The United States Endangered Species Act (ESA) of 1973 set a precedent for biodiversity conservation across the globe.1 A key requirement of protections afforded by the ESA is the accurate delimitation of imperiled species. We present a comparative reference-based taxonomic approach to species delimitation that integrates genomic and morphological data for objectively assessing the distinctiveness of species targeted for protection by governmental agencies. We apply this protocol to the Snail Darter (Percina tanasi), a freshwater fish from the Tennessee River that was discovered in 1973 and declared an endangered species under the ESA in 1975.2 Concurrently, the Snail Darter’s habitat was slated to be destroyed through the construction of the Tellico Dam by the Tennessee Valley Authority (TVA),3,4 inspiring nationwide protests advocating for the suspension of the federal project. This David versus Goliath struggle between supporters of the 3-inch fish and the TVA culminated in the first major legal conflict over protections afforded by the ESA, the US Supreme Court case Hill v. TVA, 437 U.S. 153 (1978), with a 6 to 3 ruling in favor of protecting the Snail Darter and interrupting the completion of the Tellico Dam. Here, we integrate multiple lines of evidence in a comparative framework to demonstrate that despite its legacy, the Snail Darter is not a distinct species but is a population of the Stargazing Darter (Percina uranidea) described in 1887. These results illustrate how a reference-based framework for species delimitation dramatically aids the proper direction of efforts toward protecting biodiversity.

The high levels of biodiversity supported by mountains suggest a possible link between geologic processes and biological evolution. Freshwater biodiversity is high not only in tectonically active settings but also in tectonically quiescent montane regions such as the Appalachian Mountains. We show that erosion through different rock types drove allopatric divergence between lineages of the Greenfin Darter (Nothonotus chlorobranchius), a fish species endemic to rivers draining metamorphic rocks in the Tennessee River basin in the United States. In the past, metamorphic rock preferred by N. chlorobranchius was more widespread, but as erosion exposed other rock types, lineages of this species were progressively isolated in tributaries farther upstream, where metamorphic rock remained. Our results suggest a geologic mechanism for initiating allopatric diversification in mountains long after tectonic activity ceases.

Translocation, the movement of organisms for conservation purposes, can result in unintended introgression if genetic material flows between populations in new ways. The Bluemask Darter Etheostoma akatulo is a federally endangered species of freshwater fish inhabiting the Caney Fork River system and three of its tributaries (Collins River, Rocky River, and Cane Creek) in Tennessee. The current conservation strategy for Bluemask Darters involves translocating the progeny of broodstock from the Collins River (in the west) to the Calfkiller River (in the east) where the species had been extirpated. In this study, we use ddRAD sequence data from across the extant range to assess this translocation strategy in light of population structure, phylogeny, and demography. We also include museum specimen data to assess morphological variation among extant and extirpated populations. Our analyses reveal substantial genetic and phenotypic disparities between a western population in the Collins River and an eastern population encompassing the Rocky River, Cane Creek, and upper Caney Fork, the two of which shared common ancestry more than 100,000 years ago. Furthermore, morphological analyses classify 12 of 13 Calfkiller River specimens with phenotypes consistent with the eastern population. These results suggest that current translocations perturb the evolutionary boundaries between two delimited populations. Instead, we suggest that repopulating the Calfkiller River using juveniles from the Rocky River could balance conflicting signatures of demography, diversity, and divergence. Beyond conservation, the microgeographic structure of Bluemask Darter populations adds another puzzle to the phylogeography of the hyperdiverse freshwater fishes in eastern North America.

Background and Aims Enlarged sterile flowers on the periphery of inflorescences increase the attractiveness of floral displays, and previous studies have generally demonstrated that these have positive effects on insect visitation and/or reproductive success. However, experiments have not specifically been designed to examine the benefits of sterile flowers under conditions that reflect the early stages in their evolution, i.e. when plants that produce sterile flowers are at low frequency. Methods Over three years, three experiments were performed in natural populations of Viburnum lantanoides, which produces sterile marginal flowers (SMFs). The first experiment established that fruit production in V. lantanoides increases with the receipt of outcross pollen. The second tested the role of SMFs under extant conditions, comparing fruit production in two populations composed entirely of intact plants or entirely of plants with the SMFs removed. The third was designed to mimic the presumed context in which SMFs first evolved; here, SMFs were removed from all but a few plants in a population, and rates of insect visitation and fruit set were compared between plants with intact and denuded SMFs. Key Results In comparing whole populations, the presence of SMFs nearly doubled fruit set. Under simulated ancestral' conditions within a population, plants with intact SMFs received double the insect visits and produced significantly more fruits than denuded plants. There was no significant effect of the number of inflorescences or fertile flowers on insect visitation or fruit set, indicating that the presence of SMFs accounted for these differences. Conclusions The presence of SMFs significantly increased pollinator attraction and female reproductive success both in contemporary and simulated ancestral contexts, indicating that stabilizing selection is responsible for their maintenance, and directional selection likely drove their evolution when they first appeared. This study demonstrates a novel approach to incorporating historically relevant scenarios into experimental studies of floral evolution.

