Maternal transmission of bacterial microbiota during embryonic development in a viviparous lizard.

IMPORTANCE: We investigated the presence and diversity of bacteria in the embryos of the viviparous lizard Sceloporus grammicus and their amniotic environment. We compared this diversity to that found in the maternal intestine, mouth, and cloaca. We detected bacterial DNA in the embryos, albeit with a lower bacterial species diversity than found in maternal tissues. Most of the bacterial species detected in the embryos were also found in the mother, although not all of them. Interestingly, we detected a high similarity in the composition of bacterial species among embryos from different mothers. These findings suggest that there may be a mechanism controlling the transmission of bacteria from the mother to the embryo. Our results highlight the possibility that the interaction between maternal bacteria and the embryo may affect the development of the lizards.

Living in the mountains: Thermal ecology and freezing tolerance of the lizard Abronia taeniata (Squamata: Anguidae).

The impact of daily and seasonal variation in environmental temperature on lizards is important, since their physiological processes are body temperature dependent. Lizards that occupy mountainous areas must have been favoured to colonize such habitats through selection on thermal biology traits to thermoregulate effectively. Moreover, mountain lizards may be able to maintain their activity near their minimum critical temperature and even have antifreeze mechanisms. Tolerance of freezing is related to the biosynthesis of cryoprotective molecules, such as glucose, whose concentration may increase after freezing. The aims of the present work were: (1) study the thermoregulation of the viviparous lizard Abronia taeniata, and (2) determine its survival and/or tolerance to freezing. This species occurs in pine forests, pine-oak forests, and mountain mesophilic forests in areas that reach freezing temperatures. In the field, we recorded air, substrate, and body temperatures at capture time of the lizards, and registered operative temperatures at the study area. In the laboratory, we determined thermal preferences, crystallization point, and blood glucose levels of individuals before and after freezing. We found out that A. taeniata sustains activity in a wide range of temperatures, actively avoids thermally favourable microhabitats in spring, and is a moderate thermoregulator during autumn and winter. In A. taeniata, the body temperatures are tightly linked to air and substrate temperatures. Seasonality had an effect over body temperature, preferred temperatures and thermoregulatory effectiveness indices. When exposed to temperatures below zero, A. taeniata showed an increase in blood glucose levels, which aided them in surviving freezing. Taken together, our results suggest that A. taeniata may sustain activity at low environmental temperatures, due to an effective behavioural thermoregulation, and in case temperatures of its habitat go below zero, is also capable of tolerate freezing.

Exceptional parallelisms characterize the evolutionary transition to live birth in phrynosomatid lizards.

Viviparity, an innovation enhancing maternal control over developing embryos, has evolved >150 times in vertebrates, and has been proposed as an adaptation to inhabit cold habitats. Yet, the behavioral, physiological, morphological, and life history features associated with live-bearing remain unclear. Here, we capitalize on repeated origins of viviparity in phrynosomatid lizards to tease apart the phenotypic patterns associated with this innovation. Using data from 125 species and phylogenetic approaches, we find that viviparous phrynosomatids repeatedly evolved a more cool-adjusted thermal physiology than their oviparous relatives. Through precise thermoregulatory behavior viviparous phrynosomatids are cool-adjusted even in warm environments, and oviparous phrynosomatids warm-adjusted even in cool environments. Convergent behavioral shifts in viviparous species reduce energetic demand during activity, which may help offset the costs of protracted gestation. Whereas dam and offspring body size are similar among both parity modes, annual fecundity repeatedly decreases in viviparous lineages. Thus, viviparity is associated with a lower energetic allocation into production. Together, our results indicate that oviparity and viviparity are on opposing ends of the fast-slow life history continuum in both warm and cool environments. In this sense, the 'cold climate hypothesis' fits into a broader range of energetic/life history trade-offs that influence transitions to viviparity.

Habitat thermal quality for Gopherus evgoodei in tropical deciduous forest and consequences of habitat modification by buffelgrass.

