Showing papers in "Molecular Ecology in 1994"

Analysis of population genetic structure with RAPD markers

Abstract: Recent advances in the application of the polymerase chain reaction make it possible to score individuals at a large number of loci The RAPD (random amplified polymorphic DNA) method is one such technique that has attracted widespread interest The analysis of population structure with RAPD data is hampered by the lack of complete genotypic information resulting from dominance, since this enhances the sampling variance associated with single loci as well as induces bias in parameter estimation We present estimators for several population-genetic parameters (gene and genotype frequencies, within- and between-population heterozygosities, degree of inbreeding and population subdivision, and degree of individual relatedness) along with expressions for their sampling variances Although completely unbiased estimators do not appear to be possible with RAPDs, several steps are suggested that will insure that the bias in parameter estimates is negligible To achieve the same degree of statistical power, on the order of 2 to 10 times more individuals need to be sampled per locus when dominant markers are relied upon, as compared to codominant (RFLP, isozyme) markers Moreover, to avoid bias in parameter estimation, the marker alleles for most of these loci should be in relatively low frequency Due to the need for pruning loci with low-frequency null alleles, more loci also need to be sampled with RAPDs than with more conventional markers, and some problems of bias cannot be completely eliminated

Applications of mitochondrial DNA analysis in conservation: a critical review

Abstract: Patterns of variation in mitochondrial DNA (mtDNA) increasingly are being investigated in threatened or managed species, but not always with clearly defined goals for conservation. In this review I identify uses of mtDNA analysis which fall into two different areas: (i) ‘gene conservation’ - the identification and management of genetic diversity, and (ii) ‘molecular ecology’ - the use of mtDNA variation to guide and assist demographic studies of populations. These two classes of application have different conceptual bases, conservation goals and time-frames. Gene conservation makes extensive use of phylogenetic information and is, in general, most relevant to long-term planning. Appropriate uses here include identification of Evolutionarily Significant Units and assessment of conservation priority of taxa or areas from an evolutionary perspective. Less appropriate are inferences about fitness from within-population diversity and about species boundaries. Molecular ecology makes more use of allele frequencies and provides information useful for short-term management of populations. Powerful applications are to identify Management Units and to define and use naturally occurring genetic tags. Estimating demographic parameters, e.g migration rate and population size, from patterns of mtDNA diversity is fraught with difficulty, particularly where populations are fluctuating, and is unlikely to produce quantitative estimates sufficiently accurate to be useful for practical management of contemporary populations. However, through comparative studies, mtDNA analysis can provide qualitative signals of population changes, allowing efficient targeting of resource-intensive ecological studies. Thus, there are some relatively straightforward uses of mtDNA, preferably in conjunction with assays of nuclear variation, that can make a significant contribution to the long-term planning and short-term execution of species recovery plans.

Microsatellite analysis of genetic variation in black bear populations

Abstract: Measuring levels of genetic variation is an important aspect of conservation genetics The informativeness of such measurements is related to the variability of the genetic markers used; a particular concern in species, such as bears, which are characterized by low levels of genetic variation resulting from low population densities and small effective population sizes We describe the development of microsatellite analysis in bears and its use in assessing interpopulation differences in genetic variation in black bears from three Canadian National Parks These markers are highly variable and allowed identification of dramatic differences in both distribution and amount of variation between populations Low levels of variation were observed in a population from the Island of Newfoundland The significance of interpopulation differences in variability was tested using a likelihood ratio test of estimates of theta = 4Ne mu

Molecular genetics of the most endangered canid: the Ethiopian wolf Canis simensis

Abstract: The world's most endangered canid is the Ethiopian wolf Canis simensis, which is found in six isolated areas of the Ethiopian highlands with a total population of no more than 500 individuals. Ethiopian wolf populations are declining due to habitat loss and extermination by humans. Moreover, in at least one population, Ethiopian wolves are sympatric with domestic dogs, which may hybridize with them, compete for food, and act as disease vectors. Using molecular techniques, we address four questions concerning Ethiopian wolves that have conservation implications. First, we determine the relationships of Ethiopian wolves to other wolf-like canids by phylogenetic analysis of 2001 base pairs of mitochondrial DNA (mtDNA) sequence. Our results suggest that the Ethiopian wolf is a distinct species more closely related to gray wolves and coyotes than to any African canid. The mtDNA sequence similarity with gray wolves implies that the Ethiopian wolf may hybridize with domestic dogs, a recent derivative of the gray wolf. We examine this possibility through mtDNA restriction fragment analysis and analysis of nine microsatellite loci in populations of Ethiopian wolves. The results imply that hybridization has occurred between female Ethiopian wolves and male domestic dogs in one population. Finally, we assess levels of variability within and between two Ethiopian wolf populations. Although these closely situated populations are not differentiated, the level of variability in both is low, suggesting long-term effective population sizes of less than a few hundred individuals. We recommend immediate captive breeding of Ethiopian wolves to protect their gene pool from dilution and further loss of genetic variability.

