccoloba species. We show that ECM morpho-anatomy can be used, in combination with other analyses, to delineate species within Sebacinales Group A. In addition to phylogenetic information, type B rhizomorphs observed in different Sebacinales clades have important ecological implications for this fungal group. The phylogeography of Sebacinales suggests that dispersion and host jump are important radiation mechanisms that shaped P. dipterocarpacea ECM fungal community. This study emphasizes the need for more sequence data to evaluate the hypothesis that phylogeographic relationships between neo- and paleotropical ECM fungal species could be attributed to the vicariance of cross-continental hosts such as the Dipterocarpacae.     

Divergence Times and Phylogenetic Patterns of Sebacinales,a Highly Diverse and Widespread Fungal Lineage

Patterns of geographic distribution and composition of fungal communities are still poorly understood. Widespread occurrence in terrestrial ecosystems and the unique richness of interactions of Sebacinales with plants make them a target group to study evolutionary events in the light of nutritional lifestyle. We inferred diversity patterns, phylogenetic structures and divergence times of Sebacinales with respect to their nutritional lifestyles by integrating data from fossil-calibrated phylogenetic analyses. Relaxed molecular clock analyses indicated that Sebacinales originated late Permian within Basidiomycota, and their split into Sebacinaceae and Serendipitaceae nom. prov. likely occurred during the late Jurassic and the early Cretaceous, coinciding with major diversifications of land plants. In Sebacinaceae, diversification of species with ectomycorrhizal lifestyle presumably started during the Paleocene. Lineage radiations of the core group of ericoid and cavendishioid mycorrhizal Sebacinales started probably in the Eocene, coinciding with diversification events of their hosts. The diversification of Sebacinales with jungermannioid interactions started during the Oligocene, and occurred much later than the diversification of their hosts. Sebacinales communities associated either with ectomycorrhizal plants, achlorophyllous orchids, ericoid and cavendishioid Ericaceae or liverworts were phylogenetically clustered and globally distributed. Major Sebacinales lineage diversifications started after the continents had drifted apart. We also briefly discuss dispersal patterns of extant Sebacinales.     

Sebacinales everywhere: previously overlooked ubiquitous fungal endophytes

Inconspicuous basidiomycetes from the order Sebacinales are known to be involved in a puzzling variety of mutualistic plant-fungal symbioses (mycorrhizae), which presumably involve transport of mineral nutrients. Recently a few members of this fungal order not fitting this definition and commonly referred to as 'endophytes' have raised considerable interest by their ability to enhance plant growth and to increase resistance of their host plants against abiotic stress factors and fungal pathogens. Using DNA-based detection and electron microscopy, we show that Sebacinales are not only extremely versatile in their mycorrhizal associations, but are also almost universally present as symptomless endophytes. They occurred in field specimens of bryophytes, pteridophytes and all families of herbaceous angiosperms we investigated, including liverworts, wheat, maize, and the non-mycorrhizal model plant Arabidopsis thaliana. They were present in all habitats we studied on four continents. We even detected these fungi in herbarium specimens originating from pioneering field trips to North Africa in the 1830s/40s. No geographical or host patterns were detected. Our data suggest that the multitude of mycorrhizal interactions in Sebacinales may have arisen from an ancestral endophytic habit by specialization. Considering their proven beneficial influence on plant growth and their ubiquity, endophytic Sebacinales may be a previously unrecognized universal hidden force in plant ecosystems.     

Sebacinales are associates of the leafy liverwort Lophozia excisa in the southern maritime Antarctic

The leafy liverwort Lophozia excisa, which is colonised by basidiomycete fungi in other biomes and which evidence suggests may be colonised by mycorrhizal fungi in Antarctica, was sampled from Léonie Island in the southern maritime Antarctic (67°36′ S, 68°21′ W). Microscopic examination of plants indicated that fungal hyphae colonised 78% of the rhizoids of the liverwort, apparently by entering the tips of rhizoids prior to growing into their bases, where they formed hyphal coils. Extensive colonisation of stem medullary cells by hyphae was also observed. DNA was extracted from surface-sterilised liverwort tissues and sequenced following nested PCR, using the primer set ITS1F/TW14, followed by a second round of amplification using the ITSSeb3/TW13 primer set. Neighbour-joining analyses showed that the sequences obtained nested in Sebacinales clade B as a 100% supported sister group to Sebacinales sequences from the leafy liverworts Lophozia sudetica, L. incisa and Calypogeia muelleriana sampled from Europe. Direct PCR using the fungal specific primer set ITS1F/ITS4 similarly identified fungi belonging to Sebacinales clade B as the principal colonists of L. excisa tissues. These observations indicate the presence of a second mycothallus in Antarctica and support the previous suggestion that the Sebacinales has a wide geographical distribution.     

