Molecular systematics of the Middle American genus Hypopachus (Anura: Microhylidae)

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Abstract

We present the first phylogenetic study on the widespread Middle American microhylid frog genus Hypopachus. Partial sequences of mitochondrial (12S and 16S ribosomal RNA) and nuclear (rhodopsin) genes (1275 bp total) were analyzed from 43 samples of Hypopachus, three currently recognized species of Gastrophryne, and seven arthroleptid, brevicipitid and microhylid outgroup taxa. Maximum parsimony (PAUP), maximum likelihood (RAxML) and Bayesian inference (MrBayes) optimality criteria were used for phylogenetic analyses, and BEAST was used to estimate divergence dates of major clades. Population-level analyses were conducted with the programs NETWORK and Arlequin. Results confirm the placement of Hypopachus and Gastrophryne as sister taxa, but the latter genus was strongly supported as paraphyletic. The African phrynomerine genus Phrynomantis was recovered as the sister taxon to a monophyletic Chiasmocleis, rendering our well-supported clade of gastrophrynines paraphyletic. Hypopachus barberi was supported as a disjunctly distributed highland species, and we recovered a basal split in lowland populations of Hypopachus variolosus from the Pacific versant of Mexico and elsewhere in the Mesoamerican lowlands. Dating analyses from BEAST estimate speciation within the genus Hypopachus occurred in the late Miocene/early Pliocene for most clades. Previous studies have not found bioacoustic or morphological differences among these lowland clades, and our molecular data support the continued recognition of two species in the genus Hypopachus.

Highlights

► We present a phylogenetic study of the Middle American microhylid frog genus Hypopachus. ► Results confirm the placement of Hypopachus and Gastrophryne as sister taxa. ► Gastrophryne was strongly supported as paraphyletic. ► Phrynomantis was recovered as sister to Chiasmocleis, rendering gastrophrynines as paraphyletic. ► Our molecular data support the continued recognition of two species in the genus Hypopachus.

Introduction

The microhylid genus Hypopachus (Keferstein, 1867), which currently includes two species in the lowlands and highlands of southeastern Texas, Mexico and Middle America, is a group of morphologically conservative, fossorial frogs. However, species of the genus Hypopachus vary considerably in coloration, intensity and distribution of pigmentation, and male advertisement calls (Nelson, 1973a, Nelson, 1974). Unbeknownst to earlier workers, Hypopachus is sexually dimorphic in its pattern of foot webbing (EG, unpubl. data); this intraspecific and intersexual variation may have contributed to the current 23 available synonyms for the widespread and lowland species Hypopachus variolosus (Cope, 1866) and three for the Mesoamerican highland species Hypopachus barberi Schmidt, 1939 (Frost, 2011). The former species occurs from southern Texas, USA, to northern Costa Rica on the Atlantic versant, and from southern Sonora, Mexico, to central Costa Rica on the Pacific versant (McCranie and Wilson, 2002); its habitat ranges from desert flats to rainforests (Frost, 2011, Savage, 2002).

The genus Hypopachus currently includes two species that were extensively studied by Nelson, 1973a, Nelson, 1974, who examined adult morphology (with multivariate statistical analyses), color pattern, and mating calls from dozens of localities throughout the range. However, Savage (2002, p. 395) pointed out that, “[Nelson’s] analysis left unresolved whether the nominal upland form, H. barberi, was really distinct from H. variolosus, since no diagnostic feature unequivocally separates these population systems.” Some of the characters traditionally used to distinguish the highland and lowland species include size and distance between the inner and outer metatarsal tubercles (inner and outer metatarsal tubercles separated from each other by a multiple of the width of the outer metatarsal tubercle; Köhler et al., 2006); however, we have observed intra- and interspecific variation of this character, which suggests it is not a reliable diagnostic character for the species.

