宏基因组学及其在口腔微生物研究中的应用

宏基因组是指环境中所有微生物的遗传物质总和。宏基因组学技术可以最大限度地利用环境中的微生物资源,受到了国内外微生物研究者的重点关注。口腔中寄居着大量的微生物群落,以往对口腔疾病微生物的研究大多局限于单纯的细菌培养技术,然而,由于培养技术的局限性,部分微生物很难或根本不能培养,宏基因组学技术打破了这一局限性,帮助人类发掘更丰富的口腔微生物资源。最近,以宏基因组学测序为基础的研究描绘出了口腔生态系统的图谱,越来越多的实验证明口腔微生物组在各种口腔疾病甚至全身系统性疾病中的重要作用。同时,这也为基于人类微生物组的诊断和治疗开辟了新的途径。本综述旨在说明宏基因组学是研究人类口腔疾病及全身疾病相关微生物的得力工具,而且具有广阔的发展前景,同时也讨论了宏基因组学在应用中有待克服的局限性。     

宏基因组方法揭示草地土壤微生物群落响应全球变化

草地在生物圈中发挥着重要的生态服务功能,而草地土壤微生物又是维持生态系统功能和稳定的关键要素之一。过去十几年间,宏基因组方法的进步为微生物群落分析提供了有力的工具。本文综述了宏基因组方法应用于草地土壤微生物群落响应全球变化的最新研究进展,特别是针对气候变化、大气组成变化、土地利用方式改变和外来物种入侵等条件下微生物群落的响应规律和反馈机制的研究。这些研究对于我们认识和了解微生物群落的生态功能十分重要,同时也对维持地球生态系统平衡具有积极的意义。最后,我们对未来应用宏基因组方法研究草地微生物群落进行了展望。     

土壤微生物资源管理、应用技术与学科展望

土壤中蕴藏着高度的微生物多样性,在陆地生态系统中发挥着非常重要的功能,加强对土壤微生物资源的综合管理与开发应用是提升生态系统稳定性与生产力及农产品质量的重要途径。首先,土壤微生物多样性具有全球性的重大意义,有待完善对土壤微生物的检测与监测技术研究,进而实现土壤微生物多样性与土壤功能的耦合以及对土壤质量的评定;其次,土壤微生物作为一种宝贵的生产资料和可持续资源,要加强其在土壤肥力强化与保育、土壤障碍消减与调节、土壤污染控制与修复等3个领域的应用研究。最后,未来土壤微生物学发展将会形成土壤微生物系统学、土壤微生物过程学与土壤微生物功能学3个子学科,要建立土壤微生物种质资源库与遗传信息库,推进土壤微生物生理代谢过程、生物化学过程及生态行为过程的研究,联结土壤微生物与土壤功能的关系,并从土壤中的功能微生物出发对环境变化作出积极响应和主动调控。此外,原创性方法的建立与应用是限制土壤微生物学发展的技术瓶颈,联合生物地理学与生物信息学破译重要基因的特定生态功能,并将其应用到生态模型以及生态系统未知领域的研究中去,是土壤微生物学面临的挑战。     

微生物组学的技术和方法及其应用

微生物组是指一个特定环境或生态系统中全部微生物及其遗传信息的集合, 其蕴藏着极为丰富的微生物资源。全面系统地解析微生物组的结构和功能, 将为解决人类面临的能源、生态环境、工农业生产和人体健康等重大问题带来新思路。然而, 微生物组学研究在很大程度上取决于其技术与方法的发展。在高通量测序技术出现以前, 微生物研究主要基于分离培养和指纹图谱等技术, 然而, 由于这些技术存在的缺陷, 人们对于微生物的认识十分有限。自21世纪初以来, 尽管高通量测序和质谱技术的革命性突破极大地促进了人们对于微生物的认识, 微生物组学技术在微生物组研究中的应用仍面临着诸多挑战。此外, 目前微生物组的结构和多样性等描述性研究已臻成熟, 微生物组学研究正处于从数量到质量、从结构到功能的关键转变时期。因此, 该文首先介绍了微生物组学的基本概念及其发展简史, 其次简述了微生物组学研究的相关技术和方法及其发展历程, 并进一步阐述了微生物组学的技术和方法在生态学研究中的应用及存在的主要问题, 最后从技术、理论和应用层面阐述了未来微生物组学技术和方法发展的前沿方向, 并提出了今后微生物组学研究的优先发展领域。     

