A hundred-year-old mystery-the reproductive mode and larval morphology of the enigmatic frog genus Allophryne (Amphibia; Anura; Allophrynidae).

Frogs of the Allophrynidae are an enigmatic family from South America. To date, published information is lacking regarding this group's reproductive biology and larval morphology. Here, we provide the first detailed description of the reproductive mode, developmental mode, and tadpole morphology for Allophryne ruthveni. We developed a captive breeding and rearing protocol for this species and then conducted a series of observations to describe aspects of its reproductive biology. In captivity, this species exhibits aquatic oviposition, where single eggs are laid ungrouped within a simple jelly capsule and are scattered free in the water column before sinking to develop on benthic substrates. We did not observe parental care nor any parental interactions with eggs post-fertilization. Tadpoles are characterized by an oval body, anteroventral oral disc, a labial tooth row formula of 2(2)/3, and a dextral vent tube. The buccopharyngeal cavity is marked by the presence of two pairs of infralabial papilla and four lingual papillae. Cranial morphology is characterized by the presence of the commissura quadratoorbital. This species possesses an additional slip of the m. rectus cervicis and of the m. levator arcuum branchialium III. We discuss our results in comparison with glassfrogs (Centrolenidae).

Anatomical features of the phytotelma dwelling, egg-eating, fanged tadpoles of Rhacophorus vampyrus (Anura: Rhacophoridae).

Tadpoles of the Vampire tree frog Rhacophorus vampyrus differ substantially from other rhacophorid tadpoles, by having profound modifications in external morphology. The morphological peculiarities of this species likely correlate with their arboreal microhabitat and strict oophagous diet. In this work, we examine buccal and musculoskeletal anatomy and compare them to other rhacophorid and egg-eating larvae. The shape and arrangement of cartilages of the lower jaw are unique among tadpoles, and the lack of a palatoquadrate suspensorium is only known in the distantly related macrophagous tadpoles of the dicroglossid Occidozyga baluensis. The cranial musculature is massive, and the morphology of several mandibular, hyoid, and abdominal muscles could be related to the ingestion and transit of large eggs. In the buccal cavity, conspicuous aspects are the absence of ridges and papillae, and the development of a unique glandular zone in the buccal floor. Finally, observations of the skeletal support of keratinized mouthparts allow us to present a topography-based hypothesis of homology of the conspicuous fangs of these tadpoles.

Tadpole of the Critically Endangered Sterling's Toothed Toad (Oreolalax sterlingae).

Sterling's Toothed Toad, Oreolalax sterlingae (Nguyen et al. 2013), was described from near the summit of the highest peak in Vietnam, Mount Fansipan, Lao Cai Province. The species is the only member of the genus in Vietnam and is presently known from a single stream system on Mount Fansipan at about 2800 m elevation. The closest congener occurs more than 200 km away in China (Fei et al. 2010), and the relationship of O. sterlingae within the genus is not known. Because of its extremely small range and profound habitat modifications and pollution at the site, O. sterlingae was recently assessed as Critically Endangered (IUCN SSC 2015).

Morphological clarifications of Australian hylid and limnodynastid tadpoles.

Tadpole morphology usually is consistent within taxa and ecologies (Altig & Johnston 1989), but the occurrences of unique morphologies offer informative perspectives on the potential diversity of these structures. Anstis (2013) characterized most of the tadpoles of Australia, but a thorough understanding of certain morphological features requires closer examinations. Until we understand the morphological features of anuran larvae, and preferably their ontogeny, we cannot productively discuss their evolution or ecological significance.

The reproductive biology and larvae of the first tadpole-bearing frog, Limnonectes larvaepartus.

Most of the reproductive modes of frogs include an exotrophic tadpole, but a number of taxa have some form of endotrophic development that lacks a feeding tadpole stage. The dicroglossid frog genus Limnonectes ranges from China south into Indonesia. The breeding biologies of the approximately 60 described species display an unusual diversity that range from exotrophic tadpoles to endotrophic development in terrestrial nests. There have been mentions of oviductal production of typical, exotrophic tadpoles in an undescribed species of Limnonectes from Sulawesi, Indonesia. Here we examine newly collected specimens of this species, now described as L. larvaepartus and present the first substantial report on this unique breeding mode. Typical exotrophic tadpoles that are retained to an advanced developmental stage in the oviducts of a female frog are birthed into slow-flowing streams or small, non-flowing pools adjacent to the streams.

The breeding behaviour, advertisement call and tadpole of Limnonectes dabanus (Anura: Dicroglossidae).

Fanged frogs (Limnonectes) are a group of dicroglossid frogs from Asia that often have reversed sexual dimorphism with larger males. Limnonectes dabanus is a poorly known species of fanged frog from forested habitats in southern Vietnam and eastern Cambodia. Adult males exhibit an extreme degree of megacephaly and possess bizarre head ornamentation. L. dabanus breeds in shallow, non-flowing or very slow-flowing pools, puddles, and drainage ditches. Eggs are laid as a widely spaced array, and the larvae have a morphology typical of pond-dwelling tadpoles. Although males of the species lack vocal sacs, they produce a low-pitched (0.4-0.6 kHz), single-note advertisement call that sounds like a drop of water falling into water. Given the spacing of calling males, presence of multiple females near breeding sites, and reversed sexual dimorphism, the mating system of L. dabanus may be an example of resource-defense polygyny, and the massive head of the male is likely used in male combat.

