Northern Refugia Research Articles

Spatial Distribution and Dispersal Patterns of Central North American Freshwater Crayfish (Decapoda: Cambaridae) with Emphasis on Implications of Glacial Refugia

Spatial distributions of crayfish were evaluated in relation to glacial geography and possible modes of dispersal from refugia. Species dispersal patterns were a priori hypothesized and tested using principle components analysis (PCA). PCA factor loading plots were evaluated for hypothesized crayfish dispersal patterns. Cambarus laevis was limited to the unglaciated region, while Orconectes immunis, Orconectes virilis, and Procambarus gracilis may have dispersed from western glacial refugia in the upper Missouri drainage. Fallicambarus fodiens and Procambarus acutus dispersed from south of the glacial advance within the Mississippi embayment. Previous dispersal hypotheses for Orconectes propinquus recognized that northern refugia may have been from the Driftless Area in Wisconsin and Illinois and may have invaded more than one refuge since this species was common in unglaciated areas of southwestern Indiana. Orconectes indianensis center of abundance is in the unglaciated region. Disjunct populations likely dispersed into temporary glacial lakes that, when receded, left populations in previously glaciated areas. Cambarus polychromatus possibly dispersed from southern refugia, while Cambarus sp. A cf. diogenes dispersed from southern refugia not sympatric with C. polychromatus. The glacial refugia included western and southern areas of the glacial maximum; however, northern dispersal routes may be important with global climate change.

Contrasting patterns of genetic structuring in natural populations of Arabidopsis lyrata Subsp. petraea across different regions in northern Europe.

Level and partitioning of genetic diversity is expected to vary between contrasting habitats, reflecting differences in strength of ecological and evolutionary processes. Therefore, it is necessary to consider processes acting on different time scales when trying to explain diversity patterns in different parts of species' distributions. To explore how historical and contemporary factors jointly may influence patterns of genetic diversity and population differentiation, we compared genetic composition in the perennial herb Arabidopsis lyrata ssp. petraea from the northernmost parts of its distribution range on Iceland to that previously documented in Scandinavia. Leaf tissue and soil were sampled from ten Icelandic populations of A. lyrata. Seedlings were grown from soil samples, and tissue from above-ground and seed bank individuals were genotyped with 21 microsatellite markers. Seed bank density in Icelandic populations was low but not significantly different from that observed in Norwegian populations. While within-population genetic diversity was relatively high on Iceland (H E = 0.35), among-population differentiation was low (F ST = 0.10) compared to Norwegian and Swedish populations. Population differentiation was positively associated with geographical distance in both Iceland and Scandinavia, but the strength of this relationship varied between regions. Although topography and a larger distribution range may explain the higher differentiation between mountainous Norwegian relative to lowland populations in Sweden, these factors cannot explain the lower differentiation in Icelandic compared to Swedish populations. We propose that low genetic differentiation among Icelandic populations is not caused by differences in connectivity, but is rather due to large historical effective population sizes. Thus, rather than contemporary processes, historical factors such as survival of Icelandic lineages in northern refugia during the last glacial period may have contributed to the observed pattern.

Spatial structure in European moose (Alces alces): genetic data reveal a complex population history

AbstractAimMoose, Alces alces (Linnaeus, 1758), survived the European Pleistocene glaciations in multiple southern refugia, in a northern refugium near the Carpathians and possibly in other locations. During the second millennium ad, moose were nearly extirpated in Europe and only recolonized their current range after World War II. The number and location of refugia during the Pleistocene and recent population lows may have affected the current genetic diversity. We sought to characterize the genetic diversity in European moose in order to determine its genetic structure and the location of genetic hotspots as a way of inferring its population history and the number of Last Glacial Maximum (LGM) refugia.LocationEurope.MethodsWe sequenced 538 nucleotides from the mitochondrial control region of 657 moose from throughout the species' European range. We estimated diversity within and among 16 sampling localities, and used samova to cluster sampling locations into subpopulations. We constructed phylogenetic trees and median‐joining networks to examine systematic relationships, and conducted Bayesian analysis of the coalescent and used mismatch distributions and approximate Bayesian computation to infer demographic history.ResultsEstonia had the highest nucleotide diversity, and western Belarus had the highest haplotype diversity. We observed four regional populations from the samova analysis. We found three haplogroups in European moose, probably representing lineages conserved in different refugia during the Pleistocene. European moose underwent spatial expansion after the LGM, but did not undergo demographic expansion. The effective population size has declined markedly within the last 2000 years.Main conclusionsThe current levels and distribution of genetic diversity in European moose indicate the effects both of Pleistocene glaciations and of a recent bottleneck, probably associated with anthropogenic influences such as pastoralization and hunting, and a very recent re‐expansion. We show that both historical and recent events can influence the diversity and distribution of a large mammal on a large scale.