Tortoises of the genus Gopherus evolved in North America and have survived major environmental challenges in the past 40 million years. However, this genus now faces multiple anthropogenic threats, such as the introduction of invasive plant species. Buffelgrass (Cenchrus ciliaris) is considered one of the greatest threats to arid and tropical ecosystems, where gopher tortoises inhabit, because the grass displaces native flora and fauna. Modification of the environment as a result of this invasive plant portends an alteration of the available thermal landscape. The aim of this paper is twofold: 1) to evaluate the thermal quality of the primary habitat of Gopherus evgoodei (tropical deciduous forest [TDF], and 2) determine the potential thermal changes due to habitat modification by buffelgrass. First, we obtained data on body temperature of active tortoises in semi-captivity. Second, we measured the operative environmental temperature during 5 years at three sites south of Sonora, Mexico that support G. evgoodei: a) a pristine TDF (Conserved-TDF); b) a forest patch surrounded by introduced buffelgrass pasture (Partial-TDF); and c) an introduced buffelgrass pasture area (Buffel-Pasture). Our results demonstrate that the intact microhabitats within the TDF provide G. evgoodei with high thermal quality at both spatial and temporal scales. However modified habitat by buffelgrass had higher operative temperatures for G. evgoodei than TDF. The thermal quality of the sites disturbed with buffelgrass can exceed the thermal requirements of G. evgoodei by up to 25 °C. Finally, we discussed potential collateral effects of habitat modification by invasion by buffelgrass.

Thermoregulation of Liolaemus aparicioi (Iguania: Liolaemidae) along a 1000 m elevational gradient in La Paz Valley, La Paz, Bolivia.

Lizard species have diverse behavioral and physiological responses to thermo-environmental conditions, which allow them to inhabit a broad range of latitudes and elevations. Because the availability of suitable thermal resources is limited and more variable at high-elevation environments than at lower elevations, we expect high-elevation lizards to be constrained in their thermoregulation relative to lizards at lower elevations by the fewer available thermal resources to reach optimal temperatures (colder environment). We studied the thermal biology of an endemic and Critically Endangered lizard, Liolaemus aparicioi, to assess its thermal responses along a 1000 m elevational gradient in La Paz Valley from May to August of 2015 (dry season). We took field body and microhabitat temperatures at capture sites (substrate and air above ground), and body size (snout-vent length and mass) of individuals at Taypichullo (3000 m asl), Gran Jardín de la Revolución Municipal Park (3500 m asl), and Taucachi (4000 m asl) localities. Operative temperatures were taken from calibrated models deployed in different available microhabitats. Preferred temperatures and thermal tolerance limits were determined in laboratory settings for lizards from each locality. Field body, microhabitat, and operative temperatures decreased with increasing elevation and differed between sexes. Lizards at the high elevation locality had the lowest thermoregulatory efficiency as compared with the mid and lower elevation localities. In laboratory measurements, while the preferred temperatures varied between sexes, pooled preferred temperatures and thermal tolerances were similar in all localities. Although thermal resources at high elevation can limit thermoregulatory possibilities in L. aparicioi, behavioral microhabitat use, time allocated to thermoregulation, and physiological adjustments seem to be possible strategies to counteract thermal costs along elevational gradients.

Thermal ecophysiology of Basiliscus galeritus (Squamata: Corytophanidae) in two populations at different altitudes: Does the crest participate actively in thermoregulation?

Thermoregulation is a critical process for ectotherms as non-avian reptiles, since their vital physiological processes depend on it. These organisms have a temperature range where their physiological processes are more efficient, this range is usually a requirement set phylogenetically even though they vary in the use of habitats. This research compares the thermal ecophysiology of Basiliscus galeritus (western basilisk) in two populations with altitudinal differences in the northwestern of Ecuador. The results suggest that the difference of altitude between the two locations (500 m) influences the thermal ecophysiology of the species since the body temperature (Tb), operative temperature (Te) and preferred body temperature (Tset) showed significative differences. Furthermore, B. galeritus obtains its Tb through heliothermy and it behaves as an active thermoregulator. On the other hand, the crest temperatures reach higher values than their Tset, the results suggested that the species uses the crest as an alternative to regulate its body temperature when exceeding its Tset. Besides, this thermoregulatory behavior has been suggested only in some extinct synapsids.

Environmental heterogeneity shapes physiological traits in tropical direct-developing frogs.

