New Results

Differential gene expression and gene variants drive color and pattern development in divergent color morphs of a mimetic poison frog

Adam M M Stuckert, Tyler Linderoth, Matthew D MacManes, Kyle Summers
doi: https://doi.org/10.1101/706671

Abstract

Evolutionary biologists have long investigated the ecological contexts, evolutionary forces, and proximate mechanisms that produce the diversity of animal coloration we see in the natural world. In aposematic species, color and pattern is directly tied to survival and thus understanding the origin of the phenotype has been a focus of both theoretical and empirical inquiry. In order to better understand this diversity, we examined gene expression in skin tissue during development in four different color morphs of the aposematic mimic poison frog, Ranitomeya imitator. We identified a suite of candidate color-related genes a priori and identified the pattern of expression in these genes over time, differences in expression of these genes between the mimetic morphs, and genetic variants that differ between color morphs. We identified several candidate color genes that are differentially expressed over time or across populations, as well as a number of color genes with fixed genetic variants between color morphs. Many of the color genes we discovered in our dataset are involved in the canonical Wnt signaling pathway, including several fixed SNPs between color morphs. Further, many genes in this pathway were differentially expressed at different points in development (e.g., lef1, tyr, tyrp1). Importantly, Wnt signaling pathway genes are overrepresented relative to expression in Xenopus tropicalis. Taken together, this provides evidence that the Wnt signaling pathway is contributing to color pattern production in R. imitator, and is an excellent candidate for producing some of the differences in color pattern between morphs. In addition, we found evidence that sepiapterin reductase is likely important in the production of yellow-green coloration in this adaptive radiation. Finally, two iridophore genes (arfap1, gart) draw a strong parallel to previous work in another dendrobatid, indicating that these genes are also strong candidates for differential color production. We have used high throughput sequencing throughout development to examine the evolution of coloration in a rapid mimetic adaptive radiation and found that these divergent color patterns are likely to be affected by a combination of developmental patterns of gene expression, color morph-specific gene expression, and color morph-specific gene variants.

Introduction

The diversity of animal coloration in the natural world has long been a focus of investigation in evolutionary biology. Color phenotypes are profoundly impacted by both natural and sexual selection, and color phenotypes are often under selection from multiple biotic and abiotic factors (Rudh and Qvarnström 2013). For example, in some species color pattern has evolved in the context of both predator avoidance and thermoregulation (Hegna et al. 2013). The underlying mechanisms behind color and pattern phenotypes are of general interest, particularly in systems in which color phenotypes are variable and yet likely to be under intense selection.

One such example are adaptive radiations, in which selection produces rapid phenotypic diversification within a species or group of species. Well-documented examples of adaptive radiations that involve variation in coloration include sticklebacks (Schluter 1995), cichlid fishes (Seehausen 2006), and Hawaiian spiders (Gillespie 2004). Adaptive radiations can be driven by various factors, including strong, frequency dependent selection imposed by predation (Nosil and Crespi 2006). The dendrobatid mimic poison frog (Ranitomeya imitator) underwent a rapid adaptive radiation to mimic established congenerics (R. fantastica, R. summersi, and R. variabilis) in order to gain protection from predators—a case of Müllerian mimicry (Symula et al. 2001, 2003; Stuckert et al. 2014b, a). For these frogs and other Müllerian mimics, it is clear that the comimetic species experience strong selection to maintain local color phenotypes (e.g. Heliconius butterflies (Mallet and Barton 1989), velvet ants (Wilson et al. 2015), and millipedes (Marek and Bond 2009)). Although evolutionary theory has historically predicted that mimicry (and aposematism in general) should be locally monomorphic, geographic variation in color and pattern appear to be the norm in both aposematic and mimetic species (Joron and Mallet 1998; Briolat et al. 2018).

This kind of variation has long been a focus of scientific interest at both the proximate and ultimate level. Several experiments have revealed that local predators exert purifying selection on aposematic phenotypes (Hensel and Brodie 1976; Hegna et al. 2011; Paluh et al. 2014). However, over geographic distances heterogeneity in local predator communities and genetic drift in aposematic species are likely sufficient to produce the geographical mosaics in color and pattern seen in many aposematic and mimetic species (Ruxton et al. 2004; Sherratt 2006; Nokelainen et al. 2012). Determining the underlying genetic architecture of these changes has been a primary thrust of research in recent decades, because this allows researchers to examine both selection on these genes and associated phenotypes as well as convergence at the molecular level within and between parallel adaptive radiations.