Frugivory in lizards is often assumed to be constrained by body size; only large individuals are considered capable of consuming fruits, with the potential of acting as seed dispersers. However, only one previous study has tested the correlation of frugivory with body and head size at an archipelago scale across closely related species. All nine lava lizards (Microlophus spp.) were studied on the eleven largest Galápagos islands from 2010 to 2016 to investigate whether frugivory is related to body and head size. We also tested whether fruit abundance influences fruit consumption and explored the effect of seed ingestion on seedling emergence time and percentage. Our results showed that across islands, lava lizards varied considerably in size (64-102 mm in mean snout-vent length) and level of frugivory (1-23%, i.e., percentage of droppings with seeds). However, level of frugivory was only weakly affected by size as fruit consumption was also common among small lizards. Lava lizards consumed fruits throughout the year and factors other than fruit abundance may be more important drivers of fruit selection (e.g., fruit size, energy content of pulp). From 2,530 droppings, 1,714 seeds of at least 61 plant species were identified, 76% of the species being native to the Galápagos. Most seeds (91%) showed no external structural damage. Seedling emergence time (44 versus 118 days) and percentage (20% versus 12%) were enhanced for lizard-ingested seeds compared to control (uningested) fruits. De-pulping by lizards (i.e., removal of pulp with potential germination inhibitors) might increase the chances that at least some seeds find suitable recruitment conditions. We concluded that lizards are important seed dispersers throughout the year and across the whole archipelago, regardless of body size.

Percina apina, the Tennessee Logperch, is described as a new species endemic to Tennessee and distributed in the Duck River system and Whiteoak Creek. The earliest collection records for Percina apina date to 1971 and the species was identified as Percina burtoni, the Blotchside Logperch. A phylogenetic analysis of mitochondrial DNA (mtDNA) published in 2006 showed that populations identified as Percina burtoni in the Duck River system and Whiteoak Creek were a new and undescribed species. In this study, we test the hypothesis that Percina burtoni is composed of multiple species through analyses of mtDNA, nuclear genetic variation, and traditional meristic trait morphology. Our analyses of morphological divergence, nuclear genotypes, mtDNA gene trees, and comparisons with other sister species pairs of logperches confirm the distinctiveness of Percina apina. Morphologically, Percina apina is distinguished from Percina burtoni through higher average numbers of lateral line scales (93.1 versus 89.9); pored lateral line scales (91.6 versus 88.8); rows of transverse scales (38.1 versus 33.6); and scales around the caudal peduncle (36.2 versus 33.5). The two species also differ in patterns of pigmentation; the lateral blotches in Percina apina are typically wider than high, whereas the blotches tend to be higher than wide in Percina burtoni. We recommend that future species descriptions of North American freshwater fishes leverage available genetic resources and include molecular phylogenetic assessments in analyses of taxon distinctiveness. In addition, we advocate the deposition of morphological data used in species descriptions to online data repositories to ensure that other researchers are able to evaluate and modify hypotheses of species delimitations.

