Evidence for chromosome and Pst I satellite DNA family evolutionary stasis in the Bufo viridis group (Amphibia, Anura)

The West Palearctic green toads, Bufo viridis, represent a species complex. Apart from tetraploid populations, which form at least one separate species, evidence exists for relevant differentiation among diploid populations. We present the results of a chromosomal (C-, Ag-NOR-, Replication pattern, DAPI and CMA₃ banding) and molecular study (isolation and characterization of a satellite DNA family) carried out on a number of Central Asian, European and North African populations. For comparative purposes, our molecular analysis was also extended to specimens of three additional Bufo species (B. bufo, B. mauritanicus and B. cf. regularis), as well as two rare African bufonids (Werneria mertensis and Wolterstoffina sp.). Our results demonstrate a remarkable karyological and molecular evolutionary stasis in the Bufo viridis complex. In fact, all chromatinic markers showed the same pattern and/or composition in all specimens, independently of their origin and ploidy levels. Even the NOR loci were invariably two and located on the telomeric regions of two chromosomes of the sixth pair, or quartet. Furthermore, very similar patterns of genomic hybridization of a monomeric unit of the Pst I satellite DNA family (named pBv) were observed in all diploid and tetraploid populations, as well as in B. bufo and B. mauritanicus. Finally, pBv hybridizes with monomeric units of Pst I satellite DNA in all species studied, including Werneria and Wolterstorffina, which are thought to have separated from Bufo as early as in the Mesozoic. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytogenetic studies of Hynobiidae (Urodela) XIX. Morphological variation of sex chromosomes pairing behavior of sex lampbrush chromosomes in <Emphasis Type="Italic">Hynobius quelpaertensis</Emphasis> (Mori) from Cheju Island,South Korea

Using Giemsa staining, C-banding and Ag-NOR staining techniques, we analyzed chromosomes in adult male and female Hynobius quelpaertensis and in embryos of this species in egg sacs collected from eight localities of Cheju Island, South Korea. Chromosome pair 21 was consistently homomorphic in male specimens, while it was heteromorphic in female specimens, suggesting the occurrence of ZZ/ZW sex chromosome constitution in this species. The W chromosome, being much larger than the Z chromosome, was of three morphologically distinct types: W^A, W^B and W^C. Lampbrush chromosomes examined in the oocytes of one female specimen having the W^A chromosome showed that the short arm of the W^A chromosome and the long arm of the Z chromosome paired closely and hence are genetically homologous. We also tried to analyze the structural relationship among the three types of W chromosomes based on their C-banding and Ag-NOR patterns.     

Robertsonian polymorphism, B chromosomes variation and sex chromosomes heteromorphism in the African water rat Dasymys (Rodentia, Muridae)

Chromosome banding analysis (G- and C-bands) of Dasymys rufulus from Senegal, Mali and the Ivory Coast, and D. cf. incomtus from Eastern and South-western Ethiopia was carried out. The diploid numbers (2N) in the former species range between 36 and 39 due to the presence of 0–3 small biarmed heterochromatic B chromosomes, resulting in a corresponding variation of the number of autosomal arms (NFa) between 44 and 50. The basic autosomal set was, however, constant and identical in these specimens. The karyotypes of D. cf. incomtus from Eastern and Western Ethiopia were found to be different (2N=40 and 38, respectively). Comparison of G-banding patterns of the species studied revealed that they differ from each others by 1–2 Rb fusions/fissions, one paracentric inversion and heteromorphous sex chromosomes resulting from addition/deletion of heterochromatic blocks (X) and pericentric inversion (Y). In the light of the available chromosome banding data, the significance of intraspecies karyotypic variability within D. cf. incomtus and its relevance to the systematics of the genus are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Distribution of L1-retroposons on the giant sex chromosomes of Microtus cabrerae (Arvicolidae, Rodentia): functional and evolutionary implications

