Molecular cytogenetics of tragelaphine and alcelaphine interspecies hybrids: hybridization, introgression and speciation in some African antelope.

Hybridization can occur naturally among diverging lineages as part of the evolutionary process leading to complete reproductive isolation, or it can result from range shifts and habitat alteration through global warming and/or other anthropogenic influences. Here we report a molecular cytogenetic investigation of hybridization between taxonomically distinct species of the Alcelaphini (Alcelaphus buselaphus 2n = 40 × Damaliscus lunatus 2n = 36) and the Tragelaphini (Tragelaphus strepsiceros 2n = 31/32 × Tragelaphus angasii 2n = 55/56). Cross-species fluorescence in situ hybridization provides unequivocal evidence of the scale of karyotypic difference distinguishing parental species. The findings suggest that although hybrid meiosis of the former cross would necessitate the formation of a chain of seven, a ring of four and one trivalent, the progeny follow Haldane's rule showing F1 male sterility and female fertility. The tragelaphine F1 hybrid, a male, was similarly sterile and, given the 11 trivalents and chain of five anticipated in its meiosis, not unexpectedly so. We discuss these findings within the context of the broader evolutionary significance of hybridization in African antelope, and reflect on what these hold for our views of antelope species and their conservation.

Phylogeny and vicariant speciation of the Grey Rhebok, Pelea capreolus.

A South African endemic antelope, the Grey Rhebok (Pelea capreolus), has long been an evolutionary enigma in bovid systematics-its phylogenetic intractability attributed to its curious combination of derived and primitive morphological attributes and the consequences of a rapid radiation. By using a combination of DNA sequences, chromosomal characteristics and quantitative and qualitative morphological features we show that the species is a sister taxon to a clade that comprises the waterbuck, reedbuck and allies. Our finding of few unambiguous synapomorphies reinforces suggestions of a rapid radiation and highlights the effects of incomplete lineage sorting, including the hemiplasic nature of several chromosomal rearrangements. We investigate these data to address the general question of what may have led to Pelea being both genetically and ecologically distinct from the Reduncini. We argue that its adaptation to exposed habitats, free of standing water, arose by vicariance prompted by increasing aridity of the extreme south/southwestern region of the African continent in the Miocene. Ancestral lineages leading to the extant Redunca and Kobus, on the other hand, retreated to water-abundant refugia in the north during these mostly globally cool phases. The mosaic of water-rich environments provided by the Okavango and the drainage systems in the southwestern extension of the East African Rift system are considered to have facilitated speciation and chromosomal evolution within these antelope.

A comparative study of meiotic recombination in cattle (Bos taurus) and three wildebeest species (Connochaetes gnou, C. taurinus taurinus and C. t. albojubatus).

The karyotypic evolution in the family Bovidae is based on centric fusions of ancestral acrocentric chromosomes. Here, the frequency and distribution of meiotic recombination was analyzed in pachytene spermatocytes from Bos taurus (2n = 60) and 3 wildebeest species (Connochaetes gnou, C. taurinus taurinus and C. t. albojubatus) (2n = 58) using immunofluorescence and fluorescence in situ hybridization. Significant differences in mean numbers of recombination events per cell were observed between B. taurus and members of the genus Connochaetes (47.2 vs. 43.7, p < 0.001). The number of MLH1 foci was significantly correlated with the length of the autosomal synaptonemal complexes. The average interfocus distance was influenced by interference. The male recombination maps of bovine chromosomes 2 and 25 and of their fused homologues in wildebeests were constructed. A significant reduction of recombination in the fused chromosome BTA25 was observed in wildebeests (p = 0.005). This was probably caused by interference acting across the centromere, which was significantly stronger than the intra-arm interference. This comparative meiotic study showed significant differences among the species from the family Bovidae with the same fundamental number of autosomal arms (FNa = 29) which differ by a single centric fusion.

Molecular insights into X;BTA5 chromosome rearrangements in the tribe Antilopini (Bovidae).

