The genomics of mimicry: Gene expression throughout development provides insights into convergent and divergent phenotypes in a Müllerian mimicry system.

A common goal in evolutionary biology is to discern the mechanisms that produce the astounding diversity of morphologies seen across the tree of life. Aposematic species, those with a conspicuous phenotype coupled with some form of defence, are excellent models to understand the link between vivid colour pattern variations, the natural selection shaping it, and the underlying genetic mechanisms underpinning this variation. Mimicry systems in which species share a conspicuous phenotype can provide an even better model for understanding the mechanisms of colour production in aposematic species, especially if comimics have divergent evolutionary histories. Here we investigate the genetic mechanisms by which mimicry is produced in poison frogs. We assembled a 6.02-Gbp genome with a contig N50 of 310 Kbp, a scaffold N50 of 390 Kbp and 85% of expected tetrapod genes. We leveraged this genome to conduct gene expression analyses throughout development of four colour morphs of Ranitomeya imitator and two colour morphs from both R. fantastica and R. variabilis which R. imitator mimics. We identified a large number of pigmentation and patterning genes differentially expressed throughout development, many of them related to melanophores/melanin, iridophore development and guanine synthesis. We also identify the pteridine synthesis pathway (including genes such as qdpr and xdh) as a key driver of the variation in colour between morphs of these species, and identify several plausible candidates for colouration in vertebrates (e.g. cd36, ep-cadherin and perlwapin). Finally, we hypothesise that keratin genes (e.g. krt8) are important for producing different structural colours within these frogs.

What makes a mimic? Orange, red, and black color production in the mimic poison frog (Ranitomeya imitator).

Aposematic organisms rely on their conspicuous appearance to signal that they are defended and unpalatable. Such phenotypes are strongly tied to survival and reproduction. Aposematic colors and patterns are highly variable; however, the genetic, biochemical and physiological mechanisms producing this conspicuous coloration remain largely unidentified. Here, we identify genes potentially affecting color variation in two color morphs of Ranitomeya imitator: the orange-banded Sauce and the redheaded Varadero morphs. We examine gene expression in black and orange skin patches from the Sauce morph and black and red skin patches from the Varadero morph. We identified genes differentially expressed between skin color patches, including those that are involved in melanin synthesis (e.g., mlana, pmel, tyrp1), iridophore development (e.g., paics, ppat, ak1), pteridine synthesis (e.g., gch1, recql4, xdh), and carotenoid metabolism (e.g., dgat2, rbp1, scarb2). In addition, using weighted gene network analysis, we identified the top 50 genes with high connectivity from the most significant network associated with gene expression differences between color morphs. Of these 50 genes, 14 were known to be related to color production (gch1, gmps, gpr143, impdh1, mc1r, pax3-a, pax7, ppat, rab27a, rlbp1, tfec, trpm1, xdh).

On the wings of dragons: Wing morphometric differences in the sexually dichromatic common whitetail skimmer dragonfly, Plathemis lydia (Odonata: Libellulidae).

Sexual dimorphism is common throughout the animal kingdom, leading to sex-specific phenotypic differences. The common whitetail skimmer dragonfly, Plathemis lydia (Drury, 1773), is sexually dichromatic, where males of this species display a conspicuous white abdomen and females display a dark brown abdomen. Differences in abdomen conspicuousness between male and female P. lydia are likely attributed to differences in selective pressure where males use their white conspicuous abdomen during male-male territorial chases. We hypothesized that male P. lydia would exhibit wing morphology adaptations to better offset the costs of predation and territoriality and that these adaptations would differ from females. We used field-collected images to quantify differences in body length, wing length, wing area, wing shape, and wing loading between male and female P. lydia. Our results show that male P. lydia have significantly shorter fore and hind wings relative to body size with a higher wing loading when compared to females. We also found that male P. lydia have narrower and pointier fore and hind wings compared to females. These results are consistent with the idea that males are adapted for faster flight, specifically higher acceleration capacity, and higher agility whereas females are adapted for higher maneuverability.

A histological analysis of coloration in the Peruvian mimic poison frog (Ranitomeya imitator).

