Phylogeography of Tibouchina papyrus (Pohl) Toledo (Melastomataceae), an endangered tree species from rocky savannas, suggests bidirectional expansion due to climate cooling in the Pleistocene

Many endemic species present disjunct geographical distribution; therefore, they are suitable models to test hypotheses about the ecological and evolutionary mechanisms involved in the origin of disjunct distributions in these habitats. We studied the genetic structure and phylogeography of Tibouchina papyrus (Melastomataceae), endemic to rocky savannas in Central Brazil, to test hypothesis of vicariance and dispersal in the origin of the disjunct geographical distribution. We sampled 474 individuals from the three localities where the species is reported: Serra dos Pirineus, Serra Dourada, and Serra de Natividade. Analyses were based on the polymorphisms at cpDNA and on nuclear microsatellite loci. To test for vicariance and dispersal we constructed a median-joining network and performed an analysis of molecular variance (AMOVA). We also tested population bottleneck and estimated demographic parameters and time to most recent common ancestor (TMRCA) using coalescent analyses. A remarkable differentiation among populations was found. No significant effect of population expansion was detected and coalescent analyses showed a negligible gene flow among populations and an ancient coalescence time for chloroplast genome. Our results support that the disjunct distribution of T. papyrus may represent a climatic relict. With an estimated TMRCA dated from ～836.491 ± 107.515 kyr BP (before present), we hypothesized that the disjunct distribution may be the outcome of bidirectional expansion of the geographical distribution favored by the drier and colder conditions that prevailed in much of Brazil during the Pre-Illinoian glaciation, followed by the retraction as the climate became warmer and moister.     

Seventeen novel microsatellite markers from an enriched library of the pest species Bactrocera dorsalis sensu stricto

Seventeen novel microsatellite markers were isolated and characterized from an enriched DNA library of the pest species Bactrocera dorsalis sensu stricto. The polymorphism of these loci was tested in individual fruit flies from a Thai mass‐rearing strain and three wild Asian populations: two from Thailand and one from Myanmar. Allele numbers per locus ranged from two to 15. In the natural populations, a high level of polymorphism was detected in all loci suggesting the usefulness of these markers to quantify the genetic variation in this highly invasive insect.     

Identification and characterization of 23 microsatellite loci for <Emphasis Type="BoldItalic">Chlorostoma rustica</Emphasis> based on RAD-seq

Chlorostoma rustica is an ecologically and economically important species in China, which plays an important role on the overall function of the coastal systems. Understanding of the genetic structure of C. rustica populations is vital to breeding strategies and conservation programmes. In this study, we isolated and characterized 23 microsatellite loci with high polymorphism using the restriction site-associated DNA sequencing approach. The number of alleles per locus ranged from 6 to 18, and the expected heterozygosities varied from 0. 760 to 0.936. All the polymorphism information content values of the 23 loci were greater than 0.5, indicating that these markers were highly informative and laid the foundation for further genetic analysis.     

Isolation and characterization of polymorphic microsatellite markers from fireweed,Senecio madagascariensis Poir. (Asteraceae)

Ten polymorphic microsatellite loci were isolated and characterized from invasive fireweed (Senecio madagascariensis) populations in the Hawaiian archipelago. These loci provided markers with polymorphism of six to 24 alleles per locus within 96 individuals collected from two populations from the island of Maui. The expected and observed heterozygosities ranged from 0.31 to 0.91 and from 0.056 to 1. These markers should be useful to study the importance of genetic diversity in invasion success of this species.     

Isolation and characterization of microsatellite loci from the barley scald pathogen,Rhynchosporium secalis

Fifteen primer pairs were designed for 14 polymorphic microsatellite loci, which were isolated and characterized from genomic libraries of Rhynchosporium secalis. Conditions for multiplexing and simultaneous genotyping of up to eight loci in a single run are described. The number of alleles per polymorphic locus ranged from two to 13 in populations from Switzerland and Australia. Genotypic diversity ranged from 61.5 to 66.7. Gene diversity ranged from 0.08 to 0.89 for individual polymorphic loci, with averages of 0.54 and 0.62 for the populations from Switzerland and Australia, respectively. Variable levels of polymorphism make these ideal markers for population genetic analyses.     

Isolation and characterization of microsatellite markers in an endemic Hawaiian planthopper (Nesosydne chambersi: Delphacidae)

We have isolated and characterized 17 microsatellite loci for the endemic Hawaiian planthopper Nesosydne chambersi (Delphacidae), a member of a large Hawaiian Nesosydne radiation. Thirty individuals from one population and 10 individuals from two populations across the species' range were tested to investigate polymorphism. The observed loci contained two to nine alleles per locus. Expected heterozygosity within this species ranged from 0.2 to 0.85. These markers will be used to assess intraspecific differentiation and population structure within N. chambersi.     

