The central claim of this review is deliberately restricted. We do not claim to demonstrate genomic adaptation in B. sitibundus or P. bedriagae. What is demonstrated here is a set of ecological, distributional and physiological contrasts between the two species, several of which were measured directly in Israeli animals. What is proposed here is a structured, prioritized set of candidate genes and molecular pathways that could plausibly underlie those contrasts and that can be tested by future work. The two are kept separate throughout the text, and the reader should treat every gene name in Section 7 as a prediction rather than as a finding unless direct expression, sequence or functional evidence is cited alongside it. No such direct molecular evidence currently exists for either focal species.

A second and equally important limitation concerns what an ecological contrast between two genera can and cannot show. Bufotes (Bufonidae) and Pelophylax (Ranidae) are distantly related lineages whose most recent common ancestor lies deep within the Neobatrachia [18][19]. The two genera therefore differ in a very large number of characters, most of which have nothing to do with present-day habitat use and simply reflect inherited phylogenetic history. Skin thickness and glandular architecture, limb proportions, webbing, karyotype and genome size all differ between bufonids and ranids as a general rule, irrespective of whether a particular species is arid-adapted or aquatic. Consequently, an observed difference between B. sitibundus and P. bedriagae, whether physiological or, in future, genomic, cannot by itself be attributed to habitat association: the same difference is expected under the null hypothesis of phylogenetic inertia alone.

Distinguishing habitat-driven adaptation from phylogenetic background requires designs that this review can recommend but not supply. The most informative are: 1) within-genus comparisons along the Israeli climatic gradient, for example among B. sitibundus populations from Mediterranean, semi-arid and desert-margin sites, where phylogenetic background is approximately constant and habitat varies; 2) phylogenetically replicated cross-genus comparisons, in which several independent terrestrial-aquatic species pairs are examined so that convergence can be tested statistically; and 3) explicit phylogenetic comparative methods applied to trait and sequence data across a broader anuran sample. Until such designs are implemented, statements in this review about “lineage-specific” or “convergent” adaptation should be read as hypotheses to be tested, not as conclusions.

To avoid conflating observation with inference, every substantive statement in this review belongs to one of three evidence levels. These levels are signalled in the text by explicit wording, and the reader can identify the level of any claim from that wording alone.

Level 1—Direct evidence from the focal taxa (B. sitibundusorP. bedriagae,including material published under earlier names). This is the strongest category and is comparatively small. It comprises distributional and habitat data for Israeli populations [1][2][6][14]; larval ecology and development in Israeli winter ponds [6][14]; urea accumulation during terrestrial acclimation in the green toad [20]; respirometric measurements of oxygen consumption and carbon dioxide production across ambient temperatures in hydrated and dehydrated animals of both species [21]; and mitochondrial 12S and 16S rRNA sequences generated from Israeli P. bedriagae and from Israeli green toads [16][17]. Statements resting on Level 1 evidence are introduced in the text by phrases such as “has been measured in”, “was observed in” or “direct evidence from the focal species shows”.

Level 2—Evidence from related taxa withinBufotesorPelophylax. This category supplies most of the molecular and genomic material in the review, because genomic resources for the focal species are essentially absent. It includes the chromosome-level reference genome of B. viridis [12], phylogenetic and phylogeographic analyses of the B. viridis subgroup [10] and of the P. ridibundus complex [9][13], and population-genomic work on hybridogenetic Pelophylax systems [8]. Level 2 evidence constrains what is plausible for the focal species but does not establish it, because the congeners differ from the focal taxa in ploidy, range, climate and, for several Pelophylax lineages, reproductive mode. Statements at this level are introduced by “in related species”, “in congeners” or “by extrapolation from”.

Level 3—General amphibian or vertebrate evidence. This is the weakest category with respect to the focal species and supplies the mechanistic background: aquaporin-mediated water uptake across ventral skin in semiterrestrial and aquatic anurans generally [22][23]; heat-shock and cellular stress responses across ectotherms [24]-[26]; antioxidant, hypoxia-signalling, innate-immune, metabolic and endocrine pathways characterized largely in model vertebrates [27]-[37]; and general amphibian environmental physiology [3][15][18][38]. Because these pathways are conserved across vertebrates, their presence in Bufotes and Pelophylax can be assumed, but nothing about habitat-specific regulation in either genus follows from that conservation. Statements at this level are introduced by “in amphibians generally”, “across vertebrates” or “conserved across taxa”.

