Characterization of G-banded chromosomes of a female saola (Pseudoryx nghetinhensis,2n = 50) and X chromosome i dentification by means of fluorescent in situ hybridization

The saola (Pseudoryx nghetinhensis) is a newly discovered large mammal species, belongs to the subfamily Bovinae and is listed as being endangered. Due to the limitation of the material available, no cytogenetic studies have been carried out on this species. In the present study, preliminary cytogenetic analysis was undertaken on cultured female fibroblast cells to characterize the karyotype organization of saola. An examination of 120 Giemsa stained metaphases showed the diploid chromosome number of 2n = 50, including five bi-armed chromosome pairs. The distribution of constitutive heterochromatin in saola was studied. However, the variability in the size of C-bands was not significant on all the homologous chromosomes. The X chromosome pair, corresponding to the largest telocentric chromosomes, was identified by fluorescent in situ hybridization (FISH) using a bacterial artificial chromosome clone (BAC 0577G05, which maps to BTAXq25-->q33). In comparison to the standard karyotype of cattle (ISCNDB 2000), a G-banded ideogram of saola (about 390 band level) was presented. This work, therefore, provided a basic insight into the karyotype organization of this endangered species and will be particularly useful to improve the understanding of differences of genomes between related species.     

挂榜山小鲵的核型(有尾目:小鲵科)

本文首次报道了挂榜山小鲵Hynobius guabangshanensis 的核型.其2n=56,分为4组,其中9对大型染色体,4对双臂中型染色体,5对双臂小型染色体和10对单臂小型染色体.挂榜山小鲵具有池塘类型小鲵的特征,即2n=56以及缺少中端着丝粒染色体,这与挂榜山小鲵的生态和形态特征也属池塘类型相符.     

Karyotype of a Japanese salamander Hynobius katoi and its implication on breeding ecology (Amphibia: Caudata)

Karyotype of a Japanese small salamander, Hynobius katoi, was first described. All individuals examined had 2n=58 chromosomes, consisting of nine pairs of biarmed macrochromosomes, four pairs of biarmed medium-sized chromosomes, six pairs of biarmed microchromosomes, and 10 pairs of uniarmed microchromosomes, although distinction of the second and the third groups of chromosome pairs was not clear. All pairs appeared homologous and no sexual dimorphism was found. Possession of 2n=58 chromosomes in H. katoi strongly suggests its lotic-breeding habits as was expected from the number and size of eggs and adult morphology. When compared morphology of chromosomes among lotic-breeders with 2n=58 chromosomes, metacentric nature of No. 10 seems to characterize the karyotype of H. katoi.     

Meiotic chromosomes of the tree frog Smilisca baudinii (Anura: Hylidae)

The Mexican tree frog Smilisca baudinii, is a very common frog in Central America. In spite their importance to keep the ecological equilibrium of the rainforest, its biology and genetics are poorly known. In order to contribute with its biological knowledge, we described the typical meiotic karyotype based in standard cytogenetic protocols to specimens collected in Tabasco, Mexico. The study was centered in the analysis of 131 chromosome spreads at meiotic stage from two adults of the species (one female and one male). The metaphase analysis allowed the establishment of the modal haploid number of 1n = 12 bivalent chromosomes. The chromosomic formulae from the haploid bivalent karyotype was integrated by 12 biarmed chromosomes characterized by twelve pairs of metacentric-submetacentric (msm) chromosomes. The meiotic counting gives the idea that diploid chromosome number is integrated by a complement of 2n = 24 biarmed chromosomes. The presence of sex chromosomes from female and male meiotic spreads was not observed. Current results suggest that S. baudinii chromosome structure is well shared among Hylidae family and "B" chromosomes are particular structures that have very important evolutionary consequences in species diversification.     

A new chromosomal race (2n=44) of Nannospalax xanthodon from Turkey (Mammalia: Rodentia)

A new karyotype for blind mole rats was recorded in Tunceli province in Eastern Turkey. The karyotype contained 44 chromosomes, including 13 biarmed pairs, 7 acrocentric pairs, and one heteromorphic pair with a submetacentric and an acrocentric homologue in the autosomal complement (FNa=69). The X chromosome was submetacentric and the Y chromosome medium-sized subtelocentric (FN=73). Distinct dark centromeric C-bands were observed on most of the biarmed and three pairs of the acrocentric autosomes. The NORs were detected on short arms of three subtelocentric pairs and one acrocentric pair of autosomes. The diploid number of chromosomes and the karyotype characteristics observed are obviously unique among hitherto studied populations of blind mole rats and the complement can be evaluated as a new chromosome race of Nannospalax xanthodon. The distribution ranges of individual chromosome races of the species recorded in Eastern Anatolia are revised and possible interracial hybridization is discussed in respect of the finding of a new race.     