Characterization of polymorphic microsatellite markers in the Dalmatian wall lizard Podarcis melisellensis (Squamata: Lacertidae)

We describe polymerase chain reaction primers and amplification conditions for 13 highly polymorphic microsatellite DNA loci isolated from the Dalmatian wall lizard, Podarcis melisellensis. The number of alleles per locus ranged from 12 to 41, with levels of observed heterozygosity between 0.62 and 0.94. Most of these loci were successfully cross-amplified in the closely related species P. sicula, but levels of polymorphism were always lower.     

Polymorphic microsatellite markers in the guava fruit fly, Bactrocera correcta (Diptera: Tephritidae)

Bactrocera correcta (Bezzi) is one of the most destructive insect pests of fruits and vegetables in tropical and subtropical regions. At present, this fly is primarily distributed in Southeast Asia. Twelve microsatellite loci were isolated from an enriched genomic library based on a biotin/streptavidin capture protocol. The polymorphism of these loci was tested on 74 individual flies from two natural populations. Allele number ranged from 6 to 14 and 10 loci demonstrated a polymorphic information content (PIC) greater than 0.5. The pairwise F _ST value between the two populations was 0.0048 (P > 0.05). These microsatellite loci have potential utility for studies of population genetic structure in this species.     

Characterization of polymorphic microsatellite loci in the endangered Miho spine loach (Iksookimia choii) and cross-species amplification within the Cobitidae family

The Miho spine loach (Iksookimia choii) is an endangered freshwater fish endemic to Korea. To aid a conservation programme for this species, 20 polymorphic microsatellite loci were isolated and characterized in 67 individuals from three populations. The number of alleles per locus ranged from 8 to 33. Within populations, the observed and expected heterozygosities ranged from 0.59 to 1.00 and from 0.56 to 0.96, respectively, indicating its usefulness in population genetics studies. Cross-species amplification of the loci was tested in six species of the family Cobitidae, and 18 loci were amplified in two or more related species.     

De novo development and characterization of polymorphic microsatellite markers in a schilbid catfish, <Emphasis Type="Italic">Silonia silondia</Emphasis> (Hamilton, 1822) and their validation for population genetic studies

The stock characterization of wild populations of Silonia silondia is important for its scientific management. At present, the information on genetic parameters of S. silondia is very limited. The species-specific microsatellite markers were developed in current study. The validated markers were used to genotype individuals from four distant rivers. To develop de novo microsatellite loci, an enriched genomic library was constructed for S. silondia using affinity–capture approach. The markers were validated for utility in population genetics. A total number of 76 individuals from four natural riverine populations were used to generate data for population analysis. The screening of isolated repeat sequences yielded eleven novel polymorphic microsatellite loci. The microsatellite loci exhibited high level of polymorphism, with 6–24 alleles per locus and the PIC value ranged from 0.604 to 0.927. The observed (Ho) and expected (He) heterozygosities ranged from 0.081 to 0.84 and 0.66 to 0.938, respectively. The AMOVA analysis indicated significant genetic differentiation among riverine populations (overall F_ST = 0.075; P < 0.0001) with maximum variation (92.5 %) within populations. Cross-priming assessment revealed successful amplification (35–38 %) of heterologous loci in four related species viz. Clupisoma garua, C. taakree, Ailia coila and Eutropiichthys vacha. The results demonstrated that these de novo polymorphic microsatellite loci are promising for population genetic variation and diversity studies in S. silondia. Cross-priming results indicated that these primers can help to get polymorphic microsatellite loci in the related catfish species of family Schilbidae.     

Microsatellite markers isolated from the flightless cormorant (Phalacrocorax harrisi)

Eight polymorphic microsatellite DNA loci were isolated from the flightless cormorant (Phalacrocorax harrisi) for future population genetic studies. Genetic variability was assessed using at least 38 individuals from two populations. Allele numbers ranged from three to nine per locus. Mean observed heterozygosity varied from 0.27 to 0.78. No locus deviated from Hardy-Weinberg (HW) or linkage equilibria in either population. The high levels of detected polymorphism indicate the utility of these markers for population genetic studies of this Galápagos species.     

Isolation and characterization of EST‐derived microsatellite loci from the fungal wheat pathogen Phaeosphaeria nodorum

Eleven polymorphic microsatellite loci and one minisatellite locus originating from expressed sequence tag (EST) libraries of Phaeosphaeria (syn. Stagonospora) nodorum were isolated and characterized. The satellite markers were used to genotype isolates from field populations collected in China, North America and South Africa. The number of alleles per locus ranged from two to 15. Genotype diversity ranged from 87.5 to 95.3 and gene diversity from 0.1 to 0.8. The variable levels of polymorphism within and among populations of P. nodorum renders these 12 satellite loci ideal markers for population genetic analysis of P. nodorum.     

Highly polymorphic microsatellite loci in the rice- and maize-infecting fungal pathogen Rhizoctonia solani anastomosis group 1 IA

Ten polymorphic microsatellite loci were isolated and characterized from the rice- and maize-infecting Basidiomycete fungus Rhizoctonia solani anastomosis group AG-1 IA. All loci were polymorphic in two populations from Louisiana in USA and Venezuela. The total number of alleles per locus ranged from four to eight. All 10 loci were also useful for genotyping soybean-infecting R. solani AG-1 isolates from Brazil and USA. One locus, TC06, amplified across two other AG groups representing different species, showing species-specific repeat length polymorphism. This marker suite will be used to determine the global population structure of this important pathogenic fungus.     

