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.     

Limited Phylogeographic Signal in Sex-Linked and Autosomal Loci Despite Geographically,Ecologically, and Phenotypically Concordant Structure of mtDNA Variation in the Holarctic Avian Genus Eremophila

Phylogeographic studies of Holarctic birds are challenging because they involve vast geographic scale, complex glacial history, extensive phenotypic variation, and heterogeneous taxonomic treatment across countries, all of which require large sample sizes. Knowledge about the quality of phylogeographic information provided by different loci is crucial for study design. We use sequences of one mtDNA gene, one sex-linked intron, and one autosomal intron to elucidate large scale phylogeographic patterns in the Holarctic lark genus Eremophila. The mtDNA ND2 gene identified six geographically, ecologically, and phenotypically concordant clades in the Palearctic that diverged in the Early - Middle Pleistocene and suggested paraphyly of the horned lark (E. alpestris) with respect to the Temminck''s lark (E. bilopha). In the Nearctic, ND2 identified five subclades which diverged in the Late Pleistocene. They overlapped geographically and were not concordant phenotypically or ecologically. Nuclear alleles provided little information on geographic structuring of genetic variation in horned larks beyond supporting the monophyly of Eremophila and paraphyly of the horned lark. Multilocus species trees based on two nuclear or all three loci provided poor support for haplogroups identified by mtDNA. The node ages calculated using mtDNA were consistent with the available paleontological data, whereas individual nuclear loci and multilocus species trees appeared to underestimate node ages. We argue that mtDNA is capable of discovering independent evolutionary units within avian taxa and can provide a reasonable phylogeographic hypothesis when geographic scale, geologic history, and phenotypic variation in the study system are too complex for proposing reasonable a priori hypotheses required for multilocus methods. Finally, we suggest splitting the currently recognized horned lark into five Palearctic and one Nearctic species.     

Genetic differentiation of theErigeron species (Asteraceae) in the Alps: A case of unusual allozymic uniformity

Allozyme variation was studied in all nine diploidErigeron species known from the Alps:E. alpinus, E. neglectus, E. polymorphus, E. candidus, E. uniflorus, E. atticus, E. gaudinii, E. acer, andE. angulosus. A total of 248 individuals from 24 natural populations was investigated using starch gel electrophoresis. Seven enzymes and 13 loci were assessed. Genetic variation within populations was low with the proportion of polymorphic loci ranging from 0.0–0.385, and average number of alleles per polymorphic locus from 2.0–2.5. In general, 70–100% of the genetic variation was attributed to between population differences. Mean genetic identities for pair-wise comparisons of populations averaged 0.893 within species, and 0.890 among species. Interspecific genetic variation of populations usually did not exceed intraspecific variation. It was concluded that theErigeron species from the Alps may have arisen by recent speciation probably during the epoches of glaciation. Morphological and ecological differences between species seem to be based on few gene loci.     

Isolation and characterization of di‐ and tetranucleotide microsatellite loci in the yellow‐spotted night lizard Lepidophyma flavimaculatum (Squamata: Xantusiidae)

We report the development of 17 di‐ and tetranucleotide microsatellite markers in the Yellow‐Spotted Night Lizard Lepidophyma flavimaculatum. Levels of heterozygosity ranged from 0 to 100%. Several loci also amplify in other Lepidophyma species and several species of the sister genus Xantusia. High levels of variation at some loci indicate that they will be useful for parentage assessment and population genetic studies, whereas the 15 loci that amplify across multiple Lepidophyma species suggest these will be useful in determining the origin of parthenogenetic populations.     

