Marlis R. Douglas

Holarctic phylogeography of Arctic charr (Salvelinus alpinus L.) inferred from mitochondrial DNA sequences.

Abstract This study evaluated mitochondrial DNA (mtDNA) sequence variation in a 552‐bp fragment of the control region of Arctic charr (Salvelinus alpinus) by analyzing 159 individuals from 83 populations throughout the entire range of the complex. A total of 89 (16.1%) nucleotide positions were polymorphic, and these defined 63 haplotypes. Phylogenetic analyses supported the monophyly of the complex and assigned the observed haplotypes to five geographic regions that may be associated with different glacial refugia. Most notably, a formerly defined major evolutionary lineage (S. a. erythrinus) ranging from North America across the Arctic archipelago to the Eurasian continent has now been partitioned into the Arctic group and the newly identified Siberian group. The Beringian group, formed entirely by specimens assigned to S. malma (Dolly Varden), encompassed the area formerly assigned to S. a. taranetzi. The latter, due to a unique haplotype, became the basal member of the Arctic group. Overall, the S. alpinus complex reflects divergent evolutionary groups coupled with shallow intergroup differentiation, also indicated by an analysis of molecular variance that attributed 73.7% (P < 0.001) of the total genetic variance among groups. Time estimates, based on sequence divergence, suggest a separation of the major phylogeographic groups during early to mid‐Pleistocene. In contrast, colonization of most of todays range started relatively recently, most likely late Pleistocene during the last retreat of ice sheets some 10,000–20,000 years ago. This time scale obviously is too shallow for detecting significant variation on a smaller scale using mtDNA markers. However, other studies using nuclear microsatellite DNA variation strongly suggested ongoing evolution within groups by revealing strong population‐genetic substructuring and restricted gene flow among populations. Thus, Arctic charr could serve as a model organism to investigate the linkage between historical and contemporary components of phylogeographic structuring in fish, and, with a global perspective of the distribution of genetic variation as a framework, meaningful comparisons of charr studies at a smaller geographic scale will now be possible.

Microsatellite and mitochondrial DNA assessment of population structure and stocking effects in Arctic charr Salvelinus alpinus (Teleostei: Salmonidae) from central Alpine lakes

Despite geographical isolation and widespread phenotypic polymorphism, previous population genetic studies of Arctic charr, Salvelinus alpinus, have detected low levels of intra‐ and interpopulation variation. In this study, two approaches were used to test the generality of low genetic diversity among 15 Arctic charr populations from three major drainages of the central Alpine region of Europe. First, a representative subsample of each drainage was screened by PCR–RFLP analysis of mtDNA using 31 restriction enzymes. All individuals but one shared an identical haplotype. In contrast, microsatellite DNA variation revealed high levels of genetic diversity within and among populations. The number of alleles per locus ranged from six to 49, resulting in an overall expected heterozygosity from 0.72 ± 0.09 to 0.87 ± 0.04 depending on the locus. Despite evidence for fish transfers among Alpine charr populations over centuries, genetic diversity was substantially structured, as revealed by hierarchical Φ statistics. Eighteen per cent of total genetic variance was apportioned to substructuring among Rhône, Rhine, and Danube river systems, whereas 19% was due to partitioning among populations within each drainage. Cluster analyses corroborated these results by drainage‐specific grouping of nonstocked populations, but also revealed damaging effects of stocking practices in others. However, these results suggest that long‐term stocking practices did not generally alter natural genetic partitioning, and stress the importance of considering genetic diversity of Arctic charr in the Alpine region for sound management. The results also refute the general view of Arctic charr being a genetically depauperate species and show the potential usefulness of microsatellite DNAs in addressing evolutionary and conservation issues in this species.

Do assemblages of Coregonus (Teleostei: Salmoniformes) in the Central Alpine region of Europe represent species flocks?

