Conservation genomics of an endangered montane amphibian reveals low population structure, low genomic diversity and selection pressure from disease.

Wildlife diseases are a major global threat to biodiversity. Boreal toads (Anaxyrus [Bufo] boreas) are a state-endangered species in the southern Rocky Mountains of Colorado and New Mexico, and a species of concern in Wyoming, largely due to lethal skin infections caused by the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). We performed conservation and landscape genomic analyses using single nucleotide polymorphisms from double-digest, restriction site-associated DNA sequencing in combination with the development of the first boreal toad (and first North American toad) reference genome to investigate population structure, genomic diversity, landscape connectivity and adaptive divergence. Genomic diversity (π = 0.00034-0.00040) and effective population sizes (Ne = 8.9-38.4) were low, likely due to post-Pleistocene founder effects and Bd-related population crashes over the last three decades. Population structure was also low, likely due to formerly high connectivity among a higher density of geographically proximate populations. Boreal toad gene flow was facilitated by low precipitation, cold minimum temperatures, less tree canopy, low heat load and less urbanization. We found >8X more putatively adaptive loci related to Bd intensity than to all other environmental factors combined, and evidence for genes under selection related to immune response, heart development and regulation and skin function. These data suggest boreal toads in habitats with Bd have experienced stronger selection pressure from disease than from other, broad-scale environmental variations. These findings can be used by managers to conserve and recover the species through actions including reintroduction and supplementation of populations that have declined due to Bd.

Genetic diversity and gene flow decline with elevation in montane mayflies.

Montane environments around the globe are biodiversity 'hotspots' and important reservoirs of genetic diversity. Montane species are also typically more vulnerable to environmental change than their low-elevation counterparts due to restricted ranges and dispersal limitations. Here we focus on two abundant congeneric mayflies (Baetis bicaudatus and B. tricaudatus) from montane streams over an elevation gradient spanning 1400 m. Using single-nucleotide polymorphism genotypes, we measured population diversity and vulnerability in these two species by: (i) describing genetic diversity and population structure across elevation gradients to identify mechanisms underlying diversification; (ii) performing spatially explicit landscape analyses to identify environmental drivers of differentiation; and (iii) identifying outlier loci hypothesized to underlie adaptive divergence. Differences in the extent of population structure in these species were evident depending upon their position along the elevation gradient. Heterozygosity, effective population sizes and gene flow all declined with increasing elevation, resulting in substantial population structure in the higher elevation species (B. bicaudatus). At lower elevations, populations of both species are more genetically similar, indicating ongoing gene flow. Isolation by distance was detected at lower elevations only, whereas landscape barriers better predicted genetic distance at higher elevations. At higher elevations, dispersal was restricted due to landscape effects, resulting in greater population isolation. Our results demonstrate differentiation over small spatial scales along an elevation gradient, and highlight the importance of preserving genetic diversity in more isolated high-elevation populations.

Partial support for the central-marginal hypothesis within a population: reduced genetic diversity but not increased differentiation at the range edge of an island endemic bird.

Large-scale population comparisons have contributed to our understanding of the evolution of geographic range limits and species boundaries, as well as the conservation value of populations at range margins. The central-marginal hypothesis (CMH) predicts a decline in genetic diversity and an increase in genetic differentiation toward the periphery of species' ranges due to spatial variation in genetic drift and gene flow. Empirical studies on a diverse array of taxa have demonstrated support for the CMH. However, nearly all such studies come from widely distributed species, and have not considered if the same processes can be scaled down to single populations. Here, we test the CMH on a species composed of a single population: the Island Scrub-Jay (Aphelocoma insularis), endemic to a 250 km2 island. We examined microsatellite data from a quarter of the total population and found that homozygosity increased toward the island's periphery. However, peripheral portions of the island did not exhibit higher genetic differentiation. Simulations revealed that highly localized dispersal and small total population size, but not spatial variation in population density, were critical for generating fine-scale variation in homozygosity. Collectively, these results demonstrate that microevolutionary processes driving spatial variation in genetic diversity among populations can also be important for generating spatial variation in genetic diversity within populations.

Cryptic species diversity reveals biogeographic support for the 'mountain passes are higher in the tropics' hypothesis.

