Cryptic lineages and standing genetic variation across independent cane toad introductions.

Widespread introduced species can be leveraged to investigate the genetic, ecological and adaptive processes underlying rapid evolution and range expansion, particularly the contributions of genetic diversity to adaptation. Rhinella marina, the cane toad, has been a focus of invasion biology for decades in Australia. However, their introduction history in North America is less clear. Here, we investigated the roles of introduction history and genetic diversity in establishment success of cane toads across their introduced range. We used reduced representation sequencing (ddRAD) to obtain 34,000 SNPs from 247 toads in native (French Guiana, Guyana, Ecuador, Panama, Texas) and introduced (Bermuda, southern Florida, northern Florida, Hawai'i, Puerto Rico) populations. Unlike all other cane toad introductions, we found that Florida populations were more closely related to native Central American lineages (R. horribilis), than to native Southern American lineages (R. marina). Furthermore, we found high levels of diversity and population structure in the native range, corroborating suggestions that R. marina is a species complex. We also found that introduced populations exhibit only slightly lower genetic diversity than native populations. Together with demographic analyses, this indicates founding populations of toads in Florida were larger than previously reported. Lastly, within R. marina, only one of 245 putatively adaptive SNPs showed fixed differences between native and introduced ranges, suggesting that putative selection in these introduced populations is based upon existing genetic variation. Our findings highlight the importance of genetic sequencing in understanding biological introductions and hint at the role of standing genetic variation in range expansion.

Population genomics of a predatory mammal reveals patterns of decline and impacts of exposure to toxic toads.

Mammal declines across northern Australia are one of the major biodiversity loss events occurring globally. There has been no regional assessment of the implications of these species declines for genomic diversity. To address this, we conducted a species-wide assessment of genomic diversity in the northern quoll (Dasyurus hallucatus), an Endangered marsupial carnivore. We used next generation sequencing methods to genotype 10,191 single nucleotide polymorphisms (SNPs) in 352 individuals from across a 3220-km length of the continent, investigating patterns of population genomic structure and diversity, and identifying loci showing signals of putative selection. We found strong heterogeneity in the distribution of genomic diversity across the continent, characterized by (i) biogeographical barriers driving hierarchical population structure through long-term isolation, and (ii) severe reductions in diversity resulting from population declines, exacerbated by the spread of introduced toxic cane toads (Rhinella marina). These results warn of a large ongoing loss of genomic diversity and associated adaptive capacity as mammals decline across northern Australia. Encouragingly, populations of the northern quoll established on toad-free islands by translocations appear to have maintained most of the initial genomic diversity after 16 years. By mapping patterns of genomic diversity within and among populations, and investigating these patterns in the context of population declines, we can provide conservation managers with data critical to informed decision-making. This includes the identification of populations that are candidates for genetic management, the importance of remnant island and insurance/translocated populations for the conservation of genetic diversity, and the characterization of putative evolutionarily significant units.

Captivity induces large and population-dependent brain transcriptomic changes in wild-caught cane toads (Rhinella marina).

Gene expression levels are key molecular phenotypes at the interplay between genotype and environment. Mounting evidence suggests that short-term changes in environmental conditions, such as those encountered in captivity, can substantially affect gene expression levels. Yet, the exact magnitude of this effect, how general it is, and whether it results in parallel changes across populations are not well understood. Here, we take advantage of the well-studied cane toad, Rhinella marina, to examine the effect of short-term captivity on brain gene expression levels, and determine whether effects of captivity differ between long-colonized and vanguard populations of the cane toad's Australian invasion range. We compared the transcriptomes of wild-caught toads immediately assayed with those from toads captured from the same populations but maintained in captivity for seven months. We found large differences in gene expression levels between captive and wild-caught toads from the same population, with an over-representation of processes related to behaviour and the response to stress. Captivity had a much larger effect on both gene expression levels and gene expression variability in toads from vanguard populations compared to toads from long-colonized areas, potentially indicating an increased plasticity in toads at the leading edge of the invasion. Overall, our findings indicate that short-term captivity can induce large and population-specific transcriptomic changes, which has significant implications for studies comparing phenotypic traits of wild-caught organisms from different populations that have been held in captivity.

