A comparative ultrastructural analysis of spermatozoa in Pleurodema (Anura,Leptodactylidae, Leiuperinae)

This study describes the spermatozoa of 10 of the 15 species of the Neotropical frog genus Pleurodema through transmission electron microscopy. The diversity of oviposition modes coupled with a recent phylogenetic hypothesis of Pleurodema makes it an interesting group for the study of ultrastructural sperm evolution in relation to fertilization environment and egg‐clutch structure. We found that Pleurodema has an unusual variability in sperm morphology. The more variable structures were the acrosomal complex, the midpiece, and the tail. The acrosomal complex has all the structures commonly reported in hyloid frogs but with different degree of development of the subacrosomal cone. Regarding the midpiece, the variability is given by the presence or absence of the mitochondrial collar. Finally, the tail is the most variable structure, ranging from single (only axoneme) to more complex (presence of paraxonemal rod, cytoplasmic sheath, and undulating membrane), with the absence of the typical axial fiber present in hyloid frogs, also shared with some other genera of Leiuperinae. J. Morphol. 277:957–977, 2016. © 2016 Wiley Periodicals, Inc.     

Spermatozoa of Pseudinae (Amphibia, Anura, Hylidae), with a test of the hypothesis that sperm ultrastructure correlates with reproductive modes in anurans

We describe, for the first time, the sperm ultrastructure of the two genera of Pseudinae. Based on sperm ultrastructure, the five species herein examined can be separated into three groups: one containing Pseudis paradoxa, P. bolbodactyla, and P. tocantins, the second containing P. minuta, and the third containing Lysapsus laevis. The midpiece is similar in all species and auxiliary fibers and the undulating membrane are absent. In Pseudis a subacrosomal cone and a multilaminar structure (P. minuta) or a granular material (P. paradoxa group) are seen above the nucleus. Lysapsus laevis has only remnants of the subacrosomal cone. All species have peripheral fibers associated with the outer doublets of the axoneme. We tested the hypothesis of correlation between the presence of an undulating membrane and fertilization environments in anurans using a concentrated changes test (CCT) based on the Hay et al. (Mol Biol Evol 1995;12:928-937) hypothesis of phylogenetic relationships among anuran families. Only a subset of the resolved topologies derived from the Hay et al. (1995) cladogram, where Ranoidea is the sister-group of Sooglossidae, produced significant probabilities of the CCT. Therefore, support for the correlation between sperm ultrastructure and fertilization environments in anurans is, at best, equivocal.     

Sperm morphology of five species of Colostethus (Anura, Dendrobatidae) from Brazil, with phylogenetic comments

We describe the sperm ultrastructure of five species of Colostethus from Brazil. The general structures of the spermatozoa (acrosomal complex and flagellar apparatus) were similar to species within the Bufonoidea. Colostethus brunneus, Colostethus sp. (aff. trilineatus), Colostethus nidicola and Colostethus sp. had biflagellated spermatozoa, whereas the spermatozoa of Colostethus stepheni had a single flagellum, a characteristic previously unknown for this genus. In addition, the spermatozoa of this species showed some mitochondria within the thick, undulating membrane. These characteristics are apparently common to dendrobatid species that have a single flagellum. Based on these findings, we tentatively consider biflagellarity to be an apomorphic condition in the Dendrobatidae. In addition, the results described here do not support the proposed regrouping of the ‘brunneus’ and ‘alagoanus’ groups in a monophyletic ‘trilineatus’ group.     

Phylogeny and genus-level classification of mantellid frogs (Amphibia, Anura)