The 'mountain passes are higher in the tropics' (MPHT) hypothesis posits that reduced climate variability at low latitudes should select for narrower thermal tolerances, lower dispersal and smaller elevational ranges compared with higher latitudes. These latitudinal differences could increase species richness at low latitudes, but that increase may be largely cryptic, because physiological and dispersal traits isolating populations might not correspond to morphological differences. Yet previous tests of the MPHT hypothesis have not addressed cryptic diversity. We use integrative taxonomy, combining morphology (6136 specimens) and DNA barcoding (1832 specimens) to compare the species richness, cryptic diversity and elevational ranges of mayflies (Ephemeroptera) in the Rocky Mountains (Colorado; approx. 40°N) and the Andes (Ecuador; approx. 0°). We find higher species richness and smaller elevational ranges in Ecuador than Colorado, but only after quantifying and accounting for cryptic diversity. The opposite pattern is found when comparing diversity based on morphology alone, underscoring the importance of uncovering cryptic species to understand global biodiversity patterns.

Elevational speciation in action? Restricted gene flow associated with adaptive divergence across an altitudinal gradient.

Evolutionary theory predicts that divergent selection pressures across elevational gradients could cause adaptive divergence and reproductive isolation in the process of ecological speciation. Although there is substantial evidence for adaptive divergence across elevation, there is less evidence that this restricts gene flow. Previous work in the boreal chorus frog (Pseudacris maculata) has demonstrated adaptive divergence in morphological, life history and physiological traits across an elevational gradient from approximately 1500-3000 m in the Colorado Front Range, USA. We tested whether this adaptive divergence is associated with restricted gene flow across elevation - as would be expected if incipient speciation were occurring - and, if so, whether behavioural isolation contributes to reproductive isolation. Our analysis of 12 microsatellite loci in 797 frogs from 53 populations revealed restricted gene flow across elevation, even after controlling for geographic distance and topography. Calls also varied significantly across elevation in dominant frequency, pulse number and pulse duration, which was partly, but not entirely, due to variation in body size and temperature across elevation. However, call variation did not result in strong behavioural isolation: in phonotaxis experiments, low-elevation females tended to prefer an average low-elevation call over a high-elevation call, and vice versa for high-elevation females, but this trend was not statistically significant. In summary, our results show that adaptive divergence across elevation restricts gene flow in P. maculata, but the mechanisms for this potential incipient speciation remain open.

Locally adapted traits maintained in the face of high gene flow.

Gene flow between phenotypically divergent populations can disrupt local adaptation or, alternatively, may stimulate adaptive evolution by increasing genetic variation. We capitalised on historical Trinidadian guppy transplant experiments to test the phenotypic effects of increased gene flow caused by replicated introductions of adaptively divergent guppies, which were translocated from high- to low-predation environments. We sampled two native populations prior to the onset of gene flow, six historic introduction sites, introduction sources and multiple downstream points in each basin. Extensive gene flow from introductions occurred in all streams, yet adaptive phenotypic divergence across a gradient in predation level was maintained. Descendants of guppies from a high-predation source site showed high phenotypic similarity with native low-predation guppies in as few as ~12 generations after gene flow, likely through a combination of adaptive evolution and phenotypic plasticity. Our results demonstrate that locally adapted phenotypes can be maintained despite extensive gene flow from divergent populations.

The influence of geographic heterogeneity in predation pressure on sexual signal divergence in an Amazonian frog species complex.

Sexual selection plays an important role in mating signal divergence, but geographic variation in ecological factors can also contribute to divergent signal evolution. We tested the hypothesis that geographic heterogeneity in predation causes divergent selection on advertisement call complexity within the Engystomops petersi (previously Physalaemus petersi) frog species complex. We conducted predator phonotaxis experiments at two sites where female choice is consistent with call trait divergence. Engystomops at one site produces complex calls, whereas the closely related species at the other site produces simple calls. Bats approached complex calls more than simple calls at both sites, suggesting selection against complex calls. Moreover, bat predation pressure was greater at the site with simple calls, suggesting stronger selection against complex calls and potentially precluding evolution of complex calls at this site. Our results show that geographic variation in predation may play an important role in the evolution and maintenance of mating signal divergence.

Testing the molecular and evolutionary causes of a 'leapfrog' pattern of geographical variation in coloration.

