Saprolegniaceae identified on amphibian eggs throughout the Pacific Northwest, USA, by internal transcribed spacer sequences and phylogenetic analysis

We assessed the diversity and phylogeny of Saprolegniaceae on amphibian eggs from the Pacific Northwest, with particular focus on Saprolegnia ferax, a species implicated in high egg mortality. We identified isolates from eggs of six amphibians with the internal transcribed spacer (ITS) and 5.8S gene regions and BLAST of the GenBank database. We identified 68 sequences as Saprolegniaceae and 43 sequences as true fungi from at least nine genera. Our phylogenetic analysis of the Saprolegniaceae included isolates within the genera Saprolegnia, Achlya and Leptolegnia. Our phylogeny grouped S. semihypogyna with Achlya rather than with the Saprolegnia reference sequences. We found only one isolate that grouped closely with S. ferax, and this came from a hatchery-raised salmon (Idaho) that we sampled opportunistically. We had representatives of 7-12 species and three genera of Saprolegniaceae on our amphibian eggs. Further work on the ecological roles of different species of Saprolegniaceae is needed to clarify their potential importance in amphibian egg mortality and potential links to population declines.     

Analysis of fungal flora in indoor dust by ribosomal DNA sequence analysis, quantitative PCR, and culture

In recent years increasing attention has been given to the potential health effects of fungal exposure in indoor environments. We used large-scale sequencing of the fungal internal transcribed spacer region (ITS) of nuclear ribosomal DNA to describe the mycoflora of two office buildings over the four seasons. DNA sequencing was complemented by cultivation, ergosterol determination, and quantitative PCR analyses. Sequences of 1,339 clones were clustered into 394 nonredundant fungal operational taxonomical units containing sequences from 18 fungal subclasses. The observed flora differed markedly from that recovered by cultivation, the major differences being the near absence of several typical indoor mold genera such as Penicillium and Aspergillus spp. and a high prevalence of basidiomycetes in clone libraries. A total of 55% of the total diversity constituted of unidentifiable ITS sequences, some of which may represent novel fungal species. Dominant species were Cladosporium cladosporioides and C. herbarum, Cryptococcus victoriae, Leptosphaerulina americana and L. chartarum, Aureobasidium pullulans, Thekopsora areolata, Phaeococcomyces nigricans, Macrophoma sp., and several Malassezia species. Seasonal differences were observed for community composition, with ascomycetous molds and basidiomycetous yeasts predominating in the winter and spring and Agaricomycetidae basidiomycetes predominating in the fall. The comparison of methods suggested that the cloning, cultivation, and quantitative PCR methods complemented each other, generating a more comprehensive picture of fungal flora than any of the methods would give alone. The current restrictions of the methods are discussed.     

A simple method to disperse eggs from lepidopteran scalelike egg masses and to observe embryogenesis

Various insect species lay tiny, thin- and soft-shelled eggs in a connected “egg mass”. Especially in several lepidopteran species, the structure of such clustered eggs is covered with complicated scale-like secretion, which has so far prevented analysis of individual embryos. However, few studies on methods to disperse egg clusters of such insects and to compare different methods have been carried out. To overcome these problems, we developed methods to separate egg masses into individual eggs, using two Tortricidae pests, Homona magnanima Diakonoff and Adoxophyes honmai Yasuda (Lepidoptera: Tortricidae). The eggs were successfully separated from each other using potassium hydroxide and sodium hypochlorite. Although the separated eggs no longer continued their embryogenesis, fixation with heptane–paraformaldehyde, permeabilization with heptane–methanol, and staining with several dyes enabled easy observation of embryogenesis. This protocol is expected to be applicable to other insect taxa and will facilitate further morphological and genetic studies in insects that lay egg masses.     

The importance of combining air sampling and surface analysis when studying problematic houses for mold biodiversity determination

Houses that underwent water damages are often responsible for heath problems of the occupants. Since there is no universally used protocol for the analysis, we wanted to verify the usefulness of surface sampling versus air sampling for the evaluation of mold diversity in problematic houses and the value of the number of visible mold growth zones to predict air quality. Seventeen houses were sampled for culturable molds in the air and on the surfaces showing contamination. We compared the mold taxa found in the air and on the surfaces and verified the correlation between the number of moldy surfaces and airbone mold concentration. This study demonstrated that, surprisingly, some of the so called wet spore molds (e.g. Stachybotrys) were found more often from air than surface samples whereas, some dry spore molds (e.g. Asp. fumigatus) was more easily isolated from surface samples. There was a good correlation between the number of visible mold growth zones and the concentration of airborne molds. We conclude that air and surface sampling are necessary to evaluate mold diversity in problematic houses and that the number of mold growth zones is a good predictor of airborne mold concentration.     

