Chromosome banding in Amphibia. XXVI. Coexistence of homomorphic XY sex chromosomes and a derived Y-autosome translocation in Eleutherodactylus maussi (Anura,Leptodactylidae)

A 15-year cytogenetic survey on one population of the leaf litter frog Eleutherodactylus maussi in northern Venezuela confirmed the existence of multiple XXAA male symbol /XAA(Y) female symbol sex chromosomes which originated by a centric (Robertsonian) fusion between the original Y chromosome and an autosome. 95% of the male individuals in this population are carriers of this Y-autosome fusion. In male meiosis the XAA(Y) sex chromosomes pair in the expected trivalent configuration. In the same population, 5% of the male animals still possess the original, free XY sex chromosomes. In a second population of E. maussi analyzed, all male specimens are characterized by these ancestral XY chromosomes which form normal bivalents in meiosis. E. maussi apparently represents the first vertebrate species discovered in which a derived Y-autosome fusion still coexists with the ancestral free XY sex chromosomes. The free XY sex chromosomes, as well as the multiple XA(Y) sex chromosomes are still in a very primitive (homomorphic) stage of differentiation. With no banding technique applied it is possible to distinguish the Y from the X. DNA flow cytometric measurements show that the genome of E. maussi is among the largest in the anuran family Leptodactylidae. The present study also supplies further data on differential chromosome banding and fluorescence in situ hybridization experiments in this amphibian species.     

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.     

Chromosome banding in Amphibia. XXII. Atypical y chromosomes in Gastrotheca walkeri and Gastrotheca ovifera (Anura,Hylidae)

The chromosomes of the rare South American marsupial frogs Gastrotheca walkeri and G. ovifera were extensively reexamined with various banding techniques. The karyotypes of both species are distinguished by a new category of XY female symbol /XX male symbol female sex chromosomes. The unusual Y chromosomes are characterized by containing the least amount of constitutive heterochromatin in the karyotypes. This is in contrast to all previously known amphibian Y chromosomes and does not fit the evolutionary model of early XY differentiation in vertebrates. In male meiosis, the heteromorphic XY chromosomes of both species still exhibit the same pairing configurations as the autosomes. DNA flow cytometric measurements show the nuclear DNA amount of G. walkeri to be 10.90 pg. The significance of the XY/XX sex chromosomes of these marsupial frogs, the various classes of constitutive heterochromatin detected, and the data obtained from meiotic analyses are discussed in detail.     

Chromosome banding in Amphibia. XV. Two types of Y chromosomes and heterochromatin hypervariabilty inGastrotheca pseustes (Anura,Hylidae)

The chromosomes of the newly discovered South American marsupial frogGastrotheca pseustes were analyzed by conventional methods and by various banding techniques. This species is characterized by XY/XX sex chromosomes and the existence of two different morphs of Y chromosomes. Whereas in type A males the XY^A chromosomes are still homomorphic, in type B males the Y^B chromosome displays a large heterochromatic region at the long arm telomere which is absent in the X. In male meiosis, the homomorphic XY^A chromosomes exhibit the same pairing configuration as the autosomal bivalents. On the other hand, the heteromorphic XY^B chromosomes form a sex bivalent by pairing their short arm telomeres in a characteristic end-to-end arrangement. Analysis of the karyotypes by C-banding and DNA base pair-specific fluorochromes reveals enormous interindividual size variability of the autosomal heterochromatin.     

Chromosome banding in Amphibia. XXV. Karyotype evolution and heterochromatin characterization in Australian Mixophyes (Anura,Myobatrachidae)

The mitotic chromosomes of the Australian ground frogs Mixophyes fasciolatus and M. schevilli were analyzed by means of banding techniques and restriction endonuclease digestions. Chromosomal differentiation in these two species occurred exclusively by considerable changes in the amount of telomeric and centromeric heterochromatin, whereas the sizes and locations of interstitial heterochromatic regions, the sizes of all euchromatic segments as well as the positions of centromeres remained nearly identical during karyotype evolution. The major heterochromatic regions in the karyotypes of M. fasciolatus and M. schevilli amount to 30.2% and 20.7%, respectively. They consist of AT base pair-rich repetitive DNA sequences that are brightly labeled by AT-specific fluorochromes and display quenched fluorescence after staining with GC-specific fluorochromes. The heterochromatic regions can be differentiated by treatment of metaphase chromosomes and interphase cell nuclei with various restriction enzymes which either disclose the complete set of C-band patterns in the karyotypes of both species, or else reveal several subsets of these C-bands.     

Cranial ontogeny in the direct-developing frog, Eleutherodactylus coqui (Anura: Leptodactylidae), analyzed using whole-mount immunohistochemistry.