Understanding the mechanisms accounting for the evolution of phenotypic diversity is central to evolutionary biology. We use molecular and phenotypic data to test hypotheses for 'leapfrog' patterns of geographical variation, in which phenotypically similar, disjunct populations are separated by distinct populations of the same species. Phylogenetic reconstructions revealed independent evolution of melanic plumage characters in different populations in the Neotropical avian genus Arremon. Thus, phenotypic similarities between distant populations cannot be explained by close phylogenetic affinity. Nor can they be attributed to recurring mutations in the MC1R gene, a locus involved in melanic pigmentation. A coalescent analysis indicates that plumage traits have become fixed at a faster rate than expected under genetic drift, suggesting that selection underlies their repeated evolution. In contrast to views that genetic drift drives phenotypic differentiation in Neotropical montane birds, our results imply that geographical variation preceding speciation may reflect the action of deterministic selective processes.

Genetic divergence is more tightly related to call variation than landscape features in the Amazonian frogs Physalaemus petersi and P. freibergi.

Behavioural isolation from divergence in male advertisement calls and female preferences is hypothesized to cause genetic divergence and speciation in the Amazonian frogs Physalaemus petersi and P. freibergi, yet the importance of call variation and landscape features in genetic divergence is unresolved. We tested for correlations between genetic divergence at microsatellite loci and (1) call variables; and (2) landscape variables among 10 populations of these frogs. Genetic divergence was not correlated with geographical distance, rivers or elevation. There was a strong positive relationship, however, between genetic divergence and inter-population differences in one call variable, whine dominant frequency. Effective population sizes varied among sites (range = 15-846) and were often small, suggesting that genetic drift could influence call evolution. Evidence for fine-scale genetic structure within sites was also found. Our results support the hypothesis that behavioural isolation from divergence in male calls and female preferences causes genetic divergence and speciation.

Genetic basis of variation in morphological and life-history traits of a wild population of pink salmon.

Understanding the genetic basis of phenotypic variation is essential for predicting the direction and rate of phenotypic evolution. We estimated heritabilities and genetic correlations of morphological (fork length, pectoral and pelvic fin ray counts, and gill arch raker counts) and life-history (egg number and individual egg weight) traits of pink salmon (Oncorhynchus gorbuscha) from Likes Creek, Alaska, in order to characterize the genetic basis of phenotypic variation in this species. Families were created from wild-caught adults, raised to the fry stage in the lab, released into the wild, and caught as returning adults and assigned to families using microsatellite loci and a growth hormone locus. Morphological traits were all moderately to highly heritable, but egg number and egg weight were not heritable, suggesting that past selection has eliminated additive genetic variation in egg number and egg weight or that there is high environmental variance in these traits. Genetic correlations were similar for nonadjacent morphological traits and adjacent traits. Genetic correlations predicted phenotypic correlations fairly accurately, but some pairs of traits with low genetic correlations had high phenotypic correlations, and vice versa, emphasizing the need to use caution when using phenotypic correlations as indices of genetic correlations. This is one of only a handful of studies to estimate heritabilities and genetic correlations for a wild population.

Small effective population size in the long-toed salamander.

The effective population sizes (Ne) of six populations of the long-toed salamander (Ambystoma macrodactylum) from Montana and Idaho, USA were estimated from allozyme data from samples collected in 1978, 1996 and 1997 using the temporal allele frequency method. Five of the six estimates ranged from 23 to 207 (mean = 123 +/- 79); one estimate was indistinguishable from infinity. In order to infer the actual Ne of salamander populations, we compared the frequency distribution of our observed Ne estimates with distributions obtained from simulated populations of known Ne. Our observed Ne estimate distribution was consistent with distributions from simulated populations with Ne values of 10, 25, and 50, suggesting an actual Ne for each of the six salamander populations of less than 100. This Ne estimate agrees with most other Ne estimates for amphibians. We conclude by discussing the conservation implications of small Ne values in amphibians in the context of increasing isolation of populations due to habitat fragmentation.

Requirement for microtubules in new membrane formation during cytokinesis of Xenopus embryos.

In cleaving Xenopus eggs, exposure to nocodazole or cold shock prevents the addition of new plasma membrane to the cleavage plane and causes furrows to recede, suggesting a specific role for microtubules in cytokinesis. Whole-mount confocal immunocytochemistry reveals a ring of radially arranged, acetylated microtubule bundles at the base of all advancing cleavage furrows, from the first cleavage through the midblastula stage. We hypothesize that this novel microtubular structure is involved in transporting maternal stores of membrane in the subcortex to a site of membrane addition near the leading edge of the furrow.