Performance of native grape phylloxera on host plants within and among terrestrial islands in Arizona, USA

In Arizona, USA, the canyon grape, Vitis arizonica Englemann, and grape phylloxera (Daktulosphaira vitifoliae Fitch, Homoptera, Phylloxeridae) are distributed among mountain ranges that are surrounded by expanses of desert lacking Vitis habitat, thus forming a system of terrestrial islands. Both herbivore and host populations may have diverged genetically among mountain ranges under the influence of restricted gene flow and variable selection among sites. Herbivore adaptation to local hosts would be expected to ensue, with the potential to promote divergence, both in traits under selection and by further reducing the probability of interisland colonization. To test the hypothesis that phylloxera are adapted to local hosts, demographic components of fitness of field-collected native grape phylloxera were measured in the greenhouse on vines of V. arizonica that were categorized as either natal, neighboring, and or isolated hosts. There was no evidence for greater adaptation to natal or neighboring hosts but there were significant interactions between herbivore and host treatments in one experiment. There was genetic variation for gall formation among six clones tested. Though a failure to detect local adaptation could have resulted from low statistical power, benign experimental conditions, or inadequate genetic variation, the divergence of isolated grape populations is suggested to have been insufficient to promote local adaptation in grape phylloxera at the level of isolated mountain ranges. It is further suggested that, within populations, adaptation to individual host plants could be forestalled by selection for ’general purpose genotypes’ through wind-borne displacement of colonizers into the unpredictable environment of a heterogeneous array of hosts. In addition, short-term extinction/colonization dynamics could promote gene flow such that time is insufficient for adaptive mutations or gene combination to arise. Received: 26 December 1998 / Accepted: 24 May 1999     

Metarhizium anisopliae, a potential agent for the control of grape phylloxera

This investigation deals with the control effects of the insect pathogenic fungus Metarhizium anisopliae var. anisopliae on Daktulosphaira vitifoliae. In pot experiments, the soil surrounding phylloxera-infected grapes was inoculated with barley colonised with M. anisopliae. After thirty-two days, ineight of ten Metarhizium-applied pots nofresh phylloxera infections could be observed.In two of ten plants, a few fresh nodositiessingly occupied with phylloxera or phylloxeraeggs could be found. In all untreated plants,fresh nodosities with either single (two of sixplants) or multiple (four of six plants)occupation with phylloxera could be observed.M. anisopliae could be re-isolated in aconcentration of <1 × 10³ CFU g^$minus;1 soil dry weight from those pots with phylloxera-infected plants that had been treated with the fungus. The potential role of M. anisopliae in grape phylloxera management is discussed.     

Phylogeography in a galling insect, grape phylloxera, Daktulosphaira vitifoliae (Phylloxeridae) in the fragmented habitat of the Southwest USA

Aim The Southwest USA is characterized by fragmented habitat for a range of species in a manner analogous to island systems. This can restrict gene flow among populations and may promote genetic differentiation and the evolution of endemic populations or species among the forested terrestrial islands. The region has experienced cycles of cooling and warming throughout the Pleistocene and the current isolation among populations may date back only c. 11,000 years. Thus, some studies have found genetic structure that predates the current distribution of organisms. The effect of habitat fragmentation on genetic structure was tested using DNA sequences in a highly specialized herbivorous insect, grape phylloxera, Daktulosphaira vitifoliae, that exhibits poor dispersal capacity and natal‐host associated mating that would seem to predispose it to genetic differentiation. Location Southwestern USA. Methods Phylogenetic trees and statistical parsimony networks were estimated from 458 bp of the mitochondrial cytochrome oxidase I gene sampled from 12 populations in Arizona and New Mexico. Results There was no evidence that populations were differentiated at the level of canyon or mountain range but significant structure was found at the larger level of geographic region. The pattern was confounded by the polyphyly of Southwest (SW) grape phylloxera relative to eastern populations and the coexistence of two highly divergent lineages within populations. Main conclusions An early split of grape phylloxera into the SW has resulted in some differentiation between populations in the Transition and Sonoran + Chihuahuan geographic regions. It is hypothesized that this differentiation has been overlaid by a more recent introduction of haplotypes from midwestern USA populations that occur primarily in the Transition region. The importance of sampling broadly is stressed.     