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

Abstract: 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.

Conservation genetics: beyond the maintenance of marker diversity

Abstract: One of the major problems faced by conservation biologists is the allocation of scarce resources to an overwhelmingly large number of species in need of preservation efforts. Both demographic and genetic information have been brought to bear on this problem; however, the role of information obtained from genetic markers has largely been limited to the characterization of gene frequencies and patterns of diversity. While the genetic consequences of rarity may be a contributing factor to endangerment, it is widely recognized that demographic factors often may be more important. Because patterns of genetic marker variation are influenced by the same demographic factors of interest to the conservation biologist, it is possible to extract useful demographic information from genetic marker data. Such an approach may be productive for determining plant mating systems, inbreeding depression, effective population size, and metapopulation structure. In many cases, however, data consisting only of marker frequencies are inadequate for these purposes. Development of genealogical based analytical methods coupled with studies of DNA sequence variation within and among populations is likely to yield the most information on demographic processes from genetic marker data. Indeed, in some cases it may be the only means of obtaining information on the long-term demographic properties that may be most useful for determining the future prospects of a species of interest.

Hierarchical structure of mitochondrial DNA gene flow among humpback whales Megaptera novaeangliae, world-wide.

Abstract: The genetic structure of humpback whale populations and subpopulation divisions is described by restriction fragment length analysis of the mitochondrial (mt) DNA from samples of 230 whales collected by biopsy darting in 11 seasonal habitats representing six subpopulations, or 'stocks', world-wide. The hierarchical structure of mtDNA haplotype diversity among population subdivisions is described using the analysis of molecular variance (AMOVA) procedure, the analysis of gene identity, and the genealogical relationship of haplotypes as constructed by parsimony analysis and distance clustering. These analyses revealed: (i) significant partitioning of world-wide genetic variation among oceanic populations, among subpopulations or 'stocks' within oceanic populations and among seasonal habitats within stocks; (ii) fixed categorical segregation of haplotypes on the south-eastern Alaska and central California feeding grounds of the North Pacific; (iii) support for the division of the North Pacific population into a central stock which feeds in Alaska and winters in Hawaii, and an eastern or 'American' stock which feeds along the coast of California and winters near Mexico; (iv) evidence of genetic heterogeneity within the Gulf of Maine feeding grounds and among the sampled feeding and breeding grounds of the western North Atlantic; and (v) support for the historical division between the Group IV (Western Australia) and Group V (eastern Australia, New Zealand and Tonga) stocks in the Southern Oceans. Overall, our results demonstrate a striking degree of genetic structure both within and between oceanic populations of humpback whales, despite the nearly unlimited migratory potential of this species. We suggest that the humpback whale is a suitable demographic and genetic model for the management of less tractable species of baleen whales and for the general study of gene flow among long-lived, mobile vertebrates in the marine ecosystem.

Mitochondrial DNA control region polymorphisms: genetic markers for ecological studies of marine turtles.

Abstract: We describe a rapid and sensitive method for the detection of population‐specific genetic markers in mitochondrial DNA (mtDNA) and the use of such markers to analyse population structure of marine turtles. A series of oligonucleotide primers specific for the amplification of the mtDNA control region in Cheloniid turtles were designed from preliminary sequence data. Using two of these primers, a 384–385‐bp sequence was amplified from the 5′ portion of the mtDNA control region of 15 green turtles Chelonia mydas from 12 different Indo‐Pacific rookeries. Fourteen of the 15 individuals, including some with identical whole‐genome restriction fragment patterns, had sequences that differed by one or more base substitutions. Analysis of sequence variation among individuals identified a total of 41 nucleotide substitutions and a 1‐bp insertion/deletion. Comparison with evidence from whole‐genome restriction enzyme analysis of the same individuals indicated that this portion of the control region is evolving approximately eight times faster than the average rate and that the sequence analysis detected approximately one fifth of the total variation present in the genome. Restriction enzyme analysis of amplified products from an additional 256 individuals revealed significant geographic structuring in the distribution of mtDNA genotypes among five of the 10 rookeries surveyed extensively. Additional geographic structuring of genotypes was identified through denaturing gradient gel electrophoresis (DGGE) of amplified products. Only two of the 10 rookeries surveyed could not be differentiated, indicating that the Indo‐Pacific C. mydas include a number of genetically differentiated populations, with minimal female‐mediated gene flow among them. Important applications for genetic markers in the conservation and management of marine turtles include the identification of appropriate demographic units for research and management (i.e. genetically discrete populations) and assessment of the composition of feeding and harvested populations.