Cytogenetic study of Callicebus hoffmannsii (Cebidae, Primates) and comparison with C. m. moloch.

Callicebus is a neotropical primate genus divided into four or five groups of species. Species of the moloch group are distributed in the tropical forests of the Amazon basin. The karyotype of Callicebus hoffmannsii (moloch group) was studied by means of G- and C-banding, Ag-NOR staining and in situ hybridization of telomeric probes. C. hoffmannsii had 2n = 50 chromosomes, with ten biarmed and fourteen acrocentric autosomal pairs. The X chromosome was submetacentric and the Y chromosome was a minor acrocentric. Constitutive heterochromatin was detected in the centromeric regions of all chromosomes; in pairs 7 and 10, it was found in the distal regions of the short arms, and distally in the long arm of the X chromosome. Size heteromorphism in C-bands was detected in pairs 7 and 10. Ag-NOR staining revealed a maximum of three nucleolar organizers. Telomeric probes hybridized only at the terminal regions of all chromosomes. Additionally, a comparison was carried out between C. hoffmannsii and C. m. moloch (2n = 48), as previously reported. Both species shared gross chromosomal similarities diverging by a single rearrangement of centric fusion/fission. A high similarity between C. hoffmannsii and C. donacophilus indicated a close association between the moloch and donacophilus groups.     

Polymorphism of the Sphaerium corneum (Bivalvia, Veneroida, Sphaeriidae) revealed by cytogenetic and sequence comparison

Two populations of Sphaerium corneum were sampled from River Vilnelė and small pond in Vilnius, Lithuania. The chromosomes were studied using conventional Giemsa staining and karyometric analysis. Inter- and intra-individual variation in the diploid chromosome numbers was revealed and two different sources of chromosome variability were identified: B chromosomes and the structural changes of chromosomes of the basic (A) set. The chromosome set of the more common karyotypic form, 2 n  = 30, found in both populations, consists of all biarmed metacentric and meta-submetacentric chromosomes of gradually decreasing size. Small, biarmed, mitotically unstable B chromosomes were found in the cells of this karyotypic form. Specimens with 2 n  = 36 were found only in pond. No B chromosomes were detected in their cells. The karyotype is characterized by presence of two pairs of medium telocentrics and four pairs of small subtelocentrics. The remaining chromosomes are biarmed. Robertsonian fusions appear to be involved in formation of two karyotypic forms of S. corneum . DNA sequence analyses showed that ITS1 is identical in both karyotypic forms. On the other hand, differences in 16S sequence were revealed and two haplotypes, corresponding to two karyotypic forms, were identified. The present study opens new perspectives in establishing species-specific characters for confident identification of Sphaerium species and provides insights to the genetic intraspecific variability and possible mechanisms of speciation.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 89 , 53–64.     

Description of the karyotype of Rhagomys rufescens Thomas, 1886 (Rodentia, Sigmodontinae) from Southern Brazil Atlantic forest

Rhagomys rufescens (Rodentia: Sigmodontinae) is an endemic species of the Atlantic forest from Southern and Southeastern Brazil. Some authors consider Rhagomys as part of the tribe Thomasomyini; but its phylogenetic relationships remain unclear. Chromosomal studies on eight specimens of Rhagomys rufescens revealed a diploid number of 2n = 36 and a number of autosome arms FN = 50. GTG, CBG and Ag-NOR banding and CMA(3) /DAPI staining were performed on metaphase chromosomes. Eight biarmed and nine acrocentric pairs were found in the karyotype of this species. The X and Y chromosomes were both acrocentric. Most of the autosomes and the sex chromosomes showed positive C-bands in the pericentromeric region. The X chromosome showed an additional heterochromatic block in the proximal region of the long arm. Nucleolus organizer regions (NORs) were located in the pericentromeric region of three biarmed autosomes (pairs 4, 6 and 8) and in the telomeric region of the short arm of three acrocentrics (pairs 10, 12 and 17). CMA (3) /DAPI staining produced fluorescent signals in many autosomes, especially in pairs 4, 6, and 8. This study presents cytogenetic data of Rhagomys rufescens for the first time.     

Chromosome conservation in the Bovidae.