No downregulation of immune function during breeding in two year‐round breeding bird species in an equatorial East African environment

Some equatorial environments exhibit substantial within‐location variation in environmental conditions throughout the year and yet have year‐round breeding birds. This implies that breeding in such systems are potentially unrelated to the variable environmental conditions. By breeding not being influenced by environmental conditions, we become sure that any differences in immune function between breeding and non‐breeding birds do not result from environmental variation, therefore allowing for exclusion of the confounding effect of variation in environmental conditions. This create a unique opportunity to test if immune function is down‐regulated during reproduction compared to non‐breeding periods. We compared the immune function of sympatric male and female chick‐feeding and non‐breeding red‐capped Calandrella cinerea and rufous‐naped larks Mirafra africana in equatorial East Africa. These closely‐related species occupy different niches and have different breeding strategies in the same grassland habitat. Red‐capped larks prefer areas with short grass or almost bare ground, and breed during low rainfall periods. Rufous‐naped larks prefer areas of tall grass and scattered shrubs and breed during high rainfall. We measured the following immune indices: nitric oxide, haptoglobin, agglutination and lysis, and measured total monthly rain, monthly average minimum (T_min) and maximum (T_max) temperatures. Contrary to our predictions, we found no down‐regulation of immune function during breeding; breeding birds had higher nitric oxide than non‐breeding ones in both species, while the other three immune indices did not differ between breeding phases. Red‐capped larks had higher nitric oxide concentrations than Rufous‐naped larks, which in turn had higher haptoglobin levels than red‐capped larks. T_max was higher during breeding than during non‐breeding for red‐capped larks only, suggesting potential confounding effect of T_max on the comparison of immune function between breeding and non‐breeding birds for this species. Overall, we conclude that in the two year‐round breeding equatorial larks, immune function is not down‐regulated during breeding.     

Reduced variation on the chicken Z chromosome

Understanding the population genetic factors that shape genome variability is pivotal to the design and interpretation of studies using large-scale polymorphism data. We analyzed patterns of polymorphism and divergence at Z-linked and autosomal loci in the domestic chicken (Gallus gallus) to study the influence of mutation, effective population size, selection, and demography on levels of genetic diversity. A total of 14 autosomal introns (8316 bp) and 13 Z-linked introns (6856 bp) were sequenced in 50 chicken chromosomes from 10 highly divergent breeds. Genetic variation was significantly lower at Z-linked than at autosomal loci, with one segregating site every 39 bp at autosomal loci (theta(W) = 5.8 +/- 0.8 x 10(-3)) and one every 156 bp on the Z chromosome (theta(W) = 1.4 +/- 0.4 x 10(-3)). This difference may in part be due to a low male effective population size arising from skewed reproductive success among males, evident both in the wild ancestor-the red jungle fowl-and in poultry breeding. However, this effect cannot entirely explain the observed three- to fourfold reduction in Z chromosome diversity. Selection, in particular selective sweeps, may therefore have had an impact on reducing variation on the Z chromosome, a hypothesis supported by the observation of heterogeneity in diversity levels among loci on the Z chromosome and the lower recombination rate on Z than on autosomes. Selection on sex-linked genes may be particularly important in organisms with female heterogamety since the heritability of sex-linked sexually antagonistic alleles advantageous to males is improved when fathers pass a Z chromosome to their sons.     

Genetic rescue of an inbred captive population of the critically endangered Puerto Rican crested toad (<Emphasis Type="Italic">Peltophryne lemur</Emphasis>) by mixing lineages

The Puerto Rican crested toad (Peltophryne lemur) is currently composed of a single wild population on the south coast of Puerto Rico and two captive populations founded by animals from the northern and southern coasts. The main factors contributing to its decline are habitat loss, inundation of breeding ponds during storms, and impacts of invasive species. Recovery efforts have been extensive, involving captive breeding and reintroductions, habitat restoration, construction of breeding ponds, and public education. To guide future conservation efforts, genetic variation and differentiation were assessed for the two captive colonies and the remaining wild population using the mitochondrial control region and six novel microsatellite loci. Only two moderately divergent mitochondrial haplotypes were found, with one fixed in each of the southern and northern lineages. Moderate genetic variation exists for microsatellite loci in all three groups. The captive southern population has not diverged substantially from the wild population at microsatellite loci (F _ST = 0.03), whereas there is little allelic overlap between the northern and southern lineages at five of six loci (F _ST > 0.3). Despite this differentiation, they are no more divergent than many populations of other amphibian species. As the northern breeding colony may not remain viable due to its small size and inbred nature, it is recommended that a third breeding colony be established in which northern and southern individuals are combined. This will preserve any northern adaptive traits that may exist, and provide animals for release in the event that the pure northern lineage becomes extirpated.     

