Parallel evolution of toxin resistance in a predator-prey system
Santiago Herrera-Álvareza*, Maria del Pilar Rodríguez-Ordóñeza, Peter Andolfattob and Andrew J. Crawfordacd
a Departamento de Ciencias Biológicas, Universidad de los Andes, A.A. 4976, Bogotá,
Colombia.
b Department of Ecology and Evolutionary Biology & The Lewis-Sigler Institute for
Integrative Genomics, Princeton University, New Jersey, USA.
c Smithsonian Tropical Research Institute, Apartado 0843-03092, Panamá, Republic of
Panama.
d
Círculo Herpetológico de Panamá, Apartado 0824-00122, Panamá, Republic of
Panama*
Corresponding author:
Fax: +57-1-332-4069
Tel.: +57-1-339-4949 ext. 2751
Laboratorio Biom|ics , Edif. M1-301
Departamento de Ciencias Biológicas Universidad de los Andes
Carrera 1 No. 18A – 10 A.A. 4976, Bogotá Colombia.
E-mail address: [email protected]
Introduction
How predictable is genetic evolution? Repeated use of the same underlying genes during repeated phenotypic evolution (i.e. parallel and convergent evolution) is thought to indicate biases and constraints on the type of mutations that will become fixed (Conte
et al., 2012; Gompel and Prud’homme, 2009), and, moreover, implicates natural
selection (Endler, 1986). The probability of gene reuse (i.e. the frequency that the same independently evolved phenotypes are caused by the same underlying gene(s)) can be greatly influenced by levels of standing genetic variation, mutation rates, pleiotropic effects, epistatic interactions or linkage relationships (Orr, 2005; Chevin et al., 2010; Stern, 2013). According to Conte et al. (2012), the probability of gene reuse in natural populations lies between 0.32-0.55, which is surprisingly high, and indicates that phenotypic evolution is strongly constrained towards few genetic or mutational paths (Weinreich et al., 2006; Stern and Orgogozo, 2009). In other words, evolutionarily relevant mutations tend to accumulate in hotspot genes and in specific positions within these genes (Stern and Orgogozo, 2009; Stern, 2013). Recently, it has been
demonstrated that repeated evolution of complex phenotypic traits is sometimes achieved by the independent recruitment of the same genes (eg. Reed et al., 2011; Colosimo et al., 2005; Harrison et al., 2005; Copley, 2004; Swanson et al., 1991) and, strikingly, by the same gene expression profiles (Pankey et al., 2014; Gallant et al.,
2014). All of this evidence suggests that genetic evolution may be a more predictable process than expected before.
Toxin resistance is one of the clearest examples of natural selection acting upon genes and also represents a useful scenario to test for parallel evolution (Feldman et al., 2009; Jost et al., 2008; Zhen et al., 2012). Here, we study a natural predator-prey system, which involves the ability of a variety of snake and frog species to feed on
toxin-producing toads (Anura: Bufonidae). As an antipredator strategy, toads produce a set of toxins commonly known as bufadienolides; in fact, skin secretions of toads contain levels of these toxins 25-40.000-fold higher than other anurans (Flier et al., 1980). Bufadienolides belong to the family of endogenous cardioactive steroids that can bind to and inhibit the alpha subunit of the Na+,K+-ATPase (ATPα) affecting muscle contraction, neural function and the membrane potential of cells generally (Flier et al., 1980). Moore et al. (2009) detected positive selection on specific positions within the ATPα1 protein of toads, which is not surprising given that toads should be resistant to their own toxins, but also found the first evidence for adaptive substitutions in a predator species, the Criolla Frog (Leptodactylus latrans). Within vertebrates,
amphibians and reptiles have diverged for at least 300 million years making this system an excellent scenario to test for parallel evolution.
All vertebrates have at least three paralogs of the ATPα gene and all copies are
functionally equivalent (Blanco & Mercer, 1998). ATPα1 is expressed in nearly every tissue, ATPα2 predominates in muscle, heart and brain tissues and ATPα3 is expressed in nervous tissues (Blanco & Mercer, 1998), however, all three copies retain the catalytic and ion-transporting functions. Given that bufadienolides are low molecular weight hydrophobic molecules (Rash et al., 2010) it may be possible for them to pass through the predator’s blood-brain barrier (Cserr & Bundgaard, 1984), acting as a selective pressure in all three paralogs of the gene family. There is evidence that when genes within a gene family retain their physiological function, all the paralogs may be
under the same selective pressure and subsequently show patterns of parallel adaptive evolution (Jost et al., 2008; McGlothlin et al., 2014).
Adaptive molecular evolution can follow three main paths in order produce fit phenotypes: 1) substitutions in protein-coding DNA sequence, 2) regulatory changes that modify gene expression patterns or 3) a combination of both processes (Pardo-Diaz
et al., 2015; Hofmann et al., 2009). To date, toxin resistance has been shown to be
mainly achieved by either: 1) Specific substitutions at the catalytic site of the protein that reduces the binding affinity with the toxin (eg. Feldman et al., 2009) or 2)
increasing the expression of the gene thereby counteracting the adverse effects of toxins (eg. Kondrashov and Kondrashov, 2006). It remains elusive, however, whether
structural and regulatory changes can both simultaneously shape this phenotype, and, if happens, how common it is. Nonetheless, Zhen et al., 2012 found the first clear
evidence of evolution of toxin-resistance achieved via structural and regulatory changes in a herbivore insect community, shedding light on the evolution and complexity of this phenotype.