Long interspersed nuclear elements (L1 or LINE-1) are the most abundant and active retroposons in the mammalian genome. Traditionally, the bulk of L1 sequences have been explained by the ‘selfish DNA’ hypothesis; however, recently it has been also argued that L1s could play an important role in genome and gene organizations. The non-random chromosomal distribution of these retroelements is a striking feature considered to reflect this functionality. In the present study we have cloned and analyzed three different L1 fragments from the genome of the rodent Microtus cabrerae. In addition, we have examined the chromosomal distribution of this L1 in several species of Microtus, a very interesting group owing to the presence in some species of enlarged (‘giant’) sex chromosomes. Interestingly, in all species analyzed, L1-retroposons have preferentially accumulated on both the giant- and the normal-sized sex chromosomes compared with the autosomes. Also we have demonstrated that L1-retroposons are not similarly distributed among the heterochromatic blocks of the giant sex chromosomes in M. cabrerae and M. agrestis, which suggest that L1 retroposition and amplification over the sex heterochromatin have been different and independent processes in each species. Finally, we proposed that the main factors responsible for the L1 distribution on the mammalian sex chromosomes are the heterochromatic nature of the Y chromosome and the possible role of L1 sequences during the X-inactivation process.     

Dosage compensation, the origin and the afterlife of sex chromosomes

Over the past 100 years Drosophila has been developed into an outstanding model system for the study of evolutionary processes. A fascinating aspect of evolution is the differentiation of sex chromosomes. Organisms with highly differentiated sex chromosomes, such as the mammalian X and Y, must compensate for the imbalance in gene dosage that this creates. The need to adjust the expression of sex-linked genes is a potent force driving the rise of regulatory mechanisms that act on an entire chromosome. This review will contrast the process of dosage compensation in Drosophila with the divergent strategies adopted by other model organisms. While the machinery of sex chromosome compensation is different in each instance, all share the ability to direct chromatin modifications to an entire chromosome. This review will also explore the idea that chromosome-targeting systems are sometimes adapted for other purposes. This appears the likely source of a chromosome-wide targeting system displayed by the Drosophila fourth chromosome.     

Evidence for Heteromorphic Sex Chromosomes in Males of Rana tagoi and Rana sakuraii in Nishitama District of Tokyo (Anura: Ranidae)

Karyotypes of the Tago brown frog Rana tagoi and stream Tago brown frog Rana sakuraii from a mountain region in the Nishitama district in Tokyo were examined by conventional Giemsa staining, C-banding and late replication (LR)-banding. Chromosome number was 2n = 26 in all cases. The 26 chromosomes consisted of five (1–5) pairs of large chromosomes and eight (6–13) pairs of small chromosomes. Chromosome 10 had a secondary constriction on the long arm. In all frogs, on chromosome pair 8, the XX/XY type sex chromosome was present. C-banding analysis indicated that, in R. sakuraii, neither the X nor Y chromosome possessed interstitial C-bands but each had centromere staining, while in R. tagoi, an interstitial C-band was present on the long arm of the X chromosome. The Y chromosome had no interstitial C-band. LR-banding analysis demonstrated the X and Y chromosomes to have a LR-band on the short arm and two LR-bands, each on the long arm, and the bands on both species to be essentially the same. Heteromorphic sex chromosomes in males of R. sakuraii and R. tagoi were identified for the first time in this study. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterizing the chromosomes of the platypus (Ornithorhynchus anatinus)

Like the unique platypus itself, the platypus genome is extraordinary because of its complex sex chromosome system, and is controversial because of difficulties in identification of small autosomes and sex chromosomes. A 6-fold shotgun sequence of the platypus genome is now available and is being assembled with the help of physical mapping. It is therefore essential to characterize the chromosomes and resolve the ambiguities and inconsistencies in identifying autosomes and sex chromosomes. We have used chromosome paints and DAPI banding to identify and classify pairs of autosomes and sex chromosomes. We have established an agreed nomenclature and identified anchor BAC clones for each chromosome that will ensure unambiguous gene localizations.     

FAST-FISH with laser beam microdissected DOP-PCR probe distinguishes the sex chromosomes of Silene latifolia

We present an improved FISH strategy for differentiating the sex chromosomes of the dioecious model plant, Silene latifolia. Fixed mitotic protoplasts were dropped on a polyethylene naphthalate membrane, the X or Y chromosomes were isolated using nitrogen laser beam microdissection, catapulted by laser pressure, and amplified by DOP-PCR. A modified FAST-FISH protocol based on a short hybridization time combined with a low concentration of probe was used. The success of this approach is demonstrated by the differential labeling of the X and Y chromosomes and it could represent a quick method for comparing organization of plant genomes.     

