Comparative cytogenetic analysis on four tree frog species (Anura, Hylidae, Hylinae) from Brazil

A comparative cytogenetic analysis was carried out on four Hylinae tree frogs from Brazil ((Aparasphenodon brunoi, Corythomantis greeningi, Osteocephalus langsdorffii, and Scinax fuscovarius) using Giemsa staining, BrdU replication banding, Ag-NOR staining, C-banding, DAPI and CMA(3) fluorochrome staining, and fluorescence in situ hybridization (FISH) with an rDNA probe. All the species share closely similar 2n = 24 karyotypes, almost indistinguishable by standard staining. The technique of BrdU incorporation allowed the identification of each pair of homologs and the establishment of extensive homeology for the great majority of the chromosomes, mainly of A. brunoi, C. greeningi, and O. langsdorffii. Despite highly conserved replication banding patterns, the use of the other banding techniques disclosed some minor differences, which reinforces the importance of extensive cytogenetic analyses for the karyotypic characterization of Anuran species. The present cytogenetic data confirm the closer proximity of A. brunoi, C. greeningi, and O. langsdorffii, whereas S. fuscovarius is phylogenetically more distant.     

A cladistic analysis of Scinax (Anura: Hylidae)

The hylid frog genus Scinax ranges from southern Mexico to eastern Argentina. The 84 recognized species are currently arranged in five groups: the Scinax rubra , Scinax rostrata , Scinax catharinae , Scinax staufferi , and Scinax perpusilla species groups. Although a few species groups are distinctive, compelling evidence of monophyly is lacking for three groups, and their interrelationships remain virtually unknown. With the objective of testing the monophyly of the species groups and exploring their interrelationships, a cladistic analysis was performed, employing 86 characters from osteology, myology, adult and larval external morphology, reproductive biology, and karyotypes taken from 36 representative species of the five species groups plus eight outgroup taxa. The analysis of the resulting data matrix shows moderately well-supported (as assessed through Bremer support and parsimony jackknifing) S. catharinae and S. rostrata species groups. The S. staufferi species group is polyphyletic, as currently composed, given that some of its species are nested within the S. catharinae species group. The only representative of the S. perpusilla species group is the sister species of the S. catharinae species group. Although weakly supported, the representatives of the S. rostrata and S. staufferi species groups are nested, separately, within the S. rubra species group. In order to have a taxonomy consistent with these results, it is proposed not to recognize the S. rubra group but to recognize a more inclusive rubra clade that includes all studied exemplars of the former S. rubra group, plus some of the S. staufferi group and all of the S. rostrata group. Similarly, a catharinae clade is recognized, within which all studied exemplars of the S. catharina e group (that now includes Scinax berthae and Scinax agilis ) and the one studied representative of the S. perpusilla group are included.     

The phylogeny of Dendropsophini (Anura: Hylidae: Hylinae)

The relationships of the hyline tribe Dendropsophini remain poorly studied, with most published analyses dealing with few of the species groups of Dendropsophus. In order to test the monophyly of Dendropsophini, its genera, and the species groups currently recognized in Dendropsophus, we performed a total evidence phylogenetic analysis. The molecular dataset included sequences of three mitochondrial and five nuclear genes from 210 terminals, including 12 outgroup species, the two species of Xenohyla, and 93 of the 108 recognized species of Dendropsophus. The phenomic dataset includes 46 terminals, one per species (34 Dendropsophus, one Xenohyla, and 11 outgroup species). Our results corroborate the monophyly of Dendropsophini and the reciprocal monophyly of Dendropsophus and Xenohyla. Some species groups of Dendropsophus are paraphyletic (the D. microcephalus, D. minimus, and D. parviceps groups, and the D. rubicundulus clade). On the basis of our results, we recognize nine species groups; for three of them (D. leucophyllatus, D. microcephalus, and D. parviceps groups) we recognize some nominal clades to highlight specific morphology or relationships and facilitate species taxonomy. We further discuss the evolution of oviposition site selection, where our results show multiple instances of independent evolution of terrestrial egg clutches during the evolutionary history of Dendropsophus.     