Diversity and significance of mold species in Norwegian drinking water

In order to determine the occurrence, distribution, and significance of mold species in groundwater- and surface water-derived drinking water in Norway, molds isolated from 273 water samples were identified. Samples of raw water, treated water, and water from private homes and hospital installations were analyzed by incubation of 100-ml membrane-filtered samples on dichloran-18% glycerol agar. The total count (number of CFU per 100 ml) of fungal species and the species diversity within each sample were determined. The identification of mold species was based on morphological and molecular methods. In total, 94 mold species belonging to 30 genera were identified. The mycobiota was dominated by species of Penicillium, Trichoderma, and Aspergillus, with some of them occurring throughout the drinking water system. Several of the same species as isolated from water may have the potential to cause allergic reactions or disease in humans. Other species are common contaminants of food and beverages, and some may cause unwanted changes in the taste or smell of water. The present results indicate that the mycobiota of water should be considered when the microbiological safety and quality of drinking water are assessed. In fact, molds in drinking water should possibly be included in the Norwegian water supply and drinking water regulations.     

Diversity and Significance of Mold Species in Norwegian Drinking Water

In order to determine the occurrence, distribution, and significance of mold species in groundwater- and surface water-derived drinking water in Norway, molds isolated from 273 water samples were identified. Samples of raw water, treated water, and water from private homes and hospital installations were analyzed by incubation of 100-ml membrane-filtered samples on dichloran-18% glycerol agar. The total count (number of CFU per 100 ml) of fungal species and the species diversity within each sample were determined. The identification of mold species was based on morphological and molecular methods. In total, 94 mold species belonging to 30 genera were identified. The mycobiota was dominated by species of Penicillium, Trichoderma, and Aspergillus, with some of them occurring throughout the drinking water system. Several of the same species as isolated from water may have the potential to cause allergic reactions or disease in humans. Other species are common contaminants of food and beverages, and some may cause unwanted changes in the taste or smell of water. The present results indicate that the mycobiota of water should be considered when the microbiological safety and quality of drinking water are assessed. In fact, molds in drinking water should possibly be included in the Norwegian water supply and drinking water regulations.     

Batrachology of Japan and adjacent regions - a systematic review

As is the case with most other organisms, studies of amphibians started with systematics as the basis for many other biological fields, but systematics itself has endured a slower progress than other fields of biology have enjoyed for a long time. Nevertheless, philosophical and methodological 'revolutions' in the past 30 years, combined with more recent progress in biochemical and molecular techniques, have been forcing current systematics into a great change. Stejneger's (1907) monumental book, the first thorough review of amphibians from Japan and adjacent regions, was published nearly a century ago, and since then, the increase in number of areas that have been closely surveyed, together with the application of new theories and methods, have greatly enlarged our knowledge of the amphibian fauna in this region. For example, extensive experiments by the Hiroshima School using artificial hybridization techniques greatly contributed to our understandings of relationships among some groups of amphibians that had been taxonomically confusing. In addition to this, new species discovered by more detailed field surveys in a wider area and cryptic species revealed by analyses of isozymes and acoustic properties have made for a great increment in the number of taxa in this region. Further, analyses of DNA sequences now enable us to infer the affinities of taxa with few phylogenetically informative phenotypic properties, and the resultant phylogenetic hypotheses further contribute to our understandings of biogeography and many other issues on amphibian evolution. From these advances, it has been clarified that the amphibian species diversity is far greater than was previously expected, which in turn means a high endemism within particular regions. It is expected that future studies will also reveal exact systematic relationships of wide-ranging species, both those occurring within this region and those extending outside of it. For that purpose, international and interdisciplinary studies are indispensable. But what is also necessary is to conserve amphibian populations so that they will not disappear before their true relationships are realized. Knowledge of systematics among young amphibian biologists is also a matter of high priority.     

