Molecular phylogeny of the entomopathogenic fungi of the genus Cordyceps (Ascomycota: Clavicipitaceae) and its evolutionary implications

Abstract Cordyceps is an endoparasite ascomycetous genus containing approximately 450 species with a diversity of insect hosts, traditionally included in the family Clavicipitaceae of Ascomycota. Establishing the relationships among species with a varied range of morphologies and hosts is of importance to our understanding of the phylogeny and co‐evolution of parasites and hosts in entomopathogenic ascomycetes. To this end, we used a combination of molecular index and morphological characters from 40 representative species to carry out comprehensive molecular phylogenetic analyses. Based on the phylogenetic tree, we used the program DISCRETE for inferring the rates of evolution and finding ancestral states of morphological character. The phylogenetic analyses revealed two important points. (i) Types of perithecia attached to stroma reflected an evolutionary trend in Cordyceps. The vertically immersed perithecia form was the ancestral state, superficial and obliquely immersed perithecia were derived characters, obliquely immersed was irreversible. Species with obliquely immersed perithecia were in a closely related group and were the derived group. (ii) A strong correlation between fungal relatedness and the microhabitat supported the hypothesis that the host jumps through commingling in soil microhabitats. Based on the results of these analyses, host switching explains the diversity of entomopathogenic fungi of the genus Cordyceps.     

Molecular phylogeny of the entomopathogenic fungi of the genus Cordyceps (Ascomycota:Clavicipitaceae) and its evolutionary implications

Cordyceps is an endoparasite ascomycetous genus containing approximately 450 species with a diversity of insect hosts,traditionally included in the family Clavicipitaceae of Ascomycota.Establishing the relationships among species with a varied range of morphologies and hosts is of importance to our understanding of the phylogeny and co-evolution of parasites and hosts in entomopathogenic ascomycetes.To this end,we used a combination of molecular index and morphological characters from 40 representative species to carry out comprehensive molecular phylogenetic analyses.Based on the phylogenetic tree,we used the program DISCRETE for inferring the rates of evolution and finding ancestral states of morphological character.The phylogenetic analyses revealed two important points.(i) Types of perithecia attached to stroma reflected an evolutionary trend in Cordyceps.The vertically immersed perithecia form was the ancestral state,superficial and obliquely immersed perithecia were derived characters,obliquely immersed was irreversible.Species with obliquely immersed perithecia were in a closely related group and were the derived group.(ii) A strong correlation between fungal relatedness and the microhabitat supported the hypothesis that the host jumps through commingling in soil microhabitats.Based on the results of these analyses,host switching explains the diversity of entomopathogenic fungi of the genus Cordyceps.     

Development of microsatellite markers for the endangered medicinal plant Launaea arborescens (Asteraceae)

• Premise of the study: Microsatellite loci were developed in Launaea arborescens, an endangered and medicinal Asteraceae species in North Africa, for further investigation of its conservation genetics. • Methods and Results: We isolated and characterized 10 polymorphic and nine monomorphic microsatellite loci from L. arborescens using the protocol of Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO). For the 10 polymorphic loci, the number of alleles detected per locus varied from two to six, and the observed and expected heterozygosities ranged from 0.000 to 0.833 and 0.059 to 0.713, respectively. • Conclusions: The polymorphic markers provide a useful tool for conservation genetics studies of L. arborescens, including analysis of mating system, estimating gene flow, and identifying discrete genetic units within the species.     

蛹虫草水溶性多糖含量测定方法的比较与优化

已报道文献中蛹虫草多糖含量差异非常大,通过比较分析发现差异主要在于干扰物质的去除。结合高效阴离子色谱对干扰物质的测定,对常见的几种蛹虫草多糖提取测定方法进行了分析比较,在此基础上建立了去除干扰物的蛹虫草多糖测定方法。该方法简便、快速、精确,经系统的方法学考察证明,是一种分析蛹虫草多糖较好的方法。     

