The karyotype of three Brazilian Terrarana frogs (Amphibia, Anura) with evidence of a new Barycholos species

A recent substantial rearrangement of the 882 described eleutherodactyline frog species has considerably improved the understanding of their systematics. Nevertheless, many taxonomic aspects of the South American eleutherodactyline species remain unknown and require further investigation using morphological, cytogenetic and molecular approaches. In this work, the karyotypes of the Brazilian species Ischnocnema juipoca (Atibaia and Campos do Jordão, SP), Barycholos cf. ternetzi (Uberlândia, MG, and Porto Nacional, TO), and Pristimantis crepitans (Chapada dos Guimarães and São Vicente, MT) were analyzed using Giemsa staining, Ag-NOR labeling, and C-banding techniques. All individuals had a diploid number of 22 chromosomes, but the Fundamental Numbers were different among species. The herein described low chromosome number of Pristimantis crepitans is unique within this genus, suggesting that cytogenetically this species is not closely related either to its congeneric species or to Ischnocnema. In addition, karyotype differences, mainly in the NOR position, clearly distinguished the two Barycholos populations, besides indicating the existence of a so far undescribed species in this genus. A taxonomic review could clarify the systematic position of P. crepitans and verify the hypothetic new Barycholos species.     

Meiotic chromosomes and nucleolar behavior in testicular cells of the grassland spittlebugs Deois flavopicta, Mahanarva fimbriolata and Notozulia entreriana (Hemiptera, Auchenorrhyncha)

Spittlebugs annually infest pastures and cause severe damage, representing a serious problem for the tropical American beef cattle industry. Spittlebugs are an important biotic constraint to forage production and there is a lack of cytogenetic data for this group of insects. For these reasons, we conducted this work, in which the spermatogenesis and nucleolar behavior of Deois flavopicta, Mahanarva fimbriolata and Notozulia entreriana were studied. The males possessed testes in the shape of a "bunch of grapes"; a variable number of testicular lobes per individual and polyploid nuclei composed of several heteropycnotic bodies. A heteropycnotic area was located in the periphery of the nucleus (prophase I); the chiasmata were terminal or interstitial; metaphases I were circular or linear and anaphase showed late migration of the sex chromosome. The chromosome complement had 2n = 19, except for N. entreriana (2n = 15); the spermatids were round with heteropycnotic material in the center and elongated with conspicuos chromatin. The analysis of testes after silver nitrate staining showed polyploid nuclei with three large and three smaller nucleolar bodies. Early prophase cells had an intensely stained nucleolar body located close to the chromatin and another less evident body located away from the chromatin. The nucleolar bodies disintegrated during diplotene. Silver staining occurred in two autosomes, in terminal and subterminal locations, the latter probably corresponding to the nucleolus organizer regions (NORs). The spermatids were round with a round nucleolar body and silver staining was observed in the medial and posterior region of the elongated part of the spermatid head.     

Note on the karyotype and NOR phenotype of leuciscine fish Acanthobrama marmid (Osteichthyes, Cyprinidae)

The karyotype and major ribosomal sites as revealed using silver staining of Anatolian leuciscine cyprinid fish Acanthobrama marmid were studied. The diploid chromosome number was invariably 2n = 50. Karyotype consisted of eight pairs of metacentric, 13 pairs of submetacentric and four pairs of subtelocentric to acrocentric chromosomes. The largest chromosome pair of the complement was subtelo-to acrocentric characteristically, which is a characteristic cytotaxonomic marker for representatives of the cyprinid lineage Leuciscinae. The nucleolar organizer regions (NORs) were detected in the telomeres of two pairs of medium sized submeta-to subtelocentric chromosomes. No heteromorphic sex chromosomes were found. The karyotype pattern of A. marmid is nearly identical to that found in most other representatives of the Eurasian leuciscine cyprinids, while the multiple NOR phenotype appears to be more derived as opposed to a uniform one, ubiquitous in this group.     