Unique mitochondrial DNA lineages in Irish stickleback populations: cryptic refugium or rapid recolonization?

Repeated recolonization of freshwater environments following Pleistocene glaciations has played a major role in the evolution and adaptation of anadromous taxa. Located at the western fringe of Europe, Ireland and Britain were likely recolonized rapidly by anadromous fishes from the North Atlantic following the last glacial maximum (LGM). While the presence of unique mitochondrial haplotypes in Ireland suggests that a cryptic northern refugium may have played a role in recolonization, no explicit test of this hypothesis has been conducted. The three-spined stickleback is native and ubiquitous to aquatic ecosystems throughout Ireland, making it an excellent model species with which to examine the biogeographical history of anadromous fishes in the region. We used mitochondrial and microsatellite markers to examine the presence of divergent evolutionary lineages and to assess broad-scale patterns of geographical clustering among postglacially isolated populations. Our results confirm that Ireland is a region of secondary contact for divergent mitochondrial lineages and that endemic haplotypes occur in populations in Central and Southern Ireland. To test whether a putative Irish lineage arose from a cryptic Irish refugium, we used approximate Bayesian computation (ABC). However, we found no support for this hypothesis. Instead, the Irish lineage likely diverged from the European lineage as a result of postglacial isolation of freshwater populations by rising sea levels. These findings emphasize the need to rigorously test biogeographical hypothesis and contribute further evidence that postglacial processes may have shaped genetic diversity in temperate fauna.

Multiple cryptic refugia of forest grass Bromus benekenii in Europe as revealed by ISSR fingerprinting and species distribution modelling

Despite not having been fully recognized, the cryptic northern refugia of temperate forest vegetation in Central and Western Europe are one of the most important in the Holocene history of the vegetation on the subcontinent. We have studied a forest grass Bromus benekenii in 39 populations in Central, Western and Southern Europe with the use of PCR-ISSR fingerprinting. The indices of genetic population diversity, multivariate, and Bayesian analyses, supplemented with species distribution modelling have enabled at least three putative cryptic northern refugial areas to be recognized: in Western Europe—the Central and Rhenish Massifs, in Central Europe—the Bohemia–Moravia region and in the Eastern/Western Carpathians. Central Poland is the regional genetic melting-pot where several migratory routes might have met. Southern Poland had a different postglacial history and was under the influence of an Eastern/Western Carpathian cryptic refugium. More forest species should be checked in a west–east gradient in Europe to corroborate the hypothesis on the Western European glacial refugia.

Mountain barriers and river conduits: phylogeographical structure in a large, mobile lizard (Varanidae: Varanus varius) from eastern Australia

AbstractAimAcross eastern Australia, mountain ranges (the Great Dividing Range) and river catchments (the Murray–Darling Basin) are likely to have shaped the phylogeographical structure of many species. We address how such processes have influenced the phylogeography of the lace monitor, Varanus varius, a large mobile lizard.LocationEastern and south‐eastern Australia.MethodsPhylogeographical hypotheses were tested using up to 90 museum and field‐collected samples from across the entire species' range; a 671‐bp region of the mtDNA gene ND4 was sequenced and all individuals were genotyped (eight microsatellite loci).ResultsMaximum‐likelihood analysis of sequence data revealed three geographically separate clades, with divergences estimated to have occurred during the Pleistocene. The south‐eastern clade showed an expansion pattern from northern refugia and dispersal appears to have occurred along the Murray–Darling river system. Microsatellite analyses support mtDNA clades but indicate secondary contact in the Hunter Valley, New South Wales.Main conclusionsOur results indicate that phylogeographical structure and contemporary gene flow in Varanus varius is shaped by dispersal capacity, geographical barriers and the presence of ancient river corridors. Indeed, only the most significant geological (McPherson Range) and habitat barriers (Burdekin Gap) appear to limit gene flow in this species. The expansion of the clade on the western side of the Great Dividing Range suggests that ancient riparian corridors have facilitated extensive gene flow. Our study highlights the importance of understanding a species' ecological dynamics when examining broad‐scale evolutionary patterns.