Aposematism continues to be a phenomenon of central interest in evolutionary biology. The life history of the mimic poison frog, Ranitomeya imitator, relies heavily on aposematism. In order for aposematic signals to be effective, predators must be able to learn to avoid the associated phenotype. However, in R. imitator, aposematism is associated with four different color phenotypes that mimic a complex of congeneric species occurring across the mimic frog's geographic range. Investigations of the underlying mechanics of color production in these frogs can provide insights into how and why these different morphs evolved. We used histological samples to examine divergence in the color production mechanisms used by R. imitator to produce effective aposematic signals across its geographic range. We measured the coverage of melanophores and xanthophores (the area covered by chromatophores divided by total area of the skin section) in each color morph. We find that morphs that produce orange skin exhibit a higher coverage of xanthophores and lower coverage of melanophores than those that produce yellow skin. In turn, morphs that produce yellow skin exhibit a higher coverage of xanthophores and lower coverage of melanophores than those that produce green skin. Generally, across the morphs, a high ratio of xanthophores to melanophores is associated with colors of brighter spectral reflectance. Together, our results contribute to the understanding of color production in amphibians and document divergence in the histology of a species that is subject to divergent selection associated with aposematism.

The evolution and ecology of multiple antipredator defences.

Prey seldom rely on a single type of antipredator defence, often using multiple defences to avoid predation. In many cases, selection in different contexts may favour the evolution of multiple defences in a prey. However, a prey may use multiple defences to protect itself during a single predator encounter. Such "defence portfolios" that defend prey against a single instance of predation are distributed across and within successive stages of the predation sequence (encounter, detection, identification, approach (attack), subjugation and consumption). We contend that at present, our understanding of defence portfolio evolution is incomplete, and seen from the fragmentary perspective of specific sensory systems (e.g., visual) or specific types of defences (especially aposematism). In this review, we aim to build a comprehensive framework for conceptualizing the evolution of multiple prey defences, beginning with hypotheses for the evolution of multiple defences in general, and defence portfolios in particular. We then examine idealized models of resource trade-offs and functional interactions between traits, along with evidence supporting them. We find that defence portfolios are constrained by resource allocation to other aspects of life history, as well as functional incompatibilities between different defences. We also find that selection is likely to favour combinations of defences that have synergistic effects on predator behaviour and prey survival. Next, we examine specific aspects of prey ecology, genetics and development, and predator cognition that modify the predictions of current hypotheses or introduce competing hypotheses. We outline schema for gathering data on the distribution of prey defences across species and geography, determining how multiple defences are produced, and testing the proximate mechanisms by which multiple prey defences impact predator behaviour. Adopting these approaches will strengthen our understanding of multiple defensive strategies.

Investigating signal modalities of aposematism in a poison frog.

Aposematic species combine a conspicuous signal with a secondary defence, the majority of which are studied in the context of a visual signal. While multimodality of the aposematic signal appears to be common in invertebrate species, we know very little about the presence or absence of multimodality in vertebrates. Here, we examine the possibility of multimodality of aposematism in the green-and-black poison frog, Dendrobates auratus. Using a non-visual predator (the cat-eyed snake, Leptodeira annulata) and extractions of chemicals in frog skins, we test whether there is sufficient non-visual information for predators to avoid this aposematic species without using visual cues. We found that experienced predators avoid chemicals in this poison frog's skin by olfactory cues alone in trials with live frogs and extracts from captive poison frogs, whereas extracts from wild poison frogs did not lead to avoidance behaviours in predators. Further, in our limited sampling, naïve predators demonstrate no avoidance. This not only indicates that predators can make informed decisions from the frog's odour, but also indicates that avoidance based on olfactory cue is a learned response.

Neural crest cell genes and the domestication syndrome: A comparative analysis of selection.

Neural crest cell genes control the migration of neural crest cells to multiple parts of developing vertebrate embryos. A recent hypothesis posits that the "domestication syndrome" characteristic of domesticated animals is driven by selection for tameness acting on neural crest cell genes, particularly those affecting cell migration. This is posited to explain why this syndrome involves many disparate phenotypic effects. These effects can be connected to deficits in neural crest cell migration. This hypothesis predicts that patterns of selection on these neural crest cell genes will differ between domesticated species and related wild species. Specifically, it predicts higher levels of positive selection on these genes in domesticated species, relative to closely related wild species. Here we test this prediction in a comparative framework. We obtained DNA sequences from a public database (NCBI) for eleven key neural crest cell genes from a set of thirty domesticated vertebrates and matched close relatives that remain wild. We used the program Contrast-FEL in the software suite HyPhy to compare the number of sites under positive selection (as measured by non-synonymous to synonymous nucleotide substitution rates across codons) between these two types of taxa in a phylogenetic framework. We found that domesticated lineages showed a consistently higher level of positive selection on these key genes, relative to their closely related wild counterparts. In addition, we found support for relaxation of selection and purifying selection. We argue that this result is consistent with an important role for these genes in the domestication syndrome.