Isolation and characterization of microsatellite loci in a passerine bird: the reed bunting Emberiza schoeniclus.

Abstract: Microsatellite loci consist of tandem repeats of short DNA sequences (5 5 bp), usually of less than 100 bp total length. These loci can be polymorphic due to variation between alleles in the number of repeat units. The application of such markers in molecular ecology is a recent development but it seems very likely that that they will be intensively used in the future (see Bruford & Wayne 1993 for a review). Polymorphic microsatellites have been reported in a variety of organisms including birds ( e g Ellegren 1992). Here, we report the isolation and characterization of six microsatellite loci in the reed bunting, Ernberim schoeniclus (Fringillidae). Cross-species amplification of the loci was also investigated in 13 other passerine species. Microsatellite loci were isolated after screening a reed bunting plasmid library with the dinucleotide probes (AC/GT),, and (AG/CT),, (Pharmacia) following standard procedures (Rassmann et al. 1991; Sambrook et al. 1989). Twenty-three positive clones were sequenced. Primers were designed for six loci (Escpl, Esc& Escp3, Esq.14, EscS , Escp6) (Table 1). PCR reactions were performed under the following conditions: 1 min at 94 \"C, 1 min at 55 \"C (Escpl, Escp2, Escp4, Escp5) or 52 \"C (Escp3, Escp6), and 1 min at 72 \"C (25 cycles) using a DNA thermal cycler (Techne PHC-3). Each PCR reaction (following Jeffreys et al. 1988) included 10-20 ng of total genomic DNA, 67m Tris-HC1 (pH 8.8), 16.6-m ammonium sulphate, 6.7mM MgCl,, 1 0 w 2-mercaptoethanol, 6 . 7 p ~ EDTA, 1.5 m of each nucleotide (Pharmacia), 170 mg/mL BSA (DNAase free, Pharmacia) and 10 pmoles of each primer in a total of 10 pL of distilled water. The PCR was initiated with a 'hot start' by first heating the samples for 5 min at 94 \"C and by adding the Tnq DNA polymerase (1 unit/ sample, Advanced Biotechnologies) just before the first annealing step at 55 or 52 \"C. Following this PCR, a single band of the predicted size was seen for all the loci except

Mitochondrial control region and protein coding genes sequence variation among phenotypic forms of brown trout Salmo trutta from northern Italy

Abstract: The PB River basin of northern Italy is the home of distinctive and endemic morphological forms of brown trout Salmo trutta. We used PCR-direct sequencing and RFLP techniques to study variation in the mitochondrial control region of 225 trout in order to assess genetic relatedness among 18 populations from that region. The distribution analysis of these genotypes among north Italian populations confirmed the phylogenetic differentiation of marbled trout Salmo trutta marmoratus populations and the postglacial origin of S. t. carpio. Extensive genetic heterogeneity was observed among morphologically identical S. t. faria populations. Introgression with domestic strains of Atlantic basin origin was detected in all forms. In order to assess the phylogenetic congruence detected in coding and noncoding regions of the mitochondrial genome, we also analysed sequence variation in segments of the cytochrome b and ATPase subunit VI genes among representatives of all variants detected in the analysis of the control region. Variation in protein coding genes was only slightly less than that observed in the control region of the same individuals, both in terms of number of variants detected and of pairwise sequence divergence estimates among variants. Phylogenetic analysis based on protein coding genes sequences identified the same phylogenetic groupings defined by the control region analysis and also allowed a partial resolution of their phyletic relationships that was previously unresolved. However, coding and noncoding segments differed substantially in the transition-transversion ratio (17:O in coding segments vs. 126 in control region segments).

