Testing an hypothesis of hybrid zone movement for toads in France

Hybrid zone movement may result in substantial unidirectional introgression of selectively neutral material from the local to the advancing species, leaving a genetic footprint. This genetic footprint is represented by a trail of asymmetric tails and displaced cline centres in the wake of the moving hybrid zone. A peak of admixture linkage disequilibrium is predicted to exist ahead of the centre of the moving hybrid zone. We test these predictions of the movement hypothesis in a hybrid zone between common (Bufo bufo) and spined toads (B. spinosus), using 31 nuclear and one mtDNA SNPs along a transect in the northwest of France. Average effective selection in Bufo hybrids is low and clines vary in shape and centre. A weak pattern of asymmetric introgression is inferred from cline discordance of seven nuclear markers. The dominant direction of gene flow is from B. spinosus to B. bufo and is in support of southward movement of the hybrid zone. Conversely, a peak of admixture linkage disequilibrium north of the hybrid zone suggests northward movement. These contrasting results can be explained by reproductive isolation of the B. spinosus and B. bufo gene pools at the southern (B. spinosus) side of the hybrid zone. The joint occurrence of asymmetric introgression and admixture linkage disequilibrium can also be explained by the combination of low dispersal and random genetic drift due to low effective population sizes.     

Hybrid zone formation and contrasting outcomes of secondary contact over transects in common toads

Much progress in speciation research stems from documenting patterns of morphological and genetic variation in hybrid zones. Contrasting patterns of marker introgression in different sections of the contact can provide valuable insights on the relative importance of various evolutionary mechanisms maintaining species differences in the face of hybridization and gene flow and on hybrid zone temporal and spatial dynamics. We studied species interactions in the common toads Bufo bufo and B. spinosus in France and northwestern Italy using morphological and molecular data from the mitochondrial and nuclear genomes in an extensive survey, including two independent transects west and east of the Alps. At both, we found sharp, coincident and concordant nuclear genetic transitions. However, morphological clines were wider or absent and mtDNA introgression was asymmetric. We discuss alternative, nonexclusive hypotheses about evolutionary processes generating these patterns, including drift, selection, long‐distance dispersal and spatial shifts in hybrid zone location and structure. The distribution of intraspecific mtDNA lineages supports a scenario in which B. bufo held a local refugium during the last glacial maximum. Present‐day genetic profiles are best explained by an advance of B. spinosus from a nearby Iberian refugium, largely superseding the local B. bufo population, followed by an advance of B. bufo from the Balkans, with prongs north and south of the Alps, driving B. spinosus southwards. A pendulum moving hybrid zone, first northwards and then southwards, explains the wide areas of introgression at either side of the current position of the contact zones.     

Concordant morphological and molecular clines in a contact zone of the Common and Spined toad (<Emphasis Type="Italic">Bufo bufo</Emphasis> and <Emphasis Type="Italic">B. spinosus</Emphasis>) in the northwest of France

Background

Hybrid zones are regions where individuals of two species meet and produce hybrid progeny, and are often regarded as natural laboratories to understand the process of species formation. Two microevolutionary processes can take place in hybrid zones, with opposing effects on population differentiation. Hybridization tends to produce genetic homogenization, reducing species differences, whereas the presence of mechanisms of reproductive isolation result in barriers to gene flow, maintaining or increasing differences between taxa.

Results

Here we study a contact zone between two hybridizing toad species, Bufo bufo and B. spinosus, through a combination of molecular (12 polymorphic microsatellites, four nuclear and two mitochondrial SNP markers) and morphological data in a transect in the northwest of France. The results show largely concordant clines across markers, defining a narrow hybrid zone of ca. 30 km wide. Most hybrids in the centre of the contact zone are classified as F₂ or backcrossed individuals, with no individuals assigned to the F₁ hybrid class.

Conclusions

We discuss the implications of these results for our understanding of the evolutionary history of these species. We anticipate that the toad contact zone here described will become an important asset in the study of hybrid zone dynamics and evolutionary biology because of its easy access and the abundance of the species involved.

