Reliability assessment of null allele detection: inconsistencies between and within different methods

Microsatellite loci are widely used in population genetic studies, but the presence of null alleles may lead to biased results. Here, we assessed five methods that indirectly detect null alleles and found large inconsistencies among them. Our analysis was based on 20 microsatellite loci genotyped in a natural population of Microtus oeconomus sampled during 8 years, together with 1200 simulated populations without null alleles, but experiencing bottlenecks of varying duration and intensity, and 120 simulated populations with known null alleles. In the natural population, 29% of positive results were consistent between the methods in pairwise comparisons, and in the simulated data set, this proportion was 14%. The positive results were also inconsistent between different years in the natural population. In the null‐allele‐free simulated data set, the number of false positives increased with increased bottleneck intensity and duration. We also found a low concordance in null allele detection between the original simulated populations and their 20% random subsets. In the populations simulated to include null alleles, between 22% and 42% of true null alleles remained undetected, which highlighted that detection errors are not restricted to false positives. None of the evaluated methods clearly outperformed the others when both false‐positive and false‐negative rates were considered. Accepting only the positive results consistent between at least two methods should considerably reduce the false‐positive rate, but this approach may increase the false‐negative rate. Our study demonstrates the need for novel null allele detection methods that could be reliably applied to natural populations.     

Short allele dominance as a source of heterozygote deficiency at microsatellite loci: experimental evidence at the dinucleotide locus Gv1CT in Gracilaria gracilis (Rhodophyta)

In this study, we compared the genotypes obtained at a microsatellite locus using two methods of amplification and detection of variation in a set of individuals belonging to the red alga haplo-diploid species, Gracilaria gracilis . The methods varied in their capacity to detect longer alleles in heterozygotes, resulting in an apparent heterozygote deficiency. We attributed this bias in favour of short alleles to competition leading to the preferential amplification of shorter alleles (short allele dominance). To test this hypothesis, we created artificial heterozygotes (mixtures of two haploid DNA samples) and showed that long alleles already less intense than short alleles, 'suffer' more from being in association.     

Cross‐species amplification of microsatellite loci in Aquilegia and Semiaquilegia (Ranunculaceae)

We developed 16 microsatellite loci from an F₂ hybrid between Aquilegia formosa and Aquilegia pubescens. In samples of 28 individuals, we found an average of 14 alleles per locus from each parental species. We tested these loci for cross‐amplification in 10 additional species of Aquilegia and found that all 16 loci amplified in other North American species and 12 consistently amplified in European or Asian species. Nine loci amplified in the sister species to Aquilegia, Semiaquilegia adoxoides. The success of cross‐species amplification suggests that these microsatellites should prove useful for studies in a broad range of Aquilegia species.     

梨小食心虫微卫星标记的扩增稳定性及多态性分析

【目的】筛选适合我国梨小食心虫Grapholita molesta种群遗传学研究的微卫星位点,并依据所筛选的微卫星位点进行梨小食心虫地理种群的遗传多样性分析。【方法】利用欧洲梨小食心虫和苹果蠹蛾Cydia pomonella种群中已报道的11个微卫星位点, 分析各位点在我国12个种群257头梨小食心虫样本中的扩增稳定性,再进行其多态性分析,筛选适合的位点,然后进行种群遗传多态性分析。【结果】在分析的11个微卫星位点中, 位点Gm01, Gm03, Gm04和Cyd15无法稳定扩增; 位点Gm05扩增成功率较低, 位点Gm07遗传多态性较低; 而位点Gm02, Gm06, Gm08, Gm09和Gm10等扩增效果稳定且遗传多态性丰富。这5个稳定扩增的微卫星位点平均等位基因数量（N_A）为7.417~12.500, 平均观察杂合度（H_o）为0.366~0.655, 平均期望杂合度（H_e）为0.642~0.846, 多态信息含量（PIC）为0.800~0.935。【结论】本研究成功筛选出位点Gm02, Gm06, Gm08, Gm09和Gm10等5个微卫星位点。基于这5个微卫星位点标记的结果显示, 山东和陕西不同梨小食心虫地理种群均具有丰富的遗传多样性。 这5个位点可以适用于我国梨小食心虫种群的进一步遗传分析研究。     

Isolation and characterization of polymorphic microsatellite loci in yellowtail catfish, Pangasius pangasius (Hamilton, 1822)