深海微生物的研究与开发

深海是全球最大的独立生态系统,其中蕴涵着丰富并且独特的微生物资源有待我们开发。对深海微生物及其所处生态系统的研究将提升我们对地球早期环境及其变化过程的认识,为研究生命起源,甚至探索域外生命或其他潜在生命形式提供新的线索。对深海微生物研究开发的历史和进展进行了概述,并对这一研究领域的前沿及影响进行了讨论。     

油藏微生物多样性的分子生态学研究进展

油藏微生物是一类宝贵的资源, 在油层生态系统的物质循环和能量流动中发挥着主导作用。传统上, 主要依赖纯培养技术, 使得大部分油藏微生物都没能得到充分认识。分子方法克服了纯培养方法中遇到的问题, 能更好地了解环境微生物群落多样性。近年来, 在油藏微生物群落多样性研究中的应用进展迅速, 对油藏微生物学和生态学的发展产生了重要影响。文中评述了近年来国内外在油藏微生物分子生态方面的研究进展, 并对进一步的研究提出了展望。     

油藏微生物多样性的分子生态学研究进展

油藏微生物是一类宝贵的资源,在油层生态系统的物质循环和能量流动中发着主导作用.传统上,主要依赖纯培养技术,使得大部分油藏微生物都没能得到充分认识.分子方法克服了纯培养方法中遇到的问题,能更好地了解环境微生物群落多样性.近年来,在油藏微生物群落多样性研究中的应用进展迅速,对油藏微生物学和生态学的发展产生了重要影响.文中评述了近年来国内外在油藏微生物分子生态方面的研究进展,并对进一步的研究提出了展望.     

自养微生物同化CO_2的分子生态研究及同化碳在土壤中的转化

大气中CO2浓度持续升高和全球气候变暖是亟待解决的重大环境问题。自养微生物在环境中广泛分布,能直接参与CO2的同化,因此研究自养微生物同化CO2的分子生态学机制具有重大的科学意义。以往对自养微生物的研究多针对基因组DNA,从DNA水平揭示了不同生态系统中碳同化自养微生物的种群结构和多样性,但这些微生物在生态系统中的具体功能有待进一步的研究。近年来,随着转录组学研究技术和稳定同位素探针技术(SIP)的发展,自养微生物同化CO2的生态机理研究不断深入,这些研究明确揭示了碳同化自养微生物是河流、湖泊和海洋生态系统中CO2固定作用的驱动者,并新发现了一些具有CO2同化功能的微生物群落。基于国内外有关研究进展,从DNA和RNA水平上对自养微生物同化CO2的分子机理以及稳定同位素探针技术(SIP)在碳同化微生物研究中的应用进行了分析和总结,初步展望了RNA-SIP技术在陆地生态系统碳同化微生物分子生态学研究中的前景。同时,探讨了陆地生态系统同化碳的转化和稳定性机理,以期为深入了解生态系统碳循环过程和应对气候变化提供理论依据。     

丛生盔形珊瑚共附生可培养真菌

海洋中蕴藏着目前尚无法估量的微生物未知种群和资源,在众多海洋生态系统中,珊瑚礁生态系统是海洋中物种多样性程度最高的生态类型,其中几乎生活着所有海洋生物的门类.随着海洋天然产物和海洋药物研究的不断深入,海洋共附生微生物,特别是热带珊礁无脊椎动物来源的共附生生物,引起了人们的广泛关注.珊瑚共附生真菌的研究可能揭示珊瑚的生存能力和健康状态,同时又是开发利用海洋真菌的重要种质资源.     