蛹虫草金属硫蛋白分离纯化及性质研究

研究探讨锌离子胁迫下蛹虫草Cordyceps militaris金属硫蛋白的产生及性质。蛹虫草菌丝体以15g/L Zn2+在10L发酵罐中诱导培养56h后收集,产率为每升发酵液收集12.021g菌丝体（干重）,细胞破碎取上清液通过两次凝胶柱层析,冷冻干燥得到蛹虫草金属硫蛋白纯品。利用Bradford法进行蛋白质含量测定,用银饱和分析法结合原子吸收光谱（AAS）测定MT含量,发酵终点处金属硫蛋白含量为12.876mg/g菌丝体（湿重）。用电喷雾质谱法测得金属硫蛋白的分子量为7 390Da,用Ellman’s方法和火焰原子吸收法分别测得每分子蛋白质含有14个巯基、结合5个Zn原子。氨基酸组成分析结果显示,每分子蛋白质共含57个氨基酸,其中含有13个半胱氨酸,疏水氨基酸占29.8%,且含有组氨酸。以上表明,研究中的蛹虫草金属硫蛋白与哺乳动物金属硫蛋白结构差异较大,但与酵母菌金属硫蛋白结构组成类似。     

Isolation and characterization of twelve polymorphic microsatellite markers in bottlenose dolphins (Tursiops truncatus)

We developed polymerase chain reaction primers for 12 dinucleotide microsatellite loci in the bottlenose dolphin, Tursiops truncatus. Seven markers were obtained after hybridization screening, and five following random genome sequencing. Orthologous positions were computed for nine markers on the bovine genome and for seven on the human genome. The markers are distributed across chromosomes and found in different types of DNA regions. All 12 loci are polymorphic for Tursiops. Five loci were also polymorphic in the related species Stenella frontalis and the more distantly related river dolphin, Inia geoffrensis, indicating these markers will be informative across the Delphinidae and other cetacean taxa.     

Development and characterisation of microsatellite markers for the medicinal plant Scutellaria baicalensis (Lamiaceae)

Scutellaria baicalensis is a popular medicinal plant that is on the verge of extinction due to uncontrolled harvesting, habitat destruction and deterioration of its ecosystem. We isolated and characterised 21 microsatellite loci in this species. Ninety-four individuals from six populations were used to test the polymorphism of the microsatellite loci. The number of alleles per locus ranged from 1 to 13, with a mean of 7.2. Observed and expected heterozygosities varied from 0.000 to 1.000 and 0.000 to 0.938, respectively. Among these new microsatellite markers, only two loci showed significant deviation from Hardy–Weinberg equilibrium. No locus pairs showed significant linkage disequilibrium. The 21 primer pairs were tested in other Scutellaria species. Most of these primer pairs worked successfully, except for Scut18. These new microsatellite markers could be applied to investigate the genetic diversity and population genetic structure of S. baicalensis and its closely related species.     

Development of polymorphic microsatellite markers for the zebra finch (Taeniopygia guttata)

To study the population genetics as well as the mating system of captive zebra finch (Taeniopygia guttata) populations, we developed primers for 12 microsatellite loci and screened them in 529 individuals from two successive generations of a single captive population. All markers were polymorphic with five to 14 alleles per locus. We checked all markers for Mendelian inheritance in 307 offspring whose parents were known for sure. Four markers showed evidence for the presence of null alleles. Once allowing for null alleles, we found no mismatches between offspring and parents, suggesting a very low rate of mutation. Average observed and expected heterozygosities across the eight loci showing no evidence for null‐alleles was 0.819 and 0.812, respectively.     

Development of polymorphic microsatellite markers for the sorghum plant bug, Stenotus rubrovittatus (Heteroptera: Miridae)

Nine polymorphic microsatellite DNA markers from the sorghum plant bug, Stenotus rubrovittatus, were isolated and characterized. These markers were used to analyse 22 individuals from a single field population. The number of alleles at these nine loci ranged from two to 28 (mean = 11.4) and heterozygosity ranged from 0.27 to 0.86 (mean = 0.58). Stenotus rubrovittatus has shown rapid population growth in the decades since the first report in the 1980s of serious damage to a rice crop. These microsatellite markers will be of value for studying both the population genetics and population dynamics of S. rubrovittatus.     

