Origin of a cryptic lineage in a threatened reptile through isolation and historical hybridization.

Identifying phylogenetically distinct lineages and understanding the evolutionary processes by which they have arisen are important goals of phylogeography. This information can also help define conservation units in endangered species. Such analyses are being transformed by the availability of genomic-scale data sets and novel analytical approaches for statistically comparing different historical scenarios as causes of phylogeographic patterns. Here, we use genomic-scale restriction-site-associated DNA sequencing (RADseq) data to test for distinct lineages in the endangered Eastern Massasauga Rattlesnake (Sistrurus catenatus). We then use coalescent-based modeling techniques to identify the evolutionary mechanisms responsible for the origin of the lineages in this species. We find equivocal evidence for distinct phylogenetic lineages within S. catenatus east of the Mississippi River, but strong support for a previously unrecognized lineage on the western edge of the range of this snake, represented by populations from Iowa, USA. Snakes from these populations show patterns of genetic admixture with a nearby non-threatened sister species (Sistrurus tergeminus). Tests of historical demographic models support the hypothesis that the genetic distinctiveness of Iowa snakes is due to a combination of isolation and historical introgression between S. catenatus and S. tergeminus. Our work provides an example of how model-based analysis of genomic-scale data can help identify conservation units in rare species.

Niche divergence and lineage diversification among closely related Sistrurus rattlesnakes.

Comparing niche divergence among closely related taxa can yield important insights into the ecological distinctiveness of genetically similar forms, and identify the processes that are responsible for diversification in such organisms. Here, we apply newly developed techniques for analysing niche divergence to assess how ecologically distinct a group of closely related rattlesnakes (Sistrurus sp.) are and to explore the role that niche divergence may have played in their diversification. We find that all taxa even the most recently evolved subspecies (approximately 100,000 years old) are now ecologically distinct, implying a role for ecology in the diversification process. Statistical analysis based on comparisons with null models show that niche divergence between forms is more common than niche conservation. Finally, there is nonlinear relationship between phylogenetic and niche divergence in this group whereby niche divergence develops more rapidly between recently diverged subspecies than more distantly related forms. Overall, our results argue that ecology may play an important role in the diversification process in these snakes.

Similarity of contemporary and historical gene flow among highly fragmented populations of an endangered rattlesnake.

Populations of endangered taxa in recently fragmented habitats often show high levels of genetic structure, but the role that contemporary versus historical processes play in generating this pattern is unclear. The eastern massasauga rattlesnake (Sistrurus c. catenatus) is an endangered snake that presently occurs throughout central and eastern North America in a series of populations that are isolated because of habitat fragmentation and destruction. Here, we use data from 19 species-specific microsatellite DNA loci to assess the levels of genetic differentiation, genetic effective population size, and contemporary and historical levels of gene flow for 19 populations sampled across the range of this snake. Eastern massasaugas display high levels of genetic differentiation (overall θ(Fst) = 0.21) and a Bayesian clustering method indicates that each population represents a unique genetic cluster even at regional spatial scales. There is a twofold variation in genetically effective population sizes but little genetic evidence that populations have undergone recent or historical declines in size. Finally, both contemporary and historical migration rates among populations were low and similar in magnitude even for populations located <7 km apart. A test of alternate models of population history strongly favours a model of long-term drift-migration equilibrium over a recent isolation drift-only model. These results suggest that recent habitat fragmentation has had little effect on the genetic characteristics of these snakes, but rather that this species has historically existed in small isolated populations that may be resistant to the long-term negative effects of inbreeding.

Microgeographic genetic structure in the yellow-pine chipmunk (Tamias amoenus).

While there is evidence for broad-scale genetic structure in small mammals, few studies have used variable DNA-based genetic markers to examine genetic differentiation at microgeographic (tens of kilometres) scales. Yellow-pine chipmunks (Tamias amoenus) live in the heterogeneous landscape of the Rockies in southwest Alberta and are generally restricted to areas of low elevation. We used seven microsatellite loci to determine whether chipmunks show evidence of population genetic structure among three closely situated sites (< 15 km) in the Kananaskis Valley, Alberta. We found evidence for genetic structure in the form of significant differences in allele frequencies among populations and significantly nonzero values of FST for both overall and pairwise population comparisons. However, FIS values for each population were not significantly different from zero, suggesting little evidence for inbreeding within populations. Genetic differentiation probably occurs as a result of the strong effect of drift in very small (N(e) approximately 25) populations of these animals even in the face of substantial immigration rates.

Insights into population ecology and sexual selection in snakes through the application of DNA-based genetic markers.