活性污泥微生物群落宏组学研究进展

活性污泥是全球最常用的废水生物处理人工生态系统,微生物是驱动其污染净化能力的关键。活性污泥微生物群落所有物种与基因(简称"微生物组")的研究先后经历了"显微镜观察和纯菌培养分离"(1915)、"PCR扩增-测序"(1994)和"高通量测序-宏组学分析"(2006)三个重要阶段的发展变迁。相应地,我们对活性污泥微生物组的认知经历了从最早对微型动物(如钟虫和轮虫)及其他微生物的形貌观察和纯种培养鉴定到今天对整个微生物组的全局多样性认识的飞跃。近13年来,基于高通量测序的宏组学方法被广泛应用于揭示活性污泥微生物群落组成结构和功能,我们现在充分意识到活性污泥微生物组蕴藏着大量不可培养新物种和基因多样性,驱动着各类污染物的降解与转化。目前,特异性分子标记基因的扩增子测序技术已经被广泛应用于揭示城市和工业废水处理活性污泥微生物组和典型功能种群(如硝化细菌和聚磷菌)的时空多样性和群落构建机制,进而为未来实现活性污泥微生物组功能的精准调控奠定理论基础。宏基因组学研究在群落、种群和个体基因组水平全面解析了活性污泥微生物组驱动的碳、氮、磷元素循环过程,以及有机微污染物的生物降解和转化机理。将来活性污泥微生物组学研究需要在"标准化的组学分析方法和绝对定量""高通量培养组学""高通量功能基因组学"和"多组学方法的结合及多种方法并用"4个方面取得实现精准生态基因组学所需的技术突破,以最大限度发掘活性污泥微生物组在污水处理与资源回收领域的生态学与工程学价值。     

Upper Pleistocene Panthera leo spelaea (Goldfuss, 1810) remains from the Bilstein Caves (Sauerland Karst) and contribution to the steppe lion taphonomy,palaeobiology and sexual dimorphism

Remains of the steppe lion Panthera leo spelaea (Goldfuss) from historical digs in the Bilstein Caves of Warstein (Sauerland, NW Germany) are described. Their age seems to be from the Early Weichselian periods (Upper Pleistocene). Whereas the Bilstein cave was inhabited by cave bears at that time only a few hyena prey remains, were most likely imported into the cave entrance by hyenas. Bite and crush marks on a few bones of Bison priscus, Bos primigenius, Cervus elaphus, a rhinoceros Coelodonta antiquitatis vertebra and even several chewed cave bear bones prove the hyena presence which is similar to other caves in the Sauerland hyena den cave rich region. Additionally some larger wolves subspecies Canis lupusspelaeus bones were found, but only few Crocuta crocuta spelaea remains are present. After taphonomic comparisons to six other hyena and cave bear den caves of northern Germany, this cave can be classified as a cave bear den, which was briefly used by hyenas only for food storage or commuting or cave bear predation site in one part of the Cave. The lion material refers at least to one young adult lioness, one more adult female and two male lions; therefore, at minimum, the remains of four adult individuals are represented. The absence of juvenile lion material, in contrast to cave bear cub remains in the Bilstein Caves, proves that P. leo spelaea did not use this and all other caves in the region to raise their cubs. The bone material from the Bilstein Caves would prove the same hyena-lion antagonism conflict being recently proven for the Perick Caves, Balve Cave or Martins Cave well. Other situations in caves such as the Keppler Cave and the Bilstein Cave initially show the more complex taphonomic situation of lion remains in European caves, especially in cave bear dens, where they seem to have hunted periodically cave bears, such as it is already proven for hyenas in the Sauerland Karst and other caves of Europe.     

Phylogeography of cave pseudoscorpions in southern Australia

Aim  To investigate molecular phylogenetic divergence and historical biogeography of the cave-dwelling pseudoscorpion genus Protochelifer.
Location  Caves and nearby epigean habitats in southern Australia were sampled from western Victoria, Naracoorte Caves, Flinders Ranges, Kangaroo Island, Nullarbor Plain and south-west Western Australia.
Methods  Allozyme electrophoresis (57 individuals) and a 569-base-pair section of the mtDNA COI gene (22 individuals) were used to reconstruct phylogenetic relationships among four cave species and three epigean species from 13 locations.
Results  Phylogenetic reconstruction using the allozyme and mtDNA sequence data revealed a similar topology, showing recent speciation of several Protochelifer populations in caves from Naracoorte to the Nullarbor Plain. Naracoorte Caves contained a single species, Protochelifer naracoortensis , found in four separate caves, while all other cave species appear to be restricted to single caves.
Main conclusions  At a local scale, as indicated by the four Naracoorte caves, dispersal is thought to occur via micro- and mesocaverns, and possibly by phoresy using insect or bat vectors. With current data we are unable to determine if cavernicolous species of Protochelifer have arisen from a single cave colonization event followed by phoretic dispersal on bats to other caves, or multiple cave-invasion events from independent epigean ancestors. Genetic heterogeneity among Protochelifer populations from Nullarbor caves suggest that P. cavernarum , the only species presently recorded from this region, is likely to constitute a species complex requiring further study to fully resolve its relationships.     