We provide a description of the Blueface Darter, Etheostoma cyanoprosopum, which is distributed in the upper Sipsey Fork of the Mobile Basin and the upper portion of the Bear Creek system in the Tennessee River Drainage. The distinctiveness of Etheostoma cyanoprosopum is assessed through analysis of morphological variation and molecular phylogenetic diversity within the Etheostoma zonistium species complex. In addition to analyzing disparity of morphometric and meristic traits, we present phylogenetic analyses of a mitochondrial gene and two nuclear genes and identify genetic clusters through analysis of 25 microsatellite loci. In the mitochondrial DNA (mtDNA) gene tree, Etheostoma cyanoprosopumis resolved as the sister lineage to a clade containing all other species of the Etheostoma zonistium complex. Etheostoma zonistium is paraphyletic with respect to both Etheostoma pyrrhogaster and Etheostoma cervus, which do not resolve as sister species in the mtDNA gene tree. The two nuclear gene trees are much less resolved, but the S7 ribosomal protein intron 1 (S7) gene tree resolves Etheostoma cyanoprosopum and all sampled populations of Etheostoma zonistium as a clade with strong Bayesian posterior node support. Etheostoma cyanoprosopum is morphologically differentiated from Etheostoma zonistium by a shallower body, a more elongate nape, a higher number of lateral line scales, a higher number of transverse scale rows, and differences in coloration.

The taxonomy of giant Galapagos tortoises (Chelonoidis spp.) is currently based primarily on morphological characters and island of origin. Over the last decade, compelling genetic evidence has accumulated for multiple independent evolutionary lineages, spurring the need for taxonomic revision. On the island of Santa Cruz there is currently a single named species, C. porteri. Recent genetic and morphological studies have shown that, within this taxon, there are two evolutionarily and spatially distinct lineages on the western and eastern sectors of the island, known as the Reserva and Cerro Fatal populations, respectively. Analyses of DNA from natural populations and museum specimens, including the type specimen for C. porteri, confirm the genetic distinctiveness of these two lineages and support elevation of the Cerro Fatal tortoises to the rank of species. In this paper, we identify DNA characters that define this new species, and infer evolutionary relationships relative to other species of Galapagos tortoises.

Long-term population history can influence the genetic effects of recent bottlenecks. Therefore, for threatened or endangered species, an understanding of the past is relevant when formulating conservation strategies. Levels of variation at neutral markers have been useful for estimating local effective population sizes (Ne) and inferring whether population sizes increased or decreased over time. Furthermore, analyses of genotypic, allelic frequency, and phylogenetic information can potentially be used to separate historical from recent demographic changes. For 15 populations of Galápagos giant tortoises (Chelonoidis sp.), we used 12 microsatellite loci and DNA sequences from the mitochondrial control region and a nuclear intron, to reconstruct demographic history on shallow (past ∽100 generations, ∽2500 years) and deep (pre-Holocene, >10 thousand years ago) timescales. At the deep timescale, three populations showed strong signals of growth, but with different magnitudes and timing, indicating different underlying causes. Furthermore, estimated historical Ne of populations across the archipelago showed no correlation with island age or size, underscoring the complexity of predicting demographic history a priori. At the shallow timescale, all populations carried some signature of a genetic bottleneck, and for 12 populations, point estimates of contemporary Ne were very small (i.e., < 50). On the basis of the comparison of these genetic estimates with published census size data, Ne generally represented ∽0.16 of the census size. However, the variance in this ratio across populations was considerable. Overall, our data suggest that idiosyncratic and geographically localized forces shaped the demographic history of tortoise populations. Furthermore, from a conservation perspective, the separation of demographic events occurring on shallow versus deep timescales permits the identification of naturally rare versus newly rare populations; this distinction should facilitate prioritization of management action.

Although many classic radiations on islands are thought to be the result of repeated lineage splitting, the role of past fusion is rarely known because during these events, purebreds are rapidly replaced by a swarm of admixed individuals. Here, we capture lineage fusion in action in a Galápagos giant tortoise species, Chelonoidis becki, from Wolf Volcano (Isabela Island). The long generation time of Galápagos tortoises and dense sampling (841 individuals) of genetic and demographic data were integral in detecting and characterizing this phenomenon. In C. becki, we identified two genetically distinct, morphologically cryptic lineages. Historical reconstructions show that they colonized Wolf Volcano from Santiago Island in two temporally separated events, the first estimated to have occurred ~199 000 years ago. Following arrival of the second wave of colonists, both lineages coexisted for approximately ~53 000 years. Within that time, they began fusing back together, as microsatellite data reveal widespread introgressive hybridization. Interestingly, greater mate selectivity seems to be exhibited by purebred females of one of the lineages. Forward-in-time simulations predict rapid extinction of the early arriving lineage. This study provides a rare example of reticulate evolution in action and underscores the power of population genetics for understanding the past, present and future consequences of evolutionary phenomena associated with lineage fusion.