     

Multilocus species tree analyses resolve the radiation of the widespread Bufo bufo species group (Anura, Bufonidae)

New analytical methods are improving our ability to reconstruct robust species trees from multilocus datasets, despite difficulties in phylogenetic reconstruction associated with recent, rapid divergence, incomplete lineage sorting and/or introgression. In this study, we applied these methods to resolve the radiation of toads in the Bufo bufo (Anura, Bufonidae) species group, ranging from the Iberian Peninsula and North Africa to Siberia, based on sequences from two mitochondrial and four nuclear DNA regions (3490 base pairs). We obtained a fully-resolved topology, with the recently described Bufo eichwaldi from the Talysh Mountains in south Azerbaijan and Iran as the sister taxon to a clade including: (1) north African, Iberian, and most French populations, referred herein to Bufo spinosus based on the implied inclusion of populations from its type locality and (2) a second clade, sister to B. spinosus, including two sister subclades: one with all samples of Bufo verrucosissimus from the Caucasus and another one with samples of B. bufo from northern France to Russia, including the Apennine and Balkan peninsulas and most of Anatolia. Coalescent-based estimations of time to most recent common ancestors for each species and selected subclades allowed historical reconstruction of the diversification of the species group in the context of Mediterranean paleogeography and indicated a long evolutionary history in this region. Finally, we used our data to delimit the ranges of the four species, particularly the more widespread and historically confused B. spinosus and B. bufo, and identify potential contact zones, some of which show striking parallels with other co-distributed species.     

Movement of a Heliconius hybrid zone over 30 years: A Bayesian approach

Hybrid zones have long been of interest to biologists as natural laboratories where we can gain insight into the processes of adaptation and speciation. Repeated sampling of individual hybrid zones has been particularly useful in elucidating the dynamic balance between selection and dispersal that maintains most hybrid zones. Here, we revisit a hybrid zone between Heliconius erato butterflies in Panamá for a third time over more than 30 years. We combine a novel Bayesian extension of stepped‐cline hybrid zone models with environmental data to understand the genetic and environmental causes of cline dynamics in this species. The cline has continued to move west, likely due to dominance drive, but has slowed and broadened. Environmental analyses suggest that widespread deforestation in Panamá could be leading to decreased avian predation and relaxed selection, causing the observed changes in cline dynamics.     

Stabilization of a salamander moving hybrid zone

When related species meet upon postglacial range expansion, hybrid zones are frequently formed. Theory predicts that such zones may move over the landscape until equilibrium conditions are reached. One hybrid zone observed to be moving in historical times (1950–1979) is that of the pond‐breeding salamanders Triturus cristatus and Triturus marmoratus in western France. We identified the ecological correlates of the species hybrid zone as elevation, forestation, and hedgerows favoring the more terrestrial T. marmoratus and pond density favoring the more aquatic T. cristatus. The past movement of the zone of ca. 30 km over three decades has probably been driven by the drastic postwar reduction of the “bocage” hedgerow landscape, favoring T. cristatus over T. marmoratus. No further hybrid zone movement was observed from 1979 to the present. To explain the changing dynamics of the hybrid zone, we propose that it stalled, either because an equilibrium was found at an altitude of ca. 140 m a.s.l. or due to pond loss and decreased population densities. While we cannot rule out the former explanation, we found support for the latter. Under agricultural intensification, ponds in the study area are lost at an unprecedented rate of 5.5% per year, so that remaining Triturus populations are increasingly isolated, hampering dispersal and further hybrid zone movement.     

Cross‐decades stability of an avian hybrid zone

Hybrid zones are particularly valuable for understanding the evolution of partial reproductive isolation between differentiated populations. An increasing number of hybrid zones have been inferred to move over time, but in most such cases zone movement has not been tested with long‐term genomic data. The hybrid zone between Townsend's Warblers (Setophaga townsendi) and Hermit Warblers (S. occidentalis) in the Washington Cascades was previously inferred to be moving from northern S. townsendi southwards towards S. occidentalis, based on plumage and behavioural patterns as well as a 2000‐km genetic wake of hermit mitochondrial DNA (mtDNA) in coastal Townsend's Warblers. We directly tested whether hybrid zone position has changed over 2–3 decades by tracking plumage, mtDNA and nuclear genomic variation across the hybrid zone over two sampling periods (1987–94 and 2015–16). Surprisingly, there was no significant movement in genomic or plumage cline centres between the two time periods. Plumage cline widths were narrower than expected by neutral diffusion, consistent with a ‘tension zone’ model, in which selection against hybrids is balanced by movement of parental forms into the zone. Our results indicate that this hybrid zone is either stable in its location or moving at a rate that is not detectable over 2–3 decades. Despite considerable gene flow, the stable clines in multiple phenotypic and genotypic characters over decades suggest evolutionary stability of this young pair of sister species, allowing divergence to continue. We propose a novel biogeographic scenario to explain these patterns: rather than the hybrid zone having moved thousands of kilometres to its current position, inland Townsend's met coastal Hermit Warbler populations along a broad front of the British Columbia and Alaska coast and hybridization led to replacement of the Hermit Warbler plumage with Townsend's Warbler plumage patterns along this coastline. Hence, hybrid zones along British Columbia and Alaska moved only a short distance from the inland to the coast, whereas the Hermit Warbler phenotype appears stable in Washington and further south. This case provides an example of the complex biogeographic processes that have led to the distribution of current phenotypes within and among closely related species.     