Microbial Activity in Caves−A Geological Perspective

Caves are commonly the home of diverse microbial biotas, the sites of active mineral precipitation, and/or receptacles for the deposition of sediment. Mineral precipitation is commonly considered to be abiogenic despite the fact that microbes are present in caves, especially in the twilight zone. Detailed analysis of cave substrates from a geological perspective shows that microbes can mediate constructive (microbe calcification, trapping and binding, mediation of crystal growth) and destructive (substrate etching and breakdown) processes. Potentially these processes can significantly influence the formation and preservation of any cave deposit. Preservation of microbes is possible if mineralization takes place while the microbe is alive or shortly after its demise. If not, all record of the microbe will be lost to decay. Even if the microbes are preserved, it may be difficult to determine if they played an active or passive role in the formation of the deposits in which they are entombed. For old cave deposits, such an assessment must rely on spatial relationships and comparison of textures with those known to form as a result of microbial activity. Nevertheless, available evidence indicates that microbes can play a major role in the formation and modification of cave deposits. Equally, however, it is apparent that the full scope and impact of microbial activity on cave deposits has yet to be realized. Recognition of microbial activity in old CaCO 3 cave deposits relies on (1) documentation and recognition of mineralized microbes, (2) recognition of stromatolitic structures that formed through microbial activity, and/or (3) the identification of fabrics/textures that are known to be indicative of microbial activity. All of these criteria fundamentally rely on the interpretation of fabrics preserved in the cave deposits. Virtually all of these interpretations are open to debate.     

Phylogenetic evidence from freshwater crayfishes that cave adaptation is not an evolutionary dead‐end

Caves are perceived as isolated, extreme habitats with a uniquely specialized biota, which long ago led to the idea that caves are “evolutionary dead‐ends.” This implies that cave‐adapted taxa may be doomed for extinction before they can diversify or transition to a more stable state. However, this hypothesis has not been explicitly tested in a phylogenetic framework with multiple independently evolved cave‐dwelling groups. Here, we use the freshwater crayfish, a group with dozens of cave‐dwelling species in multiple lineages, as a system to test this hypothesis. We consider historical patterns of lineage diversification and habitat transition as well as current patterns of geographic range size. We find that while cave‐dwelling lineages have small relative range sizes and rarely transition back to the surface, they exhibit remarkably similar diversification patterns to those of other habitat types and appear to be able to maintain a diversity of lineages through time. This suggests that cave adaptation is not a “dead‐end” for freshwater crayfish, which has positive implications for our understanding of biodiversity and conservation in cave habitats.     

Anthropization level of Lascaux Cave microbiome shown by regional‐scale comparisons of pristine and anthropized caves

Limestone areas across the world develop karstic caves, which are populated by a wide range of macro‐ and microorganisms. Many of these caves display Paleolithic art or outstanding speleothems, and in the last century they have been subjected to anthropization due to touristic management and intense human frequentation. Despite their cultural importance and associated conservation issues, the impact of anthropization on cave biodiversity is not known. Here, we show that anthropization is associated with specific cave biota modifications. We compared diversity in four pristine caves, four anthropized show caves, and the iconic Lascaux Cave with even stronger anthropization. The predominant microbial higher taxa were the same in all caves, but the most anthropized cave (Lascaux) was unique as it differed from the eight others by a higher proportion of Bacteroidetes bacteria and the absence of Euryarchaeota and Woesearchaeota archaea. Anthropization resulted in lower diversity and altered community structure for bacteria and archaea on cave walls, especially in Lascaux, but with a more limited effect on microeukaryotes and arthropods. Our findings fill a key gap in our understanding of the response of karstic communities to anthropization, by revealing that tourism‐related anthropization impacts on the prokaryotic microbiome rather than on eukaryotic residents, and that it shapes cave biota irrespective of cave natural features.     

