A polyphasic approach for the taxonomy of cyanobacteria: principles and applications

Taxonomic classification is the only method for understanding and exploring knowledge about organismal diversity. However, it is complicated in prokaryotic, phylogenetically old, phototrophic cyanobacteria, which contain very simple unicellular forms up to multicellular types with a differentiated and diversified thallus. Their cells are cytologically relatively simple, but variable in shape. Various genotypes are adaptable to various specialized ecosystems. The introduction and combination of modern molecular, cytomorphological and ecological methods in the taxonomy of cyanobacteria is necessary and should be accepted as the only method for the elaboration of their modern systematics. The combination of different methods should be based on molecular sequencing as the basic approach, to which must be added other criteria (morphological, ecological) if they are available and which are distinct and recognizable in cyanobacterial populations. The use of such characteristics is necessary and must be obligatorily included for the final characterization both of strains and natural populations. Application of this polyphasic, i.e. combined approach is considered as a unique, modern, unambiguous, unequivocal and a fully acceptable methodological procedure, but it is not yet commonly used, nor possible for all known cyanobacterial populations. The main principles and recent problems of this modern classification method are discussed in the following review and will be the basis of further discussion.     

分子生物学技术在昆虫系统学研究中的应用

分子生物学技术应用于昆虫系统学研究,是８０年代末新兴起来的,近几年来发展相当迅速。为了把握这个研究方向,并促进这个研究领域的发展,作者从研究方法、研究内容、研究对象等方面着手,对近１０年来分子生物学技术应用于昆虫系统学中的研究进展进行了概括和总结。介绍了ＤＮＡ序列测定、ＲＦＬＰ,分子杂交技术、ＲＦＰＬ、分子杂交技术、ＲＡＰＤ、ＳＳＣＰ及ＤＳＣＰ等几种主要方法及其应用情况,并从种及种下阶元的分类鉴定     

Cyanotoxin production and phylogeny of benthic cyanobacterial strains isolated from the northeast of Brazil

Most of the knowledge about cyanobacteria toxin production is traditionally associated with planktonic cyanobacterial blooms. However, some studies have been showing that benthic cyanobacteria can produce cyanotoxins. According to this, we aimed to evaluate the production of microcystins and saxitoxins in benthic cyanobacteria isolated from aquatic ecosystems in the Northeast of Brazil and to use a polyphasic approach for their identification. Forty-five clonal strains were isolated from rivers and water supply reservoirs, and identified using morphological and molecular phylogenetic characteristics. In order to evaluate the toxins production, the strains were screened for genes involved in the biosynthesis of microcystins and saxitoxins, positive results were confirmed and cyanotoxins quantified using HPLC. Eight species were identified belonging to the Phormidiaceae, Pseudanabaenaceae and Nostocaceae families. This is the first study in Brazil that shows that strains from the Geitlerinema genus correspond to at least three phylogenetic lineages, which possibly correspond to three distinct species to be subsequently reclassified. The strains that showed one of the genes involved in the cyanotoxins production were analyzed by HPLC and Geitlerinema amphibium, Geitlerinema lemmermannii, Cylindrospermum stagnale and Phormidium uncinatum were identified as producing one or more saxitoxins variants. Thus, this is the first report of saxitoxins production for those first three species and the first report in Brazil for P. uncinatum.     

Systematics,conservation and sustainable development

The contribution of systematics to conservation and sustainable use of species is discussed. An adequate inventory of species is required for both areas and recent field work shows that the inventory is far from complete. This supports the first mission of Agenda 2000 which is to discover described and inventory global species diversity. The importance of making these data available in data base format is stressed. Sound systematics is a necessity for conservation legislation where control depends on the ability to identify organisms correctly. The establishment of reserves is also dependent upon data from systematics about centres of diversity and endemism and examples of this are given. Molecular systematics has made new tools available to conservation such as genetic fingerprinting which is useful both to breeding programmes of rare species and for identification for legal proceedings.Systematics is also one of the foundations for programmes of sustainable development especially in the search for new crops, non-timber forest plants from extraction forests and the identification of wild relatives of crop species. Examples of the role of systematics in a fuelwood programme in Zimbabwe, a sustainable development programme in northeast Brazil and in the search for a chemical component with medicinal properties for curing AIDS are given. The more predictive a classification we can develop using modern cladistic and molecular techniques the more useful systematics will be for both conservation and sustainable development. The goal of Agenda 2000 to organize the information derived from the programme in an efficiently retrievable form that best meets the needs of science and society is a laudable target that is crucial for conservation and the sustainable use of the plant resources of the world.     

