Downloaded from rsbl.royalsocietypublishing.org on 5 August 2009 Positive Darwinian selection results in resistance to cardioactive toxins in true toads (Anura: Bufonidae) David J. Moore, Damien C. T. Halliday, David M. Rowell, Anthony J. Robinson and J. Scott Keogh Biol. Lett. 2009 5, 513-516 first published online 22 May 2009 doi: 10.1098/rsbl.2009.0281 References This article cites 13 articles, 6 of which can be accessed free http://rsbl.royalsocietypublishing.org/content/5/4/513.full.html#ref-list-1 Receive free email alerts when new articles cite this article - sign up in the box at the top Email alerting service right-hand corner of the article or click here To subscribe to Biol. Lett. go to: http://rsbl.royalsocietypublishing.org/subscriptions This journal is © 2009 The Royal Society Downloaded from rsbl.royalsocietypublishing.org on 5 August 2009 Biol. Lett. (2009) 5, 513–516 Melanophryniscus and Dendrophryniscus, cardiotonic doi:10.1098/rsbl.2009.0281 steroids produced are bufadienolides (Flier et al. Published online 22 May 2009 1980; Mebs et al. 2007). Most other anurans are not Evolutionary biology tolerant to cardioactive steroids, but the hylid Scinax ruber and leptodactylid Leptodactylus macrosternum (Leptodactylus ocellatus species group) are natural pre- Positive Darwinian dators of bufonids in South America (Chen & Chen 1933; Crossland & Azevedo-Ramos 1999). selection results in Amino acid sites that determine tolerance to cardio- active steroids have been identified in the sodium resistance to cardioactive pump polymer. The initial extracellular loop of the toxins in true toads a1 subunit of the sodium pump is a primary binding site for cardiotonic steroids. Changes within and (Anura: Bufonidae) around this region have been identified as facilitating resistance in bufonids as well as in some insects and David J. Moore1,2, Damien C. T. Halliday2, rodents (Jaisser et al. 1992; Croyle et al. 1997). David M. Rowell1, Anthony J. Robinson2 This study sequenced the initial extracellular loop, and J. Scott Keogh1,* and part of the surrounding transmembrane regions 1School of Botany and Zoology, The Australian National University, of the sodium pump, from representative anurans in Canberra, ACT 0200, Australia order to determine whether resistance, conferred by 2CSIRO Entomology, Black Mountain Laboratories, Clunies Ross Street, changes in this region, is restricted to and conserved Acton, Canberra, ACT 2601, Australia *Author for correspondence ([email protected]). within Bufonidae. Specifically, we tested the hypoth- esis that positive selection has driven change in the Members of the Family Bufonidae, true toads, binding region of the sodium pump along lineages are famous for their endogenously synthesized ancestral to, or within, Bufonidae. cardioactive steroids that serve as defensive toxins. Evolution of resistance to these toxins is not understood. We sequenced a key region of 1 1 2. MATERIAL AND METHODS the toxin’s binding site in the Na /K ATPase DNA was extracted from ethanol-preserved tissue using Puregene for relevant taxa representing Hyloidea (includ- DNA Purification Kit (Gentra). Amplification of the initial ing bufonids), Ranoidea and Archaeobatrachia extracellular loop including parts of the M1 and M2 transmembrane and tested for positive selection in a phylogenetic regions was performed using the primers ATP1_178Fwd (WGARATCCTGGCACGAGATG), or ATP1_222Fwd2a (ATGG- context. Bufonidae were distinct from other GATACGGGGCCGGA), with ATP1_178Rvs (GAGGMACCAT- Hyloidea at 4–6 of 12 sites and, with one excep- GTTCTTGAAGG), with an annealing temperature of 568C for tion, had a homologous amino acid sequence. 30 cycles. PCR products were cleaned using ExoSapIT or Qiaquick Melanophryniscus stelzneri had a distinct PCR purification kit (Qiagen) after gel electrophoresis. sequence, consistent with other independent Products were sequenced using ATP1_178_Fwd and ATP1_ evidence for a differentiated toxin. Tests within 178_Rvs with BigDye Terminator v. 3.1, with an annealing temperature of 538C for 25 cycles, or subcloned using Invitrogen Topo protocol Bufonidae detected positive selection within the with PCR@4 vectors and Top10 chemically competent cells, puri- binding region, providing, to our knowledge, fied using Purelink Quick Plasmid Miniprep kit (Invitrogen) and the first evidence of this type for positive selection sequenced with M13 forward and M13 reverse primers. Reactions within Amphibia. There was no evidence for were cleaned using sodium acetate/EDTA/ethanol and sequenced positive selection on Bufonidae or M. stelzneri with an ABI Prism 3100 Sequencer. Sequence traces were aligned lineages. Sequence change in Leptodactylus and edited using SEQUENCER 3.1. The phylogeny published by Frost et al. (2006) was used for ocellatus, a leptodactylid predator of Bufonidae, analysis. Tests for positive selection were performed on three lineages provides a molecular basis for predator resistance (figure 1) using the improved branch-sites model A implemented possibly associated with gene duplication. in PAML (Yang 1997; Zhang et al. 2005). Significance was calculated by log likelihood comparison and Bayes empirical Bayes probabilities. Keywords: Bufonidae; positive selection; Amino acid naming is consistent with Croyle et al. (1997).The cardioactive steroids; Naþ/Kþ ATPase binding region spans amino acids 111–122. 