Isolation and characterization of 11 polymerase chain reaction primers for microsatellite loci for the Chilean marine isopod Excirolana hirsuticauda

The Chilean isopod Excirolana hirsuticauda is a marine benthic brooder with wide distributional range and low potential for long-distance dispersal. Eleven microsatellite markers were developed for E. hirsuticauda using enriched libraries. Characterization of those loci in 35 individuals from Playa Blanca beach showed high allelic diversity with a mean of 10.9 alleles per locus. The average expected and observed heterozygosities were 0.65 and 0.41. These microsatellite loci are the first published for any Excirolana species and should be useful to study the genetic structure of E. hirsuticauda.     

Novel polymorphic microsatellite DNA markers from Malaysian giant freshwater prawn, <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis>

Seven single locus microsatellite markers were characterized in Malaysian giant freshwater prawn, Macrobrachium rosenbergii from an enriched genomic library Primer pairs were designed to flank the repeat sequences and the loci characterized for this species. The bands resulting from the PCR amplifications of these eight microsatellite loci were polymorphic with the number of alleles ranging from 8 to 26 alleles per locus, whereas the observed heterozygosity ranged from 0.0641 to 0.6564. These newly developed microsatellite markers should prove to be useful for population studies and in the management of genetic variations in broodstocks of freshwater prawn, M. rosenbergii.     

A large-scale stream benthic diatom database

A relational database linking benthic diatom records, taxonomic nomenclature including synonyms, and corresponding environmental data has been built in MS Access. It allowed flexible and long-term use of a relatively important amount of data (∼3000 records) gathered in the framework of the EC-funded PAEQANN project, gathering precise and documented information both about benthic diatoms and quantitative or semi-quantitative environmental data. Such a database has been shown to be a useful tool for the definition of benthic diatom typology at a multi-regional scale, the prediction of the impact of environmental characteristics on the structure of diatom communities, and additionally for a new insight on the auto-ecology of some taxa. This database could serve as a template for further work on diatoms and, after some implementation, on other freshwater communities. It could also be the basis for wider typology of stream diatoms, extended to other regions.     

基于高通量测序的达氏鲟微卫星标记筛选

达氏鲟(Acipenser dabryanus)是我国特有的珍稀鱼类,主要分布在长江上游干支流。由于人类活动的影响,其种群数量急剧下降,被列为国家Ⅰ级重点保护动物、IUCN红色名录极危(CR)物种。本研究针对当前达氏鲟群体的濒危现状,应用微卫星标记开展达氏鲟保护遗传学研究。通过高通量测序成功筛选出25个微卫星位点,采用18尾野生个体和30尾人工繁殖个体对其多样性进行了检验,结果表明,25个位点共检测出181个等位基因,每个位点的等位基因数(A)为4~11(平均值7.2),观测杂合度(HO)为0.160~1.000(平均值0.744),期望杂合度(HE)为0.346~0.875(平均值0.727)。所有位点均不偏离"Hardy-Weinberg"平衡(P0.05),各位点间也无连锁不平衡现象。除了其中一个位点以外,所有位点的多态信息含量(PIC)均大于0.5,其多态性较好。本次筛选的微卫星位点将有助于达氏鲟资源保护和群体遗传研究。     

MICROFISSURATA GEN. NOV. (BACILLARIOPHYTA), A NEW DIATOM GENUS FROM DYSTROPHIC AND INTERMITTENTLY WET TERRESTRIAL HABITATS1

A new freshwater benthic diatom genus, Microfissurata gen. nov., was identified from light and scanning electron micrographs. The most characteristic morphological feature (referred to also by the name of the new genus) is the structure of the striae/alveoli, which are simple, mostly uninterrupted, transapical slits. The combination of characteristics of the new genus is unique. It includes two new species: Microfissurata paludosa Cantonati et Lange‐Bert. sp. nov. and M. australis Van de Vijver et Lange‐Bert. sp. nov., distributed in Europe and on an austral island, respectively. The new genus occurs in freshwater dystrophic lakes, pools, seepage springs, mires, and in intermittently wet terrestrial habitats. It is not abundant but widespread wherever these habitat types are well developed, in Nordic‐alpine and sub‐Antarctic areas. Overall, the new genus appears to be mostly epiphytic (bryophilous) and capable of tolerating a wide range of moisture conditions (xerotolerant). The finding of the type species (M. paludosa) in a well‐investigated area like central Europe highlights the diatom species richness of dystrophic habitats and their importance for diatom biodiversity conservation.     

