The Birth of Aposematism: High Phenotypic Divergence and Low Genetic Diversity in a Young Clade of Poison Frogs ⇑ Rebecca D
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Molecular Phylogenetics and Evolution 109 (2017) 283–295 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev The birth of aposematism: High phenotypic divergence and low genetic diversity in a young clade of poison frogs ⇑ Rebecca D. Tarvin a,1, , Emily A. Powell a,b,1, Juan C. Santos c,d, Santiago R. Ron e, David C. Cannatella a a Department of Integrative Biology and Biodiversity Collections, University of Texas, Austin, TX, United States b Department of Biology, University of Miami, Miami, FL, United States c Department of Biology, Brigham Young University, Provo, UT, United States d Department of Biological Sciences, St. John’s University, Queens, NY, United States e Museo de Zoología, Escuela de Biología, Pontificia Universidad Católica del Ecuador, Quito, Ecuador article info abstract Article history: Rapid radiation coupled with low genetic divergence often hinders species delimitation and phylogeny Received 4 August 2016 estimation even if putative species are phenotypically distinct. Some aposematic species, such as poison Revised 30 November 2016 frogs (Dendrobatidae), have high levels of intraspecific color polymorphism, which can lead to overesti- Accepted 28 December 2016 mation of species when phenotypic divergence primarily guides species delimitation. We explored this Available online 13 January 2017 possibility in the youngest origin of aposematism (3–7 MYA) in poison frogs, Epipedobates, by comparing genetic divergence with color and acoustic divergence. We found low genetic divergence (2.6% in the 16S Keywords: gene) despite substantial differences in color and acoustic signals. While chemical defense is inferred to Species delimitation have evolved in the ancestor of Epipedobates, aposematic coloration evolved at least twice or was lost Phenotypic divergence Introgression once in Epipedobates, suggesting that it is evolutionarily labile. We inferred at least one event of introgres- Warning signals sion between two cryptically colored species with adjacent ranges (E. boulengeri and E. machalilla). We Polymorphism also find evidence for peripheral isolation resulting in phenotypic divergence and potential speciation Multispecies coalescent method of the aposematic E. tricolor from the non-aposematic E. machalilla. However, we were unable to estimate a well-supported species tree or delimit species using multispecies coalescent models. We attribute this failure to factors associated with recent speciation including mitochondrial introgression, incomplete lin- eage sorting, and too few informative molecular characters. We suggest that species delimitation within young aposematic lineages such as Epipedobates will require genome-level molecular studies. We caution against relying solely on DNA barcoding for species delimitation or identification and highlight the value of phenotypic divergence and natural history in delimiting species. Ó 2017 Elsevier Inc. All rights reserved. 1. Introduction separately evolving set of populations (metapopulation) under the General Lineage Concept (de Queiroz, 2007), there are many In the face of the rapid loss of biodiversity, efforts to attain a competing criteria and methods for delimiting species. However, more complete catalog and phylogeny of living organisms have the application of these procedures on recently diverged species intensified, especially in diverse tropical regions (Myers et al., remains particularly difficult because incomplete lineage sorting 2000; Vieites et al., 2009). Among those groups, amphibians are a (ILS) and introgression, often associated with low genetic diver- priority as they have experienced a rapid decline, with over a third gence, can obfuscate reconstructing well supported trees of species recognized as globally threatened (Stuart et al., 2004). (Fouquet et al., 2007; Hebert et al., 2004; Reid and Carstens, Although there is a general consensus that a species constitutes a 2012; Vieites et al., 2009). Multispecies coalescent methods (MSCs) bolster inferences in cases of low genetic divergence by taking into account the timing of allele coalescence across loci (Fujita et al., Abbreviations: bpp, Bayesian Posterior Probabilities; BP&P, Bayesian Phyloge- netics and Phylogeography program; ML, Maximum Likelihood; ILS, Incomplete 2012; Yang, 2015a). However, MSCs are currently limited to molec- Lineage Sorting; TNHCFS, Texas Natural History Collections Field Series; MSC, ular data (de Queiroz, 2007, 2005), but the best species- MultiSpecies Coalescent; SNP, Single Nucleotide Polymorphism; MYA, million years delimitation methods should be integrative, adducing evidence ago; HPD, Highest Posterior Density. from morphology, behavior, and ecology (Dayrat, 2005). ⇑ Corresponding author. Although species delimitation is challenging in the case of phe- E-mail address: [email protected] (R.D. Tarvin). 