Genetic structure of an isolated population of mantled howler monkeys (Alouatta palliata) on Barro Colorado Island, Panama

Habitat fragmentation may influence genetic variability through reductions in population size and the physical isolation of conspecifics. We explored the effects of these factors on genetic diversity in a population of mantled howler monkeys (Alouatta palliata) on Barro Colorado Island (BCI), Panama. The study population was established in 1914 when an unknown number of resident individuals were isolated from the surrounding mainland by damming of the Río Chagres to create the Panama Canal. Analyses of 10 microsatellite loci indicated that, despite this isolation, the howler monkeys on BCI exhibit levels of genetic diversity (H _S = 0.584 ± 0.063) among the highest reported for any species of Alouatta. These data also revealed that although relatedness among adults in a social group was significantly greater than zero, the BCI population is not highly genetically structured. Tests for genetic bottlenecks based on departures from equilibrium expectations failed to reveal evidence of a recent reduction in population size. In contrast, coalescent modeling indicated that this population has likely experienced a marked decline within the last few 100 years. These findings generate new insights into the genetic structure of A. palliata and suggest that while the formation of BCI may not have substantially reduced genetic variation in these animals, genetic diversity has been influenced by historical changes in population size.     

Microsatellite allele sequencing in population analyses of the South American cactophilic species Drosophila antonietae (Diptera: Drosophilidae)

Drosophila antonietae belongs to the Drosophila buzzatii cluster, a cactophilic group of species naturally endemic to South America. Morphological and genetic analyses indicate that its populations are the most homogenous in the cluster and that the diversity observed is mainly a result of variation within populations. Seven polymorphic microsatellite loci were described for this species and used in the present study to investigate the genetic diversity of natural populations of D. antonietae by both length and sequence variation. The study aimed to understand how homoplasy and null alleles affect inferences about the population history of this species and to obtain an accurate interpretation of population inferences where these loci could be applied. The results provide useful information on the interpretation of genetic data derived from the microsatellite loci described for D. antonietae and on evolutionary aspects of cactophilic Drosophila. Importantly, the results indicate that size homoplasy and null alleles do not represent significant problems for the population genetics analyses because the large amount of variability at microsatellite loci compensate the low frequency of these problems in the populations. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 573–584.     

Isolation and characterization of new microsatellite markers in the surfclam Mactromeris polynyma

We describe primers and amplification conditions for seven microsatellite loci that were developed and characterized in the commercially harvested surfclam Mactromeris polynyma. Five of these loci were polymorphic: we found 6 to 23 alleles per locus among 100 individuals from one population in Nova Scotia, with some large heterozygote deficits that may reflect unrecognized temporal genetic variation. We are using these markers to investigate temporal and spatial genetic structure in this species.     

Assessing natural introgression in 2 biomedical model species, the rhesus macaque (Macaca mulatta) and the long-tailed macaque (Macaca fascicularis)