The genomics of mimicry: Gene expression throughout development provides insights into convergent and divergent phenotypes in a Müllerian mimicry system.

A common goal in evolutionary biology is to discern the mechanisms that produce the astounding diversity of morphologies seen across the tree of life. Aposematic species, those with a conspicuous phenotype coupled with some form of defence, are excellent models to understand the link between vivid colour pattern variations, the natural selection shaping it, and the underlying genetic mechanisms underpinning this variation. Mimicry systems in which multiple species share the same conspicuous phenotype can provide an even better model for understanding the mechanisms of colour production in aposematic species, especially if comimics have divergent evolutionary histories. Here we investigate the genetic mechanisms by which vivid colour and pattern are produced in a Müllerian mimicry complex of poison frogs. We did this by first assembling a high-quality de novo genome assembly for the mimic poison frog Ranitomeya imitator. This assembled genome is 6.8 Gbp in size, with a contig N50 of 300 Kbp R. imitator and two colour morphs from both Ranitomeya fantastica and R. variabilis which R. imitator mimics. We identified a large number of pigmentation and patterning genes that are differentially expressed throughout development, many of them related to melanocyte development, melanin synthesis, iridophore development and guanine synthesis. Polytypic differences within species may be the result of differences in expression and/or timing of expression, whereas convergence for colour pattern between species does not appear to be due to the same changes in gene expression. In addition, we identify the pteridine synthesis pathway (including genes such as qdpr and xdh) as a key driver of the variation in colour between morphs of these species. Finally, we hypothesize that genes in the keratin family are important for producing different structural colours within these frogs.

Early extubation to noninvasive respiratory support of former preterm lambs improves long-term respiratory outcomes.

Invasive mechanical ventilation (IMV) and exposure to oxygen-rich gas during early postnatal life are contributing factors for long-term pulmonary morbidities faced by survivors of preterm birth and bronchopulmonary dysplasia. The duration of IMV that leads to long-term pulmonary morbidities is unknown. We compared two durations of IMV (3 h vs. 6 days) during the first 6-7 days of postnatal life in preterm lambs to test the hypothesis that minimizing the duration of IMV will improve long-term respiratory system mechanics and structural outcomes later in life. Moderately preterm (∼85% gestation) lambs were supported by IMV for either 3 h or 6 days before weaning from all respiratory support to become former preterm lambs. Respiratory system mechanics and airway reactivity were assessed monthly from 1 to 6 mo of chronological postnatal age by the forced oscillation technique. Quantitative morphological measurements were made for smooth muscle accumulation around terminal bronchioles and indices of alveolar formation. Minimizing IMV to 3 h led to significantly better (P < 0.05) baseline respiratory system mechanics and less reactivity to methacholine in the first 3 mo of chronological age (2 mo corrected age), significantly less (P < 0.05) accumulation of smooth muscle around peripheral resistance airways (terminal bronchioles), and significantly better (P < 0.05) alveolarization at the end of 5 mo corrected age compared with continuous IMV for 6 days. We conclude that limiting the duration of IMV following preterm birth of fetal lambs leads to better respiratory system mechanics and structural outcomes later in life.

Internal Auditory Canal Diverticula in Children: A Congenital Variant.

OBJECTIVES/HYPOTHESIS: Internal auditory diverticula in adults have been found to exist independent of otosclerosis, and in the presence of otosclerosis. We sought to determine the prevalence of internal auditory canal (IAC) diverticula in a pediatric cohort, to assess whether IAC diverticula are a risk factor for hearing loss, and the co-occurrence of otic capsule hypoattenuation. STUDY DESIGN: Retrospective review. METHODS: A single-site retrospective review of high-resolution temporal bones computed tomography (CT) scans including the presence and size of diverticula and hypoattenuation of the otic capsule. Demographic, imaging, and audiometric data were collected and descriptively analyzed. Bivariate analysis of collected variables was conducted. Comparisons between sides in unilateral cases were also performed. RESULTS: 16/600 (2.7%; 95% CI [2.0%, 3.4%]) were found to have IAC diverticula. Six were bilateral. Thirty-one patients (5.2%) were found to have hypoattenuation of the otic capsule. There were no coincident cases of IAC diverticulum and hypoattenuation of the otic capsule. There was no association between the presence of IAC diverticula and age (P = .13). In six patients with unilateral diverticula, pure tone average (P = .42), and word recognition (P = .27) scores were not significantly different when compared to the normal, contralateral side. CONCLUSIONS: The prevalence of IAC diverticula in children is lower than the prevalence in adults. IAC diverticula in children likely represent congenital variants of temporal bone anatomy. Similar to adult populations, there is evidence that IAC diverticula in children are likely not an independent risk factor for hearing loss. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:E1683-E1687, 2021.