中国灵芝属真菌的多样性与资源

灵芝属是大型真菌的一个重要类群,具有重要的经济价值、生态价值和文化价值。尽管国内外对灵芝属真菌的研究较多,但灵芝属真菌的分类一直存在诸多问题,我国过去报道的灵芝属真菌有114个分类单元,但其中很多的分类地位存在争议。本文基于凭证标本,确认我国目前发现的灵芝种类有40种,其中具有ITS分子序列的种类有39种,其他74个分类单元或为同物异名或为待定种。本文提供的中国39种灵芝的ITS序列可为今后准确鉴定灵芝的野生和栽培种类提供依据。     

Diversity and phylogenetic affinities of foliar fungal endophytes in loblolly pine inferred by culturing and environmental PCR

We examined endophytic fungi in asymptomatic foliage of loblolly pine (Pinus taeda) in North Carolina, U.S.A., with four goals: (i) to evaluate morphotaxa, BLAST matches and groups based on sequence similarity as functional taxonomic units; (ii) to explore methods to maximize phylogenetic signal for environmental datasets, which typically contain many taxa but few characters; (iii) to compare culturing vs. culture-free methods (environmental PCR of surface sterilized foliage) for estimating endophyte diversity and species composition; and (iv) to investigate the relationships between traditional ecological indices (e.g. Shannon index) and phylogenetic diversity (PD) in estimating endophyte diversity and spatial heterogeneity. Endophytes were recovered in culture from 87 of 90 P. taeda leaves sampled, yielding 439 isolates that represented 24 morphotaxa. Sequence data from the nuclear ribosomal internal transcribed spacer (ITS) for 150 isolates revealed 59 distinct ITS genotypes that represented 24 and 37 unique groups based on 90% and 95% sequence similarity, respectively. By recoding ambiguously aligned regions to extract phylogenetic signal and implementing a conservative phylogenetic backbone constraint, we recovered well supported phylogenies based on ca. 600 bp of the nuclear ribosomal large subunit (LSUrDNA) for 72 Ascomycota and Basidiomycota, 145 cultured endophytes and 33 environmental PCR samples. Comparisons with LSUrDNA-delimited species showed that morphotaxa adequately estimated total species richness but rarely corresponded to biologically meaningful groups. ITS BLAST results were variable in their utility, but ITS genotype groups based on 90% sequence similarity were concordant with LSUrDNA-delimited species. Environmental PCR yielded more genotypes per sampling effort and recovered several distinct clades relative to culturing, but some commonly cultured clades were never found (Sordariomycetes) or were rare relative to their high frequency among cultures (Leotiomycetes). In contrast to traditional indices, PD demonstrated spatial heterogeneity in endophyte assemblages among P. taeda trees and study plots. Our results highlight the need for caution in designating taxonomic units based on gross cultural morphology or ITS BLAST matches, the utility of phylogenetic tools for extracting robust phylogenies from environmental samples, the complementarity of culturing and environmental PCR, the utility of PD relative to traditional ecological indices, and the remarkably high diversity of foliar fungal endophytes in this simplified temperate ecosystem.     

Evaluating the Ribosomal Internal Transcribed Spacer (ITS) as a Candidate Dinoflagellate Barcode Marker

Background

DNA barcoding offers an efficient way to determine species identification and to measure biodiversity. For dinoflagellates, an ancient alveolate group of about 2000 described extant species, DNA barcoding studies have revealed large amounts of unrecognized species diversity, most of which is not represented in culture collections. To date, two mitochondrial gene markers, Cytochrome Oxidase I (COI) and Cytochrome b oxidase (COB), have been used to assess DNA barcoding in dinoflagellates, and both failed to amplify all taxa and suffered from low resolution. Nevertheless, both genes yielded many examples of morphospecies showing cryptic speciation and morphologically distinct named species being genetically similar, highlighting the need for a common marker. For example, a large number of cultured Symbiodinium strains have neither taxonomic identification, nor a common measure of diversity that can be used to compare this genus to other dinoflagellates.