Compared to most other New World anurans, microhylids are relatively dull-colored, globular, rotund, and small. Many species are burrowers, with secretive habits and brief reproductive seasons – factors contributing to their rareness in collections. These characteristics might help explain why New World microhylids have received relatively little taxonomic and systematic attention from herpetologists (Duellman and Trueb, 1986). Following taxonomic treatments of New World microhylines (Carvalho, 1954; Parker, 1934), a subfamily of microhylids that formerly included taxa in the New World and Southeast Asia, Donnelly et al. (1990) and Wild (1995) used morphological data to construct phylogenies of the New World microhylids. Wild (1995) used novel morphological data and larval characters from Donnelly et al. (1990) to construct a phylogeny of all New World microhyline genera. However, Wild (1995) did not specify outgroups and assumed the monophyly of the group, which has been partially refuted by recent large-scale phylogenetic analyses (e.g., Frost et al., 2006, Van der Meijden et al., 2007). The foci of these and subsequent molecular studies (e.g., Van der Meijden et al., 2004, Van der Meijden et al., 2007, Frost et al., 2006, Van Bocxlaer et al., 2006), were at the level of family or higher taxonomic categories, and included a limited number of New World microhylids (only one included data from the genus Hypopachus). Van der Meijden et al. (2007) published a global-scale phylogeny of microhylid frogs that included H. variolosus, which was recovered as sister to Gastrophryne carolinensis, the type species of both genera. No molecular study to date has included H. barberi, or examined intraspecific variation within either species of Hypopachus.

Frost and Hillis (1990) predicted that most wide-ranging polytypic species of amphibians and reptiles eventually would be found to consist of several evolutionary lineages. Recent molecular studies have modified decades-old taxonomy of Middle American herpetofauna to include more species than recognized by morphology alone (e.g., Parra-Olea et al., 2004, Shaffer et al., 2004, Mendelson et al., 2005, Pfeiler and Markow, 2008, Zarza et al., 2008, Daza et al., 2009, Douglas et al., 2010). The chaotic taxonomic history (Frost, 2011) and widespread distribution of the Middle American microhylid genus Hypopachus render it an ideal taxon for testing species boundaries with molecular data.

Section snippets

Taxon sampling

We sequenced eight samples of H. barberi and 35 samples of H. variolosus from across the entire known distribution of the species (Fig. 1). Based on previous studies of microhylid relationships (Frost et al., 2006, Van der Meijden et al., 2007), we included several outgroups in the study, including: three species of Gastrophryne (the sister group to Hypopachus), one species of Elachistocleis, three species of Chiasmocleis, one species of Phrynomantis (phrynomerine), and two arthroleptid species

Alignment, phylogenetic analyses and haplotype networks

The total alignment for the dataset included 1275 bp (12S = 394 bp; 16S = 583 bp; rhodopsin = 298 bp). Alignment of all datasets did not result in any ambiguous regions and no stop codons were detected in the rhodopsin dataset; 417 characters were parsimony-informative (32.7%) sites. Three samples of H. barberi from Honduras failed to amplify for 16S (these were excluded from NETWORK and Arlequin analyses), and two samples of Gastrophryne and seven samples of Hypopachus failed to amplify for rhodopsin (

Phylogeny and taxonomy of Hypopachus

Our results provide the first comprehensive analysis of Hypopachus relationships based on molecular data. The well-supported clades identified in this study are geographically structured and correspond to previously recognized species of Hypopachus (Frost, 2011, Nelson, 1973a, Nelson, 1974). We confirm the recognition of disjunct highland populations as H. barberi, which is consistent with current taxonomy. The well-supported basal split among lowland samples of Hypopachus variolosus from

Acknowledgments

The work for this project occurred at the University of Kansas (KU), University of Richmond (UR), Villanova University (VU), and the University of Texas at El Paso (UTEP). The senior author thanks Linda Trueb, Bill Duellman, Rafe Brown, Ed Wiley, Bruce Lieberman, Oliver Komar, Omar Torres-Carvajal (KU), Ken Yanek (UR), Aaron Bauer and Todd Jackman (VU), Max Shpak (UTEP) and Tony Gamble (University of Minnesota) for useful comments, assistance with preliminary data analyses, and access to

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