Marine and freshwater fishes of Alabama: a revised checklist and discussion of taxonomic issues.

Checklists are fundamental and important tools for organizing information about biodiversity that provide a basis for conservation and additional scientific research. While Alabama is recognized as an aquatic biodiversity hotspot with the highest native freshwater fish diversity in the contiguous United States, we currently lack an up-to-date list of the states fishes. In particular, much has changed over the past ~20 years regarding our knowledge of fishes from Alabama and the Mobile River Basin, rendering past comprehensive treatments by Mettee et al. (1996) and Boschung and Mayden (2004) out of date. Here, we provide a revised checklist of marine and freshwater fishes known from the coastal and inland waters of Alabama that includes 463 species (335 primarily freshwater fishes, and 128 marine or diadromous fishes) in 35 orders, 78 families, and 176 genera. Extant, extirpated, and extinct species are included, as are putative candidate species. The checklist is based on prior work, searches of the literature and online sources, as well as parsing a large compilation of >140,000 fish records for Alabama and the Mobile River Basin from 37 data providers in the global Fishnet2 database (www.fishnet2.net) and >4000 marine survey records from the SEAMAP database (https://www.gsmfc.org/seamap.php). After editing and quality control checks, the final combined database contained 144,215 collection records, ~95% of which were georeferenced. We discuss the species descriptions, nomenclatural changes, and updates to marine species that account for changes to the state list, and we close with a discussion of ~13 candidate species forms that remain undescribed, which represent outstanding taxonomic issues in need of further research attention.

Genome-wide ultraconserved elements resolve phylogenetic relationships and biogeographic history among Neotropical leaf-nosed bats in the genus Anoura (Phyllostomidae).

AnouraGray, 1838 are Neotropical nectarivorous bats and the most speciose genus within the phyllostomid subfamily Glossophaginae. However, Anoura species limits remain debated, and phylogenetic relationships remain poorly known, because previous studies used limited Anoura taxon sampling or focused primarily on higher-level relationships. Here, we conduct the first phylogenomic study of Anoura by analyzing 2039 genome-wide ultraconserved elements (UCEs) sequenced for 42 individuals from 8 Anoura species/lineages plus two outgroups. Overall, our results based on UCEs resolved relationships in the genus and supported (1) the monophyly of small-bodied Anoura species (previously genus Lonchoglossa); (2) monotypic status of A. caudifer; and (3) nested positions of "A. carishina", A. caudifer aequatoris, and A. geoffroyi peruana specimens within A. latidens, A. caudifer and A. geoffroyi, respectively (suggesting that these taxa are not distinct species). Additionally, (4) phylogenetic networks allowing reticulate edges did not explain gene tree discordance better than the species tree (without introgression), indicating that a coalescent model accounting for discordance solely through incomplete lineage sorting fit our data well. Sensitivity analyses indicated that our species tree results were not adversely affected by varying taxon sampling across loci. Tree calibration and Bayesian coalescent analyses dated the onset of diversification within Anoura to around âˆ¼ 6-9 million years ago in the Miocene, with extant species diverging mainly within the past âˆ¼ 4 million years. We inferred a historical biogeographical scenario for Anoura of parapatric speciation fragmenting the range of a wide-ranging ancestral lineage centered in the Central to Northern Andes, along with Pliocene-Pleistocene dispersal or founder event speciation in Amazonia and the Brazilian Atlantic forest during the last âˆ¼ 2.5 million years.

Two new species of Hypostomus suckermouth-armoured catfishes (Teleostei: Loricariidae) from central Brazil.