To examine models of evolution for Coregonus from the Central Alpine region of Europe, 20 populations from nine lakes were assessed for variation at six microsatellite DNA loci. Patterns of variation were tested against three evolutionary models: phenotypic plasticity, multiple invasions of lakes by divergent forms, and within‐lake radiation of species flocks. All sympatric and all but one allopatric pairs of populations were significantly divergent in allele frequencies. Pairwise F‐statistics indicated reduced gene flow among phenotypically divergent sympatric populations. These results reject the hypothesis that within‐lake morphological and ecological diversity reflects phenotypic plasticity within a single gene pool. Genetic similarity was higher among forms within lakes than between populations of the same form in different lakes. Among‐lake divergence was primarily a product of allele size differences. Mantel tests contrasting patterns of genetic divergence against patterns predicted from the multiple invasions and species flocks models indicated that the latter is the best explanation of the observed genetic variation. Thus, reproductively isolated species diverged within lakes, with similar patterns repeatedly emerging among lakes. While this study argues for a particular mode of evolution in Central Alpine Coregonus, the taxonomy of these forms remains unresolved.

Small Fish in a Large Landscape: Diversification of Rhinichthys osculus (Cyprinidae) in Western North America

Abstract We mapped 112 restriction sites in the mitochondrial DNA genome of the Speckled Dace (Rhinichthys osculus), a small cyprinid fish broadly distributed in western North America. These data were used to derive a molecular phylogeny that was contrasted against the hydrographic evolution of the region. Although haplotypic variation was extensive among our 59 sampled populations and 104 individuals, their fidelity to current drainage basins was a hallmark of the study. Two large clades, representing the Colorado and Snake Rivers, were prominent in our results. The Colorado River clade was divided into four cohesive and well-defined subbasins that arose in profound isolation as an apparent response to regional aridity and tectonism. The Lower and Little Colorado River subbasins are sister to one another and (with the Upper Colorado River) form a large clade of higher-elevation populations that seemingly reflect postglacial recolonization from refugia in the Middle Colorado River. The latter subbasin is sister to the Los Angeles Basin and, thus, supports the hypothesis of an ancient connection between the two. A haplotype from the Northern Bonneville was sister to the entire Colorado River clade. The Snake River clade revealed a strongly supported Lahontan group that did not share haplotypes with surrounding basins. It contained instead scattered sites from former Pluvial Lake Lahontan, as well as from eastern California. It was, in turn, sister to the Owens River, whereas Rhinichthys falcatus was sister to this larger clade. The hypothesis of a southerly, “fishhook”-configured tributary associated with a westward-draining Pliocene Snake River was manifested by the relationship of this Lahontan clade to upper Snake and northern Bonneville localities. The Klamath/Pit and Columbia Rivers were sisters in a clade basal to all the above, which in turn supported the hypothesis of a pre-Pliocene western passage of the Snake River. Our data also suggested at least three separate ichthyofaunal invasions of California, as well as a Bonneville Basin fragmented by a north-south connection between southeastern Idaho and the Colorado River. The dual western and southern movements of R. osculus from southern Idaho argued for a northern origin, possibly associated with Tertiary Lake Idaho.

Microsatellites behaving badly: empirical evaluation of genotyping errors and subsequent impacts on population studies

Microsatellites are useful tools for ecological studies because they can be used to discern population structure, dispersal patterns and genetic relationships among individuals. However, they can also yield inaccurate genotypes that, in turn, bias results, promote biological misinterpretations, and create repercussions for population management and conservation programs. We used empirical data from a large-scale microsatellite DNA study of white-tailed deer (Odocoileus virginianus) to identify sources of genotyping error, evaluate corrective measures, and provide recommendations to prevent bias in population studies. We detected unreported mutations that led to erroneous genotypes in five of 13 previously evaluated microsatellites. Of the five problematic markers, two contained mutations that resulted in null alleles, and three contained mutations that resulted in imperfect repeats. These five microsatellites had error rates that were four times greater on average than those observed in the remaining eight. Methodological corrections, such as primer redesign, reduced errors up to 5-fold in two problematic loci, although analytical corrections (computational adjustment for errors) were unable to fully prevent bias and, consequently, measures of genetic differentiation and kinship were negatively impacted. Our results demonstrate the importance of error evaluation during all stages of population studies, and emphasize the need to standardize procedures for microsatellite analyses. This study facilitates the application of microsatellite technology in population studies by examining common sources of genotyping error, identifying unreported problems with microsatellites, and offering solutions to prevent error and bias in population studies.