Cryptic lineages in a small frog: the post-glacial history of the spring peeper,Pseudacris crucifer (Anura: Hylidae)

The spring peeper (Pseudacris crucifer) is believed to have been a primary herpetological invader of eastern North America following the most recent period of glacial retreat. We examined the phylogeographic pattern and population structure of P. crucifer to determine whether the distribution of haplotypic variants reflect post-Pleistocene recolonization dynamics. A number of geographically isolated evolutionary lineages were supported by both maximum parsimony and neighbor-joining analyses, and by coalescence approaches applied to mtDNA. South-western Ontario represents a high level of genotypic diversity (pi) due to the presence of two divergent lineages. The geographic distribution of these lineages are interpreted as reflecting post-glacial recolonization dynamics from separate, isolated refugia during the late Pleistocene that have come into secondary contact in SW Ontario. The phylogenetic placement of haplotypes from the range of P. crucifer bartramiana (Florida and South Carolina) does not allow for monophyly of P. crucifer crucifer, and therefore the bartramiana subspecies designation does not reflect a separate evolutionary lineage.     

Observations on the polyphyodont dentition of Hemiphractus proboscideus (Anura: Hylidae)

In Hemiphractus fang–like teeth are ankylosed to the premaxilla, maxilla and prevomer, and bony odontoids are found on the dentary, angular and palatine bones. The odontoids are small, but a larger pair at the front of the lower jaw project upwards and backwards into the mouth and fit into a diastema between the anterior premaxillary teeth when the mouth is closed.
The teeth are unipartite and monocuspid, and each consists of a strongly recurved and elongated cone of orthodentine, capped at the tip by a thin layer of enamel. The inner circumpulpal layer of the dentine is tubular, but no tubules are present in the outer pallial layer. During tooth development, dentine is formed before the enamel matrix is produced, and the tooth germs lie horizontally beneath the ventral surface of each dentigerous bone. On eruption, the tooth germs migrate horizontally and become ankylosed to the outer edge of the jaw bone by a layer of cellular cementum.
During tooth replacement, the vast majority of the dentine of each tooth, and the cementum at the tooth base, are resorbed by osteoclasts. It is not clear whether the tips of the teeth are shed or not.     

The tadpole of Scinax aromothyella (Anura: Hylidae) from Uruguay

We describe the tadpole of Scinax aromothyella from the surroundings of Quebrada de los Cuervos, Departamento de Treinta y Tres, Uruguay. It shares with other larvae of the Scinax catharinae clade the slightly depressed body, a dextral vent tube that reaches the free margin of the ventral fin, and the oral disc with a dorsal gap in the marginal row of papillae and with a concave posterior edge. It is most similar to the tadpole of Scinax berthae, from which it differs in the larger total length of S. aromothyella.

Describimos la larva de Scinax aromothyella de los alrededores de la Quebrada de los Cuervos, Departamento de Treinta y Tres, Uruguay. Comparte con las larvas de otras especies del clado de Scinax catharinae el tener el cuerpo levemente deprimido, el tubo proctodeal dextro que alcanza el borde libre de la aleta ventral, y el disco oral con un claro rostral en la hilera de papilas marginales y con su borde posterior cóncavo. Es muy parecida a la larva de Scinax berthae, de la que difiere en la mayor longitud total de S. aromothyella.     

Chromosome banding in Amphibia. XXIV. The B chromosomes of Gastrotheca espeletia (Anura,Hylidae)

The mitotic chromosomes of an Ecuadorian population of the marsupial frog Gastrotheca espeletia were analyzed by means of banding techniques and fluorescence in situ hybridization. This species is characterized by unusual supernumerary (B) chromosomes. The maximum number of B chromosomes is 9 and they occur in three different morphological types. Banding analyses show that the B chromosomes are completely heterochromatic, consist of AT base pair-rich repeated DNA sequences, replicate their DNA in very late S-phase of the cell cycle, and are probably derived from a centromeric or paracentromeric region of a standard (A) chromosome. Exceptionally, the B chromosomes carry 18S + 28S ribosomal RNA genes and the conserved vertebrate telomeric DNA sequence appears to be underrepresented. Flow cytometric measurements of the nuclear DNA content differentiate between individuals with different numbers of B chromosomes. Significantly more B chromosomes are present in female than in male animals.     

Genotoxicity evaluation of the insecticide imidacloprid on circulating blood cells of Montevideo tree frog Hypsiboas pulchellus tadpoles (Anura,Hylidae) by comet and micronucleus bioassays