Methodology/Principal Findings

The purpose of this study was to evaluate the Internal Transcribed Spacer units 1 and 2 (ITS) of the rDNA operon, as a high resolution marker for distinguishing species dinoflagellates in culture. In our study, from 78 different species, the ITS barcode clearly differentiated species from genera and could identify 96% of strains to a known species or sub-genus grouping. 8.3% showed evidence of being cryptic species. A quarter of strains identified had no previous species identification. The greatest levels of hidden biodiversity came from Scrippsiella and the Pfiesteriaceae family, whilst Heterocapsa strains showed a high level of mismatch to their given species name.

Conclusions/Significance

The ITS marker was successful in confirming species, revealing hidden diversity in culture collections. This marker, however, may have limited use for environmental barcoding due to paralogues, the potential for unidentifiable chimaeras and priming across taxa. In these cases ITS would serve well in combination with other markers or for specific taxon studies.     

Free fatty acids and sterols in the benthic spawn of aquatic molluscs, and their associated antimicrobial properties

The free lipid content of extracts from the spawn of 17 molluscs were analysed by gas chromatography/mass spectrometry. These extracts encompass the encapsulated embryos and extraembryonic structures from benthic gelatinous egg masses and leathery egg capsules covering five taxonomic groups. Palmitic and stearic acids were the dominant saturated fatty acids and oleic acid was the principal unsaturated acid found in the spawn. Cholesterol was the dominant sterol and the only sterol found in the spawn from every species. Extracts from gelatinous egg masses were found to contain proportionally more fatty acids compared to leathery egg capsules. No unsaturated fatty acids were found in any of the leathery egg capsules, including five neogastropods and one littorinimorph. Unsaturated fatty acids were present in all of the gelatinous egg masses, including two other littorinimorphs. This is the first study to demonstrate that unsaturated fatty acids possess significant bacteriolytic activity against four aquatic pathogens. Encapsulated Anaspidea egg masses contain relatively high concentrations of these unsaturated fatty acids and a lipid mixture modeled on these extracts was strongly bacteriolytic at concentrations down to 0.0001 mg/ml. By comparison, lipid mixtures modeled on extracts from the spawn of four other molluscan taxa with higher proportions of saturated fatty acid and cholesterol, were only partially active against some of the bacteria at 0.1 mg/ml. Thus, unsaturated fatty acids could explain the antimicrobial activity previously reported in lipid extracts of some, but not most, molluscan spawn. MDS ordination and ANOSIM revealed significant taxonomic differences in the composition of free lipids from molluscan spawn, suggesting that lipid analyses may be useful in future systematic studies of the Mollusca.     

A global meta-analysis of Tuber ITS rDNA sequences: species diversity, host associations and long-distance dispersal

Truffles (Tuber) are ectomycorrhizal fungi characterized by hypogeous fruitbodies. Their biodiversity, host associations and geographical distributions are not well documented. ITS rDNA sequences of Tuber are commonly recovered from molecular surveys of fungal communities, but most remain insufficiently identified making it difficult to determine whether these sequences represent conspecific or novel taxa. In this meta-analysis, over 2000 insufficiently identified Tuber sequences from 76 independent studies were analysed within a phylogenetic framework. Species ranges, host associates, geographical distributions and intra- and interspecific ITS variability were assessed. Over 99% of the insufficiently identified Tuber sequences grouped within clades composed of species with little culinary value (Maculatum, Puberulum and Rufum). Sixty-four novel phylotypes were distinguished including 36 known only from ectomycorrhizae or soil. Most species of Tuber showed 1-3% intraspecific ITS variability and >4% interspecific ITS sequence variation. We found 123 distinct phylotypes based on 96% ITS sequence similarity and estimated that Tuber contains a minimum of 180 species. Based on this meta-analysis, species in Excavatum, Maculatum and Rufum clades exhibit preference for angiosperm hosts, whereas those in the Gibbosum clade are preferential towards gymnosperms. Sixteen Tuber species (>13% of the known diversity) have putatively been introduced to continents or islands outside their native range.     