The Apennine Peninsula is one of Europe's main glacial refugial areas and harbors a large number of lineages and species. Here, a pattern of higher genetic diversity in the south compared to that of the north is characteristic of most vertebrates; however, most studies that have produced these results have relied only on inferences based on mitochondrial DNA. The spectacled salamanders (genus Salamandrina) are endemic to the Apennine Peninsula and have diverged into two sibling species: S. terdigitata (in the south) and S. perspicillata (in the north), presumably in the late Miocene or early Pliocene. By sequencing one mitochondrial (cytb) and two nuclear genes (RAG1 and POMC) and genotyping 10 microsatellite loci, we traced the evolution of these sibling species from their divergence to their contemporary population structure at a fine scale. Using a multilocus coalescent-based approach, we estimated the temporal divergence of both species at approximately 2.25 mya (million years ago), which, hence, is much younger than previous estimates. The classical pattern of high genetic diversity in the south and lower diversity in the north was confirmed only for some markers, and the demographic histories of the two species differed substantially. Whereas S. perspicillata (north) expanded from a single major refugium in the center of the Apennine Peninsula, populations of S. terdigitata (south) persisted through cooler periods in multiple refugia. Further, the fine-scale population genetic structure of 16 S. perspicillata populations revealed significant genetic differentiation, even across short geographic distances. The results of our study stress that for a better understanding of phylogeographic patterns and past demographic processes, both mitochondrial and multiple nuclear loci should be analyzed to avoid gene-specific, and possibly biased results.

Genes from recently extinct species can live on in the genomes of extant individuals of mixed ancestry. Recently, genetic signatures of the giant Galápagos tortoise once endemic to Floreana Island (Chelonoidis elephantopus) were detected within eleven hybrid individuals of otherwise pure Chelonoidis becki on Volcano Wolf, Isabela Island [1]. Movement of tortoises between islands by pirate and whaling ships was not uncommon during the 1800s [2], representing a likely mechanism by which individuals from Floreana were translocated to northern Isabela, despite being presumed extinct soon after Charles Darwin's historic voyage to the Galápagos Islands in 1835. These eleven hybrid individuals with C. elephantopus ancestry were thought to be the last genetic vestiges of a unique evolutionary lineage in the wild. Here, we report that reproductively mature purebred tortoises of the recently ‘extinct’ C. elephantopus from Floreana Island are very likely still alive today, as identified and tracked through the genetic footprints left in the genomes of very recent hybrid offspring on Volcano Wolf. If found, these purebred C. elephantopus individuals could constitute core founders of a captive breeding program directed towards resurrecting this species.

Genetic tools have become a critical complement to traditional approaches for meeting short- and long-term goals of ex situ conservation programs. The San Diego Zoo (SDZ) harbors a collection of wild-born and captive-born Galápagos giant tortoises (n = 22) of uncertain species designation and unknown genealogical relationships. Here, we used mitochondrial DNA haplotypic data and nuclear microsatellite genotypic data to identify the evolutionary lineage of wild-born and captive-born tortoises of unknown ancestry, to infer levels of relatedness among founders and captive-born tortoises, and assess putative pedigree relationships assigned by the SDZ studbook. Assignment tests revealed that 12 wild-born and five captive-born tortoises represent five different species from Isabela Island and one species from Santa Cruz Island, only five of which were consistent with current studbook designations. Three wild-born and one captive-born tortoise were of mixed ancestry. In addition, kinship analyses revealed two significant first-order relationship pairs between wild-born and captive-born tortoises, four second-order relationships (half-sibling) between wild-born and captive tortoises (full-sibs or parent-offspring), and one second-order relationship between two captive-born tortoises. Of particular note, we also reconstructed a first-order relationship between two wild-born individuals, violating the founder assumption. Overall, our results contribute to a worldwide effort in identifying genetically important Galápagos tortoises currently in captivity while revealing closely related founders, reconstructing genealogical relationships, and providing detailed management recommendations for the SDZ tortoises.