Rhesus macaque (Macaca mulatta) and long-tailed macaque (Macaca fascicularis) are the 2 most commonly used primate model species in biomedical sciences. Although morphological studies have revealed a weak hybridization at the interspecific contact zone, in the north of Indochina, a molecular study has suggested an ancient introgression from rhesus to long-tailed macaque into the Indo-Chinese peninsula. However, the gene flow between these 2 taxa has never been quantified using genetic data and theoretical models. In this study, we have examined genetic variation within and between the parapatric Chinese rhesus macaque and Indo-Chinese long-tailed macaque populations, using 13 autosomal, 5 sex-linked microsatellite loci and mitochondrial DNA sequence data. From these data, we assessed genetic structure and estimated gene flow using a Bayesian clustering approach and the "Isolation with Migration" model. Our results reveal a weak interspecific genetic differentiation at both autosomal and sex-linked loci, suggesting large population sizes and/or gene flow between populations. According to the Bayesian clustering, Chinese rhesus macaque is a highly homogeneous gene pool that contributes strongly to the current Indo-Chinese long-tailed macaque genetic makeup, whether or not current admixture is assumed. Coalescent simulations, which integrated the characteristics of the loci, pointed out 1) a higher effective population size in rhesus macaque, 2) no mitochondrial gene flow, and 3) unilateral and male-mediated nuclear gene flow of approximately 10 migrants per generation from rhesus to long-tailed macaque. These patterns of genetic structure and gene flow suggest extensive ancient introgression from Chinese rhesus macaque into the Indo-Chinese long-tailed macaque population.     

Antagonistic pleiotropy may help population-level selection in maintaining genetic polymorphism for transmission rate in a model phytopathogenic fungus

It has been shown theoretically that the conditions for the maintenance of polymorphism at pleiotropic loci with antagonistic effects on fitness components are rather restrictive. Here, we use a metapopulation model to investigate whether antagonistic pleiotropy could help maintain polymorphism involving common deleterious alleles in the phytopathogenic fungus Microbotryum violaceum. This fungus causes anther smut disease of the Caryophyllaceae. A previous model has shown that the sex-linked deleterious alleles can be maintained under a metapopulation structure, when intra-tetrad selfing (mating between products of the same meiosis) is high, due to founder effects and selection at the population level. Here, we add two types of pleiotropic advantages to the metapopulation model. A competitive advantage for strains carrying the sex-linked deleterious alleles did not facilitate their maintenance because competitive situations were too rare. In contrast, higher spore production did facilitate the maintenance of the deleterious alleles at low intra-tetrad mating rates and with a large advantage for spore production. These results show that antagonistic pleiotropy may promote the persistence of genetic variation, in combination with other selective forces.     

GATA tandem repeats detect minisatellite regions in blowfly DNA (Diptera: Calliphoridae)

A DNA probe containing GATA tandem repeats detected numerous dispersed minisatellite regions in the genomes of the blowflies Chrysomya rufifacies and Calliphora erythrocephala. These regions seemed to be actively transcribed into poly(A)⁺ RNA in a tissue-specific manner. When genomic DNA of blastoderm embryos was compared with adult genomic DNA some loci hybridizing to GATA displayed a marked stage-specific variation in length. In Calliphora, a small sex-linked dimorphism of GATA minisatellite associated restriction fragments was observed.     

Mimicry as a novel pathway linking biodiversity functions and individual behavioural performances

The feedback of biodiversity on individual trait variation is a poorly explored mechanistic pathway in ecological research. We analysed the relationship between biodiversity and individual performance by focusing on vocal mimicry, a widespread interaction that may serve in intra- and interspecific communication. We studied the songs of two lark species (genus Galerida) that increase the complexity of their song displays by mimicking other birds, and analysed the influence of bird species richness on individual song performance. The diversity of mimicked species and the prevalence of mimicry increased in areas characterized by great α and γ diversity (i.e. where larks experience more diverse encounters with community members, many of them being highly vocal owing to breeding). Conversely, the variability in species-specific song components peaked where larks were abundant, probably matching the complexity of conspecific social milieu. Some trade-offs existed between homo- and heterospecific complexity, suggesting that larks could change from population- to community-driven song variation by tracking the composition of the auditory environment. Mimicry, which serves communication with conspecifics or predators, may mediate interactions, ultimately cascading to aspects of ecological diversity other than those promoting its complexity.     