Direct development in amphibians is an evolutionarily derived life-history mode that involves the loss of the free-living, aquatic larval stage. We examined embryos of the direct-developing anuran Eleutherodactylus coqui (Leptodactylidae) to evaluate how the biphasic pattern of cranial ontogeny of metamorphosing species has been modified in the evolution of direct development in this lineage. We employed whole-mount immunohistochemistry using a monoclonal antibody against the extracellular matrix component Type II collagen, which allows visualization of the morphology of cartilages earlier and more effectively than traditional histological procedures; these latter procedures were also used where appropriate. This represents the first time that initial chondrogenic stages of cranial development of any vertebrate have been depicted in whole-mounts. Many cranial cartilages typical of larval anurans, e.g., suprarostrals, cornua trabeculae, never form in Eleutherodactylus coqui. Consequently, many regions of the skull assume an adult, or postmetamorphic, morphology from the inception of their development. Other components, e.g., the lower jaw, jaw suspensorium, and the hyobranchial skeleton, initially assume a mid-metamorphic configuration, which is subsequently remodeled before hatching. Thirteen of the adult complement of 17 bones form in the embryo, beginning with two bones of the jaw and jaw suspensorium, the angulosplenial and squamosal. Precocious ossification of these and other jaw elements is an evolutionarily derived feature not found in metamorphosing anurans, but shared with some direct-developing caecilians. Thus, in Eleutherodactylus cranial development involves both recapitulation and repatterning of the ancestral metamorphic ontogeny.(ABSTRACT TRUNCATED AT 250 WORDS)     

.Chromosome sex determination and Y-autosome fusion in Blennius tentacularis Brunnich, 1765 (Pisces, Blennidae)

Diploid modal numbers of 2n =48 for females, and 2 n =48 and 2 n =47 for males of Blennius tentacularis caught in the Gulf of Palermo (Sicily) are established. Chromosome sex-determination is proposed for this species in which a Y-autosome fusion has been found.     

The late replication banding patterns of chromosomes are highly conserved in the genera Rana,Hyla, and Bufo (Amphibia: Anura)

Late replication banding and C-banding analyses were performed on the metaphase chromosomes of six species and one subspecies of Palearctic water frogs, genus Rana. Although C-banding patterns showed interspecific or intersubspecific variation, late replication banding patterns of all 13 chromosome pairs of these species were homologous. Minor differences of banding patterns were observed only in chromosomes 2, 7 and 13. Close comparison of the late replication banding patterns with those of three non-water frog species of Rana, and one each of Hyla and Bufo, provided important information on interspecific and intergeneric variability. In the Rana species, the banding patterns of all 13 pairs were homologous except for those some regions of 8 pairs. In one species each of Hyla and Bufo that was examined, the six large chromosome pairs (Nos. 1-6) showed banding homologies. Furthermore, among the Rana, Hyla and Bufo species the four large chromosome pairs (Nos. 1-3, 5 of Rana and Hyla, and Nos. 1, 3–5 of Bufo) shared banding homologies. These results show that the large chromosomes have been highly conserved in the evolutionary history of the three genera.     

Spermiogenesis in Odontophrynus cultripes (Amphibia,Anura, Leptodactylidae): Ultrastructural and cytochemical studies of proteins using E-PTA

Spermiogenesis in the South American leptodactylid frog Odontophrynus cultripes was analyzed ultrastructurally. The spermatids undergo morphological modification while still enclosed in microtubule-rich processes of Sertoli cells. Electron-dense plates resembling junctional structures appear in regions at which the spermatids lie in close contact with the surface of Sertoli cell processes. Spermatid differentiation can be divided into five distinct stages based mainly on chromatin condensation. In the late stages, the densely compacted chromatin loses reactivity to ethanolic phosphotungstic acid (E-PTA). Helical arrangements of microtubules appear in the cytoplasm that surrounds the spermatid nucleus after the second stage. The acrosomal vesicle differentiates into a cone-shaped acrosome that caps the anterior region of the nucleus. The connecting piece, located in the flagellum implantation zone, has transverse striations, and is continuous with the axial rod. The tail is formed by a 9 + 2 axoneme, an undulating membrane, and an axial rod that is rich in basic proteins as demonstrated by E-PTA staining.     