All candidate-gene assignments in Section 7 rest on Level 3 evidence, occasionally supported at Level 2. None currently rests on Level 1. They are accordingly labelled as predictions throughout, and Section 7.9 sets out what would be required to raise them to Level 1.

Nomenclature in both genera has changed repeatedly, and several of the primary datasets used here were published under names that are no longer valid. To avoid ambiguity, the following conventions are applied consistently throughout this review.

Pelophylax bedriagae (Camerano, 1882) is used for the Levant water frog of Israel and the adjacent eastern Mediterranean. Material published as Rana ridibunda, Rana bedriagae, Rana levantina or Pelophylax ridibundus from Israeli localities is referred here to P. bedriagae, and the original name is given in parentheses at first mention of each dataset. Pelophylax ridibundus (Pallas, 1771) is retained in its restricted modern sense for the marsh frog of central and eastern Europe and western Asia and is treated as a distinct species, not as a senior synonym of the Israeli taxon. Where a cited study analysed a broader assemblage, the term “P. ridibundus species complex” is used and the Israeli taxon is not assumed to be represented unless the study sampled Levantine localities.

Bufotes sitibundus (Pallas, 1771) is used for the green toad of Israel. Material published as Bufo viridis, Pseudepidalea viridis or Bufotes variabilis from Israeli localities is referred here to B. sitibundus, again with the original name given in parentheses at first mention. Bufotes viridis is retained for the European green toad in its restricted sense, including the individual from which the reference genome was assembled [12]; that genome is therefore Level 2 evidence for B. sitibundus, not Level 1.

Assignment of the principal datasets used here is as follows. The respirometry of Degani and Meltzer [21], published as Bufo viridis and Rana ridibunda, was performed on animals collected in northern Israel and is assigned to B. sitibundus and P. bedriagae respectively. The urea-accumulation study of Degani et al. [20], published as Bufo viridis, is assigned to B. sitibundus. The mitochondrial sequence data of Degani et al. [16], published as Pseudepidalea viridis (syn. Bufo viridis), are assigned to B. sitibundus; those of Degani et al. [17], published as Rana bedriagae, are assigned to P. bedriagae and are listed in Table 1. The larval-ecology studies [6][14] were conducted in Israeli winter ponds and are assigned to the focal species accordingly. By contrast, the phylogeographic and population-genomic datasets of Dubey et al. [8], Ualiyeva et al. [13] and Dufresnes et al. [9] sample predominantly non-Levantine lineages; they are used here as Level 2 evidence and are not treated as data on the focal Israeli populations.

Mitochondrial markers, including cytochrome b (cytb), 12S rRNA, 16S rRNA, cytochrome c oxidase subunit I (COI), NADH dehydrogenase subunit 2 (ND2) and the control region (D-loop), are widely used to reconstruct phylogenetic relationships and historical biogeography because they are maternally inherited and generally lack recombination [1][2][39]. In the Pelophylax ridibundus species complex, analyses of ND2, COI, cytb and 16S sequences have revealed deeply divergent mitochondrial lineages associated with historical isolation, recolonization and secondary contact [9][13]. Studies of the Bufotes viridis species complex similarly support B. sitibundus as a distinct evolutionary lineage distributed across the Levant, Anatolia, Iran and Central Asia [8]-[10]; comparable patterns of refugial isolation and secondary contact have been described in other west-Palearctic toads [11] (Figure 2). Mitochondrial 12S and 16S rRNA sequences generated directly from Israeli P. bedriagae (published as Rana bedriagae) constitute Level 1 evidence for the focal taxon and are listed with their GenBank accession numbers in Table 1 [17].

Figure 2. Comparative mitochondrial DNA (mtDNA) markers and phylogeographic patterns of Pelophylax bedriagae and Bufotes sitibundus.

The figure summarizes the major mitochondrial DNA markers (12S, 16S, ND2, COI, cytb and D-loop) commonly used in amphibian phylogenetic studies. It compares the mitochondrial lineages and geographic distributions of P. bedriagae (25 samples from 5 sites) and B. sitibundus (50 samples from 24 sites), illustrating their evolutionary history and population divergence. The lower panel highlights the main applications of each mtDNA marker for phylogenetic, phylogeographic and population-genetic analyses [8]-[10].

Table 1. Accession numbers of mitochondrial 12S and 16S rRNA sequences of Pelophylax bedriagae (published as Rana bedriagae), from Degani et al. [17].

