Patterns of species richness for blackflies (Diptera: Simuliidae) in the Nearctic and Neotropical regions

Abstract.  1. Patterns of simuliid species richness were examined over a variety of scales at 532 stream sites in the Nearctic (394) and Neotropical (138) regions. In Nearctic streams, species richness of immature blackflies both within and across ecoregions and over two seasons was examined. Stream variables at each site included seston, width, depth, velocity, discharge, conductivity, pH, dissolved oxygen, water temperature, dominant streambed-particle size, canopy cover, and riparian vegetation. These variables were subjected to a principal component analysis and derived principal components were related back to richness, using regression analysis. At the level of the stream reach, richness was not highly correlated with single-point measurements of stream conditions.
2. Using data from both Nearctic and Neotropical sites, the effect of regional richness on local richness was examined. As regional richness increased, local diversity reached an asymptote in which further increases in regional richness were not matched by increases in local richness. Hence, simuliid communities are best described as saturated (type II) communities, consistent with the current view of lotic communities as non-equilibrium systems.
3. The well-established pattern of greater species richness in tropical regions was not observed in this study. To the contrary, blackfly richness is higher in temperate streams than in tropical streams at both local and regional scales.     

Different life history strategies in lake-outlet blackflies (Diptera: Simuliidae)

The life histories of Simulium noelleri Fried. and S. truncatum Ldstr. were studied at a lake-outlet site in Finland. A detailed study was made of the second summer generation of both species and this consisted of a single, synchronized cohort in S. noelleri, and overlapped cohorts in S. truncatum. Larvae of the two species also show differences in their habitat preference: those of S. noelleri predominate on the sluice which marks the beginning of the river, and S. truncatum larvae just downstream from the sluice. In winter, the latter site was dominated by immature individuals of two other species, S. erythrocephalum de Geer and S. equinum L., and these were found downstream from the two ‘lake-outlet’ species during the summer. Adult females of S. noelleri are able to mature a first batch of eggs without taking a blood meal but those of S. truncatum need to feed on blood before eggs can be fully developed. The consequences of these different reproductive strategies on the ecology of individuals of the two species is discussed.     

Genetic relationship among species in the genus Sonneratia in China as revealed by inter-simple sequence repeat (ISSR) markers

Inter-simple sequence repeat (ISSR) analysis was used to establish the genetic relationship among six Sonneratia species in China. A total of 100 primers were screened, of which 11 polymorphic and informative patterns were selected to determine the genetic relationship. Four hundred and eighty five DNA bands were amplified, among which 481 bands (99.18%) were polymorphic. The average number of DNA bands amplified by each primer was 44. Similarity coefficients were calculated and a dendrogram was constructed by using UPGMA algorithm. The six Sonneratia species were divided into two major groups. Group I consisted of Sonneratia caseolaris, Sonneratia × gulngai, Sonneratia alba, and Group II included Sonneratia × hainanensis, Sonneratia ovata and Sonneratia apetala. In Group I, S. × gulngai was close to S. alba, and in Group II, S. × hainanensis was close to S. ovata. The genetic relationships estimated by the polymorphism of ISSR markers are basically in agreement with those previously inferred by morphological data. Thus, ISSR approach is a reliable marker system that can be used to study genetic relationship in the genus Sonneratia.     

Assessment of a silhouette trap for sampling zoophilic blackflies (Diptera: Simuliidae)

A collapsible animal-type silhouette trap was designed to catch zoophilic female blackflies (Diptera: Simuliidae) which feed on the head or ventral surface of cattle. The trap was assessed for its ability to sample attacking blackflies by comparison with simultaneous manual catches from a bait cow, in an enzootic bovine onchocerciasis area in North Wales. In thirteen 1-hour collections, the trap provided a representative sample of the blackfly population in terms of the relative abundance of species. There was a strong correlation between the catch sizes from both methods (r = +0.73), and the body site feeding preferences for all species were similar with both trap and cow. There was no significant difference between the Onchocerca spp. infection rates of flies caught by either method.     