The main goals of this study were to determine the genetic basis of bufadienolide resistance in a group of evolutionary independent toad-eating species, and determine how predictable is the evolution of the ATPα genes in terms of sequence evolution and expression patterns. For these aims we used two approaches: 1) among-lineage
evolution and 2) within-lineage evolution. The former focuses on patterns of substitution throughout the ATPα1 orthologs, first within vertebrates, i.e. between anurans and snakes (including gene expression patterns), and then between vertebrates (our system) and invertebrates, i.e. insects that feed on cardenolide-producing plants (Zhen et al., 2012). The latter approach focuses on patterns of substitution between the three differentially expressed paralogs (ATPα1, ATPα2 and ATPα3) within species.
Methodology
Sample collection
Individuals from each species in the study were collected from different geographic locations in Colombia. Transciptome data from each individual was used in order to find ATPα genes implicated in toxin resistance in snakes and frogs. The genes were sampled from sequenced transcriptomes obtained from three different tissues from four snake species and two frog species that either: i) feed on toads, i.e.Leptodeira annulata,
Xenodon sp., Leptodactylus pentadactylusand Ceratophrys calcarata, ii) presumably
feed on toads, i.e. Leptodeira septentrionalis, or iii) controls that do not feed on toads, i.e. Atractus crassicaudatus (Table 1).
cDNA library construction and Illumina Sequencing
Transcriptome data was obtained from three tissues (brain, muscle and stomach) of four snake species and two frog species (for a total of 14 transcriptomes) through RNA isolation and next-generation sequencing (Table 2). Tissues were preserved in
RNAlater® (Qiagen, Hilden, Germany) to avoid RNA degradation. RNA was extracted
with standard TRI Reagent® Solution (Ambion Inc., Austin, Texas, USA) and then
cleaned with the RNA Cleanup protocol of RNeasy Plus Mini Kit (Qiagen. Hilden, Germany) and concentrated to a final volume of 30µl with nuclease-free water. Quantity
of extracted RNA for library construction was measured with Qubit® RNA HS Assay kit. Complementary DNA (cDNA) libraries were constructed with the Illumina TruSeq v. 2 kit using half reactions. Quality of cDNA libraries was assessed using Agilient 2100 BioAnalyzer and Agilient High Sensitivity DNA kit. The 14 libraries were barcoded and run together (single-end mode) in one lane of an Illumina Hiseq 2000.
Bioinformatic analyses
Demultiplexing, sequences trimming and de-novo assembly
Demultiplexing of reads by barcodes was conducted with a custom python pipeline. Reads size and quality was assessed with the software FastQC v.0.10.1 (Simon 2011). The raw reads were cleaned using Trimmomatic v. 0.33 (Bolger et al., 2014) with the following parameters: Phred score cut off of 33 (99% confidence) and default values for ILLUMINACLIP, LEADING, TRAILING, SLIDINGWINDOW and MINLEN
arguments.
Since all study species lacked a reference genome to which the reads could be mapped, we used the software Trinity v. r2013-02-25 (Haas et al., 2013) to perform de-novo
assembly of the reads and generate full-length (or nearly full-length) transcripts for each tissue. The software was run with the default parameters and a minimum contig length of 200. In order to avoid assembly artifacts that could affect further analyses, each assembly was cleaned-up using the abundance estimation pipeline available at Trinity’s webpage (http://trinityrnaseq.github.io/analysis/abundance_estimation.html). In this pipeline, the reads are re-mapped to each assembly in order to estimate a relative abundance value (FPKM: Fragments Per Kilobase of transcript per Million fragments mapped) for each transcript and then poorly supported transcripts (FPKM<1) are filtered.
Assessing assembly quality
As a metric for assembly contiguity we calculated the N50 for each assembly, which represents the size of the smallest contig such that at least half of the sum of the lengths of the total sequence data is contained in contigs of size N50 or greater. To evaluate the quality of the assemblies we used CEGMA (Core Eukaryotic Genes Mapping
Approach; Parra et al., 2007) to determine the number of complete Core Eukaryotic Genes (CEGs) recovered by each assembly given that a good assembly should recover a high proportion of the 248 ultra-conserved CEGs regardless of the cell type or
organism. Further, to examine the number of transcripts that appeared to be full-length or nearly full-length we used the full-length transcript analysis pipeline available at Trinity’s webpage (http://trinityrnaseq.github.io/analysis/full_length_transcript
_analysis.html). This pipeline uses blastx to align each transcript against a database of known proteins (Uniprot-sprot Database), thus scoring the number of proteins that match with Trinity transcripts by more than 80% of their lengths.
Searching for ATPα genes
ATPα1 genes were obtained for all species surveyed, while all three ATPα paralogs were obtained only for those species that had all three tissues (brain, muscle and stomach) sequenced and assembled using either blastn or tblastn. Each assembled library was used as a blast database and ATPα1 sequences of Python molurus (GenBank
sequences of Python molurus (Genbank A.N. XP_007429144) and Xenopus laevis
(GenBank A.N. NP_001083112) and ATPα3 sequences of Python molurus (GenBank
A.N. XM_007437572) and Xenopus (Silurana) tropicalis (GenBank A.N.
NP_001120366) were used as query in order to find the three ATPα paralogs in each species.