Tropical ectotherm species tend to have narrower physiological limits than species from temperate areas. As a consequence, tropical species are considered highly vulnerable to climate change since minor temperature increases can push them beyond their physiological thermal tolerance. Differences in physiological tolerances can also be seen at finer evolutionary scales, such as among populations of ectotherm species along elevation gradients, highlighting the physiological sensitivity of such organisms.Here, we analyze the influence of elevation and bioclimatic domains, defined by temperature and precipitation, on thermal sensitivities of a terrestrial direct-developing frog (Craugastor loki) in a tropical gradient. We address the following questions: (a) Does preferred temperature vary with elevation and among bioclimatic domains? (b) Do thermal tolerance limits, that is, critical thermal maximum and critical thermal minimum vary with elevation and bioclimatic domains? and (c) Are populations from high elevations more vulnerable to climate warming?We found that along an elevation gradient body temperature decreases as environmental temperature increases. The preferred temperature tends to moderately increase with elevation within the sampled bioclimatic domains. Our results indicate that the ideal thermal landscape for this species is located at midelevations, where the thermal accuracy (db ) and thermal quality of the environment (de ) are suitable. The critical thermal maximum is variable across elevations and among the bioclimatic domains, decreasing as elevation increases. Conversely, the critical thermal minimum is not as variable as the critical thermal maximum.Populations from the lowlands may be more vulnerable to future increases in temperature. We highlight that the critical thermal maximum is related to high temperatures exhibited across the elevation gradient and within each bioclimatic domain; therefore, it is a response to high environmental temperatures.

Thermoregulatory strategies of three reclusive lizards (genus Xantusia) from the Baja California peninsula, Mexico, under current and future microenvironmental temperatures.

The thermal quality of the habitat is key for the regulation of body temperature in terrestrial ectotherms and, therefore, permits them to carry out their fundamental biological activities. In thermally heterogeneous environments, ectotherms might follow different behavioral or physiological strategies to maintain their body temperature within biologically adequate boundaries, for which they depend on microhabitat selection. These aspects are, thus, relevant in the context of habitat degradation and land-use change. In this study, we characterized the thermal ecology of three lizard species (genus Xantusia) that differ in microhabitat use along the Baja California peninsula, Mexico. We made three predictions: (1) the three species will follow different thermoregulatory strategies according to habitat thermal quality; (2) the thermal requirements and tolerances of these species will match the environmental or microenvironmental thermal conditions; and (3) due to their habitat and range restriction, the species studied will be highly vulnerable to climate change. Our results indicate the existence of thermoregulatory mechanisms in Xantusia to face thermal heterogeneity, including behavioral thermoregulation by choosing different microhabitats, shifts in activity periods, and adaptation to particular high thermal quality microhabitats. Furthermore, despite their association to specific microhabitats and specialized physiology, the studied species will not be adversely affected by climate change, as the increased microenvironmental temperatures will lead to a higher habitat thermal quality and lower costs of thermoregulation. However, we do not discard other indirect adverse effects of climate change not considered in this study.

How will climate change impact fossorial lizard species? Two examples in the Baja California Peninsula.

Global climate change and the associated erosion of habitat suitability are pervasive threats to biodiversity. It is critical to identify specific stressors to assess a species vulnerability to extinction, especially in species with distinctive natural histories. Here, we present a combination of field, laboratory, and modeling approaches to evaluate the potential consequences of climate change on two endemic, fossorial lizards species (Anniella geronimensis and Bipes biporus) from Baja California, Mexico. We also include soil type in our models to refine the suitable areas using our mechanistic models. Results suggest that both species are at high risk of extinction by global climate change based on the thermal habitat suitability. The forecast for species persistence is most grave under the RCP8.5 scenario. On the one hand, suitable habitat for A. geronimensis diminishes at its southern distribution, but potential suitable expands towards the north. On the other hand, the suitable habitat for B. biporus will contract significantly with a concomitant reduction in its potential distribution. Because both species have low mobility and are restricted to low elevation, the potential for elevational and latitudinal dispersal to mitigate extinction risk along the Baja California Peninsula is unlikely. In addition each species has specialized thermal requirements (i.e., stenothermic) and soil type preferences to which they are adapted. Our ecophysiological models in combination with the type of soil are fundamental in developing conservation strategies.

Thermal physiology responds to interannual temperature shifts in a montane horned lizard, Phrynosoma orbiculare.