Researchers have been able to pin down some key genetic loci in Heliconius butterfly mimicry systems (e.g., WntA (Martin et al. 2012) and optix (Reed et al. 2011; Supple et al. 2013)), though there are many others likely involved as well (Kronforst and Papa 2015). Interestingly, it seems that only a handful of loci control the different phenotypes in certain mimetic complexes, and that supergenes may be critically important in the diversity of mimetic phenotypes seen in Heliconius Müllerian mimicry systems and Papilio Batesian mimicry systems (Kunte et al. 2014; Kronforst and Papa 2015; Nishikawa et al. 2015). While the generality of this trend remains unclear, preliminary evidence from an analogous mimetic radiation of poison frogs indicates that this pattern may be found in this system as well (Vestergaard et al. 2015).

Amphibian coloration is linked to three structural chromatophore types (melanophores, iridophores, and xanthophores) and the pigments deposited in them (Mills & Patterson 2009). Dark coloration is produced by melanophores and the melanin pigments found inside them, including blacks, browns, and dark greens (Duellman and Trueb 1986). While the melanophores contribute to how dark a green color is, greens and blues are largely structurally produced, with hue mainly determined by the reflection of light from iridophores (Bagnara et al. 2007), which depends on the presence and orientation of guanine platelets, where thicker platelets tend to reflect longer wavelengths of light (Ziegler 2003; Bagnara et al. 2007; Saenko et al. 2013). Yellow, orange and red coloration are dependent on pigments such as pteridines and carotenoids that absorb different wavelengths of light, and are deposited within xanthophores (Duellman and Trueb 1986).

Here we characterize the genetic architecture of coloration in this adaptive radiation by examining gene expression and its timing across a developmental time series of the skin of the Peruvian mimic poison frog. This poison frog has evolved to converge on the appearance of sympatric congeners and is therefore polytypic with substantial geographic variation in the color phenotype (Symula et al. 2001, 2003). Thus, this species provides a good opportunity to examine gene expression as it relates to color and pattern in an adaptive radiation. Color in this species begins to appear early during development while individuals are still tadpoles, which is consistent with observations that chromatophores develop from the neural crest early in embryonic development (DuShane 1935). We examined gene expression patterns using RNA sequencing from four different mimetic color populations of R. imitator (Figure 1), each at four different time points during development. These different color morphs represent a variety of both colors and patterns, providing an opportunity to examine the underlying genetic basis of these traits. First, we consider overall gene expression patterns during development and across color morphs. Then we examine expression, timing, and morph-based differences of candidate color genes compiled from previous research on other taxa. This approach allows us to carefully examine the genomics underlying mimicry in poison frogs. Further, our approach provides insight into the genetic architecture of color and pattern in amphibians, which has historically been a challenge due to their large genomes. We discuss our findings in the context of what is known about color production in amphibians as well as the adaptive radiation that produced these color phenotypes.

Figure 1.
Figure 1.

Representatives of the four color morphs of Ranitomeya imitator used in this study. Clockwise from top left: orange-banded morph from Sauce, yellow-striped morph from Baja Huallaga, redheaded morph from Varadero, and the green-spotted morph from Tarapoto.

Methods

Tadpole collection

The initial breeding stock of Ranitomeya imitator was purchased from Understory Enterprises, LLC (Chatham, Canada). Frogs used in this project represent captive-bred individuals sourced from the following wild populations: Baja Huallaga (yellow-striped morph), Sauce (orange-banded), Tarapoto (green-spotted), and Varadero (redheaded; see Figure 1). Breeding R. imitator pairs were placed in 5-gallon terraria containing small (approximately 13 cm) PVC pipes capped on one end and filled halfway with water. We removed tadpoles from the tanks after the male transported them into the pools of water and hand reared them. Although in the wild female R. imitator feed their tadpoles unfertilized eggs, tadpoles can survive and thrive on other food items (Brown et al. 2008). We raised experimental tadpoles on a diet of Omega One Marine Flakes fish food mixed with Freeze Dried Argent Cyclop-Eeze, which they received three times a week, with full water changes twice a week until sacrificed for analyses at 2, 4, 7, and 8 weeks of age. At two weeks, tadpoles are limbless, patternless, and a light gray color with two dark black eyeballs. At 4 weeks tadpoles are a slightly darker gray and have back limb buds. Tadpoles had developed their pattern and some coloration as well as reached the onset of metamorphosis at around week 7, and had metamorphosed, were resorbing the tail, and had their froglet patterns at 8 weeks old. Pattern development continues as juveniles and subadults frogs as they grow into the ultimate pattern they possess as adults. Our four sampling periods correspond to roughly Gosner stages 25, 27, 42, and 44 (Gosner 1960). We sequenced a minimum of three individuals at each time point from the Sauce, Tarapoto, and Varadero populations (except for Tarapoto at 8 weeks), and two individuals per time point from the Huallaga population. Individuals within the same time points were sampled from different family groups (Table 1).