Assessing Genetic Diversity of Brazilian Reef Fishes by Chromosomal and DNA Markers

Little is known on genetics of Brazilian coral reef fish and most of this information is limited to chromosome characterization of major representative species. The diploid chromosome number in marine fish varies from 2n= 22–26 to 2n = 240–260. Despite of this apparent diversity, most studied marine species have a diploid complement with 48 acrocentric chromosomes. This latter trend is mostly observed among Perciformes, an important major taxon of coral reef fishes. Studies in the families Pomacentridae, Pomacanthidae and Chaetodontidae, for example, have shown a common karyotype pattern entirely formed by 48 uniarmed chromosomes. However, rare numerical and structural chromosome polymorphisms and cryptic chromosome rearrangements involving heterochromatin segments and/or nucleolar organizing sites have been reported among such fishes. Although new chromosome forms can contribute to the establishment of genetically isolated populations, their role in reef fish speciation at marine realm still is an open question. More recently, genomic DNA analyses using RAPD and microsatellites, and sequencing and RFLP of mitochondrial DNA have increasingly been used in Atlantic reef fish species. Genetic homogeneity over wide geographical ranges has been reported for different fish groups, in contrast to several cases of population substructuring related to environmental constraints or evolutionary history. Amazonas outflow and upwelling on the Southeastern coast of Brazil are believed to be strong barriers to dispersal of some reef species. Moreover, it is suggested that the pattern of speciation and population structure at South Atlantic is quite distinctive from Pacific Ocean, even when comparing closely related taxa. Further genetic studies are strongly encouraged in Brazilian reef fishes in order to provide a reliable scenario of the genetic structure in this important and diverse fish group.     

Laser‐assisted microdissection of polytene chromosomes from Diptera for the development of molecular markers

The structural details visible under the light microscope have made polytene chromosomes from Diptera much used in studies of the taxonomy, evolution and genetics of important biological models such as Drosophila and Chironomus, and the medical entomology of important disease vectors such as blackflies and mosquitoes. This paper describes the isolation of sections of polytene chromosomes from preserved wild‐caught blackflies using off‐the‐shelf laser microdissection microscopy and subsequent analysis of the DNA using polymerase chain reaction. This allows a direct link between the visible structure of the genome and the unknown DNA sequence, facilitating the development of molecular markers for population cytogenetics and cytotaxonomy.     

Differentiated ZZ/ZW sex chromosomes in Apareiodon ibitiensis (Teleostei,Parodontidae): cytotaxonomy and biogeography

Conventional and molecular chromosomal analyses were carried out on three populations of Apareiodon ibitiensis sampled from the hydrographic basins of the São Francisco River and Upper Paraná River (Brazil). The results reveal a conserved diploid number (2n = 54 chromosomes), a karyotype formula consisting of 50 m‐sm + 4st and a ZZ/ZW sex chromosome system that has not been previously identified for the species. C‐banding analysis with propidium iodide staining revealed centromeric and terminal bands located in the chromosomes of the specimens from the three populations and allowed the identification of heteromorphism of heterochromatin regions in the Z and W chromosomes. The number of 18S sites located through fluorescent in situ hybridization (FISH) varied between the populations of the São Francisco and Upper Paraná Rivers. The location of 5S rDNA sites proved comparable in one pair of metacentric chromosomes. Thus, the present study proposes a ZZ/ZW sex chromosome system for A. ibitiensis among the Parodontidae, and a hypothesis is presented regarding possible W chromosome differentiation stages in this species through DNA accumulation, showing geographical variations for this characteristic, possibly as a consequence of geographical reproductive isolation.     