Evidence of reproductive isolation among closely related sympatric species of Serrasalmus (Ostariophysii, Characidae) from the Upper Madeira River, Amazon, Bolivia

The delimitation of the Serrasalmus species (Characiformes, Serrasalminae) from the Bolivian Amazon (Amazonas, Madeira) was examined using intron length polymorphism assessed by EPIC-PCR. The six pairs of primers provided 16 polymorphic loci across the species of the region and the allelic diversity ranged between two and 10 alleles per locus. For each locus, the lack of departure from Hardy-Weinberg expectations in a large number of the populations analysed and the homogenous distribution of linkage disequilibrium between paralogous loci and loci belonging to different intronic systems provided strong lines of evidence that the 16 amplified loci constituted independent neutral markers. Furthermore, allelic diversity was size-dependent, thereby indicating that insertion-deletions occurred frequently but randomly in introns, and that intron length polymorphism was a valid marker for investigating the systematics of piranhas. EPIC-PCR demonstrated that eight of the nine nominal species of piranha of the Upper Madeira were reproductively isolated and identified a new species that differed from its closest morphological and genetic relatives by seven diagnostic or semi-diagnostic loci. By contrast, no diagnostic or semi-diagnostic locus was found between S. spilopleura and S. eigenmanni , nor were their allelic frequencies different, thereby questioning the validity of their biological species status, at least in the Upper Madeira. This study, which was one of the first applications of EPIC-PCR to a large-scale molecular systematic purpose, demonstrates that it is a rapid, reliable and cost-effective tool for elucidating issues pertaining to fish systematics.     

Isolation and characterization of polymorphic microsatellite loci in Asiatic sand sedge,Carex kobomugi Ohwi (Cyperaceae)

Fourteen polymorphic microsatellite loci were isolated from Asiatic sand sedge, Carex kobomugi Ohwi (Cyperaceae), a clonal perennial herb predominant in sandy coast vegetation in Japan. Polymorphism was surveyed for 24 ramets within a single population and for eight distinct individuals from six populations. Within a population, between two and eight alleles were found per locus, and expected heterozygosity ranged from 0.041 to 0.720. Among populations, a higher level of polymorphism was observed. The microsatellite loci were shown to be useful for the study of spatial genetic structure at fine scale and genetic differentiation among populations.     

Development of polymorphic microsatellite markers for Dalbergia nigra (Papilionoideae), an endangered tree from the Brazilian Atlantic Forest

Dalbergia nigra is an endangered tree restricted to the Brazilian Atlantic Forest. Ten polymorphic microsatellite loci were isolated and characterized in 47 trees from two populations. The total number of alleles per locus ranged from three to 12 alleles. The levels of observed and expected heterozygosities ranged from 0.304 to 0.740 and from 0.278 to 0.872, respectively. Significant deviations from Hardy-Weinberg were detected for only three loci in each population. No pair of loci exhibited significant linkage disequilibrium. These microsatellites provide an efficient tool to investigate genetic structure in forest remnants with the purpose of conservation of this species.     

Isolation and characterization of microsatellites in Rattus rattus

We isolated and characterized 10 microsatellite loci in the black rat Rattus rattus (Muridae, Rodentia), a widespread invasive species largely known to cause serious problems in agriculture and human health. Polymorphism was studied in two populations, one from Madagascar and one from Senegal. It ranged from three to 12 alleles in Madagascar, and from two to five alleles in Senegal. Together with the loci previously adapted from Rattus norvegicus, this set of markers should allow the conduct of thorough studies on the genetic structure of natural populations of R. rattus.     

Twelve microsatellite loci in Manilkara huberi (Ducke) Standl (Sapotaceae), an Amazonian timber species

Manilkara huberi is a timber species, found and intensely exploited in the Amazonian forest. Twelve highly polymorphic microsatellite loci were developed from a genomic library enriched for AG/TC repeats. Levels of polymorphism were evaluated using a total of 12 adult trees from a natural population. An average of 6.43 alleles per locus were detected, and expected heterozygosity ranged from 0.721 to 0.862. These loci represent a powerful tool in investigating the mating system, gene flow, parentage and population dynamics in natural populations of M. huberi, all of which are needed to implement sound management.     

Microsatellite loci from the ectomycorrhizal basidiomycete Suillus pictus associated with the genus Pinus subgenus Strobus

Seven microsatellite loci were isolated and characterized from ectomycorrhizal basidiomycete Suillus pictus associated with soft pine species (Pinus subgenus Strobus) using a dual‐suppression‐PCR technique. Microsatellite variation was assessed using 38 isolates of S. pictus sampled from three different local populations in Japan. The number of alleles per locus ranged from three to 12 within populations. These microsatellite loci can be used for studying the allelic variation and genetic structure in/among local populations of S. pictus.