Freshwater diatom and macroinvertebrate diversity of coastal permanent ponds along a gradient of human impact in a Mediterranean eco-region

Mediterranean coastal areas are characterised by heavily transformed landscapes and an ever-increasing number of ponds are subjected to strong alterations. Although benthic diatoms and macroinvertebrates are widely used as indicators in freshwater ecosystems, little is still known about the diatom communities of lowland freshwater ponds in the Mediterranean region, and, furthermore, there are few macroinvertebrate-based methods to assess their ecological quality, especially in Italy. This article undertakes an analysis of benthic diatom and macroinvertebrate communities of permanent freshwater ponds, selected along a gradient of anthropogenic pressures, to identify community indicators (taxa and/or metrics) useful to evaluate the effect of human impacts. A series of 21 ponds were sampled along Tyrrhenian coast in central Italy. Five of these ponds, in a good conservations status and surrounded by woodland were selected as ‘reference sites’ for macroinvertebrates and epipelic diatoms. The remaining sixteen ponds were located in an agricultural landscape subject to different levels of human impact. The total number of macroinvertebrate taxa found in each pond was significantly higher in reference sites than in both the intermediate and heavily degraded ones, whereas the diatom species richness did not result in a good community variable to evaluate the pond ecological quality. The analysis revealed a substantial difference among the compositions of diatom communities between reference ponds and degraded ponds. The former were characterised by the presence of several species belonging to genera, such as Pinnularia sp., Eunotia sp., Stauroneis sp., Neidium sp., all of which were mostly absent from degraded ponds. Furthermore, the taxonomic richnesses of some macroinvetebrate groups (Odonata, Ephemeroptera, Trichoptera, Coleoptera), and taxa composition attributes of macroinvertebrate communities (total abundance, percentages of top three dominant taxa, percentages of Pleidae, Ancylidae, Hirudinea, Hydracarina) significantly correlated with variables linked with anthropogenic pressures. The results of the investigation suggested that diatoms tended more to reflect water chemistry through changes in community structure, whereas invertebrates responded to physical habitat changes primarily through changes in taxonomic richness. The methodologies developed for the analysis of freshwater benthic diatom and macroinvertebrate communities may have a considerable potential as a tool for assessing the ecological status of this type of water body, complying with the European Union Water Framework Directive 2000/60/EC. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Guest editors: B. Oertli, R. Cereghino, A. Hull & R. Miracle Pond Conservation: From Science to Practice. 3rd Conference of the European Pond Conservation Network, Valencia, Spain, 14–16 May 2008     

Microsatellite DNA markers in the giant freshwater prawn, Macrobrachium rosenbergii: a tool for genetic analysis

Eight microsatellite loci were identified and characterized in the commercially important giant freshwater prawn, Macrobrachium rosenbergii. The microsatellite loci were detected by the random screening for dinucleotide repeat units within a partial genomic library developed for the species with biotinylated probes (CA)(15) , (AT)(15) and (GA)(15) . All the eight loci were found to be polymorphic. The number of alleles and observed heterozygosities per locus ranged between three to 16 and 0.22 to 0.71, respectively. These microsatellite markers will be useful for the conservation and management of wild and cultured stocks and population genetic studies of freshwater prawn.     

Positive relationships between genetic diversity and abundance in fishes

Molecular markers, such as mitochondrial DNA and microsatellite loci, are widely studied to assess population genetics and phylogeography; however, the selective neutrality of these markers is increasingly being questioned. Given the importance of molecular markers in fisheries science and conservation, we evaluated the neutrality of both mtDNA and microsatellite loci through their associations with population size. We surveyed mtDNA and microsatellite data from the primary literature and determined whether genetic diversity increased with abundance across a total of 105 marine and freshwater fishes, with both global fisheries catch data and body size as proxies for abundance (with an additional 57 species for which only body size data were assessed). We found that microsatellite data generally yielded higher associations with abundance than mtDNA data, and within mtDNA analyses, number of haplotypes and haplotype diversity were more strongly associated with abundance than nucleotide diversity, particularly for freshwater fishes. We compared genetic diversity between freshwater and marine fishes and found that marine fishes had higher values of all measures of genetic diversity than freshwater fishes. Results for both mtDNA and microsatellites generally conformed to neutral expectations, although weaker relationships were often found between mtDNA nucleotide diversity and ‘abundance’ compared to any other genetic statistic. We speculate that this is because of historical events unrelated to natural selection, although a role for selection cannot be ruled out.     