Is dispersal guided by the environment? A comparison of interspecific gene flow estimates among differentiated regions of a newt hybrid zone

How frequently genes pass through a hybrid zone may be influenced by the environment. Accordingly, in long hybrid zones that span more than one environmental setting, different patterns may emerge. The varied conditions allow testing of hypotheses on dispersal as a function of the environment. We reconstruct the amount and direction of gene flow across a heterogeneous hybrid zone of two species of marbled newts (Triturus marmoratus and Triturus pygmaeus), in four widely separated areas of the Iberian Peninsula from one mitochondrial and three nuclear genes. The main variables associated with the position of the contact zone are precipitation, rivers, altitude and relief. In some sections of the contact zone, however, its position is not correlated with any environmental factor and is instead determined by the shortest geographical distance between fixed positions at either side (mountains in the East and river in the West). In areas where the position of the zone is stable, gene flow was bidirectional. External data show that T. pygmaeus has superseded T. marmoratus over a large area and here gene flow was unidirectional. The prediction that a major river would reduce gene flow was not confirmed.     

Hybridization and mitochondrial genome introgression between Rana chensinensis and R. kukunoris

Mitochondrial genome (mito‐genome) introgression among metazoans is commonplace, and several biological processes may promote such introgression. We examined two proposed processes for the mito‐genome introgression between Rana chensinensis and R. kukunoris: natural hybridization and sex‐biased dispersal. We sampled 477 individuals from 28 sites in the potential hybrid zone in the western Tsinling Mountains. Mitochondrial gene (cyt‐b) trees were used to examine the introgression events. Microsatellite DNA loci, cyt‐b and morphological data were used to identify hybrids and to examine the extent of natural hybridization. We detected rampant bidirectional introgressions, both ancient and recent, between the two species. Furthermore, we found a wide hybrid zone, and frequent and asymmetric hybridization. The hybrid zone cline analysis revealed a clear mitochondrial–nuclear discordance; while most nuclear markers displayed similar and steep clines, cyt‐b had a displaced cline centre and a more gradual and wider cline. We also detected strong and asymmetric historical maternal gene flow across the hybrid zone. This widespread hybridization and detected low mito‐nuclear conflicts may, at least partially, explain the high frequency of introgression. Lastly, microsatellite data and population genetic methods were used to assess sex‐biased dispersal. A weak pattern of female‐biased dispersal was detected in both species, suggesting it may not play an important role in the observed introgression. Our data are consistent with the hybridization hypothesis, but support for the sex‐biased dispersal hypothesis is weak. We further suggest that selective advantages of the R. kukunoris‐type mito‐genome in thermal adaptation may also contribute to the introgression between the two species.     

Tracing species replacement in Iberian marbled newts

Secondary contact between closely related species can lead to the formation of hybrid zones, allowing for interspecific gene flow. Hybrid zone movement can take place if one of the species possesses a competitive advantage over the other, ultimately resulting in species replacement. Such hybrid zone displacement is predicted to leave a genomic footprint across the landscape in the form of asymmetric gene flow (or introgression) of selectively neutral alleles from the displaced to the advancing species. Hybrid zone movement has been suggested for marbled newts in the Iberian Peninsula, supported by asymmetric gene flow and a distribution relict (i.e., an enclave) of Triturus marmoratus in the range of T. pygmaeus. We developed a panel of nuclear and mitochondrial SNP markers to test for the presence of a T. marmoratus genomic footprint in the Lisbon peninsula, south of the enclave. We found no additional populations of T. marmoratus. Analysis with the software Structure showed no genetic traces of T. marmoratus in T. pygmaeus. A principal component analysis showed some variation within the local T. pygmaeus, but it is unclear if this represents introgression from T. marmoratus. The results may be explained by (a) species replacement without introgressive hybridization and (b) displacement with hybridization followed by the near‐complete erosion of the footprint by purifying selection. We predict that testing for a genomic footprint north of the reported enclave would confirm that species replacement in these marbled newts occurred with hybridization.     