Phylogeography of North American herbaceous Smilax (Smilacaceae): Combined AFLP and cpDNA data support a northern refugium in the Driftless Area

The genetic structure of North American herbaceous Smilax, a clade of closely related understory herbs that has a wide and disjunct distribution, was investigated to test the hypothesis of whether a northern refugium in the upper Midwest may have existed for these plants during the last glaciation. We analyzed 33 populations sampled from California and throughout the eastern United States using AFLP and chloroplast DNA sequence data. Individuals of S. jamesii from northern California formed a clade sister to the eastern North American species, and they likely diverged from each other during the Pleistocene. Among the eastern species, two lineages were found on opposite sides of the Appalachian Mountains. The populations near the "Driftless Area" contained most of the chlorotypes found in the Midwest, including a unique one endemic to this area, and the AFLP data indicated that one population from this area has the highest frequency-down-weighted-marker value. This study, and others that have focused on mammals, amphibians, and woody plants, provides further evidence for the debate over whether the upper Midwest's "Driftless Area" may have hosted a biologically diverse northern glacial refugium. Herbaceous species of Smilax from eastern North American exhibit an Appalachian discontinuity. They appear to have persisted in multiple refugia both east and west of the Appalachians, with several populations persisting in situ through the Last Glacial Maximum (LGM). We suggest that they experienced localized expansion after the LGM, possibly followed by subsequent contact between the Midwest and the East Coastal lineages.

Montane refugia predict population genetic structure in theLarge‐blotchedEnsatina salamander

Understanding the biotic consequences of Pleistocene range shifts and fragmentation remains a fundamental goal in historical biogeography and evolutionary biology. Here, we combine species distribution models (SDM) from the present and two late Quaternary time periods with multilocus genetic data (mitochondrial DNA and microsatellites) to evaluate the effect of climate-induced habitat shifts on population genetic structure in the Large-blotched Ensatina (Ensatina eschscholtzii klauberi), a plethodontid salamander endemic to middle and high-elevation conifer forest in the Transverse and Peninsular Ranges of southern California and northern Baja California. A composite SDM representing the range through time predicts two disjunct refugia, one in southern California encompassing the core of the species range and the other in the Sierra San Pedro Mártir of northern Baja California at the southern limit of the species range. Based on our spatial model, we would expect a pattern of high connectivity among populations within the northern refugium and, conversely, a pattern of isolation due to long-term persistence of the Sierra San Pedro Mártir population. Our genetic results are consistent with these predictions based on the hypothetical refugia in that (i) historical measures of population connectivity among stable areas are correlated with gene flow estimates; and (ii) there is strong geographical structure between separate refugia. These results provide evidence for the role of recent climatic change in shaping patterns of population persistence and connectivity within the Transverse and Peninsular Ranges, an evolutionary hotspot.

Comparative phylogeography of the ants Myrmica ruginodis and Myrmica rubra

AbstractAimHere we compare the phylogeographies of the ants Myrmica ruginodis and Myrmica rubra. We test the hypotheses that owing to differences in their cold tolerance and life‐history traits, M. ruginodis has less differentiated populations, a weaker phylogeographical structure and some Pleistocene refugia further north than M. rubra.LocationThe Palaearctic region, from western Europe to Siberia and from Fennoscandia to the northern parts of the Mediterranean peninsulas and Turkey.MethodsWe sequenced mitochondrial DNA from M. ruginodis samples collected in 53 localities, and used the GenBank sequences of M. rubra individuals from 95 localities, presented in previous work. We studied the genetic diversity of M. ruginodis, the differentiation of its populations, and the geographical distribution and expansion of its haplogroups.ResultsPopulations of M. rubra were genetically slightly more differentiated than those of M. ruginodis, but we did not obtain differences in their phylogeographical structures. Both species had a west–east phylogeographical structure with secondary contact zone in between, and many haplogroups were widely distributed.Main conclusionsThe different life histories of the species (monogyne–monocalic versus polygyne–polycalic) seem to have affected population differentiation on a local rather than a phylogeographical scale. Myrmica ruginodis survived the last glaciation in several refugia in the Mediterranean peninsulas and probably in Siberia – as did M. rubra – but also in Turkey. Circumstantial evidence supported the hypothesis of additional, more northerly refugia of the cold‐tolerant M. ruginodis close to the southern edge of the permafrost of the Last Glacial Maximum, whereas for M. rubra a northern refugium in western Europe may have also existed. Post‐glacial recolonization of both species took place from both the south‐west and south‐east, and probably also from the east. The origins of the eastern lineages of M. ruginodis were, however, difficult to localize, which stresses the importance of extensive sampling in the east when studying the phylogeography of European taxa.