This study describes two new endemic Hypostomus species from central Brazil, which were previously identified as genetically distinct lineages in a recent genomic study that recommended their testing and potential description based on morphological data. A machine learning classification procedure (random forest) was used to investigate morphological variation and identify putatively diagnostic characters for these candidate species and revealed that each is morphologically distinct. The new species Hypostomus cafuringa is characterized by small size, dark spots under a light background, deeper caudal peduncle and shorter first ray of the pectoral fin and base of the dorsal fin when compared to congeneric species from the region. H. cafuringa is known from the headwaters of the Maranhão River, upper Tocantins River basin, Distrito Federal, Brazil. The second new species, Hypostomus crulsi, is characterized by dark spots under a light background, absence of plates along the abdomen region, shorter first ray of the pelvic fin, shorter first ray of the pectoral fin and smaller body size. H. crulsi is known from the headwaters of the São Bartolomeu River, upper Paraná River basin, Distrito Federal, Brazil. The rapid conversion of natural habitats for agricultural development and the isolation of protected areas represent a serious threat to the continued existence of these two newly described endemic species, which warrant conservation assessment.

Adaptive evolution in a conifer hybrid zone is driven by a mosaic of recently introgressed and background genetic variants.

Extant conifer species may be susceptible to rapid environmental change owing to their long generation times, but could also be resilient due to high levels of standing genetic diversity. Hybridisation between closely related species can increase genetic diversity and generate novel allelic combinations capable of fuelling adaptive evolution. Our study unravelled the genetic architecture of adaptive evolution in a conifer hybrid zone formed between Pinus strobiformis and P. flexilis. Using a multifaceted approach emphasising the spatial and environmental patterns of linkage disequilibrium and ancestry enrichment, we identified recently introgressed and background genetic variants to be driving adaptive evolution along different environmental gradients. Specifically, recently introgressed variants from P. flexilis were favoured along freeze-related environmental gradients, while background variants were favoured along water availability-related gradients. We posit that such mosaics of allelic variants within conifer hybrid zones will confer upon them greater resilience to ongoing and future environmental change and can be a key resource for conservation efforts.

Genotyping-by-sequencing and ecological niche modeling illuminate phylogeography, admixture, and Pleistocene range dynamics in quaking aspen (Populus tremuloides).

Populus tremuloides is the widest-ranging tree species in North America and an ecologically important component of mesic forest ecosystems displaced by the Pleistocene glaciations. Using phylogeographic analyses of genome-wide SNPs (34,796 SNPs, 183 individuals) and ecological niche modeling, we inferred population structure, ploidy levels, admixture, and Pleistocene range dynamics of P. tremuloides, and tested several historical biogeographical hypotheses. We found three genetic lineages located mainly in coastal-Cascades (cluster 1), east-slope Cascades-Sierra Nevadas-Northern Rockies (cluster 2), and U.S. Rocky Mountains through southern Canadian (cluster 3) regions of the P. tremuloides range, with tree graph relationships of the form ((cluster 1, cluster 2), cluster 3). Populations consisted mainly of diploids (86%) but also small numbers of triploids (12%) and tetraploids (1%), and ploidy did not adversely affect our genetic inferences. The main vector of admixture was from cluster 3 into cluster 2, with the admixture zone trending northwest through the Rocky Mountains along a recognized phenotypic cline (Utah to Idaho). Clusters 1 and 2 provided strong support for the "stable-edge hypothesis" that unglaciated southwestern populations persisted in situ since the last glaciation. By contrast, despite a lack of clinal genetic variation, cluster 3 exhibited "trailing-edge" dynamics from niche suitability predictions signifying complete northward postglacial expansion. Results were also consistent with the "inland dispersal hypothesis" predicting postglacial assembly of Pacific Northwestern forest ecosystems, but rejected the hypothesis that Pacific-coastal populations were colonized during outburst flooding from glacial Lake Missoula. Overall, congruent patterns between our phylogeographic and ecological niche modeling results and fossil pollen data demonstrate complex mixtures of stable-edge, refugial locations, and postglacial expansion within P. tremuloides. These findings confirm and refine previous genetic studies, while strongly supporting a distinct Pacific-coastal genetic lineage of quaking aspen.

Utility of targeted sequence capture for phylogenomics in rapid, recent angiosperm radiations: Neotropical Burmeistera bellflowers as a case study.