A total of nine polymorphic microsatellite loci were obtained from a genomic library of Pangasius pangasius (order Siluriformes, family Pangasiidae). Samples from rivers Bhagirathi (n = 22) and Mahanadi (n = 20) were genotyped for each of the nine microsatellite loci to determine genetic variation. The mean number of alleles per locus was 5.22 in Bhagirathi and 5.78 in Mahanadi; and expected heterozygosity ranged from 0.567 (Bhagirathi) to 0.578 (Bhagirathi). Significant deviation (P < 0.003) from Hardy–Weinberg expectations was evident at three loci, Ppa2 (Bhagirathi), Ppa14 (Mahanadi) and Ppa28 (Bhagirathi and Mahanadi). The identified microsatellite loci were found to be promising for population genetics studies of P. pangasius.     

Isolation and characterization of microsatellite loci in the flat‐headed bat (Tylonycteris pachypus)

Microsatellite loci were developed in the flat‐headed bat (Tylonycteris pachypus) from genomic DNA using an enriched library method. Nine loci were tested on 48 individuals sampled from Guangxi Province, China. The mean number of observed alleles per locus was 6.4 (range 4–12). Observed and expected heterozygosity values ranged from 0.24 to 0.83 and from 0.30 to 0.89, respectively. One locus revealed significant departure from Hardy–Weinberg equilibrium and no significant linkage disequilibrium was detected between loci pairs. These markers will be used to examine genetic structure and parentage analysis in this species.     

Microsatellite allele ladders in two species of Pacific salmon: preparation and field‐test results

To support microsatellite data communication, we have developed a convenient method for creating locus‐specific microsatellite allele ladders used to align data from different laboratories. The ladders were constructed by pooling polymerase chain reaction (PCR) products to create a template for amplification. Four ladders were field‐tested in six different laboratories using different genotyping platforms. Despite substantial differences in absolute size estimates of DNA fragments, each laboratory correctly scored unknown sample genotypes according to the ladder designations. The results indicate that our simple preparation method provides reliable allele ladders in a time‐efficient manner for verifying microsatellite genotypes across platforms.     

Isolation and characterization of 125 microsatellite DNA markers in the blacklip abalone,Haliotis rubra

Australian abalone species are subject to wild harvest and aquaculture production. This study characterized 125 polymorphic microsatellite markers in the blacklip abalone, Haliotis rubra, and evaluated cross‐species amplification in Haliotis laevigata and Haliotis coccoradiata. Segregation analysis of a mapping family revealed non‐amplifying polymerase chain reaction null alleles at 34 loci. Cross‐species amplification was achieved for 89 loci.     

Isolation and characterization of microsatellite loci in a marine fish species,the tusk (Brosme brosme)

We developed polymerase chain reaction primers for nine dinucleotide microsatellite loci in the marine deep‐sea fish, the tusk (Brosme brosme). All markers were obtained from a partial genomic DNA library, and characterized in 100 unrelated individuals from one putative population. The number of alleles ranged from two to 60 with an average of 15/locus. The observed heterozygosity ranged from 0.020 to 0.826 (average 0.438). Several of the markers amplified multiple alleles from the two other gadoid species tested.     

Isolation and characterization of polymorphic microsatellite loci in the Chagas’ disease vector Triatoma infestans (Hemiptera: Reduviidae)

Microsatellites were identified and characterized from Triatoma infestans, the principal vector of Chagas’ disease in the Southern Cone of South American countries. Ninety‐three microsatellite loci were isolated from partial genomic libraries, of which 30 were amplified and 10 were selected for genotyping. The degree of intrapopulation variation in these loci was determined using 34 specimens from the locality of Chancaní (Córdoba, Argentina). The number of alleles per locus ranged from five to 19 and the expected heterozygosity ranged from 0.608 to 0.941. The variability of these microsatellite markers provides a valuable molecular tool for population genetic studies in T. infestans.     

Isolation and characterization of microsatellite DNA loci in the toad‐headed lizards,Phrynocephalus przewalskii complex

To assess mechanisms of hybridization and speciation, we isolated and characterized 12 dinucleotide microsatellite DNA loci for the toad‐headed lizards, Phrynocephalus przewalskii complex. A total of 48 specimens were examined and all loci were polymorphic with seven to 25 alleles per locus. The observed and expected heterozygosities ranged from 0.282 to 0.946 and 0.400 to 0.937, respectively. These loci are therefore suitable for a wide range of population level studies within the P. przewalskii complex.     