Phylogeography and genetic diversity of East Asian Neolitsea sericea (Lauraceae) based on variations in chloroplast DNA sequences

Neolitsea sericea is an evergreen broad leaved tree in the warm-temperate regions of East Asia. This area is a hotspot for plant species richness and endemism caused by dynamic changes in land configuration during the Quaternary. However, the historical migration of such evergreen tree species is still poorly understood. In an attempt to reconstruct the phylogeographic history of N. sericea during the Quaternary, we identified the chloroplast DNA haplotypes of 287 individuals from 33 populations covering almost all of its geographic range. Analyses were based on sequence data from the trnL-F, psbC-trnS, and rps16 regions. Nine haplotypes were identified. The majority included ancestral types in the southwestern part of the main islands of Japan, with other region-specific haplotypes being found in populations on the Korean Peninsula, Taiwan (Isl. Lanyu), and elsewhere in Japan. A statistical parsimony network revealed two lineages derived from Japanese main islands. One was represented on the Korean Peninsula, the other on Isl. Lanyu. The current distribution of N. sericea has been shaped by colonization via land bridges. During the glacial periods, two primary, but separate migration routes were followed--from the southwestern part of the Japanese main islands to either the Korean Peninsula or Taiwan. In addition, we believe the Zhoushan populations were shaped by post-glacial processes through an ECS land bridge (East China Sea basin) from northern refugia that existed during the late Pleistocene.

One species, three Pleistocene evolutionary histories: phylogeography of the Italian crested newt, Triturus carnifex.

Phylogeographic patterns of temperate species from the Mediterranean peninsulas have been investigated intensively. Nevertheless, as more phylogeographies become available, either unique patterns or new lines of concordance continue to emerge, providing new insights on the evolution of regional biotas. Here, we investigated the phylogeography and evolutionary history of the Italian crested newt, Triturus carnifex, through phylogenetic, molecular dating and population structure analyses of two mitochondrial gene fragments (ND2 and ND4; overall 1273 bp). We found three main mtDNA lineages having parapatric distribution and estimated divergence times between Late Pliocene and Early Pleistocene. One lineage (S) was widespread south of the northern Apennine chain and was further geographically structured into five sublineages, likely of Middle Pleistocene origin. The second lineage (C) was widespread throughout the Padano–Venetian plain and did not show a clear phylogeographic structure. The third lineage (N) was observed in only two populations located on western Croatia/Slovenia. Results of analysis of molecular variance suggested that partitioning populations according to the geographic distribution of these lineages and sublineages explains 76% of the observed genetic variation. The phylogeographic structure observed within T. carnifex and divergence time estimates among its lineages, suggest that responses to Pleistocene environmental changes in this single species have been as diverse as those found previously among several codistributed temperate species combined. Consistent with the landscape heterogeneity, physiographic features, and palaeogeographical evolution of its distribution range, these responses encompass multiple refugia along the Apennine chain, lowland refugia in large peri-coastal plains, and a ‘cryptic’ northern refugium.

A northern refugium of the Mediterranean water shrew Neomys anomalus in Słowiński National Park (N Poland)

Abstract The influence of recent anthropogenic and climatic changes on the distribution or population size of individual species should be especially pronounced at the boundaries of their geographical ranges. Therefore, in summers 2008-2010, we live-trapped small mammals and analysed habitat features on 14 study plots comprising various wet habitats of Słowiński National Park (at the Baltic coast in northern Poland) in order to recognize the current distribution and population size of Neomys anomalus Cabrera, 1907 in its northernmost population in Europe. N. anomalus was, however, captured only on one plot - a wet, unmown meadow crossed by unkempt drainage ditches, situated on the Gać Peninsula (Półwysep Gacki). This result, compared to historical data, suggests that neither the abundance of N. anomalus has increased nor its range has been extended. We give a detailed description of the habitat in this study plot. The information can be useful in the protection of this rare shrew species with very specific habitat requirements.