Note. Sequences were obtained from Israeli populations and are assigned here to P. bedriagae following the conventions in Section 1.3. This dataset is Level 1 evidence for the focal taxon.

Figure 3. Nuclear DNA markers complement mitochondrial analyses for resolving phylogeny, hybridization, and population structure in Pelophylax ridibundus and Bufotes sitibundus.

Nuclear markers complement mitochondrial data because they are inherited from both parents and provide information on recombination, recent gene flow, hybridization and introgression. Commonly used markers include RAG1, RAG2, serum albumin intron 1 (SAI-1), rhodopsin, microsatellites and single-nucleotide polymorphisms (SNPs) [19], together with genome-wide reduced-representation approaches such as RAD-seq [40][41]. Nuclear analyses have helped resolve taxonomic uncertainty within the Pelophylax ridibundus complex and have detected hybridization and introgression that may be obscured by mitochondrial data alone [9][13]. Combined mitochondrial and nuclear analyses have likewise clarified species boundaries within Bufotes and demonstrated the contribution of historical hybridization to present-day phylogeographic patterns [8][10] (Figure 3).

The complementarity of the two marker systems is summarized in Figure 3. Hybridization and introgression can remain undetected when only maternally inherited mtDNA is analysed but are readily identified using biparentally inherited nuclear loci; recent gene flow and fine-scale population connectivity are likewise resolved more accurately with nuclear markers, because these reflect recombination and inheritance from both parents, whereas mtDNA records historical maternal lineages only. No RAD-seq or SNP dataset has yet been published for Israeli populations of either focal species, so all statements in this subsection concerning the focal taxa are Level 2 extrapolations.

In Figure 3, nuclear DNA markers complement mitochondrial analyses for resolving phylogeny, hybridization and population structure in Pelophylax bedriagae (25 samples from 5 sites) and Bufotes sitibundus (25 samples from 5 sites). 1) Nuclear markers used in phylogenetic and population-genetic studies. RAG1 and RAG2 are conserved protein-coding genes commonly used for deep phylogenetic analyses; SAI-1 (serum albumin intron 1) is an intron marker useful for species identification and population differentiation; rhodopsin is a nuclear gene applied in phylogenetic and adaptive studies; microsatellites (SSR) are highly polymorphic markers suitable for assessing genetic diversity, kinship and population structure; SNPs provide thousands of genome-wide markers for fine-scale population-genomic analyses; and RAD-seq enables large-scale SNP discovery across the genome and is widely used for phylogeography, adaptation and evolutionary genomics. 2) Nuclear markers reveal patterns not detected by mitochondrial DNA, including hybridization, introgression, recent gene flow and fine-scale population structure. When combined with mitochondrial markers they provide a more complete reconstruction of species boundaries and evolutionary history [8][19][40][41].

Maintaining water balance is one of the greatest physiological challenges for amphibians. In terrestrial species, water uptake and conservation are regulated primarily through aquaporin (AQP) water channels expressed in the pelvic skin, urinary bladder and kidneys, which facilitate rapid water transport while limiting dehydration during prolonged exposure to dry environments. Vasotocin-regulated AQP2-like proteins contribute to water reabsorption, whereas AQP5-like proteins contribute to skin permeability [22][23]. These mechanisms are characterized in anurans generally (Level 3) and have not been examined in either focal species. Direct evidence from B. sitibundus is limited to the whole-organism level: green toads from Israel accumulate urea during terrestrial acclimation, which reduces the osmotic gradient driving evaporative water loss [20]. This is a Level 1 observation of a physiological outcome; the transporters and channels responsible have not been identified in this species.

Pelophylax bedriagae inhabits permanent freshwater habitats, where dehydration risk is generally lower than in terrestrial environments [3]. The principal prediction is therefore not the absence of osmoregulatory genes, which are broadly conserved across amphibians, but habitat-associated differences in their regulation, tissue-specific expression or sequence variation [23]. In particular, aquaporins and other genes involved in transepithelial water and ion transport are predicted to exhibit differential expression or regulation in response to contrasting hydric environments. Direct comparative genomic and transcriptomic analyses of P. bedriagae and terrestrial Bufotes species are required to test this hypothesis. The most informative test would be quantitative comparison of AQP transcript abundance in pelvic skin and urinary bladder between hydrated and dehydrated animals of both species, ideally replicated across the Israeli climatic gradient so that habitat and phylogeny are not confounded.