Isolation and characterization of microsatellite loci in the Malaysian giant freshwater prawn, Macrobrachium rosenbergii

Eight single locus microsatellite markers were developed to characterize the Malaysian giant freshwater prawn, Macrobrachium rosenbergii. These microsatellites were isolated from an enriched genomic library contained by using a 5'-anchored polymerase chain reaction technique. Primers were designed to flank the repeat sequences and subsequently used to characterize 30 unrelated individuals of the giant freshwater prawn. The polymerase chain reaction amplification products of these eight microsatellite loci were polymorphic with the number of alleles ranging from two to 10 alleles per locus while the levels of heterozygosity ranged from 0.6333 to 0.8667.     

Sex and outcrossing in a sessile freshwater invertebrate

SUMMARY 1. The freshwater bryozoan Cristatella mucedo , in common with other sessile, benthic freshwater taxa, has an unusual life history: sex occurs during a relatively brief period near the start of the growing season, and overwintering occurs in the form of asexually produced dormant propagules (statoblasts). Consistent observed heterozygosity (Ho) deficits in C. mucedo populations have previously suggested that inbreeding is common, although a possible contribution of a Wahlund effect to low Ho could not be discounted.
2. We have used microsatellite data in the first study based on codominant markers to genetically characterise maternal colonies and larval offspring of C. mucedo . The 'population' represented by the larvae was in Hardy–Weinberg equilibrium, which has previously been found in only one of 39 populations of C. mucedo . At least 64% of larvae were the products of outcrossing. We suggest that the unusual early timing of sex may be a strategy to maximise rates of outcrossing within populations of sessile freshwater invertebrates.     

Influence of Algal Community Structure on Denitrification Rates in Periphyton Cultivated on Artificial Substrata

We conducted a field survey of periphyton cultivated on benthic mesh installations in freshwater aquatic systems, including two constructed wetlands and a pond, and also studied periphyton grown on a benthic mesh in laboratory mesocosms. The objectives of this study were to (1) determine if periphyton cultivated on benthic mesh denitrifies at higher rates than the underlying sediments and (2) determine if denitrification rates within periphyton vary with characteristics such as algal and bacterial community structure and biomass. We measured denitrification potential rates of field and laboratory periphyton by the acetylene inhibition method. We characterized algal community composition by algal identification and bacterial community composition by terminal restriction fragment length polymorphisms. Periphyton collected on benthic mesh from our field sites denitrified at significantly higher rates than the underlying sediments, regardless of sampling site or season. Results from both our field survey and laboratory studies indicated a significant, positive correlation between diatom presence and denitrification rate. In our laboratory studies, we found that periphyton with the highest diatom abundance showed the highest denitrification rates as well as a distinct bacterial community composition. These results suggest a synergistic relationship between diatoms and denitrifying bacteria that warrants further study.     

New polymorphic microsatellite markers for the masu salmon (Oncorhynchus masou masou) from Korea and their application to wild and hatchery populations

Masu salmon, Oncorhynchus masou masou, is an economically important fish species in the Far East and occurs in two life history forms: sea-run migratory (anadromous) and freshwater resident (non-anadromous). The non-anadromous form has recently become a popular freshwater food and game fish during a well-known Korean winter festival. However, the genetic background of this species remains largely unknown, partly due to a lack of molecular genetic markers. In this study, we developed new polymorphic microsatellite markers for masu salmon using next-generation sequencing technology. From 40 primer sets, 11 primer sets (27.5% of the primer sets selected) were successfully amplified with 106 alleles (range 2–9) in 64 individuals from different populations: two wild and one hatchery. Observed and expected heterozygosities ranged from 0.304 to 0.947 and 0.278 to 0.865, respectively. Significant departures from the Hardy–Weinberg equilibrium were detected for four markers (OMM11, OMM17, OMM28, and OMM33) in a single population. All pair-wise F_ST values were highly significant between the wild and hatchery populations (range 0.084–0.183, P < 0.0001). We identified a set of robust microsatellite markers that worked well even in formalin-fixed samples, which will be suitable for biogeographical and population structure analyses of the masu salmon.     