PERMANENT GENETIC RESOURCES: Isolation, characterization and multiplex genotyping of 11 autosomal and four sex-linked microsatellite loci in the reed bunting, Emberiza schoeniclus (Emberizidae, Aves)

Fifteen highly polymorphic microsatellite loci were characterized in the reed bunting, Emberiza schoeniclus. Eleven loci were autosomal and four linked to the Z chromosome. All loci were characterized and tested in 45 unrelated reed buntings from a Swiss population. Autosomal loci displayed seven to 17 and sex-linked loci displayed four to 13 alleles with heterozygosities ranging from 0.756 to 0.933 and from 0.478 to 0.957, respectively. These loci will be used in population genetic and mating system studies of reed buntings.     

GENETIC DIVERSITY AND POPULATION STRUCTURE IN CAMELLIA JAPONICA L. (THEACEAE)

Camellia japonica is a widespread and morphologically diverse tree native to parts of Japan and adjacent islands. Starch gel electrophoresis was used to score allelic variation at 20 loci in seeds collected from 60 populations distributed throughout the species range. In comparison with other plant species, the level of genetic diversity within C. japonica populations is very high: 66.2% of loci were polymorphic on average per population, with a mean number of 2.16 alleles per locus; the mean observed and panmictic heterozygosities were 0.230 and 0.265, respectively. Genotypic proportions at most loci in most populations fit Hardy-Weinberg expectations. However, small heterozygote deficiencies were commonly observed (mean population fixation index = 0.129). It is suggested that the most likely cause of the observed deficiencies is population subdivision into genetically divergent subpopulations. The overall level of population differentiation is greater than is typically observed in out-breeders: The mean genetic distance and identity (Nei's D and I) between pairs of populations were 0.073 and 0.930, respectively, and Wright's Fst was 0.144. Differences among populations appeared to be manifested as variation in gene frequencies at many loci rather than variation in allelic composition per se. However, the patterns of variation were not random. Reciprocal clinal variation of gene frequencies was observed for allele pairs at six loci. In addition, principal components analysis revealed that populations tended to genetically cluster into four regions representing the geographic areas Kyushu, Shikoku, western Honshu, and eastern Honshu. There was a significant relationship between genetic and geographic distance (r = 0.61; P < 0.01). Analysis of variance on allozyme frequencies showed that there was approximately four times as much differentiation among populations within regions, as among regions. It is likely that the observed patterns of population relationships result from the balance between genetic drift in small subpopulations and gene flow between them.     

Are habitat fragmentation,local adaptation and isolation‐by‐distance driving population divergence in wild rice Oryza rufipogon?

Habitat fragmentation weakens the connection between populations and is accompanied with isolation by distance (IBD) and local adaptation (isolation by adaptation, IBA), both leading to genetic divergence between populations. To understand the evolutionary potential of a population and to formulate proper conservation strategies, information on the roles of IBD and IBA in driving population divergence is critical. The putative ancestor of Asian cultivated rice (Oryza sativa) is endangered in China due to habitat loss and fragmentation. We investigated the genetic variation in 11 Chinese Oryza rufipogon populations using 79 microsatellite loci to infer the effects of habitat fragmentation, IBD and IBA on genetic structure. Historical and current gene flows were found to be rare (m_h = 0.0002–0.0013, m_c = 0.007–0.029), indicating IBD and resulting in a high level of population divergence (F_ST = 0.343). High within‐population genetic variation (H_E = 0.377–0.515), relatively large effective population sizes (N_e = 96–158), absence of bottlenecks and limited gene flow were found, demonstrating little impact of recent habitat fragmentation on these populations. Eleven gene‐linked microsatellite loci were identified as outliers, indicating local adaptation. Hierarchical AMOVA and partial Mantel tests indicated that population divergence of Chinese O. rufipogon was significantly correlated with environmental factors, especially habitat temperature. Common garden trials detected a significant adaptive population divergence associated with latitude. Collectively, these findings imply that IBD due to historical rather than recent fragmentation, followed by local adaptation, has driven population divergence in O. rufipogon.     