Application of the methods of molecular systematics to classification and identification of bacteria of the genus Bifidobacterium

The methods of molecular systematics currently used in the classification and identification of bifidobacteria are reviewed. The sequencing of the 16S rRNA gene and some other genes considered to be phylogenetic markers is a universal and effective approach to taxonomic characterization of members of the genus Bifidobacterium and to reliable identification of new isolates. Various techniques of obtaining DNA fingerprints (PFGE, RAPD, rep-PCR) are widely used for solving particular problems in identifying bifidobacteria. Bacteria of the genus Bifidobacterium are important organisms in biotechnology and medicine. The research into molecular systematics of bifidobacteria provides a basis not only for the solution of taxonomic problems, but also for monitoring of individual species in the environment and for more detailed study of the genetics and ecology of this group of microorganisms.     

植物遗传多样性和系统学研究中的等位酶分析*

本文对“等位酶”概念、等位酶分析方法在植物遗传多样性和系统学研究中应用的范围（包括种上和种下）进行了介绍和讨论,并对其优点和缺点进行了评论。认为等位酶分析是研究生物遗传多样性的重要方法,并为系统学和进化研究进入分子水平开辟了广阔而实用的前景,但使用分子资料并不意味着可以抛弃形态、细胞等其他方面的资料,它们的关系是互相补充,而决不是互相代替。     

How diverse a genus can be: An integrated multi-layered analysis into Desmonostoc (Nostocaceae,Cyanobacteriota)

Cyanobacteria (Phylum Cyanobacteriota) are Gram-negative bacteria capable of performing oxygenic photosynthesis. Although the taxonomic classification of cyanobacteria was for a long time based primarily on morphological characters, the application of other techniques (e.g. molecular phylogeny), especially in recent decades, has contributed to a better resolution of cyanobacteria systematics, leading to a revision of the phylum. Although Desmonostoc occurs as a new genus/cluster and some species have been described recently, relatively few studies have been carried out to elucidate its diversity, which encompasses strains from different ecological origins, or examine the application of new characterization tools. In this context, the present study investigated the diversity within Desmonostoc, based on morphological, molecular, metabolic, and physiological characteristics. Although the usage of physiological parameters is unusual for a polyphasic approach, they were efficient in the characterization performed here. The phylogenetic analysis based on 16S rRNA gene sequences put all studied strains (25) into the D1 cluster and indicated the emergence of novel sub-clusters. It was also possible to observe that nifD and nifH exhibited different evolutionary histories within the Desmonostoc strains. Collectively, metabolic and physiological data, coupled with the morphometric data, were in general, in good agreement with the separation based on the phylogeny of the 16S rRNA gene. Furthermore, the study provided important information on the diversity of Desmonostoc strains collected from different Brazilian biomes by revealing that they were cosmopolitan strains, acclimatized to low luminous intensities, with a large metabolic diversity and great biotechnological potential.     

The molecular genetics of cyanobacterial toxicity as a basis for monitoring water quality and public health risk

Toxic cyanobacteria pose a significant hazard to human health and the environment. The recent characterisation of cyanotoxin synthetase gene clusters has resulted in an explosion of molecular detection methods for these organisms and their toxins. Conventional polymerase chain reaction (PCR) tests targeting cyanotoxin biosynthesis genes provide a rapid and sensitive means for detecting potentially toxic populations of cyanobacteria in water supplies. The adaptation of these simple PCR tests into quantitative methods has additionally enabled the monitoring of dynamic bloom populations and the identification of particularly problematic species. More recently, DNA microarray technology has been applied to cyanobacterial diagnostics offering a high-throughput option for detecting and differentiating toxic genotypes in complex samples. Together, these molecular methods are proving increasingly important for monitoring water quality.     

Methods to detect cyanobacteria and their toxins in the environment

Cyanobacteria blooms are since early times a cause for environmental concern because of their negative impact through the release of odors, water discoloration, and more dangerously through the release of toxic compounds (i.e. the cyanotoxins) that can affect both human and animal welfare. Surveillance of the aquatic ecosystems is therefore obligatory, and methods to achieve such require a prompt answer not only regarding the species that are producing the blooms but also the cyanotoxins that are being produced and/or released. Moreover, besides this well-known source of possible intoxication, it has been demonstrated the existence of several other potential routes of exposure, either for humans or other biota such as through food additives and in terrestrial environments (in plants, lichens, biological soil crusts) and the recognition of their harmful impact on less studied ecosystems (e.g. coral reefs). Nowadays, the most frequent approaches to detect toxic cyanobacteria and/or their toxins are the chemical-, biochemical-, and molecular-based methods. Above their particular characteristics and possible applications, they all bring to the environmental monitoring several aspects that are needed to be discussed and scrutinized. The end outcome of this review will be to provide newer insights and recommendations regarding the methods needed to apply in an environmental risk assessment program. Therefore, a current state of the knowledge concerning the three methodological approaches will be presented, while highlighting positive and negative aspects of each of those methods within the purpose of monitoring or studying cyanobacteria and their toxins in the environment.     