Hypervariable genetic markers have revolutionized studies of kinship, behavioral ecology, and population biology in vertebrate groups such as birds, but their use in snakes remains limited. To illustrate the value of such markers in snakes, we review studies that have used microsatellite DNA loci to analyze local population differentiation and parentage in snakes. Four ecologically distinct species of snakes all show evidence for differentiation at small spatial scales (2-15 km), but with substantial differences among species. This result highlights how genetic analysis can reveal hidden aspects of the natural history of difficult-to-observe taxa, and it raises important questions about the ecological factors that may contribute to restricted gene flow. A 3-year study of genetic parentage in marked populations of the northern water snake showed that (1) participation in mating aggregations was a poor predictor of genetic-based measures of reproductive success; (2) multiple paternity was high, yet there was no detectable fitness advantage to multiple mating by females; and (3) the opportunity for selection was far higher in males than in females due to a larger variance in male reproductive success, and yet this resulted in no detectable selection on morphological variation in males. Thus genetic markers have provided accurate measures of individual reproductive success in this species, an important step toward resolving the adaptive significance of key features including multiple paternity and reversed sexual size dimorphism. Overall these studies illustrate how genetic analyses of snakes provide previously unobtainable information of long-standing interest to behavioral ecologists.

Limited differentiation in microsatellite DNA variation among northern populations of the yellow warbler: evidence for male-biased gene flow?

Comparisons of the patterns of differentiation among genetic markers with different modes of inheritance can provide insights into patterns of sex-biased dispersal and gene flow. Here, we compare the patterns of differentiation in six microsatellite loci among eight northern breeding populations of the yellow warbler (Dendroica petechia) with results obtained with mitochondrial DNA. Significant but low levels of differentiation (overall FST = 0.014; overall RST = 0.015) were present across all populations. The level of differentiation is substantially less than that observed in the same samples based on mitochondrial DNA control region variation. The presence of low population imbalance index values and significant isolation-by-distance relationships for both FST and RST suggests that these populations are at evolutionary equilibrium and that the high degree of similarity between populations may be due to high levels of male-biased gene flow. This suggests that there may be significant but previously unappreciated differences in the long-distance and/or episodic dispersal behaviour of males and females in these birds.

Genetic evidence for female host-specific races of the common cuckoo.

The common cuckoo Cuculus canorus is divided into host-specific races (gentes). Females of each race lay a distinctive egg type that tends to match the host's eggs, for instance, brown and spotted for meadow pipit hosts or plain blue for redstart hosts. The puzzle is how these gentes remain distinct. Here, we provide genetic evidence that gentes are restricted to female lineages, with cross mating by males maintaining the common cuckoo genetically as one species. We show that there is differentiation between gentes in maternally inherited mitochondrial DNA, but not in microsatellite loci of nuclear DNA. This supports recent behavioural evidence that female, but not male, common cuckoos specialize on a particular host, and is consistent with the possibility that genes affecting cuckoo egg type are located on the female-specific W sex chromosome. Our results also support the ideas that common cuckoos often switched hosts during evolution, and that some gentes may have multiple, independent origins, due to colonization by separate ancestral lineages.

Phylogeography and genetic structure of northern populations of the yellow warbler (Dendroica petechia).

Phylogeographic patterns of intraspecific variation can provide insights into the population-level processes responsible for speciation and yield information useful for conservation purposes. To examine phylogeography and population structure in a migratory passerine bird at both continental and regional geographical scales, we analysed 344 bp of mitochondrial DNA (mtDNA) control region sequence from 155 yellow warblers (Dendroica petechia) collected from seven locations across Canada and from Alaska. There is a major subdivision between eastern (Manitoba to Newfoundland) and western (Alaska and British Columbia) populations which appears to have developed during the recent Pleistocene. Some localities within these two regions also differ significantly in their genetic composition, suggesting further subdivision on a regional geographical scale. Eastern and western birds form distinct phylogeographic entities and the clustering of all western haplotypes with two eastern haplotypes suggests that the western haplotypes may be derived from an eastern lineage. Analyses based on coalescent models support this explanation for the origin of western haplotypes. These results are consistent with important features of Mengel's model of warbler diversification. From a conservation perspective they also suggest that individual populations of migrant birds may form demographically isolated management units on a smaller scale than previously appreciated.

A comparison of RAPD versus microsatellite DNA markers in population studies of the massasauga rattlesnake.

We compared genetic differentiation among populations of the threatened massasauga rattlesnake (Sistrurus c. catenatus) using two types of nuclear molecular markers: randomly amplified polymorphic DNA (RAPD) markers and microsatellites. Analyses of molecular variance (AMOVA) and G(ST) and F(ST) analyses indicated that levels of among-population differentiation between regional populations (>100 km) were comparable for both markers. However, microsatellites were superior in population assignment tests and at discerning fine-scale genetic differentiation between subpopulations separated by tens of kilometers. These results argue that both types of markers are suitable for defining broad-scale genetic structures in snake populations and can provide important inputs into conservation initiatives of focal taxa. However, our analyses suggest that microsatellites 3re better for detecting structure at limited spatial scales.