Melosis in holocentric chromosomes: Kinetic activity is randomly restricted to the chromatid ends of sex univalents inGraphosoma italicum (Heteroptera)

We have determined the number and location of the nucleolar organizing regions in spermatocytes ofGraphosoma italicum (2n=12A+ XY/XX) by means of silver impregnation, chromomycin A₃/distamycin A staining and fluorescencein situ hybridization. The identification of only one nucleolar organizing region located at one of the X chromosome ends has provided a suitable cytological marker to analyse the segregation of this univalent and that of the XY pseudobivalent during the first and second meiotic divisions respectively. Our results clearly show that at first meiotic metaphase the chromatids of the X chromosome are orientated with their long axes perpendicular to the polar axis. Although the kinetic activity is restricted to only one end in both X chromatids during the first meiotic division, both ends of the same chromatid have the same probability of showing such kinetic activity. In this sense, we also report that the chromatid segregation maybe initiated either at the same sister chromatid ends or at opposite ends in each chromatid. Thus, this indicates a sex chromatid independence as regards to the chromatid segregation during the first meiotic division. Throughout the second meiotic division both ends of the X chromatid are involved with the same probability in the end-to-end association to conform the XY pseudobivalent. This also implies a random localization of the kinetic activity at the ends opposite to those involved in the end-to-end association.accepted for publication by J. S. (Pat) Heslop-Harrison     

The evolution of sex chromosomes in the genus <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae): Identification of a new species with heteromorphic sex chromosomes

The structural features and evolutionary state of the sex chromosomes of the XX/XY species of Rumex are unknown. Here, we report a study of the meiotic behaviour of the XY bivalent in Rumex acetosella and R. suffruticosus, a new species which we describe cytogenetically for the first time in this paper, and also that of the XY₁Y₂ trivalent of R. acetosa by both conventional cytogenetic techniques and analysis of synaptonemal complex formation. Fluorescent in situ hybridization with satellite DNA and rDNA sequences as probes was used to analyse the degree of cytogenetic differentiation between the X and Y chromosomes in order to depict their evolutionary stage in the three species. Contrasting with the advanced state of genetic differentiation between the X and the Y chromosomes in R. acetosa, we have found that R. acetosella and R. suffruticosus represent an early stage of genetic differentiation between sex chromosomes. Our findings further demonstrate the usefulness of the genus Rumex as a model for analysing the evolution of sex chromosomes in plants, since within this genus it is now possible to study the different levels of genetic differentiation between the sex chromosomes and to analyse their evolutionary history from their origin.     

An XX/XY sex microchromosome system in a freshwater turtle, Chelodina longicollis (Testudines: Chelidae) with genetic sex determination

Heteromorphic sex chromosomes are rare in turtles, having been described in only four species. Like many turtle species, the Australian freshwater turtle Chelodina longicollis has genetic sex determination, but no distinguishable (heteromorphic) sex chromosomes were identified in a previous karyotyping study. We used comparative genomic hybridization (CGH) to show that C. longicollis has an XX/XY system of chromosomal sex determination, involving a pair of microchromosomes. C-banding and reverse fluorescent staining also distinguished microchromosomes with different banding patterns in males and females in ∼70% cells examined. GTG-banding did not reveal any heteromorphic chromosomes, and no replication asynchrony on the X or Y microchromosomes was observed using replication banding. We conclude that there is a very small sequence difference between X and Y chromosomes in this species, a difference that is consistently detectable only by high-resolution molecular cytogenetic techniques, such as CGH. This is the first time a pair of microchromosomes has been identified as the sex chromosomes in a turtle species.     

Inverted and satellited Y chromosome in the orangutan (Pongo pygmaeus)

An inverted and satellited Y chromosome of almost acrocentric appearance was detected in seven of 14 male orangutans. In the remaining seven animals a submetacentric Y chromosome without NORs occurred. The high frequency with which the satellited Y chromosomes were associated with acrocentric autosomes and the positive AgNO₃-staining of their satellite stalks clearly indicate the active state of the NOR on the Y chromosomes. DNA fingerprinting in two orangutan families showed that the inverted and satellited Y chromosomes in carrier orangutan males do not interfere with normal fertility. Within our sample of male orangutans studied, the inverted and satellited Y chromosome is restricted to Sumatran animals; all Bornean specimens possessed the submetacentric Y chromosome. The question arises whether these two kinds of Y chromosome differ constitutively between thePongo pygmaeus subpopulations.We dedicate this paper to Professor Ulrich Wolf on the occasion of his 60th birthday.     