Segregation of male‐sterility alleles across a species boundary

Hybrid zones may serve as bridges permitting gene flow between species, including alleles influencing the evolution of breeding systems. Using greenhouse crosses, we assessed the likelihood that a hybrid zone could serve as a conduit for transfer of nuclear male‐sterility alleles between a gynodioecious species and a hermaphroditic species with very rare females in some populations. Segregation patterns in progeny of crosses between rare females of hermaphroditic Schiedea menziesii and hermaphroditic plants of gynodioecious Schiedea salicaria heterozygous at the male‐sterility locus, and between female S. salicaria and hermaphroditic plants from the hybrid zone, were used to determine whether male‐sterility was controlled at the same locus in the parental species and the hybrid zone. Segregations of females and hermaphrodites in approximately equal ratios from many of the crosses indicate that the same nuclear male‐sterility allele occurs in the parent species and the hybrid zone. These rare male‐sterility alleles in S. menziesii may result from gene flow from S. salicaria through the hybrid zone, presumably facilitated by wind pollination in S. salicaria. Alternatively, rare male‐sterility alleles might result from a reversal from gynodioecy to hermaphroditism in S. menziesii, or possibly de novo evolution of male sterility. Phylogenetic analysis indicates that some species of Schiedea have probably evolved separate sexes independently, but not in the lineage containing S. salicaria and S. menziesii. High levels of selfing and expression of strong inbreeding depression in S. menziesii, which together should favour females in populations, argue against a reversal from gynodioecy to hermaphroditism in S. menziesii.     

Evidence for concerted movement of nuclear and mitochondrial clines in a lizard hybrid zone

Moving hybrid zones provide compelling examples of evolution in action, yet long‐term studies that test the assumptions of hybrid zone stability are rare. Using replicated transect samples collected over a 10‐year interval from 2002 to 2012, we find evidence for concerted movement of genetic clines in a plateau fence lizard hybrid zone (Sceloporus tristichus) in Arizona. Cline‐fitting analyses of SNP and mtDNA data both provide evidence that the hybrid zone shifted northward by approximately 2 km during the 10‐year interval. For each sampling period, the mtDNA cline centre is displaced from the SNP cline centre and maintaining an introgression distance of approximately 3 km. The northward expansion of juniper trees into the Little Colorado River Basin in the early 1900s provides a plausible mechanism for hybrid zone formation and movement, and a broadscale quantification of recent land cover change provides support for increased woody species encroachment at the southern end of the hybrid zone. However, population processes can also contribute to hybrid zone movement, and the current stability of the ecotone habitats in the centre of the hybrid zone suggests that movement could decelerate in the future.     

Introgression between invasive and native blue mussels (genus Mytilus) in the central California hybrid zone

The ecological and genetic factors determining the extent of introgression between species in secondary contact zones remain poorly understood. Here, we investigate the relative importance of isolating barriers and the demographic expansion of invasive Mytilus galloprovincialis on the magnitude and the direction of introgression with the native Mytilus trossulus in a hybrid zone in central California. We use double‐digest restriction‐site‐associated DNA sequencing (ddRADseq) to genotype 1337 randomly selected single nucleotide polymorphisms and accurately distinguish early and advanced generation hybrids for the first time in the central California Mytilus spp. hybrid zone. Weak levels of introgression were observed in both directions but were slightly more prevalent from the native M. trossulus into the invasive M. galloprovincialis. Few early and advanced backcrossed individuals were observed across the hybrid zone confirming the presence of strong barriers to interbreeding. Heterogeneous patterns of admixture across the zone of contact were consistent with the colonization history of M. galloprovincialis with more extensive introgression in northern localities furthest away from the putative site of introduction in southern California. These observations reinforce the importance of dynamic spatial and demographic expansions in determining patterns of introgression between close congeners, even in those with high dispersal potential and well‐developed reproductive barriers. Our results suggest that the threat posed by invasive M. galloprovincialis is more ecological than genetic as it has displaced, and continues to displace the native M. trossulus from much of central and southern California.     