Chromosome banding in Cacopsylla mali (Schmidberger) and Cacopsylla sorbi (Linnaeus) (Homoptera, Psyllidae) with polymorphic sex chromosomes

C-banding and Ag staining were applied to Cacopsylla sorbi and C. mali. The aim was to discover some additional cytological markers to follow the pathways of the sex determination system transformation in the evolution of the species. Of three karyotype patterns so far described in the literature for these species--a type A (XO), B (neoXY) and C (neoX1X2Y), only the last two types (B and C) were found. All 6 studied Cacopsylla sorbi from Finland had a karyotype of type B (2n = 20 + XY), while C. mali had both types. Type B (2n = 22 + XY) was observed in 31 males, whereas type C (2n = 20 + neoX1X2Y) in the remaining four. The karyotype of C. sorbi was found to be characterized by a very small amount of C-positive material, localized in a telomere of the Y chromosome. The karyotype of C. mali was also characterized by a very small amount of C-banded material. Both the sex chromosomes and the autosomes displayed a marked polymorphism of C-positive bands within different individuals and even the same individual. In both species the nucleolus was located in the telomere of a middle sized autosomal till diplotene inclusive. The C-banding and Ag staining in the studied Cacopsylla species did not provide any additional cytological markers for an understanding of the pathways of sex determination system transformation in the evolution of the species.     

Cytological evidence for population-specific sex chromosome heteromorphism in Palaearctic green toads (Amphibia,Anura)

A chromosome study was carried out on a number of European and Central Asiatic diploid green toad populations by means of standard and various other chromosome banding and staining methods (Ag-NOR-, Q-, CMA₃-, late replicating [LR] banding pattern, C- and sequential C-banding + CMA₃ + DAPI). This study revealed the remarkable karyological uniformity of specimens from all populations, with the only exception being specimens from a Moldavian population, where one chromosome pair was heteromorphic. Though similar in shape, size and with an identical heterochromatin distribution, the difference in the heteromorphic pair was due to a large inverted segment on its long arms. This heteromorphism was restricted to females, suggesting a female heterogametic sex chromosome system of ZZ/ZW type at a very early step of differentiation.     

Heteromorphic Z and W sex chromosomes in Physalaemus ephippifer (Steindachner, 1864) (Anura, Leiuperidae)

Heteromorphisms between sex chromosomes are rarely found in anurans and sex chromosome differentiation is considered to be a set of recent recurrent events in the evolutionary history of this group. This paper describes for the first time heteromorphic sex chromosomes Z and W in the leiuperid genus Physalaemus. They were found in P. ephippifer, a species of the P. cuvieri group, and corresponded to the eighth pair of its karyotype. The W chromosome differed from the Z chromosome by the presence of an additional segment in the short arm, composed of a distal NOR and an adjacent terminal DAPI-positive C-band. The identification of this sex chromosome pair may help in future investigations into the sex determining genes in the genus Physalaemus.     

Evolution of reproduction in the Rhacophoridae (Amphibia, Anura)

Rhacophorid treefrogs have different reproductive modes: some go through a tadpole stage and some have direct development, and the adults of some species produce foam nests. Philautus is the only genus characterized by direct development. The production of foam nests has been reported in the genera Polypedates, Rhacophorus, Chiromantis and Chirixalus. Recent molecular studies did not provide a robust hypothesis concerning the origin of these reproductive modes in the Rhacophoridae. In order to better understand the evolution of these reproductive modes, we tried to clarify relationships within this group, using DNA sequencing. Our data set consists in a large number of new sequences (1676 base pairs corresponding to threee genes) for five outgroup ranoids and 48 Rhacophoridae, including 16 undescribed species from Sri Lanka and southern India, and all homologous data available in Genbank. After the inclusion of Philautus from India, our data show that the separation of Philautus into clades does not coincide with their geographic distribution. Our data point to the existence of a clade, including the genera Rhacophorus, Polypedates, Chiromantis and Chirixalus, which confirms the results of Wilkinson et al. (2002) and suggests that the ability to produce foam nests has emerged only once in the Rhacophoridae, as already stated by these authors.     

Chromosomal rearrangements as the source of variation in the number of chromosomes in Pseudis (Amphibia, Anura)

A cytogenetic study of Pseudis specimens from three localities in Rio Grande do Sul State, the southernmost Brazilian, was performed to identify karyotypic characteristics that could account for differences in vocalization pattern and body size. Individuals from around Tainhas were compared to those of São Jerônimo and Eldorado do Sul. Specimens from these latter two localities were identified as Pseudis minuta, while those from the former were classified as Pseudis sp. (aff. minuta). The populations from São Jerônimo and Eldorado do Sul had 2n?=?24 chromosomes, classified as metacentric, submetacentric and subtelocentric. The population from Tainhas had 2n?=?28 chromosomes, with four pairs of telocentric chromosomes. Modelling of these 28 chromosomes and testing for fusion in the centromeric/telomeric regions yielded a karyotype of 2n?=?24 chromosomes, similar to that of the other populations. The similarity was reinforced by the location of the NORs and heterochromatin. The Tainhas population showed an increase in heterochromatin, as seen by the presence of additional C-bands, especially in the telocentric chromosomes. These data suggest that the two karyotypes described in this work had a common ancestry. There is evidence that the differentiation of these karyotypes may have occurred by chromosome fission and heterochromatin addition. Based on the present karyotype (2n?=?28) and on morphological and vocalization studies by other researchers, we conclude that the Tainhas population may represent a new species.     