Stream hierarchy defines riverscape genetics of a North American desert fish

Global climate change is apparent within the Arctic and the south‐western deserts of North America, with record drought in the latter reflected within 640 000 km2 of the Colorado River Basin. To discern the manner by which natural and anthropogenic drivers have compressed Basin‐wide fish biodiversity, and to establish a baseline for future climate effects, the Stream Hierarchy Model (SHM) was employed to juxtapose fluvial topography against molecular diversities of 1092 Bluehead Sucker (Catostomus discobolus). MtDNA revealed three geomorphically defined evolutionarily significant units (ESUs): Bonneville Basin, upper Little Colorado River and the remaining Colorado River Basin. Microsatellite analyses (16 loci) reinforced distinctiveness of the Bonneville Basin and upper Little Colorado River, but subdivided the Colorado River Basin into seven management units (MUs). One represents a cline of three admixed gene pools comprising the mainstem and its lower‐gradient tributaries. Six others are not only distinct genetically but also demographically (i.e. migrants/generation <9.7%). Two of these (i.e. Grand Canyon and Canyon de Chelly) are defined by geomorphology, two others (i.e. Fremont‐Muddy and San Raphael rivers) are isolated by sharp declivities as they drop precipitously from the west slope into the mainstem Colorado/Green rivers, another represents an isolated impoundment (i.e. Ringdahl Reservoir), while the last corresponds to a recognized subspecies (i.e. Zuni River, NM). Historical legacies of endemic fishes (ESUs) and their evolutionary potential (MUs) are clearly represented in our data, yet their arbiter will be the unrelenting natural and anthropogenic water depletions that will precipitate yet another conservation conflict within this unique but arid region.

Conservation phylogenetics of helodermatid lizards using multiple molecular markers and a supertree approach

We analyzed both mitochondrial (mt-) and nuclear (n) DNAs in a conservation phylogenetic framework to examine deep and shallow histories of the Beaded Lizard (Heloderma horridum) and Gila Monster (H. suspectum) throughout their geographic ranges in North and Central America. Both mtDNA and intron markers clearly partitioned each species. One intron and mtDNA further subdivided H. horridum into its four recognized subspecies (H. n. alvarezi, charlesbogerti,exasperatum, and horridum). However, the two subspecies of H. suspectum (H. s. suspectum and H. s. cinctum) were undefined. A supertree approach sustained these relationships. Overall, the Helodermatidae is reaffirmed as an ancient and conserved group. Its most recent common ancestor (MRCA) was Lower Eocene [35.4 million years ago (mya)], with a approximately 25 my period of stasis before the MRCA of H. horridum diversified in Lower Miocene. Another approximately 5 my passed before H. h. exasperatum and H. h. horridum diverged, followed by approximately 1.5 my before H. h. alvarezi and H. h. charlesbogerti separated. Heloderma suspectum reflects an even longer period of stasis (approximately 30 my) before diversifying from its MRCA. Both H. suspectum (México) and H. h. alvarezi also revealed evidence of historic range expansion following a recent bottleneck. Our conservation phylogenetic approach emphasizes the origin and diversification of this group, yields information on the manner by which past environmental variance may have impacted its populations and, in turn, allows us to disentangle historic from contemporary impacts that might threaten its long-term persistence. The value of helodermatid conservation resides in natural services and medicinal products, particularly venom constituents, and these are only now being realized.

Genealogical Concordance between Mitochondrial and Nuclear DNAs Supports Species Recognition of the Panamint Rattlesnake (Crotalus mitchellii stephensi)

