Jonathan A. Campbell

Systematic review of the frog family Hylidae, with special reference to Hylinae: Phylogenetic analysis and taxonomic revision

Abstract Hylidae is a large family of American, Australopapuan, and temperate Eurasian treefrogs of approximately 870 known species, divided among four subfamilies. Although some groups of Hylidae have been addressed phylogenetically, a comprehensive phylogenetic analysis has never been presented. The first goal of this paper is to review the current state of hylid systematics. We focus on the very large subfamily Hylinae (590 species), evaluate the monophyly of named taxa, and examine the evidential basis of the existing taxonomy. The second objective is to perform a phylogenetic analysis using mostly DNA sequence data in order to (1) test the monophyly of the Hylidae; (2) determine its constituent taxa, with special attention to the genera and species groups which form the subfamily Hylinae, and c) propose a new, monophyletic taxonomy consistent with the hypothesized relationships. We present a phylogenetic analysis of hylid frogs based on 276 terminals, including 228 hylids and 48 outgroup taxa. Included are exemplars of all but 1 of the 41 genera of Hylidae (of all four nominal subfamilies) and 39 of the 41 currently recognized species groups of the species-rich genus Hyla. The included taxa allowed us to test the monophyly of 24 of the 35 nonmonotypic genera and 25 species groups of Hyla. The phylogenetic analysis includes approximately 5100 base pairs from four mitochondrial (12S, tRNA valine, 16S, and cytochrome b) and five nuclear genes (rhodopsin, tyrosinase, RAG-1, seventh in absentia, and 28S), and a small data set from foot musculature. Concurring with previous studies, the present analysis indicates that Hemiphractinae are not related to the other three hylid subfamilies. It is therefore removed from the family and tentatively considered a subfamily of the paraphyletic Leptodactylidae. Hylidae is now restricted to Hylinae, Pelodryadinae, and Phyllomedusinae. Our results support a sister-group relationship between Pelodryadinae and Phyllomedusinae, which together form the sister taxon of Hylinae. Agalychnis, Phyllomedusa, Litoria, Hyla, Osteocephalus, Phrynohyas, Ptychohyla, Scinax, Smilisca, and Trachycephalus are not monophyletic. Within Hyla, the H. albomarginata, H. albopunctata, H. arborea, H. boans, H. cinerea, H. eximia, H. geographica, H. granosa, H. microcephala, H. miotympanum, H. tuberculosa, and H. versicolor groups are also demonstrably nonmonophyletic. Hylinae is composed of four major clades. The first of these includes the Andean stream-breeding Hyla, Aplastodiscus, all Gladiator Frogs, and a Tepuian clade. The second clade is composed of the 30-chromosome Hyla, Lysapsus, Pseudis, Scarthyla, Scinax (including the H. uruguaya group), Sphaenorhynchus, and Xenohyla. The third major clade is composed of Nyctimantis, Phrynohyas, Phyllodytes, and all South American/ West Indian casque-headed frogs: Aparasphenodon, Argenteohyla, Corythomantis, Osteocephalus, Osteopilus, Tepuihyla, and Trachycephalus. The fourth major clade is composed of most of the Middle American/Holarctic species groups of Hyla and the genera Acris, Anotheca, Duellmanohyla, Plectrohyla, Pseudacris, Ptychohyla, Pternohyla, Smilisca, and Triprion. A new monophyletic taxonomy mirroring these results is presented where Hylinae is divided into four tribes. Of the species currently in “Hyla”, 297 of the 353 species are placed in 15 genera; of these, 4 are currently recognized, 4 are resurrected names, and 7 are new. Hyla is restricted to H. femoralis and the H. arborea, H. cinerea, H. eximia, and H. versicolor groups, whose contents are redefined. Phrynohyas is placed in the synonymy of Trachycephalus, and Pternohyla is placed in the synonymy of Smilisca. The genus Dendropsophus is resurrected to include all former species of Hyla known or suspected to have 30 chromosomes. Exerodonta is resurrected to include the former Hyla sumichrasti group and some members of the former H. miotympanum group. Hyloscirtus is resurrected for the former Hyla armata, H. bogotensis, and H. larinopygion groups. Hypsiboas is resurrected to include several species groups—many of them redefined here—of Gladiator Frogs. The former Hyla albofrenata and H. albosignata complexes of the H. albomarginata group are included in Aplastodiscus. New generic names are erected for (1) Agalychnis calcarifer and A. craspedopus; (2) Osteocephalus langsdorffii; the (3) Hyla aromatica, (4) H. bromeliacia, (5) H. godmani, (6) H. mixomaculata, (7) H. taeniopus, (8) and H. tuberculosa groups; (9) the clade composed of the H. pictipes and H. pseudopuma groups; and (10) a clade composed of the H. circumdata, H. claresignata, H. martinsi, and H. pseudopseudis groups.