Microgeographic population genetic structure in the northern water snake, Nerodia sipedon sipedon detected using microsatellite DNA loci.

We describe the isolation and genetic characterization of eight microsatellite DNA loci from the northern water snake, Nerodia sipedon sipedon and use these loci to analyse levels of genetic differentiation between local (< 2 km apart) populations of these snakes in Ontario. These loci are variable, with expected heterozygosities ranging from 0.28 to 0.91, and can correctly exclude nonsires in parentage analyses with a high probability (0.998). Population analyses reveal significant deviation from expected heterozygosity levels for one population, probably a result of a null allele(s) at a single locus and small but significant levels of genetic differentiation among all three populations. This demonstrates that microgeographic genetic structure exists in this species, possibly due to limited dispersal.

Parentage and kinship studies in an obligate brood parasitic bird, the brown-headed cowbird (Molothrus ater), using microsatellite DNA markers.

Recent studies suggest that single-locus microsatellite DNA markers have the potential to unambiguously resolve parentage among individuals in natural populations where maternity is known. However, their power for determining parentage when neither parent is known is unclear. Here we investigate the usefulness of microsatellite DNA markers to determine parentage in a brood parasitic bird, the brown-headed cowbird (Molothrus ater), where, for a given offspring, no a priori knowledge of either parent is available. Seven polymorphic microsatellite DNA markers isolated from brown-headed cowbirds and yellow warblers (Dendroica petechia) were used to genetically characterize an individually marked breeding population of male and female cowbirds at Delta Marsh, Manitoba. Forty-four males, 21 females, and 61 cowbird chicks were genotyped at seven loci using DNA amplified from blood and tissue samples. The mean exclusion probabilities pooled across all seven loci were 0.9964 for males and 0.9948 for females. Two null (non-amplifying) alleles at one locus were discovered and accounted for by constructing alternate nonoverlapping primer sets. Exclusion analyses performed using all individuals determined both paternity and maternity for 43 chicks and paternity only for 4 chicks. Another microsatellite locus was then used to determine paternity for three additional chicks. Relatedness analyses placed 12 of the 18 remaining chicks not assigned both maternity and paternity into four unique full sibling groups. Overall, 90.16% (55 of 61) of all offspring examined were placed into distinct parent/sibling groups, demonstrating that this marker set is extremely useful for parentage studies in this species.

Isolation of microsatellite DNA markers from a passerine bird, Dendroica petechia (the yellow warbler), and their use in population studies.

We describe the isolation and genetic characterization of five microsatellite loci in a passerine bird, the yellow warbler Dendroica petechia, and assess their use for various types of population-level analysis using data from two breeding populations. All five loci show levels of variability comparable to those observed in other vertebrates (Hexp = 0.388-0.989). One locus, Dp mu 05, is highly variable with 46 alleles detected in 41 individuals. All loci appeared to segregate in a Mendelian fashion as judged by patterns of inheritance in known families. However, one locus showed a significant heterozygote deficiency in one population suggesting the possible presence of null alleles at this locus. These markers provide a highly accurate system for determination of parentage in this species: the probability of detecting extrapair fertilization by males given known maternity was 0.999 in each of two separate populations. Comparison of allele frequencies and genetic distances between the two populations showed no evidence for significant differences in allele frequencies at individual loci, whereas the overall genetic distance and FST-value are significantly different from zero suggesting weak differentiation. Finally, cross-species amplification experiments showed that at least one locus appears to amplify products in a wide range of birds including nonpasserine species. Thus, our results demonstrate that these loci will provide a useful set of genetic information for addressing a wide range of population-level analyses in this and other bird species.

Genetic structure of populations of the threatened eastern massasauga rattlesnake, Sistrurus c. catenatus: evidence from microsatellite DNA markers.