Novel polymorphic microsatellite DNA markers from Malaysian giant freshwater prawn, Macrobrachium rosenbergii

Seven single locus microsatellite markers were characterized in Malaysian giant freshwater prawn, Macrobrachium rosenbergii from an enriched genomic library Primer pairs were designed to flank the repeat sequences and the loci characterized for this species. The bands resulting from the PCR amplifications of these eight microsatellite loci were polymorphic with the number of alleles ranging from 8 to 26 alleles per locus, whereas the observed heterozygosity ranged from 0.0641 to 0.6564. These newly developed microsatellite markers should prove to be useful for population studies and in the management of genetic variations in broodstocks of freshwater prawn, M. rosenbergii.     

Diatom (Bacillariophyceae) assemblages in tidal environments of Vancouver Island,British Columbia,Canada

To understand distributions of coastal diatoms along Vancouver Island, British Columbia, Canada, this paper describes diatom assemblages observed in 47 surface sediment samples from intertidal environments. One hundred and eighty-four diatom taxa were identified from five transects crossing tidal flats, salt marshes, and freshwater forests in Tofino, Ucluelet, and Port Alberni. Distributions of the diatom assemblages were consistent with those reported elsewhere in the Pacific Northwest, but a few diatom taxa show different trends in their distributions. For example, one benthic species Denticula subtilis shows widespread distributions along the transect in Tofino. An ordination shown by Detrended Correspondence Analysis (DCA) using a combined dataset indicated overlapped scatter plots of diatom assemblages, suggesting that assemblages with similar species compositions are observed in more than one location. Hierarchical and k-means clustering analyses using Euclidean distance recognized unique small groups along each transect. Rank abundance curves show different trends for richness and evenness of diatom assemblages among the five transects.     

Improved genetic markers for monitoring recruitment dynamics in the endangered Mary River cod (Maccullochella mariensis)

The Mary River cod (Maccullochella mariensis) is a large predatory freshwater fish identified as a potential flagship for freshwater ecosystem conservation in Australia. The species is endemic to the Mary River catchment in eastern Australia, and is listed as Endangered. Previous conservation genetic assessment of Mary River cod was based on a small set of microsatellite loci developed for congeneric Maccullochella species. Here we develop a novel set of 15 microsatellite loci specific to M. mariensis, and demonstrate that these markers exhibit higher variability than those used previously. Mean number of alleles per locus was 4 and mean expected heterozygosity was 0.57. We genotyped 35 Mary River cod larvae belonging to a single cohort using the 15 novel loci and eight previously used loci, and found 10 full‐sib family groups along with clear genetic differentiation between individuals collected from the two sub‐catchments – Tinana Creek and Mary River. Microsatellites presented here will be useful for cost‐effective monitoring of genetic diversity and recruitment dynamics in this endangered fish species.     

Development of 18 microsatellite loci for the freshwater snail Bellamya aeruginosa (Mollusca, Gastropoda)

Although it is a major freshwater gastropod species, genetic diversity of Bellamya aeruginosa was completely unknown. Eighteen microsatellite loci were isolated and characterized from (AC)(15)-enriched genomic libraries of the freshwater snail B. aeruginosa. Most of the 18 loci were successfully amplified and high polymorphic information content values were found, ranging from 0.244 to 0.792 (mean 0.541). The number of alleles per locus ranged from 5 to 13 (mean 8.8), the expected heterozygosity varied from 0.347 to 0.950 (mean 0.815) and the observed heterozygosity varied from 0.087 to 0.782 (mean 0.431). Eight loci showed significant deviation from Hardy-Weinberg equilibrium after Bonferroni's correction and no significant genotypic linkage disequilibrium was detected between most locus pairs, except for TXH79-TXH97 and TXH113-TXH121. These 18 polymorphic microsatellite loci should be useful for population genetics analysis and species identification of Bellamya.     