Brain transcriptome analysis reveals gene expression differences associated with dispersal behaviour between range-front and range-core populations of invasive cane toads in Australia.

Understanding the mechanisms allowing invasive species to adapt to novel environments is a challenge in invasion biology. Many invaders demonstrate rapid evolution of behavioural traits involved in range expansion such as locomotor activity, exploration and risk-taking. However, the molecular mechanisms that underpin these changes are poorly understood. In 86 years, invasive cane toads (Rhinella marina) in Australia have drastically expanded their geographic range westward from coastal Queensland to Western Australia. During their range expansion, toads have undergone extensive phenotypic changes, particularly in behaviours that enhance the toads' dispersal ability. Common-garden experiments have shown that some changes in behavioural traits related to dispersal are heritable. At the molecular level, it is currently unknown whether these changes in dispersal-related behaviour are underlain by small or large differences in gene expression, nor is known the biological function of genes showing differential expression. Here, we used RNA-seq to gain a better understanding of the molecular mechanisms underlying dispersal-related behavioural changes. We compared the brain transcriptomes of toads from the Hawai'ian source population, as well as three distinct populations from across the Australian invasive range. We found markedly different gene expression profiles between the source population and Australian toads. By contrast, toads from across the Australian invasive range had very similar transcriptomic profiles. Yet, key genes with functions putatively related to dispersal behaviour showed differential expression between populations located at each end of the invasive range. These genes could play an important role in the behavioural changes characteristic of range expansion in Australian cane toads.

Transposable elements expression in Rhinella marina (cane toad) specimens submitted to immune and stress challenge.

Transposable elements (TEs) are important components of eukaryotic genomes and compose around 30% of the genome of Rhinella marina, an invasive toad species. Considering the possible role of TEs in the adaptation of populations, we have analyzed the expression of TEs in publicly available spleen tissue transcriptomic data generated for this species after immune and stress challenge. By analyzing the transcriptome assembly, we detected a high number of TE segments. Moreover, some distinct TE families were differentially expressed in some conditions. Our result shows that several TEs are capable of being transcribed in R. marina and they could help to generate a rapid response of specimens to the environment. Also, we can suggest that these TEs could be activated in the germinative cells as well producing variability to be selected and shaped by the evolutionary processes behind the success of this invasive species. Thus, the TEs are important targets for investigation in the context of R. marina adaptation.

Intergenerational effects of manipulating DNA methylation in the early life of an iconic invader.

In response to novel environments, invasive populations often evolve rapidly. Standing genetic variation is an important predictor of evolutionary response but epigenetic variation may also play a role. Here, we use an iconic invader, the cane toad (Rhinella marina), to investigate how manipulating epigenetic status affects phenotypic traits. We collected wild toads from across Australia, bred them, and experimentally manipulated DNA methylation of the subsequent two generations (G1, G2) through exposure to the DNA methylation inhibitor zebularine and/or conspecific tadpole alarm cues. Direct exposure to alarm cues (an indicator of predation risk) increased the potency of G2 tadpole chemical cues, but this was accompanied by reductions in survival. Exposure to alarm cues during G1 also increased the potency of G2 tadpole cues, indicating intergenerational plasticity in this inducible defence. In addition, the negative effects of alarm cues on tadpole viability (i.e. the costs of producing the inducible defence) were minimized in the second generation. Exposure to zebularine during G1 induced similar intergenerational effects, suggesting a role for alteration in DNA methylation. Accordingly, we identified intergenerational shifts in DNA methylation at some loci in response to alarm cue exposure. Substantial demethylation occurred within the sodium channel epithelial 1 subunit gamma gene (SCNN1G) in alarm cue exposed individuals and their offspring. This gene is a key to the regulation of sodium in epithelial cells and may help to maintain the protective epidermal barrier. These data suggest that early life experiences of tadpoles induce intergenerational effects through epigenetic mechanisms, which enhance larval fitness. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

Got Glycogen?: Development and Multispecies Validation of the Novel Preserve, Precipitate, Lyse, Precipitate, Purify (PPLPP) Workflow for Environmental DNA Extraction from Longmire's Preserved Water Samples.