The chromosomes of 12 bovid species were harvested from fibroblast cultures after incorporation of bromodeoxyuridine into early replicating DNA. Q-band karyotypes were constructed, and, when possible, autosomal arms were numbered according to the cattle standard karyotype. Diploid chromosome number ranged from 30 to 60, yet, based on band similarity, chromosome-arm homologies were extensive. Employing the cattle karyotype as the standard, autosomal-arm differences indicative of possible syntenic disruption were noted for only chromosomes 3, 9, and 14. While chromosome-arm homologies were extensive, shared homologous biarmed chromosomes were rare. The commonness of monobrachially homologous biarmed chromosomes among some bovids (e.g., Antilopinae) suggested that reproductive isolation and speciation in some instances might have resulted from centric fusion events.     

Citogenetic characterization of the tropical freshwater fish Parachromis managuensis (Pisces: Cichlidae)

To describe the cytogenetics of the jaguar cichlid fish Parachromis managuensis, we collected eight males and 13 females in Villahermosa, Tabasco, México. The specimens were processed with standard cytogenetic techniques (slightly modified), and high quality fields of chromosomes in mitosis and meiosis were obtained; 14 of these fields were analyzed by meristics and statistics methods. The specimens presented a diploid modal number of 2n = 48 chromosomes, which is similar to the number reported for others Central American cichlids; five pairs were submetacentric-metacentrics (biarmed) and 19 were subtelocentric-telocentric (uni-armed), giving a fundamental number (NF) of 58. The haploid number was confirmed by counting meiotic fields in metaphase I. There was not evidence of heteromorphism: sexual chromosomes were not identifiable.     

Chromosomal evolution in the Brazilian lizards of genus Leposoma (Squamata, Gymnophthalmidae) from Amazon and Atlantic rain forests: banding patterns and FISH of telomeric sequences

An extensive karyotype differentiation was found among three species of gymnophthalmid lizard genus Leposoma which occur in the tropical forest areas of Brazil. We examined the chromosomes of the Amazonic species L. guianense (LOU) and L. oswaldoi (LOS) and the Atlantic forest species L. scincoides (LSC) after conventional and differential staining, and FISH of telomeric sequences. Both Amazonic species shared very similar 2n = 44 karyotypes, including 20 biarmed macrochromosomes and 24 microchromosomes (20 M + 24 m). However, the location of Ag-NORs and the amount of constitutive heterochromatin differed in these karyotypes. The Atlantic forest species L. scincoides has a very distinct karyotype with 52 acrocentric and subtelocentric chromosomes of decreasing size. Comparative R-banding analysis revealed complete homeology of the macrochromosomes of LGU and LOS and correspondence of banding patterns between LSC acrocentrics and subtelocentrics and some arms of biarmed LGU and LOS chromosomes. Pair 1 had similar banding patterns in the three species, implying the occurrence of a pericentric inversion. Interstitial telomeric bands (ITBs) detected by FISH at the pericentromeric region of some biarmed LGU and LOS chromosomes could be remnants of chromosomal rearrangements occurred during the differentiation of the karyotypes. Robertsonian rearrangements as well as pericentric inversions events probable were involved in the karyotype evolution of these Amazon and Atlantic forests species of Leposoma.     

A comparative chromosome study of twenty killifish species of the genus Fundulus (Teleostei: Cyprinodontidae)

Female karyotypes from ovarian cell cultures of 20 species of killifish (Fundulus) ranged in diploid number from 32 to 48, but in arm number (NF) from 48 to 52. The small F chromosomes, which constituted the fundamental elements in the karyotype, were evenly graded in length. The large biarmed chromosomes (L), which were about twice the length of the average Fs, characterized only those species with 2N less than 48 chromosomes. And among these species, an increase in complement by a pair of L's was always accompanied by a decrease of two pairs of A's, indicating Robertsonian changes by the centric fusion of two A's to form one L chromosome. Other diagnostic chromosome characters included: the number and structure of biarmed and satellited F chromosomes and the percentage of F's with relatively short short-arms (SSA). Besides centric fusion, mechanisms of chromosomal evolution in Fundulus probably included pericentric inversion, producing biarmed F chromosomes from acrocentric F's and partial loss of a chromosome segment producing smaller biarmed F chromosomes from larger ones. The percentage of SSA chromosomes generally decreases from relatively primitive to specialized species. The presumably most primitive species have only SSA type acrocentric F chromosomes. The 20 Fundulus species were classified into 2 major groups according to the percentage of SSA chromosomes: the SSA group, including 3 subgroups, had more than 50% SSA's; the LSA group, including 2 subgroups, had fewer than 50% SSA's. This classification based only on karyotypic characters generally agreed with others based on gross morphological characters. A possible evolutionary scheme is proposed to account for the derived killifish karyotypes.     