Mechanisms for Color Convergence in a Mimetic Radiation of Poison Frogs.

In animals, bright colors often evolve to mimic other species when a resemblance is selectively favored. Understanding the proximate mechanisms underlying such color mimicry can give insights into how mimicry evolves-for example, whether color convergence evolves from a shared set of mechanisms or through the evolution of novel color production mechanisms. We studied color production mechanisms in poison frogs (Dendrobatidae), focusing on the mimicry complex of Ranitomeya imitator. Using reflectance spectrometry, skin pigment analysis, electron microscopy, and color modeling, we found that the bright colors of these frogs, both within and outside the mimicry complex, are largely structural and produced by iridophores but that color production depends crucially on interactions with pigments. Color variation and mimicry are regulated predominantly by iridophore platelet thickness and, to a lesser extent, concentration of the red pteridine pigment drosopterin. Compared with each of the four morphs of model species that it resembles, R. imitator displays greater variation in both structural and pigmentary mechanisms, which may have facilitated phenotypic divergence in this species. Analyses of nonmimetic dendrobatids in other genera demonstrate that these mechanisms are widespread within the family and that poison frogs share a complex physiological "color palette" that can produce diverse and highly reflective colors.

Cognitive Phenotype and Differential Gene Expression in a Hippocampal Homologue in Two Species of Frog.

The complexity of an animal's interaction with its physical and/or social environment is thought to be associated with behavioral flexibility and cognitive phenotype, though we know little about this relationship in amphibians. We examined differences in cognitive phenotype in two species of frog with divergent natural histories. The green-and-black poison frog (Dendrobates auratus) is diurnal, displays enduring social interactions, and uses spatially distributed resources during parental care. Túngara frogs (Physalaemus=Engystomops pustulosus) are nocturnal, express only fleeting social interactions, and use ephemeral puddles to breed in a lek-type mating system. Comparing performance in identical discrimination tasks, we find that D. auratus made fewer errors when learning and displayed greater behavioral flexibility in reversal learning tasks than túngara frogs. Further, túngara frogs preferred to learn beacons that can be used in direct guidance whereas D. auratus preferred position cues that could be used to spatially orient relative to the goal. Behavioral flexibility and spatial cognition are associated with hippocampal function in mammals. Accordingly, we examined differential gene expression in the medial pallium, the amphibian homolog of the hippocampus. Our preliminary data indicate that genes related to learning and memory, synaptic plasticity, and neurogenesis were upregulated in D. auratus, while genes related to apoptosis were upregulated in túngara frogs, suggesting that these cellular processes could contribute to the differences in behavioral flexibility and spatial learning we observed between poison frogs and túngara frogs.

The neural basis of tadpole transport in poison frogs.

Parental care has evolved repeatedly and independently across animals. While the ecological and evolutionary significance of parental behaviour is well recognized, underlying mechanisms remain poorly understood. We took advantage of behavioural diversity across closely related species of South American poison frogs (Family Dendrobatidae) to identify neural correlates of parental behaviour shared across sexes and species. We characterized differences in neural induction, gene expression in active neurons and activity of specific neuronal types in three species with distinct care patterns: male uniparental, female uniparental and biparental. We identified the medial pallium and preoptic area as core brain regions associated with parental care, independent of sex and species. The identification of neurons active during parental care confirms a role for neuropeptides associated with care in other vertebrates as well as identifying novel candidates. Our work is the first to explore neural and molecular mechanisms of parental care in amphibians and highlights the potential for mechanistic studies in closely related but behaviourally variable species to help build a more complete understanding of how shared principles and species-specific diversity govern parental care and other social behaviour.

A cognitive map in a poison frog.