Genotypic diversity and structure of Satureja mutica revealed by inter-simple sequence repeat markers

Satureja mutica (Lamiaceae) is an herbaceous medicinal plant which grows in Iran. The objective of the study was to obtain an overview of the genetic relatedness among and within seven populations of this species using inter-simple sequence repeat (ISSR). Fourteen ISSR primers amplified a total of 197 DNA fragments of which 176 (88.91%) were polymorphic. All ISSR primers were highly effective in discriminating among the populations. Genetic similarity coefficients ranged from 0.45 to 0.94, indicating considerable distance and diversity in the germplasm and were confirmed by clustering analysis. The dendrogram showed a clear clustering pattern of plants indicating a significant association between genetic similarity and geographical distance. Analysis of molecular variance revealed that a greater proportion of total genetic variation existed within populations (75%) rather than among populations (25%). The study indicated that ISSR markers were effective and reliable for assessing the degree of genetic variation of S. mutica. These findings can support future research on the selection of S. mutica for breeding and medicinal plant development.     

樟科濒危植物思茅木姜子遗传多样性的ISSR分析

本文采用ISSR标记对中国特有且仅在云南南部狭域分布的樟科濒危植物思茅木姜子(Litseaszemaois)现存8个居群的遗传多样性进行了研究。从96条引物中筛选出了10条,对103个个体进行了扩增,共扩增出77条条带,其中多态性条带为67条。分析结果表明:(1)思茅木姜子的遗传多样性水平很高。在物种水平上,多态位点百分率PPB=87.01%,平均每个位点的有效等位基因数Ne=1.4006,Nei’s基因多样度指数H=0.2466,Shannon多样性信息指数Hsp=0.3826;在居群水平上,PPB=37.99%,Ne=1.2500,H=0.1418,Shannon多样性信息指数Hpop=0.2088。(2)居群间的遗传分化较低。基于Nei’s遗传多样性分析得出的居群间遗传分化系数Gst=0.3700;Shannon’s居群分化系数((Hsp–Hpop)/Hsp)为0.45。AMOVA分析显示:思茅木姜子的遗传变异主要存在于居群内,占总变异的72.99%,居群间的遗传变异占27.01%,表明思茅木姜子属于异交种。(3)两两居群间的Nei’s遗传一致度(I)的范围为0.8233–0.9761。经Mantel检测,居群间的遗传距离和地理距离之间不存在显著的正相关关系(r=0.0925,P=0.6931)。我们推断人类活动的干扰和生境的片断化是导致思茅木姜子濒危现状的主要因素。考虑到目前其遗传多样性水平虽然很高,但各居群个体数量很少,因此应该对思茅木姜子各居群的所有个体实施及时的就地保护;而遗传变异大部分存在于居群内的个体间,所以在迁地保护时应在各居群内大量采样。     

Genetic variation in five Mediterranean populations of Juniperus phoenicea as revealed by inter-simple sequence repeat (ISSR) markers

BACKGROUND AND AIMS: The assessment of the genetic variability and the identification of isolated populations within a given species represent important information to plan conservation strategies on a genetic basis. In this work, the genetic variability in five natural populations of Juniperus phoenicea, three from Sardinia, one from Cyprus and the last one in the Maritime Alps was analysed by means of ISSRs, on the hypothesis that the latter could have been a refugial one during the last glaciation. METHODS: ISSRs were chosen because of their ability to detect variation without any prior sequence information. The use of three primers yielded 45 reproducible, polymorphic bands, which were utilized to estimate the basic parameters of genetic variability and diversity. KEY RESULTS: All of the populations analysed harboured an adequate amount of genetic variability, with H(S) = 0.1299. The proportion of genetic diversity between populations has been estimated by G(ST) = 0.12. The three Sardinian populations are separated, as tested by AMOVA, from the Cyprus and the continental ones. CONCLUSIONS: The results indicate that geographical isolation has represented a major barrier to gene flow in Juniperus phoenicea. This work represents a first step towards a full genetic characterization of a conifer from the Mediterranean, a world biodiversity hotspot confronted with climate change, and thus contributes towards the planning of genetics-informed conservation strategies.     

Genotyping of Ganoderma species by improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis

Species of Ganoderma are used in traditional medicines. An improved random amplified polymorphic DNA (RAPD) analysis, where the RAMP time is prolonged, has been used to characterize the genetic variation in some well known species of Ganoderma. The DNA materials were collected from ten Ganoderma strains, amplified with randomly selected 24 RAPD primers and evaluated by agarose gel electrophoresis. A cluster dendrogram was constructed for genetic analysis on the basis of amplification results. The improved RAPD amplified DNA with consistent and clear banding patterns. A total of 316 bands were found with 93% polymorphism. There was a significant genetic distance between the different strains of Ganoderma, with an index of similarity coefficient in the range of 0.52–0.74. The inter-simple sequence repeat (ISSR) analysis of the Ganoderma DNA samples showed similar trend results to the RAPD analysis with 0.49–0.81 similarity coefficients. This study reports the high level of genetic differences between different species or strains of a single species of Ganoderma and confirms the significance of the improved RAPD method in genetic characterization of organisms. Therefore, the improved RAPD combined with ISSR techniques might be used for the genetic characterization of organisms.     