Candidate genes (predicted):AQP1,AQP2-like (AQP-h2),AQP3,AQP5-like(AQP-h3), AVPR2-like,SLC9A3, ATP1A1. Evidence level: 3, with no supporting Level 1 or Level 2 data in either genus.

Semi-arid habitats expose Bufotes to elevated temperatures and prolonged solar radiation. Heat-shock proteins protect cellular proteins from denaturation and maintain protein folding under thermal stress, and HSP70 and HSP90 are induced by thermal stress in many ectotherms, including amphibians [24]-[26]. This is Level 3 evidence. Terrestrial Bufotes populations are predicted to show stronger or more rapidly inducible heat-shock responses than aquatic Pelophylax populations, but this prediction has not been tested in the focal species, and no heat-shock expression data exist for either. A direct test would compare induction kinetics and threshold temperatures for HSP70 and HSP90 transcripts and proteins in tissue from both species under controlled thermal ramps.

Candidate genes (predicted):HSP70(HSPA1A),HSP90AA1,HSF1. Evidence level: 3.

This subsection differs from the remainder of Section 7 in that it reports measured data from the two focal species rather than predictions. It is the principal body of Level 1 physiological evidence available for the comparison.

Degani and Meltzer [21] examined the effects of environmental temperature and dehydration on energy metabolism in two amphibian species occupying contrasting ecological habitats: the terrestrial green toad, Bufotes sitibundus (published as Bufo viridis), and the semi-aquatic marsh frog, Pelophylax bedriagae (published as Rana ridibunda). Oxygen consumption was measured across a range of ambient temperatures before and after the animals were exposed to dehydration.

At moderate ambient temperatures ranging from 14˚C to 27˚C, the two species exhibited generally similar metabolic rates, indicating that their metabolic responses were comparable under relatively mild thermal conditions. Clear interspecific differences emerged as environmental temperatures increased.

At higher temperatures, between 27˚C and 36˚C, P. bedriagae (the semi-aquatic frog) showed a greater metabolic rate than B. sitibundus (the terrestrial toad). This difference was evident in both hydrated and dehydrated animals, suggesting that the contrasting metabolic responses were associated primarily with species-specific physiological characteristics rather than with hydration status alone [21].

Measurements of carbon dioxide production were consistent with the oxygen-consumption results. At elevated temperatures, CO₂ production was also higher in P. bedriagae than in B. sitibundus. Together, these findings indicate that the terrestrial toad maintains a comparatively lower metabolic rate when exposed to high environmental temperatures, whereas the semi-aquatic frog exhibits a stronger increase in metabolic activity [21].

These physiological differences are consistent with adaptation to the contrasting ecological conditions occupied by the two species. The relatively reduced metabolic response of B. sitibundus at high temperatures may contribute to its ability to tolerate terrestrial environments in which elevated temperatures and limited water availability frequently occur. In contrast, the higher metabolic activity observed in P. bedriagae is consistent with its greater dependence on aquatic or semi-aquatic habitats, where access to water reduces some of the physiological constraints associated with dehydration and exposure to high temperatures.

Two qualifications should be emphasized. First, the observed difference in thermal metabolic response is a demonstrated phenotypic contrast; the genes or regulatory mechanisms responsible for it were not investigated and remain entirely unknown. No inference about metabolic gene sequence or expression in either species follows from these measurements. Second, because the comparison involves a single bufonid and a single ranid, the difference cannot be attributed to habitat association rather than to family-level physiological differences (Section 1.1). Replication across additional terrestrial-aquatic species pairs, and across B. sitibundus populations spanning the Israeli climatic gradient, would be required to establish that the contrast is habitat-driven.

Thermal stress, dehydration and fluctuating oxygen availability increase the production of reactive oxygen species (ROS). Amphibians counteract oxidative damage through antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GSR) and peroxiredoxins detoxify ROS and protect lipids, proteins and DNA [26][27]. These functions are conserved across vertebrates (Level 3). The prediction that terrestrial B. sitibundus sustains higher constitutive or inducible antioxidant capacity than aquatic P. bedriagae is untested; enzyme activity assays and transcript quantification in liver, muscle and skin under controlled heat and dehydration treatments would provide the first direct test.

Candidate genes (predicted):SOD1,SOD2,CAT,GPX1,GSR,PRDX1. Evidence level: 3.

