Analysis of microsatellite variation in Pinus radiata reveals effects of genetic drift but no recent bottlenecks

Most conifer species occur in large continuous populations, but radiata pine, Pinus radiata, occurs only in five disjunctive natural populations in California and Mexico. The Mexican island populations were presumably colonized from the mainland millions of years ago. According to Axelrod (1981), the mainland populations are relicts of an earlier much wider distribution, reduced some 8,000 years ago, whereas according to Millar (1997, 2000), the patchy metapopulation-like structure is typical of the long-term population demography of the species. We used 19 highly polymorphic microsatellite loci to describe population structure and to search for signs of the dynamics of population demography over space and time. Frequencies of null alleles at microsatellite loci were estimated using an approach based on the probability of identity by descent. Microsatellite genetic diversities were high in all populations [expected heterozygosity (H(e)) = 0.68-0.77], but the island populations had significantly lower estimates. Variation between loci in genetic differentiation (F(ST)) was high, but no locus deviated statistically significantly from the rest at an experiment wide level of 0.05. Thus, all loci were included in subsequent analysis. The average differentiation was measured as F(ST) = 0.14 (SD 0.012), comparable with earlier allozyme results. The island populations were more diverged from the other populations and from an inferred common ancestral gene pool than the mainland ones. All populations showed a deficiency of expected heterozygosity given the number of alleles, the mainland populations more so than the island ones. The results thus do not support a recent important contraction in the mainland range of radiata pine.     

Adult survival and microsatellite diversity in possums: effects of major histocompatibility complex-linked microsatellite diversity but not multilocus inbreeding estimators

Adult survival is perhaps the fitness parameter most important to population growth in long-lived species. Intrinsic and extrinsic covariates of survival are therefore likely to be important drivers of population dynamics. We used long-term mark-recapture data to identify genetic, individual and environmental covariates of local survival in a natural population of mountain brushtail possums (Trichosurus cunninghami). Rainfall and intra-individual diversity at microsatellite DNA markers were associated with increased local survival of adults and juveniles. We contrasted the performance of several microsatellite heterozygosity measures, including internal relatedness (IR), homozygosity by loci (HL) and the mean multilocus estimate of the squared difference in microsatellite allele sizes within an individual (mean d ²). However, the strongest effect on survival was not associated with multilocus microsatellite diversity (which would indicate a genome-wide inbreeding effect), but a subset of two loci. This included a major histocompatibility complex (MHC)-linked marker and a putatively neutral microsatellite locus. For both loci, diversity measures incorporating allele size information had stronger associations with survival than measures based on heterozygosity, whether or not allele frequency information was included (such as IR). Increased survival was apparent among heterozygotes at the MHC-linked locus, but the benefits of heterozygosity to survival were reduced in heterozygotes with larger differences in allele size. The effect of heterozygosity on fitness-related traits was supported by data on endoparasites in a subset of the individuals studied in this population. There was no apparent density dependence in survival, nor an effect of sex, age or immigrant status. Our findings suggest that in the apparent absence of inbreeding, variation at specific loci can generate strong associations between fitness and diversity at linked markers.     

Heterozygosity-fitness correlations in a bottlenecked island species: a case study on the Seychelles warbler

We used capture-mark-recapture models to investigate the effects of both individual and parental heterozygosity, measured at microsatellite loci on the survival of Seychelles warblers (Acrocephalus sechellensis), an endemic island species which went through a severe population bottleneck in the middle of the last century. We found that an individual's survival was not correlated with multilocus heterozygosity, or with heterozygosity at any specific locus. However, maternal, but not paternal, multilocus heterozygosity was positively associated with offspring survival, but only in years with low survival probabilities. A nestling cross-fostering experiment showed that this was a direct maternal effect as there was an effect of the genetic mother's, but not of the social mother's, heterozygosity. Heterozygosity-fitness correlations at microsatellite markers were generally assumed to reflect genome-wide effects. Although this might be true in partially inbred populations, such correlations may also arise as a result of local effects with specific markers being closely linked to genes which determine fitness. However, heterozygosity at the individual microsatellite loci was not correlated and therefore does not seem to reflect genome-wide heterozygosity. This suggests that even in a small bottlenecked population, heterozygosity-fitness correlations may not be caused by genome-wide effects. Support for the local effects hypothesis was also equivocal; although three specific loci were associated with offspring survival, including all single-locus heterozygosities as independent predictors for the variation in survival was not supported by the data. Furthermore, in contrast to the local effects hypothesis, the loci which contributed most to the heterozygosity-survival relationship were not more polymorphic than the other loci. This study highlights the difficulties in distinguishing between the two hypotheses.     