Diversity, distribution and larval habitats of North Swedish blackflies (Diptera: Simuliidae)

1. Blackfly species richness and community structure were analysed at fifty-six sites in northern Sweden in two seasons. The sites were situated in a wide range of streams and rivers from small springbrooks, bog streams and lake-outlet streams to medium-sized forest rivers and large rivers draining montane regions.
2. Thirty-nine blackfly species were found, with between two and thirteen species per site. Neither species richness nor abundance could be related to the environmental variables measured.
3. An analysis of labral fan size of blackflies indicated a clear trend for the prevalence of larvae with small fans in large rivers and larvae with larger fan size in small streams. Similarly, fan size related to current velocities so that large fans were associated with slow current velocities and small fans with high velocities.
4. A strong relationship existed between species composition and habitat, as seen in ordination by non-metric multidimensional scaling. The relationship found between fan size and habitat size-related variables, such as channel width, depth, velocity and substratum particle size, along with longitude and altitude, in partial least squares regression analysis offered an explanation of the species composition–habitat relationship.
5. In addition to testing that distributions of blackfly larvae reflect morphological traits, we tested two general hypotheses pertaining to distribution patterns: (a) that blackfly communities show bimodal distributions; and (b) that their distributions are nested. Neither of these two hypotheses was supported by our observations. However, widespread blackfly species were locally more abundant than those found at relatively few sites, thus showing a positive abundance–occupancy relationship.     

Molecular phytogeny and typing of blackflies (Diptera: Simuliidae) that serve as vectors of human or bovine onchocerciasis

Abstract. A subregion of the mitochondrial large subunit (16s) rRNA gene was amplified by polymerase chain reaction (PCR) from nine species of blackflies (Diptera: Simuliidae) which serve as natural or experimental vectors of human or bovine Onchocerca parasites. PCR products from each species of blackfly were tested by directed heteroduplex analysis (DHDA), and their genotypes established according to diagnostic banding patterns of the heteroduplex products. Three alleles of mitochondrial 16s rRNA were found to exist in members of the Simulium (Ewardsellum) damnosum sensu lato complex from West Africa, and two alleles were found in the Neotropical Simulium (Psilopelmia) ochraceum Walker complex and the Simulium (Simulium) metallicum Bellardi complex. Different single alleles were detected in Austrosimulium bancrofti , in English S.(S.)noelleri and in two North American laboratory vectors: Simulium (Psilozia) vittatum Zetterstedt and S.(S.)decorum Walker. Phylogenetic analysis of 16s sequences indicated that blackflies from West Africa and the Americas formed distinct clades. Neotropical onchocerciasis vectors were found to be more closely related to Nearctic and Palaearctic non-vector Simulium species than to the African vectors of onchocerciasis.     

Difference in carbon weight of the immature stages of two co-existing species of blackflies (Diptera: Simuliidae) with contrasting reproductive strategies

Two species of blackflies (Diptera: Simuliidae) co-exist at a lake-outlet site in Finland. The adult females of one of them, Simulium noelleri Fried., are able to produce eggs without taking a blood meal; females of the other species, S. truncalum Ldstr., must suck blood before they can mature eggs. Female pupae, pharate adults and adults of S. noelleri are markedly heavier in carbon weight than males of this species, and of males and females of S. truncatum. Females of the former species develop a fat body during the larval stadia.     