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Table 1.

Sample sizes by color morph and tadpole age.

Tadpoles were anesthetized with 20% benzocaine (Orajel), then sacrificed via pithing. The entirety of the skin was removed and stored in RNA later (Ambion) at −20° C until RNA extraction. RNA was extracted from the whole skin using a standardized Trizol protocol, cleaned with DNAse and RNAsin, and purified using a Qiagen RNEasy mini kit. Libraries were prepared using standard poly-A tail purification with Illumina primers, and individually barcoded using a New England Biolabs Ultra Directional kit as per the manufacturer’s protocol. Individually barcoded samples were pooled and sequenced using 50 bp paired end reads on three lanes of the Illumina HiSeq 2500 at the New York Genome Center. This yielded on average 24.45M reads per library ± 8.6M sd (range: 10.1-64.M).

Transcriptome assembly

Choosing a single individual or treatment to assemble a transcriptome could plausibly influence the quality of our transcriptome and bias our results. Therefore, in order to generate a reference transcriptome we assembled 40 M randomly subsampled forward and reverse reads sampled across morphs and time points using seqtk (https://github.com/lh3/seqtk) and used the Oyster River Protocol version 1.1.1 (MacManes 2017) to assemble this dataset. Evidence indicates that there is a substantial diminishment of returns in terms of transcriptome assembly completeness from using over 20-30 million reads (MacManes 2017). Initial error correction was done using RCorrector 1.01, followed by adaptor removal and quality trimming using trimmomatic version 0.36 at a Phred score of ≤ 3 (Bolger et al. 2014) since over-rigorous quality trimming has been shown to reduce assembly completeness (MacManes 2014). The Oyster River Protocol (MacManes 2017) assembles a transcriptome by merging multiple assemblies constructed using a series of different transcriptome assemblers and kmer lengths. We constructed the Independent assemblies with Trinity version 2.4.0 (Grabherr et al. 2011), Shannon version 0.0.2 (Kannan et al. 2016), and SPAdes assembler version 3.11 using 35-mers (Bankevich et al. 2012). This deviates slightly from the Oyster River Protocol specified in MacManes (2017), which specifies kmer lengths of 55 and 75 for SPAdes assemblies, but that exceeds our 50 bp sequencing read length. We then merged these individual assemblies using OrthoFuser (MacManes 2017). Finally, we assessed transcriptome quality using BUSCO version 3.0.1 (Simão et al. 2015) and TransRate 1.0.3 (Smith-Unna et al. 2016).

Downstream analyses

We used Diamond version 0.9.10 (Buchfink et al. 2015) to annotate our transcriptome with the peptide databases for Xenopus tropicalis. We then pseudo-quantified alignments for each library and technical replicate using Kallisto version 0.43.0 (Bray et al. 2016) and tested for differential gene expression in R version 3.4.2 (R Core Team 2017) using Sleuth version 0.29.0 (Pimentel et al. 2017). We accounted for the fact that samples were sequenced on three separate lanes by treating the lane that each sample was sequenced on as a fixed effect in our linear models. We tested for a significant change in gene expression driven by tadpole age with a likelihood ratio test. The alternative model involving tadpole age was fit with the sequencing lane on which libraries were sequenced as fixed effects using the R ‘splines’ library. Similarly, the null model that excluded tadpole age was fit with sequencing lane treated as a fixed effect. We also tested for differential gene expression between color morphs using likelihood ratio tests, correcting for multiple comparisons with a Benjamini-Hochberg-adjusted False Discovery Rate and using a significance threshold of α = 0.05. Specifically, for each gene we compared a model involving color morph to one without color morph while controlling for batch effects by treating sequencing lane as a fixed effect. In addition to examining overall differential expression, we examined differential expression in an a priori group of candidate color genes which we collated from the literature on other taxa (see code repository). We also used PANTHER version 14.0 (Mi et al. 2017) to determine if any pigmentation or color gene pathways were statistically overrepresented in our dataset.