We propose a novel classification of frogs in the family Mantellidae, based on published phylogenetic information and on a new analysis of molecular data. Our molecular tree for 53 mantellid species is based on 2419 base pairs of the mitochondrial 12S rRNA, 16S rRNA, tRNAVal and cytochrome b genes, and of the nuclear rhodopsin gene. Because the genus Mantidactylus Boulenger sensu lato is confirmed to be paraphyletic with respect to Mantella Boulenger, and is highly diverse in morphology and reproductive biology, we propose to partition Mantidactylus into seven genera by elevating four subgenera to genus rank (Blommersia Dubois, Guibemantis Dubois, Spinomantis Dubois, and Gephyromantis Methuen) and creating two new genera (Boehmantis gen. n. and Wakea gen. n.). In addition, we create the new subgenera Boophis (Sahona) subgen. n., Gephyromantis (Duboimantis) subgen. n., G. (Vatomantis) subgen. n., and Mantidactylus (Maitsomantis) subgen. n. The following species are transferred to Spinomantis, based on their phylogenetic relationships: S. elegans (Guibé) comb. n. (formerly in Mantidactylus subgenus Guibemantis); S. bertini (Guibé) comb. n. and S. guibei (Blommers-Schlösser) comb. n. (both formerly in Mantidactylus subgenus Blommersia); S. microtis (Guibé) comb. n. (formerly in Boophis Tschudi). Within Boophis, the new B. mandraka species group and B. albipunctatus species group are established. Boophis rhodoscelis (Boulenger) is transferred to the B. microtympanum group. The following five species are revalidated: Mantidactylus bellyi Mocquard and M. bourgati Guibé (not junior synonyms of M. curtus (Boulenger)); M. cowanii (Boulenger) (not syn. M. lugubris (Duméril)); M. delormei Angel (not syn. M. brevipalmatus Ahl); Mantella ebenaui (Boettger) (not syn. M. betsileo (Grandidier)). The new classification accounts for recent progress in the understanding of the phylogeny and natural history of these frogs, but it is still tentative for a number of species. Future modifications may be necessary, especially as concerns species now included in Gephyromantis and Spinomantis.Full article published online at: http://www.senckenberg.de/odes/06-11.htm     

The sperm of Hylodinae species (Anura, Leptodactylidae): Ultrastructural characteristics and their relevance to interspecific taxonomic relationships

Aaabstract Hylodinae leptodactylids(sensu Lynch 1971) form a group of diurnal frogs, which is hypothesized on the basis of morphological traits to be the closest relatives of the dendrobatid frogs. Our study describes ultrastructural characteristics of sperm from three hylodine species(Hylodes phyllodes, Crossodactylus sp. n. andMegaelosia massarti) to reassess the intergeneric relationships within the Hylodinae, as well as the supposed relationship between the Hylodinae and Dendrobatidae. The ultrastructure of the sperm is very similar among the three species and is indicative of its conserved nature within the Hylodinae. The structure of the acrosomal complex was very similar to that of other leptodactylid species, to most of the remaining species included in the Bufonoidea lineage, and also to that observed in the dendrobatid species examined so far. Since such a structure has been considered a plesiomorphic trait, it contributes little to our understanding of the relationships between the Hylodinae and Dendrobatidae. The flagellar apparatus ofCrossodactylus sp. n. is very similar to that of most leptodactylids. The sperm ofMegaelosia massarti andHylodes phyllodes display a distinctive condition in their axial and juxtaxonemal fibers. This distinctive flagellar condition expands the already known variability in sperm structure within the Leptodactylidae.     

Ultrastructure of poison glands of South American frogs: a comparison between Physalaemus albonotatus and Leptodactylus chaquensis (Anura: Leptodactylidae)

Serous (poison) cutaneous glands of the leptodactylid species Physalaemus albonotatus and Leptodactylus chaquensis were compared using light and transmission electron microscopy. Glands in the two species share structural traits common in anurans, including the peripheral contractile sheath (myoepithelium) and the syncytial secretory unit that produces, stores, and modifies the poison. At the ultrastructural level, early steps of poison production are also similar and fit the usual path of proteosynthesis, involving rough endoplasmic reticulum (RER) and Golgi stacks (dictyosomes) in the peripheral syncytial cytoplasm. However, several differences are obvious during the maturational processes that lead post-Golgian products to their ultimate ultrastructural traits. In P. albonotatus, the dense product released from the dictyosomes acquires a thick repeating substructure, which, however, becomes looser in the inner portion of the syncytium. In L. chaquensis, serous maturation involves gradual condensation, and opaque, somewhat "vacuolized" granules are formed. These different maturational paths expressed during poison manufacturing in the two species agree with the polyphyletic origin of the family Leptodactylidae. On the other hand, data collected for P. albonotatus fit previous findings from P. biligonigerus and stress the view that poisons produced by congeneric species share similar (or identical) ultrastructural features.     