A fundamental question in cognitive science is whether an animal can use a cognitive map. A cognitive map is a mental representation of the external world, and knowledge of one's place in this world, that can be used to determine efficient routes to any destination. Many birds and mammals are known to employ a cognitive map, but whether other vertebrates can create a cognitive map is less clear. Amphibians are capable of using beacons, gradients and landmarks when navigating, and many are proficient at homing. Yet only one prior study directly tested for a cognitive map in amphibians, with negative results. Poison frogs exhibit unusually complex social and spatial behaviors and are capable of long-distance homing after displacement, suggesting that they may be using complex spatial navigation strategies in nature. Here, we trained the poison frog Dendrobates auratus in a modified Morris water maze that was designed to suppress thigmotaxis to the maze wall, promoting exploration of the arena. In our moat maze, the poison frogs were able to use a configuration of visual cues to find the hidden platform. Moreover, we demonstrate that they chose direct paths to the goal from multiple random initial positions, a hallmark of a cognitive map. The performance of the frogs in the maze was qualitatively similar to that of rodents, suggesting that the potential to evolve a cognitive map is an evolutionarily conserved trait of vertebrates.

Variation in pigmentation gene expression is associated with distinct aposematic color morphs in the poison frog Dendrobates auratus.

BACKGROUND: Color and pattern phenotypes have clear implications for survival and reproduction in many species. However, the mechanisms that produce this coloration are still poorly characterized, especially at the genomic level. Here we have taken a transcriptomics-based approach to elucidate the underlying genetic mechanisms affecting color and pattern in a highly polytypic poison frog. We sequenced RNA from the skin from four different color morphs during the final stage of metamorphosis and assembled a de novo transcriptome. We then investigated differential gene expression, with an emphasis on examining candidate color genes from other taxa. RESULTS: Overall, we found differential expression of a suite of genes that control melanogenesis, melanocyte differentiation, and melanocyte proliferation (e.g., tyrp1, lef1, leo1, and mitf) as well as several differentially expressed genes involved in purine synthesis and iridophore development (e.g., arfgap1, arfgap2, airc, and gart). CONCLUSIONS: Our results provide evidence that several gene networks known to affect color and pattern in vertebrates play a role in color and pattern variation in this species of poison frog.

Preferential generation of Ca2+-permeable AMPA receptors by AKAP79-anchored protein kinase C proceeds via GluA1 subunit phosphorylation at Ser-831.

AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission in the mammalian central nervous system. Preferential AMPAR subunit assembly favors heteromeric GluA1/GluA2 complexes. The presence of the GluA2 subunit generates Ca2+-impermeable (CI) AMPARs that have linear current-voltage (I-V) relationships. However, diverse forms of synaptic plasticity and pathophysiological conditions are associated with shifts from CI to inwardly rectifying, GluA2-lacking, Ca2+-permeable (CP) AMPARs on time scales ranging from minutes to days. These shifts have been linked to GluA1 phosphorylation at Ser-845, a protein kinase A (PKA)-targeted site within its intracellular C-terminal tail, often in conjunction with protein kinase A anchoring protein 79 (AKAP79; AKAP150 in rodents), which targets PKA to GluA1. However, AKAP79 may impact GluA1 phosphorylation at other sites by interacting with other signaling enzymes. Here, we evaluated the ability of AKAP79, its signaling components, and GluA1 phosphorylation sites to induce CP-AMPARs under conditions in which CI-AMPARs normally predominate. We found that GluA1 phosphorylation at Ser-831 is sufficient for the appearance of CP-AMPARs and that AKAP79-anchored protein kinase C (PKC) primarily drives the appearance of these receptors via this site. In contrast, other AKAP79-signaling components and C-terminal tail GluA1 phosphorylation sites exhibited a permissive role, limiting the extent to which AKAP79 promotes CP-AMPARs. This may reflect the need for these sites to undergo active phosphorylation/dephosphorylation cycles that control their residency within distinct subcellular compartments. These findings suggest that AKAP79, by orchestrating phosphorylation, represents a key to a GluA1 phosphorylation passcode, which allows the GluA1 subunit to escape GluA2 dominance and promote the appearance of CP-AMPARs.

Conserved transcriptomic profiles underpin monogamy across vertebrates.