As climate change marches on, rapidly rising temperatures shatter records every year, presenting ever-growing physiological challenges to organisms worldwide. Ectotherms rely on behavioral and physiological plasticity to contend with environmental fluctuations. Nonetheless, our understanding of thermal plasticity has been largely limited to laboratory settings. Here, we test whether aspects of thermal physiology respond to interannual shifts in thermal environment in a natural population of Phrynosoma orbiculare, a montane horned lizard, from Hidalgo, Mexico. At our field site, 2019 was markedly warmer than the year that preceded it. We detected population-level increases in three key thermal physiological traits: preferred temperature, the critical thermal minimum, and the critical thermal maximum. Thus, thermal phenotypes appear to shift in tandem in response to environmental fluctuations. A subset of individuals were resampled across years, allowing insight into plastic shifts within an organism's lifetime. We detected parallel increases in these lizards for the preferred temperature and the critical thermal minimum, but not for the critical thermal maximum. Our results support a growing body of literature indicating that preferred conditions and cold tolerance can be highly labile over the course of an organism's lifetime, whereas hardening over shorter time periods is more common for heat tolerance. Given that heat tolerance increased at the population-level, but not in resampled individuals, it is possible that rapid evolution occurred due to temperature increases. In short, physiological shifts can be observed in natural populations over relatively short timespans, and these shifts might reflect a combination of evolutionary and acclimatory responses.

Comparative thermal ecology parameters of the mexican dusky rattlesnake (Crotalus triseriatus).

Montane habitats exhibit a high degree of thermal heterogeneity, and thus provide considerable thermoregulatory challenges for ectotherms. Comparative analyses provide an opportunity to understand how variation in abiotic factors (e.g., operative temperatures, thermal quality) can affect life history traits within species. We studied the thermal ecology of three populations of the rattlesnake Crotalus triseriatus inhabiting different volcanoes in the central region of Mexico using the Hertz et al. (1993) protocol. The average body temperature of dusky rattlesnakes from the three study sites was 22.4 °C; mean active body temperature was higher in site 2 than in sites 1 and 3, but no differences between females, males and juveniles nor an interaction among site and sex was found. The thermal quality was low in the three sites, particularly in sites 1 and 3. Thermoregulation accuracy statistically differed among populations: individuals from site 2 were more accurate thermoregulating, while individuals from site 1 were the least accurate. Compared to other snakes, dusky rattlesnakes can be considered as a eurythermic species, which can often be active at relatively low body temperatures.

Sex determination systems in reptiles are related to ambient temperature but not to the level of climatic fluctuation.

BACKGROUND: Vertebrates exhibit diverse sex determination systems and reptiles stand out by having highly variable sex determinations that include temperature-dependent and genotypic sex determination (TSD and GSD, respectively). Theory predicts that populations living in either highly variable or cold climatic conditions should evolve genotypic sex determination to buffer the populations from extreme sex ratios, yet these fundamental predictions have not been tested across a wide range of taxa. RESULTS: Here, we use phylogenetic analyses of 213 reptile species representing 38 families (TSD = 101 species, GSD = 112 species) and climatic data to compare breeding environments between reptiles with GSD versus TSD. We show that GSD and TSD are confronted with the same level of climatic fluctuation during breeding seasons. However, TSD reptiles are significantly associated with warmer climates. We found a strong selection on the breeding season length that minimises exposure to cold and fluctuating climate. Phylogenetic path analyses comparing competing evolutionary hypotheses support that transitions in sex determination systems influenced the ambient temperature at which the species reproduces and nests. In turn, this interaction affects other variables such as the duration of the breeding season and life-history traits. CONCLUSIONS: Taken together, our results challenge long-standing hypotheses about the association between sex determination and climate variability. We also show that ambient temperature is important during breeding seasons and it helps explain the effects of sex determination systems on the geographic distribution of extant reptile species.

Viviparous Reptile Regarded to Have Temperature-Dependent Sex Determination Has Old XY Chromosomes.

The water skinks Eulamprus tympanum and Eulamprus heatwolei show thermally induced sex determination where elevated temperatures give rise to male offspring. Paradoxically, Eulamprus species reproduce in temperatures of 12-15 °C making them outliers when compared with reptiles that use temperature as a cue for sex determination. Moreover, these two species are among the very few viviparous reptiles reported to have thermally induced sex determination. Thus, we tested whether these skinks possess undetected sex chromosomes with thermal override. We produced transcriptome and genome data for E. heatwolei. We found that E. heatwolei presents XY chromosomes that include 14 gametologs with regulatory functions. The Y chromosomal region is 79-116 Myr old and shared between water and spotted skinks. Our work provides clear evidence that climate could be useful to predict the type of sex determination systems in reptiles and it also indicates that viviparity is strictly associated with sex chromosomes.

Novel placental structure in the Mexican gerrhonotine lizard, Mesaspis viridiflava (Lacertilia; Anguidae).