Among-lineage evolutionary and statistical analyses
Alignments of ATPα1 cDNA and protein sequences were performed using Geneious
Pro 8.3.1 software with manual adjustment. Due to their poor alignment, the first 40 amino acids (relative to sheep and pig sequences) were removed prior to analysis. We used previous studies to establish phylogenetic relationships among the surveyed taxa (Anurans: Pyron and Wiens, 2011; Snakes: Pyron et al., 2011; Table 3). We separated the taxa into separate orders (Serpentes and Anura) and did a separate analysis for each. Branch lengths were obtained with the Bayesian posterior consensus phylogeny of the
ATPα1 gene using MrBayes (Huelsenbeck & Ronquist, 2001) under the HKY + G
model (with a gamma distribution of four rate categories) allowing for rate variation across lineages (relaxed clock). The search was started from a random topology, for 10 million generations, sampling every 10.000 generations. Generations sampled before the chain reached stationarity (burn-in) were discarded (100 generations). We focused on patterns of substitution at a subset of sites that were previously identified as having a functional role in ouabain/cardenolide affinity (Supplemental Material of the study of Zhen et al., 2012) either with site-directed mutagenesis or in silico (molecular docking) studies. For each site we used standard parsimony criteria to establish the state of ancestral nodes.
Beneficial substitutions occurring more often on lineages exposed to a certain selective pressure can imply a signal of adaptive protein evolution. Thus, for statistical analyses of clustering of mutations, we formulated a two-tailed binomial test to evaluate the probability that X or more of 14 phylogenetically independent substitutions (see
Results, Fig. 1) occur on lineages associated with toads (either toads or predators) using branch lengths as expected proportions. To evaluate whether substitutions were
occurring randomly (in amino acid space) in the gene we combined our results of the patterns of substitutions on the ATPα1 with the ones obtained by Zhen et al., 2012 in order to map when and where in the phylogeny the specific substitutions appeared (see Results, Fig. 3). We then established the number of independent (parallel) substitutions in each site of the protein (see Results, Fig. 4). Then we performed a χ2 test to
determine if there is a departure from a null expectation of a Poisson distribution
of the substitutions occurring throughout the gene.
Within-lineage evolutionary and statistical analyses
First, to evaluate that each of the three paralogs of the ATPα gene form a monophyletic clade we reconstructed a maximum likelihood tree based on predicted protein sequences of different vertebrate species available on GenBank and UniProt (including sequences of the species surveyed in the present study; Table 3). Protein alignments were
performed using Geneious Pro 8.3.1 software. The tree was conducted in the online Phylogeny.fr platform (Dereeper et al., 2008) with the following parameters: WAG + Gamma (0.641) + I (0.404) model with a gamma distribution of four rate categories, and 500 bootstrap replicates. The tree was rooted with the cnidarian Hydra vulgaris
(GenBank A.N. M75140). We chose H. vulgaris as the outgroup because apparently it only has one copy of the gene, resembling the ancestral state (Sáez et al., 2009), thus, reduces possible noise in the phylogenetic inference between paralogs. We wanted to discard non-independent evolutionary processes occurring between paralogs (such as gene conversion) in order to test whether substitutions in each copy (if any) arose independently. To test for the possibility of gene conversion between gene copies of ATPα we did an alignment of L. annulata, Xenodon sp. and C. calcarata paralogs (separated by species) using MUSCLE with default parameters, and then each
alignment was analyzed in GENECONV (Sawyer, 1989) with default parameters. The species were chosen according to two conditions: 1) they should be toad-eating species to test for adaptive substitutions in all paralogs and 2) they should have transcriptomic data for the three tissues (brain, muscle and stomach) to sample the three ATPα copies.
Given that all three copies are functionally equivalent, we focused on patterns of
substitutions at the same subset of sites used for ATPα1, assuming that the substitutions have similar effects on ouabain/cardenolide affinity in all copies. Then, amino acid substitution events in each ATPα copy were optimized onto branches of the phylogeny using the parsimony criterion minimizing the number of independent changes required to explain the phylogenetic pattern of distribution of variable sites. To evaluate whether substitutions are clustered in the H1-H2 extracellular domain in ATPα paralogs we performed a two-tailed binomial test to determine the probability that 9 of 9
substitutions (see Results, Fig. 5) occur at positions 111 through 122 of the protein, which correspond to the H1-H2 extracellular loop.
Identification of two putative ATPα1 duplication events
We detected two putative independent duplication events of the ATPα1 gene in
Leptodactylus latrans and L. pentadactylus (see Results, Fig. 1). To discard the
possibility of concerted evolution between the copies in each lineage that could account for the observed pattern, we performed a test of gene conversion (GENECONV;
Sawyer, 1989). We did two separate DNA alignments of the two putative copies per species with the whole gene sequences using MUSCLE with default parameters, and
then each alignment was analyzed with GENECONV. According to Shibata and
Yamazaki (1995), if there is evidence of gene conversion in a certain portion of the gene (which might cause incongruence in phylogenetic inference), one could
reconstruct a phylogeny using the portion that do not show gene conversion to obtain the real phylogenetic pattern. Thus, we constructed two Bayesian trees using either the whole gene sequence or only the regions that are evolving independently according to the gene conversion test (i.e. from position 1 to 427). Both trees were conducted in
MrBayes (Huelsenbeck & Ronquist, 2001) under the HKY + G model (with a gamma distribution of four rate categories) allowing for rate variation across lineages (relaxed clock). The search was started from a random topology, for 10 million generations, sampling every 10.000 generations. Generations sampled before the chain reached stationarity (burn-in) were discarded.