Identification of closely related citrus cultivars with inter-simple sequence repeat markers

 Inter-simple sequence repeat (ISSR) markers generated by 22 primers were tested for their ability to distinguish among samples from 94 trees of 68 citrus cultivars. Within each of the six cultivar groups studied, most of these cultivars are so closely related that they are difficult to distinguish by other molecular-marker techniques. ISSR markers involve PCR amplification of DNA using a single primer composed of a microsatellite sequence anchored at the 3′ or 5′ end by 2–4 arbitrary, often degenerate, nucleotides. The amplification products were separated on non-denaturing polyacrylamide gels and detected by silver staining. ISSR banding profiles were very repeatable on duplicate samples. Different citrus species had very different fingerprint patterns. Within Citrus sinensis (L.) Osbeck and C. paradisi Macf., in which all cultivars have originated by the selection of mutants, ISSR markers distinguished 14 of 33 sweet orange and 1 of 7 grapefruit cultivars. Five of six lemon cultivars were discriminated by ISSR markers. Many differences were found among mandarin cultivars; however, all five satsuma cultivars analyzed had identical ISSR fingerprints. Four of five citrange cultivars were distinguishable, but ‘Troyer’ and ‘Carrizo’ had identical ISSR fingerprints. ‘Kuharske Carrizo’ citrange, which has better citrus nematode resistance than other ‘Carrizo’ citrange accessions, had unique ISSR fingerprints. Three ISSR markers that differentiated certain sweet orange cultivars were hybridized to Southern blots of sweet orange DNA digested with different restriction endonucleases. The sweet orange cultivars tested could be distinguished by these ISSR-derived RFLP markers. Moreover, one ISSR marker unique to ‘Ruby’ blood orange was observed in its progeny trees. Received: 9 September 1996 / Accepted: 4 April 1997     

Genetic diversity in Arabidopsis thaliana L. Heynh. investigated by cleaved amplified polymorphic sequence (CAPS) and inter-simple sequence repeat (ISSR) markers

In this study, we investigated genetic diversity among 37 accessions in Arabidopsis thaliana from Eurasia, North Africa and North America using morphological traits and two polymerase chain reaction (PCR)-based marker systems: cleaved amplified polymorphic sequences (CAPS) and inter-simple sequence repeats (ISSR). Cluster analysis based on genetic similarities calculated from CAPS data grouped the accessions roughly according to their geographical origin: one large group contained accessions from Western, Northern and Southern Europe as well as North Africa, a second group consisted of Eastern European and Asian continental accessions. North American accessions were interspersed into these groups. Contrary to the CAPS analysis, the dendrogram obtained from the ISSR data did not reflect the geographical origin of the accessions, and the calculated genetic distances did not match the CAPS results. This could be attributable to an uneven genomic distribution of ISSR markers as substantiated by a database search for ISSR binding sites in A. thaliana genomic DNA sequence files, or to the ISSR's different mode of evolution. We recommend CAPS markers for diversity analysis in A. thaliana because a careful selection of markers can ascertain an even representation of the entire genome.     

Emergence of blackflies (Diptera: Simuliidae) from a small forested stream in Ontario

Three species of blackflies were found in emergence-trap samples taken overa period of 8 months from a second-order, forested, cold-stenothermal stream in southern Ontario. The emergence phenologies of the two common species, Prosimulium mixtum and Stegopterna mutata, are described and compared with their phenologies in other streams in North America. Hypotheses are presented for the poor faunal diversity and prolonged emergence of the two species in the stream studied. Emergence data are used to describe the pupal distribution in the stream.Wing-length measurements showed a distinct dimorphism in P. mixtum: females were larger than males. Adult size (except of P. mixtum females) varied among sampling sites in the stream and, in the case of S. mutata, this variation was time dependent.     

Genetic relationships among species of the genus Diplotaxis (Brassicaceae) using inter-simple sequence repeat markers

Inter-simple sequence repeat (ISSR) amplification was evaluated for its applicability as a genetic marker system to establish relationships among ten Diplotaxis species. ISSR amplification generated multiple banding profiles with the 12 primers from all DNA samples, with an average of 41.2 fragments per primer. This average was clearly higher for the 5′ triple-anchored primers than for other primers. The banding profiles were highly repeatable across separate PCR runs. DNA mixing procedures were found to be appropriate strategies to generate banding patterns representative of each species studied. Similarity values were calculated considering 494 ISSR bands, and a dendrogram was constructed based on the similarity matrix. The ten Diplotaxis species were clustered into two major groups. The first group consists of five species, Diplotaxis tenuifolia and Diplotaxis cretacea, and Diplotaxis muralis with their putative parents (D. tenuifolia and Diplotaxis viminea). In the second group three species are clustered that are closely related (Diplotaxis virgata, Diplotaxis catholica and Diplotaxis siettiana), in addition to Diplotaxis harra, and Diplotaxis erucoides, which has lowest similarity values with the rest of the species studied. The two groups defined in the present work may be concordant with the idea suggested by several authors of a biphyletic origin for Diplotaxis. The genetic relationships among the ten Diplotaxis species estimated by the polymorphism of ISSR markers are in agreement with those previously inferred by other morphological, biochemical and molecular data, indicating the reliability of the ISSR approach for this purpose. Received: 3 January 2000 / Accepted: 31 March 2000     