Unfiltered and filtered water samples can be used to collect environmental DNA (eDNA). We developed the novel "Preserve, Precipitate, Lyse, Precipitate, Purify" (PPLPP) workflow to efficiently extract eDNA from Longmire's preserved unfiltered and filtered water samples (44-100% recovery). The PPLPP workflow includes initial glycogen-aided isopropanol precipitation, guanidium hypochlorite and Triton X-100-based lysis, terminal glycogen-aided polyethylene glycol precipitation, and inhibitor purification. Three novel eDNA assays that exclusively target species invasive to Australia were also developed: Tilapia_v2_16S concurrently targets Oreochromis mossambicus (Mozambique tilapia) and Tilapia mariae (spotted tilapia) while R.marina_16S and C.caroliniana_matK discretely target Rhinella marina (cane toad) and Cabomba caroliniana (fanwort), respectively. All 3 assays were validated in silico before in vitro and in situ validations using PPLPP workflow extracted samples. PPLPP workflow was concurrently validated in vitro and in situ using all 3 assays. In vitro validations demonstrated that 1) glycogen inclusion increased extracellular DNA recovery by ∼48-fold compared with glycogen exclusion, 2) swinging-bucket centrifugation for 90 min at 3270 g is equivalent to fixed-angle centrifugation for 5-20 min at 6750 g, and 3) Zymo OneStep Inhibitor Removal Kit, Qiagen DNeasy PowerClean Pro Cleanup Kit, and silica-Zymo double purification provide effective inhibitor removal. In situ validation demonstrated 95.8 ± 2.8% (mean ± SEM) detectability across all 3 target species in Longmire's preserved unfiltered and filtered water samples extracted using the PPLPP workflow (without phenol:chloroform:isoamyl alcohol purification) after 39 d of incubation at room temperature and 50°C. PPLPP workflow is recommended for future temperate and tropical eDNA studies that use Longmire's to preserve unfiltered or filtered water samples.

The roles of vicariance and dispersal in the differentiation of two species of the Rhinella marina species complex.

The high levels of Neotropical biodiversity are commonly associated with the intense Neogene-Quaternary geological events and climate dynamics. Here, we investigate the evolutionary history of two species of Neotropical closely related amphibians (R. horribilis and R. marina). We combine published data with new mitochondrial DNA sequences and multiple nuclear markers, including 12 microsatellites. The phylogenetic analyses showed support for grouping the samples in two main clades; R. horribilis (Central America and Mexico) and R. marina (South America east of the Andes). However, the phylogenetic inferences also show an evident mito-nuclear discordance. We use Approximate Bayesian Computation (ABC) to test the role of different events in the diversification between the two groups recovered. We found that both species were affected primarily by a recent Pleistocene divergence, which was similar to the divergence estimate revealed by the Isolation-with-Migration model, under persistent bidirectional gene flow through time. We provide the first evidence that R. horribilis is differentiated from the South American R. marina at the nuclear level supporting the taxonomic status of R. horribilis, which has been controversial for more than a century.

May the (selective) force be with you: Spatial sorting and natural selection exert opposing forces on limb length in an invasive amphibian.

Spatial sorting on invasion fronts drives the evolution of dispersive phenotypes, and in doing so can push phenotypes in the opposite direction to natural selection. The invasion of cane toads (Rhinella marina) through tropical Australia has accelerated over recent decades because of the accumulation of dispersal-enhancing traits at the invasion front, driven by spatial sorting. One such trait is the length of the forelimbs: invasion-front toads have longer arms (relative to body length) in comparison with populations 10-20 years after invasion. Such a shift likely has fitness consequences: an increase of forearm length would decrease the strength with which a male could cling to a female during amplexus and so render such a male less competitive in competition for mates, compared to short-armed conspecifics. Our laboratory trials of attachment strength confirmed that males with relatively longer arms were easier to displace, and competition trials show higher duration of amplexus for males with shorter arms. Together with the sharp cline in limb length observed behind the invasion front, these results imply an opposition of selective forces: spatial sorting optimizes dispersal, but as this force wanes behind the invasion front, we see the primacy of natural selection reassert itself.