Quantification in soil ofBacillus thuringiensisvar.kurstakiδ-endotoxin from transgenic plants

Abstract: Transgenic plants that produce pesticidal proteins have the potential to release these products into the environment when the plants are incorporated into soil. This could result in novel exposure of soil organisms to these pesticidal proteins. There is a lack of knowledge about the fate and persistence of transgenic pesticidal products in the soil. A model system of transgenic cotton, which produces Bacillus thuringiensis kurstakiδ‐endotoxin (Bt toxin), was used to address this issue. Methods were developed to quantify Btk toxin in soil and soil/plant litter by extraction of the Btk toxin with an aqueous buffer and quantification by ELISA. The highest recovery of Btk toxin from soil was obtained with a high salt, high pH buffer. In addition, for certain soil types, addition of a non‐ionic detergent, Tween‐20, was needed for optimal recovery. Recovery of Btk toxin from soil ranged from 60% for a low clay content, low organic matter soil to 27% for a high clay content, high organic matter soil. The limit of detection of this method is 0.5 ng of extractable toxin per g dry weight soil. The method was shown to be useful in tracking over time the persistence of both purified and transgenic Btk toxin in laboratory experiments.

Genetic stability in a population of a plant pathogenic fungus over time

Abstract: Collections of the plant pathogenic fungus Mycosphaerella graminicola were made from the same field of wheat over a 3-year period. The field was planted with small plots containing four varieties of wheat grown in pure stand and in all possible two-, three- and four-way mixtures. In each year, the wheat field was recolonized by a local source of inoculum of unknown origin. Allele frequencies at 10 RFLP loci were compared at two different times within a growing season and over the 3-year period. No significant differences in allele frequencies were found for any of the RFLP loci over any of the time periods. DNA fingerprints were used to identify clones produced by asexual reproduction. Genotypic diversity based on the frequency of each clone was compared for each collection. No significant changes in genotypic diversity were found within a year or between years. Identical genotypes were found in the field at different times within a season, but no clones were conserved between years. No clone existed in a high frequency in any year, suggesting that selection for particular asexual lineages was weak. The founding population each year probably originated from wind-borne ascospores of the teleomorph, which may exist as an indigenous population on alternative hosts, such as Poa annua (annual bluegrass).

Paternity exclusion in a community of wild chimpanzees using hypervariable simple sequence repeats

Abstract: We report the use of hypervariable simple sequence repeat (SSR) nuclear loci to study paternity in a community of wild chimpanzees (Pan troglodytes schweinfurthii) in Gombe National Park, Tanzania All 43 living members of a habituated community were sampled and 35 were genotyped at 8 SSR loci using DNA amplified from hair Paternity exclusions were performed for 25 chimpanzees including 10 for whom the mother was also genotyped In each case 12–20 males were potential fathers based on their age and/ or direct observation of sexual behaviour 179 tests involving potential father/offspring combinations were performed In four cases the data permit the probable identification of the previously undetermined father; these are the first such determinations for free-ranging chimpanzees, and the first based on non-invasive sampling In another four cases we were able to exclude all but two to five potential fathers, and in the remaining cases we were able to exclude all living males For molecular ecologists SSR genotype databases offer important advantages over currently popular minisatellite DNA fingerprinting: they can be analysed unequivocally using traditional population genetics techniques and they can be expanded through time and space by other researchers

Population differentiation in randomly amplified polymorphic DNA of red-cockaded woodpeckers Picoides borealis.

Abstract: Random amplified polymorphic DNA (RAPD) phenotypes generated by 13 primers were scored for 101 individuals in 14 populations of the endangered red-cockaded woodpecker Picoides borealis. Although no population-specific markers were found, the frequencies of several markers differed significantly among populations. Application of the recently developed AMOVA method (analysis of molecular variance; Excoffier, Smouse & Quattro 1992) showed that more than 90% of phenotypic variance occurred among individuals within populations; of the remaining variance, half was attributed among groups of geographically adjacent populations and half among populations within those groups. The statistical significance of these patterns was supported by Monte Carolo sampling simulations and permutation tests. Estimation of allele frequencies from phenotypes provided somewhat weaker evidence for population structure, although among-population variance in allele frequencies was detectable (Fst = 0.19; chi 2(169) = 509.3, P < 0.0001). UPGMA cluster analyses based on Rogers' (1972) genetic distance revealed grouping of some geographically proximate populations. A Mantel test indicated a positive (r = 0.16), although not significant, correlation between geographic and genetic distances. We compared a subset of our RAPD data with data from a previous study that used allozymes (Stangel, Lennartz & Smith 1992). RAPD (n = 75) and allozyme (n = 245) results based on samples from the same ten populations showed similar patterns. Our study indicates that RAPDs can be helpful in differentiating populations at the phenotypic level even when small sample sizes, estimation bias, and inability to test for Hardy-Weinberg equilibrium complicate the genotypic interpretation. Lack of large differences among populations of red-cockaded woodpeckers may allow flexibility in interpopulation translocations, provided factors such as habitat preference, latitudinal direction of translocation, and status of donor populations are considered.

The establishment of a hybrid zone between red and sika deer (genus Cervus).