The breeding behavior of Glyphoglossus molossus and the tadpoles of Glyphoglossus molossus and Calluella guttulata (Microhylidae).

The breeding behavior of Glyphoglossus molossus is described from still and video images taken in Cambodia. These large, burrowing frogs follow the general theme of microhylids that deposit aquatic eggs: explosive breeding in ephemeral water and performing multiple amplectic dips to oviposit surface films of pigmented eggs. A portion of a clutch is released with each dip, a dip lasts for about 6 s, 200-300 eggs are released per dip, and about 5 s pass between dips. The ova have a dark black animal pole and yellow vegetal pole. Expanded datasets on the morphology of the tadpoles of Glyphoglossus from Vietnam and Calluella from Myanmar are presented. 

A novel sample preparation method that enables nucleic acid analysis from ultrathin sections.

The ability to isolate and perform nucleic acid analyses of individual cells is critical to studying the development of various cell types and structures. We present a novel biological sample preparation method developed for laser capture microdissection-assisted nucleic acid analysis of ultrathin cell/tissue sections. We used cells of the mitotic bed of the tadpole teeth of Lithobates sphenocephalus (Southern Leopard Frog). Cells from the mitotic beds at the base of the developing teeth series were isolated and embedded in the methacrylate resin, Technovit® 9100®. Intact cells of the mitotic beds were thin sectioned and examined by bright-field and transmission electron microscopy. The cytological and ultrastructural anatomy of the immature and progressively more mature tooth primordia appeared well preserved and intact. A developmental series of tooth primordia were isolated by laser capture microdissection (LCM). Processing of these cells for RNA showed that intact RNA could be isolated. The study demonstrates that Technovit® 9100® can be used as an embedding medium for extremely small tissues and from individual cells, a prerequisite step to LCM and nucleic acid analyses. A relatively small amount of sample material was needed for the analysis, which makes this technique ideal for cell-specific analyses when the desired cells are limited in quantity.

Configurations of the rectus abdominis muscle of anuran tadpoles.

Configurations of the rectus abdominis in tadpoles of 60 anuran species in 13 families were examined. This muscle is present by Gosner stage 25 and does not change until late in metamorphosis. The anterior terminus of the r. abdominis usually is a straight, transverse front or fan-shaped array only loosely associated with the rectus cervicis. In some suspension feeders (Rhinophrynus, Xenopus, but not microhylids), macrophagous suction feeders (Lepidobatrachus) and bromeliad inhabitants (Hyla bromeliacia) the r. abdominis is contigous with the r. cervicis which continues anterolaterally and dorsally from the wall of the spiracular cavity. Suctorial forms (Scutiger), those that live in confined spaces (burrowers, Centrolenella; bromeliad inhabitants, Hyla bromeliacia), and the taxa that have the r. cervicis and r. abdominis contiguous all have closed myosepta; the myosepta of other taxa; except for ones with large tadpoles (e.g., Rana catesbeiana), have large gaps between at least anterior myotomes. These initial data suggest that the configurations of the r. abdominis have responded to selection based on ecomorphological function and convergence among lineages are noted. © 1992 Wiley-Liss, Inc.

Oral disc muscles of anuran tadpoles.

The presence and configurations of the small, extrinsic muscle fibers of the M. mandibulolabialis superior in the upper labium and the M. inferior in the lower labium are described for 24 species in 9 families. Although several species possess only the M. inferior, or both the M. inferior and superior, none has only the M. superior. Some species without tooth rows possess the M. inferior, and microhylid tadpoles have a single-stranded muscle different than the M. mandibulolabialis in each oral flap. The extrinsic oral muscles insert near the bottom of the inter-row valleys behind each tooth ridge and the bases of marginal papillae lateral to the ends of tooth rows. Muscles do not extend to marginal papillae beyond the most distal row of teeth. Muscle contraction is presumed to rotate the teeth distally so that they contact the substrate at the proper working angle. This action probably occurs at full extension of the oral disc just before disc closure starts.

Sequence of ontogenetic development and atrophy of the oral apparatus of six anuran tadpoles.

The development of the oral structures of six species of anuran tadpoles with four different types of mouth parts and the metamorphic atrophy of these structures in two species with different mouth parts are described. The oral labia of typical tadpoles, oral flaps of microhylids, and lateral oral folds of Rhinophrynus are assumed to be homologous. We also suggest that the barbels of the tadpoles of Rhinophrynus are homologs of the marginal papillae of species with an oral disc. Developmental patterns and sequences of the oral structures of all tadpoles examined follow a common pattern: stomodeal invagination, oral pad development, jaw sheeth delimitation, tooth row ridge development, jaw sheath keratinization, and labial tooth keratinization. Developmental patterns remain constant, while interspecific differences are apparent because of truncations of ontogeny at specific stages. Metamorphic atrophy of oral structures occurs roughly in the reverse order of development, although the procedure is rapid and more haphazard than development.