We conducted two separate overrepresentation tests, one with the genes differentially expressed over time and a second with the genes differentially expressed between color morphs. Tests were conducted using Xenopus tropicalis as a reference, and a Fisher’s exact test with a Benjamini–Hochberg False Discovery Rate correction for multiple comparisons. Finally, we generated hypotheses about candidate genes by examining SNPs that were fixed between color morphs. To do this we used ANGSD to identify SNPs showing fixed differences between morphs (Korneliussen et al. 2014). For SNP calling we only retained reads with a minimum base quality score of 20, sites with a minimum depth of 100 reads and at least six individuals, and used a p value threshold of 0.000001. Following SNP calling, we examined SNPS that were fixed in at least one color morph and were in our candidate color gene list. We then used BLAST translated nucleotide to protein searches (tblastx) to align the color morph specific gene variants to the best amino acid sequence match in the model species genome (either Xenopus or Nanorana). We confirmed codon frame by aligning the specific protein sequence from the model species (Xenopus or Nanorana) to the matching translated nucleotide sequence for each candidate gene in R. imitator. We then determined whether the color morph specific fixed variants produced synonymous or non-synonymous changes or introduced stop codons.

Results

Data and code availability

All read data are archived with the European Nucleotide Archive (accession number PRJEB28312). Code for transcriptome assembly, annotation, and downstream analyses are all available on GitHub (https://github.com/AdamStuckert/Ranitomeya-imitator-Developmental-Series). Our candidate color genes are available in the same GitHub repository, and for the purposes of review, our assembled transcriptome is publicly available in our GitHub repository.

Transcriptome assembly

The Oyster River Protocol (MacManes 2017), produced a a transcriptome assembly that contained 88,182 total transcripts. When we ran BUSCO against the eukaryote database, our BUSCO score was 93.1% ([S:74.9%,D:18.2%],F:5.6%,M:1.3%,n:303), indicating that our transcriptome contained the majority of conserved genes that we would expect to see in a eukaryotes. The transrate score for the assembly, which measures contig accuracy, completeness, and non-redundancy, was 0.32505. Transrate scores in the 0.2-0.3 range are considered good, and scores above 0.3 are considered very good (Smith-Unna et al. 2016). We pseudoaligned reads from each library to our reference transcriptome with a mapping rate of 88.2% ± 1.1% SD. Using Xenopus tropicalis (NCBI Resource Coordinators 2016) as our annotation database we successfully annotated 35,014 transcripts (39.7% of our total transcriptome), which corresponds to roughly 10,300 unique genes.

Differential expression and gene variants

We found a total of 3,619 genes differentially expressed between time points in development. Among these genes, 111 of them were in our a priori color gene list of 501 genes (color gene list collated in the code repository). We also found 837 genes that were differentially expressed between color morphs of Ranitomeya imitator. Of these, we found 15 genes (Figure 2) in our a priori color gene list. Three genes (recql4, sema3c, and tspan36) were differentially expressed over time and between color morph.

Figure 2.
Figure 2.

Expression patterns of genes which were differentially expressed between color morphs. Data represented is the log of normalized transcripts per million from each individual. Trend line shows the mean produced via loess smoothing across all samples and shaded gray areas represent 95% confidence intervals.

View this table:
Table 2.

Statistically significant color genes in this study. Columns represent differentially expressed genes across time, differentially expressed genes across color morphs, and finally the number of fixed SNPs in that gene between color morphs. An “X” represents a gene is significant, numbers in the final column represent the number of fixed SNPs.

We identified a total of 194,445 SNPs on 6,507 genes. We found 115 SNPs among contigs annotated as potential color genes according to our a priori list that were fixed between color morphs; these represented 58 unique candidate color genes. Of these SNPs, 63 represented synonymous changes, while 52 represented non-synonymous changes (Supplemental Table 1). Overall, our results illuminate 150 candidate color genes that vary over time, between morphs, or have SNP differences between morphs.

