Phylogeny of the túngara frog genus Engystomops (=Physalaemus pustulosus species group; Anura: Leptodactylidae)

Abstract

We present a phylogeny of the Neotropical genus Engystomops (=Physalaemus pustulosus species group) based on sequences of ∼2.4 kb of mtDNA, (12S rRNA, valine-tRNA, and 16S rRNA) and propose a phylogenetic nomenclature. The phylogeny includes all described taxa and two unnamed species. All analyses indicate that Engystomops is monophyletic and contains two basal allopatric clades. Clade I (Edentulus) includes E. pustulosus and the Amazonian E. petersi + E. cf. freibergi. Clade II (Duovox) includes all species distributed in W Ecuador and NW Peru. Brevivox, a clade of small-sized species is strongly supported within Duovox. Populations of Engystomops pustulosus fall into two well-supported clades, each of which occupies two disjunct portions of the species range. Overall, our phylogeny is congruent with most previous hypotheses. This study is among the few published species-level phylogenies of Neotropical amphibians derived from molecular datasets. A review of the proportion of new species detected by similar studies suggests that the increasing use of molecular techniques will lead to the discovery of a vast number of species of Neotropical amphibians.

Introduction

Physalaemus and Engystomops are closely related genera of frogs of the subfamily Leptodactylinae; until recently these were allocated into a single genus (Physalaemus) with 49 species (updated from Frost, 2004) and four species groups (Cannatella and Duellman, 1984, Lynch, 1970): P. biligonigerus, P. cuvieri, P. pustulosus, and P. signifer group. In a taxonomic review, Nascimento et al. (2005) resurrected the genus Engystomops for the species of the P. pustulosus group and defined seven species groups within Physalaemus. Engystomops is distributed from central Veracruz (Mexico) to the Amazon Basin and the lowlands of western Ecuador and NW Peru.

Engystomops has been a model system in studies of sexual selection and animal communication since the 1980s (e.g., Bosch et al., 2000, Cannatella et al., 1998, Ryan, 1983, Ryan and Drewes, 1990, Ryan and Rand, 1995, Tarano and Ryan, 2002, Wilczynski et al., 2001). The systematics of Engystomops was reviewed by Cannatella and Duellman (1984), who recognized four species and provided morphological evidence for the group’s monophyly. Sister species status was established for (E. petersi + E. pustulosus) and (E. coloradorum + E. pustulatus). Ryan and Rand (1993) presented a phylogeny based on unpublished morphological characters, allozyme variation and 12S mtDNA sequences (pers. com. from D.C. Cannatella et al.; Fig. 1B). Their phylogeny differed from that implied by Cannatella and Duellman (1984) in placing E. pustulosus as sister taxon to the remaining species instead of to E. petersi. Cannatella et al. (1998) included two additional species and analyzed morphology, behavior, allozyme variation and 12S rRNA and COI mtDNA sequences. The combined analysis of all characters placed E. pustulosus as sister taxon to the clade (E. petersi + E. cf. freibergi). However, their COI mtDNA data partition supported E. pustulosus as sister taxon to all species of the group (Figs. 1C and D). A recent phylogeny based on COI mtDNA shows the same basal position for E. pustulosus (Weigt et al., 2005). To demonstrate the taxonomic status of the cryptic E. guayaco, Ron et al. (2005) included a brief phylogeny based on a subset of the mtDNA data presented here (five species). Because that analysis is congruent with our results, we will not discuss it further.

Taxon sampling influences tree topology (Zwickl and Hillis, 2002) and the interpretation of character evolution (Ackerly, 2000). Comprehensive taxon sampling for phylogenetic inference is particularly important in model systems, like Engystomops, where large datasets need to be analyzed in an evolutionary framework. The earliest studies on communication and sexual selection in Engystomops had the virtue of being among the first comparative analyses of behavioral characters that used explicit phylogenetic methods (e.g., Ryan and Rand, 1995). Unfortunately, the phylogenies used in those studies have incomplete taxon sampling and/or conflicting topologies (Fig. 1). For example, the influential concept of the Sensory Exploitation Hypothesis, which posits that a male secondary sexual trait can evolve to take advantage of pre-existing female sensory biases (Ryan, 1990) was based on character reconstructions on a phylogeny that only included the four species of Engystomops known at the time (Fig. 1A). Since then, the number of species of Engystomops has more than doubled. The addition of these new taxa could plausibly compromise support for the Sensory Exploitation Hypothesis, depending on the resulting new topology and character state distributions of the male secondary sexual trait and female mate choice in the added species.

The existence of undescribed species of Engystomops has been previously reported (e.g., Cannatella et al., 1998, Ryan and Rand, 2001) and recent fieldwork has confirmed and expanded the list of new species (Ron et al., 2004, Ron et al., 2005). The present study is an effort to provide a complete phylogeny for all extant species of Engystomops (described as well as new, but as yet undescribed, species; 10 or 11 in total). The phylogeny is based on analyses of ∼2.4 kb from three mitochondrial genes (ribosomal RNA genes, and the valine tRNA gene). In combination with the wealth of available data on call evolution and female mate preference, this new phylogeny presents new opportunities to expand, complement, and reevaluate previous analyses of sexual selection and the evolution of communication in this model clade.

As exemplified by the recent discovery of morphologically cryptic species in Engystomops (Ron et al., 2004, Ron et al., 2005), the use of genetic markers in systematics has an enormous potential to facilitate the global inventory of biodiversity. The revolution that systematics is experiencing will be crucial for management and conservation of biotic resources considering that probably <10% of species on the planet have been discovered and as few as <1% are known beyond a succinct anatomical description (Wilson, 2005). Although the taxonomic deficit seems to be less severe among terrestrial vertebrates, sampling of amphibians and reptiles inhabiting highly diverse regions in the Neotropics is far from complete (Duellman, 1999, Rodrigues, 2005). Although it is clear that the inventory of species of tropical amphibians and reptiles is still inadequate, the extent of this inadequacy is unknown. In this paper, we also combine our results with those from other species-level phylogenies of Neotropical amphibians to estimate the potential impact of molecular systematics on the discovery of new species in the tropics.