Conserved ZZ/ZW sex chromosomes in Caribbean croaking geckos (Aristelliger: Sphaerodactylidae)

Current understanding of sex chromosome evolution is largely dependent on species with highly degenerated, heteromorphic sex chromosomes, but by studying species with recently evolved or morphologically indistinct sex chromosomes we can greatly increase our understanding of sex chromosome origins, degeneration and turnover. Here, we examine sex chromosome evolution and stability in the gecko genus Aristelliger. We used RADseq to identify sex‐specific markers and show that four Aristelliger species, spanning the phylogenetic breadth of the genus, share a conserved ZZ/ZW system syntenic with avian chromosome 2. These conserved sex chromosomes contrast with many other gecko sex chromosome systems by showing a degree of stability among a group known for its dynamic sex‐determining mechanisms. Cytogenetic data from A. expectatus revealed homomorphic sex chromosomes with an accumulation of repetitive elements on the W chromosome. Taken together, the large number of female‐specific A. praesignis RAD markers and the accumulation of repetitive DNA on the A. expectatus W karyotype suggest that the Z and W chromosomes are highly differentiated despite their overall morphological similarity. We discuss this paradoxical situation and suggest that it may, in fact, be common in many animal species.     

Genome evolution in a Mediterranean species complex: phylogeny and cytogenetics of Helictotrichon (Poaceae) allopolyploids based on nuclear DNA sequences (rDNA,topoisomerase gene) and FISH

We examined the molecular phylogeny and chromosomal features of European Helictotrichon species to explore the relationships within the genus and to investigate the origin of several polyploids. Using both approaches, molecular and cytogenetic, revealed the strong impact of allopolyploidization on genome organization from chromosome structure to sequence level. Our research focused on Mediterranean and endemic species of the Alps. Altogether, the molecular phylogenetic analyses include a sample of 17 Helictotrichon species and subspecies, used DNA sequences from the nuclear ribosomal (nr) internal transcribed spacer region (ITS) and the single copy gene topoisomerase 6 (Topo6), and were analysed by maximum parsimony and Bayesian methods. Karyotype structures were investigated by fluorescence in situ hybridization (FISH) and fluorochrome banding. Cytogenetic characters were mapped on the combined phylogenetic tree. The absence or comparatively rare occurrence of different ITS sequence types in some (allo-) polyploid species of Helictotrichon suggests frequent intergenomic homogenization of ribosomal DNA (rDNA) loci due to the phenomenon of concerted evolution. This result implies that the ITS region is not an ideal marker to study polyploid evolution of these grasses. The phylogenetic analysis of the Topo6 region revealed three major clades that concur with three different copy types (termed SAR, SET, PAR), representing the major genome groups in Helictotrichon. A comparison of the molecular phylogenetic trees with the chromosome and karyotype structure supports allopolyploidy of several Helictotrichon species and identifies potential genome donors. A correlation between molecular phylogenetic/cytogenetic results and geographic distribution is expressed by a west-east disjunction, in the narrower or wider sense, of the analysed species. While SAR represents a geographically narrowly distributed southwest Mediterranean genome group, PAR and SET are very widespread (Mediterranean to Asia) and encompass several instances of west-east disjunctions.     

Cytogenetic characterisation of the Caspian Pond Turtle,Mauremys caspica in Golestan and Mazandaran provinces,Iran (Reptilia: Testudines)

Cytogenetic characteristics of the Caspian Pond Turtle, Mauremys caspica, in Golestan and Mazandaran provinces in the northern part of Iran show that the chromosome number is 2n?=?52 and the arm number NF?=?78. The karyotype consisted of 9 metacentric (M), 1 submetacentric (SM), 3 subtelocentric (ST) and 13 telocentric (T) chromosome pairs. The Centromeric Index ranges from 11.79 to 45.68, the arm ratio between 1.18 and 7.47, the relative length between 1.60 and 11.46, and the length variation between 1.05 and 7.48. Average total length of the chromosomes is 65.27 µm. The largest chromosome in this species is a pair of the metacentric chromosome. Location of NOR was determined on chromosome pair no. 10.     

The role of the Giemsa stain in cytogenetics

Abstract

In just half a century since the human diploid chromosome number was correctly identified as 46, there has been a rapid expansion in our understanding of both the genetic foundation of normal human development and the development of various constitutional and acquired abnormalities. The ability to detect numerical and structural chromosomal abnormalities was made possible by the Giemsa stain. Despite the recent advent of powerful molecular-based cytogenetic techniques (e.g., fluorescence in situ hybridization, array-based comparative genomic hybridization), Giemsa-based chromosomal banding and staining techniques retain their crucial role in cytogenetics.