We also looked for evidence of statistical overrepresentation in pathways that contribute to color and pattern production. When we examined overrepresentation across time, we found no enrichment of pathways related to iridophore or xanthophore production or function. We did, however, find overrepresentation in the Wnt signaling pathway over developmental time. The canonical Wnt signaling pathway exhibited 2.11-fold enrichment (GO category: 0060070, p value = 0.00139, FDR corrected p value = 0.0184), the regulation of Wnt signaling pathway exhibited a 1.76-fold enrichment (GO category: 0030111, p = 0.00208, FDR corrected p value = 0.0259), the Wnt signaling pathway exhibited a 1.59-fold enrichment (GO category: 0016055, p = 0.00112, FDR corrected p value = 0.0155), and cell-cell signaling by Wnt exhibited a 1.59-fold enrichment (GO category: 0198738,, p = 0.00112, FDR corrected p value = 0.0155). Additionally, we found that the generic pigmentation pathway exhibited 2.02-fold enrichment (GO category: 0043473, p = 0.000407, FDR corrected p value = 0.00653). When we examined overrepresentation across color morphs, we found no overrepresented gene groups that were linked to pigmentation. Results for overrepresentation over time and between color morphs can be found in Supplemental Table 2 and 3 respectively.

Figure 3.
Figure 3.

Expression patterns of selected genes in the Wnt3 signaling pathway. Data represented is the log of normalized transcripts per million from each individual. Trend line shows the mean produced via loess smoothing across all samples and shaded gray areas represent 95% confidence intervals.

Figure 4.
Figure 4.

Expression patterns of selected pteridine genes. Data represented is the log of normalized transcripts per million from each individual. Trend line shows the mean produced via loess smoothing across all samples and shaded gray areas represent 95% confidence intervals.

Figure 5.
Figure 5.

Venn diagram of genes differentially expressed over time (top left), differentially expressed between color morphs (right), and genes with fixed SNPs between color morphs (bottom).

Discussion

The genetic, biochemical, cellular, physiological and morphological mechanisms that control coloration in adaptive radiations are of interest because of the obvious implications for survival and selection. Further, these mechanisms in amphibians are poorly characterized, particularly compared to better known groups like mammals and fish. Our results illuminate 150 candidate color genes that may be contributing to different color phenotypes between populations in a highly variable, polytypic poison frog. Specifically, we find differences in the developmental timing of gene expression (i.e., when these genes may be contributing to color and pattern formation) as well as differences in expression between different color morphs. Furthermore, we examined differences in coding region between morphs in order to generate hypotheses for sequence variants contributing to differentiation in color and pattern. We found fixed differences between color genes that are plausibly contributing to color and pattern differentiation between color morphs. Many of these are very promising candidates for controlling color and pattern, but the limited sample sizes in our study provide low power to accurately identify highly divergent sites and so this merits further sampling in future studies.

Wnt signaling pathway

The canonical Wnt signaling pathway is important for the production of coloration, and Wnt genes have been shown to control differential color patterns in certain species (e.g., Kronforst et al. 2006; Martin et al. 2012; Gallant et al. 2014; Martin and Reed 2014). We found that several gene ontology pathways involving the Wnt signaling pathways were overrepresented among genes showing differential expression over time (although not between color morphs), indicating that this is likely an important pathway for color production. Additionally, we found several genes in the Wnt signaling pathway with fixed allele differences. Perhaps the most intriguing of these is wnt3 (Wingless-related integration site 3). Wnt3 is a gene with major effects on melanocyte-related coloration, as it is part of the canonical Wnt signaling pathway. This gene has been shown to upregulate many genes related to melanocyte production and melanin synthesis, including mitf (microphthalmia-associated transcription factor), tyr (tyrosinase), tyrp1 (tyrosinase-like protein 1), lef1 (lymphoid enhancer binding factor 1), and sox10 (SRY-Box 10)(Takeda et al. 2000; Guo et al. 2012). We found four color morph specific SNPs in wnt3, two of which had non-synonymous changes. Although we did not find any color morph specific expression of wnt3 or the aforementioned downstream targets, we did find that expression patterns of genes affected by wnt3 (lef1, tyr, tyrp1) change throughout development, which is further corroborated by evidence that Wnt signaling pathway genes are overrepresented throughout development. Other genes with fixed SNPs that fall into the Wnt signaling pathway are sox10, erbb3, axin2, and apc. The presence of genomic variants fixed between color morphs in wnt3 and other Wnt signaling genes, significant changes in downstream targets throughout development, and the overrepresentation of these pathways in our dataset provides evidence that the Wnt signaling pathway is involved in pigment production and is putatively involved in the development of different color phenotypes in poison frogs. However, this requires validation, as Wnt and related genes are involved in early general development and therefore the overrepresentation of this pathway may or may not be a driving agent of color and pattern production/divergence in this species.