For a clade that includes Antilope, Gazella,Nanger and Eudorcas (Antilopinae), X;BTA5 translocation is a synapomorphy. Using a combination of fluorescence in situ hybridization (FISH) probes and polymerase chain reaction techniques, we provide (i) the first insight into the X;BTA5 architecture which differs in the species under study: Antilope cervicapra (genus Antilope), Gazella leptoceros (genus Gazella) and Nanger dama ruficollis (genus Nanger), (ii) determination of interstitial satellite DNA at the X;BTA5 junctions, and (iii) determination of repetitive sequences occupying constitutive heterochromatin of Xp arms in the studied species. The distribution of 2 repetitive DNA families in the centromeric regions of all chromosomes has been investigated by FISH with probes representing satellite I and satellite II DNA in all studied species. In this context, we discuss a markedly smaller centromere in the BTA5 (Y2) unfused chromosomes in males in the XY1Y2 determining system in comparison with other acrocentrics. An analysis of karyotypic data described in current published studies revealed a disparity with the data determined by FISH. In this report, we document chromosomal fusions in the 3 species mentioned resulting from FISH with painting probes prepared from cattle (Bos taurus). The number and chromosomal location of nucleolus organizer regions were determined by FISH. In the present study, we emphasize the importance of chromosomal rearrangement verification, particularly, if they are used for phylogenetic analysis.

Comparative molecular cytogenetics in Cetartiodactyla.

Cetartiodactyla comprises Artiodactyla (even-toed ungulates) and Cetacea (whales, dolphins and porpoises). Artiodactyla is a large taxon represented by about 200 living species ranked in 10 families. Cetacea are classified into 13 families with almost 80 species. Many publications concerning karyotypic relationships in Cetartiodactyla have been published in previous decades. Formerly, the karyotypes of closely related species were compared by chromosome banding. Introduction of molecular cytogenetic methods facilitated comparative mapping between species with highly rearranged karyotypes and distantly related species. Such information is a prerequisite for the understanding of karyotypic phylogeny and the reconstruction of the karyotypes of common ancestors. This study summarizes the data on chromosome evolution in Cetartiodactyla, mainly derived from molecular cytogenetic studies. Traditionally, phylogenetic relationships of most groups have been estimated using morphological data. However, the results of some molecular studies of mammalian phylogeny are discordant with traditional conceptions of phylogeny. Cetartiodactyls provide several examples of incongruence between traditional morphological and molecular data. Such cases of conflict include the relationships of the major clades of artiodactyls, the relationships among the extant families of the suborder Ruminantia or the phylogeny of the family Bovidae. The most unexpected aspect of the molecular phylogeny was the recognition that Cetacea is a deeply nested member of Artiodactyla. The largest living order of terrestrial hoofed mammals is the even-toed hoofed mammals, or Artiodactyla. The artiodactyls are composed of over 190 living species including pigs, peccaries, hippos, camels, llamas, deer, pronghorns, giraffes, sheep, goats, cattle and antelopes. Cetacea is an order of wholly aquatic mammals, which include whales, dolphins and porpoises. Cetartiodactyla has become the generally accepted name for the clade containing both of these orders.

Analysis of morphological disorders and ploidy in domestic cat blastocysts.

This study describes, for the first time, the relationship between morphology and ploidy in domestic cat embryos. Blastocyst morphology and quality were assessed using time-lapse recordings, while ploidy was analyzed using fluorescence in situ hybridization. Out of 54 blastocysts, clear fluorescence signals for all the molecular probes used were observed in 24 (44.4%) blastocysts, while in another 14 (25.9%) blastocysts, fluorescence signals only allowed for sex assessment. No clear signals were observed in the remaining 16 blastocysts (29.7%). Of the 24 blastocysts with clear signals, normal ploidy was detected in 10 (41.4%), 7 (29.2%) were diagnosed as haploid, and the remaining 7 blastocysts (29.2%) were mosaics. Additionally, results showed the distribution of diploid, haploid, and mosaic blastocysts in relation to the occurrence of morphological disorders and to embryo quality. The presence of abnormal embryo morphology and karyotype disorders may affect further development and the pregnancy rate. Due to the comparable proportion of good and poor quality blastocysts with disturbed ploidy, it is important to implement new methods of embryo assessment, especially when techniques used in humans, such as pronuclear observation, cannot be used.

Cytotypes of Kirk's dik-dik (Madoqua kirkii,Bovidae) show multiple tandem fusions.