Comparative phylogeography of the two pink salmon broodlines: an analysis based on a mitochondrial DNA genealogy

Over most of their natural northern Pacific Ocean range, pink salmon (Oncorhynchus gorbuscha) spawn in a habitat that was repeatedly and profoundly affected by Pleistocene glacial advances. A strictly two-year life cycle of pink salmon has resulted in two reproductively isolated broodlines, which spawn in alternating years and evolved as temporal replicates of the same species. To study the influence of historical events on phylogeographical and population genetic structure of the two broodlines, we first reconstructed a fine-scale mtDNA haplotype genealogy from a sample of 80 individuals and then determined the geographical distribution of the major genealogical assemblages for 718 individuals sampled from nine Alaskan and eastern Asian even- and nine odd-year pink salmon populations. Analysis of restriction site states in seven polymerase chain reaction (PCR)-amplified mtDNA regions (comprising 97% of the mitochondrial genome) using 13 endonucleases resolved 38 haplotypes, which clustered into five genealogical lineages that differed from 0.065 to 0.225% in net sequence divergence. The lineage sorting between broodlines was incomplete, which suggests a recent common ancestry. Within each lineage, haplotypes exhibited star-like genealogies indicating recent population growth. The depth of the haplotype genealogy is shallow ( approximately 0.5% of nucleotide sequence divergence) and probably reflects repeated decreases in population size due to Pleistocene glacial advances. Nested clade analysis (NCA) of geographical distances showed that the geographical distribution observed for mitochondrial DNA (mtDNA) haplotypes resulted from alternating influences of historical range expansions and episodes of restricted dispersal. Analyses of molecular variance showed weak geographical structuring of mtDNA variation, except for the strong subdivision between Asian and Alaskan populations within the even-year broodline. The genetic similarities observed among and within geographical regions probably originated from postglacial recolonizations from common sources rather than extensive gene flow. The phylogeographical and population genetic structures differ substantally between broodlines. This can be explained by stochastic lineage sorting in glacial refugia and perhaps different recolonization routes in even- and odd-year broodlines.     

Founder contribution and pedigree inference in a captive breeding colony of lion-tailed macaques,using mitochondrial DNA and DNA fingerprint analyses

Captive colony genetic management is dependent on knowledge of the colony pedigree. In most zoos, records for colonies held prior to the past decade are incomplete or nonexistent. We have used a nested set of molecular techniques to infer genetic relationships in a colony of 15 lion-tailed macaques after twenty years of unmonitored reproduction. The troop was first divided into lines of matrilineal descent using mitochondrial DNA RFLP patterns. Paternity and maternity were then assigned on the basis of DNA fingerprint analysis. As mtDNA is maternally inherited, it is of no use in paternity assessment, but enabled subdivision of the colony into maternal lines. DNA fingerprinting would have been of little use by itself because of high levels of relatedness in several generations of offspring within the colony. The combination of the two techniques, however, enabled strong inference of both paternity and maternity in all cases. These data were instrumental in the inclusion of this troop in the AAZPA Species Survival Plan.     

Population genetics of Scirtothrips perseae: tracing the origin of a recently introduced exotic pest of Californian avocado orchards, using mitochondrial and microsatellite DNA markers

The Californian avocado industry has recently been impacted by the establishment of three exotic arthropod pests that are native to Mexico and Central America. Establishment of all three pests is thought to have resulted from illegal movement of host plants (avocado) into California. To identify likely sources and routes of entry of such pest invasions, we examined the population genetics of the most recent invader, avocado thrips [Scirtothrips perseae Nakahara (Thysanoptera: Thripidae)], using mitochondrial DNA (mtDNA) and microsatellite markers. The mtDNA sequences revealed three geographically distinct and divergent lineages, of which the mtDNA haplotypes of Californian individuals were most closely related to populations in the center of the pest's native range. Analysis of allele frequencies at four microsatellite loci added resolution, indicating Coatepec Harinas, Mexico, as the most likely source of the Californian population. Statistically, we did not detect any bottleneck in population size associated with the invasion of California. However, estimates of the effective population size of the invading population suggest that a severe bottleneck occurred, indicating that the quantity of host‐plant material entering California was small. Our findings implicate Coatepec Harinas, a large avocado germplasm and breeding center, as the most likely source of the introduced Californian population of S. perseae and as a likely source of previous and future avocado pest introductions. Efforts to identify natural enemies of S. perseae for biological control should focus on Coatepec Harinas and immediate surrounds. Moreover, identification of the source of invasive pests enables the establishment and enforcement of plant quarantine and free‐trade protocols.     