Survey of repetitive sequences in <Emphasis Type="Italic">Silene latifolia</Emphasis> with respect to their distribution on sex chromosomes

We carried out a global survey of all major types of transposable elements in Silene latifolia, a model species with sex chromosomes that are in the early stages of their evolution. A shotgun genomic library was screened with genomic DNA to isolate and characterize the most abundant elements. We found that the most common types of elements were the subtelomeric tandem repeat X-43.1 and Gypsy retrotransposons, followed by Copia retrotransposons and LINE non-LTR elements. SINE elements and DNA transposons were less abundant. We also amplified transposable elements with degenerate primers and used them to screen the library. The localization of elements by FISH revealed that most of the Copia elements were accumulated on the Y chromosome. Surprisingly, one type of Gypsy element, which was similar to Ogre elements known from legumes, was almost absent on the Y chromosome but otherwise uniformly distributed on all chromosomes. Other types of elements were ubiquitous on all chromosomes. Moreover, we isolated and characterized two new tandem repeats. One of them, STAR-C, was localized at the centromeres of all chromosomes except the Y chromosome, where it was present on the p-arm. Its variant, STAR-Y, carrying a small deletion, was specifically localized on the q-arm of the Y chromosome. The second tandem repeat, TR1, co-localized with the 45S rDNA cluster in the subtelomeres of five pairs of autosomes. FISH analysis of other Silene species revealed that some elements (e.g., Ogre-like elements) are confined to the section Elisanthe while others (e.g. Copia or Athila-like elements) are present also in more distant species. Similarly, the centromeric satellite STAR-C was conserved in the genus Silene whereas the subtelomeric satellite X-43.1 was specific for Elisanthe section. Altogether, our data provide an overview of the repetitive sequences in Silene latifolia and revealed that genomic distribution and evolutionary dynamics differ among various repetitive elements. The unique pattern of repeat distribution is found on the Y chromosome, where some elements are accumulated while other elements are conspicuously absent, which probably reflects different forces shaping the Y chromosome. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparative chromosome mapping of sex-linked genes and identification of sex chromosomal rearrangements in the Japanese wrinkled frog (Rana rugosa, Ranidae) with ZW and XY sex chromosome systems

There are regional variations of sex chromosome morphologies in the Japanese wrinkled frog, Rana rugosa (2n = 26): heterogametic ZZ/ZW-type and XX/XY-type sex chromosomes, and two different types of homomorphic sex chromosomes. To search for homology between the ZW and XY sex chromosomes and the chromosome rearrangements that have occurred during sex chromosomal differentiation in R. rugosa, we performed chromosome mapping of sexual differentiation genes for R. rugosa by FISH. Three genes, AR, SF-1/Ad4BP and Sox3, were localized to both the ZW and XY chromosomes, and their locations were all different between the Z and W and between the X and Y. AR and SF-1/Ad4BP were located on the short arms of the W and X and the long arms of Z and Y, and Sox3 was mapped to the different locations on the long arms between the Z and W and between the X and Y, probably as a result of multiple rearrangements that occurred during the process of sex chromosome differentiation. However, the chromosomal locations of three genes were almost consistent between the Z and Y and between the W and X, indicating that the Z and Y chromosomes and the W and X chromosomes were respectively derived from the same origins. Dmrt1, which is located on avian sex chromosomes, was localized to autosomes in R. rugosa with both the ZW and XY sex chromosomes, suggesting that Dmrt1 might not be related to sex determination in this species.     

Helminth community structure of the oven frog Leptodactylus latinasus (Anura, Leptodactylidae) from Corrientes, Argentina

A total of 36 adult individuals of Leptodactylus latinasus were collected near the city of Corrientes, Province of Corrientes in Argentina. The main goals of this study were: (1) to determine the helminth parasite fauna of L. latinasus; (2) to determine the richness and diversity of parasites at the component and infracommunity levels; (3) to analyze the relationships between helminth, frog body size and sex, and (4) to identify and examine species affinity of helminth communities. The helminth component community of this frog’s population consisted of 17 species. The predominant groups of parasites were the trematodes (adults: Glypthelmins repandum, Catadiscus inopinatus and Haematoloechus longiplexus; larvae: Travtrema aff. stenocotyle, Bursotrema aff. tetracotyloides, Styphlodora sp., unknown opisthogonimid species, Petasiger sp. and unknown strigeid species), followed by the nematodes (Cosmocerca podicipinus, C. parva, C. rara, C. cruzi, Schrankiana schranki and Aplectana hylambatis); other groups of parasites were represented by only one species (unknown larval cestode species and Centrorhynchus sp.). All parasite helminth species showed an aggregated pattern of distribution. The most infected organs were kidneys, small intestine, large intestine and pharyngeal zone. The host body size was important in determining the parasites abundance of G. repandum. At the level of component community G. repandum was the species with highest prevalence of infection and Bursotrema aff. tetracotyloides was the dominant species. Helminth species showed four significant pairs of covariation and two significant pairs of association in the infracommunities of Leptodactylus latinasus.     