Mitochondrial genes reveal cryptic diversity in plant-breeding frogs from Madagascar (Anura, Mantellidae, Guibemantis)

One group of mantellid frogs from Madagascar (subgenus Pandanusicola of Guibemantis) includes species that complete larval development in the water-filled leaf axils of rainforest plants. This group consists of six described species: G. albolineatus, G. bicalcaratus, G. flavobrunneus, G. liber, G. pulcher, and G. punctatus. We sequenced the 12S and 16S mitochondrial rRNA genes ( approximately 1.8 kb) from multiple specimens (35 total) of all six species to assess phylogenetic relationships within this group. All reconstructions strongly supported G. liber as part of the Pandanusicola clade, even though this species does not breed in plant leaf axils. This result confirms a striking reversal of reproductive specialization. However, all analyses also indicated that specimens assigned to G. liber include genetically distinct allopatric forms that do not form a monophyletic group. Most other taxa that were adequately sampled (G. bicalcaratus, G. flavobrunneus, and G. pulcher) likewise consist of several genetically distinct lineages that do not form monophyletic groups. These results suggest that many of the recognized species in this group are complexes of cryptic species.     

Phylogenetic analyses of mtDNA sequences reveal three cryptic lineages in the widespread neotropical frog Leptodactylus fuscus (Schneider, 1799) (Anura, Leptodactylidae)

Leptodactylus fuscus is a neotropical frog ranging from Panamá to Argentina, to the east of the Andes mountains, and also inhabiting Margarita, Trinidad, and the Tobago islands. We performed phylogenetic analyses of 12S rRNA, 16S rRNA, tRNA-Leu, and ND1 mitochondrial (mt) DNA sequences from specimens collected across the geographic distribution of L. fuscus to examine two alternative hypotheses: (i) L. fuscus is a single, widely distributed species, or (ii) L. fuscus is a species complex. We tested statistically for geographic association and partitioning of genetic variation among mtDNA clades. The mtDNA data supported the hypothesis of several cryptic species within L. fuscus. Unlinked mtDNA and nuclear markers supported independently the distinctness of a 'northern' phylogenetic unit. In addition, the mtDNA data divided the southern populations into two clades that showed no sister relationship to each other, consistent with high differentiation and lack of gene flow among southern populations as suggested by allozyme data. Concordance between mtDNA and allozyme patterns suggests that cryptic speciation has occurred in L. fuscus without morphological or call differentiation. This study illustrates a case in which lineage splitting during the speciation process took place without divergence in reproductive isolation mechanisms (e.g. advertisement call in frogs), contrary to expectations predicted using a biological species framework.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87 , 325–341. No claim to original US government works.     

Postmetamorphic ontogenetic allometry and the evolution of skull shape in Nest‐building frogs Leptodactylus (Anura: Leptodactylidae)

Allometry constitutes an important source of morphological variation. However, its influence in head development in anurans has been poorly explored. By using geometric morphometrics followed by statistical and comparative methods we analyzed patterns of allometric change during cranial postmetamorphic ontogeny in species of Nest‐building frogs Leptodactylus (Leptodactylidae). We found that the anuran skull is not a static structure, and allometry plays an important role in defining its shape in this group. Similar to other groups with biphasic life‐cycle, and following a general trend in vertebrates, ontogenetic changes mostly involve rearrangement in rostral, otoccipital, and suspensorium regions. Ontogenetic transformations are paralleled by shape changes associated with evolutionary change in size, such that the skulls of species of different intrageneric groups are scaled to each other, and small and large species show patterns of paedomorphic/peramorphic features, respectively. Allometric trajectories producing those phenotypes are highly evolvable though, with shape change direction and magnitude varying widely among clades, and irrespective of changes in absolute body size. These results reinforce the importance of large‐scale comparisons of growth patterns to understand the plasticity, evolution, and polarity of morphological changes in different clades.     