Acute toxicity and genotoxicity of imidacloprid (IMI) was evaluated on Hypsiboas pulchellus (Anura: Hylidae) tadpoles exposed under laboratory conditions. A lethal effect was used as the end point for lethality, whereas the frequency of micronuclei (MNs) and DNA single-strand breaks evaluated by the single cell gel electrophoresis assay were employed as end points for genotoxicity. Experiments were performed on tadpoles at stage 36 (range, 35–37) according to the classification proposed by Gosner. Mortality studies revealed an LC₅₀ (96 h) value of 84.91 mg/L IMI (95% confidence limits, 77.20–93.04). While increased frequency of MNs was observed when 15 and 30 mg/L were assayed for 48 h, only 15 mg/L increased the frequency of MNs in tadpoles exposed for 96 h. Furthermore, other nuclear abnormalities, i.e., binucleated cells and blebbed and notched nuclei, were induced in tadpoles exposed for both 48 h when treated with 15 mg/L and 96 h when treated with 15 and 30 mg/L. An increase in the genetic damage index was observed in tadpoles treated with 30 mg/L for 48 and 96 h. This study represents the first evidence of acute lethal and sublethal effects exerted by IMI on tadpoles of an amphibian species native to Argentina. Finally, our findings highlight the hazardous properties of this insecticide for nontarget living species exposed to this agrochemical.     

Phylogenetic relationships among marsupial frog genera (Anura: Hylidae: Hemiphractinae) based on evidence from morphology and natural history

We reassess the phylogenetic relationships of genera of hemiphractine hylid frogs (Marsupial Treefrogs) and discuss the evolution of several distinctive characters within this group using parsimony analysis. Fifty-one morphological and life-history characters were sampled from two species of Cryptobatrachus , three species of Flectonotus , 17 species of Gastrotheca , all five species of Hemiphractus , and one species of Stefania as the ingroup and three hyline, one phyllomedusine, and one pelodryadine species as outgroups. Our results support the mon-ophyly of Flectonotus, Cryptobatrachus , and Hemiphractus. Gastrotheca is paraphyletic with respect to Hemiphractus , dorsal pouches were lost in the ancestor of Hemiphractus. Direct development is a synapomorphy for Hemiphractinae and tadpoles were regained independently several times. These results stand in stark contrast to the prevailing paradigm regarding marsupial frog relationships.     

The submandibular musculature of phyllomedusinae (Anura: Hylidae): A reappraisal

The submandibular musculature of 37 species of the five currently recognized genera of the subfamily Phyllomedusinae (Anura: Hylidae) is described; observations are made on the variation and ontogeny of these muscles. Supplementary apical elements of the m. intermandibularis occur in all phyllomedusines studied, in addition to the supplementary posterolateral elements previously reported. Our observations are discussed in the context of 1) the proposed homology between supplementary apical and posterolateral elements; 2) the homology with the apical elements reported for Pelodryadinae (sister taxon of Phyllomedusinae); and 3) the implications for our understanding of the relationships between Phyllomedusinae and Pelodryadinae. Anatomical differences between the apical and posterolateral elements and their co‐occurrence in phyllomedusines indicate that these supplementary elements are not homologous. Despite differences between phyllomedusines and pelodryadines in the adhesion of supplementary fibers to the principal element of the m. intermandibularis and the occurrence of a broad aponeurosis or a medial raphe, the extensive morphological and developmental resemblances of the apical elements indicate that these structures are homologous, and that the presence of apical elements is a synapomorphy of Phyllomedusinae + Pelodryadinae. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Muscle development in the abdominal region of Larval Hylidae (Amphibia: Anura)

Larval muscle development in the abdominal region of five species of hylid frogs (Scinax nasicum, S. fuscovarium, Hyla andina, Phyllomedusa boliviana, Gastrotheca gracilis) was studied using differential staining techniques. These five species represent three major hylid subfamilies. The development of the main abdominal muscles, the rectus abdominis, the two lateral muscles (obliquus externus and transversus), and the lateral pectoralis abdominalis is described. The number of myotomes of the rectus abdominis varies between five and six, and the abdominal muscles associated with the rectus abdominis (obliquus externus, pectoralis abdominalis, and rectus cervicis) vary interspecifically in time of appearance and configuration. The presence of gaps in the configuration of the rectus abdominis has been related to the lotic habits of the larvae. However, our observations indicate the presence of such gaps in larvae that inhabit lentic environments as well. These results suggest that the presence of these gaps is unrelated to larval habitat. There are relatively small differences in muscle morphology among these closely related species, which apparently cannot be explained by morphological adaptations related to their ecology. In the species studied, the number of elements that form the abdominal musculature in larvae is equal to that observed in adults. Likewise, the general morphology of the muscles is ontogenetically conserved. This suggests that both the axial skeleton and musculature are more ontogenetically conserved in relation to the substantial changes that are observed in the skull and head muscles of developing anurans. J. Morphol. 241:275–282, 1999. © 1999 Wiley‐Liss, Inc.     