Molecular markers provide insights into contemporary and historic gene flow for a non‐migratory species

Hairy woodpeckers Picoides villosus are a common, year round resident with distinct plumage and morphological variation across North America. We genotyped 335 individuals at six variable microsatellite loci and analyzed 322 mtDNA control region sequences in order to examine the role of contemporary and historical barriers to gene flow. In addition we combined genetic analyses with ecological niche modelling to test if hairy woodpeckers were isolated in northern refugia (Alaska, Newfoundland and the Queen Charlotte Islands) during the last glacial maximum. Genetic analyses revealed that gene flow among North American hairy woodpecker populations is restricted, but not to the extent predicted for a sedentary species. Populations clustered into two main genetic groups, east and west of the Great Plains in the south and the Rocky Mountains in the north. Contact zones between the two main genetic groups exist in central British Columbia and Washington, but are narrow. Within each group we found additional population structure with genetic breaks between subgroups in the geographic west corresponding to breaks in forested habitat and physical barriers like open expanses of water. Population genetic patterns for hairy woodpeckers have resulted from isolation in multiple southern refugia with the current distribution of genetic groups resulting from post‐glacial expansion and subsequent reduction in gene flow. While populations in Alaska, Newfoundland and the Queen Charlotte Islands are genetically distinct from other populations, we found no evidence of these areas acting as refugia throughout the Pleistocene. Atlantic Canada populations contained unique haplotypes raising the possibility of a separate colonization from the rest of eastern Canada. The endemic subspecies on the island of Newfoundland is not genetically distinct from their closest mainland population unlike the Queen Charlotte Island subspecies.

High latitudes and high genetic diversity: Phylogeography of a widespread boreal bird, the gray jay (Perisoreus canadensis)

We describe range-wide phylogeographic variation in gray jays (Perisoreus canadensis), a boreal Nearctic corvid that occurs today primarily in recently glaciated regions. Phylogenetic analysis of mitochondrial DNA (1041 base pairs ND2 gene; N=205, 50 localities) revealed four reciprocally monophyletic groups. One widespread clade occurs across the North American boreal zone, from Newfoundland to Alaska and southwest into Utah. Three other clades occur at lower latitudes in the montane West in Colorado, the northern Rocky Mountains, and the Pacific Northwest respectively. The geographic distribution of clades in gray jays corresponds with a general pattern that is emerging for boreal taxa, having one widespread northern clade and one or more geographically restricted southwestern clades. Population genetic analyses indicate that the larger boreal clade is genetically structured and harbors significantly more genetic diversity than those clades occurring at lower latitudes. Species distribution modeling (SDM) revealed multiple putative Pleistocene refugia including several occurring at higher latitudes. We suggest that multiple post-glacial colonization routes, some of which originate from these northern refugia, are responsible for the relatively high genetic diversity at high latitudes. Conversely, lower latitude clades show little variation, probably as a result of historical restriction to smaller geographical areas with smaller long-term population sizes. This ‘upside-down’ pattern of genetic diversity contrasts with the conventional view that populations of north-temperate species occupying previously glaciated habitats should possess lower levels of diversity than their southern counterparts.

Northern refugia and recent expansion in the North Sea: the case of the wrasse Symphodus melops (Linnaeus, 1758)

Pleistocene climate changes have imposed extreme conditions to intertidal rocky marine communities, forcing many species to significant range shifts in their geographical distributions. Phylogeographic analyses based on both mitochondrial and nuclear genetic markers provide a useful approach to unravel phylogeographic patterns and processes of species after this time period, to gain general knowledge of how climatic changes affect shifts in species distributions. We analyzed these patterns on the corkwing wrasse (Symphodus melops, Labridae), a rocky shore species inhabiting North Sea waters and temperate northeastern Atlantic Ocean from Norway to Morocco including the Azores, using a fragment of the mitochondrial control region and the first intron of the nuclear S7 ribosomal protein gene. We found that S. melops shows a clear differentiation between the Atlantic and the Scandinavian populations and a sharp contrast in the genetic diversity, high in the south and low in the north. Within each of these main geographic areas there is little or no genetic differentiation. The species may have persisted throughout the last glacial maximum in the southern areas as paleotemperatures were not lower than they are today in North Scandinavia. The North Sea recolonization most likely took place during the current interglacial and is dominated by a haplotype absent from the south of the study area, but present in Plymouth and Belfast. The possibility of a glacial refugium in or near the English Channel is discussed.

