云南大白蚁亚科白蚁分飞情况及与蚁巢伞关系

在云南绿春观察大白蚁亚科4种土白蚁的分飞活动,研究大白蚁亚科白蚁和蚁巢伞之间的关系,得知:大白蚁亚科不同种类白蚁分飞孔形状不相同,分飞时分布于孔口周围的白蚁型、数目和扩散范围存在差异,这些蚁巢的外露特征可以作为白蚁种类识别的辅助依据。采集32巢白蚁以及其巢上生长的蚁巢伞,结果显示,大白蚁亚科每一个白蚁巢仅生长一种蚁巢伞Termitomyces子实体。与小果蚁巢伞共生的白蚁也与其他蚁巢伞共生,推测真菌能改变白蚁的行为。对白蚁种类和蚁巢伞种类对应关系的研究表明在小地域内,白蚁和蚁巢伞不仅存在属间高度专一性,而且种间也显示较高的共生专一性。     

培菌白蚁菌圃和粪便微生物多样性分析

【背景】培菌白蚁是属于白蚁科的一类与鸡枞菌属真菌共生的高等白蚁,其与体内肠道微生物和体外菌圃微生物形成三维共生体系。【目的】分析培菌白蚁菌圃和粪便的微生物多样性,并与肠道微生物进行比较。【方法】通过Illumina MiSeq高通量测序方法对培菌白蚁菌圃和粪便样品进行细菌16S rRNA基因和真菌ITS测序分析。【结果】高通量测序获得培菌白蚁菌圃和粪便样品细菌和真菌的有效序列和OTU数目。5个样品细菌OTU数目在90-199之间,而真菌OTU在10-58之间,细菌的种类多样性明显大于真菌。不论是细菌还是真菌,粪便样品的OTU数目多于菌圃样品。经物种分类分析,菌圃样品主要优势细菌是变形菌门(Proteobacteria),其相对含量超过82.4%;其次是拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes);粪便样品中优势细菌为拟杆菌门,其次是变形菌门,粪便优势菌属为别样杆菌属和营发酵单胞菌属,这与培菌白蚁肠道菌多样性组成一致。培菌白蚁菌圃和粪便样品共生真菌主要为担子菌门(Basidiomycota)和子囊菌门(Ascomycota)。菌圃优势真菌为鸡枞菌属(Termitomyces),相对含量在51.83%以上,菌圃中还鉴定到炭角菌属(1%,Xylaria)。【结论】为今后培菌白蚁-体内外微生物共生关系研究以及微生物的分离培养提供了依据和参考。     

Fungus-growing termites originated in African rain forest

Fungus-growing termites (subfamily Macrotermitinae, Isoptera) cultivate fungal crops (genus Termitomyces, Basidiomycotina) in gardens inside their colonies. Those fungus gardens are continuously provided with plant substrates, whereas older parts that have been well decomposed by the fungus are consumed (cf.). Fungus-growing termites are found throughout the Old World tropics, in rain forests and savannas, but are ecologically dominant in savannas. Here, we reconstruct the ancestral habitat and geographical origin of fungus-growing termites. We used a statistical model of habitat switching repeated over all phylogenetic trees sampled in a Bayesian analysis of molecular data. Our reconstructions provide strong evidence that termite agriculture originated in African rain forest and that the main radiation leading to the extant genera occurred there. Because extant savanna species are found in most genera, this moreover suggests that the savanna has repeatedly been colonized by fungus-growing termites. Furthermore, at least four independent "out-of-Africa" migrations into Asia, and at least one independent migration to Madagascar, have occurred. Although fungus growing by termites is ecologically most successful under the variable, unfavorable conditions of the savanna, it seems to have evolved under the more constant and favorable conditions of the rain forest.     

Levels of specificity of Xylaria species associated with fungus-growing termites: a phylogenetic approach

Fungus-growing termites live in obligate mutualistic symbiosis with species of the basidiomycete genus Termitomyces , which are cultivated on a substrate of dead plant material. When the termite colony dies, or when nest material is incubated without termites in the laboratory, fruiting bodies of the ascomycete genus Xylaria appear and rapidly cover the fungus garden. This raises the question whether certain Xylaria species are specialised in occupying termite nests or whether they are just occasional visitors. We tested Xylaria specificity at four levels: (1) fungus-growing termites, (2) termite genera, (3) termite species, and (4) colonies. In South Africa, 108 colonies of eight termite species from three termite genera were sampled for Xylaria . Xylaria was isolated from 69% of the sampled nests and from 57% of the incubated fungus comb samples, confirming high prevalence. Phylogenetic analysis of the ITS region revealed 16 operational taxonomic units of Xylaria , indicating high levels of Xylaria species richness. Not much of this variation was explained by termite genus, species, or colony; thus, at level 2–4 the specificity is low. Analysis of the large subunit rDNA region, showed that all termite-associated Xylaria belong to a single clade, together with only three of the 26 non-termite-associated strains. Termite-associated Xylaria thus show specificity for fungus-growing termites (level 1). We did not find evidence for geographic or temporal structuring in these Xylaria phylogenies. Based on our results, we conclude that termite-associated Xylaria are specific for fungus-growing termites, without having specificity for lower taxonomic levels.     