Genetic variation of microsatellite loci in a bottlenecked species: the northern hairy-nosed wombat Lasiorhinus krefftii

We investigate the utility of hypervariable microsatellite loci to measure genetic variability remaining in the northern hairy-nosed wombat, one of Australia's rarest mammals. This species suffered a dramatic range and population reduction over the past 120 years and now exists as a single colony of about 70 individuals at Epping Forest National Park, central Queensland. Because our preliminary research on mitochondrial DNA and multilocus DNA fingerprints did not reveal informative variation in this population, we chose to examine variation in microsatellite repeats, a class of loci known to be highly polymorphic in mammals. To assess the suitability of various wombat populations as a reference for comparisons of genetic variability and subdivision we further analysed mitochondrial DNA cytochrome b sequence, using phylogenetic methods. Our results show that appreciable levels of variation still exist in the Epping Forest colony although it has only 41% of the heterozygosity shown in a population of a closely-related species. From museum specimens collected in 1884, we also assessed microsatellite variation in an extinct population of the northern hairy-nosed wombat, from Deniliquin, New South Wales, 2000 km to the south of the extant population. The apparent loss of variation in the Epping Forest colony is consistent with an extremely small effective population size throughout its 120-year decline.     

A panel of polymorphic microsatellite markers in Himalayan monal Lophophorus impejanus developed by cross-species amplification and their applicability in other Galliformes

Isolation and development of new microsatellite markers for any species is still labour-intensive and requires substantial inputs of time, money and expertise. Therefore, cross-species microsatellite amplification can be an effective way in obtaining microsatellite loci for closely related taxa in bird species. We have reported microsatellite loci for Himalayan monal for the first time. Fifteen microsatellite markers developed for chicken were cross-amplified in Himalayan monal. All the tested 15 microsatellite markers were polymorphic, with mean (± s.e.) allelic number of 4 ± 1.51, ranging 2–7 per locus. The observed heterozygosity in the population ranged between 0.285 and 0.714, with mean (± s.e.) of 0.499 ± 0.125, indicating considerable genetic variation in this population. While 12 loci conformed to Hardy–Weinberg equilibrium (P > 0.05), 3 loci, i.e. MCW0295, MCW0081, MCW0330 deviated from it (P < 0.05). No evidence for linkage disequilibrium was observed among pair of loci. Our study show that these 15 microsatellites loci could be employed in population genetic studies for Himalayan monal and their applicability in Jungle Bush Quail, Grey francolin and Kalij pheasant.

     

Characterization of microsatellite loci for the Argentine ant,Linepithema humile,and their potential for analysis of colony structure in invading Hawaiian populations

We developed primers for five polymorphic microsatellite loci to analyse the genetic structure of colonies in an invading Argentine ant population located in Haleakala National Park on the island of Maui, Hawaii. Microsatellite loci were isolated using both a polymerase chain reaction‐based and a cloning‐based method. With a range of 3–18 alleles and expected levels of heterozygosity of 0.46–0.77, these loci provide useful markers for the detection of colony and population structure in new or expanding populations of this species.     