The Boxgrove,England, middle Pleistocene herpetofauna: Paleogeographic,evolutionary, stratigraphic,and paleoecological relationships

Triturus vulgaris (smooth newt), Triturus helveticus or T. vulgaris (palmate or smooth newt), Triturus sp. (newt), Pelobates fuscus (common spadefoot), Bufo bufo (common toad), Bufo calamita (natterjack toad), Bufo sp. (toad), Rana arvalis (moor frog), Rana temporaria (common frog), Rana sp. (frog), Anguis fragilis (slow worm), Lacerta cf. L. vivipara (common lizard), Natrix natrix (grass snake), and Natrix sp. (grass, viperine, or dice snake) were identified at the Middle Pleistocene Boxgrove Site, West Sussex, England. This is the first British fossil record of Pelobates fuscus and the earliest fossil record in Britain for the endangered species Bufo calamita. All of these herpetological species are extant and all of them occur in Britain today with the exception of Pelobates fuscus and Rana arvalis that presently live on the European continent.

The Boxgrove, Westbury, and West Runton British pre‐Anglian Middle Pleistocene herpetofaunas show no apparent differences among themselves in patterns of species composition, diversity, or number of exotics. But these three herpetofaunas together have [1] less species diversity and [2] fewer exotic continental species than in the Cudmore Grove British post‐Anglian Middle Pleistocene herpetofauna.

Only the Terrestral Sequence Unit at Boxgrove yielded enough herpetological species for paleoecological interpretation. These taxa indicate a quiet pool surrounded by a somewhat humid vegetated area that gave way to a more xerophytic sandy area, and a paleoclimate at least as warm and perhaps somewhat warmer than occurs in the area today.     

Genomic replacement of native Cobitis lutheri with introduced C. tetralineata through a hybrid swarm following the artificial connection of river systems

River connections via artificial canals will bring about secondary contacts between previously isolated fish species. Here, we present a genetic consequence of such a secondary contact between Cobitis fish species, C. lutheri in the Dongjin River, and C. tetralineata in the Seomjin River in Korea. The construction of water canals about 80 years ago has unidirectionally introduced C. tetralineata into the native habitat of C. lutheri, and then these species have hybridized in the main stream section of the Dongjin River. According to the divergence population genetic analyses of DNA sequence data, the two species diverged about 3.3 million years ago, which is interestingly coincident with the unprecedented paleoceanographic change that caused isolations of the paleo‐river systems in northeast Asia due to sea‐level changes around the late Pliocene. Multilocus genotypic data of nine microsatellites and three nuclear loci revealed an extensively admixed structure in the hybrid zone with a high proportion of various post‐F1 hybrids. Surprisingly, pure native C. lutheri was absent in the hybrid zone in contrast to the 7% of pure C. tetralineata. Such a biased proportion must have resulted from the dominant influence of continually introducing C. tetralineata on the native C. lutheri which has no supply of natives from other tributaries to the hybrid zone due to numerous low‐head dams. In addition, mating experiments indicated that there is no discernible reproductive isolation between them. All the results suggest that the gene pool of native C. lutheri is being rapidly replaced by that of continually introducing C. tetralineata through a hybrid swarm for the last 80 years, which will ultimately lead to the genomic extinction of natives in this hybrid zone.     

Fitness of natural willow hybrids in a pioneer mosaic hybrid zone

Hybrid fitness is an important parameter to predict the evolutionary consequences of a hybridization event and to characterize hybrid zones. We studied fitness parameters of F₁ and later‐generation hybrids between the lowland species Salix purpurea and the alpine S. helvetica that have recently emerged during colonization of an alpine glacier forefield. Fruit production (number of capsules per catkin and fruit set) did not differ between hybrids and parents, but the number of seeds per capsule of F₁ hybrids was slightly lower than that of later‐generation hybrids and of the parents. Germination rates and seedling growth were tested on three substrates (pH 4.5, 7.0, and 8.0). Germination rates of seeds collected from F₁ hybrids were lower on acid and neutral substrates, but equal at pH 8.0 compared to all other groups, while the seeds from later‐generation hybrids performed as well as the parents on all three substrates. In seedling growth, the colonizer S. purpurea performed better than all other taxa on all three substrates, while hybrids resembled the subalpine species S. helvetica. Results suggest that endogenous selection acts against F₁ hybrids, but favors fitter genotypes in later‐generation hybrids. Exogenous selection via soil pH appears to be weak during seedling establishment. The pioneer vegetation on the glacier forefield may offer sufficient niche space for hybrid seedlings. Owing to the relatively high fitness of the hybrids and the scattered distribution of hybrids and parental individuals on the glacier forefield, this hybrid zone can be assigned to a mosaic model, probably facilitating gene flow and introgression between the parental species. As establishment of the hybrid zone appears to be linked to a colonization process, we propose to call it a pioneer mosaic hybrid zone.     