Abstract: Japanese sika deer (Cervus nippon nippon) were introduced to Scotland around 80 years (20 generations) ago. The sika phenotype is expanding its range and hybridizing extensively with native red deer (Cervus elaphus) leading to the establishment of a hybrid zone. This zone is currently moving and cannot be considered to be at equilibrium. Cervid genotypes and mitochondrial haplotypes were mapped across the sika phenotype range, using diagnostic protein isozymes, microsatellite nuclear DNA markers and RFLPs in mtDNA. These were analysed to estimate heterozygote deficits and nuclear linkage disequilibria and cytonuclear disequilibria in relation to gene frequencies and time since contact. Introgression was found in both taxa and strong linkage disequilibria and heterozygote deficits characterize the populations longest exposed to hybridization. Populations further from the introduction site, where hybridization is facilitated by the dispersal of sika-like stages, show low values for linkage disequilibria and heterozygote deficit. The observed patterns in genotype are explained in terms of assortative mating and a selective advantage of the sika genotype. The genetic integrity of the Scottish mainland red deer is shown to be at risk from the invasion of sika.

A molecular analysis of relationships and biogeography within a species complex of Holarctic fish (genus Osmerus)

Abstract: Episodes of trans-Arctic faunal exchange and isolation between the north Pacific and Atlantic ocean basins have been implicated as important historic geological events contributing to extant patterns of genetic diversity and structure in Holarctic faunas. We made a further test of the significance of such biogeographic events by examining mitochondrial DNA (mtDNA) restriction fragment length and cytochrome b sequence polymorphism among north Pacific and Arctic, north-western Atlantic (north-eastern North American), and north-eastern Atlantic (European) regional forms of the boreal smelt, genus Osmerus. Our analyses also assessed whether the regional forms within this 'species complex': (i) represent a single widely distributed and polytypic species, or is composed of three geographically distinct species, and (ii) resulted from a single split from north Pacific ancestral Osmerus or two independent Pacific-Atlantic divergences. MtDNA sequence divergence estimates among forms ranged from 5.6-8.9% and from 6.1-8.5% based on restriction fragment and 300 base pairs of cytochrome b sequencing, respectively. Divergence within forms averaged less than 0.5% for fragment analysis and no differences were detected from sequence analysis. Provisional dating of lineage separations in Osmerus based on our sequence divergence estimates suggested a mid-Pliocene to early Pleistocene time frame for diversification among the forms. These estimated lineage separation dates support the idea that geological events in 'Beringia' and the surrounding trans-Arctic area (e.g. opening of the Bering Seaway, Pleistocene glacial advances), occurring over a similar time frame, have influenced radiation in Osmerus. Phenetic and parsimony analyses of the sequence divergence estimates and of sequence polymorphisms suggested that the north Pacific/Arctic form and the north-western Atlantic form shared a common ancestor more recently than either has with the north-eastern Atlantic form, thus supporting the hypothesis that the species complex has arisen from two independent Pacific-Atlantic divergences probably beginning during the mid-Pliocene.

Fluorescent Pseudomonas species categorized by using polymerase chain reaction (PCR)/restriction fragment analysis of 16S rDNA.

Abstract: A rapid procedure for the identification of fluorescent pseudomonads, based on the polymerase chain reaction (PCR) and restriction fragment analysis of 16S rDNA genes is described. Thirty-one strains belonging to 10 different Pseudomonas species of the Pseudomonas fluorescens rRNA branch were characterized. Amplified rDNA was digested with 13 different restriction endonucleases. The combined data from restriction analysis enabled the definition of 17 different 16S rDNA genotypes. All type strains belonging to different species were differentiated. The good correlation between grouping obtained using restriction analysis with other molecular classification criteria demonstrates the value of the described method to characterize rapidly fluorescent Pseudomonas strains at the species level.

Mate fidelity and parentage in Cory's shearwater Calonectris diomedea ‐ field studies and DNA fingerprinting

Abstract: Field studies on Cory's shearwater Calonectris diomedea, which were carried out in a breeding colony in the Aegean Sea between 1989 and 1993, revealed that almost all breeding mates stay together over many consecutive years. Mates usually changed when one partner disappeared (e.g. through death), whereas ‘divorce’ occurred at a rate of 2.7%. Since birds are nesting at very close quarters, the potential for extrapair copulation (EPC) and subsequent extrapair fertilization (EPF) seems to be high. Multilocus DNA fingerprints were used to determine the true parentage of 46 offspring (broods contain a single chick only) from 29 pairs (few pairs were studied in two and three successive years). There were no cases of extrapair paternity.