Structural diversity of nuptial pads in Phyllomedusinae (Amphibia: Anura: Hylidae)

We studied the morphological variation of the nuptial pads using light microscopy and scanning electron microscopy (SEM) in 26 species of phyllomedusines (Anura: Hylidae), representing the five currently recognized genera. All phyllomedusines have single nuptial pads with dark colored epidermal projections (EPs). Spine‐shaped EPs occur in Cruziohyla calcarifer, Phrynomedusa appendiculata and in one species of Phasmahyla. The other species have roundish EPs. The density of the EPs on the pad is variable. Species in the Phyllomedusa hypochondrialis Group have EPs with a density that varies between 764 ± 58/mm² and 923 ± 160/mm². In all other studied species (including the Phyllomedusa burmeisteri and Phyllomedusa perinesos groups, Phyllomedusa camba, Phyllomedusa boliviana, Phyllomedusa sauvagii, Phyllomedusa bicolor, and Phyllomedusa tomopterna) the density of EPs varies between 108 ± 20/mm² and 552 ± 97/mm². Pores were observed with SEM in C. calcarifer, Agalychnis lemur, Agalychnis moreletii, but its presence is confirmed through histological sections on several other species. Its visibility using SEM seems to be related with the level of separation between adjacent EPs. The pores in the four studied species of Agalychnis are shown with SEM and histological sections to have a characteristic epidermal rim, that is absent in the otherphyllomedusines. Unlike most previous reports on breeding glands, those of phyllomedusines are alcian blue positive, indicating the presence of acidic mucosubstances on its secretions. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

A new species of Osteocephalus (Anura: Hylidae) from Guyana.

A new member of the genus Osteocephalus is described from the Pakaraima mountains of western Guyana. This species is the smallest known member of the genus and is probably closely related to O. subtilis. Both share a small size (less than 40 mm snout-vent length), large and bulgy eyes directed somewhat rostrally, green bones, smooth and brownish dorsal skin, relatively short and truncate snout, small tympanum, subgular and laterally expanded vocal sac, poorly developed subarticular and supernumerary tubercles, a supra-anal glandular ridge, and cream-white venter and subocular region. The new species can be distinguished from O. subtilis by the Buff iris (vs black), smaller overall size (32.7 vs 35.8-38.8 mm snout-vent length), relatively larger toe disks, and less developed foot webbing. The cranium of the new species is well ossified, relatively reduced in width between the orbits, without an exposed frontoparietal fontanelle and with the anterior arm of the squamosal extending to about half the distance to the maxillary. The vocal sac is subgularly poorly developed and possess lateral extensions to the area behind the jaw angles. Well developed supraocular and suprasquamosal cartilages give support to the enlarged eyes of this species.     

The hyolaryngeal apparatus of three species of Scinax (Scinaxinae: Hylidae: Anura) and comparison with other Hylidae species

The hyolaryngeal apparatus includes the arytenoid, cricoid and hyoid cartilages, and varied sizes and shapes can be distinguished between species. Although the larynx is an important morphological structure for sound production in frogs, few studies describe the structures and processes of its parts in hylids. We studied the hyolaryngeal anatomy of both males and females of Scinax ruber (Laurenti, 1768), using clearing and double staining methods, comparing it with that of Scinax wandae (Pyburn and Fouquette, 1971), Scinax kennedyi (Pyburn, 1973) and other hylid species. We found that S. ruber has the largest arytenoid cartilage of any species of the subfamily Scinaxinae studied to date. We described both the laryngeal differences among three Scinax species and the interspecific variability in the shapes of the projections of the arytenoid in the males of these species. The taxonomic characters (presence/absence of processes of the cartilage of Santorini, oesophageal process or bronchial processes) described here can play a key role in differentiating these species from Scinax species and from other species. In addition, we want to contribute to the natural history and comparison of the larynges in Scinax and within the genus and the Hylidae.     

Paracosmocercella rosettae n. gen. et n. sp. (Nematoda: Cosmocercoidea: Cosmocercidae) collected from the Japanese tree frog, Hyla japonica (Anura: Hylidae), in Japan

A new genus, Paracosmocercella (Nematoda: Cosmocercoidea: Cosmocercidae), is proposed with monotypic Paracosmocercella rosettae n. sp. from the Japanese tree frog, Hyla japonica Günther, 1859, in Oita, Kyushu Island, Japan. Paracosmocercella resembles Cosmocercella and Cosmocercoides by having 2 rows of rosette papillae without plectanes ventrally in preanal region of male, but it is readily distinguished from Cosmocercella by lacking clear vesicle supporting rosette papillae and from Cosmocercoides by lacking rosette papillae in perianal region and having large-sized eggs, which hatch in uterus.