Differential gene expression to an LPS challenge in relation to exogenous corticosterone in the invasive cane toad (Rhinella marina).

The cane toad (Rhinella marina) is an invasive amphibian in several parts of the world. Much of the research performed on assessing the dispersal potential of invasive species has focused immunity. Invaders are predicted to rely less on pro-inflammatory immunity, allowing them to allocate energy to dispersal. Elevated stress may play a role in regulation of immune responses used by invasive species. RNA sequencing of spleen tissue from cane toads subjected to an acute LPS challenge revealed genes coding for cytokines involved in typical innate responses such as phagocytic cell recruitment, extravasation, inflammation, and lymphocyte differentiation were significantly upregulated, while toads receiving transdermal application of corticosterone in addition to an LPS injection showed downregulation of genes involved with cell mediated immunity. These results indicate hormonal changes associated with acute stress may alter investment into mounting cell-mediated or humoral responses while allowing for prolonged phagocytic innate responses in this invasive species.

Genetic damage in Rhinella marina populations in habitats affected by agriculture in the middle region of the Sinú River, Colombia.

Contamination with pesticide residues affects the environmental health of agroecosystems, especially the amphibian fauna that lives in these environments. The objective of the present study was to determine pesticides concentrations in sediments of agroecosystems and to evaluate genetic damage in Rhinella marina populations living in these zones. A total of 91 individuals were collected, 51 in the group exposed in different areas of the middle region of the Sinú River (Irrigation District of Mocari 16, Irrigation District of Aguas Negras 21, Irrigation District of Cerete 14) and 40 in a control group; at the same time, 36 subsamples of sediments were taken at each sampled station to determine pesticides organochlorine by means of chromatography coupled with ISQ Thermo Scientific mass spectrometer. The micronucleus test was applied in erythrocytes of the individuals collected. Results showed the presence of persistent organochlorine pesticides (POPs) in the sediment samples (p,p'-DDT, p,p'-DDE, and p,p'-DDD) of agricultural soils. Two individuals were registered with abnormalities in their limbs at the Mocari station, representing 12.5% of the morphological malformations to this sector. Micronucleus analysis revealed statistically significant genetic damage in exposed individuals (Mocari 9.87 ± 5.1, Cerete 7.7 ± 1.7, Aguas Negras 5.6 ± 3.6) with respect to the control group (2.4 ± 1.9) (p < 0.05). Spearman correlation analysis revealed a positive association between genetic damage and POP concentrations (p < 0.05). In addition, cellular alterations such as nuclear buds, and pyknosis (cell death), were statistically significant in the exposed group compared to the control group (p < 0.05). This study suggests that there is evidence for morphological and genotoxic effects in R. marina populations inhabiting areas influenced by agriculture, possibly associated with the presence of p,p'-DDT, p,p'-DDD, and p,p'-DDE.

The cane or marine toad, Rhinella marina (Anura, Bufonidae): two genetically and morphologically distinct species.

Rhinella marina is a Neotropical toad that has been introduced widely worldwide. Its toxic effects to frog-eating predators threaten the native and domestic fauna of some regions where it has been introduced. Despite previous studies suggesting two genetically distinct cryptic species within R. marina, one east and one west of the Andes, its taxonomic status remained unresolved due to the absence of morphological complementary evidence. For the first time, data from two mitochondrial genes (ND3 and CR) and 23 morphometric landmarks are combined to evaluate the taxonomic status of this species. Our results support the hypothesis of two separate evolutionary lineages within R. marina and demonstrate that these lineages have significantly diverged in skull shape. We identified two distinct morphotypes, one eastern and one Andean western, with no overlapping morphospaces. The geographic pattern of genetic variation was consistent with a stable structured population with no evidence of recent demographic or geographic expansions. The concordance between the observed geographic patterns in morphometric and genic traits calls for the recognition of two species under R. marina name.