Pseudogenization of the Humanin gene is common in the mitochondrial DNA of many vertebrates

In the human the peptide Humanin is produced from the small Humanin gene which is embedded as a gene-within-a-gene in the 16S ribosomal molecule of the mitochondrial DNA (mtDNA).The peptide itself appears to be significant in the prevention of cell death in many tissues and improve cognition in animal models.By using simple data mining techniques,it is possible to show that 99.4％ of the human Humanin sequences in the GenBank database are unaffected by mutations.However,in other vertebrates,pseudogenization of the Humanin gene is a common feature;occurring apparently randomly in some species and not others.The persistence,or loss,of a functional Humanin gene may be an important factor in laboratory animals,especially if they are being used as animal models in studies of Alzheimer's disease (AD).The exact reason why Humanin underwent pseudogenization in some vertebrate species during their evolution remains to be determined.This study was originally planned to review the available information about Humanin and it was a surprise to be able to show that pseudogenization has occurred in a gene in the mtDNA and is not restricted solely to chromosomal genes.     

Intraspecific phylogeography of the Cape galaxias from South Africa: evidence from mitochondrial DNA sequences

Mitochondrial cytochrome b gene sequences were obtained from representatives of five populations of the Cape galaxias Galaxias zebratus . Four highly distinct genotypes were revealed, with sequence divergence values ranging from 5.8 to 13.8%. Some of the pairwise divergences are the highest yet reported for cytochrome b within a fish species, and are more typical of interspecific and intergeneric comparisons. In contrast, the low genetic divergence (0.3%) between two recently recorded populations from the Krom and Gamtoos (Kouga) Rivers indicates recent gene flow. Parsimony analysis consistently produced a tree with the population from the Olifants River on the west coast as ancestral and eastern populations as progressively more derived. Estimated divergence times for the Olifants population range from 4.4 to 6.6 million years ago. The findings suggest that another detailed revision is required to determine whether these divergent populations represent a single species or a species complex.     

Mitochondrial DNA genealogy of chimpanzees in the Nimba mountains and Bossou, West Africa

The chimpanzee populations of the Bossou and Nimba regions in West Africa were genetically surveyed to 1) reveal the genetic relationship between the Bossou and Nimba populations, and 2) elucidate the evolutionary relationship between the Bossou-Nimba and other West African populations. The chimpanzee group at Bossou is characterized by its small population size, no evidence of contact with neighboring populations, and no female immigration. It is believed that most females and adolescent males emigrate from this population. To reveal the genetic signature of these characteristics, we examined the genetic diversity of Bossou and two neighboring populations (Seringbara and Yealé) in the Nimba Mountains by sequencing approximately 605 bp of the mitochondrial DNA (mtDNA) control region. A total of 20 distinct mtDNA variants were observed from 56 sequences of noninvasively collected, anonymous samples. Nucleotide diversity in the Nimba Mountain populations was 0.03-0.04, and did not differ significantly from that in the Bossou population. Very few mitochondrial variants are shared among the sites sampled, which suggests that there is little gene flow involving mtDNA. Nevertheless, no clear population structures were revealed in either population. A comparison with published sequences from West African chimpanzees (Pan troglodytes verus) indicates that the variants observed in the Bossou and Nimba regions are scattered throughout the subspecies, rather than clustered according to geographic region. This suggests that the Bossou-Nimba populations derived only recently from the common ancestral population of the West African chimpanzees, and did not pass through a bottleneck.     