Chromosome painting of Z and W sex chromosomes in Characidium (Characiformes, Crenuchidae)

Some species of the genus Characidium have heteromorphic ZZ/ZW sex chromosomes with a totally heterochromatic W chromosome. Methods for chromosome microdissection associated with chromosome painting have become important tools for cytogenetic studies in Neotropical fish. In Characidium cf. fasciatum, the Z chromosome contains a pericentromeric heterochromatin block, whereas the W chromosome is completely heterochromatic. Therefore, a probe was produced from the W chromosome through microdissection and degenerate oligonucleotide-primed polymerase chain reaction amplification. FISH was performed using the W probe on the chromosomes of specimens of this species. This revealed expressive marks in the pericentromeric region of the Z chromosome as well as a completely painted W chromosome. When applying the same probe on chromosome preparations of C. cf. gomesi and Characidium sp., a pattern similar to C. cf. fasciatum was found, while C. cf. zebra, C. cf. lagosantense and Crenuchus spilurus species showed no hybridization signals. Structural changes in the chromosomes of an ancestral sexual system in the group that includes the species C. cf. gomesi, C. cf. fasciatum and Characidium sp., could have contributed to the process of speciation and could represent a causal mechanism of chromosomal diversification in this group. The heterochromatinization process possibly began in homomorphic and homologous chromosomes of an ancestral form, and this process could have given rise to the current patterns found in the species with sex chromosome heteromorphism.     

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     

Origins and introductions of the Caribbean frog, Eleutherodactylus johnstonei (Leptodactylidae): management and conservation concerns

Johnstone's Whistling Frog, Eleutherodactylus johnstonei, is a highly successful colonizer that has become widely distributed throughout the Caribbean region. It has been introduced both purposefully and unintentionally by humans, and it continues to expand its range locally and regionally. Its current distribution and recent expansion do not support the hypothesis that E. johnstonei is expanding into new habitats exclusively by outcompeting native species. Instead, its range expansion progresses mainly parallel to human expansion (habitat disturbance through land development) and extreme climatic events (habitat disturbance through hurricanes and volcanism). Once a habitat has been disturbed and E. johnstonei has arrived, any previously existing endemic Eleutherodactylus species tend not to be found again at their previous ranges or population densities. The most probable explanation for this is that the broader physiological tolerance of the ecological generalist E. johnstonei allows it to become permanently established in a disturbed biota, whereas ecologically specialized endemics are prevented from recolonizing such habitats. Invasion of E. johnstonei can result in a parapatric distribution with endemics (e.g. E. euphronides, E. shrevei) or in sympatry (e.g. E. martinicensis), and habitats include areas with widely divergent climatic conditions (e.g. xeric: Anguilla, Barbuda; mesic: Grenada, St Vincent). Management for this species includes prevention of further or repeat introductions, close monitoring of ranges, and preservation of native habitats to ensure survival of local endemics.     

Habitat selection in tadpoles of Ranidella signifera and R. riparia (Anura: Leptodactylidae)

Summary Two leptodactylid frog species Ranidella signifera and R. riparia occupy adjacent but different habitat types in the Flinders Ranges of South Australia. R. signifera is found in slow-flowing creeks with mud or sand substrates. R. riparia occupies fast-flowing rocky creeks. The two coexist in a narrow overlap zone with heterogeneous habitats. Laboratory experiments were used to test habitat preferences of tadpoles in a tub with sand and rock substrates. R. riparia was most commonly found on the rock substrate. R. signifera showed no distinct preference for sand, but when mixed with R. riparia increased its use of the habitat in the spaces between and under rocks. This was particularly apparent in flowing water where 81% of all R. signifera tadpoles were observed in this sheltered habitat.The data suggest that R. signifera tadpoles have reduced fitness in flowing water because they cannot exploit exposed feeding sites where most of the algae grow, and that interspecific interactions with R. riparia tadpoles reduce their fitness further. This helps to explain why R. signifera do not extend their distribution into R. riparia occupied creeks. The data do not explain why R. riparia do not colonize the slow muddy creeks occupied by R. signifera.