Genetic consequences of fragmentation in “arbor vitae,” eastern white cedar (Thuja occidentalis L.), toward the northern limit of its distribution range

We tested the hypothesis that marginal fragmented populations of eastern white cedar (EWC) are genetically isolated due to reduced pollen and gene flow. In accordance with the central-marginal model, we predicted a decrease in population genetic diversity and an increase in differentiation along the latitudinal gradient from the boreal mixed-wood to northern coniferous forest. A total of 24 eastern white cedar populations were sampled along the north-south latitudinal gradient for microsatellite genotyping analysis. Positive F_is values and heterozygote deficiency were observed in populations from the marginal (F_is = 0.244; P_HW = 0.0042) and discontinuous zones (F_is = 0.166; P_HW = 0.0042). However, populations from the continuous zone were in HW equilibrium (F_is = −0.007; P_HW = 0.3625). There were no significant latitudinal effects on gene diversity (H_s), allelic richness (AR), or population differentiation (F_st). Bayesian and NJT (neighbor-joining tree) analyses demonstrated the presence of a population structure that was partly consistent with the geographic origins of the populations. The impact of population fragmentation on the genetic structure of EWC is to create a positive inbreeding coefficient, which was two to three times higher on average than that of a population from the continuous zone. This result indicated a higher occurrence of selfing within fragmented EWC populations coupled with a higher degree of gene exchange among near-neighbor relatives, thereby leading to significant inbreeding. Increased population isolation was apparently not correlated with a detectable effect on genetic diversity. Overall, the fragmented populations of EWC appear well-buffered against effects of inbreeding on genetic erosion.     

Cross‐species amplification of microsatellite loci in aphids: assessment and application

Despite the relative ease of isolating microsatellites, their development still requires substantial inputs of time, money and expertise. For this reason there is considerable interest in using existing microsatellites on species from which markers were not cloned. We tested cross‐species amplification of 48 existing aphid loci in species of the following genera: Aphidinae: Aphidini: Aphis and Rhopalosiphum; Aphidinae: Macrosiphini: Acyrthosiphum, Brevicoryne, Diuraphis, Illinoia, Macrosiphoniella, Macrosiphum, Metopeurum, Metapolophium, Myzus, Phorodon, Sitobion and Uroleucon and Neuquenaphidinae: Neuquenaphis. Our results show cross‐species application of known microsatellite loci is a highly promising source of codominant markers for population genetic and evolutionary studies in aphids.     

Development of microsatellite markers for an extremely limited distributed rare diving beetle species,Acilius kishii,and a widely distributed species,Acilius japonicus (Coleoptera: Dytiscidae)

Acilius kishii Nakane, 1963 (Coleoptera: Dytiscidae) is an endangered diving beetle species distributed in only one location, Lake Yashaga‐Ike, Honshu Island, Japan. Acilius japonicus, which is related to A. kishii, is distributed widely in northern Honshu Island and Hokkaido Island in Japan. In this study, we identified 14 microsatellite loci for A. kishii and A. japonicus, including both polymorphic and monomorphic loci, using the next‐generation sequencing method. We observed that 5 and 10 loci showed polymorphisms in 31 and 32 individuals of A. kishii and A. japonicus, respectively. The observed and expected heterozygosities were 0.00–1.00 and 0.00–0.74, respectively. These microsatellite loci could be useful for future conservation genetic studies, including monitoring of genetic diversity and extinction risk of A. kishii.     

The tangled link between β‐ and γ‐diversity: a Narcissus effect weakens statistical inferences in null model analyses of diversity patterns

Understanding the structure of and spatial variability in the species composition of ecological communities is at the heart of biogeography. In particular, there has been recent controversy about possible latitudinal trends in compositional heterogeneity across localities (β‐diversity). A gradient in the size of the regional species pool alone can be expected to impose a parallel gradient on β‐diversity, but whether β‐diversity also varies independently of the size of the species pool remains unclear. A recently suggested methodological approach to correct latitudinal β‐diversity gradients for the species pool effect is based on randomization null models that remove the effects of gradients in α‐ and γ‐diversity on β‐diversity. However, the randomization process imposes constraints on the variability of α‐diversity, which in turn force γ‐ and β‐diversity to become interdependent, such that any change in one is mirrored in the other. We argue that simple null model approaches are inadequate to discern whether correlations between α‐, β‐ and γ‐diversity reflect processes of ecological interest or merely differences in the size of the species pool among localities. We demonstrate that this kind of Narcissus effect may also apply to other metrics of spatial or phylogenetic species distribution. We highlight that Narcissus effects may lead to artificially high rejection rates for the focal pattern (Type II errors) and caution that these errors have not received sufficient attention in the ecological literature.     