The distribution of cave twilight‐zone spiders depends on microclimatic features and trophic supply

Caves are not closed systems. Trophic dynamics in these habitats are driven by resource availability, and species that move between cave and outdoor environments may play a major role in resource availability. Spiders are among the most abundant invertebrates in caves; however, very few studies have tested factors hypothesized to affect the distribution of spiders among caves, and it is not known whether the trophic features of caves play a role in determining the occurrence, abundance, or breeding success of spiders. We assessed the distribution of the cave‐dwelling orb‐weaver spider Meta menardi in Italy, in a Mediterranean and in a Pre‐alpine area during summer and winter. We analyzed the relationships between spider distribution and multiple cave features, describing both the abiotic and the biotic environment. Using visual encounter surveys, the detection probability of this species was high, indicating that this technique provides reliable information on spider distribution. In Mediterranean caves, spider presence was more likely in cold and wet caves with abundant dipterans. In Pre‐alpine caves, spider presence was more likely in deep caves with abundant dipterans. Dipteran abundance was the variable best explaining spider distribution when pooling all sampled caves. This study shows that adults of M. menardi do not occur randomly among caves, but select caves with specific features. Prey availability and abiotic features are major determinants of habitat suitability for cave spiders. The strong relationship between spider distribution and prey availability suggests that the distribution of these spiders might be an indicator of the resources available in the twilight zones of caves.     

Sampling adequacy in an extreme environment: species richness patterns in Slovenian caves

Caves harbor a rich fauna unique to subterranean environments. Although extensive records of cave animals are available, only a small fraction of known caves in any region have been biologically assessed. We investigated the impact of incomplete sampling using one of the richest, best documented cave faunas in the world – that of the Dinaric karst of Slovenia. We utilized time snapshots (1940, 1970, and 2000) of the caves and cave fauna to analyze stability of hotspots, spatial pattern, and relationship between number of species and number of caves. Using data aggregated into 100km² hexagons, the location of hotspots, black–white joins, Moran's I, and spatial autocorrelation all remained constant, at least from 1970 on. The linear regression coefficient of the relationship between number of caves and number of species declined with time. Most hexagons had been sampled, but there was no indication that any hexagon had been sampled intensively enough for the accumulation curve of number of caves versus number of species within a hexagon to reach an asymptote. This appeared to be the result of a highly skewed distribution of species richness among caves. Number and position of hotspots can be predicted from information on these few high diversity caves.     

ECOMORPHOLOGICAL CONVERGENCE OF CAVE COMMUNITIES

Extreme selective environments are commonly believed to funnel evolution toward a few predictable outcomes. Caves are well‐known extreme environments with characteristically adapted faunas that are similar in appearance, physiology, and behavior all over the world, even if not closely related. Morphological diversity between closely related cave species has been explained by difference in time since colonization and different ecological influence from the surface. Here, we tested a more classical hypothesis: morphological diversity is niche‐based, and different morphologies reflect properties of microhabitats within caves. We analyzed seven communities with altogether 30 species of the subterranean amphipod (crustacean) genus Niphargus using multivariate morphometrics, multinomial logit models cross‐validation, and phylogenetic reconstruction. Species clustered into four distinct ecomorph classes—small pore, cave stream, cave lake, and lake giants—associated with specific cave microhabitats and of multiple independent phylogenetic origins. Traits commonly regarded as adaptations to caves, such as antenna length, were shown to be related to microhabitat parameters, such as flow velocity. These results demonstrate that under the selection pressure of extreme environment, the ecomorphological structure of communities can converge. Thus, morphological diversity does not result from adaptive response to temporal and ecological gradients, but from fine‐level niche partitioning.     

Cave invertebrate assemblages differ between native and exotic leaf litter

Abstract Allochtonous leaf litter is an important source of energy and nutrients for invertebrates in cave ecosystems. A change to the quality or quantity of litter entering caves has the potential to disrupt the structure and function of cave communities. In this study, we adopted an experimental approach to examine rates of leaf litter decomposition and the invertebrate assemblages colonizing native and exotic leaf litter in limestone caves in the Jenolan Caves Karst Conservation Reserve, New South Wales, Australia. We deployed traps containing leaf litter from exotic sycamore (Acer pseudoplatanus) and radiata pine (Pinus radiata) trees and native eucalypts (Eucalyptus spp.) in twilight zones (near the cave entrance) and areas deep within the caves for 3 months. Thirty‐two invertebrate morphospecies were recorded from the litter traps, with greater richness and abundance evident in the samples from the twilight zone compared with areas deep within the cave. Sycamore litter had significantly greater richness and abundance of invertebrates compared with eucalypt and pine litter in samples from the twilight zone, but there was no difference in richness or abundance among litter samples placed deep within the cave. Relative rates of decay of the three litters were sycamore > eucalypt > pine. We discuss the potential for the higher decomposition rates and specific leaf area in sycamores to explain their higher invertebrate diversity and abundance. Our findings have important implications for the management of exotic plants and the contribution of their leaf litter to subterranean ecosystems.