Targeted sequence capture is a promising approach for large-scale phylogenomics. However, rapid evolutionary radiations pose significant challenges for phylogenetic inference (e.g. incomplete lineages sorting (ILS), phylogenetic noise), and the ability of targeted nuclear loci to resolve species trees despite such issues remains poorly studied. We test the utility of targeted sequence capture for inferring phylogenetic relationships in rapid, recent angiosperm radiations, focusing on Burmeistera bellflowers (Campanulaceae), which diversified into ~130 species over less than 3 million years. We compared phylogenies estimated from supercontig (exons plus flanking sequences), exon-only, and flanking-only datasets with 506-546 loci (~4.7 million bases) for 46 Burmeistera species/lineages and 10 outgroup taxa. Nuclear loci resolved backbone nodes and many congruent internal relationships with high support in concatenation and coalescent-based species tree analyses, and inferences were largely robust to effects of missing taxa and base composition biases. Nevertheless, species trees were incongruent between datasets, and gene trees exhibited remarkably high levels of conflict (~4-60% congruence, ~40-99% conflict) not simply driven by poor gene tree resolution. Higher gene tree heterogeneity at shorter branches suggests an important role of ILS, as expected for rapid radiations. Phylogenetic informativeness analyses also suggest this incongruence has resulted from low resolving power at short internal branches, consistent with ILS, and homoplasy at deeper nodes, with exons exhibiting much greater risk of incorrect topologies due to homoplasy than other datasets. Our findings suggest that targeted sequence capture is feasible for resolving rapid, recent angiosperm radiations, and that results based on supercontig alignments containing nuclear exons and flanking sequences have higher phylogenetic utility and accuracy than either alone. We use our results to make practical recommendations for future target capture-based studies of Burmeistera and other rapid angiosperm radiations, including that such studies should analyze supercontigs to maximize the phylogenetic information content of loci.

Tracing the footprints of a moving hybrid zone under a demographic history of speciation with gene flow.

A lack of optimal gene combinations, as well as low levels of genetic diversity, is often associated with the formation of species range margins. Conservation efforts rely on predictive modelling using abiotic variables and assessments of genetic diversity to determine target species and populations for controlled breeding, germplasm conservation and assisted migration. Biotic factors such as interspecific competition and hybridization, however, are largely ignored, despite their prevalence across diverse taxa and their role as key evolutionary forces. Hybridization between species with well-developed barriers to reproductive isolation often results in the production of offspring with lower fitness. Generation of novel allelic combinations through hybridization, however, can also generate positive fitness consequences. Despite this possibility, hybridization-mediated introgression is often considered a threat to biodiversity as it can blur species boundaries. The contribution of hybridization towards increasing genetic diversity of populations at range margins has only recently gathered attention in conservation studies. We assessed the extent to which hybridization contributes towards range dynamics by tracking spatio-temporal changes in the central location of a hybrid zone between two recently diverged species of pines: Pinus strobiformis and P. flexilis. By comparing geographic cline centre estimates for global admixture coefficient with morphological traits associated with reproductive output, we demonstrate a northward shift in the hybrid zone. Using a combination of spatially explicit, individual-based simulations and linkage disequilibrium variance partitioning, we note a significant contribution of adaptive introgression towards this northward movement, despite the potential for differences in regional population size to aid hybrid zone movement. Overall, our study demonstrates that hybridization between recently diverged species can increase genetic diversity and generate novel allelic combinations. These novel combinations may allow range margin populations to track favourable climatic conditions or facilitate adaptive evolution to ongoing and future climate change.

DNA barcode and minibarcode identification of freshwater fishes from Cerrado headwater streams in Central Brazil.