Differential ATPα1 expression
Relative expression level of the ATPα1 gene in each tissue was approximated using the FPKM value obtained through the abundance estimation pipeline available at Trinity’s webpage (http://trinityrnaseq.github.io/analysis/abundance_estimation.html). To
identify differential expression between libraries we normalized the FPKM value by the number of trimmed reads of each library to avoid possible bias of sequencing coverage on expression level. We then compared: 1) differential expression of ATPα1 associated with diet, i.e. between toad-eating snakes (L. annulata, L. septentrionalis and Xenodon sp.) and the non-toad-eating snake (Atractus crassicaudatus) using only the expression levels in the stomach, and 2) differential expression of ATPα1 between brain, muscle and stomach of toad-eating snakes. Given the low number of species surveyed, no statistical analyses were attempted.
Results
De-novo assembly and assembly quality
Previous studies have shown that Trinity software outperforms other assemblers (Singhal, 2013; Clarke et al., 2013) in a variety of quality metrics. In fact, all metrics analyzed indicated that assembled libraries were of good quality for further analyses (Table 2). Although N50 is not as important for transcriptomes as for genomes (Clarke
et al., 2013), this statistic provides a valuable measure in terms of contiguity. All
libraries had a N50 between 1100-1800 bp, CEGMA analyses showed that all libraries recovered over 75% of the 248 CEGs and the full-length transcript analysis revealed that all libraries had over 4300 full-length transcripts (match ≥ 80% of protein length; Table 2). Moreover, no significant differences in any quality measure were seen among tissues (N50: ANOVA, F=2.41, df=2, P>0.05; CEGs: ANOVA, F=0.83, df=2, P>0.05; Number of full-length transcripts: ANOVA, F=3.54, df=2, P>0.05), indicating that all libraries were of similar high quality, and were good enough to proceed searching for ATPα genes.
Among-lineage evolution
Except for seven amino acid substitutions (T797I in L. annulata and L. septentrionalis, V314I + R792K in T. elegans, T114S in D.rerio and toads, Y108F in several sanke species and Y108H in C. calcarata), the rest of observed amino acid substitutions (Fig. 1) corresponded to substitutions of known effect on ouabain-binding affinity based on site-directed mutagenesis studies (C104Y, Q111H, Q111R, D121N, N122H, N122D, and the combination Q111R+N122D; Croyle et al., 1997; Holzinger & Wink, 1996), or based on molecular docking (Q111L and P118A; Zhen et al., 2012). These substitutions range from 6.3-fold (for C104Y) to an incredibly 1250-fold increased resistance (for Q111R+N122D) relative to the wild-type enzyme (Croyle et al., 1997).
We found eight different substitutions: C104Y, Q111H, Q111R, D121N, N122H, N122D, P118A and Q111L, with different individual effects on ouabain-binding
affinity (Fig. 1). Moreover, substitutions are found in different combinations across taxa (e.g. P118A+N122H and Q111H+N122H) sugesting that there is a relative high
diversity of evolutionary responses to a similar selective pressure.
We detected two putative independent duplication events of the ATPα1 gene in
Leptodactylus latrans and L. pentadactylus (Fig. 1). Note that L. latrans ATPα1 copies
and L. pentadactylus ATPα1 copies form a monophyletic clade, respectively, with
posterior probabilities of 1 in each case (Fig. 2), and in both cases, the duplication events generated a susceptible copy and a resistant copy of the ATPα1. A test of gene
conversion (GENECONV; Sawyer, 1989) was performed in order to discard the
possibility of concerted evolution between the copies in each lineage that could account for the observed pattern; in other words, we wanted to distinguish between a single ancestral duplication or two independent duplications. Gene conversion test revealed an excess of shared sequence between the two copies in each lineage (P<0.05), occurring after the H1-H2 domain from position 428 to 3075. In both species, substitutions that reduce ouabain-binding affinity were found in the first extracellular domain of the protein (i.e. H1-H2 domain). These results suggest that despite gene conversion is homogenizing the majority of the molecule, natural selection may be avoiding the homogenization of the H1-H2 domain, thus maintaining the susceptible and resistant
forms. Following Shibata and Yamazaki (1995), we constructed a Bayesian tree (see
Materials and Methods) using only the regions that are evolving independently according to the gene conversion test (i.e. from position 1 to 427) to obtain the real
pattern. This tree (not shown) showed the exact same topology as the tree based on whole gene sequence (Fig. 2) thus, this suggests that there where actually two independent duplications.
Our test of clustering of substitutions revealed evidence of a signature of adaptive protein evolution in lineages related to toads (either toads or predators; Fig. 1). Specifically, 12 out of the 14 inferred phylogenetically independent amino acid substitutions occured in lineages associated with toads (binomial test, P= 8.5 x 10-6, accounting for branch lengths sampled). Note that the substitution N122H observed in snakes seemed to be fixed at the base of the Dipsadinae family and may not correspond to a substitution directly associated with feeding on a toxic prey. If this substitution is excluded from the analysis (11 out of 14 substitutions) the signature of adaptive evolution remains significant (binomial test, P= 9.5 x 10-5, accounting for branch lengths sampled).