PCR-RFLP和多重PCR技术检测常见病原性丝状真菌的实验研究

目的建立检测常见丝状真菌感染病原菌的PCR-RFLP和多重PCR方法。方法建立以PCR技术为基础的限制片段长度多态性(RFLP)方法 ,首先用真菌通用引物扩增丝状真菌的ITS区,然后用限制性核酸内切酶对PCR产物进行酶切。用4种丝状真菌的特异性引物建立多重PCR体系,用该体系检测单模板、双模板和三模板的扩增情况,并测定该体系的特异性和敏感性。结果用PCR-RFLP技术能够鉴别5种常见丝状真菌,多重PCR能够根据扩增片段的不同鉴别菌种,在合适的反应条件下,对单模板、双模板和三模板均能扩增出目的片段。结论 PCR-RFLP和多重PCR技术能够快速鉴定丝状真菌感染病原菌,有临床应用的良好前景。     

Ground-feeding migratory songbirds as cellular slime mold distribution vectors

Cosmopolitan species of cellular slime molds occur continents apart in both tropical and temperate zones of the world though the spore masses are too heavy to be wind borne, and water dispersal is limited to the watercourses. A highly mobile distribution vector was found in ground-feeding migratory song birds. Nine ubiquitous species and 2 ecologicially distinct species of dictyostelid cellular slime molds were isolated from the feces of ground-feeding eastern North American migratory thrushes, finches, sparrows and warblers, both on breeding and winter grounds. Three propagules of slime molds, amoebae, spores and macrocysts survive passage through the avian digesive tract and remain in the gut long enough to be transported by major bird migrations. Habitats with the greates species diversity of both cellular slime molds and ground-feeding passerines concur in both eastern North America and Central America. Birds actively seek their prefered habitats; the cellular slime molds have arrived at these habitats as passengers. Rare slime molds can serve as a marker to the habitats that migratory birds have visited, or birds with known habitats can provide clues as to the distribution of rare species of cellular slime molds.     

Application of DNA barcodes in Hedyotis L.(Spermacoceae, Rubiaceae)

The potential application of DNA barcodes of plastid (matK, trnH-psbA, petD, and rbcL) and nuclear (internal transcribed spacer (ITS) of rDNA) DNA regions was investigated for 25 Hedyotis taxa. The ITS showed the best species discrimination by resolving 23 of the species as exclusive lineages with no shared alleles between any of the 24 distinct species (H. Assimilis and H. Mellii are not supported as distinct species based on our molecular and morphological data). Conversely, rbcL performed the worst and only resolved 10 of the species as exclusive lineages, and 10 species with shared alleles. Using ITS has the advantage of high PCR amplification success and it provides good intra- and interspecific variation distribution patterns. The most powerful plastid markers were petD and trnH-psbA, but we could amplify and sequence trnH-psbA for only 83% of the accessions sampled. Combination of ITS and petD performed extremely well, with all 24 of the distinct species resolved as exclusive lineages and no shared alleles between any of the distinct species. We therefore recommend ITS, or a combination of ITS and petD, as the standard DNA barcode in Hedyotis, but acknowledge that there are no shared alleles between distinct species for marK and rbcL combined.     

Evaluation of the DNA Barcodes in Dendrobium (Orchidaceae) from Mainland Asia

DNA barcoding has been proposed to be one of the most promising tools for accurate and rapid identification of taxa. However, few publications have evaluated the efficiency of DNA barcoding for the large genera of flowering plants. Dendrobium, one of the largest genera of flowering plants, contains many species that are important in horticulture, medicine and biodiversity conservation. Besides, Dendrobium is a notoriously difficult group to identify. DNA barcoding was expected to be a supplementary means for species identification, conservation and future studies in Dendrobium. We assessed the power of 11 candidate barcodes on the basis of 1,698 accessions of 184 Dendrobium species obtained primarily from mainland Asia. Our results indicated that five single barcodes, i.e., ITS, ITS2, matK, rbcL and trnH-psbA, can be easily amplified and sequenced with the currently established primers. Four barcodes, ITS, ITS2, ITS+matK, and ITS2+matK, have distinct barcoding gaps. ITS+matK was the optimal barcode based on all evaluation methods. Furthermore, the efficiency of ITS+matK was verified in four other large genera including Ficus, Lysimachia, Paphiopedilum, and Pedicularis in this study. Therefore, we tentatively recommend the combination of ITS+matK as a core DNA barcode for large flowering plant genera.     