Nondestructive DNA extraction from blackflies (Diptera: Simuliidae): retaining voucher specimens for DNA barcoding projects

A nondestructive, chemical-free method is presented for the extraction of DNA from small insects. Blackflies were submerged in sterile, distilled water and sonicated for varying lengths of time to provide DNA which was assessed in terms of quantity, purity and amplification efficiency. A verified DNA barcode was produced from DNA extracted from blackfly larvae, pupae and adult specimens. A 60-second sonication period was found to release the highest quality and quantity of DNA although the amplification efficiency was found to be similar regardless of sonication time. Overall, a 66% amplification efficiency was observed. Examination of post-sonicated material confirmed retention of morphological characters. Sonication was found to be a reliable DNA extraction approach for barcoding, providing sufficient quality template for polymerase chain reaction amplification as well as retaining the voucher specimen for post-barcoding morphological evaluation.     

Molecular characterization of Jatropha genetic resources through inter-simple sequence repeat (ISSR) markers

The genetic diversity among eight Jatropha species and three Jatropha curcas accessions were analyzed using ISSR-PCR. Nine ISSR primers generated reproducible amplification banding pattern of 61 polymorphic bands out of 64 scored accounting for 98.14% polymorphism across the species. The ISSR primers viz., I1, I2, I3, I4, I5, I6, I7 and I10 generated 100% polymorphic patterns. Jaccard’s coefficient of similarity varied from 0.346 to 0.807, indicative of high level of genetic variation among the genotypes studied. The UPGMA cluster analysis indicated three distinct clusters, one comprising all accessions of J. curcas L (TNMJ1, TNMJ 22 and TNMJ 23), while second included four species viz., J. tanjorensis J. L. Ellis et Saroja., J. gossypifolia L., J. podagrica Hook and J. maheshwarii Subrum and M.P. Nayer and the third cluster included another four species viz., J. villosa Wight J. multifida L., J. integerrima Jacq and J. glandulifera Roxb. The overall grouping pattern of clustering corresponds well with principal component analysis (PCA) confirming patterns of genetic diversity observed among the species. So far, there are no reports on the molecular diversity of the Jatropha species through ISSR marker. This study provides valid guidelines for collection, conservation and characterization of Jatropha genetic resources and also for further breeding programme towards biodiesel production.     

甘肃省冬虫夏草遗传多样性的ISSR分析

对采自甘肃省3个冬虫夏草主产区域6个具有代表性地方的18个样本进行ISSR分析。15条ISSR引物共扩增得到条带清晰并呈多态性的95条谱带,每一引物扩增获得的ISSR条带数在3－9条之间,扩增片段集中在300－3,000bp。基于遗传相似性系数（GS）、不加权成对群算术平均法（UPGMA）构建的系统树分析,可以看出：分离自同一株虫草上不同部位的样本无显著遗传差异;同一地点样本间的遗传分化较小;不同地域的样本间存在着较大的遗传分化,遗传多样性较丰富。同时6个地方的冬虫夏草明显地分为3个区域,而在同一区域不同     

Species richness and spatial distribution of blackflies (Diptera: Simuliidae) in streams of Central Amazonia, Brazil

1. The spatial distribution and species richness of blackflies were evaluated at 58 stream sites in Central Amazonia, Brazil. Samples were taken along a north–south axis of approximately 130 km and a east–west axis of approximately 220 km.
2. Based on stream-site characteristics, the occurrence of larvae of the six most frequently collected species was highly predictable (79.3–91.5% accuracy in prediction of occurrence). The predictive value of stream size and the presence of impoundments agrees with results of similar work in the Holarctic Region, suggesting a general responses of blackflies to environmental parameters.
3. Although only 19.0% of interstream variation in species richness was explained by a regression model, results suggested that species richness was greater in larger, cooler, faster, covered streams with rocky beds than in smaller, warmer, slower, open streams with sandy bottoms. Overall, the species richness of blackflies (11 species in total) was lower than in the temperate zone suggesting, for some taxa at least, that aquatic communities do not follow the terrestrial pattern of greater species richness in the tropics.