Genetic variation of symbiotic fungi cultivated by the macrotermitine termite Odontotermes formosanus (Isoptera: Termitidae) in the Ryukyu Archipelago

Fungus-growing termites have a mutualistic relationship with their cultivated fungi. To improve understanding of genetic aspects of this relationship, we examined molecular markers in the fungus-growing termite Odontotermes formosanus and its fungi Termitomyces spp. from the Ryukyu Archipelago. Based on the polymorphic band patterns obtained from arbitrarily primed polymerase chain reaction methods, we constructed cladograms for related colonies of the termites and fungi. The resulting trees indicated that the termites display little genetic variation among the colonies, while the symbiotic fungi consist of two major genetic types. In addition, molecular phylogenetic trees of the symbiotic fungi based on internal transcribed spacer and 18S rDNA suggested that these two types of fungi are different species. We also demonstrated that the fungi comprising the fruiting bodies and fungus combs are identical, and that fungus combs are probably a monoculture within a single termite colony. Our results indicate that horizontal transmission of symbiotic fungi among termite colonies occurred during the evolutionary history of this symbiosis.     

Identifying the core microbial community in the gut of fungus‐growing termites

Gut microbes play a crucial role in decomposing lignocellulose to fuel termite societies, with protists in the lower termites and prokaryotes in the higher termites providing these services. However, a single basal subfamily of the higher termites, the Macrotermitinae, also domesticated a plant biomass‐degrading fungus (Termitomyces), and how this symbiont acquisition has affected the fungus‐growing termite gut microbiota has remained unclear. The objective of our study was to compare the intestinal bacterial communities of five genera (nine species) of fungus‐growing termites to establish whether or not an ancestral core microbiota has been maintained and characterizes extant lineages. Using 454‐pyrosequencing of the 16S rRNA gene, we show that gut communities have representatives of 26 bacterial phyla and are dominated by Firmicutes, Bacteroidetes, Spirochaetes, Proteobacteria and Synergistetes. A set of 42 genus‐level taxa was present in all termite species and accounted for 56–68% of the species‐specific reads. Gut communities of termites from the same genus were more similar than distantly related species, suggesting that phylogenetic ancestry matters, possibly in connection with specific termite genus‐level ecological niches. Finally, we show that gut communities of fungus‐growing termites are similar to cockroaches, both at the bacterial phylum level and in a comparison of the core Macrotermitinae taxa abundances with representative cockroach, lower termite and higher nonfungus‐growing termites. These results suggest that the obligate association with Termitomyces has forced the bacterial gut communities of the fungus‐growing termites towards a relatively uniform composition with higher similarity to their omnivorous relatives than to more closely related termites.     

Open relationships in the castles of clay: high diversity and low host specificity of Termitomyces fungi associated with fungus-growing termites in Africa

In the African and Asian tropics, termites of the subfamily Macrotermitinae play a major role in the decomposition of dead plant material. Their ecological success lies in the obligate mutualism of the termites with fungi of the genus Termitomyces. Before the advent of molecular studies, the interaction with these fungi was poorly understood. Here, we combined available ITS sequence data from West, Central, and South Africa with data of 39 new samples from East Africa to achieve the most comprehensive view of the diversity and host specificity of Termitomyces symbionts across Africa to date. A high amount of sequence divergence in the ITS sequences was found; 11 different Termitomyces lineages in East Africa and >30 lineages across Africa were identified, and the expected diversity is estimated to be about 41 lineages. The fungal lineages belong to four major clades, each almost exclusively associated with one termite host genus. Analysis of molecular variance revealed that 40% of the ITS sequence variation occurred between host genera, indicating close co-evolution at this level. However, within host genera, fungal lineages and haplotypes were frequently shared among host species and sampling localities, except for fungal symbionts of Odontotermes. Horizontal transmission of fungal symbionts may facilitate the transfer of haplotypes and species among hosts. However, at present, we have little understanding of the maintenance of specificity at the genus level. Possible explanations range from substrate specificity of fungi to an active selection of fungi by termites.     