Social monogamy, typically characterized by the formation of a pair bond, increased territorial defense, and often biparental care, has independently evolved multiple times in animals. Despite the independent evolutionary origins of monogamous mating systems, several homologous brain regions and neuropeptides and their receptors have been shown to play a conserved role in regulating social affiliation and parental care, but little is known about the neuromolecular mechanisms underlying monogamy on a genomic scale. Here, we compare neural transcriptomes of reproductive males in monogamous and nonmonogamous species pairs of Peromyscus mice, Microtus voles, parid songbirds, dendrobatid frogs, and Xenotilapia species of cichlid fishes. We find that, while evolutionary divergence time between species or clades did not explain gene expression similarity, characteristics of the mating system correlated with neural gene expression patterns, and neural gene expression varied concordantly across vertebrates when species transition to monogamy. Our study provides evidence of a universal transcriptomic mechanism underlying the evolution of monogamy in vertebrates.

Genomic Takeover by Transposable Elements in the Strawberry Poison Frog.

We sequenced the genome of the strawberry poison frog, Oophaga pumilio, at a depth of 127.5× using variable insert size libraries. The total genome size is estimated to be 6.76 Gb, of which 4.76 Gb are from high copy number repetitive elements with low differentiation across copies. These repeats encompass DNA transposons, RNA transposons, and LTR retrotransposons, including at least 0.4 and 1.0 Gb of Mariner/Tc1 and Gypsy elements, respectively. Expression data indicate high levels of gypsy and Mariner/Tc1 expression in ova of O. pumilio compared with Xenopus laevis. We further observe phylogenetic evidence for horizontal transfer (HT) of Mariner elements, possibly between fish and frogs. The elements affected by HT are present in high copy number and are highly expressed, suggesting ongoing proliferation after HT. Our results suggest that the large amphibian genome sizes, at least partially, can be explained by a process of repeated invasion of new transposable elements that are not yet suppressed in the germline. We also find changes in the spliceosome that we hypothesize are related to permissiveness of O. pumilio to increases in intron length due to transposon proliferation. Finally, we identify the complement of ion channels in the first genomic sequenced poison frog and discuss its relation to the evolution of autoresistance to toxins sequestered in the skin.

Searching for hormonal facilitators: Are vasotocin and mesotocin involved in parental care behaviors in poison frogs?

Although the involvement of peptide hormones in parental care behaviors is well investigated in vertebrates, in amphibians the physiological basis of parental care is largely unknown. This is all the more surprising as parental care behaviors in these tetrapods are remarkably diverse. The poison frog Ranitomeya imitator performs biparental care, including clutch guarding, tadpole transportation and nutrient provisioning. Here we tested whether the nonapeptides arginine-vasotocin (AVT) and mesotocin (MT) are involved in clutch guarding and tadpole transportation in these frogs. In ex-sito experiments we injected males and females after clutch deposition and before tadpole transport with AVT and MT, respectively, as well as their antagonist or a control. We measured two types of egg caring behavior (intense and general care) and compared the success rate of tadpole transportation after treatments. Surprisingly we found that AVT did not trigger, but decreased intense egg care behaviors in males and females. However, there was a trend for general care behavior to increase, which might explain the adverse effect regarding intense care. MT did not have an effect on egg caring behaviors, but after administration of this hormone males were less likely to transport their offspring later on. Our results indicate that AVT might be partly involved in egg caring behaviors in R. imitator, while MT does not appear to play any role in behaviors prior to tadpole transportation in males. This implies that other hormones, such as steroids or prolactin are likely to be important for early parental care behaviors in poison frogs.

Mimetic Divergence and the Speciation Continuum in the Mimic Poison Frog Ranitomeya imitator.

While divergent ecological adaptation can drive speciation, understanding the factors that facilitate or constrain this process remains a major goal in speciation research. Here, we study two mimetic transition zones in the poison frog Ranitomeya imitator, a species that has undergone a Müllerian mimetic radiation to establish four morphs in Peru. We find that mimetic morphs are strongly phenotypically differentiated, producing geographic clines with varying widths. However, distinct morphs show little neutral genetic divergence, and landscape genetic analyses implicate isolation by distance as the primary determinant of among-population genetic differentiation. Mate choice experiments suggest random mating at the transition zones, although certain allopatric populations show a preference for their own morph. We present evidence that this preference may be mediated by color pattern specifically. These results contrast with an earlier study of a third transition zone, in which a mimetic shift was associated with reproductive isolation. Overall, our results suggest that the three known mimetic transition zones in R. imitator reflect a speciation continuum, which we have characterized at the geographic, phenotypic, behavioral, and genetic levels. We discuss possible explanations for variable progress toward speciation, suggesting that multifarious selection on both mimetic color pattern and body size may be responsible for generating reproductive isolation.