Invasive species are a key factor contributing to the global decline of biodiversity, and understanding the underlying mechanisms is crucial to mitigate detrimental effects []. One such mechanism is the introduction of invasive species with defensive strategies, such as novel toxins, that can disrupt native predator communities []. Disruption of such communities can produce trophic cascades, impacting a diverse array of taxa []. Madagascar, a globally significant biodiversity hotspot, has recently experienced the introduction of a toxic bufonid amphibian, the Asian common toad (Duttaphrynus melanostictus) []. Since its invasion, the toad population has expanded rapidly, making control efforts problematic and eradication extremely difficult []. Previous cases of bufonid introductions, such as the ongoing spread of the cane toad (Rhinella marina) in Australia, have resulted in the decimation of many indigenous species [], prompting fears that Madagascar may be similarly impacted []. Here we show that these fears are warranted: we demonstrate that many Malagasy vertebrates are likely to be susceptible to the toxins of this invasive toad.

The new toad in town: Distribution of the Asian toad, Duttaphrynus melanostictus, in the Toamasina area of eastern Madagascar.

Main Text

5 Flier J.

Edwards M.

Daly J.

Myers C. Widespread occurrence in frogs and toads of skin compounds interacting with the ouabain site of Na+, K+-ATPase. +/K+-ATPase) of cells, resulting in the inhibition of ion transport, causing cardiotoxic effects and, ultimately, death [ 6 Lingrel J.B. The physiological significance of the cardiotonic steroid/ouabain-binding site of the Na, K-ATPase. 7 Ujvari B.

Casewell N.R.

Sunagar K.

Arbuckle K.

Wüster W.

Lo N.

O’Meally D.

Beckmann C.

King G.F.

Deplazes E.

Madsen T. Widespread convergence in toxin resistance by predictable molecular evolution. +/K+-ATPase perturb binding interactions with the bufadienolides, resulting in target-site insensitivity [ 7 Ujvari B.

Casewell N.R.

Sunagar K.

Arbuckle K.

Wüster W.

Lo N.

O’Meally D.

Beckmann C.

King G.F.

Deplazes E.

Madsen T. Widespread convergence in toxin resistance by predictable molecular evolution. Bufonid toads secrete potent forms of cardiac glycosides known as bufadienolides to defend themselves from predators []. These molecules bind to the sodium-potassium pump (Na/K-ATPase) of cells, resulting in the inhibition of ion transport, causing cardiotoxic effects and, ultimately, death []. Although bufadienolides are highly toxic to naïve predators, many species from diverse animal lineages (for example, certain reptiles, amphibians and mammals) have evolved resistance and readily consume toads without suffering ill effects []. Resistant species are phylogenetically diverse, yet the adaptations that confer tolerance are remarkably consistent and represent a fascinating example of convergent molecular evolution (see Supplemental Discussion ). In each case, two amino acid replacements, with at least one adding charge, in the first extracellular domain (H1–H2) of the alpha 1 or alpha 3 isoforms of the Na/K-ATPase perturb binding interactions with the bufadienolides, resulting in target-site insensitivity []. The universality of this resistance mechanism means that by sequencing a short portion of the relevant gene, we can reliably predict a species’ vulnerability to bufadienolides.

3 Moore M.

Fidy J.F.S.N.

Edmonds D. The new toad in town: Distribution of the Asian toad, Duttaphrynus melanostictus, in the Toamasina area of eastern Madagascar. 4 McClelland, P., Reardon, J.T., Kraus, F., Raxworthy, C.J., and Randrianantoandro, C. (2015). Asian Toad Eradication Feasibility Report for Madagascar. (Te Anau, New Zealand). 8 Ujvari B.

Mun H.C.

Conigrave A.D.

Bray A.

Osterkamp J.

Halling P.

Madsen T. Isolation breeds naivety: Island living robs Australian varanid lizards of toad-toxin immunity via four-base-pair mutation. 9 Mohammadi S.

Gompert Z.

Gonzalez J.

Takeuchi H.

Mori A.

Savitzky A.H. Toxin-resistant isoforms of Na+/K+-ATPase in snakes do not closely track dietary specialization on toads. 9 Mohammadi S.

Gompert Z.

Gonzalez J.

Takeuchi H.

Mori A.

Savitzky A.H. Toxin-resistant isoforms of Na+/K+-ATPase in snakes do not closely track dietary specialization on toads. While most recent authors have assumed that all potential Malagasy toad predators are likely sensitive to bufadienolides [], the distribution of resistance cannot be easily predicted from evolutionary origin or diet. For example, Australian monitor lizards appear to be descended from bufadienolide-resistant Asian species but have lost that resistance after a prolonged period of allopatry with bufonids []. However, recent work on snakes has demonstrated that resistance to bufadienolides is far more widespread than bufophagy [], suggesting phylogenetic conservatism. Since we cannot rely on dietary studies and/or evolutionary relatedness to predict resistance [], the assumption that the Malagasy fauna will be vulnerable to bufadienolides due to lack of prior coexistence with toads needs to be explicitly tested.