Evolutionary trend of phylogenetic diversity of nitrogen fixation genes in the gut community of wood-feeding termites

Nitrogen fixation by gut microorganisms is one of the crucial aspects of symbiosis in wood-feeding termites since these termites thrive on a nitrogen-poor diet. In order to understand the evolution of this symbiosis, we analysed the nitrogenase structural gene nifH in the gut microbial communities. In conjunction with the published sequences, we compared approximately 320 putatively functional NifH protein sequences obtained from a total of 19 termite samples that represent all the major branches of their currently proposed phylogeny, and from one species of the cockroach Cryptocercus that shares a common ancestor with termites. Using multivariate techniques for clustering and ordination, a phylogeny of NifH protein sequences was created and plotted variously with host termite families, genera, and species. Close concordance was observed between NifH communities and the host termites at genus level, but family level relationships were not always congruent with accepted termite clade structure. Host groups examined included basal families (Mastotermitidae, Termopsidae, Kalotermitidae, as well as Cryptocercus), the most derived lower termite family Rhinotermitidae, and subfamilies representing the advanced and highly diverse apical family Termitidae (Macrotermitinae, Termitinae, and Nasutitermitinae). This selection encompassed the major nesting and feeding styles recognized in termites, and it was evident that NifH phylogenetic divergence, as well as the occurrence of alternative nitrogenase-type NifH, was to some extent dependent on host lifestyle as well as phylogenetic position.     

Molecular phylogeny of symbiotic basidiomycetes of fungus-growing termites in Thailand and their relationship with the host

Termitomyces-related symbiotic basidiomycetes in the nests of fungus-growing termites (Macrotermitinae) of several genera in Thailand were cultivated and analyzed phylogenetically based on the DNA sequence of nuclear ribosomal RNA genes. The relationships of the symbiotic fungi with host termites and their locality were apparently complex, supporting intricate mechanisms for the termites to acquire the symbionts.     

Uncovering cryptic species diversity of a termite community in a West African savanna

To uncover the termite species diversity of a natural African savanna ecosystem, we combined morphological analyses and sequencing of three gene fragments (cytochrome oxidase I, cytochrome oxidase II and 28SrDNA, total length about 2450 bp) to infer putative species from phylogenetic trees. We identified 18 putative species clusters with high support values and which we retrieved consistently. Samples from two genera (Ancistrotermes and Microcerotermes) were excluded from the mitochondrial phylogenetic analyses as they might represent nuclear mitochondrial sequences (NUMTs). In total, our data suggest a species richness of at least 20 species, all but one belonging to the Termitidae (higher termites), and among them the fungus-growing Macrotermitinae were most prevalent with at least nine putative species. Within the fungus-growers the most species-rich genus was Microtermes and its four putative species were all cryptic species. Their abundance in the samples suggests that they play an important ecological role which is completely unstudied also due to the lack of reliable identification means. Our study shows that morphological traits are unreliable means of species identification for several termite taxa. Yet reliable and consistent identification is necessary for studying the functional role of termites in ecosystem and global processes.     

Exploring the Potential for Actinobacteria as Defensive Symbionts in Fungus-Growing Termites

In fungus-growing termites, fungi of the subgenus Pseudoxylaria threaten colony health through substrate competition with the termite fungus (Termitomyces). The potential mechanisms with which termites suppress Pseudoxylaria have remained unknown. Here we explore if Actinobacteria potentially play a role as defensive symbionts against Pseudoxylaria in fungus-growing termites. We sampled for Actinobacteria from 30 fungus-growing termite colonies, spanning the three main termite genera and two geographically distant sites. Our isolations yielded 360 Actinobacteria, from which we selected subsets for morphological (288 isolates, grouped in 44 morphotypes) and for 16S rRNA (35 isolates, spanning the majority of morphotypes) characterisation. Actinobacteria were found throughout all sampled nests and colony parts and, phylogenetically, they are interspersed with Actinobacteria from origins other than fungus-growing termites, indicating lack of specificity. Antibiotic-activity screening of 288 isolates against the fungal cultivar and competitor revealed that most of the Actinobacteria-produced molecules with antifungal activity. A more detailed bioassay on 53 isolates, to test the specificity of antibiotics, showed that many Actinobacteria inhibit both Pseudoxylaria and Termitomyces, and that the cultivar fungus generally is more susceptible to inhibition than the competitor. This suggests that either defensive symbionts are not present in the system or that they, if present, represent a subset of the community isolated. If so, the antibiotics must be used in a targeted fashion, being applied to specific areas by the termites. We describe the first discovery of an assembly of antibiotic-producing Actinobacteria occurring in fungus-growing termite nests. However, due to the diversity found, and the lack of both phylogenetic and bioactivity specificity, further work is necessary for a better understanding of the putative role of antibiotic-producing bacteria in the fungus-growing termite mutualistic system.     