Madoqua kirkii, a miniature African antelope, is noted for extensive chromosomal variation that has been categorized in four distinct cytotypes (A-D). In this investigation, we analyzed the A cytotype (2n = 46, FN = 48) using a suite of molecular cytogenetic approaches that entailed (i) whole chromosome and subchromosomal painting by fluorescence in situ hybridization (FISH), (ii) the study of Madoqua centromeric-specific DNA derived from pooled DNA obtained from the centromeric regions of the acrocentric chromosomes, and (iii) DNA from the telomere:centromere junctions of tandemly fused chromosomes. DNA from these sources was used to probe for the persistence of interstitial satellite DNA and residual centromeric sequences in the tandem and centric fusion junctions by PCR and FISH. The analyses show centromeric sequences at two of the six tandem fusion junctions. These data, and those of hybrid specimens (A × B cytotypes) in conjunction with published information permitted an interpretation of the probable sequence of chromosomal rearrangements among the M. kirkii cytotypes. We discuss the findings in the context of chromosomal evolution in these antelopes, and the implications that these hold for ex-situ breeding programs of the species.

Sperm chromosome segregation of rob(4;16) and rob(4;16)inv(4) in the brown brocket deer (Mazama gouazoubira).

The genus Mazama stands out among the Neotropical deer due to their wide intra and interspecific karyotypic diversification, which is associated with an accentuated chromosomal fragility. There are reports of heterozygous Robertsonian translocation (RT) carriers in a free-range population of Mazama gouazoubira (brown brocket deer), as well as in captive animals of this and other species of the genus. To analyze possible negative impacts of heterozygous chromosome rearrangements on reproductive fitness of the carriers, we performed an analysis of sperm meiotic segregation in four brown brocket bucks, carriers of a rob(4;16), and compared the results with those of a normal buck. We established a reliable FISH and sperm-FISH protocol for the brown brocket deer using bovine (Bos taurus; diploid number, 2n = 60) whole chromosome painting (WCP) and BAC probes. Using BAC probes, we revealed the presence of a paracentric inversion (PAI) of the fused chromosome 4 in two of the four analyzed RT carriers. The mean frequency of normal/balanced sperm in the translocation carriers was significantly lower than in the normal buck (94.78% vs 98.40%). The mean value of total unbalanced spermatozoa was almost doubled in the RT/PAI carriers (6.68%) when compared to RT carriers (3.76%), but the difference was not statistically significant. This study demonstrated the efficiency of FISH with bovine WCP and BAC probes in the characterization of chromosome rearrangements and gametic segregation patterns in brown brocket deer. Our results indicate a low to moderate increase in the rates of unbalanced meiotic segregation products in brown brocket bucks heterozygous for RT and RT/PAIs.

Different fusion configurations of evolutionarily conserved segments in karyotypes of Potamochoerus porcus and Phacochoerus africanus.

The karyotype of the red river hog Potamochoerus porcus (2n = 34) differs from that of the domestic pig by the presence of 2 fusion chromosomes homologous to pig chromosomes 13/16 and 15/17. Moreover, chromosomes corresponding to pig chromosomes 13/16 and 1 are both acrocentric. Hybridization with region-specific painting probes confirmed tandem fusion of pig chromosomes 13 and 16, and a pericentric inversion of the pig chromosome 1p equivalent in P. porcus. The chromosome complement of the wart hog Phacochoerus africanus (2n = 34) differs from the pig karyotype in 2 centric fusions, 13/16 and 15/17. Karyotypic relationships among different Suidae species are discussed in the article. Besides fusions 13/16 and 15/17, which are common to several suids, another fusion of pig chromosomes 14 and 18 is suggested to exist in the karyotype of Sus cebifrons.

Reciprocal translocation t(4;7)(q14;q28) in cattle: molecular characterization.

Cytogenetic analysis of a phenotypically normal young bull from the Marchigiana breed revealed the presence of an abnormal chromosome. The finding of one oversize chromosome in all metaphases, associated with a 2n = 60, XY karyotype, suggested that a reciprocal translocation had occurred. RBG-banding and FISH analyses, using specific bovine BAC probes, identified a de novo reciprocal translocation t(4;7)(q14;q28). The presence of rcp(4;7) was confirmed by FISH experiments using BTA4 and BTA7 whole chromosome probes. An array-CGH analysis (Agilent 244A) using a bovine custom design was performed to investigate if the translocation was associated with loss or gain of genetic material. The absence of a concomitant deletion or duplication at the break points allowed the balanced state of the translocation to establish. The analysis also revealed the presence of several CNVs throughout the genome. To our knowledge this is the first time the balanced condition of a cattle RCP has been ascertained using the array-CGH approach.