Characterization of 10 microsatellite loci in an avian louse, Degeeriella regalis (Phthiraptera: Ischnocera: Philopteridae)

We isolated and characterized 10 polymorphic microsatellite loci in an ischnoceran louse, Degeeriella regalis, which parasitizes the threatened Galápagos hawk (Buteo galapagoensis) and other falconiform birds. The loci were screened across 30 individuals from two island populations in the Galápagos Islands. The number of alleles per locus ranged from two to 28. Polymorphic information content ranged from 0.14 to 0.94 and observed heterozygosity ranged from 0 to 0.67. These markers will be valuable in comparative population genetics studies in this species, which is the focus of a long-term population and disease ecology research program.     

Characterization of polymorphic microsatellite loci for the Tawny Pipit Anthus campestris and their utility in other steppe birds

New microsatellite loci for Tawny Pipit Anthus campestris were isolated from a genomic library. We were able to unambiguously score six loci: two were dinucleotide, one trinucleotide, two tetranucleotide and one pentanucleotide that turned out to be sex-linked. Four out of six loci were polymorphic with 7–23 alleles in our population and an observed heterozygosity ranging between 0.286 and 0.936. Cross-utility of these markers was tested in other 17 steppe-bird species of six families. In addition, 16 microsatellite loci developed for other species were tested for cross-species amplification in A. campestris. Eight microsatellite markers were successfully amplified; seven of them were polymorphic with 2–43 alleles and an observed heterozygosity of 0.040–0.863. Overall, 14 functional locus markers have been characterized for A. campestris that could be useful for future studies of paternity, genetic variability and population structure.     

Characterization of microsatellite loci in fulvous fruit bat Rousettus leschenaulti

Rousettus leschenaulti is an abundant species in many countries of South‐East Asia, including south China. We isolated seven microsatellite loci in R. leschenaulti from genomic DNA enriched for CA repeats with the enriched library method. A total of 56 samples from a population in the Guangxi Province of China were tested with these microsatellite markers. The polymorphism ranged from seven to 16 alleles, and the observed heterozygosity was 84–94%. It is the first time microsatellite markers were characterized from R. leschenaulti, and these markers can be an important tool for analysing population structure and genotypic diversity.     

Heterozygosity-fitness correlations of conserved microsatellite markers in Kentish plovers Charadrius alexandrinus

Heterozygosity-fitness correlations (HFCs) are frequently used to examine the relationship between genetic diversity and fitness. Most studies have reported positive HFCs, although there is a strong bias towards investigating HFCs in genetically impoverished populations. We investigated HFCs in a large genetically diverse breeding population of Kentish plovers Charadrius alexandrinus in southern Turkey. This small shorebird exhibits highly variable mating and care systems, and it is becoming an ecological model species to understand breeding system evolution. Using 11 conserved and six anonymous microsatellite markers, we tested whether and how heterozygosity was associated with chick survival, tarsus and body mass growth controlling for nongenetic effects (chick sex, hatching date, length of biparental care and site quality) that influence survival and growth. There was no genome-wide effect of heterozygosity on fitness, and we did not find any significant effects of heterozygosity on growth rates. However, two of the 11 conserved markers displayed an association with offspring survival: one marker showed a positive HFC, whereas the other marker showed a negative HFC. Heterozygosity at three further conserved loci showed significant interaction with nongenetic variables. In contrast, heterozygosity based on anonymous microsatellite loci was not associated with fitness or growth. Markers that were correlated with chick survival were not more likely to be located in exons or introns than other markers that lacked this association.     

Characterization of six microsatellite loci in the African wild silk moth (Gonometa postica,Lasiocampidae)

Six microsatellite markers were developed for the African wild silk moth, Gonometa postica (Lasiocampidae), using an enrichment protocol. The total number of alleles ranged from three to 17 for a sample of 130 individuals across the distribution of the species. Observed levels of heterozygosity ranged from 0.17 to 0.78. Deviation from Hardy–Weinberg equilibrium detected in some loci is probably the result of large interannual population size fluctuations characteristic of this species. No evidence of linkage disequilibrium was detected among loci. These loci will be useful for the inference of demographic processes in a moth species that is of potential economic importance.     