The extraordinary species diversity of the Neotropical freshwater fish fauna is world renown. Yet, despite rich species diversity, taxonomic and genetic resources for its Cerrado ichthyofauna remain poorly developed. We provide a reference library of 149 DNA barcodes for 39 species/lineages of Cerrado headwater stream fishes from the Brazilian Distrito Federal and nearby areas and test the utility of distance-based criteria, tree-based criteria and minibarcodes for specimen identification. Mean Kimura 2-parameter genetic distances within species to orders ranged 1·8-12·1%. However, mean intraspecific v. congeneric-interspecific distances (0·9-1·3%) overlapped extensively and distance-based barcoding failed to achieve correct identifications due to c. 4-12·1% error rates and 19·5% ambiguous identifications related to the presence of singletons. Overlap was reduced and best-match success rates improved drastically to 83·5% when Characidium barcodes representing potential misidentifications or undescribed species were removed. Tree-based monophyly criteria generally performed similarly to distance methods, correctly differentiating up to c. 85% of species/lineages despite neighbour-joining and Bayesian tree errors (random lineage-branching events, long-branch attraction). Five clusters (Ancistrus aguaboensis, Characidium spp., Eigenmannia trilineata, Hasemania hanseni and Hypostomus sp. 2) exhibited deep intraspecific divergences or para-/polyphyly and multiple Barcode Index Number assignments indicative of putative candidate species needing taxonomic re-examination. Sliding-window analyses also indicated that a 200 bp minibarcode region performed just as well at specimen identification as the entire barcode gene. Future DNA barcoding studies of Distrito Federal-Cerrado freshwater fishes will benefit from increased sampling coverage, as well as consideration of minibarcode targets for degraded samples and next-generation sequencing.

Phylogeny and divergence times of suckers (Cypriniformes: Catostomidae) inferred from Bayesian total-evidence analyses of molecules, morphology, and fossils.

Catostomidae ("suckers") is a diverse (76 species) and broadly distributed family of Holarctic freshwater fishes with a rich fossil record and a considerable number (∼35%) of threatened and imperiled species. We integrate DNA sequences (three mitochondrial genes, three nuclear genes), morphological data, and fossil information to infer sucker phylogenetic relationships and divergence times using Bayesian "total-evidence" methods, and then test hypotheses about the temporal diversification of the group. Our analyses resolved many nodes within subfamilies and clarified Catostominae relationships to be of the form ((Thoburniini, Moxostomatini), (Erimyzonini, Catostomini)). Patterns of subfamily relationships were incongruent, but mainly supported two placements of the Myxocyprininae; distinguishing these using Bayes factors lent strongest support to a model with Myxocyprininae sister to all remaining sucker lineages. We improved our Bayesian total-evidence dating analysis by excluding problematic characters, using a clock-partitioning scheme identified by Bayesian model selection, and employing a fossilized birth-death tree prior accommodating morphological data and fossils. The resulting chronogram showed that suckers evolved since the Late Cretaceous-Eocene, and that the Catostomini and Moxostomatini clades have accumulated species diversity since the early to mid-Miocene. These results agree with the fossil record and confirm previous hypotheses about dates for the origins of Catostomide and catostomine diversification, but reject previous molecular hypotheses about the timing of divergence of ictiobines, and between Asian-North American lineages. Overall, our findings from a synthesis of multiple data types enhance understanding of the phylogenetic relationships, taxonomic classification, and temporal diversification of suckers, while also highlighting practical methods for improving Bayesian divergence dating models by coupling phylogenetic informativeness profiling with relaxed-clock partitioning.

The role of hybridization during ecological divergence of southwestern white pine (Pinus strobiformis) and limber pine (P. flexilis).

Interactions between extrinsic factors, such as disruptive selection and intrinsic factors, such as genetic incompatibilities among loci, often contribute to the maintenance of species boundaries. The relative roles of these factors in the establishment of reproductive isolation can be examined using species pairs characterized by gene flow throughout their divergence history. We investigated the process of speciation and the maintenance of species boundaries between Pinus strobiformis and Pinus flexilis. Utilizing ecological niche modelling, demographic modelling and genomic cline analyses, we illustrated a divergence history with continuous gene flow. Our results supported an abundance of advanced generation hybrids and a lack of loci exhibiting steep transition in allele frequency across the hybrid zone. Additionally, we found evidence for climate-associated variation in the hybrid index and niche divergence between parental species and the hybrid zone. These results are consistent with extrinsic factors, such as climate, being an important isolating mechanism. A build-up of intrinsic incompatibilities and of coadapted gene complexes is also apparent, although these appear to be in the earliest stages of development. This supports previous work in coniferous species demonstrating the importance of extrinsic factors in facilitating speciation. Overall, our findings lend support to the hypothesis that varying strength and direction of selection pressures across the long lifespans of conifers, in combination with their other life history traits, delays the evolution of strong intrinsic incompatibilities.