Wnt3 is capable of inhibiting erbb3, and this inhibition contributes to increased Wnt signaling (Jullien et al. 2012). Potentially, the SNP in erbb3 is associated with similar effects on Wnt signaling. Additionally, beta-catenin is a key part of the Wnt signaling pathway. In the absence of Wnt signaling beta-catenin is bound by a multiprotein complex involving apc (adenomatous polyposis coli), axin (axis inhibition protein), and gsk3beta (glycogen synthase kinase 3 beta) which then degrades beta-catenin. In fact, apc escorts beta-catenin to be degraded (Larue and Delmas 2006). However, this multiprotein complex is dissociated when Wnt signaling occurs. Thus, the presence of fixed variants in wnt3, apc, and axin2 suggest that color variants in Ranitomeya imitator may be mechanistically controlled via the maintenance of beta-catenin. Extracellular heparan sulfate proteoglycans which bind to Wnt ligands, and modification of these by sulf1 and sulf2 (sulfatase 1 and sulfatase 2) can promote or reduce Wnt signaling (Ai et al. 2003; Kleinschmit et al. 2010). We found a fixed SNP in the sulf1 gene, which may also be associated with the development of distinct color patterns. We acknowledge that SNP calling RNA-seq data with our sample sizes does not indicate with certainty that these genes are contributing to functional differences in phenotype between color morphs. However, we view these genes, especially those within the Wnt signaling pathway, as particularly good candidates for future examination and validation. More in-depth population level sampling could elucidate whether these genes are worth pursuing functional validation.

The four morphs of Ranitomeya imitator used in this study have pattern elements on top of a generally black dorsum and legs. In vertebrates, black coloration is caused by light absorption by melanin in melanophores or (in mammals and birds) in the epidermis (Sköld et al. 2016). Melanophores (and the other chromatophores) originate from populations of cells in the neural crest early in development (Park et al. 2009). Given the timing of melanin synthesis and our sampling scheme, it is logical that many of our differentially expressed candidate genes are in this pathway. Melanin is synthesized from tyrosine, and this synthesis is influenced by a variety of different signaling pathways (e.g., Wnt, cAMP, and MAPK), many of which influence mitf (known as the “master regulator gene” of melanogenesis), a gene which encodes the melanogenesis associated transcription factor (Videira et al. 2013; D’Mello et al. 2016). It therefore makes sense that mitf is constitutively expressed across populations and time in our study. The gene creb1 (cAMP responsive element binding protein 1) is a binding protein in the cAMP pathway, which ultimately influences the transcriptional factor mitf, and the expression of this gene increases dramatically over time in R. imitator tadpoles as they show increasing pigmentation. The upregulation of creb1 causes mitf to increase melanin synthesis (D’Mello et al. 2016). Intriguingly, frogs from the Varadero population typically have the lowest amount of black overall (see Figure 1), and they also exhibit the lowest level of mitf expression. This, coupled with evidence that mitf plays a role in the production of black versus brown coloration in the poison frog Dendrobates auratus (Stuckert et al. 2019), indicates that this gene likely plays a critical role in melanin synthesis and the relative darkness of pigmentation in amphibians generally. This is logical, as mitf is highly conserved throughout vertebrates (Lister et al. 1999).