The evolution of viviparity alters the physical relationship between mothers and offspring and the prevalence of viviparity among squamate reptiles presents an opportunity to uncover patterns in the evolution of placental structure. Understanding the breadth of this diversity is limited because studies of placental structure and function have emphasized a limited number of lineages. We studied placental ontogeny using light microscopy for an embryological series of the Mexican gerrhonotine lizard, Mesaspis viridiflava. This species develops an elaborate yolk sac placenta, an omphaloplacenta, which receives vascular support arising in a structure known only from other gerrhonotine lizards. A prominent feature of the omphaloplacenta is a zone of uterine and embryonic epithelial cell hyperplasia located at the upper shoulder of the yolk mass, often extending above the yolk mass. The omphaloplacenta covers more than one-half of the surface area of maternal-embryonic contact. The chorioallantoic placenta has a more restricted distribution because the allantois remains in the embryonic hemisphere of the egg throughout development and lies internal to the vascular support for the omphaloplacenta in areas where they overlap. The structural profile of the chorioallantoic placenta indicates a potential for respiratory exchange and/or hemotrophic nutritive transport, while that of the omphaloplacenta suggests that nutritive transfer is primarily via histotrophy. An eggshell is present in the earliest embryonic stages examined but regresses relatively early in development. Placental specializations of this species are consistent with a pattern of matrotrophic embryonic nutrition and have evolved in a unique lineage specific developmental pattern.

Inferring introgression using RADseq and DFOIL : Power and pitfalls revealed in a case study of spiny lizards (Sceloporus).

Introgression is now commonly reported in studies across the Tree of Life, aided by recent advancements in data collection and analysis. Nevertheless, researchers working with nonmodel species lacking reference genomes may be stymied by a mismatch between available resources and methodological demands. In this study, we demonstrate a fast and simple approach for inferring introgression using RADseq data, and apply it to a case study involving spiny lizards (Sceloporus) from northeastern México. First, we find evidence for recurrent mtDNA introgression between the two focal species based on patterns of mito-nuclear discordance. We then test for nuclear introgression by exhaustively applying the "five-taxon" D-statistic (DFOIL ) to all relevant individuals sampled for RADseq data. In our case, this exhaustive approach (dubbed "ExDFOIL ") entails testing up to ~250,000 unique four-taxon combinations of individuals across species. To facilitate use of this ExDFOIL approach, we provide scripts for many relevant tasks, including the selection of appropriate four-taxon combinations, execution of DFOIL tests in parallel and visualization of introgression results in phylogenetic and geographic space. Using ExDFOIL , we find evidence for ancient introgression between the focal species. Furthermore, we reveal geographic variation in patterns of introgression that is consistent with patterns of mito-nuclear discordance and with recurrent introgression. Overall, our study demonstrates that the combination of DFOIL and RADseq data can effectively detect introgression under a variety of sampling conditions (for individuals, populations and loci). Importantly, we also find evidence that batch-specific error and linkage in RADseq data may mislead inferences of introgression under certain conditions.

Climate change, thermal niches, extinction risk and maternal-effect rescue of toad-headed lizards, Phrynocephalus, in thermal extremes of the Arabian Peninsula to the Qinghai-Tibetan Plateau.

Determining the susceptibility of species to changing thermal niches is a major goal for biologists. In this paper we develop an eco-physiological model of extinction risk under climate change premised on behavioral thermoregulation. Our method downscales operative environmental temperatures, which restrict hours of activity of lizards, hr , for present-day climate (1975) and future climate scenarios (2070). We apply our model using occurrence records of 20 Phrynocephalus lizards (or taxa in species complexes) drawn from literature and museum records. Our analysis is phylogenetically informed, because some clades may be more sensitive to rising temperatures. The limits for computed hr predict local extirpations among Phrynocephalus lizards at continental scales and delineate upper boundaries of thermal niches as defined by Extreme Value Distributions. Under the 8.5 Representative Concentration Pathway scenario, we predict extirpation of 64% of local populations by 2070 across 20 Phrynocephalus species, and 12 are at high risk of total extinction due to thermal limits being exceeded. In tandem with global strategies of lower CO2 emissions, we propose regional strategies for establishing new national parks to protect extinction-prone taxa by preserving high-elevation climate refugia within existing sites of species occurrence. We propose that evolved acclimatization - maternal plasticity - may ameliorate risk, but is poorly studied. Previous studies revealed that adaptive maternal plasticity by thermoregulating gravid females alter progeny thermal preferences by ±1 °C. We describe plasticity studies for extinction-prone species that could assess whether they might be buffered from climate warming - a self-rescue. We discuss an epigenetic framework for studying such maternal-effect evolution.