Mixed population genomics support for the central marginal hypothesis across the invasive range of the cane toad (Rhinella marina) in Australia.

Understanding factors that cause species' geographic range limits is a major focus in ecology and evolution. The central marginal hypothesis (CMH) predicts that species cannot adapt to conditions beyond current geographic range edges because genetic diversity decreases from core to edge due to smaller, more isolated edge populations. We employed a population genomics framework using 24 235-33 112 SNP loci to test major predictions of the CMH in the ongoing invasion of the cane toad (Rhinella marina) in Australia. Cane toad tissue samples were collected along broad-scale, core-to-edge transects across their invasive range. Geographic and ecological core areas were identified using GIS and habitat suitability indices from ecological niche modelling. Bayesian clustering analyses revealed three genetic clusters, in the northwest invasion-front region, northeast precipitation-limited region and southeast cold temperature-limited region. Core-to-edge patterns of genetic diversity and differentiation were consistent with the CMH in the southeast, but were not supported in the northeast and showed mixed support in the northwest. Results suggest cold temperatures are a likely contributor to southeastern range limits, consistent with CMH predictions. In the northeast and northwest, ecological processes consisting of a steep physiological barrier and ongoing invasion dynamics, respectively, are more likely explanations for population genomic patterns than the CMH.

Molecular characterization of MHC class II in the Australian invasive cane toad reveals multiple splice variants.

The cane toad has gained notoriety for its invasion across the Australian landscape, with significant impacts on the native Australian fauna. The invasion has accelerated over time, with invading cane toads adapted for highly dispersive traits. This, however, has come at the cost of the immune system, with lower investment in some immune functions. To investigate the cane toad's immunogenetics, we characterized four major histocompatibility complex (MHC) class IIA and three MHC class IIB loci. Preliminary observations suggest very low allelic diversity at all loci. We also observed various splice isoforms. One isoform seen at one class IIA and two class IIB loci was missing exon 2, which is essential to peptide binding and presentation. The other isoform, observed at a class IIA locus, is likely to be a soluble MHC product. These results may suggest a significant role of alternative splicing of MHC loci in the Australian cane toad.

Patterns of Genetic Variability in Island Populations of the Cane Toad (Rhinella marina) from the Mouth of the Amazon.

The Amazonian coast has several unique geological characteristics resulting from the interaction between drainage pattern of the Amazon River and the Atlantic Ocean. It is one of the most extensive and sedimentologically dynamic regions of the world, with a large number of continental islands mostly formed less than 10,000 years ago. The natural distribution of the cane toad (Rhinella marina), one of the world's most successful invasive species, in this complex Amazonian system provides an intriguing model for the investigation of the effects of isolation or the combined effects of isolation and habitat dynamic changes on patterns of genetic variability and population differentiation. We used nine fast-evolving microsatellite loci to contrast patterns of genetic variability in six coastal (three mainlands and three islands) populations of the cane toad near the mouth of the Amazon River. Results from Bayesian multilocus clustering approach and Discriminant Analyses of Principal Component were congruent in showing that each island population was genetically differentiated from the mainland populations. All FST values obtained from all pairwise comparisons were significant, ranging from 0.048 to 0.186. Estimates of both recent and historical gene flow were not significantly different from zero across all population pairs, except the two mainland populations inhabiting continuous habitats. Patterns of population differentiation, with a high level of population substructure and absence/restricted gene flow, suggested that island populations of R. marina are likely isolated since the Holocene sea-level rise. However, considering the similar levels of genetic variability found in both island and mainland populations, it is reliable to assume that they were also isolated for longer periods. Given the genetic uniqueness of each cane toad population, together with the high natural vulnerability of the coastal regions and intense human pressures, we suggest that these populations should be treated as discrete units for conservation management purposes.