Throughout its distribution in North America, the threatened eastern massasauga rattlesnake (Sistrurus c. catenatus) persists in a series of habitat-isolated disjunct populations of varying size. Here, we use six microsatellite DNA loci to generate information on the degree of genetic differentiation between, and the levels of inbreeding within populations to understand how evolutionary processes operate in these populations and aid the development of conservation plans for this species. Samples were collected from 199 individuals from five populations in Ontario, New York and Ohio. Our results show that all sampled populations: (i) differ significantly in allele frequencies even though some populations are < 50 km apart, and may contain genetically distinct subpopulations < 2 km apart; (ii) have an average of 23% of alleles that are population specific; and (iii) have significant FIS values (mean overall FIS = 0.194) probably due to a combination of Wahlund effects resulting from fine-scale genetic differentiation within populations and the presence of null alleles. Our results imply that massasauga populations may be genetically structured on an extremely fine scale even within continuous populations, possibly due to limited dispersal. Additional information is needed to determine if dispersal and mating behaviour within populations can account for this structure and whether the observed differentiation is due to random processes such as drift or to local adaptation. From a conservation perspective, our results imply that these massasauga populations should be managed as demographically independent units and that each has high conservation value in terms of containing unique genetic variation.

Analysis of genetic differentiation of host races of the common cuckoo Cuculus canorus using mitochondrial and microsatellite DNA variation.

It has long been argued that populations of the parasitic common cuckoo Cuculus canorus consist of sympatric host-specific female races, each of which lays eggs that match, to varying degrees, those of their chosen hosts. We tested this hypothesis by comparing rapidly evolving DNA markers among the cuckoo chicks reared by the three most common hosts in the United Kingdom. Comparing cuckoos from different hosts, we found no significant differences in the number of repeats in the control region of the mtDNA nor in the allele frequencies of three microsatellite loci. Given that cuckoos parasitizing the three different hosts do lay different eggs, these results suggest that either: (i) egg-colour variation in cuckoos is facultative, which is unlikely; (ii) gene flow between races occurs because female cuckoos sporadically successfully parasitize alternative hosts; or (iii) the presumably neutral markers in this study have not have not tracked the rapid and/or recent evolution of host races in this species. Studies of the laying and mating patterns of female cuckoos in marked populations in the wild will help evaluate which of these interpretations is most likely.

Sizing bands on autoradiograms: a study of precision for scoring DNA fingerprints.

We replicated DNA fingerprints of snapping turtles (Chelydra serpentina) and hypervariable restriction fragments of red-winged blackbirds (Agelaius phoeniceus) to estimate the between-blot and between-lane components of variance in molecular weights of restriction fragments. Molecular weight standards were included in every lane, and bands were sized using a sonic digitizer. In both studies, a strong positive correlation was found between band size and coefficient of variation (CV; mean = 0.7%). In the DNA fingerprint study, 26% of the variance in estimates of band size was due to differences between blots, 10% due to differences between lanes on the same blot, and 64% due to error in the digitizing process. In the restriction fragment length polymorphism (RFLP) study, 16% of the variance was due to difference between lanes, and 84% to digitizing. Statistical models were developed to measure the effect of sizing error on identifying identical fragments in different lanes or on different blots, in categorizing distinct alleles, and in determining the size of bins in operational allele definitions. We suggest that the distance between bands be at least 2.8 standard deviations (SD) before they are declared different at alpha = 0.05, and 3.7 SD for alpha = 0.01. A variation in CVs strongly indicates that empirical relationships between SD and band size must be used to decide if two bands represent the same allele. Alleles must be at least 3.9 SD apart before the chance of assigning new observations in error falls below 0.05. We suggest that a minimum bin width of 16 SD is necessary before the chances of assigning a band to the wrong bin falls below 0.05.

Realized reproductive success of polygynous red-winged blackbirds revealed by DNA markers.

Hypervariable genetic markers, including a novel locus-specific marker detected by a mouse major histocompatibility complex probe, reveal that multiple paternity is common in families of polygynous red-winged blackbirds (Agelaius phoeniceus). Almost half of all nests contained at least one chick resulting from an extra-pair fertilization, usually by a neighboring male. Genetically based measures of reproductive success show that individual males realize more than 20% of their overall success from extra-pair fertilizations, on average, and that this form of mating behavior confounds traditional measures of male success. The importance of alternative reproductive tactics in a polygynous bird is quantified, and the results challenge previous explanations for the evolution of avian polygny.

Recurrent patterns of natural selection in a population of Darwin's finches.

The adaptive significance of morphological traits can be assessed by measuring and identifying the forces of selection acting on them. Boag and Grant documented directional selection in a small population of Darwin's medium ground finches, Geospiza fortis, on I. Daphne Major, Galápagos, in 1977. Large beak and body size were favoured at a time of diminishing food supply and high adult mortality. We show here that in two subsequent periods of moderate to high adult mortality (1980 and 1982), the population was subject to the same selection. We have used a recently developed technique to ascertain the targets of direct selection. Beak depth and body weight were commonly under direct selection to increase but, surprisingly, beak width was directly selected to decrease, over all three periods of mortality. The results have implications for our understanding of evolutionary change in morphological traits of Darwin's finches.