Assessment of phenotypic and genetic diversity in the yeast genusMetschnikowia

The taxonomy of the yeast genusMetschnikowia has undergone profound changes over the past century. Major developments, from the capacity to obtain pure cultures of parasitic species to progress associated with the extensive use of molecular biology tools in yeast systematics, are briefly reviewed. Results from past work and new data are combined to evaluate evolutionary relationships and clarify the classification of both terrestrial and aquatic species. Recent physiological studies, including the utilization of non-conventional carbon and nitrogen sources, and characteristics like lipolytic activity and maximum temperatures for growth, are presented. The assessment of the genetic diversity within the genus by restriction analysis of the mitochondrial DNA and by the production of specific DNA probes has been explored. The results indicate the potential application of the latter in rapid identification procedures.     

分子标记在食（药）用真菌遗传多样性研究中的应用

食（药）用真菌在经济和生态方面都具有重要意义,其遗传多样性研究是资源可持续利用和生物保护学研究的基础,有利于食（药）用真菌种质资源的收集、保存、评价和利用,也有助于其分类学、系统学及进化等的研究。遗传多样性的研究方法很多,分子标记是目前最常用最有效的方法之一。综合分析了分子标记在食（药）用真菌遗传多样性研究中的应用,比较了各种标记的应用范围、优缺点,探讨了分子标记用于食（药）用真菌遗传多样性评价的前景及问题。     

串珠藻目分子系统学研究进展

串珠藻目（Batrachospermales）是淡水红藻中最主要的类群。近年来,应用DNA序列分析探讨串珠藻目的系统发育,并与传统的形态学和生态学特征相结合,为串珠藻目系统学研究拓展了新的思路。本文回顾了串珠藻目的建立及其所含类群的研究历史,归纳了目前在串珠藻目系统发育与进化研究中常用的分子标记方法,其中包括核基因组的18SrDNA、26SrDNA和ITS序列,叶绿体基因组的rbcL序列,线粒体基因组的cox9．-3序列,以及新兴的ISSR技术,并对各种分子标记的特点及适用范围做了评述。结果表明,ITS序列多适用于种群分化及相近种间遗传分析,ISSR标记适用于种下分类群间及同一种群不同个体间基因多态性分析,cox2-3序列在一定程度上也可用于同一种群不同个体间的基因多态性分析,而18SrDNA与rbcLFF列既可用于种问关系分析,又可用于更高水平分析的构建系统树。这些分子枥对己已被证明在研究串珠藻目系统地理、物种起源和散布机制方面有着广泛的应用前景。同时,本文对串珠藻目分子系统学研究的最新进展也进行了概述,并对今后的研究方向做了展望。     

串珠藻目分子系统学研究进展

串珠藻目(Batrachospermales)是淡水红藻中最主要的类群。近年来, 应用DNA序列分析探讨串珠藻目的系统发育, 并与传统的形态学和生态学特征相结合, 为串珠藻目系统学研究拓展了新的思路。本文回顾了串珠藻目的建立及其所含类群的研究历史, 归纳了目前在串珠藻目系统发育与进化研究中常用的分子标记方法, 其中包括核基因组的18S rDNA、26S rDNA和ITS序列, 叶绿体基因组的rbcL序列, 线粒体基因组的cox2-3序列, 以及新兴的ISSR技术, 并对各种分子标记的特点及适用范围做了评述。结果表明, ITS序列多适用于种群分化及相近种间遗传分析, ISSR标记适用于种下分类群间及同一种群不同个体间基因多态性分析, cox2-3序列在一定程度上也可用于同一种群不同个体间的基因多态性分析, 而18S rDNA 与rbcL序列既可用于种间关系分析, 又可用于更高水平分析的构建系统树。这些分子标记已被证明在研究串珠藻目系统地理、物种起源和散布机制方面有着广泛的应用前景。同时, 本文对串珠藻目分子系统学研究的最新进展也进行了概述,并对今后的研究方向做了展望。     

Metabolic fingerprinting as an indicator of biodiversity: towards understanding inter-specific relationships among Homoscleromorpha sponges