Monitoring the persistence of Laccaria bicolor as an ectomycorrhizal symbiont of nursery‐grown Douglas fir by PCR of the rDNA intergenic spacer

Abstract: The large-scale inoculation of selected beneficial ectomycorrhizal fungi in forest nurseries has generated renewed interest in the ecology of these symbiotic fungi. However, information on the dissemination and persistence of introduced symbionts is scarce due to the limitation of the current identification methods. To identify ectomycorrhizal fungi on single root tips, we investigated the polymorphism of the PCR-amplified ribosomal DNA intergenic spacer (IGS) from a wide range of ectomycorrhizal fungi. To investigate the reliability of this molecular approach in large-scale surveys, the dissemination and persistence on Douglas fir seedlings of the introduced Laccaria bicolor S238N were assessed in a forest nursery in the Massif Central (France). Several hundred ectomycorrhizas and fruiting bodies were sampled from plots where control and L. bicolor inoculated-Douglas fir seedlings were grown for 1.5 years. PCR typing of mycorrhizas indicated that trees inoculated with L. bicolor S238N remained exclusively colonized by that isolate (or sexually derived isolates) for the entire test period. In contrast, control seedlings were infected by indigenous isolates of Laccaria laccata and Thelephora terrestris. The molecular evidence for the persistence of the introduced mycobiont despite the competition from indigenous isolates of the same species provides further illustration of the potential of exotic species for large-scale microbial application.

Genetic analysis of populations of threatened snake species using RAPD markers

Abstract: Snakes are a particularly threatened vertebrate taxon, with distributions of many species and populations becoming increasingly fragmented. At present, little is known about the degree of genetic differentiation that exists between isolated populations even though such information may be critical to their survival and conservation. As an example of how recently developed RAPD genetic markers can be used in conservation genetics, we present preliminary results from a study which used these DNA-based markers to assess population divergence in two threatened Canadian snakes, the black rat snake (Elaphe o. obsoleta) and the eastern massasauga rattlesnake (Sistrurus c. catenatus). We present information on the levels of variation and reliability of amplification for fragments generated from five primers. We then use a recently developed analytical technique to estimate levels of nucleotide diversity within populations and sequence divergence between populations. Our results show that intrapopulation levels of divergence as estimated by the methods of Clark & Lanigan (Molecular Biology and Evolution 1993, 10, 1096–1111) approximate those found for mtDNA in vertebrates and that diversity between snake populations is small and non-significant when tested using randomization procedures. Thus, our study provides an example of how RAPDs can be applied to conservation genetic studies of vertebrates and suggest that the snake populations we examined have only recently become isolated and maybe considered genetically equivalent from a conservation perspective, although this conclusion needs to be confirmed with other DNA-based markers.

DNA sequencing with arbitrary primer pairs

Abstract: Keywords: molecular population genetics; nucleotide diversity; nucleotide polymorphism; PCR; RAPDs; SWAPP

Variation in mitochondrial DNA and population structure of the Taipei treefrog Rhacophorus taipeianus in Taiwan

Abstract: Mitochondrial DNA (mtDNA) from 40 samples of the Taipei treefrog Rhacophorus taipeianus collected from seven populations in Taiwan were sequenced to document the DNA sequence variation in anuran mtDNA and to elucidate the phylogeographic population structure in the Taipei treefrog. Sequences of 722-764 bases in length, including a 108-bp segment of the cytochrome b gene and a 614-656-bp D-loop segment, were obtained by direct sequencing using polymerase chain reaction (PCR). The variation in length was due to a 40-bp region that tandemly repeated four to five times in the D-loop region. The first repeat is the most conserved one among the five repeats because there are no variable sites in this repeat. Besides the 40-bp length variation, 28 positions in the 764-bp sequences are variable and distributed evenly in the cytochrome b gene fragment and D-loop region. Variation in the D-loop of the Taipei treefrog is comparable to those of other vertebrates. Two well-differentiated lineages (northern and central) differing by mean sequence divergence of 1.7% are identified and concordant with their geographic distributions. The two lineages are inferred to have split from a common ancestral population in the early Pleistocene. However, the interpopulation divergence of the northern lineage (< 0.33%) is apparently lower than that of the central lineage (1.11%), implying that the two lineages evolved independently and had different demographic histories after divergence. This study reveals that anuran D-loop has potential as a genetic marker in phylogenetic and population genetic analyses of anurans.