Heterologous amplification of microsatellite markers from colubroid snakes in European natricines (serpentes: natricinae)

Eighteen microsatellite loci developed for a range of snake species (New World natricines, elapids, crotalids) were tested against European natricines (Natrix natrix, N. maura, and N. tessellata) in cross-species amplification experiments. Five loci were polymorphic (average expected heterozygosity 0.749 for a population of N. natrix in Amsterdam, mean sample size 47.8) and three loci were monomorphic. The remainder could not be consistently scored or failed to amplify. Further tests on single individuals of a diverse set of eight species of colubroid snakes showed that 15 of the 18 loci could be cross-amplified in at least one of these species. We conclude that our results show promise for the utilization of these markers for experimental assessments of genetic variation in the phylogenetically closely related group of European natricine snakes with emphasis on N. natrix. The full suite of microsatellite markers now available for snakes may show additional potential for subsequent investigation across a broader range of colubroid snakes.     

Cloning and characterization of microsatellite loci in channel catfish, Ictalurus punctatus

Short tandem repeat (microsatellite) loci were cloned from the channel catfish, Ictalurus punctatus , genome for use as molecular markers for genetic improvement of this important agricultural species. Plasmid clones containing catfish genomic DNA inserts were identified, by hybridization with tandem repeat DNA probes, and sequenced using automated laser fluorescence. A feral population of catfish displayed levels of heterozygosity greater than 0·7 for 13 of 22 loci and heterozygosity greater than 0·5 for 20 of 22 loci. Allelic polymorphism ranged from three to 17 alleles per locus in the feral population. Populations of domestic, farm-raised catfish and a research strain displayed levels of heterozygosity similar to the feral population. Non-invasive tissue sampling provided abundant material for the polymerase chain reaction-based genotype assay. The microsatellite loci will be useful in the molecular characterization and genetic improvement of channel catfish populations.     

Isolation and characterization of microsatellite loci in the finless porpoise (Neophocaena phocaenoides)

Eight dinucleotide microsatellite loci were isolated from the finless porpoise (Neophocaena phocaenoides). Analysis of 30 individuals showed the number of alleles ranged from four to 21 with observed heterozygosity ranging from 0.300 to 0.833, and expected heterozygosity ranging from 0.437 to 0.932. Cross‐species amplification was tested in four other cetacean species. These microsatellite markers would be valuable tools for population genetic studies of finless porpoises and other cetacean species.     

Power of microsatellite markers for fingerprinting and parentage analysis in Eucalyptus grandis breeding populations

We report the genetic analysis of 192 unrelated individuals of an elite breeding population of Eucalyptus grandis (Hill ex Maiden) with a selected set of six highly polymorphic microsatellite markers developed for species of the genus Eucalyptus. A full characterization of this set of six loci was carried out generating allele frequency distributions that were used to estimate parameters of genetic information content of these loci, including expected heterozygosity, polymorphism information content (PIC), power of exclusion, and probability of identity. The number of detected alleles per locus ranged from 6 to 33, with an average of 19.8 +/- 9.2. The average expected heterozygosity was 0.86 +/- 0.11 and the average PIC was 0.83 +/- 0.16. Using only three loci, it was possible to discriminate all 192 individuals. The overall probability of identity considering all six EMBRA microsatellite markers combined was lower than 1 in 2 billion. An analysis of the sample size necessary to estimate expected heterozygosity with minimum variance indicated that at least 64 individuals have to be genotyped to characterize this parameter with adequate accuracy for most microsatellites in Eucalyptus. The high degree of multiallelism and the clear and simple codominant Mendelian inheritance of the set of microsatellites used provide an extremely powerful system for the unique identification of Eucalyptus individuals for fingerprinting purposes and parentage testing.     

Characterization of novel microsatellite loci in the great leaf-nosed bat, Hipposideros armiger and cross-amplification in other related species

Eight microsatellite loci were isolated from an enriched genomic library of the great leaf-nosed bat, Hipposideros armiger. The polymorphism of these loci was tested on a population of 48 individuals from Anhui Province, China. All loci revealed the polymorphism ranging from three to 12 alleles. The observed heterozygosity values were from 0.213 to 0.875 and expected heterozygosity from 0.232 to 0.820. No significant linkage disequilibrium was detected. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. In addition, successful cross-amplification also suggested that these microsatellite markers will facilitate research on the population genetics and gene flow of H. armiger and other related species.     