Sponges are an important source of secondary metabolites showing a great diversity of structures and biological activities. Secondary metabolites can display specificity on different taxonomic levels, from species to phylum, which can make them good taxonomic biomarkers. However, the knowledge available on the metabolome of non-model organisms is often poor. In this study, we demonstrate that sponge chemical diversity may be useful for fundamental issues in systematics or evolutionary biology, by using metabolic fingerprints as indicators of metabolomic diversity in order to assess interspecific relationships. The sponge clade Homoscleromorpha is particularly challenging because its chemistry has been little studied and its phylogeny is still debated. Identification at species level is often troublesome, especially for the highly diversified Oscarella genus which lacks the fundamental characters of sponge taxonomy. An HPLC–DAD–ELSD–MS metabolic fingerprinting approach was developed and applied to 10 Mediterranean Homoscleromorpha species as a rapid assessment of their chemical diversity. A first validation of our approach was to measure intraspecific variability, which was found significantly lower than interspecific variability obtained between two Oscarella sister-species. Interspecific relationships among Homoscleromorpha species were then inferred from the alignment of their metabolic fingerprints. The resulting classification is congruent with phylogenetic trees obtained for a DNA marker (mitochondrial COI) and demonstrates the existence of two distinct groups within Homoscleromorpha. Metabolic fingerprinting proves a useful complementary tool in sponge systematics. Our case study calls for a revision of Homoscleromorpha with further phylogenetic studies and identification of additional chemical synapomorphic characters.     

Use of qPCR for the study of hepatotoxic cyanobacteria population dynamics

Toxic cyanobacteria blooms are increasingly frequent and object of greater concern due to its ecological and health impacts. One important lack in the toxic cyanobacteria research field is to understand which parameters influence most and how they operate to regulate the overall levels of cyanotoxins in a body of water. MC concentration is believed to be influenced by changes in several seasonal environmental factors that influence the succession of toxic cyanobacteria. In the last years, qPCR (quantitative polymerase chain reaction) has been applied to determine the seasonal and temporal shifts in the proportions of MC-producing and non-MC-producing subpopulations by quantifying both mcy genotypes and total population numbers. We discuss the most prominent and recent studies using qPCR to address hepatotoxic cyanobacteria population dynamics and evaluate how they helped understanding the factors promoting the growth of toxic strains in situ and the succession of hepatotoxin-producing genera in natural populations.     

直翅目昆虫分子系统学研究新进展

对1994年以来国内外在直翅目昆虫种群遗传变异及进化、种及种下阶元的分类鉴定、种上阶元的系统发育分析及分子进化等分子系统学方面的研究进展进行了综述。近年来,蝗亚目昆虫分子系统学方面的研究成果较为丰富,而有关螽亚目的分子系统学研究较少。线粒体基因和核基因序列联合分析、整个基因组全序列分析以及分子数据与形态学的密切结合将是分子系统学未来发展的主要研究手段。     

根瘤菌分类研究进展及存在的争议

根瘤菌是一类与农业生产关系密切的细菌。近年来, 现代分子生物学技术的发展和应用使根瘤菌分类研究有了突破性进展, 新的根瘤菌资源的不断发掘, 根瘤菌的分类体系也不断地被完善。文中主要阐述了根瘤菌分类系统的演化历史、目前存在争议及相关的研究进展。     

Biosynthesis of cyclic carotenoids: Biochemistry and molecular genetics of the reaction sequence

Cyclic carotenids are found in the photosynthetic apparatus of higher plants. The biosynthesis of these pigments has been under investigation for two decades. Especially with in vitro systems from chromoplasts and unicellular cynaobacteria. new information has been obtained on the enzymatic reactions in carotenogenesis. Very recently, progress in the identification and cloning of several genes from the carotenogenic pathway has been made with bacteria, cyanobacteria and fungi. The new developments in biochemistry and molecular biology of cyclic carotenoids are reviewed, and the consequences of the results obtained by molecular genetics on the general understanding of the biosynthetic pathway of carotenoids are discussed, especially the desaturation and isomerization reactions.     

植物遗传多样性和系统学研究中的等位酶分析*

本文对“等位酶”的概念、等位酶分析方法在植物遗传多样性和系统学研究中应用的范围（包括种上和种下）进行了介绍和讨论,并对其优点和缺点进行了评论。认为等位酶分析是研究生物遗传多样性的重要方法,并为系统学和进化研究进入分子水平开辟了广阔而实用的前景,但使用分子资料并不意味着可以抛弃形态、细胞等其他方面的资料,它们的关系是互相补充,而决不是互相代替。     

DNA条形码在鞘翅目昆虫分子系统学研究中的应用

近年来,DNA条形码(DNA Barcoding)技术已经成为生物分类学研究中备受关注的新型技术,并在鞘翅目昆虫系统发育研究中得到广泛应用。本文总结了鞘翅目昆虫DNA条形码研究所用COⅠ基因序列,概述了DNA条形码在鞘翅目昆虫的物种分类鉴定、发现新种和隐存种、系统发育关系研究等方面的应用,并对DNA条形码研究技术新进展和标准序列筛选需要注意的问题进行了讨论。