Aquatic anurans encounter hypoxic conditions in eutrophic ponds, beneath ice and during prolonged submergence, and oxygen-sensing pathways centred on hypoxia-inducible factor 1α mediate the transcriptional response to low oxygen availability in vertebrates generally, regulating anaerobic glycolysis, angiogenesis and erythropoiesis [28][29]. The prediction arising from the ecological contrast is that P. bedriagae relies more heavily on these pathways than does the predominantly terrestrial B. sitibundus, which instead encounters desiccation and hyperthermia as its principal stressors. This prediction rests entirely on Level 3 evidence; no hypoxia-response data exist for either focal species. A direct test would compare HIF1A target-gene induction and blood lactate accumulation during controlled submergence or progressive hypoxia in the two species.

Candidate genes (predicted):HIF1A,VEGFA,EPO,LDHA,EGLN1. Evidence level: 3.

Amphibian skin serves simultaneously as a respiratory surface, an osmoregulatory organ and the primary barrier against pathogens. Antimicrobial peptides, pattern-recognition receptors and complement components constitute the principal innate defences of anuran skin [30][31], and gross differences in skin architecture between bufonids and ranids are well documented [18]. The thick, keratinized skin of Bufotes is expected to reduce evaporative water loss and to provide mechanical protection, whereas the thin, highly permeable skin of Pelophylax facilitates cutaneous respiration but presents a more exposed surface to waterborne pathogens. These structural differences are, however, family-level characters (Section 1.1) and cannot be read as habitat adaptations within this two-species comparison. Whether the associated immune gene repertoires differ between the focal species, and whether any such difference tracks habitat rather than phylogeny, is unknown; skin transcriptome comparison under standardized pathogen challenge would be the appropriate first test.

Candidate genes (predicted):TLR2,TLR4,β-defensins,cathelicidins,lysozyme(LYZ),IL1B,TNF,C3. Evidence level: 3.

Habitat differences are expected to influence energy metabolism in amphibians. Terrestrial B. sitibundus experiences intermittent food availability, dehydration and prolonged periods of inactivity, which would favour metabolic flexibility, efficient glycogen storage, lipid utilization and energy conservation. In contrast, aquatic P. bedriagae generally inhabits more stable environments with continuous access to water and relatively predictable food resources, reducing the need for extreme metabolic adjustment [38]. The measured thermal metabolic contrast reported in Section 7.3 [21] is the one element of this picture that has been demonstrated in the focal species; the remainder is inference.

In vertebrates generally, such physiological differences are regulated by genes involved in energy sensing, glycolysis, mitochondrial respiration and fatty-acid oxidation [33][43]. AMPK (PRKAA1) acts as the major cellular energy sensor, promoting ATP production while suppressing energy-consuming processes. PPARGC1A (PGC-1α) regulates mitochondrial biogenesis and oxidative metabolism [32]. CPT1A controls the transport of long-chain fatty acids into mitochondria for β-oxidation. HK1 catalyses the first step of glycolysis, LDHA supports anaerobic ATP production during oxygen limitation and COX1 encodes a key subunit of cytochrome c oxidase in the mitochondrial electron transport chain.

Comparative analyses of these genes may reveal differences in expression, regulation, enzyme activity or sequence variation between B. sitibundus and P. bedriagae. No such comparison has been performed, and the gene list below is therefore a prediction generated by the phenotypic contrast in Section 7.3 rather than a molecular explanation of it. Direct comparative genomic, transcriptomic, proteomic and physiological studies are required to determine whether these pathways have undergone habitat-specific modification.

Candidate genes (predicted):PRKAA1(AMPK),PPARGC1A(PGC-1α),CPT1A,HK1,LDHA,COX1. Evidence level: 3, with a supporting Level 1 whole-organism phenotype (Section 7.3) that does not by itself implicate any of these loci.

Endocrine pathways integrate environmental signals with physiological responses that regulate water balance, growth, metabolism, development and stress adaptation in amphibians [3][34]. Arginine vasotocin (AVT) regulates osmoregulation by stimulating aquaporin-mediated water transport in the skin and urinary bladder [23]. Corticosterone, acting through the glucocorticoid receptor (NR3C1), coordinates physiological responses to dehydration and other environmental stresses [34]. Thyroid hormones, acting through THRA and THRB, control metamorphosis and tissue differentiation [35]. Growth hormone (GH1), insulin-like growth factor 1 (IGF1) and prolactin (PRL) regulate somatic growth, energy metabolism, osmoregulation and tissue maintenance [36][37]. All of this is Level 3 evidence from amphibians and vertebrates generally.