Detection of bisphenol A using a novel surface plasmon resonance biosensor.

We present a compact surface plasmon resonance (SPR) biosensor for the detection of bisphenol A (BpA), an endocrine-disrupting chemical. The biosensor is based on an SPR sensor platform (SPRCD) and the binding inhibition detection format. The detection of BpA in PBS and wastewater was performed at concentrations ranging from 0.05 to 1,000 ng/ml. The limit of detection for BpA in PBS and wastewater was estimated to be 0.08 and 0.14 ng/ml, respectively. It was also demonstrated that the biosensor can be regenerated for repeated use. Results achieved with the SPR biosensor are compared with those obtained using ELISA and HPLC methods.

Detection of translocation rob(1;29) in bull sperm using a specific DNA probe.

The Robertsonian translocation rob(1;29), connected with reduced fertility, is widespread in different cattle breeds all over the world. After laser microdissection, DOP-PCR, cloning and sequencing, a highly sensitive translocation-specific DNA probe, suitable for detection of rob(1;29) in cattle metaphase and interphase cells, including spermatozoa was designed. Sperm samples of five heterozygous translocation carriers were analyzed using this probe and a control probe for chromosome 6. One thousand decondensed spermatozoa from each bull were scored. Signals of the translocation-specific probe were detected in 48.8, 50.9, 50.1, 51.8, and 54.8% of spermatozoa, respectively. In contrast, semen samples from five chromosomally normal bulls showed only signals of the control probe for chromosome 6. Semen from a chimeric (XX/XY) bull, showing 57.5% of 59,XX,rob(1;29) and 42.5% of 60,XY cells in cultured peripheral lymphocytes, was also examined using this probe. No sperm head with signal of the translocation-specific probe was observed among 1,000 spermatozoa analyzed in this bull, demonstrating that female cells do not pass through the process of spermatogenesis.

Identification of a new reciprocal translocation in an AI bull by synaptonemal complex analysis, followed by chromosome painting.

An AI Ayrshire bull was subjected to cytogenetic examination due to lowered fertility. Preliminary Giemsa staining revealed a normal chromosome complement (60,XY) and G-banding did not allow us to draw a clear conclusion concerning an occurrence of chromosome rearrangement. Testicles were collected at slaughter and synaptonemal complex (SC) analysis revealed a large cross-shaped tetravalent configuration in pachytene spreads. No association between the tetravalent and XY bivalent was observed. Chromosome painting, with the use of bovine whole chromosome painting probes, conjugated with DAPI staining, facilitated a detailed description of the translocation rcp(2;4)(q45;q34). This study shows that post mortem analysis of synaptonemal complexes is a simple and useful tool for the preliminary detection of reciprocal translocation carriers.

Karyotype, centric fusion polymorphism and chromosomal aberrations in captive-born mountain reedbuck (Redunca fulvorufula).

Chromosomes of fourteen captive-born mountain reedbucks (Redunca fulvorufula) have been investigated. The diploid chromosome number was 2n = 56 (FN = 60). The mountain reedbuck karyotype consists of 26 acrocentric and two biarmed chromosome pairs resulting from two centric fusions involving chromosomes 2 and 25, and 6 and 10, respectively. In some animals, 57 chromosomes were detected. Variation in the diploid number was found to be due to polymorphism for the centric fusion 6;10. Both X and Y chromosomes are large and acrocentric. The entire Y chromosome and the proximal part of the X chromosome consist of heterochromatin. The chromosomes X, 9 and 14 appeared to be of caprine type. Chromosome aberrations have been detected in two of the 14 animals investigated. A de novo formed Robertsonian translocation rob(6;13) was found in one female heterozygous for the fusion 6;10. CBG-banding revealed one block of centromeric heterochromatin in the de novo formed translocation rob(6;13) and also in the evolutionarily fixed centric fusions 6;10 and 2;25. One examined male homozygous for fusion 6;10, had a mosaic 56,XY/57,XYY karyotype, with 11% of analyzed cells containing two Y chromosomes. The findings were confirmed by cross-species fluorescence in situ hybridization (FISH) with bovine (Bos taurus L.) chromosome painting probes. The study demonstrates the relevance of cytogenetic screening in captive animals from zoological gardens.