The death of Lonesome George, the last known purebred individual of Chelonoidis abingdoni native to Pinta Island, marked the extinction of one of 10 surviving giant tortoise species from the Galapagos Archipelago. Using a DNA reference dataset including historical C abingdoni and >1600 living Volcano Wolf tortoise samples, a site on Isabela Island known to harbor hybrid tortoises, we discovered 17 individuals with ancestry in C abingdoni. These animals belong to various hybrid categories, including possible first generation hybrids, and represent multiple, unrelated individuals. Their ages and relative abundance suggest that additional hybrids and conceivably purebred C. abingdoni individuals still occur on Volcano Wolf. Spatial analyses suggest locations where additional individuals with C abingdoni ancestry are most likely to be recovered, consistent with historical records of human movement of tortoises. These results provide an opportunity for species recovery of Pinta Island tortoises using individuals with C abingdoni ancestry. (C) 2012 Elsevier Ltd. All rights reserved.

Introgressive hybridization and incomplete lineage sorting complicate the inference of phylogeny, and available species-tree methods do not simultaneously account for these processes. Both hybridization and ancestral polymorphism have been invoked to explain divergent phylogenies inferred from different datasets for Stigmacerca, a clade of 11 North American darter species. Species of Stigmacerca are characterized by a mating system involving parental care with males guarding nesting territories and fertilized eggs. Males of four species of Stigmacerca develop egg-mimic nuptial structures on their second dorsal fins during the breeding season. Previous phylogenies suggest contrasting scenarios for the evolution of this nuptial trait. Using a combination of coalescent-based methods, we analyzed a dataset comprising a mitochondrial gene and 15 nuclear loci to estimate relationships and simultaneously test for introgressive hybridization. Our analyses identified several instances of interspecific gene flow involving both cytoplamsmic haplotypes and nuclear alleles. The new phylogeny was used to infer a single origin and recent loss of egg-mimic structures in Stigmacerca and led to the discovery of a phylogenetically distinct species. Our results highlight the limited strategies available to account for introgressive hybridization in the inference of species relationships and the likely effects of this process on reconstructing trait evolution.

We studied patterns of genetic diversity within and among 5 populations (318 individuals) of Galápagos marine iguanas (Amblyrhynchus cristatus) from the island Santa Fé. Populations were separated by distances of 0.2 to 9.9 km. We sequenced 1182 base pairs of the mitochondrial control region and screened 13 microsatellite loci for variability. We also added data from 5 populations (397 individuals) sampled on 4 neighboring islands (Santa Cruz, Floreana, Espanola, and San Cristobal). The 5 Santa Fé populations, revealed as genetically distinct from populations on other islands, present relatively low levels of genetic diversity, which are similar for both microsatellite (average observed heterozygosity from 0.7686 to 0.7773) and mitochondrial DNA (mtDNA) markers (haplotypic and nucleotide diversity from 0.587 to 0.728 and from 0.00079 to 0.00293, respectively), and comparable with those observed in similar-sized sampling sites on other islands. There was frequency-based evidence of genetic structure between northern and southern sites on Santa Fé (F(st) of 0.0027-0.0115 for microsatellite and 0.0447-0.2391 for mtDNA), but the 4 southern sites showed little differentiation. Most of the intra-island genetic variation was allocated within rather than between sites. There was no evidence of sex-biased dispersal or population substructuring due to lek-mating behavior, suggesting that these 2 observed behaviors are not strong enough to leave an evolutionary signal on genetic patterns in this species.

Background Although not unusual to find captive relicts of species lost in the wild, rarely are presumed extinct species rediscovered outside of their native range. A recent study detected living descendents of an extinct Galápagos tortoise species (Chelonoidis elephantopus) once endemic to Floreana Island on the neighboring island of Isabela. This finding adds to the growing cryptic diversity detected among these species in the wild. There also exists a large number of Galápagos tortoises in captivity of ambiguous origin. The recently accumulated population-level haplotypic and genotypic data now available for C. elephantopus add a critical reference population to the existing database of 11 extant species for investigating the origin of captive individuals of unknown ancestry. Methodology/Findings We reanalyzed mitochondrial DNA control region haplotypes and microsatellite genotypes of 156 captive individuals using an expanded reference database that included all extant Galápagos tortoise species as well as the extinct species from Floreana. Nine individuals (six females and three males) exhibited strong signatures of Floreana ancestry and a high probability of assignment to C. elephantopus as detected by Bayesian assignment and clustering analyses of empirical and simulated data. One male with high assignment probability to C. elephantopus based on microsatellite genotypic data also possessed a “Floreana-like” mitochondrial DNA haplotype. Significance Historical DNA analysis of museum specimens has provided critical spatial and temporal components to ecological, evolutionary, taxonomic and conservation-related research, but rarely has it informed ex situ species recovery efforts. Here, the availability of population-level genotypic data from the extinct C. elephantopus enabled the identification of nine Galápagos tortoise individuals of substantial conservation value that were previously misassigned to extant species of varying conservation status. As all captive individuals of C. elephantopus ancestry currently reside at a centralized breeding facility on Santa Cruz, these findings permit breeding efforts to commence in support of the reestablishment of this extinct species to its native range.