Diversity of cultivable actinobacteria in geographically widespread marine sediments

Reports describing actinobacteria isolated from marine environments have been dominated by Micromonospora, Rhodococcus and Streptomyces species. Recent culture-independent studies have shown that marine environments contain a high diversity of actinobacterial species that are rarely, if at all, recovered by cultivation-based methods. In this study, it is shown that cultivation-independent methods can be used to guide the application of selective isolation methods. The detection of marine-derived actinobacterial species that have previously only been reported from terrestrial habitats is highlighted. This study provides good evidence that the previously described low diversity of actinobacterial species isolated from marine environments does not reflect an actual low species diversity, and that the use of informed selective isolation procedures can aid in the isolation of members of novel taxa.     

Amphibian diversity in Polish cities: Taxonomic diversity,functional diversity and evolutionary distinctiveness

As a process affecting animal communities, urbanization has been the subject of numerous studies. However, amphibians are still among the least studied vertebrate groups in urbanized landscapes. Generally, it has been found that the process of loss of amphibian diversity is nonrandom, with species from older evolutionary lines at greater risk. Regional data on amphibian assemblages in urban areas is a very useful tool for assessing how these assemblages react to changes.The aim of the present paper is to assess the diversity of amphibians in Polish cities based on data in the relevant literature, exploring different metrics (e.g., taxonomic, functional, and evolutionary diversity) calculated in amphibian species assemblages. We used data from 18 articles (including grey literature), characterized by comparable research methods and published between 1999 and 2017.Overall, amphibian species richness (ASR) amounted to an average of 9 species, ranging from 5 to 11 species per city. The higher species richness occurred in Białystok in North-west of Poland. Functional evenness (FEve), evolutionary distinctiveness (ED sum), and functional richness (FRic) were strongly positively correlated with ASR. However, ED mean was not significantly correlated with the total number of species in the community. Three taxa, the hybridogenetic water frogs Pelophylax esculentus complex, the common toad Bufo bufo, and the common frog Rana temporaria, occurred in all analyzed amphibian assemblages. Our study is one of the first attempts to compare urban amphibian assemblages, using different and complementary diversity metrics on a large spatial scale. In conclusion, we highlight that urban areas play an important role for conservation of amphibians, because they support amphibian assemblages characterized by a high level of overall diversity.     

Application of DNA barcodes in Hedyotis L. (Spermacoceae,Rubiaceae)

The potential application of DNA barcodes of plastid (matK, trnH–psbA, petD, and rbcL) and nuclear (internal transcribed spacer (ITS) of rDNA) DNA regions was investigated for 25 Hedyotis taxa. The ITS showed the best species discrimination by resolving 23 of the species as exclusive lineages with no shared alleles between any of the 24 distinct species (H. assimilis and H. mellii are not supported as distinct species based on our molecular and morphological data). Conversely, rbcL performed the worst and only resolved 10 of the species as exclusive lineages, and 10 species with shared alleles. Using ITS has the advantage of high PCR amplification success and it provides good intra- and interspecific variation distribution patterns. The most powerful plastid markers were petD and trnH–psbA, but we could amplify and sequence trnH–psbA for only 83% of the accessions sampled. Combination of ITS and petD performed extremely well, with all 24 of the distinct species resolved as exclusive lineages and no shared alleles between any of the distinct species. We therefore recommend ITS, or a combination of ITS and petD, as the standard DNA barcode in Hedyotis, but acknowledge that there are no shared alleles between distinct species for matK and rbcL combined.     

A Comparison of ITS Nuclear rDNA Sequence Data and AFLP Markers for Phylogenetic Studies in Phyllostachys (Bambusoideae, Poaceae)

Phyllostachys , a large, economically important genus of woody bamboos. DNA sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) were used in a parsimony analysis. Phyllostachys was well supported as monophyletic with Chimonobambusa as its closest allied genus. The 5S spacer region of nrDNA was investigated but found unsuitable for this purpose. The AFLP analysis showed much higher discriminating power between species and was more useful for phylogenetic reconstruction at this taxonomic level. The combined data were used to review the previous infra-generic classifications. Section Heteroclada Wang & Ye is strongly supported and can be further divided into sub-groups. A group within section Phyllostachys is strongly supported, but a further group of taxa previously included in this section is difficult to place. The ability of the methods to help separate species such as P. sulphurea and investigate genetic diversity at the infra-specific level was also assessed. It is argued that AFLPs could often be the method of choice for phylogenetic studies of closely related taxa for which DNA sequence data provide insufficient resolution. Received 18 April 2000/ Accepted in revised form 10 June 2000