1 Authors contributed equally. notypically similar (‘‘cryptic”) species, it may also be difficult if the http://dx.doi.org/10.1016/j.ympev.2016.12.035 1055-7903/Ó 2017 Elsevier Inc. All rights reserved. 284 R.D. Tarvin et al. / Molecular Phylogenetics and Evolution 109 (2017) 283–295 metapopulations of a putative species have recently diverged phe- 17 individuals; E. tricolor from 2 populations, 16 individuals; notypically. For example, aposematic species that use visual sig- E. machalilla from 2 populations, 16 individuals; E. anthonyi from nals to warn predators of their defenses often co-opt these 3 populations, 23 individuals) were collected by RDT and SRR signals for intraspecific communication, leading to high intraspeci- under the permit N° 001-11 IC-FAU-DNB/MA from the Ecuadorian fic color variation (Cummings and Crothers, 2013; Merrill et al., Ministry of the Environment at various sites across Western 2014). Consequently, taxonomists may divide one polymorphic Ecuador in July 2012 (see Supplementary Table S1 for voucher species into multiple species (over-splitting) or describe a new information; Fig. 1). However, we did not collect individuals of species based on a newly observed color morph (e.g., Cisneros- E. darwinwallacei, E. espinosai, and E. narinensis, all of which Heredia and Yánez-Muñoz, 2010; Perez-Peña et al., 2010; Posso- have restricted ranges. We followed protocols approved by the Terranova and Andrés, 2016). Such delimitations may or may not University of Texas at Austin (IACUC No. AUP-2012-00032). reflect reproductive isolation and might introduce human percep- Liver tissue samples were removed from euthanized animals tion bias in classification of visually distinct populations (e.g., color and stored in 95% ethanol. All voucher specimens are deposited morphs) even if different morphs can sometimes interbreed (Brusa in the Museo de Zoologia de Pontificia Universidad Católica et al., 2013; Hauswaldt et al., 2011; Medina et al., 2013; Summers del Ecuador (QCAZ at PUCE). Genetic material from 11 et al., 2004; Yang et al., 2016). Thus, the taxonomic status of puta- other species was obtained from the Genetic Diversity Collection tive species with small ranges, low genetic divergence, and no evi- (part of the Biodiversity Collections) of University of Texas at dence of reproductive isolation should be questioned. Here we Austin. explore this problem by addressing molecular and phenotypic divergence in the youngest (3–7MYA) clade of aposematic poison 2.2. DNA sequencing and alignment frogs, Epipedobates (Santos et al., 2009). Epipedobates is a small clade of seven nominal species that DNA was extracted from liver tissues using Qiagen DNeasy occupy lowlands west of the Andes from southern Colombia to Blood & Tissue kits (Valencia, CA). Two mitochondrial genes were northern Peru (Fig. 1). Chemical defense occurs via dietary uptake amplified using published protocols and primer sequences of alkaloids, along with other phenotypic adaptations including (Goebel et al., 1999; Santos et al., 2003; Santos and Cannatella, high aerobic capacity and conspicuous coloration (Santos and 2011); the primer names follow Santos et al. (2003). The first gene, Cannatella, 2011). Four of the seven Epipedobates species exhibit a segment overlapping the end of 12S and the beginning of 16S, bright coloration associated with defense; only one species (E. was amplified with primers 12L1-L (50-AAAAAGCTTCAAACTGGGAT machalilla) is considered to be undefended and inconspicuous TAGATACCCCACTAT-30) and 16SH-H (50-GCTAGACCATGATG (Santos et al., 2009; Santos and Cannatella, 2011). This genus pre- CAAAAGGTA-30) using a 2-min initial denaturation at 94 °C, fol- sents a challenging case for species delimitation because despite lowed by 40 cycles of 30 s at 94 °C, 30 s at 48 °C, 1 min at 72 °C, divergent mating calls and phenotypic differences that readily and a final extension time of 7 min at 72 °C. The second, Cyto- identify the species (Graham et al., 2004; Santos et al., 2014), all chrome B, was sequenced with primers Den3-L (50-AAYATYTC previous studies have recovered inconsistent phylogenies with CRYATGATGRAAYTTYGG) and Den1-H, (50-GCRAANAGRAAGTAT low node support, failing to resolve species-level relationships CATTCNGGYTTRAT) using the same protocol. Annealing tempera- (Clough and Summers, 2000; Grant et al., 2006; Santos, 2012; tures were lowered to 46.5 °C for individuals that were difficult Santos et al., 2014, 2009, 2003; Vences et al., 2003); however, none to amplify. The nuclear gene encoding voltage-gated potassium of these sampled more than three populations or a handful of channel 1.3 (Kv1.3)