Behavioral and physiological polymorphism in males of the austral lizard Liolaemus sarmientoi.

Integrative behavioral studies show that the interplay between individual physiology and social behavior influences the ecology of the species, ultimately affecting individual fitness. Particularly in lizards, color polymorphism is associated with differential behaviors and reproductive strategies, which are evident in mature males during the mating season. Dominant males generally have greater endurance, higher body temperature, and larger bodies than submissive males, so they can acquire and defend larger territories and have greater access to females for mating. We studied whether the color morphs observed in males of one of the world's southernmost reptiles, Liolaemus sarmientoi, are related to behavioral variation during agonistic interactions, thermal physiology, morphology, and/or locomotor stamina. Liolaemus sarmientoi males exhibit three color morphs: red (RR), red-yellow (RY), and yellow (YY). These lizards exhibit subtle behavioral displays and we did not observe stamina differences among morphs. However, we found that RR males are more aggressive than YY males during agonistic encounters. In addition, greater body temperature change during trials, higher field body temperatures, and greater head sizes of RR males compared to RY or YY indicate that RR is a dominant morph, which may influence their ability to acquire and defend territory and tactics for achieving reproductive success.

Thermoregulation of two sympatric species of horned lizards in the Chihuahuan Desert and their local extinction risk.

Thermoregulatory studies of ectothermic organisms are an important tool for ecological physiology, evolutionary ecology and behavior, and recently have become central for evaluating and predicting global climate change impacts. Here, we present a novel combination of field, laboratory, and modeling approaches to examine body temperature regulation, habitat thermal quality, and hours of thermal restriction on the activity of two sympatric, aridlands horned lizards (Phrynosoma cornutum and Phrynosoma modestum) at three contrasting Chihuahuan Desert sites in Mexico. Using these physiological data, we estimate local extinction risk under predicted climate change within their current geographical distribution. We followed the Hertz et al. (1993, Am. Nat., 142, 796-818) protocol for evaluating thermoregulation and the Sinervo et al. (2010, Science, 328, 894-899) eco-physiological model of extinction under climatic warming. Thermoregulatory indices suggest that both species thermoregulate effectively despite living in habitats of low thermal quality, although high environmental temperatures restrict the activity period of both species. Based on our measurements, if air temperature rises as predicted by climate models, the extinction model projects that P. cornutum will become locally extinct at 6% of sites by 2050 and 18% by 2080 and P. modestum will become extinct at 32% of sites by 2050 and 60% by 2080. The method we apply, using widely available or readily acquired thermal data, along with the modeling, appeared to identify several unique ecological traits that seemingly exacerbate climate sensitivity of P. modestum.

Estimating the temporal and spatial extent of gene flow among sympatric lizard populations (genus Sceloporus) in the southern Mexican highlands.

Interspecific gene flow is pervasive throughout the tree of life. Although detecting gene flow between populations has been facilitated by new analytical approaches, determining the timing and geography of hybridization has remained difficult, particularly for historical gene flow. A geographically explicit phylogenetic approach is needed to determine the overlap of ancestral populations. In this study, we performed population genetic analyses, species delimitation, simulations and a recently developed approach of species tree diffusion to infer the phylogeographic history, timing and geographic extent of gene flow in lizards of the Sceloporus spinosus group. The two species in this group, S. spinosus and S. horridus, are distributed in eastern and western portions of Mexico, respectively, but populations of these species are sympatric in the southern Mexican highlands. We generated data consisting of three mitochondrial genes and eight nuclear loci for 148 and 68 individuals, respectively. We delimited six lineages in this group, but found strong evidence of mito-nuclear discordance in sympatric populations of S. spinosus and S. horridus owing to mitochondrial introgression. We used coalescent simulations to differentiate ancestral gene flow from secondary contact, but found mixed support for these two models. Bayesian phylogeography indicated more than 60% range overlap between ancestral S. spinosus and S. horridus populations since the time of their divergence. Isolation-migration analyses, however, revealed near-zero levels of gene flow between these ancestral populations. Interpreting results from both simulations and empirical data indicate that despite a long history of sympatry among these two species, gene flow in this group has only recently occurred.