Karyotype, evolution and phylogenetic reconstruction in Micronycterinae bats with implications for the ancestral karyotype of Phyllostomidae.

BACKGROUND: The Micronycterinae form a subfamily of leaf-nosed bats (Phyllostomidae) that contains the genera Lampronycteris Sanborn, 1949, and Micronycteris Gray, 1866 (stricto sensu), and is characterized by marked karyotypic variability and discrepancies in the phylogenetic relationships suggested by the molecular versus morphological data. In the present study, we investigated the chromosomal evolution of the Micronycterinae using classical cytogenetics and multidirectional chromosome painting with whole-chromosomes probes of Phyllostomus hastatus and Carollia brevicauda. Our goal was to perform comparative chromosome mapping between the genera of this subfamily and explore the potential for using chromosomal rearrangements as phylogenetic markers. RESULTS: The Micronycterinae exhibit great inter- and intraspecific karyotype diversity, with large blocks of telomere-like sequences inserted within or adjacent to constitutive heterochromatin regions. The phylogenetic results generated from our chromosomal data revealed that the Micronycterinae hold a basal position in the phylogenetic tree of the Phyllostomidae. Molecular cytogenetic data confirmed that there is a low degree of karyotype similarity between Lampronycteris and Micronycteris specimens analyzed, indicating an absence of synapomorphic associations in Micronycterinae. CONCLUSIONS: We herein confirm that karyotypic variability is present in subfamily Micronycterinae. We further report intraspecific variation and describe a new cytotype in M. megalotis. The cytogenetic data show that this group typically has large blocks of interstitial telomeric sequences that do not appear to be correlated with chromosomal rearrangement events. Phylogenetic analysis using chromosome data recovered the basal position for Micronycterinae, but did not demonstrate that it is a monophyletic lineage, due to the absence of common chromosomal synapomorphy between the genera. These findings may be related to an increase in the rate of chromosomal evolution during the time period that separates Lampronycteris from Micronycteris.

New insights into sex chromosome evolution in anole lizards (Reptilia, Dactyloidae).

Anoles are a clade of iguanian lizards that underwent an extensive radiation between 125 and 65 million years ago. Their karyotypes show wide variation in diploid number spanning from 26 (Anolis evermanni) to 44 (A. insolitus). This chromosomal variation involves their sex chromosomes, ranging from simple systems (XX/XY), with heterochromosomes represented by either micro- or macrochromosomes, to multiple systems (X1X1X2X2/X1X2Y). Here, for the first time, the homology relationships of sex chromosomes have been investigated in nine anole lizards at the whole chromosome level. Cross-species chromosome painting using sex chromosome paints from A. carolinensis, Ctenonotus pogus and Norops sagrei and gene mapping of X-linked genes demonstrated that the anole ancestral sex chromosome system constituted by microchromosomes is retained in all the species with the ancestral karyotype (2n = 36, 12 macro- and 24 microchromosomes). On the contrary, species with a derived karyotype, namely those belonging to genera Ctenonotus and Norops, show a series of rearrangements (fusions/fissions) involving autosomes/microchromosomes that led to the formation of their current sex chromosome systems. These results demonstrate that different autosomes were involved in translocations with sex chromosomes in closely related lineages of anole lizards and that several sequential microautosome/sex chromosome fusions lead to a remarkable increase in size of Norops sagrei sex chromosomes.

Gestational experience alters sex allocation in the subsequent generation.

Empirical tests of adaptive maternal sex allocation hypotheses have presented inconsistent results in mammals. The possibility that mothers are constrained in their ability to adjust sex ratios could explain some of the remaining variation. Maternal effects, the influence of the maternal phenotype or genotype on her developing offspring, may constrain sex allocation through physiological changes in response to the gestational environment. We tested if maternal effects constrain future parental sex allocation through a lowered gestational stress environment in laboratory mice. Females that experienced lowered stress as embryos in utero gave birth to female-biased litters as adults, with no change to litter size. Changes in offspring sex ratio was linked to peri-conceptual glucose, as those females that had increasing blood glucose peri-conceptionally gave birth to litters with a higher male to female sex ratio. There was, however, no effect of the lowered prenatal stress for developing male embryos and their sperm sex ratio when adult. We discuss the implications of maternal effects and maternal stress environment on the lifelong physiology of the offspring, particularly as a constraint on later maternal sex allocation.

Chromosome painting reveals multiple rearrangements between Gymnotus capanema and Gymnotus carapo (Gymnotidae, Gymnotiformes).