Microdissection of the Y chromosome and fluorescencein situ hybridization analysis of the sex chromosomes of lake trout,Salvelinus namaycush

Lake trout,Salvelinus namaycush, is one of the few salmonids with morphologically differentiated sex chromosomes. Genetic analysis suggested that the sex-determining region of this species lies on the short arm of the Y chromosome. The differential arm of the Y chromosome was microdissected and the resulting DNA amplified in a sequence-independent manner. Amplified DNA was biotin labeled as a probe for fluorescencein situ hybridization (FISH). Strong hybridization signals were seen covering defined regions of both the Y and X chromosomes. Homeologous chromosomes of the ancestrally tetraploid genome were not identified by FISH with the Y probe, indicating diploidization of this region of the genome.     

Banding techniques on tardigrade chromosomes: the karyotype of Macrobiotus richtersi (Eutardigrada, Macrobiotidae)

This work represents the first attempt to define tardigrade chromosomes using banding techniques. Macrobiotus richtersi, a eutardigrade morphospecies with amphimictic diploid and thelytokous triploid cytotypes, was used as a model. Prime consideration was given to oocyte chromosomes because they are larger than those of spermatocytes and of mitotic chromosomes. With Giemsa staining, the chromatids of the 6 bivalents of the diploid cytotypes and those of the 17–18 univalents of the triploid cytotypes were very similar to each other and appeared rod- or flame-shaped. In the amphimictic strain, a chiasma was generally present in each bivalent at diplotene, whereas there were no chiasmata in the oocyte prophase chromosomes of the triploid strain. Both in diploid and triploid cytotypes, C-banding and fluorescence showed a heterochromatic centromeric band on the telomere of each chromosome oriented towards the spindle pole, indicating that all of them were acrocentric. Silver staining showed the presence of a NOR in only a pair of chromosomes, close to the centromeric C-banded site. NOR was particularly evident in the oocyte prophases. Other silver positive regions, corresponding to the kinetochore, were located on all other chromosomes on the telomeres towards the spindle pole. This revised version was published online in July 2006 with corrections to the Cover Date.     

A new species of Cosmocerca (Nematoda, Cosmocercidae) and other helminths from Genyophryne thomsoni (Anura, Microhylidae) from Papua New Guinea

Cosmocerca tyleri sp. nov. (Ascaridida, Cosmocercidae) from the large intestine of Genyophryne thomsoni (Anura, Microhylidae) is described and illustrated. Cosmocerca tyleri sp. nov. represents the 23rd species assigned to the genus and the 6th from the Australian realm. Of the 5 Australian species previously described, C. tyleri sp. nov. differs from C. limnodynastes and C. novaeguineae in number of plectanes, 4 pairs in C. tyleri, 5 pairs in C. limnodynastes and C. novaeguineae. Cosmocerca australis has 3–4 pairs of plectanes, C. archeyi and C. zugi each have 4 pairs of plectanes; however, in each species the plectanes lie in the fourth quarter of the body and just anterior to the cloaca. In C. tyleri sp. nov. the plectanes lie in the third quarter of the body and there is significant space between the cloaca and the posterior pair of plectanes.     

Localization of uridine monophosphate synthase (UMPS) gene to river buffalo chromosomes by FISH

Uridine monophosphate synthase plays an important role in pyrimidine synthesis, converting orotic acid to uridine 5 monophosphate. In cattle,UMPS deficiency is inherited as a monogenic autosomal recessive trait. While heterozygous carriers are phenotypically normal, homozygotes are lethalin utero (Shankset al. 1992).UMPS has been mapped to human chromosome 3q13 (Qumsiyehet al. 1989), sheep chromosome 1q (Burkinet al. 1993) and cattle chromosome 1q31 (Ryanet al. 1994). In the present study we used a cattle genomic probe to localizeUMPS to river buffalo chromosomes by fluorescencein situ hybridization.