When the results of this study were analyzed together with the results of the study of Zhen et al., 2012, we observed that the majority of substitutions are occurring at amino acid positions 111 and 122 (Fig. 4a; χ2 test of Poisson distribution, X2 =6.77, df=1, P= 9.2 x 10-3) suggesting that these sites may be hotspots for substitution in lineages that feed on organisms with cardioactive steroids. Moreover, there is a high proportion of sites that do not show substitutions (Fig. 4b), even though site-directed mutagenesis studies identifies them as residues in which a substitution can reduce the affinity of ATPα1 for ouabain, suggesting that there might be some functional restrictions for those sites to change.
Within-lineage evolution
Evidence of parallel adaptive protein evolution across the entire gene family is apparent, with each paralog having at least one putative substitution causing resistance to
bufadienolides (Fig. 5; ATPα2 for C. calcarata is absent because the assembler failed to reconstruct the N-terminal portion of the gene and the C-terminal portion was identical to the reference). The phylogenetic topology of the ATPα paralogs (Fig. 6) indicates that each copy has an independent evolutionary history; moreover, there was no significant evidence of gene conversion between paralogs in each species (P>0.05). These suggest that putative resistance in each copy arose through independent mutations. Strikingly, inferred substitutions are significantly clustered in the H1-H2
extracellular loop along the three paralogs (i.e. 9 of 9 substitutions occur at positions 111 through 122; binomial test, P= 3.9 x 10-3).
Differential ATPα1 expression
The data suggest evidence for an up-regulation of ATPα1 gene in the stomach of toad-eating snakes compared to the non-toad-toad-eating snake (Fig. 7). Although we could not perform a meaningful statistical test due to small sample size, expression level (FPKM) of ATPα1 in toad-eating snakes is ~2.6-fold higher than in the non-toad-eating snake, suggesting a possible increase in ATPα1 gene expression in the digestive tract that is likely associated with diet.
In the toad-eating snakes we found a pattern of differential expression of the ATPα1 gene between the brain, muscle and stomach tissues (Fig. 8). Although we could not perform a statistical test due to low sample size, the limited data suggested an up-regulation of the ATPα1 gene in the stomach compared to the brain and muscle (~13-fold and ~8-(~13-fold higher, respectively; Fig. 8). Thus, in addition to structural changes in the protein (i.e. specific substitutions that affect toxin-binding affinity), it appears that regulatory changes may also be an important strategy contributing to the evolution of toxin resistance.
Discussion
Our results suggest that structural and regulatory changes can both simultaneously shape the evolution of toxin resistance and that phenotypic evolution and, specifically, adaptive evolutionary trajectories are, to some extent, predictable. Among and within-lineage approaches demonstrate that ATPα is a genetic hotspot for adaptive molecular evolution (Martin and Orgogozo, 2013) in species with a cardioactive steroid-rich diet as shown by repeated de novo mutations occurring at orthologous and paralogous loci, repeated gene duplication and apparent parallel changes in gene regulation.
The role of gene duplication in evolution has been recognized as an important source of evolutionary innovation (Zhang, 2003). Evolutionary novelties after gene duplication can arise either by neo-functionalization (Ohno, 1970), sub-functionalization (Force et al., 1999) or to escape from adaptive conflict (Des Marias and Rausher, 2008). Here, we found evidence for two independent events of gene duplication in toad-eating frogs, where one of the copies retained the ancestral susceptible form and the other copy evolved a resistant form consistent with the neo-functionalization model. Strikingly, in lineages where duplications occurred, both resistant copies evolved the same amino acid substitutions (Q111R+N122D). Either substitution Q111R or N122D alone causes a 12.5-fold increase in resistance (Price and Lingrel, 1988; Lingrel et al., 1991), but when combined they produce a 1250-fold increase (Lingrel et al., 1991) meaning that there is an epistatic interaction between the two sites. This suggests a pattern of parallel
epistatic evolution within the genus Leptodactylus.
Specific amino acid substitutions at the protein-coding sequence of a gene are not the only mechanism of dealing with toxins. Other mechanisms such as increased enzyme or receptor activity can also contribute to toxin-resistance by counteracting the effects of the toxin input concentration. Increased activity of a target molecule can be achieved by either up-regulation via cis or trans-acting regulatory factors (Taylor and Feyereisen,
1996) or increased gene dosage via gene duplication (Widholm et al., 2001). It has been shown that independently evolved toad-eating snakes exhibit an enlargement of adrenal glands (Mohammadi et al., 2013) which is consistent with the apparent pattern of up-regulation of ATPα1 gene found for toad-eating snakes (L. annulata, L. septentrionalis
and Xenodon sp.). Adrenal glands produce hormones that help maintain the function of
the sodium-potassium pump by regulating gene expression via specific intracellular mineralcorticoid receptors (Therien and Blostein, 2000), thus, enlarged glands can counteract the negative effects of bufadienolides by increasing the amount of adrenergic hormones and thereby rising gene expression levels (Mohammadi et al., 2013). Further, the pattern of differential expression where ATPα1 appears to be up-regulated in the stomach seems to be reasonable given that the digestive tract would be the tissue that receives the higher quantity of bufadienolides when the prey is ingested.