Geissleria gereckei sp. nov. (Bacillariophyta) from leaf-litter covered stones of very shaded carbonate mountain springs with extremely low discharge

A new benthic freshwater diatom, Geissleria gereckei sp. nov., was identified from light and scanning electron micrographs. The most characteristic morphological features are the valve outline (elliptical, but with lateral margins that can range from slightly convex to slightly concave and also straight, and protracted, almost capitate ends), the size and length/width ratio, and the pattern of the striae around the central area. The new species was found to be epilithic, and characteristic of leaf-litter covered stones of very shaded carbonate mountain springs of the Italian Alps with extremely-low discharge (likely to be affected by seasonal desiccation). Very-low-discharge springs host a highly specialized diatom microflora, and are likely to increase in number in the near future due to the reduction of water, which is predicted to be caused by climate change in many regions.     

Testing of the applicability of European diatom indices in the tropical rift valley lake,Lake Hawassa,in Ethiopia

Quantitative ecological monitoring of African lakes is needed to understand growing human pressures on ecosystems. Diatom-based indices are routinely used for this purpose elsewhere in the world, but have not yet been produced for the flora of African freshwater lakes. Here we tested the applicability of the European diatom indices on the biomonitoring system of Lake Hawassa, Ethiopia. Physico-chemical and benthic diatom sampling was done at nine sites with different degrees of human disturbance along the lakeshore area from February to November 2015 and 2016. A percentage disturbance score (PDS) was calculated at each site and categorised from no evident disturbance (0–25%) to high disturbance (75–100%). Based on this criterion and selected physico-chemical parameters, the sampling sites categorized into minimal, moderate and high disturbance. Seventeen diatom indices were calculated using Omnidia software version 5.3. Out of a total of 17 indices that were calculated using the Omnidia software, six were selected as potential metrics. The diatom indices had a high discrimination efficiency and were significantly correlated with most the environmental parameters (r > 0.6; p < 0.05). Among these, the trophic diatom index (TDI) and generic diatom index (IDG) showed the best potential to discriminate the three clustered sites, based on their ecological classification. Accordingly, although robust locally based indices are needed, the TDI and IDG diatom indices could be used in monitoring of water quality in tropical African rift lakes.     

Development and characterization of 43 microsatellite markers for the critically endangered primrose Primula reinii using MiSeq sequencing

Primula reinii (Primulaceae), a perennial herb belonging to the Primula section Reinii, occurs on wet, shaded rocky cliffs in the mountains of Japan. This threatened species comprises four varieties; these plants are very localized and rare in the wild. In this study, 43 microsatellite markers were developed using MiSeq sequencing to facilitate conservation genetics of these critically endangered primroses. We developed novel microsatellite markers for three varieties of P. reinii, and tested its polymorphism and genetic diversity using natural populations. These novel markers displayed relatively high polymorphism; the number of alleles and expected heterozygosities ranged from 2 to 6 (mean=3.2) and 0.13 to 0.82 (mean=0.45), respectively. All loci were in HardyeWeinberg equilibrium. These microsatellite markers will be powerful tools to assess P. reinii genetic diversity and develop effective conservation and management strategies.     

青鱼微卫星标记的开发与特性分析

青鱼(Mylopharyngodon piceus)是中国最为重要的淡水养殖鱼类。开发青鱼的微卫星标记能为青鱼的遗传多样性分析提供更多工具。本研究使用磁珠富集法,利用生物素探针(CA)10和(GACA)6,富集得到青鱼基因组微卫星片段,进一步通过设计微卫星引物检验其在青鱼原种群体中的有效性和多态性水平。结果显示,所构建文库中849个克隆含有微卫星序列,通过利用PCR技术在吴江原种青鱼36个个体中进行多态性筛选,获得了25个多态性微卫星位点。其平均等位基因数(Na)和有效等位基因数(Ne)分别为7.08和3.526,平均观测杂合度(Ho)和期望杂合度(He)分别为0.602和0.619,平均多态信息含量(PIC)为0.568。其中,Mp23、Mp27和Mp35这3个位点极显著偏离哈迪-温伯格平衡(P 0.01)。本研究开发的微卫星标记能为青鱼种质资源的评价和保护等研究提供工具。