Take time to smell the frogs: vocal sac glands of reed frogs (Anura: Hyperoliidae) contain species‐specific chemical cocktails

Males of all reed frog species (Anura: Hyperoliidae) have a prominent, often colourful, gular patch on their vocal sac, which is particularly conspicuous once the vocal sac is inflated. Although the presence, shape, and form of the gular patch are well‐known diagnostic characters for these frogs, its function remains unknown. By integrating biochemical and histological methods, we found strong evidence that the gular patch is a gland producing volatile compounds, which might be emitted while calling. Volatile compounds were confirmed by gas chromatography–mass spectrometry in the gular glands in 11 species of the hyperoliid genera Afrixalus, Heterixalus, Hyperolius, and Phlyctimantis. Comparing the gular gland contents of 17 specimens of four sympatric Hyperolius species yielded a large variety of 65 compounds in species‐specific combinations. We suggest that reed frogs might use a complex combination of at least acoustic and chemical signals in species recognition and mate choice. © 2013 The Authors. Biological Journal of the Linnean Society published by John Wiley & Sons Ltd on behalf of The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 828–838.     

Fine structure of the spermatozoon of Marthasterias glacialis (Linnaeus) (Echinodermata; Asteroidea), with special reference to acrosomal morphology

The sperm of Marthasterias glacialis (Linnaeus) was studied by light and electron microscopy. It is a long uniflagellated cell of the “primitive” type. The head has a spherical shape and contains a nucleus with a spheroid acrosome lying in a cup-shaped anterior fossa. The acrosome is formed by an acrosomal vesicle surrounded by the periacrosomal material. The basal specializations of the acrosomal vesicle show a clear differentiation of its constituents resembling the structure of membrane. The midpiece contains a very large annular mitochondrion which encircles two perpendicular centrioles. The distal centriole is in close association with a pericentriolar radial complex. The tail, containing a common microtubular axoneme, is projected to a variable position.     

Molecular phylogeny and biogeography of West Indian frogs of the genus Leptodactylus (Anura, Leptodactylidae)

Three endemic species of the aquatic-breeding frog genus Leptodactylus are recognized from the West Indies: Leptodactylus albilabris (Puerto Rico and the Virgin Islands), Leptodactylus dominicensis (Hispaniola), and Leptodactylus fallax (Lesser Antilles). DNA sequences were obtained from several mitochondrial genes to resolve taxonomic questions involving these species and to provide insights into their origin and distribution in the islands. We found low levels of sequence divergence between L. dominicensis and L. albilabris, supporting morphological evidence that the former species is a junior synonym of the latter species. Phylogenetic analysis supported previous species-group allocations, finding that L. albilabris is a member of the fuscus group and L. fallax is a member of the pentadactylus group. Molecular time estimates for the divergence of L. albilabris from its closest relative in South America (24-58 million years ago, Ma) and for L. fallax from its closest relative in South America (23-34Ma) indicate that they colonized the West Indies independently by over-water dispersal in the mid-Cenozoic. The absence of detectable sequence divergence between the two extant populations of L. fallax (Dominica and Montserrat), a species used for human food and now critically endangered, suggests that one or both arose by human introduction from an island or islands where that species originated. The relatively minor genetic differentiation of populations of L. albilabris can be explained by vicariance and dispersal in the Pleistocene and Holocene, although human introduction of some populations cannot be ruled out.     

Fine structure of the spermatozoon in the mite Parasitus niveus (Mesostigmata,Acari)

The spermatozoa of the mite, Parasitus niveus, are rod-shaped cells possessing a very elongated and zig-zag shaped nucleus. The cytoplasm is filled by so-called “striated bodies” and mitochondria. The plasmalemma forms five complicated structures, called stiff bands. In the peripheral cytoplasm lie flattened canaliculi and flattened cisternae. The morphology of the spermatozoa is compared with that of other mite spermatozoa described in the literature.     