The "lava lizards" (Microlophus) are distributed throughout the Galápagos Archipelago, and consist of radiations derived from two independent colonizations. The "Eastern Radiation" includes M. bivittatus and M. habeli endemic to San Cristobal and Marchena Islands. The "Western Radiation" includes five to seven historically recognized species distributed across almost the entire Archipelago. We combine dense geographic sampling and multilocus sequence data to estimate a phylogenetic hypothesis for the Western Radiation, to delimit species boundaries in this radiation, and to estimate a time frame for colonization events. Our phylogenetic hypothesis rejects two earlier topologies for the Western Radiation and paraphyly of M. albemarlensis, while providing strong support for single colonizations on each island. The colonization history implied by our phylogeny is consistent with general expectations of an east-to-west route predicted by the putative age of island groups, and prevailing ocean currents in the Archipelago. Additionally, combined evidence suggests that M. indefatigabilis from Santa Fe should be recognized as a full species. Finally, molecular divergence estimates suggest that the two colonization events likely occurred on the oldest existing islands, and the Western Radiation represents a recent radiation that, in most cases, has produced species that are considerably younger than the islands they inhabit.

The delimitation of species is a major issue in systematic biology and has been a re-emerging discipline in the last decade. A number of studies have shown that the use of multiple data sets is critical for the identification of cryptic species, particularly in groups with complex evolutionary histories. Liolaemus monticola is a montane lizard species distributed in central Chile (32 degrees-42 degrees S), with four described subspecies in a latitudinal gradient from north to south: L. m. monticola, L. m. chillanensis, L. monticola ssp. and L. m. villaricensis. In order to test the systematic status and phylogenetic relationships of the taxa included in the L. monticola group, we analysed morphological (morphometric and meristic) and molecular (allozyme and mitochondrial DNA) data sets. The results of the morphological analyses showed that meristic variables correctly assigned individuals with higher accuracy than did morphometric characters. The results of the analyses of allozyme data revealed eight diagnostic loci that are evidence for significant differences among the four L. monticola subspecies. Phylogenetic analyses with mitochondrial DNA data, including additional species, showed that the L. monticola group is polyphyletic. We postulate that the four current subspecies represent independent evolutionary lineages and must be raised to the specific level as L. monticola, L. chillanensis and L. villaricensis. The taxonomic status of the unnamed L. monticola ssp. remains unresolved, although we provide a preliminary proposal. (C) 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 635-650.

In this study, we used a recently developed supertrees method to test for shared phylogeographical signal in partially overlapping geographical ranges of lizards of the genus Liolaemus from the Andean Range in south-central Chile. We reconstruct mtDNA gene trees for three partially codistributed species (Liolaemus tenuis, L. lemniscatus and L. pictus), and our sampling effort is sufficient to allow statistical tests of shared signal between the combinations L. tenuis-L. pictus, and L. tenuis-L. lemniscatus. For both combinations, standardized maximum agreement subtrees scores showed statistically significant signal for shared pattern in regions of overlap, as evaluated by randomization tests (P < 0.001 and < 0.05, respectively). The matrix representation with parsimony tree obtained from the combination of the three different gene trees revealed concordant phylogeographical associations of all species, and was consistent with the geographical association of intraspecific haploclades with three Chilean bioclimatic zones. A multidimensional scaling analysis of several climate variables showed highly significant differences among these zones, which further suggests that they may have contributed to similar patterns of intraspecific divergence across all three species. In the mesomorphic zone in Central Chile, the species L. tenuis and L. lemniscatus may have codiverged in response to shared orogenic vicariant events, which likely predominated over climatic events associated with cycles of glacial advance and retreat. In the hygromorphic zone in southern Chile, however, glacial cycles likely predominated in structuring the phylogeographical histories of L. tenuis and L. pictus, although important ecological differences between these two caution against broad generalizations at this point.