Development and characterization of eight microsatellite markers in bullet tuna (Auxis rochei)

The bullet tuna (Auxis rochei) is a worldwide species of high commercial value in Spain. Nevertheless, little information is currently available about the genetic characteristics of wild A. rochei populations. In this survey, we have developed eight new microsatellites for the bullet tuna using an enriched genome library protocol. Primers were screened on a total of 78 individuals from three wild populations (Mediterranean, Atlantic, and Pacific), revealing seven to 27 alleles per locus with expected heterozygosities ranging from 0.63 to 0.97. These markers can be potentially useful tools for use in population genetic studies.     

Cross-species microsatellite markers for elucidating population genetic structure in Arabidopsis and Arabis (Brassicaeae)

Species closely related to model organisms present the opportunity to efficiently apply molecular and functional tools developed by a large research community to taxa with different ecological and evolutionary histories. We complied 42 microsatellite loci that amplify under common conditions in four closely related Arabidopsis: A. thaliana; A. halleri; A. lyrata ssp. lyrata; and A. lyrata ssp. petraea, as well as in one more distantly related crucifer; Arabis drummondii. Variation at these loci is amenable to a diversity of applications including population genetics, phylogeographical analyses, mapping of inter and intraspecific crosses, and recombination mapping. Our analysis of microsatellite variation illustrates significant differences in population genetic parameters among three Arabidopsis species. A population of A. thaliana, an inbreeding annual plant associated with disturbed habitats, was highly monomorphic (P = 8% percent polymorphic loci) and only 0.2% heterozygous for 648 locus-by-individual combinations. A population of the self-incompatible perennial herb, A. halleri, was more genetically variable (P = 71%) and had an excess of heterozygosity that may reflect a recent population bottleneck associated with human-mediated founder events. A population of the self-incompatible perennial herb, A. lyrata ssp. petraea, was even more genetically variable (P = 86%) and appeared to be at mutation-drift equilibrium. Population structure estimated from neutrally evolving loci provides an empirical expectation against which hypotheses of adaptive evolution at functional loci can be tested.     

Development and characterization of eight polymorphic microsatellite markers for Daphnia atkinsoni (Crustacea: Ctenodaphnia)

A microsatellite‐enriched genomic library was developed for the water flea Daphnia atkinsoni Baird, 1859, a dominant species of intermittent wetlands in Europe. Eight polymorphic microsatellite markers were successfully optimized. Characterization of 77 individuals from Belgium and Spain showed moderate (in the former) to high (in the latter) levels of polymorphism with two to 11 alleles per locus and an observed heterozygosity ranging from 0 to 0.87. Some of these microsatellite markers were successfully amplified in three other Daphnia species (D. magna n = 4, D. similis n = 6; D. obtusa n = 6).     

Isolation and characterization of microsatellite loci from the entomopathogenic fungus Beauveria bassiana (Ascomycota: Hypocreales)

Beauveria bassiana, an entomogenous fungus used for the biological control of pest insects, comprises a globally‐distributed species complex of regionally endemic lineages. In order to study the population genetics of B. bassiana, detail species boundaries, conduct ecological studies of natural populations and track fates of experimentally‐released strains, sensitive genetic markers are required. We describe the isolation and characterization of eight microsatellite loci that amplify successfully from strains representative of the phylogenetic diversity in the B. bassiana complex.     

Development of polymorphic microsatellite markers for the human botfly, Dermatobia hominis (Diptera: Oestridae)

In this report, we describe the development of 17 polymorphic microsatellite markers for the human botfly, Dermatobia hominis, an obligatory parasite of mammals of great veterinary importance in Latin America. The number of alleles ranged from 5 to 21 per locus, with a mean of 12.2 alleles per locus. The expected heterozygosity ranged from 0.2571 to 0.9206 and from 0.2984 to 0.9291 in two populations from Brazil. These markers should provide a high resolution tool for assessment of the fine-scale genetic structure of natural populations of the human botfly.     