There were at least eight different amino acid substitutions (and in different combinations) directly involved in ouabain-binding affinity, which points to the plurality of evolutionary responses to a similar selective pressure (Bock, 1959; Kryazhimskiy et al., 2014). Despite this apparent pattern of unpredictability at the sequence level, a high predictable pattern of substitution is observed related to where in the gene substitutions are occurring (Fig. 4 and Fig. 5). Clustering of mutations at specific positions within genes can result from functional tradeoffs between proper protein function and toxin resistance (Feldamn et al., 2012) or epistatic interactions among amino acids or with other genes or gene products in the genome (Stern, 2011). In
the ATPα1 protein, sites E312, V314, G319, L330, A331, T338 and F863, did not show
any substitutions despite having been experimentally validated as having a functional role in ouabain-binding affinity. These sites are located in domains that are also involved in ion-transport of Na+ and K+ and interact with the beta subunit (ATPβ; Ogawa and Toyoshima, 2002; Croyle et al., 1997; Qui et al., 2005; Sweadner and Donnet, 2001). Thus, functional constraints to avoid negative pleiotropic effects may be driving the predictable pattern of evolutionary parallelism in the ATPα gene.
In families of genes that retain the same physiological functions, adaptation to a
selective agent may involve adaptive changes in all genes across the family (Jost et al., 2008; McGlothlin et al., 2014). In anurans and reptiles, ATPα gene family consists of three differentially expressed genes (Blanco & Mercer, 1998). We found evidence for parallel adaptive substitutions in all three genes, with each paralog having at least one putative substitution causing resistance to bufadienolides. This represents an example of natural selection acting upon a complete gene family and suggests that resistance to bufodienolides might have a polygenic basis and be more complex than previously expected. Further, all substitutions across the three paralogs were clustered in the H1-H2 domain, consistent with the pattern of spatial predictability found for ATPα1 across vertebrates and invertebrates.
For animals, adaptive mutations affecting morphological traits are more likely to occur in the cis-regulatory regions of genes due to their modular nature which reduces pleiotropic effects (Carroll, 2008; Hoekstra and Coyne, 2007; Streisfeld and Rausher, 2011), in contrast, adaptive mutations affecting physiological traits (such as toxin resistance) are more likely to occur in protein-coding regions (Streisfeld and Rausher, 2011; Stearn, 2011). However, our results partially suggest that, at least for snakes, structural and regulatory changes are both likely to be involved in physiological adaptation. These results are consistent with those of Zhen et al. (2012), where
adaptation in insects that feed on cardenolide-producing plants is achieved by structural changes in the ATPα gene as well as regulatory changes that modify expression patterns that may have facilitated the functional specialization of the protein along lineages of herbivorous insects.
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Figures and Tables
Table 1. List of species surveyed with locations and tissues used for transcriptome libraries.
Table 2. Summary of quality metrics for each library per species
Table 3. GenBank and UniProt accession numbers for species used in evolutionary analyses of ATPα
Fig. 1. Parallel evolution of toxin resistance at the ATPα1 gene across frogs and snakes. The phylogenetic pattern of amino-acid substitutions at sites implicated in ouabain binding for ATPα1 in toads and toad-eating species. Numbered columns correspond to sites for which site-directed mutagenesis or protein structure analysis has suggested a role in ouabain-binding; only are shown the sites which have a specific substitution related to toxin resistance. Red lineages are either toads or toad-eaters. Green circles correspond to inferred duplications of the ATPα1. Letters in red correspond to specific
substitutions known to affect ouabain-binding affinity based on site-directed mutagenesis studies. Letters in bold are substitutions whose effect on ouabain-binding affinity has only been tested with molecular docking simulations. The resistance is given as the increase resistance (-fold) calculated relative to the wild-type enzyme (after Croyle et al., 1997); substitutions without a resistance value correspond to studies which had no comparable reference value to the other ones or that where identified by molecular docking. The cladogram represents the accepted branching order but with arbitrary branch lengths.