Characterisation of major histocompatibility complex class I in the Australian cane toad, Rhinella marina.

The Major Histocompatibility Complex (MHC) class I is a highly variable gene family that encodes cell-surface receptors vital for recognition of intracellular pathogens and initiation of immune responses. The MHC class I has yet to be characterised in bufonid toads (Order: Anura; Suborder: Neobatrachia; Family: Bufonidae), a large and diverse family of anurans. Here we describe the characterisation of a classical MHC class I gene in the Australian cane toad, Rhinella marina. From 25 individuals sampled from the Australian population, we found only 3 alleles at this classical class I locus. We also found large number of class I alpha 1 alleles, implying an expansion of class I loci in this species. The low classical class I genetic diversity is likely the result of repeated bottleneck events, which arose as a result of the cane toad's complex history of introductions as a biocontrol agent and its subsequent invasion across Australia.

Behavioural flexibility allows an invasive vertebrate to survive in a semi-arid environment.

Plasticity or evolution in behavioural responses are key attributes of successful animal invasions. In northern Australia, the invasive cane toad (Rhinella marina) recently invaded semi-arid regions. Here, cane toads endure repeated daily bouts of severe desiccation and thermal stress during the long dry season (April-October). We investigated whether cane toads have shifted their ancestral nocturnal rehydration behaviour to one that exploits water resources during the day. Such a shift in hydration behaviour could increase the fitness of individual toads by reducing exposure to desiccation and thermal stress suffered during the day even within terrestrial shelters. We used a novel method (acoustic tags) to monitor the daily hydration behaviour of 20 toads at two artificial reservoirs on Camfield station, Northern Territory. Remarkably, cane toads visited reservoirs to rehydrate during daylight hours, with peaks in activity between 9.00 and 17.00. This diurnal pattern of rehydration activity contrasts with nocturnal rehydration behaviour exhibited by adult toads in their native geographical range and more mesic parts of Australia. Our results demonstrate that cane toads phase shift a key behaviour to survive in a harsh semi-arid landscape. Behavioural phase shifts have rarely been reported in invasive species but could facilitate ongoing invasion success.

Z and W sex chromosomes in the cane toad (Bufo marinus).

The cane toad (Bufo marinus) is one of the most notorious animal pests encountered in Australia. Members of the genus Bufo historically have been regarded as having genotypic sex determination with male homogamety/female heterogamety. Nevertheless, as with many toads, karyotypic analyses of the cane toad have so far failed to identify heteromorphics sex chromosomes. In this study, we used comparative genomic hybridization, reverse fluorescence staining, C-banding, and morphometric analyses of chromosomes to characterize sex chromosome dimorphism in B. marinus. We found that females consistently had a length dimorphism associated with a nucleolus organizer region (NOR) on one of the chromosome 7 pair. A strong signal over the longer NOR in females, and the absence of a signal in males indicated sex-specific DNA sequences. All females were heterozygous and all males homozygous, indicating a ZZ/ZW sex chromosomal system. Our study confirms the existence of sex chromosomes in this species. The ability to reliably identify genotypic sex of cane toads will be of value in monitoring and control efforts in Australia and abroad.

Cloning and expression of candidate sexual development genes in the cane toad (Bufo marinus).

The development of the reproductive system in bufonids (true toads) is unique in several respects: sexual differentiation occurs later than in other anurans, and toads develop a Bidder's organ, a rudimentary ovary that can be manipulated in males to produce mature oocytes. To illuminate the genesis of this unusual reproductive system, we isolated from the cane toad (Bufo marinus) the orthologues of several known vertebrate sex-determining genes, determined their primary structure, and studied their expression by reverse transcriptase-polymerase chain reaction and in situ hybridization of tissue sections. We report here that cane toad Sox9, Dmrt1, and p450aromatase (Cyp19a1) are highly homologous to their counterparts in other vertebrates. They show profiles of expression that generally follow patterns observed in other taxa, but with some novel features. Our data suggest that these genes likely play key roles in sex determination and early gonad development in bufonids.