Strategies for improving approximate Bayesian computation tests for synchronous diversification.

BACKGROUND: Estimating the variability in isolation times across co-distributed taxon pairs that may have experienced the same allopatric isolating mechanism is a core goal of comparative phylogeography. The use of hierarchical Approximate Bayesian Computation (ABC) and coalescent models to infer temporal dynamics of lineage co-diversification has been a contentious topic in recent years. Key issues that remain unresolved include the choice of an appropriate prior on the number of co-divergence events (Ψ), as well as the optimal strategies for data summarization. METHODS: Through simulation-based cross validation we explore the impact of the strategy for sorting summary statistics and the choice of prior on Ψ on the estimation of co-divergence variability. We also introduce a new setting (ß) that can potentially improve estimation of Ψ by enforcing a minimal temporal difference between pulses of co-divergence. We apply this new method to three empirical datasets: one dataset each of co-distributed taxon pairs of Panamanian frogs and freshwater fishes, and a large set of Neotropical butterfly sister-taxon pairs. RESULTS: We demonstrate that the choice of prior on Ψ has little impact on inference, but that sorting summary statistics yields substantially more reliable estimates of co-divergence variability despite violations of assumptions about exchangeability. We find the implementation of ß improves estimation of Ψ, with improvement being most dramatic given larger numbers of taxon pairs. We find equivocal support for synchronous co-divergence for both of the Panamanian groups, but we find considerable support for asynchronous divergence among the Neotropical butterflies. CONCLUSIONS: Our simulation experiments demonstrate that using sorted summary statistics results in improved estimates of the variability in divergence times, whereas the choice of hyperprior on Ψ has negligible effect. Additionally, we demonstrate that estimating the number of pulses of co-divergence across co-distributed taxon-pairs is improved by applying a flexible buffering regime over divergence times. This improves the correlation between Ψ and the true variability in isolation times and allows for more meaningful interpretation of this hyperparameter. This will allow for more accurate identification of the number of temporally distinct pulses of co-divergence that generated the diversification pattern of a given regional assemblage of sister-taxon-pairs.

Assessing species boundaries using multilocus species delimitation in a morphologically conserved group of neotropical freshwater fishes, the Poecilia sphenops species complex (Poeciliidae).

Accurately delimiting species is fundamentally important for understanding species diversity and distributions and devising effective strategies to conserve biodiversity. However, species delimitation is problematic in many taxa, including 'non-adaptive radiations' containing morphologically cryptic lineages. Fortunately, coalescent-based species delimitation methods hold promise for objectively estimating species limits in such radiations, using multilocus genetic data. Using coalescent-based approaches, we delimit species and infer evolutionary relationships in a morphologically conserved group of Central American freshwater fishes, the Poecilia sphenops species complex. Phylogenetic analyses of multiple genetic markers (sequences of two mitochondrial DNA genes and five nuclear loci) from 10/15 species and genetic lineages recognized in the group support the P. sphenops species complex as monophyletic with respect to outgroups, with eight mitochondrial 'major-lineages' diverged by ≥2% pairwise genetic distances. From general mixed Yule-coalescent models, we discovered (conservatively) 10 species within our concatenated mitochondrial DNA dataset, 9 of which were strongly supported by subsequent multilocus Bayesian species delimitation and species tree analyses. Results suggested species-level diversity is underestimated or overestimated by at least ~15% in different lineages in the complex. Nonparametric statistics and coalescent simulations indicate genealogical discordance among our gene tree results has mainly derived from interspecific hybridization in the nuclear genome. However, mitochondrial DNA show little evidence for introgression, and our species delimitation results appear robust to effects of this process. Overall, our findings support the utility of combining multiple lines of genetic evidence and broad phylogeographical sampling to discover and validate species using coalescent-based methods. Our study also highlights the importance of testing for hybridization versus incomplete lineage sorting, which aids inference of not only species limits but also evolutionary processes influencing genetic diversity.