! 4.0 Leptodactylus_pentadactylus_B Anaxyurus_americanus Mixophyes_fasciolatus Pristimantis_orestes Limnodynastes_tasmaniensis Danio_rerio Phyomantis_bifasciatus Leptodactylus_pentadactylus_A Leptodactylus_ocellatus_A Scinax_ruber Leptodeira_annulata Rhinella_granulosa Ophiophagus_hannah Squalus_acanthias Hyalinobatrachium_fleischmannii Leptodactylus_ocellatus_B Atelopus_spumarius Tomopterna_cryptotis Xenodon_rabdocephalus Melanophryniscus_stelzneri Leptodeira_septentrionalis Crotalus_horridus Thamnophis_elegans Xenopus_laevis Python_molurus Micrurus_fulvius Crotalus_adamanteus Atelopus_zeteki Rhinella_marina Anaxyurus_cognatus Xenopus_tropicalis Atractus_crassicaudatus Rhinella_crucifer Ceratophrys_calcarata Anaxyurus_exsul H3#H4
104 108 111 118 121 122 308 312 314 315 319 322 323 330 331
Reference C Y Q P D N Y E V I G V A L A
D.#rerio ; ; L ; ; ; ; ; ; ; ; ; ; ; ;
S.#acanthias / ; ; ; ; ; ; ; ; ; ; ; ; ; ;
L.#annulata ; F . A ; H ; ; ; ; ; ; ; ; ;
L.#septentrionalis ; F . A ; H ; ; ; ; ; ; ; ; ;
A.#crassicaudatus / / ; ; ; H ; ; ; ; ; ; ; ; ;
Xenodon#sp. ; ; H ; ; H ; ; ; ; ; ; ; ; ;
T.#elegans Y F ; ; ; ; ; ; I ; ; ; ; ; ;
M.#fulvius / F ; ; ; ; ; ; ; ; ; ; ; ; ;
O.#hannah / F ; ; ; ; ; ; ; ; ; ; ; ; ;
C.#adamanteus / F ; ; ; ; ; ; ; ; ; ; ; ; ;
C.#horridus / F ; ; ; ; ; ; ; ; ; ; ; ; ;
P.#molurus / F ; ; ; ; ; ; ; ; ; ; ; ; ;
X.#tropicalis ; ; T ; ; ; ; ; ; ; ; ; ; ; ;
X.#laevis ; ; ; ; ; ; ; ; ; ; ; ; ; ; ;
L.#ocellatus#A ; ; . ; ; ;
L.#ocellatus#B ; ; R ; ; D
L.#pentadactylus#A ; ; . ; ; ; ; ; ; ; ; ; ; ; ;
L.#pentadactylus#B ; ; R ; ; D ; ; ; ; ; ; ; ; ;
H.#fleischmannii ; ; ; ;
R.crucifer R ; ; ;
R.#granulosa R ; ; ;
R.#marina ; ; R ; ; ;
A.#exsul R ; ; ;
A.#cognatus R ; ; ;
A.#americanus R ; ; ;
A.#zeteki R ; ; ;
A.#spumarius R ; ; ;
M.#stelxneri L ; ; ;
C.#calcarata ; H L ; N ; ; ; ; ; ; ; ; ; ;
S.#ruber ; ; ; ; ; ;
P.#orestes ; ; ; ;
L.#tasmaniensis ; ; ; ;
M.#fasciolatus ; ; ; ;
T.#cryptotis ; ; ; ;
P.#bifasciatus ; ; ; ;
H1 H1#H2 Resistance!! H4
(after!Croyle!et!al.1997)! 1fold! 6.3! 1250! 1250! 12.5! 65!(D121N)!
Fig. 2. Bayesian consensus tree of the ATPα1 genes found in the species surveyed in the study and species with available sequences in GenBank or UniProt (Table 3). Branch support values are
posterior probabilities. Note that both copies of the ATPα1 in Leptodactylus pentadactylus (blue)
and L. latrans (green) form monophyletic clades, respectively, with a posterior probability of 1 in
each case. The Bayesian tree inferred using only the regions that are evolving independently (i.e. from
Fig. 3. The cladogram represents the accepted branching order for the three clades surveyed (insects, frogs and snakes) and branch lengths are arbitrary. Each box contains specific
substitutions associated with toxin-‐resistance observed across taxa only in the ATPα1 protein and the arrows point to the branch where those substitutions likely appeared, inferred by parsimony. ! 2.0 Xenodon_rabdocephalus Heliconius_melpomene Leptodactylus_pentadactylus_A Danaus_plexippus R.lineaticolis_A Thamnophis_elegans Leptodactylus_ocellatus_A Ceratophrys_calcarata Rhinella_marina C.auratus_A Limenitis_archippus D.citri Micrurus_fulvius L.clivicolis Xenopus_tropicalis L.calmii_B Papilio_glaucus C.lectularius Lycorea_halia T.tetraophtalmus Leptodactylus_pentadactylus_B Xenopus_laevis Melanophryniscus_stelzneri Danaus_glippus Leptodeira_septentrionalis Crotalus_horridus E.egle Squalus_acanthias O.fasciatus_C T.legitima Danaus_erisimus C.auratus_B C.castaneus Hyalinobatrachium_fleischmannii L.caryae P.chalceus R.lineaticolis_B Atelopus_zeteki B.mori T.castaneum Atractus_crassicaudatus Python_molurus Scinax_ruber Danio_rerio M.robiniae Ophiophagus_hannah B.trivittata L.calmii_A Rhinella_granulosa L.calmii_C C.tenera Leptodeira_annulata A.nerii O.fasciatus_A Crotalus_adamanteus Caenorhabditis_elegans Leptodactylus_ocellatus_B O.fasciatus_B P.versicolora P118A% Q111H% N122H% C104Y% Q111R% N122D% Q111R% Q111L% Q111L% D121N% N122H% Q111V% Q111L% Q111L% Q111V% P118A% N122H% I315L%
R880S% Q111V%N122H% % Q111L% I315V% Q111T% C104Y% N122Y% P118A% D121N% T797A% R972Q% Q111T% I315V% F786N% N122H% P118S% T797S% N122Y% Y308C% T797A% Serpentes Anura Insecta
Fig. 4. A) Number of parallel substitutions at each site implicated in ouabain-‐binding affinity from site-‐directed mutagenesis studies. * indicate sites having a significant higher proportion of substitutions. Dashed line indicates the expected number o substitutions under a uniform model.
B) χ2 test of a null expectation of a Poisson distribution of the substitutions occurring throughout the
gene. Observed (gray bars) vs. expected (solid line) number of substitutions per site under a
random (poisson) model across the gene. Poisson analysis reveals that substitutions are occurring at a significant higher proportion at sites 111 and 122.
Fig. 5. Parallel evolution of toxin resistance across ATPα1, ATPα2 and ATPα3 genes. The cladogram represents the phylogenetic relationships of ATPα paralogs (based on the reconstructed phylogeny of Fig. 6) for three toad-‐eating species and non-‐toad-‐eating outgroups (branch lengths !
!