白蚁菌圃真菌多样性研究进展

白蚁菌圃存在于白蚁巢中,具有硬而脆的多孔结构,是特殊的真菌生存环境。当有白蚁在白蚁巢内活动时,蚁巢伞Termitomyces是菌圃上的优势菌;当白蚁巢被废弃,炭角菌Xylaria成为菌圃上的优势真菌。菌圃中还存在其他微生物如无性型真菌（anamorphic fungi）和酵母等。菌圃中的真菌很多具有潜在药用价值或其他经济价值。从蚁巢伞、炭角菌等主要真菌类群出发,结合分子生态学研究菌圃真菌多样性的方法,综述了白蚁菌圃真菌多样性的研究进展,揭示了目前的研究热点及存在的问题,并针对这些问题提出可能的发展方向。     

Symbiotic fungi produce laccases potentially involved in phenol degradation in fungus combs of fungus-growing termites in Thailand

Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.     

Intracolony variation of bacterial gut microbiota among castes and ages in the fungus-growing termite Macrotermes gilvus

The fungus-growing termites Macrotermes cultivate the obligate ectosymbiontic fungi, Termitomyces. While their relationship has been extesively studied, little is known about the gut bacterial symbionts, which also presumably play a crucial role for the nutrition of the termite host. In this study, we investigated the bacterial gut microbiota in two colonies of Macrotermes gilvus, and compared the diversity and community structure of bacteria among nine termite morphotypes, differing in caste and/or age, using terminal restriction fragment length polymorphism (T-RFLP) and clonal analysis of 16S rRNA. The obtained molecular community profiles clustered by termite morphotype rather than by colony, and the clustering pattern was clearly more related to a difference in age than to caste. Thus, we suggest that the bacterial gut microbiota change in relation to the food of the termite, which comprises fallen leaves and the fungus nodules of Termitomyces in young workers, and leaves degraded by the fungi, in old workers. Despite these intracolony variations in bacterial gut microbiota, their T-RFLP profiles formed a distinct cluster against those of the fungus garden, adjacent soil and guts of sympatric wood-feeding termites, implying a consistency and uniqueness of gut microbiota in M. gilvus. Since many bacterial phylotypes from M. gilvus formed monophyletic clusters with those from distantly related termite species, we suggest that gut bacteria have co-evolved with the termite host and form a microbiota specific to a termite taxonomic and/or feeding group, and furthermore, to caste and age within a termite species.     

Vertical transmission as the key to the colonization of Madagascar by fungus-growing termites?

The mutualism between fungus-growing termites (Macrotermitinae) and their mutualistic fungi (Termitomyces) began in Africa. The fungus-growing termites have secondarily colonized Madagascar and only a subset of the genera found in Africa is found on this isolated island. Successful long-distance colonization may have been severely constrained by the obligate interaction of the termites with fungal symbionts and the need to acquire these symbionts secondarily from the environment for most species (horizontal symbiont transmission). Consistent with this hypothesis, we show that all extant species of fungus-growing termites of Madagascar are the result of a single colonization event of termites belonging to one of the only two groups with vertical symbiont transmission, and we date this event at approximately 13 Mya (Middle/Upper Miocene). Vertical symbiont transmission may therefore have facilitated long-distance dispersal since both partners disperse together. In contrast to their termite hosts, the fungal symbionts have colonized Madagascar multiple times, suggesting that the presence of fungus-growing termites may have facilitated secondary colonizations of the symbiont. Our findings indicate that the absence of the right symbionts in a new environment can prevent long-distance dispersal of symbioses relying on horizontal symbiont acquisition.     