Drift,selection and adaptive variation in small populations of a threatened rattlesnake

An important goal of conservation genetics is to determine if the viability of small populations is reduced by a loss of adaptive variation due to genetic drift. Here, we assessed the impact of drift and selection on direct measures of adaptive variation (toxin loci encoding venom proteins) in the eastern massasauga rattlesnake (Sistrurus catenatus), a threatened reptile that exists in small isolated populations. We estimated levels of individual polymorphism in 46 toxin loci and 1,467 control loci across 12 populations of this species, and compared the results with patterns of selection on the same loci following speciation of S. catenatus and its closest relative, the western massasauga (S. tergeminus). Multiple lines of evidence suggest that both drift and selection have had observable impacts on standing adaptive variation. In support of drift effects, we found little evidence for selection on toxin variation within populations and a significant positive relationship between current levels of adaptive variation and long‐ and short‐term estimates of effective population size. However, we also observed levels of directional selection on toxin loci among populations that are broadly similar to patterns predicted from interspecific selection analyses that pre‐date the effects of recent drift, and that functional variation in these loci persists despite small short‐term effective sizes. This suggests that much of the adaptive variation present in populations may represent an example of “drift debt,” a nonequilibrium state where present‐day levels of variation overestimate the amount of functional genetic diversity present in future populations.     

Microsatellite polymorphism and genetic differentiation in three Norwegian populations of Elymus alaskanus (Poaceae)

 Variation at seven microsatellite loci was investigated in three local E. alaskanus populations from Norway and microsatellite variation was compared with allozyme variation. The percentage of polymorphic loci was 81%, the mean number of alleles per polymorphic locus was 5.7 and expected heterozygosity was 0.37. An F-statistic analysis revealed an overall 48% deficit of heterozygotes over Hardy-Weinberg expectations. Gene diversity is mainly explained by the within population component. The averaged between population differentiation coefficient, F _st , over 7 loci is only 0.13, which accounts for only 13% of the whole diversity and was contrary to allozyme analysis. The mean genetic distance between populations was 0.12. However, a χ² -test showed that allele frequencies were different (p < 0.05) among the populations at 5 of the 7 loci. In comparison with the genetic variation detected by allozymes, microsatellite loci showed higher levels of genetic variation. Microsatellite analysis revealed that population H10576 possesses the lowest genetic variation among the tested three populations, which concur with allozyme analysis. The dendrogram generated by microsatellites agreed very well with allozymic data. Our results suggest that natural selection may be an important factor in shaping the genetic diversity in these three local E. alaskanus populations. Possible explanations for deficit heterozygosity and incongruence between microsatellites and allozymes are discussed. Received November 6, 2001; accepted April 24, 2002 Published online: November 14, 2002 Addresses of the authors: Genlou Sun (e-mail: Genlou.sun@STMARYS.CA), Biology Department, Saint Mary's University, Halifax. Nova Scotia, B3H 3C3, Canada. B. Salomon, R. von Bothmer, Department of Crop Science, The Swedish University of Agricultural Sciences, P.O. Box 44, SE-230 53, Alnarp, Sweden.     

Survival probabilities for some multitype branching processes in genetics

Consider a positively regular, slightly supercritical branching process with K types. An approximation to the probability of survival of a line descended from a single individual of type i has recently been derived by Hoppe. If K is large, however, this approximation may not be easy to compute. A further approximation that is easily computable is given. The result is used to estimate probabilities of survival of an allele A that is originally present in one male or one female in a large, random mating, age-structured population. Both autosomal and sex-linked loci are considered. Another application of the approximation is also discussed.Journal paper no. J-13183 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, project 2588