The melanogenesis transcription factor increases melanin synthesis through an interaction with the enzymes tyrosinase (tyr), tyrosinase-like protein 1 (tyrp1) and dopachrome tautomerase (dct), which are key elements in melanin biosynthesis (Park et al. 2009). Although tyr is expressed even in our youngest tadpoles, there is a dramatic increase in tyr expression over the course of development. During this time, tadpoles go from a very light, almost transparent gray color to a much darker background color with red, orange, yellow or green colored regions overlaying this black color. The phenotype and correlated expression of tyr indicate that tyrosinase is likely a key component of melanin biosynthesis in poison frogs. Similar to tyr expression, tyrp1 expression substantially increased over time, a pattern which is driven largely by high expression levels in the latest stages of the red-headed Varadero tadpoles. Although we cannot say why this is with certainty, it may be because tyrp1 seems to play a role in switching melanin synthesis from the production of black eumelanin to more reddish pheomelanin (note however that the evidence that tyrp1 promotes this switch is controversial). Less controversial however is that tyrp1 has a strong influence on the ultimate color phenotype, particularly in the presence of allelic variants in tyrp1 (Rieder et al. 2001; Li et al. 2014). Further, upregulation of tyrp1 has been linked to the production of maroon rather than black plumage in quails and horses (Xu et al. 2013; Li et al. 2014). Similarly, tyrp1 is differentially expressed between color morphs of another poison frog (Stuckert et al. 2019) in which frogs with lighter brown backgrounds have higher levels of tyrp1 expression than those of frogs with black backgrounds, providing some evidence that an increase in expression of tyrp1 may be related to the production of pheomelanin over eumelanin. However, this is speculative, as to date pheomelanin has only been identified in one species of frog, Pachymedusa dacnicolor (Wolnicka-Glubisz et al. 2012). Given that tyrp1 has been associated with pheomelanin and red-brown colors, its expression in the redheaded Varadero population indicates that pheomelanin may be contributing to red coloration in this population.

Similar to tyrp1, expression of lef1 is associated with the production of pheomelanin, a pigment associated with lighter color phenotypes (Song et al. 2017; Stuckert et al. 2019). We see early expression of lef1 which rapidly drops off until there is functionally no expression by the end of development when melanic coloration becomes most obvious in tadpoles. The gene sox9 (sex determining region Y – box 9) also influences the transcription factor mitf. However, unlike lef1 which leads to lighter pigmentation, sox9 is upregulated during melanocyte differentiation and can be activated by UVB exposure (Cheung and Briscoe 2003). In our dataset, sox9 is constitutively expressed across color morphs and times points, but with a substantial decline in the later time points. Although tadpoles are still becoming darker, and thus seem to be producing melanin at the late stages, the decrease in sox9 expression in our dataset is likely linked to a decrease in mitf activation and melanocyte differentiation. Further, sox9 is expressed in higher levels in darker color morphs of other frog species (Stuckert et al. 2019). This evidence indicates that sox9 may be an important determinant of color in amphibians generally, and poison frogs specifically. Just as sox9 is expressed most intensely in the populations with the most black skin, we see the same pattern in kit (KIT proto-oncogene receptor tyrosine kinase), a membrane receptor that is involved in one of the earliest steps of the melanogenesis pathway (D’Mello et al. 2016). Ultimately the pathway involving kit influences the same transcription factor as sox9 (mitf), so these may be complementary genetic mechanisms that produce similar effects.

In addition to the interaction between fixed SNPs and gene expression of downstream targets, wnt3a is of note because it has analogous effects on coloration in the Heliconius butterflies of Central and South America. Both the frogs and the butterflies are classic Neotropical examples of aposematism. Furthermore, our study species Ranitomeya imitator is also involved with Müllerian mimicry complexes just as Heliconius butterflies are. It has repeatedly been established that WntA controls melanization in the forewing in both Heliconius and Limnetis butterflies and delineates lighter-colored patches. Furthermore, other Wnt genes (e.g., wg, Wnt6, Wnt10) play a role in wing pigment patterning in butterflies (Kronforst et al. 2006; Martin et al. 2012; Gallant et al. 2014; Martin and Reed 2014). Our data indicates that a Wnt gene is also a good candidate for producing differential colors and patterns in poison frogs, and future work should aim to test this hypothesis. The possibility that organisms as evolutionarily distant as poison frogs and butterflies show the same patterns of diversity, extremely similar mimicry patterns, in the same exact geographic locations, with color phenotypes controlled by differences in the same family of genes is intriguing.