Larger rearranged mitochondrial genomes in Dekkera/Brettanomyces yeasts are more closely related than smaller genomes with a conserved gene order

Summary Mitochondrial genomes from yeasts in the Dekkera/Brettanomyces/Eeniella group vary in size from 28 to 101 kb. Mapping of genes has shown that the three smallest genomes, of 28–42 kb, have the same gene order, whereas the three larger mitochondrial DNAs of 57–101 kb are rearranged relative to the smaller molecules and between themselves. To examine the relationships between these genomes, a phylogenetic tree has been constructed by sequence comparison of the mitochondrialencoded cytochrome oxidase subunit gene (COX2) from the six species. Contrary to expectation, the tree shows that the larger rearranged genomes are more closely related than the smaller mtDNAs. This result indicates that the gene order of the smaller mtDNAs (28–42 kb) is ancestral and that larger mtDNA molecules (57–101 kb) are more prone to rearrangement than smaller forms.Offprint requests to: G.D. Clark-Walker     

Nucleotide sequence of a Trichophyton mentagrophytes HindIII mitochondrial DNA fragment containing at RNA gene cluster

Abstract A 0.85-kb Hin dIII mitochondrial DNA fragment of the dermatophytic fungus Trichophyton mentagrophytes has been sequenced. The fragment contains eight complete genes which corresponds to a tRNA gene cluster. From 5' to 3', the sequenced genes code for tRNA^thr, tRNA^glu, tRNA^val, tRNA^met1, tRNA^met3, tRNA^leu, tRNA^ala, and tRNA^phe. This tRNA gene cluster is located downstream of the larger ribosomal RNA gene. The particularities ofthe sequenced genes and their comparison with other fungal tRNA mitochondrial genes are reported.     

Extent of mitochondrial DNA sequence variation in Atlantic cod from the Faroe Islands: a resolution of gene genealogy

Variation in a 250 base pair (bp) fragment of the mitochondrial cytochrome b (cyt b) has been used extensively for population studies in Atlantic cod Gadus morhua. To study the shape of the gene genealogy and the nature of the polymorphism, sequences of another region of the cyt b gene and the TP intergenic spacer were added, making a total of 566 bp from 74 cod from the Faroe Islands. A total of 44 segregating sites defined 41 haplotypes, many at frequencies greater than 5%. Haplotype diversity was 0.97 and nucleotide diversity 0.73% per base. A topology referred to as a constellation gene genealogy was observed with four major haplotypes at high frequencies, from each of which a number of rare variants were derived. A young relative age of the haplotypes was gauged from the structure of the genealogy. The variation was mostly at synonymous sites within the coding region and thus likely to be neutral or under weak purifying selection. By comparative analysis this also applies to the TP spacer. Applying the locus to study population variation in the Faroe Islands by AMOVA revealed that the overall areas and localities within areas accounted for none of the variation, and all the variation was due to differences among individuals.     

Phylogeography of the endangered Cathaya argyrophylla (Pinaceae) inferred from sequence variation of mitochondrial and nuclear DNA

Cathaya argyrophylla is an endangered conifer restricted to subtropical mountains of China. To study phylogeographical pattern and demographic history of C. argyrophylla, species-wide genetic variation was investigated using sequences of maternally inherited mtDNA and biparentally inherited nuclear DNA. Of 15 populations sampled from all four distinct regions, only three mitotypes were detected at two loci, without single region having a mixed composition (G(ST) = 1). Average nucleotide diversity (theta(ws) = 0.0024; pi(s) = 0.0029) across eight nuclear loci is significantly lower than those found for other conifers (theta(ws) = 0.003 approximately 0.015; pi(s) = 0.002 approximately 0.012) based on estimates of multiple loci. Because of its highest diversity among the eight nuclear loci and evolving neutrally, one locus (2009) was further used for phylogeographical studies and eight haplotypes resulting from 12 polymorphic sites were obtained from 98 individuals. All the four distinct regions had at least four haplotypes, with the Dalou region (DL) having the highest diversity and the Bamian region (BM) the lowest, paralleling the result of the eight nuclear loci. An AMOVA revealed significant proportion of diversity attributable to differences among regions (13.4%) and among populations within regions (8.9%). F(ST) analysis also indicated significantly high differentiation among populations (F(ST) = 0.22) and between regions (F(ST) = 0.12-0.38). Non-overlapping distribution of mitotypes and high genetic differentiation among the distinct geographical groups suggest the existence of at least four separate glacial refugia. Based on network and mismatch distribution analyses, we do not find evidence of long distance dispersal and population expansion in C. argyrophylla. Ex situ conservation and artificial crossing are recommended for the management of this endangered species.     