First record of Trypanosoma chattoni in Brazil and occurrence of other Trypanosoma species in Brazilian frogs (Anura, Leptodactylidae)

The present study provides the first record of Trypanosoma chattoni Mathis and Leger, 1911, in a new host, Leptodactylus fuscus Schneider, 1799 (Anura, Leptodactylidae), and the occurrence of Trypanosoma rotatorium-like species in Leptodactylus chaquensis Cei, 1950. The anurans were captured in the State of Mato Grosso, Brazil. Blood samples were obtained by cardiac puncture, and blood smears were examined for the presence of hemoparasites. The Trypanosoma rotatorium-like species in this study refers to a short-bodied trypomastigote that has a conspicuous undulating membrane but lacks a free flagellum; T. chattoni refers to a monomorphic parasite that has a rounded body, a kinetoplast adjacent to the nucleus, and a short flagellum.     

Comparative analysis of the sperm ultrastructure of three species of Phyllomedusa (Anura, Hylidae)

We describe the sperm ultrastructure of three species of frogs in the genus Phyllomedusa . According to micrographs, total size of the spermatozoon of Phyllomedusa hypochondrialis is significantly smaller than those of Phyllomedusa bicolor and Phyllomedusa tarsius . The acrosome complex consists of two conical structures covering the nucleus, the acrosome vesicle and the subacrosomal cone. The subacrosomal cone of P. bicolor and P. tarsius is less electron-dense and appears more granular in transverse section than in P. hypochondrialis . In P. bicolor and P. tarsius , the nuclear space is reduced and the subacrosomal cone fills most of the space between the acrosome vesicle and nucleus. The anterior region of the nucleus in the spermatozoa of P. bicolor and P. tarsius ends abruptly, while in P . hypochondrialis it is sharp-ended. In P. bicolor and P. tarsius , the axial fibre is much larger than in P . hypochondrialis . The sperm ultrastructure of Phyllomedusa appears conservative at the intrageneric level. Future studies on the sperm ultrastructure of hylids can provide new insights on the systematics of the group and a larger database for a cladistic analysis.     

Sperm ultrastructure of the Brazilian Amazon poison frogs Epipedobates trivittatus and Epipedobates hahneli (Anura, Dendrobatidae)

The sperm ultrastructure of Epipedobates trivittatus and E. hahneli is described. The spermatological characteristics are identical in both species, except for the extension of the acrosomal vesicle. The structure of the acrosomal complex is shared with species of the Bufonoidea neobatrachian lineage, which differs from the condition observed in species included in the ranoid lineage. The phylogenetic significance of the mitochondrial collar, also seen in Eubufonoidea groups, is still unclear. Also the significance of the alternative mitochondrial arrangement seen here is unclear. The perpendicular arrangement of the centrioles is also shared with the Bufonoidea and is considered a synapomorphic condition. An expanded undulating membrane and the absence of a juxtaxonemal fibre appear to be apomorphic characteristics of dendrobatid species, as also observed (homoplastically) for species of Litoria and Cyclorana. However, the notable expansion of the undulating membrane appears to be an autapomorphy of Dendrobatidae frogs. The ultrastructural data presented here do not support the proposed placement of E. trivittatus in a separate genus.     

Serous cutaneous glands in the paludiculine frog Physalaemus biligonigerus (Anura, Leptodactylidae): patterns of cytodifferentiation and secretory activity in premetamorphic specimens

The development of serous cutaneous glands is described in tadpoles of the leptodactylid frog Physalaemus biligonigerus , with attention to the cytodifferentiation processes of the secretory unit (adenomere). Secretory differentiation causes the adenomere to assume the structure of a syncytium and triggers biosynthesis of the serous product (poison), consisting of granules with peculiar repeating substructure. The secretory granules resemble glomerular-like aggregates of randomly orientated tubular subunits. This complex substructure derives from the rearrangement of the dense, compact material released by the Golgi apparatus in the form of small, membrane-bounded particles. Both early and advanced features of P. biligonigerus poison closely resemble those found during serous gland development in the Italian treefrog Hyla intermedia . The similar aspects described during serous biosynthesis in these leptodactylid and hylid frogs suggest they are phylogenetically informative. On the other hand, the peculiar features of their poisons may reflect the differential patternings of their secretory repertories along the divergent phylogenetic paths, which led these frogs to be currently included in different families.