Members of Sebacinales subgroup B form mycorrhizae with epiphytic orchids in a neotropical mountain rain forest

Previous investigations revealed that epiphytic orchids in a mountain rain forest in southern Ecuador formed mycorrhizae with diverse members of Tulasnellales. Using specific primers, we now show that the same orchids are also associated with Sebacinales. Ultrastructural observations confirmed the Sebacinales mycobionts in situ. Mycorrhizae of flowering individuals of Stelis hallii, S. superbiens, S. concinna and Pleurothallis lilijae were sampled in different forest types of the mountain rain forest of southern Ecuador along an altitudinal gradient between 1,850 and 2,100 m a.s.l. Phylogenetic analysis of fungal nuclear rDNA sequences coding for the ribosomal large subunit (nucLSU) showed the presence of eight sequence types based on proportional differences of <1% bp. All sequence types clustered in the Sebacinales subgroup B which also contained sequences of mycobionts from ericads and terrestrial orchids. Sequences of the nuclear rDNA 5.8S subunit, including parts of the internal transcribed spacers ITS1 and ITS2 (5.8-ITS) from the mycobionts of the epiphytic orchids, were distinct from published sequences of sebacinoid mycobionts of green terrestrial orchids and ericads. Sebacinales sequences from different epiphytic orchid species differed at least by 1% bp as was previously found for Tulasnella sequences. Sebacinales occurred less frequently and with a lower number of sequence types than Tulasnellales, but distribution along the altitudinal gradient was similar.     

Molecular and phenotypic characterization of Sebacina vermifera strains associated with orchids, and the description of Piriformospora williamsii sp. nov

Sebacinales was described in 2004 and is currently recognized as the earliest diverging lineage of mycorrhizal Basidiomycota. In addition, recent research has demonstrated that no other known fungal order harbours a broader spectrum of mycorrhizal types. Yet because of the character poor morphology of these inconspicuous fungi, a reliable systematic framework for Sebacinales is still out of reach. In order to increase the body of comparative data on Sebacinales, we followed a polyphasic approach using a sampling of seven diverse Sebacinales strains, including several isolates of Australian orchid mycorrhizae, Piriformospora indica, and a multinucleate rhizoctonia isolated from a pot culture of Glomus fasciculatum (Williams 1985) with clover. We performed molecular phylogenetic analyses from candidate barcoding regions [rDNA: internal transcribed spacer (ITS)1-5.8-ITS2, 28S; translation elongation factor 1-α (TEF)], enzymatic profiling, genome size estimation by quantitative polymerase chain reaction (PCR), and karyotype analysis using pulsed field gel electrophoresis. Here, we report significant differences in the physiological and molecular parameters inferred from these morphologically very similar strains. Particularly, our results indicate that intron sequences of the TEF gene are useful markers for Sebacinales at the species level. As a first taxonomic consequence, we describe Piriformospora williamsii as a new member of the so far monotypic genus Piriformospora and show that this genus contains still undescribed species that were recently discovered as endophytes of field-collected specimens of Anthyllis, Medicago, and Lolium in Germany.     

Detection and identification of bacteria intimately associated with fungi of the order Sebacinales

Because of their beneficial impact on plants, the highly diverse mycorrhizal fungi grouped in the order Sebacinales lay claim to high ecological and agricultural significance. Here, we describe for the first time associations of Sebacinoid members with bacteria. Using quantitative PCR, denaturating gradient gel electrophoresis and fluorescence in situ hybridization, we detected an intimate association between Piriformospora indica and Rhizobium radiobacter, an alpha-Proteobacterium. The stability of the association, vertical transmission of the bacteria during asexual fungal reproduction and fungal plant colonization was monitored using R. radiobacter-specific primers. Treatment of mycelium or fungal protoplasts with antibiotics highly efficient against the free bacteria failed to cure the fungus. Barley seedlings dip-inoculated with R. radiobacter showed growth promotion and systemic resistance to the powdery mildew fungus Blumeria graminis comparable to P. indica inoculation. By screening additional isolates of the Sebacina vermifera complex, three species-specific associations with bacteria from the genera Paenibacillus, Acinetobacter and Rhodococcus were found. These findings suggest that Sebacinales species regularly undergo complex interactions involving host plants and bacteria reminiscent of other ectomycorrhizal and endomycorrhizal associations.     