104108111118121122308312314315319330331338786797863880972
Determinant sites of Ouabain sensitivity
F re cu en cy (Pa ra lle l su bst itu tio ns) 0 5 10 15
Histogram of observed_freq
Number of substitutions per site
F
re
qu
en
cy
0 5 10 15
0 2 4 6 8 P=0.0092 *" *" A" B" ! 2.0 Xenodon_sp2 Xenodon_sp3 Xenodon_sp1 Python_molurus1 Xenopus_laevis3 Leptodeira_annulata2 Python_molurus2 Leptodeira_annulata1 Leptodeira_annulata3 Python_molurus3 Ceratophrys_calcarata3 Ceratophrys_calcarata1 Xenopus_laevis1 ATPα1 All Muscle ATPα2
ATPα3 Brain ! ! ! ! ! ! ! !
! ! N!
! ! N122H ! ! ! ! P118A Q111H Q111L D121N
are not meaningful). Each colored circle represents a specific substitution. Colored circles along branches indicate the most parsimonius reconstruction for the origin of the change. Each substitution is shown in the 3D-‐structure of the ATPα; only the domain 1 (H1), domain 2 (H2) and first extracellular loop (H1-‐H2) are shown (all the other sites were either the same to the reference
or had substitutions not associated with resistance to ouabain). ATPα2 for C. calcarata is absent
because the assembler failed to reconstruct the N-‐terminal portion of the gene and the C-‐terminal portion was identical to the reference, so it was excluded from the analysis.
Fig. 6. Phylogenetic relationships of ATPα paralogs. Shown is a maximum likelihood tree for predicted protein sequences. Branches are labeled with bootstrap proportions based on 500
bootstrap replicates. The tree is rooted with the cnidarian Hydra vulgaris (GenBank Acc #
M75140). Note that each of the ATPα copies forms a monophyletic clade indicating that the duplications occurred before the diversification of vertebrates.
! 0.0 ATP1A3_G.GALLUS ATP1A3_L_annulata_brain ATP1A2_G.GALLUS ATP1A3_RAT ATP1A2_H.SAPIENS ATP1A2_PIG ATP1A2_L_annulata_muscle ATP1A1_Leptodeira_annulata_muscle1_5_3_Frame2 ATP1A3_Xenodon_brain ATP1A1_Atractus_crassicaudatus_stomach_3_5_Frame2 ATP1A1_L_septentrionalis_brain_3_5_Frame3 ATP1A1_SHEEP ATP1A1_C.calcarata_stomach_5_3_Frame1 ATP1A1_X.LAEVIS ATP1A1_PIG ATP1A1_C.HORRIDUS ATP1A1_X.TROPICALIS ATP1A2_P.BIVITTATUS ATP1A2_RAT ATP1A2_O.HANNAH ATP1A1_C.ADAMANTEUS ATP1A1_Xenodon_stomach_5_3_Frame2 ATP1A3_H.SAPIENS ATP1A1_O.HANNAH ATP1A1_Xenodon_muscle_5_3_Frame2 ATP1A1_Leptodeira_annulata_muscle2_5_3_Frame3 ATP1A3_P.BIVITTATUS ATP1A2_ALLIGATOR ATPA_H.VULGARIS_A.N.M75140 ATP1A1_M.FULVIUS ATP1A1_L.septentrionalis_stomach_5_3_Frame3 ATP1A1_Leptodeira_annulata_stomach_3_5_Frame3 ATP1A1_Xenodon_brain_5_3_Frame1 ATP1A1_L.pentadactylus_stomach_5_3_Frame2 ATP1A1_T.ELEGANS ATP1A1_H.SAPIENS ATP1A1_L.pentadactylus_brain_3_5_Frame1 ATP1A1_P.BIVITTATUS ATP1A1_L.pentadactylus_brain2_3_5_Frame3 Leptodeira_annulata_brain_ATP1A1_3_5_Frame2 ATP1A2_Xenodon_muscle ATP1A2_B.TAURUS ATP1A1_ANOLIS 0.256 0.67 0.956 0.998 0.992 0.448 0.14 0.96 0.998 0.892 1 1 0.78 0.996 0.998 0.496 0.336 0.812 1 1 1 0.58 0.756 1 0.264 0.958 0.934 1 0.95 0.948 0.756 0.726 0.988 0.954 0.944 0.77 0.62 0.912 1 0.51 ATPα1& ATPα2& ATPα3&
Fig. 7. Relative expression level of ATPα1 gene in stomach of toad-‐eating vs. non-‐toad-‐eating snake species. Expression levels for the ATPα1 gene were calculated using the FPKM value normalized by the number of trimmed reads per library (see Materials and Methods). For the non-toad-eating species we used Atractus crassicaudatus (n=1). For the toad-eating species we used Leptodeira
annulata, L. septentrionalis and Xenodon sp (n=3).
Fig. 8. Relative expression level of ATPα1 gene in brain, muscle and stomach of toad-‐eating snake species. Expression levels for the ATPα1 gene were calculated using the FPKM value
normalized by the number of trimmed reads per library (see Materials and Methods). For the toad-eating species we used Leptodeira annulata, L. septentrionalis and Xenodon sp (n=3 per tissue).
Differential expression of ATP1A1 in snakes
Diet Exp re ssi on le ve l (F PKM/ re ad s) Non-bufofagous Bufofagous 0e +0 0 1e -0 5 2e -0 5 3e -0 5 4e -0 5 5e -0 5 6e -0 5
Differential expression of ATP1A1 between tissues in bufofagous species
Tissue Exp re ssi on le ve l (F PKM/ re ad s)
Brain Muscle Stomach
0e +0 0 1e -0 5 2e -0 5 3e -0 5 4e -0 5 5e -0 5 6e -0 5