+/K+-ATPase from 77 Malagasy species, including 27 snakes, 2 lizards, 12 frogs, 8 mammals and 28 birds (GenBank accessions MH094669-MH094740, see Figure 1 The diversity of Madagascar’s fauna exhibiting non-resistance to bufotoxins. Show full caption Dated molecular phylogeny of the sampled diversity of taxa tested for bufadienolide-resistant Na+/K+-ATPase genotypes, demonstrating a lack of resistance across almost the entire breadth of the Malagasy vertebrate fauna. Representative resistant non-Malagasy taxa have been included for phylogenetic context. We therefore sequenced the H1–H2 extracellular domain of the Na/K-ATPase from 77 Malagasy species, including 27 snakes, 2 lizards, 12 frogs, 8 mammals and 28 birds (GenBank accessions MH094669-MH094740, see Supplemental Information ), to examine the amino acid composition in the bufadienolide-binding site. In addition, we analysed data from the genomes of 11 previously sequenced species found on Madagascar ( Figure 1 ).

10 Glaw F.

Vences M. A Field Guide to the Amphibians and Reptiles of Madagascar. +/K+-ATPase, matching other non-resistant snakes [ 7 Ujvari B.

Casewell N.R.

Sunagar K.

Arbuckle K.

Wüster W.

Lo N.

O’Meally D.

Beckmann C.

King G.F.

Deplazes E.

Madsen T. Widespread convergence in toxin resistance by predictable molecular evolution. 9 Mohammadi S.

Gompert Z.

Gonzalez J.

Takeuchi H.

Mori A.

Savitzky A.H. Toxin-resistant isoforms of Na+/K+-ATPase in snakes do not closely track dietary specialization on toads. 7 Ujvari B.

Casewell N.R.

Sunagar K.

Arbuckle K.

Wüster W.

Lo N.

O’Meally D.

Beckmann C.

King G.F.

Deplazes E.

Madsen T. Widespread convergence in toxin resistance by predictable molecular evolution. 8 Ujvari B.

Mun H.C.

Conigrave A.D.

Bray A.

Osterkamp J.

Halling P.

Madsen T. Isolation breeds naivety: Island living robs Australian varanid lizards of toad-toxin immunity via four-base-pair mutation. 10 Glaw F.

Vences M. A Field Guide to the Amphibians and Reptiles of Madagascar. We sampled all three macrostomatan snake colonisations of Madagascar []. All showed identical amino acid sequences in the H1–H2 extracellular domain of the Na/K-ATPase, matching other non-resistant snakes [] and providing strong evidence that the Malagasy species are likely to be highly sensitive to the toxins of D. melanostictus. The two studied gerrhosaurid lizards (Zonosaurus spp.) also possessed the susceptible genotype, which matches that of demonstrably non-resistant Australian lizards []. Existing dietary studies lead us to suggest that many of the sequenced reptile species will likely be directly impacted via poisoning, as they are known to feed on amphibians []. However, the exact nature of the effects on different species may be difficult to predict due to the complexity of ecosystem-level trophic interactions (see Supplemental Discussion ).

7 Ujvari B.

Casewell N.R.

Sunagar K.

Arbuckle K.

Wüster W.

Lo N.

O’Meally D.

Beckmann C.

King G.F.

Deplazes E.

Madsen T. Widespread convergence in toxin resistance by predictable molecular evolution. Of the 12 frog species sequenced, 11 showed genotypes highly similar to non-resistant frogs. We found a few species with amino acid replacements in the middle of the H1–H2 extracellular domain, but the nature of these replacements seems unlikely to confer resistance to bufadienolides, as none add charged amino acids, nor are any positioned at sites previously associated with resistance [].

+/K+-ATPase genotype of the brown rat (Rattus norvegicus, see Among mammals we also identified likely vulnerability in lemurs and tenrecs. Only one native Malagasy species, the white-tailed antsangy (Rodentia: Brachytarsomys albicauda) shared the resistant Na/K-ATPase genotype of the brown rat (Rattus norvegicus, see Table S1 ). These data suggest retention of ancestral rodent resistance, indicating either little cost of maintaining resistance or continued consumption of cardiac glycoside-producing plants.

We examined sequences of 34 bird taxa, 31 of which have Na+/K+-ATPase H1–H2 domains with no evidence of amino acid replacements likely to confer resistance to bufadienolides. While some of the endemic birds sampled are not at risk due to their diets, the 15 sampled species likely to consume amphibians are probably vulnerable to toad poisoning since, in the absence of bufonids, they are unlikely to have evolved behavioural mechanisms to avoid them as food.

7 Ujvari B.

Casewell N.R.

Sunagar K.

Arbuckle K.

Wüster W.

Lo N.

O’Meally D.

Beckmann C.

King G.F.

Deplazes E.

Madsen T. Widespread convergence in toxin resistance by predictable molecular evolution. Our results for the remaining mammals and birds, specifically the endemic mammalian carnivores (Eupleridae: Malagasy civet Fossa fossana, Eastern fanalouc Eupleres goudoti, and fossa Cryptoprocta ferox) and three bird species (cuckoo roller Leptosomus discolor, Madagascar bulbul Hypsipetes madagascariensis and Madagascar mannakin Lonchura nana), are more equivocal: their sequences display one of the two substitutions that could potentially perturb bufadienolide binding. However, resistance has thus far only been identified in vertebrates that harbour two substitutions, one towards each end of the H1–H2 extracellular domain [], suggesting that these Malagasy predators are likely to be sensitive to toad toxins.