MOLECULAR SYSTEMATICS OF THE MIDDLE AMERICAN JUMPING PITVIPERS (GENUS ATROPOIDES) AND PHYLOGEOGRAPHY OF THE ATROPOIDES NUMMIFER COMPLEX

We used 1400 bp of mitochondrial DNA sequence from two gene fragments (ND4 and cyt-b) to investigate phylogenetic relationships within Atropoides, with emphasis on the subspecies of A. nummifer. Although many relationships within the genus are strongly supported, monophyly of Atropoides was never supported, although it could not be rejected with statistical confidence. In most analyses, the genus was paraphyletic with respect to Porthidium and Cerrophidion, due to the problematic placement of A. picadoi. Our results suggest that the current taxonomy may underestimate species diversity within this group. Atropoides nummifer was found to comprise three distinct phylogroups, generally coinciding with the current subspecies recognized under A. nummifer but paraphyletic with respect to A. olmec. Additionally, disjunct populations previously thought to represent A. nummifer in Oaxaca, Mexico, and Baja Verapaz, Guatemala, appear to represent A. olmec. We use the phylogeny recovered for A. nummifer and A. olmec to discuss geological and climatic events that may historically have affected gene flow within this complex.

Is The Amphibian Tree of Life really fatally flawed

Wiens (2007 , Q. Rev. Biol. 82, 55–56) recently published a severe critique of Frost et al.s (2006, Bull. Am. Mus. Nat. Hist. 297, 1–370) monographic study of amphibian systematics, concluding that it is “a disaster” and recommending that readers “simply ignore this study”. Beyond the hyperbole, Wiens raised four general objections that he regarded as “fatal flaws”: (1) the sampling design was insufficient for the generic changes made and taxonomic changes were made without including all type species; (2) the nuclear gene most commonly used in amphibian phylogenetics, RAG‐1, was not included, nor were the morphological characters that had justified the older taxonomy; (3) the analytical method employed is questionable because equally weighted parsimony “assumes that all characters are evolving at equal rates”; and (4) the results were at times “clearly erroneous”, as evidenced by the inferred non‐monophyly of marsupial frogs. In this paper we respond to these criticisms. In brief: (1) the study of Frost et al. did not exist in a vacuum and we discussed our evidence and evidence previously obtained by others that documented the non‐monophyletic taxa that we corrected. Beyond that, we agree that all type species should ideally be included, but inclusion of all potentially relevant type species is not feasible in a study of the magnitude of Frost et al. and we contend that this should not prevent progress in the formulation of phylogenetic hypotheses or their application outside of systematics. (2) Rhodopsin, a gene included by Frost et al. is the nuclear gene that is most commonly used in amphibian systematics, not RAG‐1. Regardless, ignoring a study because of the absence of a single locus strikes us as unsound practice. With respect to previously hypothesized morphological synapomorphies, Frost et al. provided a lengthy review of the published evidence for all groups, and this was used to inform taxonomic decisions. We noted that confirming and reconciling all morphological transformation series published among previous studies needed to be done, and we included evidence from the only published data set at that time to explicitly code morphological characters (including a number of traditionally applied synapomorphies from adult morphology) across the bulk of the diversity of amphibians (Haas, 2003, Cladistics 19, 23–90). Moreover, the phylogenetic results of the Frost et al. study were largely consistent with previous morphological and molecular studies and where they differed, this was discussed with reference to the weight of evidence. (3) The claim that equally weighted parsimony assumes that all characters are evolving at equal rates has been shown to be false in both analytical and simulation studies. (4) The claimed “strong support” for marsupial frog monophyly is questionable. Several studies have also found marsupial frogs to be non‐monophyletic. Wiens et al. (2005, Syst. Biol. 54, 719–748) recovered marsupial frogs as monophyletic, but that result was strongly supported only by Bayesian clade confidence values (which are known to overestimate support) and bootstrap support in his parsimony analysis was < 50%. Further, in a more recent parsimony analysis of an expanded data set that included RAG‐1 and the three traditional morphological synapomorphies of marsupial frogs, Wiens et al. (2006, Am. Nat. 168, 579–596) also found them to be non‐monophyletic. Although we attempted to apply the rule of monophyly to the naming of taxonomic groups, our phylogenetic results are largely consistent with conventional views even if not with the taxonomy current at the time of our writing. Most of our taxonomic changes addressed examples of non‐monophyly that had previously been known or suspected (e.g., the non‐monophyly of traditional Hyperoliidae, Microhylidae, Hemiphractinae, Leptodactylidae, Phrynobatrachus, Ranidae, Rana, Bufo; and the placement of Brachycephalus within “Eleutherodactylus”, and Lineatriton within “Pseudoeurycea”), and it is troubling that Wiens and others, as evidenced by recent publications, continue to perpetuate recognition of non‐monophyletic taxonomic groups that so profoundly misrepresent what is known about amphibian phylogeny.