Testing for shared biogeographic history in the lower Central American freshwater fish assemblage using comparative phylogeography: concerted, independent, or multiple evolutionary responses?

A central goal of comparative phylogeography is determining whether codistributed species experienced (1) concerted evolutionary responses to past geological and climatic events, indicated by congruent spatial and temporal patterns ("concerted-response hypothesis"); (2) independent responses, indicated by spatial incongruence ("independent-response hypothesis"); or (3) multiple responses ("multiple-response hypothesis"), indicated by spatial congruence but temporal incongruence ("pseudocongruence") or spatial and temporal incongruence ("pseudoincongruence"). We tested these competing hypotheses using DNA sequence data from three livebearing fish species codistributed in the Nicaraguan depression of Central America (Alfaro cultratus, Poecilia gillii, and Xenophallus umbratilis) that we predicted might display congruent responses due to co-occurrence in identical freshwater drainages. Spatial analyses recovered different subdivisions of genetic structure for each species, despite shared finer-scale breaks in northwestern Costa Rica (also supported by phylogenetic results). Isolation-with-migration models estimated incongruent timelines of among-region divergences, with A. cultratus and Xenophallus populations diverging over Miocene-mid-Pleistocene while P. gillii populations diverged over mid-late Pleistocene. Approximate Bayesian computation also lent substantial support to multiple discrete divergences over a model of simultaneous divergence across shared spatial breaks (e.g., Bayes factor [B 10] = 4.303 for Ψ [no. of divergences] > 1 vs. Ψ = 1). Thus, the data support phylogeographic pseudoincongruence consistent with the multiple-response hypothesis. Model comparisons also indicated incongruence in historical demography, for example, support for intraspecific late Pleistocene population growth was unique to P. gillii, despite evidence for finer-scale population expansions in the other taxa. Empirical tests for phylogeographic congruence indicate that multiple evolutionary responses to historical events have shaped the population structure of freshwater species codistributed within the complex landscapes in/around the Nicaraguan depression. Recent community assembly through different routes (i.e., different past distributions or colonization routes), and intrinsic ecological differences among species, has likely contributed to the unique phylogeographical patterns displayed by these Neotropical fishes.

Phylogeography and biogeography of the lower Central American Neotropics: diversification between two continents and between two seas.

Lower Central America (LCA) provides a geologically complex and dynamic, richly biodiverse model for studying the recent assembly and diversification of a Neotropical biota. Here, we review the growing literature of LCA phylogeography studies and their contribution to understanding the origins, assembly, and diversification of the LCA biota against the backdrop of regional geologic and climatic history, and previous biogeographical inquiry. Studies to date reveal that phylogeographical signal within taxa of differing distributions reflects a diversity of patterns and processes rivalling the complexities of LCA landscapes themselves. Even so, phylogeography is providing novel insights into regional diversification (e.g. cryptic lineage divergences), and general evolutionary patterns are emerging. Congruent multi-taxon phylogeographic breaks are found across the Nicaraguan depression, Chorotega volcanic front, western and central Panama, and the Darién isthmus, indicating that a potentially shared history of responses to regional-scale (e.g. geological) processes has shaped the genetic diversity of LCA communities. By contrast, other species show unique demographic histories in response to overriding historical events, including no phylogeographic structure at all. These low-structure or incongruent patterns provide some evidence for a role of local, ecological factors (e.g. long-distance dispersal and gene flow in plants and bats) in shaping LCA communities. Temporally, comparative phylogeographical structuring reflects Pliocene-Pleistocene dispersal and vicariance events consistent with the timeline of emergence of the LCA isthmus and its major physiographic features, e.g. cordilleras. We emphasise the need to improve biogeographic inferences in LCA through in-depth comparative phylogeography projects capitalising on the latest statistical phylogeographical methods. While meeting the challenges of reconstructing the biogeographical history of this complex region, phylogeographers should also take up the critical service to society of applying their work to the conservation of its fascinating biodiversity.

Paleoclimatic modeling and phylogeography of least killifish, Heterandria formosa: insights into Pleistocene expansion-contraction dynamics and evolutionary history of North American Coastal Plain freshwater biota.