Molecular phylogeny of the actinorhizal Hamamelidae and relationships with host promiscuity towards Frankia

Abstract: Several of the most studied actinorhizal symbioses involve associations between host plants in the subclass Hamamelidae of the dicots and actinomycetes of the genus Frankia. These actinorhizal plants comprise eight genera distributed among three families of ‘higher’ Hamamelidae, the Betulaceae, Myricaceae, and Casuarinaceae. Contrasting promiscuity towards Frankia is encountered among the different actinorhizal members of these families, and a better assessment of the evolutionary history of these actinorhizal taxa could help to understand the observed contrasts and their implications for the ecology and evolution of the actinorhizal symbiosis. Complete DNA sequences of the chloroplast gene coding for the large subunit of ribulose 1,5-bisphosphate carboxylase (rbcL) were obtained from taxa representative of these families and the Fagaceae. The phylogenetic relationships among and within these families were estimated using parsimony and distance-matrix approaches. All families appeared monophyletic. The Myricaceae appeared to derive first before the Betulaceae and the Casuarinaceae. In the Casuarinaceae, the genus Gymnostoma derived before the genera Casuarina and Allocasuarina, which were found closely related. The analysis of character-state changes in promiscuity along the consensus tree topology suggested a strong relationship between the evolutionary history of host plants and their promiscuity toward Frankia. Indeed, the actinorhizal taxa that diverged more recently in this group of plants were shown to be susceptible to a narrower spectrum of Frankia strains. The results also suggest that the ancestor of this group of plant was highly promiscuous, and that evolution has proceeded toward narrower promiscuity and greater specialization. These results imply that a tight relationship between the phytogenies of both symbiotic partners should not be expected, and that host promiscuity is likely to be a key determinant in the establishment of an effective symbiosis.

The local distribution of highly divergent mitochondrial DNA haplotypes in toque macaques Macaca sinica at Polonnaruwa, Sri Lanka

Abstract: Surveys of mitochondrial DNA (mtDNA) variation in macaque monkeys have revealed extremely high levels of intraspecific divergence among haplotypes. One consistent pattern that has emerged from these studies is that divergent haplotypes are geographically segregated so that sampling a few matrilines from a given region shows them to be identical, or a closely related subset of haplotypes. Geographically structured mtDNA variation has also been commonly observed in other taxa. In this study, haplotype variation and distribution are studied in detail within a local population of toque macaques. The results show that highly divergent haplotypes, differing by 3.1% in their nucleotide sequences, coexist in this population and that they may be spatially segregated even on this micro-geographic scale. Furthermore, these differences are maintained between social groups that exchange male migrants, and thus nuclear genes, frequently.

Population genetic structure of Yushania niitakayamensis (Bambusoideae, Poaceae) in Taiwan

Abstract: Yushania niitakayamensis is distributed in Taiwan, south-west China and northern Philippines. In Taiwan, the species occurs in the central mountain ranges from 1500 to 3500 m in altitude. Morphological variation, especially in terms of plant height, is large, with plants ranging from 10 cm to 5 m in height. The species appears to spread mainly by rhizomes and flowers rarely, leading to the prediction that most populations are comprised of a single or a few clonal genotypes and that the observed morphological variation is primarily due to phenotypic plasticity. The purpose of the present study was to investigate the genetic structure of this species on Mt Hohuan in central Taiwan. Ten plants from a single clone and ten plants of unknown genetic background were surveyed at one site in order to select RAPD primers useful for clone identification. Plants at a second site were collected at 1-m intervals across a 50-m transect through the population. Plants at one extreme (exposed portion) of the transect were approximately 15–30 cm in height, whereas plants up to 410 cm in height were found at the other shaded end of the transect. Comparison of amplification profiles for 12 primers revealed that in contrast to our predictions of genetic uniformity, many samples had reproducibly different RAPD amplification profiles, with the 51 samples representing 31 clones. These data imply that the clone size is relatively small, and the population is actually highly diverse genetically. The genetic variation in this population may be due to a higher frequency of sexual reproduction during the evolutionary history of the species and/or a high somatic mutation rate for RAPD loci in clones of Yushania.

Transduction of a freshwater microbial community by a new Pseudomonas aeruginosa generalized transducing phage, UT1

Abstract: A pseudolysogenic, generalized transducing bacteriophage, UT1, isolated from a natural freshwater habitat, is capable of mediating the transfer of both chromosomal andplasmid DNA between strains of Pseudomonas aeruginosa. Several chromosomal alleles from three different P. aeruginosa strains were found to transduce at frequencies from 10-8 to 10-10 transductants per PFU at multiplicities of infection (MOD between 0.1 and 1. Transduction frequencies of certain alleles increased up to 1000-fold as MOIs were decreased to 0.01. UT1 is also capable of transducing plasmid DNA to indigenous populations of microorganisms in natural lake-water environments. Data obtained in this study suggest that environmentally endemic bacteriophages such as UT1 are formidable transducers of naturally occurring microbial communities. It should be possible to develop model systems to test transduction in freshwater environments using components derived exclusively from these environments.