Karyotypes of Rana tagoi Okada with diploid number 28 in the Chausu Mountains of the Minamishinshu district of Nagano Prefecture, Japan (Anura: Ranidae)

Karyotypes of Tago's brown frog Rana tagoi from the Chausu mountains in Minamishinshu of Nagano Prefecture were examined by conventional Giemsa staining, C-banding and late replication (LR)-banding. Chromosome number was 2n = 28 in all cases. The 28 chromosomes consisted of four pairs (1-4) of large biarmed chromosomes, two pairs (5-6) of telocentric chromosomes and eight pairs (7-14) of small biarmed chromosomes. Chromosome pair 11 had a secondary constriction on the long arm. In females, the C-band on the long arm of chromosome pair 6 was detected in both homologs, but was absent from the arms of the homologs of chromosome pairs 5 and 9. In males, C-bands were found in the long arms of both homologs of chromosome pairs 5 and 6, were present only in one homolog of chromosome pair 5 for certain male specimens and found in only one homolog of chromosome pair 9. Specimens of R. tagoi (2n = 28) should thus have two pairs of telocentric chromosomes to provide the same number of chromosome arms, these originating quite likely from chromosome pair 1 in the 26-chromosome specimens by centric fission. Heteromorphic sex chromosomes of the XX-XY type in R. tagoi (2n = 28) in the Chausu mountains were identified. Karyotypes of tail-tip cells from a hybrid tadpole between female R. tagoi (2n = 26) from the Hinohara village in Tokyo and male R. tagoi (2n = 28) from the Chausu mountain population were examined by squash preparation. Chromosome number was 2n = 27 in all tadpoles. The 27 chromosomes consisted of one chromosome set of R. tagoi (2n = 28) and one of R. tagoi (2n = 26).     

Identification of the sex chromosomes in the bald eagle.

Karyotypes of five American bald eagles (Haliaeetus leucocephalus and H. alacanus) are compared. All had 2n=66 chromosomes which fell into 3 size groups: A, 20 pairs of biarmed chromosomes; B, 9 pairs of acrocentric chromosomes and C, 4 pairs of microchromosomes. C-banding was done in two eagles and a heterochromatic W chromosome was identified in a presumptive female. The ZZ and ZW chromosomes could be identified in the karyotypes.     

Heterochromatin differentiation shows the pathways of karyotypic evolution in Israeli mole rats (Spalax, Spalacidae, Rodentia)

C-banding, base-specific fluorochrome staining (CMA3/DA/DAPI), and comparative genomic hybridization (CGH) were used to analyze the constitutive heterochromatin in two Israeli Spalax species, S. galili (2n = 52) and S. judai (2n = 60). It was shown that C-positive centromeric heterochromatin and some telomeric sites comprise GC-rich DNA sequences in both species. Comparative genomic in situ hybridization revealed slight qualitative differences in highly repetitive sequences in the two Spalax species. Eight acrocentric pairs in S. judai that are involved in Robertsonian rearrangements, possessed composite heterochromatin with a preference of S. judai highly repetitive sequences in the proximal region. Heterochromatin of the sex chromosomes, two biarmed homologous pairs (4 and 5) in both species, and acrocentric chromosomes from the group with a variable centromere position in S. judai was entirely species-specific. The high level of homology in the composition of heterochromatin may relate to the recent divergence of Israeli Spalax. Interspecies heterochromatin differences are discussed in the context of possible mechanisms in the Spalax chromosome evolution.     

Phylogenetic position of the saola (Pseudoryx nghetinhensis) inferred from cytogenetic analysis of eleven species of Bovidae