Iridophore genes

A number of candidate color genes related to iridophores showed sequence or expression differences in our analyses. We found fixed, color morph specific SNPs in the gene gart, a gene which catalyzes a number of steps in the de novo synthesis of purine (Ng et al. 2009). In zebrafish, mutations in this gene show a drastic reduction in the overall number of iridophores and a much lighter phenotype (Ng et al. 2009). Additionally, this gene has also been implicated in the differential production of green and blue phenotypes in another poison frog (Stuckert et al. 2019). That same study also found that arfgap1 is differentially expressed between color morphs and likely contributes to differences in iridophore-related coloration (Stuckert et al. 2019), and other work has indicated that arfgap1 likely contributes to guanine synthesis within iridophores in fish (Higdon et al. 2013). We found a fixed SNP between color morphs in both arfgap1 and arfgap2 within our dataset. Intriguingly, the Varadero population, which has substantial amounts of blue coloration on the legs and venter has a different SNP than the Huallaga and Sauce morphs.

Pteridine synthesis

Pteridines are pigments that are deposited into the xanthophores, the outermost layer of chromatophores in the skin, which are thought to contribute to orange, red, yellow, and even green coloration in amphibians (Duellman and Trueb 1986). Sepiapterin reductase (spr) is expressed primarily in the xanthophores (Negishi et al. 2003) and has been shown to only be expressed in late stages of the fire salamander tadpoles when yellowish color begins to appear (Sanchez et al. 2018). We found that spr was almost exclusively expressed in the yellow-green Tarapoto morph and the yellow Huallaga morph, which is a strong indication that this gene is playing a role in color pigmentation and color differences between morphs in R. imitator. Furthermore, spr has been shown to be an important determinant in the yellow or orange throat coloration in wall lizards (Andrade et al. 2019). In addition, we found color morph specific genomic variants in the gene quinoid dihydropteridine reductase (qdpr), a gene which is involved in the same pteridine synthesis pathway and is known to alter patterns of sepiapterin production (Ponzone et al. 2004). We additionally found differences in the expression of qdpr over time, with a fairly stark decline over development. The qdpr gene was also differentially expressed across populations in another species of poison frog (Stuckert et al. 2019). In combination, this evidence indicates that qdpr likely plays an important role in poison frog coloration. Similar to qdpr, xanthine dehydrogenase (xdh) is known to influence amphibian coloration (Frost 1978; Frost and Bagnara 1979; Thorsteinsdottir and Frost 1986), exhibited a color morph specific variant in Ranitomeya imitator, and has been implicated in another poison frog species (Stuckert et al. 2019). Xdh appears to influence pterins, and as such this is another good candidate color gene for the greens, yellows, and oranges that poison frogs exhibit. We view qdpr, spr, and xdh as excellent candidates for the control of color in this system, and future work should aim to verify this.

Conclusions

The genomics of adaptive radiations are of interest because of the strong selection imposed on phenotypes in these radiations. Further, both the specific mechanisms of color production and their genomic architecture have been poorly characterized in many groups of animals, particularly amphibians. We have produced a high-quality skin transcriptome for the polytypic poison frog Ranitomeya imitator which underwent a rapid mimetic radiation and used this transcriptome to characterize color gene expression patterns across color morphs and throughout development, as well as to characterize SNPs in color genes. We found fixed SNPs in wnt3 and other genes in the Wnt signaling pathway, as well as several color genes that were differentially expressed over time which are in the same pathway. Amongst these genes, many of these are transcription factors which are important in melanocyte stem cells or melanocyte differentiation. These genes are excellent candidates for further examination via population genetics of wild frogs. We also found that pteridine genes are likely playing a role in xanthophore-related coloration. These data will provide both genomic resources for future studies of the development and the production of color and can inspire future investigations into the specific impacts that these genes have in this species and across other taxa.

Acknowledgements

Animal use and research comply with East Carolina University’s IACUC (AUP #D281). Funding for this project was provided by NSF DEB 165536 and an East Carolina University Thomas Harriot College of Arts and Sciences Advancement Council Distinguished Professorship to K Summers. We are grateful to many individuals for their help with frog husbandry in the lab, including but not limited to M Yoshioka, C Meeks, A Sorokin, K Weinfurther, R Sen, N Davison, M Johnson, M Pahl, N Aramburu. We are also grateful to Laura Bauza-Davila for her work doing RNA extractions, and Andrew Lang for guidance converting RNA to cDNA and preparing samples for sequencing.

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Differential gene expression and gene variants drive color and pattern development in divergent color morphs of a mimetic poison frog
Adam M M Stuckert, Tyler Linderoth, Matthew D MacManes, Kyle Summers
bioRxiv 706671; doi: https://doi.org/10.1101/706671
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