BACKGROUND: Climatic and sea-level fluctuations throughout the last Pleistocene glacial cycle (~130-0 ka) profoundly influenced present-day distributions and genetic diversity of Northern Hemisphere biotas by forcing range contractions in many species during the glacial advance and allowing expansion following glacial retreat ('expansion-contraction' model). Evidence for such range dynamics and refugia in the unglaciated Gulf-Atlantic Coastal Plain stems largely from terrestrial species, and aquatic species Pleistocene responses remain relatively uninvestigated. Heterandria formosa, a wide-ranging regional endemic, presents an ideal system to test the expansion-contraction model within this biota. By integrating ecological niche modeling and phylogeography, we infer the Pleistocene history of this livebearing fish (Poeciliidae) and test for several predicted distributional and genetic effects of the last glaciation. RESULTS: Paleoclimatic models predicted range contraction to a single southwest Florida peninsula refugium during the Last Glacial Maximum, followed by northward expansion. We inferred spatial-population subdivision into four groups that reflect genetic barriers outside this refuge. Several other features of the genetic data were consistent with predictions derived from an expansion-contraction model: limited intraspecific divergence (e.g. mean mtDNA p-distance = 0.66%); a pattern of mtDNA diversity (mean Hd = 0.934; mean π = 0.007) consistent with rapid, recent population expansion; a lack of mtDNA isolation-by-distance; and clinal variation in allozyme diversity with higher diversity at lower latitudes near the predicted refugium. Statistical tests of mismatch distributions and coalescent simulations of the gene tree lent greater support to a scenario of post-glacial expansion and diversification from a single refugium than to any other model examined (e.g. multiple-refugia scenarios). CONCLUSIONS: Congruent results from diverse data indicate H. formosa fits the classic Pleistocene expansion-contraction model, even as the genetic data suggest additional ecological influences on population structure. While evidence for Plio-Pleistocene Gulf Coast vicariance is well described for many freshwater species presently codistributed with H. formosa, this species demography and diversification departs notably from this pattern. Species-specific expansion-contraction dynamics may therefore have figured more prominently in shaping Coastal Plain evolutionary history than previously thought. Our findings bolster growing appreciation for the complexity of phylogeographical structuring within North America's southern refugia, including responses of Coastal Plain freshwater biota to Pleistocene climatic fluctuations.

Molecular phylogeny and phylogeography of the Australian freshwater fish genus Galaxiella, with an emphasis on dwarf galaxias (G. pusilla).

The freshwater fauna of Southern Australia is primarily restricted to the southwestern and southeastern corners of the continent, and is separated by a large, arid region that is inhospitable to this biota. This geographic phenomenon has attracted considerable interest from biogeographers looking to explain evolutionary diversification in this region. Here, we employed phylogenetic and phylogeographic approaches to evaluate the effect of this barrier on a group of four galaxiid fish species (Galaxiella) endemic to temperate Southern Australia. We also tested if continental shelf width has influenced connectivity among populations during low sea levels when rivers, now isolated, could have been connected. We addressed these questions by sampling each species across its range using multiple molecular markers (mitochondrial cytochrome b sequences, nuclear S7 intron sequences, and 49 allozyme loci). These data also allowed us to assess species boundaries, to refine phylogenetic affinities, and to estimate species ages. Interestingly, we found compelling evidence for cryptic species in G. pusilla, manifesting as allopatric eastern and western taxa. Our combined phylogeny and dating analysis point to an origin for the genus dating to the early Cenozoic, with three of the four species originating during the Oligocene-Miocene. Each Galaxiella species showed high levels of genetic divergences between all but the most proximate populations. Despite extensive drainage connections during recent low sea levels in southeastern Australia, populations of both species within G. pusilla maintained high levels of genetic structure. All populations experienced Late Pleistocene-Holocene population growth, possibly in response to the relaxation of arid conditions after the last glacial maximum. High levels of genetic divergence and the discovery of new cryptic species have important implications for the conservation of this already threatened group of freshwater species.