Abstract The Speckled Rattlesnake (Crotalus mitchellii) is a polytypic taxon presently composed of five subspecies that range across southwestern North America, including the Baja Peninsula and islands in the Pacific Ocean and Sea of Cortés. The principles of genealogical concordance were employed to test the taxonomic status of three of the five subspecies (C. m. mitchellii, C. m. pyrrhus, and C. m. stephensi). We used two molecular marker systems: mitochondrial (mt) DNA ATPase 8 and 6 genes (675 base pairs, bp), and introns 5 and 6 of the nuclear (n) DNA ribosomal protein (RP) gene (449 bp). These markers were evaluated across 104 individuals of C. mitchellii: C. m. mitchellii (n  =  3), C. m. pyrrhus (n  =  83), C. m. stephensi (n  =  18), with Sistrurus c. catenatus as the distant outgroup. Deep phylogenetic splits were detected in the subspecies of C. mitchellii, with 5.0–6.4% mtDNA sequence divergence (SD) separating C. m. mitchellii and C. m. pyrrhus, while C. m. mitchellii and C. m. stephensi had SD values of 6.4–7.3%. Similarly, C. m. pyrrhus and C. m. stephensi had SD values of 5.2–6.7%. In addition, C. m. mitchellii and C. m. pyrrhus were identical in all 449 intron bp, but C. m. stephensi differed from both at a single nucleotide polymorphism. Our molecular results diagnose C. m. stephensi as sister to mainland subspecies of the C. mitchellii complex, a result consistent with certain head scalation characters and its northernmost geographic distribution in this complex. Furthermore, four morphological synapomorphies (supraocular scales prominently ridged and/or creased, contact between rostral and prenasal scales, ground coloration of tail congruent with that of body, and black rings in the distal 15% of the tail) also diagnose C. m. stephensi from all other subspecies of C. mitchellii. We hypothesize that the northern distribution of C. m. stephensi likely resulted from two vicariant events: Pliocene expansion of the Sea of Cortés as the Salton Trough, and Pliocene development of the lacustrine Bouse Embayment along the Colorado River drainage. Despite earlier conclusions based on morphology, our molecular results showed no evidence of intergradation between C. m. pyrrhus and C. m. stephensi. Based on the principles of genealogical concordance, we advocate that C. m. stephensi be elevated to a full species, which renders a minimum of two species within the C. mitchellii clades we examined.

Evolutionary Homoplasy among Species Flocks of Central Alpine Coregonus (Teleostei: Salmoniformes)

Abstract Evolution of phenotype and mode of speciation were examined for 19 Coregonus populations within and among eight lakes in the Central Alpine region of Europe. These populations reflect a mosaic of morphological, ecological, and life history traits, and thus represent numerous forms (qualitatively described according to relative body size and ecology). Each population had been previously evaluated for six microsatellite DNA loci, and herein for five meristic counts, four fin pigmentation characters, three body color variables, three measures relating to spawning ecology, an estimate of breeding tubercles, and average weight (= size). The two matrices (genotype vs. phenotype/ecology) were then tested for significance amongst themselves, and against evolutionary and geographic models. Microsatellite data associated significantly with a species flock model in which in situ diversification occurred from a common ancestor in each lake or cluster of neighboring lakes. However, phenotype/ecology associated significantly with a model invoking multiple invasions of lakes by pre-existing forms. The latter supports historic perspectives on the evolution of Central Alpine Coregonus and suggests forms within lakes have adapted to specific within-lake environmental niches that are replicated across lakes. This convergence of form and function has long clouded an understanding of coregonine biodiversity, and we suggest it represents a homoplasious condition (i.e., a similarity due not to genealogy but to iteration). This compendium of homoplasious and homologous characters is actually quite unique in nature, and the evolutionary diversification of Central Alpine Coregonus can now be evaluated according to the relative contribution of each character type.

Linking extinction–colonization dynamics to genetic structure in a salamander metapopulation

Theory predicts that founder effects have a primary role in determining metapopulation genetic structure. However, ecological factors that affect extinction–colonization dynamics may also create spatial variation in the strength of genetic drift and migration. We tested the hypothesis that ecological factors underlying extinction–colonization dynamics influenced the genetic structure of a tiger salamander (Ambystoma tigrinum) metapopulation. We used empirical data on metapopulation dynamics to make a priori predictions about the effects of population age and ecological factors on genetic diversity and divergence among 41 populations. Metapopulation dynamics of A. tigrinum depended on wetland area, connectivity and presence of predatory fish. We found that newly colonized populations were more genetically differentiated than established populations, suggesting that founder effects influenced genetic structure. However, ecological drivers of metapopulation dynamics were more important than age in predicting genetic structure. Consistent with demographic predictions from metapopulation theory, genetic diversity and divergence depended on wetland area and connectivity. Divergence was greatest in small, isolated wetlands where genetic diversity was low. Our results show that ecological factors underlying metapopulation dynamics can be key determinants of spatial genetic structure, and that habitat area and isolation may mediate the contributions of drift and migration to divergence and evolution in local populations.