Population genetic structure and phylogeography of Mesokalliapseudes macsweenyi (Crustacea: Tanaidacea) in the northwestern Atlantic and Gulf of Mexico

Various studies of Gulf of Mexico/Atlantic populations have detected a phylogeographic break created by the Florida peninsula despite the fact that many of these species possess planktonic larvae and/or highly vagile adults. In light of these results, one would predict that the genetic structure in a species with limited dispersal ability would be heavily influenced by the geologic history of this region. Mesokalliapseudes macsweenyi is a direct developing benthic crustacean that occurs in coastal regions of the southeastern United States, ranging from South Carolina to Mississippi. Mitochondrial cytochrome oxidase subunit I (COI) sequences were obtained from 159 specimens representing eight collection sites from across a large portion of its known range to assess the population genetic and phylogeographic structure. Significant genetic structure existed among populations and between the Atlantic/Gulf of Mexico regions, consistent with expectations based on its life history of direct larval development and a sedentary adult stage. Demographic analyses inferred a population expansion in the Gulf of Mexico, which might reflect a Pleistocene post-glacial range expansion. The significant population structure exhibited by the northern Gulf of Mexico populations is likely the product of isolation among populations post expansion, but the diversity of haplotypes might also reflect undersampled variation or the persistence of individuals in northern refugia during the Pleistocene.

Range-wide chloroplast and mitochondrial DNA imprints reveal multiple lineages and complex biogeographic history for Douglas-fir

The contemporary genetic structure of species offers key imprints of how organisms responded to past geological and climatic events, which have played a crucial role in shaping the current geographical distribution of north-temperate organisms. In this study, range-wide patterns of genetic variation were examined in Douglas-fir (Pseudotsuga menziesii), a dominant forest tree species distributed from Mexico to British Columbia in western North America. Two organelle DNA markers with contrasting modes of inheritance were genotyped for 613 individuals from 44 populations. Two mitotypes and 42 chlorotypes were recovered in this survey. Both genomes showed significant population subdivision, indicative of limited gene flow through seeds and pollen. Three distinct cpDNA lineages corresponding to the Pacific Coast, the Rocky Mountains, and Mexico were observed. The split time of the two lineages from the Rockies lineage was dated back to 8.5 million years (Ma). The most recent common ancestors of Mexican and coastal populations were estimated at 3.2 and 4.8 Ma, respectively. The northern populations of once glaciated regions were characterized by a high level of genetic diversity, indicating a large zone of contact between ancestral lineages. A possible northern refugium was also inferred. The Mexican lineage, which appeared established by southward migration from the Rockies lineage, was characterized by the lowest genetic diversity but highest population differentiation. These results suggest that the effects of Quaternary climatic oscillations on the population dynamics and genetic diversity of Douglas-fir varied substantially across the latitudinal section. The results emphasize the pressing need for the conservation of Mexican Douglas-fir.

Comment: A Last Glacial Maximum pollen record from Bodmin Moor showing a possible cryptic northern refugium in southwest England. (Kelly et al., 2010)

Comment: A Last Glacial Maximum pollen record from Bodmin Moor showing a possible cryptic northern refugium in southwest England. (Kelly <i>et al.</i>, 2010)

Reply to Comment: A Last Glacial Maximum pollen record from Bodmin Moor showing a possible cryptic northern refugium in southwest England. (Kelly et al., 2010)

Reply to Comment: A Last Glacial Maximum pollen record from Bodmin Moor showing a possible cryptic northern refugium in southwest England. (Kelly <i>et al.</i>, 2010)

Phylogeography of endemic toads and post-Pliocene persistence of the Brazilian Atlantic Forest

The Plio–Pleistocene refugia hypothesis recently gained support in explaining Brazilian Atlantic Forest megadiversity from combined analyses of species paleodistributions and genetic diversity. Here we examine genetic differentiation and historical distributions in the Rhinella crucifer group of toads, endemic to and widely distributed within this biome. We analyzed sequences of mitochondrial (control region, ND1, and ND2) and nuclear (β-crystallin and rhodopsin) DNA markers from 65 individuals representing five species. We found deep structure across the range at mitochondrial markers; genetic diversity is geographically structured in four main haplotype clades with the oldest divergence, dated to the Pliocene, between the southernmost populations and other regions of the species’ range. Remaining populations are distributed in haplotype clades that may have diverged throughout the Pleistocene. Our paleoecological distribution models support a scenario of habitat fragmentation associated with glacial cycling, but we found limited congruence of phylogeographic patterns with the refugia. We found that some genetic breaks geographically coincide with putative barriers associated to neotectonic activity, but finer-scale sampling will be necessary to test the relative importance of distinct isolation mechanisms. Overall, the data refute the recently proposed hypothesis of a southern Holocene colonization of the Atlantic Forest from northern refugia, suggesting instead persistence of forested habitats in the south. Our unexpected results underscore the need to consider distinct organismal histories in planning biome-level conservation. We discuss species correspondence to clades recovered in our phylogenetic analyses.