Effects of natural habitat fragmentation on an endemic scrub lizard (Sceloporus woodi): an historical perspective based on a mitochondrial DNA gene genealogy

The Florida scrub lizard, Sceloporus woodi, is endemic to scrub habitat patches along the central portion of the Florida peninsula and xeric coastal regions. Scrub ecosystems are the patchily distributed remnants of previously widespread habitats formed during the Pleiocene and early Pleistocene. Scrub lizards appear to have limited dispersal capabilities due to high habitat specificity and low mobility. To assess the population structure and phylogeography of S. woodi, 135 samples were collected from 16 patches on five major ridges in Florida, USA. Analysis of 273 bp of mitochondrial DNA (mtDNA) cytochrome b reveals a very strong geographic distribution of genetic diversity. Haplotype frequencies are significantly different in 63 of 66 comparisons between patches. With one exception, samples from the five major ridges are characterized by fixed differences in haplotype distribution and deep evolutionary separations (3-10%). Fixed genetic differences were also observed between northern and southern segments of several ridges. Analysis of molecular variance (AMOVA) shows an estimated 10.4% total genetic variation within patches, 17.5% among patches (within ridges), and 72.1% among ridges. This strong population structure among patches within ridges indicates that the distribution of S. woodi is tightly linked to sandy scrub habitat and that the discontinuous distribution of scrub habitats significantly inhibits dispersal and gene flow. Phylogeographic analyses indicate a pattern of dispersal down the Florida peninsula during the late Pliocene-early Pleistocene, followed by habitat fragmentation and variant isolation events. Therefore, the deep genetic structuring among scrub lizard populations on separate ridges is attributed to ancient isolation events induced by a shift from dry (xeric) to wet (mesic) conditions on the Florida peninsula. These findings indicate that some scrub lizard populations have persisted in isolation for time frames in excess of 1 Myr, providing a case history on the genetic consequences of habitat fragmentation.     

The mitochondrial genome, a growing interest inside an organelle

Mitochondria are semi-autonomously reproductive organelles within eukaryotic cells carrying their own genetic material, called the mitochondrial genome (mtDNA). Until some years ago, mtDNA had primarily been used as a tool in population genetics. As scientists began associating mtDNA mutations with dozens of mysterious disorders, as well as the aging process and a variety of chronic degenerative diseases, it became increasingly evident that the information contained in this genome had substantial potential applications to improve human health. Today, mitochondria research covers a wide range of disciplines, including clinical medicine, biochemistry, genetics, molecular cell biology, bioinformatics, plant sciences and physiology. The present review intends to present a summary of the most exiting fields of the mitochondrial research bringing together several contributes in terms of original prospective and future applications.     

Isolation,physical map and gene map of mitochondrial DNA from the cryptomonad Pyrenomonas salina

Mitochondrial DNA (mtDNA) from the cryptomonad Pyrenomonas salina was isolated by CsCl-buoyant density centrifugation of whole-cell DNA in the presence of Hoechst dye 33258. mtDNA consists of circular molecules about 47 kb in size as estimated from restriction enzyme analysis. A physical map for six restriction enzymes (Bam HI, Bge I, Eco RI, Pst I, Sac I and Sac I) has been constructed. Genes coding for the small subunit of rRNA, cytochrome oxidase subunits I and II, and apocytochrome b were localized on this map using Southern blot hybridization with heterologous gene probes from Oenothera. Genes for 5S rRNA and NADH dehydrogenase subunit 5 are absent from P. salina mtDNA. The mitochondrial genome, being the first analysed to this extent in chromophytic algae, should be valuable for taxonomic and phylogenetic studies.     

Biosurveillance for invasive insect pest species using an environmental DNA metabarcoding approach and a high salt trap collection fluid