Guilds of mycorrhizal fungi and their relation to trees,ericads, orchids and liverworts in a neotropical mountain rain forest

Mycorrhizas of vascular plants and mycorrhiza-like associations of liverworts and hornworts are integral parts of terrestrial ecosystems, but have rarely been studied in tropical mountain rain forests. The tropical mountain rain forest area of the Reserva Biológica San Francisco in South Ecuador situated on the eastern slope of the Cordillera El Consuelo is exceptionally rich in tree species, ericads and orchids, but also in liverworts. Previous light and electron microscopical studies revealed that tree roots are well colonized by structurally diverse Glomeromycota, and that epiphytic, pleurothallid orchids form mycorrhizas with members of the Tulasnellales and the Sebacinales (Basidiomycota). Sebacinales also occurred in mycorrhizas of hemiepiphytic ericads and Tulasnellales were found in liverworts belonging to the Aneuraceae. On the basis of these findings, we hypothesized that symbiotic fungi with a broad host range created shared guilds or even fungal networks between different plant species and plant families. To test this hypothesis, molecular phylogenetic studies of the fungi associated with roots and thalli were carried out using sequences of the nuclear rDNA coding for the small subunit rRNA (nucSSU) of Glomeromycota and the large subunit rRNA (nucLSU) of Basidiomycota. Sequence analyses showed that Sebacinales and Tulasnellales were only shared within but not between ericads and orchids or between liverworts and orchids, respectively. Regarding arbuscular-mycorrhiza-forming trees, however, 18 out of 33 Glomus sequence types were shared by two to four tree species belonging to distinct families. Nearly all investigated trees shared one sequence type with another tree individual. Host range and potential shared guilds appeared to be restricted to the plant family level for Basidiomycota, but were covering diverse plant families in case of Glomeromycota. Given that the sequence types as defined here correspond to fungal species, our findings indicate potential fungal networks between trees.     

Two widespread green Neottia species (Orchidaceae) show mycorrhizal preference for Sebacinales in various habitats and ontogenetic stages

Plant dependence on fungal carbon (mycoheterotrophy) evolved repeatedly. In orchids, it is connected with a mycorrhizal shift from rhizoctonia to ectomycorrhizal fungi and a high natural ¹³C and ¹⁵N abundance. Some green relatives of mycoheterotrophic species show identical trends, but most of these remain unstudied, blurring our understanding of evolution to mycoheterotrophy. We analysed mycorrhizal associations and ¹³C and ¹⁵N biomass content in two green species, Neottia ovata and N. cordata (tribe Neottieae), from a genus comprising green and nongreen (mycoheterotrophic) species. Our study covered 41 European sites, including different meadow and forest habitats and orchid developmental stages. Fungal ITS barcoding and electron microscopy showed that both Neottia species associated mainly with nonectomycorrhizal Sebacinales Clade B, a group of rhizoctonia symbionts of green orchids, regardless of the habitat or growth stage. Few additional rhizoctonias from Ceratobasidiaceae and Tulasnellaceae, and ectomycorrhizal fungi were detected. Isotope abundances did not detect carbon gain from the ectomycorrhizal fungi, suggesting a usual nutrition of rhizoctonia‐associated green orchids. Considering associations of related partially or fully mycoheterotrophic species such as Neottia camtschatea or N. nidus‐avis with ectomycorrhizal Sebacinales Clade A, we propose that the genus Neottia displays a mycorrhizal preference for Sebacinales and that the association with nonectomycorrhizal Sebacinales Clade B is likely ancestral. Such a change in preference for mycorrhizal associates differing in ecology within the same fungal taxon is rare among orchids. Moreover, the existence of rhizoctonia‐associated Neottia spp. challenges the shift to ectomycorrhizal fungi as an ancestral pre‐adaptation to mycoheterotrophy in the whole Neottieae.