Thousands of microsatellite loci from the venomous coralsnake Micrurus fulvius and variability of select loci across populations and related species

Studies of population genetics increasingly use next‐generation DNA sequencing to identify microsatellite loci in nonmodel organisms. There are, however, relatively few studies that validate the feasibility of transitioning from marker development to experimental application across populations and species. North American coralsnakes of the Micrurus fulvius species complex occur in the United States and Mexico, and little is known about their population structure and phylogenetic relationships. This absence of information and population genetics markers is particularly concerning because they are highly venomous and have important implications on human health. To alleviate this problem in coralsnakes, we investigated the feasibility of using 454 shotgun sequences for microsatellite marker development. First, a genomic shotgun library from a single individual was sequenced (approximately 7.74 megabases; 26 831 reads) to identify potentially amplifiable microsatellite loci (PALs). We then hierarchically sampled 76 individuals from throughout the geographic distribution of the species complex and examined whether PALs were amplifiable and polymorphic. Approximately half of the loci tested were readily amplifiable from all individuals, and 80% of the loci tested for variation were variable and thus informative as population genetic markers. To evaluate the repetitive landscape characteristics across multiple snakes, we also compared microsatellite content between the coralsnake and two other previously sampled snakes, the venomous copperhead (Agkistrodon contortrix) and Burmese python (Python molurus).

A New Subspecies of Beaded Lizard, Heloderma horridum, from the Motagua Valley of Guatemala

A new subspecies of beaded lizard, Heloderma horridum charlesbogerti, is described from the xeric middle Motagua Valley of eastern Guatemala. It differs from the other subspecies of H. horridum in scalation, body proportions, coloration, and pattern. The Motagua population of H. horridum is widely disjunct from other conspecific populations. The probable vicariance event that shaped present distribution of the Motagua Valley population was the formation of the volcanic cordillera in southern Guatemala during the Quaternary. With the formation of this chain of high mountains, the Pacific coast and piedmont of Guatemala became more mesic, fragmenting Heloderma populations and restricting H. horridum to the

Evolutionary relationships amongst polymorphic direct-developing frogs in the Craugastor rhodopis Species Group (Anura: Craugastoridae)

The Craugastor rhodopis Species Group includes two leaf-litter frog species (C. loki and C. rhodopis). These direct-developing frogs inhabit tropical regions of Mexico and northern Central America. Characterizing diversity within the group has been difficult due to high levels of phenotypic polymorphism within and between species. Because of these polymorphisms, each taxon has junior synonyms. Using a fragment of mitochondrial DNA (mtDNA), we investigated genetic diversity in the C. rhodopis Species Group. We then examined type specimens (including types of junior synonyms) to match nomenclature to geographically circumscribed genetic clusters. Our molecular analyses revealed four major lineages within the C. rhodopis Species Group: (1) a widely distributed clade in western Mexico, (2) a highland clade in eastern Mexico, (3) a widely distributed lowland clade occurring in eastern Mexico, Guatemala and El Salvador, and (4) a haplotype from Volcán San Martín in Veracruz, Mexico. We identified the first clade as C. occidentalis, a taxon currently placed in the ecologically similar but phylogenetically distant C. mexicanus Species Series. In light of this we place C. occidentalis in the C. rhodopis Species Group and designate a lectotype and paralectotype for the species. The second and third clades inhabiting eastern Mexico and northern Central America correspond to C. rhodopis and C. loki, respectively. Additionally, we examined the taxonomic distribution of certain colour pattern traits and compensatory mutations in Domain III of the mtDNA 12S ribosomal RNA gene. Our recovery of the divergent Veracruz haplotype and extensive mtDNA structure within species indicates that additional taxonomic revision will be necessary.