Egg production and individual genetic diversity in lesser kestrels

Fecundity is an important component of individual fitness and has major consequences on population dynamics. Despite this, the influence of individual genetic variability on egg production traits is poorly known. Here, we use two microsatellite-based measures, homozygosity by loci and internal relatedness, to analyse the influence of female genotypic variation at 11 highly variable microsatellite loci on both clutch size and egg volume in a wild population of lesser kestrels (Falco naumanni). Genetic diversity was associated with clutch size, with more heterozygous females laying larger clutches, and this effect was statistically independent of other nongenetic variables such as female age and laying date, which were also associated with fecundity in this species. However, egg volume was not affected by female heterozygosity, confirming previous studies from pedigree-based breeding experiments which suggest that this trait is scarcely subjected to inbreeding depression. Finally, we explored whether the association between heterozygosity and clutch size was due to a genome-wide effect (general effect) or to single locus heterozygosity (local effect). Two loci showed a stronger influence but the correlation was not fully explained by these two loci alone, suggesting that a main general effect underlies the association observed. Overall, our results underscore the importance of individual genetic variation for egg production in wild bird populations, a fact that could have important implications for conservation research and provides insights into the study of clutch size evolution and genetic variability maintenance in natural populations.     

Isolation and characterization of DNA microsatellite from cotton bollworm (Helicoverpa armigera,Hübner)

Five microsatellite loci of Helicoverpa armigera were isolated from a partial genomic library screened by oligonucleotide probes. Primers were designed to detect allelic variability and heterozygosity in 60 individuals collected from different host species. All loci were found to be polymorphic, have 8–11 alleles with expected heterozygosity ranging from 0.81 to 0.88. Our results indicate that the five microsatellite loci could provide valuable markers for population genetic and ecological studies of the cotton bollworm.     

Founder events and variation at microsatellite loci in an insular passerine bird, the Laysan finch (Telespiza cantans)

Historically documented founder events provide opportunities to assess the effects of population size reductions on genetic variation, but the actual magnitude of genetic change can be measured only when direct comparisons can be made to the source or ancestral population. We assayed variation at nine microsatellite loci in the translocated population of the Laysan finch ( Telespiza cantans ) at Pearl and Hermes reef (PHR), and compared the level of variation to that in the source population on Laysan Island. Heterogeneity in allele frequencies was highly significant at eight of the nine loci, primarily as a result of fluctuations in allele frequencies in the three PHR populations. Intra- and interpopulational measures of genetic diversity generally matched predictions based on the well-documented history of three islet populations at PHR: significantly lower numbers of alleles and polymorphic loci, as well as higher pairwise F _ST values and genetic distance, were observed for the two populations that underwent severe size reductions. Changes in heterozygosity at single loci were unpredictable, as both significant increases and decreases were observed in founder populations. A significant excess of heterozygotes was found in two populations and was highly significant over all four finch populations ( P < 0.003). Estimates of effective population size from temporal changes in heterozygosity and allele frequencies were very small ( N _e≤ 30) as a result of the founding events and the constraints of islet area on population numbers. We concluded that the PHR population is not adequate as a secondary genetic reserve for T. cantans , and an alternative refuge needs to be established.     

Characterization of ten microsatellite marker loci in the Komodo Monitor (<Emphasis Type="Italic">Varanus komodoensis</Emphasis>)

The Komodo monitor (Varanus komodoensis) is a classic example of a species that has been fragmented into small isolated populations over a limited range. This species, classified as endangered under Appendix I of the Convention on the International Trade of Endangered Species. We describe 10 novel species-specific microsatellite loci characterized in representatives from three of the endemic island populations. One locus, 12HDZ820 appears to be fixed in one population at an allele size not found in the other two. This microsatellite suite should be helpful in augmenting the marker selection currently available for Komodo Monitor population studies.