Comparative analyses of these genes between B. sitibundus and P. bedriagae may reveal habitat-associated differences in endocrine regulation, gene expression or sequence variation. No endocrine measurements or expression data are available for either focal species, so the assignment below is predictive only. Plasma corticosterone and AVT profiling during standardized dehydration, coupled with receptor-transcript quantification, would supply the first direct evidence.

Candidate genes (predicted):AVPR2-like,GH1,IGF1,PRL,THRA,THRB,NR3C1. Evidence level: 3.

High-quality genomic resources remain unavailable for B. sitibundus and P. bedriagae. Most molecular inference therefore relies on related species, including B. viridis, B. variabilis, P. ridibundus, P. lessonae and P. kurtmuelleri [8][10][12][13]. Genes involved in water transport, stress responses, immunity, metabolism and endocrine regulation are broadly conserved across anurans, but conservation alone does not demonstrate adaptive differentiation between the focal species (Figure 7).

Figure 7. Comparative molecular pathways proposed to underlie habitat-specific adaptation in the green toad (Bufotes sitibundus) and the Levant water frog (Pelophylax bedriagae).

Four priorities follow directly from the evidence structure set out in Section 1.2. First, chromosome-level genome assemblies for B. sitibundus and P. bedriagae, anchored to the existing B. viridis assembly [12], would convert much of the Level 2 material in this review into Level 1 evidence. Second, population-genomic sampling of B. sitibundus across the Mediterranean-to-desert gradient would allow selection scans in which phylogenetic background is held approximately constant while habitat varies, which is the single most direct way to separate adaptation from phylogenetic inertia. Third, tissue-specific transcriptomics under controlled dehydration, heat and hypoxia challenge would test the expression predictions in Sections 7.1, 7.2 and 7.4 - 7.8 without requiring a complete assembly. Fourth, phylogenetically replicated comparisons across additional terrestrial-aquatic anuran pairs would establish whether any difference recovered between the focal genera represents convergent habitat adaptation or a lineage-specific peculiarity. Until at least the second and third of these are undertaken, the candidate pathways proposed here remain untested hypotheses.

All pathways shown are candidate predictions, not demonstrated species-specific differences. 1)Water conservation and osmoregulation: the terrestrial green toad is predicted to rely on efficient water conservation through aquaporins and ion transporters regulating water uptake across the pelvic skin, urinary bladder and kidneys, whereas the aquatic frog experiences lower dehydration stress [3][26]-[28]. 2)Heat stress and cellular protection: terrestrial habitats expose Bufotes to elevated temperatures and solar radiation, predicting stronger induction of heat-shock proteins; candidate genes include HSP70 (HSPA1A), HSP90AA1 and HSF1 [3][24][26]. 3)Oxidative stress defence: heat and dehydration increase reactive oxygen species, predicting enhanced antioxidant defences in the terrestrial species; candidate genes include SOD1, SOD2, CAT, GPX1, GSR and PRDX1 [3][26][27]. 4)Hypoxia responses: aquatic frogs encounter hypoxic conditions and are predicted to depend more strongly on oxygen-sensing pathways; candidate genes include HIF1A, VEGFA, EPO and LDHA [3][28][29]. 5)Skin-barrier function and innate immunity: the thick, keratinized skin of Bufotes reduces evaporative water loss and provides protection against pathogens, whereas the thin, permeable skin of Pelophylax facilitates cutaneous respiration; candidate immune genes include TLR2, TLR4, β-defensins, cathelicidins, lysozyme, IL1B, TNF and C3 [3][30][31]. 6)Energy metabolism: the green toad is predicted to show greater metabolic flexibility in fluctuating terrestrial environments; candidate genes include PRKAA1, PPARGC1A, CPT1A, HK1, LDHA and COX1 [3][26][33]. 7)Endocrine regulation: hormonal pathways coordinate water balance, stress responses, growth, reproduction and metamorphosis; candidate genes include AVPR2-like, GH1, IGF1, PRL, THRA, THRB and NR3C1 [3][34]. 8)Current genomic evidence and research priorities: genomic resources for the focal species remain unavailable, and the proposed pathways are inferred from related Eurasian species and from vertebrates generally; future whole-genome sequencing, population-genomic analyses and functional studies are required to validate them [8]-[10][12][13][18].