1. With the increase in global trade and warming patterns, the movement, introduction, and establishment of non‐native insect species has increased. A rapid and effective early detection biosurveillance program to identify species of concern is needed to reduce future impacts and costs associated with introduced non‐native species. One of the challenges facing insect surveillance trapping methods is the sheer volume of individual specimens in the collections. Although molecular identification methods are improving, they currently have limitations (e.g., destructive processing of specimens) and a protocol addressing these limitations can support regulatory applications that need morphological evidence to corroborate molecular data.
2. The novel protocol presented here uses a metabarcoding approach to amplify environmental DNA from a saturated salt solution trap fluid, which retains trap specimens for downstream morphological identifications. The use of a saturated salt solution to preserve specimens in traps addresses issues with the high evaporation rate of ethanol in traps, and public safety concerns with other fluid preservation options with unattended traps in public settings.
3. Using a metabarcoding approach, a 407‐nucleotide segment of the cytochrome c oxidase subunit 1 (COI) animal barcode region was successfully amplified from Lindgren funnel trap collection fluids. These traps were placed in forested areas to survey for wood‐boring beetles of regulatory concern. Our results displayed successful amplification of target taxa, including the molecular identification of the Japanese Beetle Popillia japonica, a species regulated in Canada. A second species, Anisandrus maiche, recently introduced to North America, was identified in every trap. The genus Lymantria, which contains numerous species of concern to North American woodlands, was also detected. Also, there were six other species identified of interest due to their potential impacts on native and crop flora and fauna.
4. Our results show how this protocol can be used as an efficient method for the surveillance of insects using a trap with a saturated salt solution and eDNA metabarcoding to detect species of regulatory concern.

     

Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA

Maintenance of mitochondrial DNA (mtDNA) during cell division is required for progeny to be respiratory competent. Maintenance involves the replication, repair, assembly, segregation, and partitioning of the mitochondrial nucleoid. MGM101 has been identified as a gene essential for mtDNA maintenance in S. cerevisiae, but its role is unknown. Using liquid chromatography coupled with tandem mass spectrometry, we identified Mgm101p as a component of highly enriched nucleoids, suggesting that it plays a nucleoid-specific role in maintenance. Subcellular fractionation, indirect immunofluorescence and GFP tagging show that Mgm101p is exclusively associated with the mitochondrial nucleoid structure in cells. Furthermore, DNA affinity chromatography of nucleoid extracts indicates that Mgm101p binds to DNA, suggesting that its nucleoid localization is in part due to this activity. Phenotypic analysis of cells containing a temperature sensitive mgm101 allele suggests that Mgm101p is not involved in mtDNA packaging, segregation, partitioning or required for ongoing mtDNA replication. We examined Mgm101p's role in mtDNA repair. As compared with wild-type cells, mgm101 cells were more sensitive to mtDNA damage induced by UV irradiation and were hypersensitive to mtDNA damage induced by gamma rays and H2O2 treatment. Thus, we propose that Mgm101p performs an essential function in the repair of oxidatively damaged mtDNA that is required for the maintenance of the mitochondrial genome.     

The nucleotide sequence of the mitochondrial DNA molecule of the grey seal,Halichoerus grypus,and a comparison with mitochondrial sequences of other true seals

The sequence of the mtDNA of the grey seal, Halichoerus grypus, was determined. The length of the molecule was 16,797 base pairs. The organization of the molecule conformed with that of other eutherian mammals but the control region was unusually long due to the presence of two types of repeated motifs. The grey seal and the previously reported harbor seal, Phoca vitulina, belong to different but closely related genera of family Phocidae, true (or earless) seals. In order to determine the degree of differences that may occur between mtDNAs of closely related mammalian genera, the 2 rRNA genes, the 13 peptide coding genes, and the 22 tRNA genes of the 2 species were compared. Total nucleotide difference in the peptide coding genes was 2.0–6.1%. The range of conservative difference was 0.0–1.5%. In the inferred peptide sequences the amino acid difference was 0.0–4.5%, and the difference with respect to chemical properties of amino acids was 0.0–3.0%. A gene that showed a limited degree of difference in one mode of comparison did not necessarily show a corresponding limited difference in another mode. The ratio for differences in codon positions 1, 2, and 3 was 2.7:1:16. The corresponding ratio for conservative differences was 1.8:1. l:1. The evolutionary separation of the two species was calculated to have taken place 2–2.5 million years ago. This dating gives the figure 8 × 10^–9 as the mean rate of substitution per site and year in the entire mtDNA molecule. Comparison with the cytochrome b gene of the Hawaiian monk seal and the Weddell seal suggested that the lineage of these two species and that of the grey and harbor seals separated 8 million years ago. Correspondence to: Ú. Árnason     

Genetic stock structure of bigeye tuna in the Indian Ocean using mitochondrial DNA and microsatellites

Genetic differentiation was minimal and overall non-significant among five collections of bigeye tuna Thunnus obesus from the Indian Ocean, examined for variation at mitochondrial DNA (mtDNA) and at seven microsatellite loci.