Previous morphological and molecular analyses failed to resolve the phylogenetic position of the critically endangered saola (Pseudoryx nghetinhensis) with respect to its placement in Bovina (cattle, bison, and yak) or Bubalina (Asian and African buffaloes). In the present study, G- and C-banding, Ag-staining and FISH with 28S and telomeric probes was undertaken for 17 bovid species. An analysis of these data allowed us to identify 49 structural rearrangements that included autosomes, gonosomes and 17 different NOR sites. The combined data set was subjected to a cladistic analysis aimed at: (i) providing new insights on phylogenetic relationships of the saola and other species within the subfamily Bovinae, and (ii) testing the suitability of different classes of chromosomal characters for phylogenetic reconstruction of the family Bovidae. The study revealed that nucleolar organizing regions (NORs) are phylogenetically informative. It was shown that at least one, or sometimes two of these characters punctuate divergences that include nodes that are the most basal in the tree, to those that are the most recent. In this context, the shared presence of three NORs in saola and species of Syncerus and Bubalus strongly suggests the saola's placement within the subtribe Bubalina. This contrasts with Robertsonian rearrangements which are informative only at the generic level. These findings suggest that NORs are an important and frequently overlooked source of additional phylogenetic information within the Bovidae that may also have applicability at higher taxonomic levels, possibly even for Pecora.     

The karyotypes of Triaenophorus nodulosus and T. crassus (Cestoda: Pseudophyllidea)

Two species of the genus Triaenophorus were found to have widely different chromosome sets. The karyotype of T. nodulosus consists of 26 biarmed chromosomes ranging from 1.45 to 6.75 microns long. The diploid set of T. crassus contains 18 chromosomes with a well-distinguished first pair of large metacentric homologues. All the chromosomes with the exception of the last pair of acrocentric elements are biarmed. Their absolute length ranges from 1.50 to 8.50 microns. The possible pathways of karyotype differentiation and the evolution of these species are discussed.     

A new karyotype in Callicebus torquatus (Cebidae, Primates)

We describe a new karyotype of Callicebus torquatus using conventional staining, G-banding with Wright Stain, CBG, Ag-NOR staining and fluorescence in situ hybridization (FISH) with human telomere probes and comparative analysis with the previously reported karyotype of C. torquatus torquatus (2n = 20). We studied a female specimen maintained in captivity at the Centro Nacional de Primatas (Para, Brazil). This titi monkey presented 2n = 22, with four large biarmed and six acrocentric autosome pairs; the X chromosome is a medium submetacentric. C-bands were revealed at the centromeric region of all acrocentrics and X chromosome; punctual C-bands also are visualized at the centromeric region in the large biarmed pairs. The NOR site was located at the long arm of pair 4, at the position of a conspicuous secondary constriction. Hybridization signals were detected exclusively at the terminal region of all chromosomes. The karyotype described here has one acrocentric pair more than that found in the literature and also differs by amount and distribution of constitutive heterochromatin. Our data support the notion that the torquatus group may be composed of distinct species, each with its own karyotype.     

Investigation of X- and Y-specific single nucleotide polymorphisms in taurine (Bos taurus) and indicine (Bos indicus) cattle

Initially, domesticated African cattle were of taurine type. Today, we find both African Bos taurus and Bos indicus cattle, as well as their crossbreeds, on the continent of Africa and they all share the same set of African taurine mitochondrial DNA haplogroups. In this study, we report genetic variation as substitutions and insertions/deletions (indels) on both the X and Y chromosomes, and use the variation to assess hybridization between taurine and indicine cattle. Six African cattle breeds (four Sanga breeds, including Raya Azebu, Danakil, Caprivi, Nguni; and two Zebu breeds, including Kilimanjaro Zebu and South Kavirondo Zebu) were screened for six new X-chromosomal markers, specifically three single nucleotide polymorphisms and three indels in the DDX3X (previously DBX ) and ZFX genes, and five previously identified Y-chromosomal markers in the DDX3Y (previously DBY ) and ZFY genes. In total, 90 (57 bulls and 33 cows) samples from the African breeds were analysed. We identify five diagnostic haplotypes of indicine and taurine origins on both the X and Y chromosomes. For each breed, the level of indicine introgression varies; in addition to pure taurine, indicine and hybrid X-chromosome individuals, recombinant X-chromosome variants were also detected. These markers are useful molecular tools for assessing the level of indicine admixture in African cattle breeds.     

Karyotype similarities among Pimelodidae (Pisces, Siluriformes) from the Brazilian amazon region.

Three neotropical species of freshwater fish (Pseudoplatystoma fasciatum, P. tigrinum and Sorubim lima) of the Amazon region (Brazil) were cytogenetically analysed by means of conventional, silver staining and C-banding techniques. All showed 2n = 56, with a predominance of biarmed chromosomes. The NORs were located in the terminal position on the short arms, and only two chromosomes bearing NORs were detected. Heterochromatin was found to be equilocally distributed in some chromosomes of the three species, which may share homologous chromosomes. The present data indicate a somewhat conservative karyotypic evolution in this fish group.