3. RESULTS 1. INTRODUCTION One hundred and eighty-six base pairs of exon sequence Finding robust evidence for adaptive evolution of func- were generated for 27 hyloid samples, 19 Bufonidae, one tional traits is among the foremost challenges of Centrolenidae, one Strabomantidae, two Hylidae, two modern genetics. A promising approach is to use Leptodactylidae, one Limnodynastidae and one Myo- molecular and functional data to develop robust batrachidae, and two ranoid outgroups, one Microhylidae hypotheses of positive selection testable in a phyloge- and one Pyxicephalidae (GenBank accession numbers netic context ( Jost et al. 2008). We apply this FJ976618–FJ976647). Rhinella marinus (Z11798.2), approach to test for positive selection for the resistance Xenopus laevis (NM_001090595.1) and Xenopus of true toads (Anura: Bufonidae) to defensive toxins. tropicalis (NM_204076.1) were obtained from GenBank. Endogenous cardioactive steroids that inhibit Most non-bufonid hyloid frogs had a typical non- activity of the Naþ/Kþ ATPase (sodium pump) are resistant amino acid sequence at the binding site, synthesized by all bufonids. Skin secretions of bufonid homologous to sheep (Ovis aries; figure 1). Exceptions frogs contain levels of endogenous cardioactive steroids were Hyalinobatrachium fleischmanni (Centrolenidae), 25–40 000-fold higher than other anurans (Flier et al. with an M at site 119, and L. ocellatus (Leptodactyli- 1980), providing an effective defence against most dae). Direct sequencing of L. ocellatus found predators. With the exception of the genera an unusual density of ‘heterozygotic sites’ in the Received 5 April 2009 Accepted 1 May 2009 513 This journal is q 2009 The Royal Society Downloaded from rsbl.royalsocietypublishing.org on 5 August 2009 514 D. J. Moore et al. Evolution of resistance 111 122 Ovis aries (sheep) BovidaeQ A A T E E E P Q N D N Rattus norvegicus (rat) Muridae R S P D Xenopus tropicalis Pipidae T T Xenopus laevis Pipidae M Phynomantis bifasciatus Microhylidae D Tomopterna cryptotis Pyxicephalidae G D Limnodynastes tasmaniensis Limnodynastidae Mixophyes fasciolatus Myobatrachidae Pristimantis orestes Strabomantidae Hyloidea Scinax ruber Hylidae Litoria raniformis Hylidae Hyalinobatrachium fleischmannii Centrolenidae M Lo1 Leptodactylus ocellatus Leptodactylidae Lo2 R T D D Leptodactylus lineatus Leptodactylidae C Melanophryniscus stelzneri Bufonidae L V D N A Atelopus spumarius Bufonidae R K S D L D B Rhinella marinus Bufonidae R K S D L D Figure 1. Phylogeny and sequences of the binding site. Sequences of sheep and rat are included for comparison. Lineages tested for positive selection are A: Bufonidae, B: Bufonidae excluding Melanophryniscus and C: Melanophryniscus. Sites detected are highlighted in grey. M1–M2 extracellular loop. Cloning subsequently Bufonidae from other hyloid anurans; the molecular showed that there were two distinct conformations of basis for cardioactive steroid resistance of M. stelzneri the binding site. The nucleotide sequence outside the is distinct from that of other bufonids studied and posi- binding site was identical between the two sequences, tive selection on at least four sites in the M1–M2 however, within the binding site, one had a typical extracellularloophascontributedtotheevolutionofcar- non-resistant sequence and the other had four non- dioactive steroid resistance. Evidence that resistance in a synonymous changes: Q111R, A112T, E116D and member of Leptodactylidae has evolved independently N122D (figure 1). With the exception of Melanophry- of resistance in Bufonidae is also presented. niscus stelzneri, the amino acid sequence of the binding Resistance is not ancestral in hyloid frogs. Bufoni- site of all bufonids studied was homologous to dae were highly differentiated from other hyloid frogs R. marinus. Melanophryniscus stelzneri was differen- at 4–6 of 12 amino acids in the binding site. Binding tiated both from other Bufonidae and non-resistant site amino acid sequence of non-bufonid hyloid frogs hyloid frogs (figure 1). Both ranoid outgroups had a studied, except H. fleischmanni (Centrolenidae) and Datsite117,andTomopterna cryptotis had