Genetic diversity of Asian water buffalo (Bubalus bubalis): microsatellite variation and a comparison with protein-coding loci

Twenty-one microsatellite loci in 11 populations of Asian water buffalo (eight swamp, three river type) were analysed and, within and among populations, genetic variability was compared with results from 25 polymorphic protein-coding loci. Within-population mean heterozygosity ranged from 0·380–0·615, approximately twice that estimated from the protein-coding loci (0·184– 0·346). Only eight significant departures from Hardy–Weinberg equilibrium (involving four loci) were detected; global tests showed significant heterozygote deficiencies for these four loci. Non-amplifying alleles are likely to be segregating in some or all populations for one of these loci, and probably for the other three. There was significant differentiation between the swamp and river types of water buffalo, and among populations within each buffalo type. Estimates of θ (measure of population differentiation) for each locus for the eight swamp populations were all highly significant (mean θ = 0·168 ± 0·018). Mean θ for protein-coding loci was not significantly different (0·182 ± 0·041). The variance among protein-coding loci was significantly higher than among microsatellite loci, suggesting balancing selection affecting allele frequencies at some protein-coding loci. Genetic distances show clear separation of the swamp and river types, which were estimated to have diverged at least 10 000–15 000 years ago. The topology of the swamp populations’ microsatellite tree is consistent with their geographical distribution and their presumed spread through south-east Asia. By contrast, the tree based on the protein-coding loci distances is quite different, being clearly distorted by a bottleneck effect in one population, and possibly in at least two others. As many domestic livestock breeds are possibly descended from small numbers of founders, microsatellite-based trees are to be preferred in assessing breed genetic relationships.     

Isolation and characterization of highly polymorphic microsatellite loci in Schreibers’ long‐fingered bat,Miniopterus schreibersii (Chiroptera: Vespertilionidae)

Five polymorphic, dinucleotide microsatellite loci have been identified and characterized in Schreibers’ long‐fingered bat, Miniopterus schreibersii (Chiroptera: Vespertilionidae). These include three uninterrupted (CA)_n repeats, ranging in length from (CA)₁₇ to (CA)₁₉, one interrupted repeat (CA)₄(CG)₂(CA)₁₃, and one uninterrupted (GA)₂₇ repeat. All loci were highly polymorphic, with 17–20 alleles identified per locus. Observed heterozygosity levels (0.66–0.82) are lower than expected due to homozygote excess, probably caused by pooling of populations, resulting in population substructuring. All five polymorphic loci were also successfully amplified in the closely related M. fraterculus. Two pairs of primers additionally amplified polymorphic microsatellites in Chaerophon sp.     

Nuclear microsatellite and mitochondrial DNA analyses reveal the regional genetic structure and phylogeographical history of a sanguivorous land leech,Haemadipsa japonica,in Japan

Recent molecular studies have indicated that phylogeographical history of Japanese biota is likely shaped by geohistory along with biological events, such as distribution shifts, isolation, and divergence of populations. However, the genetic structure and phylogeographical history of terrestrial Annelida species, including leech species, are poorly understood. Therefore, we aimed to understand the genetic structure and phylogeographical history across the natural range of Haemadipsa japonica, a sanguivorous land leech species endemic to Japan, by using nine polymorphic nuclear microsatellites (nSSR) and cytochrome oxidase subunit one (COI) sequences of mitochondrial DNA (mtDNA). Analyses using nSSR revealed that H. japonica exhibited a stronger regional genetic differentiation among populations (G'_ST = 0.77) than other animal species, probably because of the low mobility of land leech. Analyses using mtDNA indicated that H. japonica exhibited two distinct lineages (A and B), which were estimated to have diverged in the middle Pleistocene and probably because of range fragmentation resulting from climatic change and glacial and interglacial cycles. Lineage A was widely distributed across Japan, and lineage B was found in southwestern Japan. Analyses using nSSR revealed that lineage A was roughly divided into two population groups (i.e., northeastern and southwestern Japan); these analyses also revealed a gradual decrease in genetic diversity with increasing latitude in lineage A and a strong genetic drift in populations of northeastern Japan. Combined with the largely unresolved shallow polytomies from the mtDNA phylogeny, these results implied that lineage A may have undergone a rapid northward migration, probably during the Holocene. Then, the regional genetic structure with local unique gene pools may have been formed within each lineage because of the low mobility of this leech species.