白蚁巢菌圃真菌多样性研究

培菌性白蚁能在存在于蚁巢或分散在其周围土壤中的菌圃上培养真菌。菌圃在无白蚁存在下培养会生长出炭角菌的子实体。对分别采集自我国西南四川、云南两省的4个土白蚁属菌圃采用原位培养法分离并纯化得到40株炭角菌,划分为13个形态型,ITS1-5.8S-ITS2序列分析确定为两种炭角菌。采用建立ITS基因文库的方法分析了白蚁菌圃真菌群落多样性,结果表明有白蚁存在的菌圃,蚁巢伞为单一优势菌;废弃的蚁巢中的菌圃,木霉、炭角菌等其他真菌成为优势菌。     

Diversity of fungus‐growing termites (Macrotermes) and their fungal symbionts (Termitomyces) in the semiarid Tsavo Ecosystem,Kenya

Fungus‐growing termites of the subfamily Macrotermitinae together with their highly specialized fungal symbionts (Termitomyces) are primary decomposers of dead plant matter in many African savanna ecosystems. The termites provide crucial ecosystem services also by modifying soil properties, translocating nutrients, and as important drivers of plant succession. Despite their obvious ecological importance, many basic features in the biology of fungus‐growing termites and especially their fungal symbionts remain poorly known, and no studies have so far focused on possible habitat‐level differences in symbiont diversity across heterogeneous landscapes. We studied the species identities of Macrotermes termites and their Termitomyces symbionts by excavating 143 termite mounds at eight study sites in the semiarid Tsavo Ecosystem of southern Kenya. Reference specimens were identified by sequencing the COI region from termites and the ITS region from symbiotic fungi. The results demonstrate that the regional Macrotermes community in Tsavo includes two sympatric species (M. subhyalinus and M. michaelseni) which cultivate and largely share three species of Termitomyces symbionts. A single species of fungus is always found in each termite mound, but even closely adjacent colonies of the same termite species often house evolutionarily divergent fungi. The species identities of both partners vary markedly between sites, suggesting hitherto unknown differences in their ecological requirements. It is apparent that both habitat heterogeneity and disturbance history can influence the regional distribution patterns of both partners in symbiosis.     

Fungal partnerships stimulate growth of Termitomyces clypeatus stalk mycelium in vitro

The symbiotic relationship between termites and Termitomyces fungi, which allows the termite to digest cellulose-rich food sources, is poorly understood. In this study, in vitro mixed symbiotic relationships between Termitomyces clypeatus and fungi isolated from individual fungus-comb communities using a culture-dependent method were analyzed. Twenty-day-old stalk cultures of three T. clypeatus isolates were co-cultured with cellulase-producing fungi on potato dextrose agar. The high cellulase-producing fungal isolate no. 18, which showed 99 % ITS sequence identity to Sordariomycetes endophyte isolate 2171 (EU687039), increased growth of T. clypeatus 18/50 by 85.7 %. The high xylanase-producing isolate no. 13, which showed 88 % ITS sequence identity to Arthrinium sacchari isolate L06 (HQ115662), stimulated T. clypeatus 18/50 growth by 58.6 %. The high cellulase- and xylanase-producing isolate no. 50, which showed 90 % ITS sequence identity to the fungal endophyte isolate 2196 (EU687056), improved T. clypeatus 18/50 growth by 45.7 %. A Gigantropanus sp. promoted the growth of T. clypeatus 18/50 and 20/50 by 45.7 and 44.1 %, respectively, and that of T. clypeatus 19/50 by 10.6 %. These results indicated the most beneficial potential partnership of T. clypeatus might involve cellulase-producing fungi isolated from the same ecological niche. The Gigantropanus sp. is a potential partner of T. clypeatus but is likely to be less common than cellulase-producing fungi isolated from fungus combs owing to the lower host specificity of the Gigantropanus sp. This study provides an interesting method to culture Termitomyces using an in vitro mixed culture method for production of Termitomyces fruiting bodies in the future.     

培菌白蚁与鸡枞菌研究进展

培菌白蚁起源于非洲,蚁巢内具有复杂的社会分工.培菌白蚁依靠独特的蚁巢结构维持内部稳态和气体循环.菌圃是白蚁培育鸡枞菌的场所.鸡枞菌隶属于担子菌亚门,但其传播方式和生活史具有区别于其它担子菌的特点.鸡枞菌协助白蚁进行植物纤维的消化,白蚁则为鸡枞菌提供合适的生长环境,并控制鸡枞菌的遗传结构.培菌白蚁和鸡枞菌形成紧密的共生关系,二者缺少任何一方都不能独立生存.本文综述了培菌白蚁的分类、品级、蚁巢结构,鸡枞菌的传播方式和生活史,白蚁与鸡枞菌的共生关系等,以期望为培菌白蚁生物学及鸡枞菌的研究提供有益参考.     

Intra- and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host

We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera Microcerotermes and Reticulitermes, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from Microcerotermes spp. and 367 phylotypes from Reticulitermes spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic "termite clusters" comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.