Challenges in analysis and interpretation of microsatellite data for population genetic studies

Advancing technologies have facilitated the ever‐widening application of genetic markers such as microsatellites into new systems and research questions in biology. In light of the data and experience accumulated from several years of using microsatellites, we present here a literature review that synthesizes the limitations of microsatellites in population genetic studies. With a focus on population structure, we review the widely used fixation (F_ST) statistics and Bayesian clustering algorithms and find that the former can be confusing and problematic for microsatellites and that the latter may be confounded by complex population models and lack power in certain cases. Clustering, multivariate analyses, and diversity‐based statistics are increasingly being applied to infer population structure, but in some instances these methods lack formalization with microsatellites. Migration‐specific methods perform well only under narrow constraints. We also examine the use of microsatellites for inferring effective population size, changes in population size, and deeper demographic history, and find that these methods are untested and/or highly context‐dependent. Overall, each method possesses important weaknesses for use with microsatellites, and there are significant constraints on inferences commonly made using microsatellite markers in the areas of population structure, admixture, and effective population size. To ameliorate and better understand these constraints, researchers are encouraged to analyze simulated datasets both prior to and following data collection and analysis, the latter of which is formalized within the approximate Bayesian computation framework. We also examine trends in the literature and show that microsatellites continue to be widely used, especially in non‐human subject areas. This review assists with study design and molecular marker selection, facilitates sound interpretation of microsatellite data while fostering respect for their practical limitations, and identifies lessons that could be applied toward emerging markers and high‐throughput technologies in population genetics.     

Characterization of polymorphic microsatellite markers for the blowfly Chrysomya albiceps (Diptera: Calliphoridae)

Chrysomya albiceps is a blowfly of great medical, sanitary and forensic importance widely distributed in the Afrotropical, southern Palaearctic, northern Oriental regions and, recently, in Central and South Americas. Here, we report the characterization of 13 polymorphic microsatellite markers for C. albiceps. The number of alleles ranged from three to 13 alleles with expected heterozygosities ranging from 0.4668 to 0.8408. These markers will be extremely useful for investigating many important aspects of this species such as population structure, dispersal and colonization dynamics.     

Development of polymorphic microsatellite markers for the genetic characterisation of Knoxdaviesia proteae (Ascomycota: Microascales) using ISSR-PCR and pyrosequencing

Knoxdaviesia proteae is one of the first native ophiostomatoid fungi discovered in South Africa, where it consistently occurs in the infructescences of the iconic Cape Biome plant, Protea repens. Although numerous studies have been undertaken to better understand the ecology of K. proteae, many questions remain to be answered, particularly given its unique niche and association with arthropods for dispersal. We describe the development and distribution of microsatellite markers in K. proteae through Interspersed Simple Sequence Repeat-Polymerase Chain Reaction (ISSR-PCR) enrichment and pyrosequencing. A large proportion of the 31492 sequences obtained from sequencing the enriched genomic DNA were characterised by microsatellites consisting of short tandem repeats and di- and tri-nucleotide motifs. Seventeen percent of these microsatellites contained flanking regions sufficient for primer design. Twenty-three primer pairs were tested, of which 12 amplified and 10 generated polymorphic fragments in K. proteae. Half of these could be transferred to the sister species, K. capensis. The developed markers will be used to investigate the reproductive system, genetic diversity and dispersal strategies of K. proteae.     

Development and characterization of 10 microsatellite markers for the semi-evergreen tree species, Ligustrum ovalifolium (Oleaceae)

Ligustrum ovalifolium is a semi-evergreen tree distributed in the western part of Japan and southern Korea. This species contains an insular endemic variety, L. ovalifolium var. pacificum; this variety occurs only on the Izu Islands located south of the Japanese mainland Honshu. We isolated 10 polymorphic microsatellite loci from the species, and characterized them for 21 individuals from a population of L. ovalifolium. The primers developed in this study yielded an average 12.2 alleles per locus and an average expected heterozygosity of 0.78. These markers will be powerful tools for studying the genetic differentiation within the species.     

Development and characterization of eight new microsatellite markers for the haremic sandperch,Parapercis cylindrica (family Pinguipedidae)

Eight di‐, tri‐ and tetranucleotide microsatellite markers were developed for the haremic sandperch Parapercis cylindrica using a linker‐ligated, magnetic bead enrichment protocol. Screening of at least 17 individuals showed these markers to be polymorphic with observed heterozygosity ranging from 0.381 to 1.000 (mean = 0.742) and the numbers of alleles ranging from three to 18. The average polymorphic information content for these eight loci was 0.723. These markers may be used for parentage studies aimed at exploring the complex mating strategies employed by this haremic coral reef fish and will be valuable for population genetic studies.