Genetic evidence for cuckoldry in bluegill Lepomis macrochirus

Abstract: In colonies of bluegill Lepomis macrochirus, some males provide parental care for broods in nests, whereas other males steal fertilizations and do not provide parental care. Using experimental pond populations of bluegill of known genotype (determined through protein electrophoresis), we demonstrate that cuckolder males successfully fertilize eggs in parental male nests. Using electrophoretic techniques to assess the fertilization success of nesting parental male bluegill in Lake Opinicon, Ontario, we demonstrate that paternity of these males ranges from 41% to 100% among four colonies studied. This difference among colonies is related to the density of cuckolder males.

Development of RAPD markers in two introduced fire ants, Solenopsis invicta and S. richteri, and their application to the study of a hybrid zone

Abstract: We developed RAPD DNA markers useful in distinguishing the fire ants Solenopsis invicta and S. richteri. An initial survey of 200 primers revealed seven informative markers; family studies allowed us to determine expression patterns and to confirm Mendelian inheritance of these markers. The seven RAPD markers, one of which is inherited as a codominant marker, were employed along with three allozyme markers to describe the structure of a hybrid zone that has formed between the two species in the USA, where they have been introduced. The data suggest minor introgression of alleles from one parental species (S. richteri) into the other (S. invicta), which most likely reflects the documented recent movement of this hybrid zone. This pattern is interpreted as interspecific introgression rather than shared intraspecific polymorphism on the basis of comparisons with samples from native, non-hybridizing populations in South America that lack such polymorphism. The data further reveal that the structure of the hybrid zone in the USA varies geographically. One parental species (S. invicta) and the hybrids exhibit a mosaic distribution in the east; a gradual transition between the parental species occurs in the centre, with a large intervening area of hybrid genotypes only; and there is apparent contact between parental populations with a small or no intervening zone of hybridization in the west. These differing patterns in the structure of the hybrid zone presumably reflect the unique histories of colonization in different parts of the range of introduced fire ants.

Identification of kin structure among Guam rail founders: a comparison of pedigrees and DNA profiles

Abstract: Kin structure among founders can have a significant effect on subsequent population structure. Here we use the correlation between DNA profile similarity and relatedness calculated from pedigrees to test hypotheses regarding kin structure among founders to the captive Guam rail (Rallus owstoni) population. Five different pedigrees were generated under the following hypotheses: (i) founders are unrelated; (ii) founders are unrelated except for same-nest chicks; (iii) founders from the same major site are siblings; (iv) founders from the same local site are siblings; and (v) founders are related as defined by a UPGMA cluster analysis of DNA similarity data. Relatedness values from pedigrees 1, 2 and 5 had the highest correlation with DNA similarity but the correlation between relatedness and similarity were not significantly different among pedigrees. Pedigree 5 resulted in the highest correlation overall when using only relatedness values that changed as a result of different founder hypotheses. Thus, founders were assigned relatedness based on pedigree 5 because it had the highest correlations with DNA similarity, was the most conservative approach, and incorporated all field data. The analyses indicated that estimating relatedness using DNA profiles remains problematic, therefore we compared mean kinship, a measure of genetic importance, with mean DNA profile similarity to determine if genetic importance among individuals could be determined via use of DNA profiles alone. The significant correlation suggests this method may provide more information about population structure than was previously thought. Thus, DNA profiles can provide a reasonable explanation for founder relatedness and mean DNA profile similarity may be helpful in determining relative genetic importance of individuals when detailed pedigrees are absent.

DNA fingerprinting reveals significant amounts of genetic variation in a wild raspberryRubus idaeuspopulation

Abstract: Establishing the genotypic distribution in natural plant populations is an important part of ecological studies concerning plant growth, reproduction and turn-over. Restriction enzyme-digested DNA samples, isolated from 24 plants of a natural Rubus idaeus population, were analysed with DNA fingerprinting using the M13 repeat sequence as well as a synthetic (AC)/(TG) polydinucleotide as hybridization probes. All the examined samples exhibit unique DNA fingerprint patterns, suggesting that vegetative reproduction may be considerably more restricted in wild R. idaeus populations than previously assumed. By comparison, all samples of the apomictic blackberry species Rubus nessensis, collected on the same location, were completely identical.