Rapid range expansion in the Great Plains narrow-mouthed toad (Gastrophryne olivacea) and a revised taxonomy for North American microhylids

We investigated genetic variation within the Great Plains narrow-mouthed toad, Gastrophryne olivacea, across its geographic range in the United States and Mexico. An analysis of mitochondrial DNA (mtDNA) from 105 frogs revealed remarkably low levels of genetic diversity in individuals inhabiting the central United States and northern Mexico. We found that this widespread matrilineal lineage is divergent (ca. 2% in mtDNA) from haplotypes that originate from the western United States and western coast of Mexico. Using a dataset that included all five species of Gastrophryne and both species of the closely related genus Hypopachus, we investigated the phylogenetic placement of G. olivacea. This analysis recovered strong support that G. olivacea, the tropically distributed G. elegans, and the temperately distributed G. carolinensis constitute a monophyletic assemblage. However, the placement of G. pictiventris and G. usta render Gastrophryne paraphyletic with respect to Hypopachus. To complement our mitochondrial analysis, we examined a small fragment of nuclear DNA and recovered consistent patterns. In light of our findings we recommend (1) the resurrection of the nomen G. mazatlanensisTaylor (1943) for the disjunct western clade of G. olivacea and (2) the tentative placement of G. pictiventris and G. usta in Hypopachus. To explore possible scenarios leading to low levels of genetic diversity in G. olivacea, we used mismatch distributions and Bayesian Skyline plots to examine historic population expansion and demography. Collectively these analyses suggest that G. olivacea rapidly expanded in effective population size and geographic range during the late Pleistocene or early Holocene. This hypothesis is consistent with fossil data from northern localities and contemporary observations that suggest ongoing northern expansion. Given our findings, we suspect that the rapid range expansion of G. olivacea may have been facilitated by ecological associations with open habitats and seasonal water bodies.

A New Long-Tailed Rattlesnake (Viperidae) From Guerrero, Mexico

A distinctive new species of rattlesnake is described from the western versant of the Sierra Madre del Sur of Guerrero, Mexico. This long-tailed rattlesnake cannot be confused with any other species of rattlesnake and is most similar to Crotalus stejnegeri and C. lannomi. The Guerrero species possesses a strikingly distinct color pattern and differs from all other rattlesnakes in aspects of lepidosis. Mexico continues to be the origin of newly discovered species that provide important insights into the evolution or ecology of particular groups. A few examples from recent decades include Exiliboa placata, a monotypic, relictual dwarf boa (Bogert, 1968), Rhadinophanes monticola, a monotypic, highland colubrid (Myers and Campbell, 1981), and Pseudoeurycea aquatica, the only aquatic bolitoglossine salamander (Wake and Campbell, 2001).

Molecular systematics of the genus Sonora (Squamata: Colubridae) in central and western Mexico

Mexico possesses high levels of endemic biodiversity, especially for squamate reptiles. However, the evolutionary relationships among many reptiles in this region are not well known. The closely related genera of Sonora Baird and Girard 1853 and Procinura Cope 1879 are coralsnake mimics found from the central and western United States to southwestern Mexico and Baja California. Although species delimitation in this group has historically relied upon colour pattern and other morphological characters, many populations of these species display colour pattern polymorphism, which may confound taxonomy. We used molecular phylogenetics to assess the evolutionary relationships and delimit species within Sonora, focusing on the phylogenetic position of Procinura and the validity of S. mutabilis and aequalis. We sequenced two mitochondrial (ND4 and cytb) and two nuclear (c-mos and RAG-1) genes for the single species of Procinura and each of the four species of Sonora. We analysed these sequences using maximum parsimony, maximum likelihood and Bayesian phylogenetic analyses on separately concatenated mitochondrial and nuclear datasets. Additionally, we used Bayesian coalescent methods to build a species tree (Bayesian species tree analysis) and delimit species boundaries (Bayesian species delimitation). All methods indicated that Procinura is deeply nested within Sonora, and most individual species are well supported. However, we found that one taxon (S. aequalis) is paraphyletic with regard to another (S. mutabilis). We recommend that the genus Procinura be synonymised with Sonora and that S. aequalis be synonymised with S. mutabilis. Additionally, the phylogenetic patterns that we document are broadly congruent with a Miocene or Pliocene divergence between S. michoacanensis and S. mutabilis along the Trans-Mexican Volcanic Belt. Finally, our data are consistent with the early evolution of coralsnake mimicry and colour pattern polymorphism within the genus Sonora.

A NEW MONTANE RATTLESNAKE (VIPERIDAE) FROM MICHOACAN, MEXICO

A new species of rattlesnake is described from the upper elevations of Cerro Tancítaro in Michoacán, in the western portion of the Transverse Volcanic Cordillera. This diminutive rattlesnake appears to be most closely related to several species also occurring at high elevations in Mexico and the southwestern United States including Crotalus intermedius, C. pricei, and C. transversus. The Tancítaro species is most similar to C. transversus, but differs in aspects of lepidosis and color pattern.