Variation in complex mating signals in an “island” hybrid zone between Stenobothrus grasshopper species

Two grasshopper species Stenobothrus rubicundus and S. clavatus were previously shown to meet in a narrow hybrid zone on Mount Tomaros in northern Greece. The species are remarkable for their complex courtship songs accompanied by conspicuous movements of antennae and wings. We analyzed variations in forewing morphology, antenna shape, and courtship song across the hybrid zone using a geographic information system, and we documented three contact zones on Mount Tomaros. All male traits and female wings show abrupt transitions across the contact zones, suggesting that these traits are driven by selection rather than by drift. Male clines in antennae are displaced toward S. clavatus, whereas all clines in wings are displaced toward S. rubicundus. We explain cline discordance as depending on sexual selection via female choice. The high covariance between wings and antennae found in the centers of all contact zones results from high levels of linkage disequilibria among the underlying loci, which in turn more likely results from assortative mating than from selection against hybrids. The covariance is found to be higher in clavatus‐like than rubicundus‐like populations, which implies asymmetric assortative mating in parental‐like sites of the hybrid zone and a movement of the hybrid zone in favor of S. clavatus.     

Interploidy hybridization in sympatric zones: the formation of Epidendrum fulgens × E. puniceoluteum hybrids (Epidendroideae,Orchidaceae)

Interspecific hybridization is a primary cause of extensive morphological and chromosomal variation and plays an important role in plant species diversification. However, the role of interploidal hybridization in the formation of hybrid swarms is less clear. Epidendrum encompasses wide variation in chromosome number and lacks strong premating barriers, making the genus a good model for clarifying the role of chromosomes in postzygotic barriers in interploidal hybrids. In this sense, hybrids from the interploidal sympatric zone between E. fulgens (2n = 2x = 24) and E. puniceoluteum (2n = 4x = 56) were analyzed using cytogenetic techniques to elucidate the formation and establishment of interploidal hybrids. Hybrids were not a uniform group: two chromosome numbers were observed, with the variation being a consequence of severe hybrid meiotic abnormalities and backcrossing with E. puniceoluteum. The hybrids were triploids (2n = 3x = 38 and 40) and despite the occurrence of enormous meiotic problems associated with triploidy, the hybrids were able to backcross, producing successful hybrid individuals with broad ecological distributions. In spite of the nonpolyploidization of the hybrid, its formation is a long‐term evolutionary process rather than a product of a recent disturbance, and considering other sympatric zones in Epidendrum, these events could be recurrent.     

The basis of niche separation during terrestrial life between two species of toad (Bufo bufo and Bufo calamita): competition or specialisation?

Two congeneric anurans with highly overlapping geographical ranges, Bufo bufo and B. calamita, were investigated with respect to isolating mechanisms during the terrestrial phase of life in the three habitat types in which both species occur in Britain. Marked spatial niche separations were observed in all three habitats (coastal dunes, upper saltmarshes and lowland heaths). B. bufo was associated with complex, dense vegetation structures and had a relatively wide niche, while B. calamita had a narrower niche and occurred mostly on open, poorly-vegetated ground. Food niche overlap was high at all three study sites. Animals translocated into atypical habitats returned to their preferred type whenever possible, and suffered increased mortality or loss of condition if prevented from doing so. B. calamita operated at significantly higher body temperatures (by an average of 1.4° C) than B. bufo when hunting at night. Experiments in outdoor vivaria indicated that B. calamita survived in open habitats because individuals escaped desiccation in daytime by burrowing into the substrate, whereas B. bufo lacked this behaviour and perished on the inhospitable surface. By contrast, B. calamita individuals lost mass and became hyperactive in densely-vegetated conditions because their hunting efficiency was selectively reduced relative to that of B. bufo. Taken together, the data indicated that spatial niche separation between these species during the terrestrial phase of life was dictated primarily by behavioural and physiological factors, and not by competition.