The genus Gymnotus (Gymnotiformes) is a group of fishes with karyotypic plasticity, demonstrated by cytogenetic studies using whole chromosome probes of G. carapo (GCA, 2n = 42) that were obtained by flow-sorting from fibroblast cultures. In the present work we undertook comparative mapping of the karyotype of G. capanema (GCP, 2n = 34) with GCA, 2n = 42 painting probes. The results demonstrate that the karyotype of G. capanema is extensively rearranged when compared to G. carapo. From the 12 chromosome pairs of G. carapo that can be individually differentiated (GCA1-3, 6, 7, 9, 14, 16 and 18-21), only 4 pairs (GCA6, 7, 19, and 20) maintained conserved synteny in G. capanema. From these 4, GCA6 and GCA20 correspond to individual chromosomes (GCP8 and GCP15), while the other 2 share homology with parts of GCP1 and GCP2, respectively. The remaining GCP chromosomes showed more complex hybridization patterns with homologies to other GCA pairs. These results demonstrate that the level of reorganization in the genome of G. capanema is much greater than in GCA, 2n = 42 and in karyomorph GCA, 2n = 40 which was previously analyzed by chromosome painting.

A phylogenetic analysis using multidirectional chromosome painting of three species (Uroderma magnirostrum, U. bilobatum and Artibeus obscurus) of subfamily Stenodermatinae (Chiroptera-Phyllostomidae).

The species of genera Uroderma and Artibeus are medium-sized bats belonging to the family Phyllostomidae and subfamily Stenodermatinae (Mammalia, Chiroptera) from South America. They have a wide distribution in the Neotropical region, with two currently recognized species in Uroderma and approximately 20 species in Artibeus. These two genera have different rates of chromosome evolution, with Artibeus probably having retained the ancestral karyotype for the subfamily. We used whole chromosome paint probe sets from Carollia brevicauda and Phyllostomus hastatus on Uroderma magnirostrum, Uroderma bilobatum, and Artibeus obscurus. With the aim of testing the previous phylogenies of these bats using cytogenetics, we compared these results with published painting maps on Phyllostomidae. The genome-wide comparative maps based on chromosome painting and chromosome banding reveal the chromosome forms that characterize each taxonomic level within the Phyllostomidae and show the chromosome evolution of this family. Based on this, we are able to suggest an ancestral karyotype for Phyllostomidae. Our cladistic analysis is an independent confirmation using multidirectional chromosome painting of the previous Phyllostomidae phylogenies.

Cytogenetic mapping of H1 histone and ribosomal RNA genes in hybrids between catfish species Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum.

A physical chromosome mapping of the H1 histone and 5S and 18S ribosomal RNA (rRNA) genes was performed in interspecific hybrids of Pseudoplatystoma corruscans and P. reticulatum. The results showed that 5S rRNA clusters were located in the terminal region of 2 chromosomes. H1 histone and 18S ribosomal genes were co-localized in the terminal portion of 2 chromosomes (distinct from the chromosomes bearing 5S clusters). These results represent the first report of association between H1 histone and 18S genes in fish genomes. The chromosome clustering of ribosomal and histone genes was already reported for different organisms and suggests a possible selective pressure for the maintenance of this association.

A short introduction to cytogenetic studies in mammals with reference to the present volume.

Genome diversity has long been studied from the comparative cytogenetic perspective. Early workers documented differences between species in diploid chromosome number and fundamental number. Banding methods allowed more detailed descriptions of between-species rearrangements and classes of differentially staining chromosome material. The infusion of molecular methods into cytogenetics provided a third revolution, which is still not exhausted. Chromosome painting has provided a global view of the translocation history of mammalian genome evolution, well summarized in the contributions to this special volume. More recently, FISH of cloned DNA has provided details on defining breakpoint and intrachromosomal marker order, which have helped to document inversions and centromere repositioning. The most recent trend in comparative molecular cytogenetics is to integrate sequencing information in order to formulate and test reconstructions of ancestral genomes and phylogenomic hypotheses derived from comparative cytogenetics. The integration of comparative cytogenetics and sequencing promises to provide an understanding of what drives chromosome rearrangements and genome evolution in general. We believe that the contributions in this volume, in no small way, point the way to the next phase in cytogenetic studies.

Extensive homology of chicken macrochromosomes in the karyotypes of Trachemys scripta elegans and Crocodylus niloticus revealed by chromosome painting despite long divergence times.

We report extensive chromosome homology revealed by chromosome painting between chicken (Gallus gallus domesticus, GGA, 2n = 78) macrochromosomes (representing 70% of the chicken genome) and the chromosomes of a turtle, the red-eared slider (Trachemys scripta elegans, TSC, 2n = 50), and the Nile crocodile (Crocodylus niloticus, CNI, 2n = 32). Our data show that GGA1-8 arms seem to be conserved in the arms of TSC chromosomes, GGA1-2 arms are separated and homologous to CNI1p, 3q, 4q and 5q. In addition to GGAZ homologues in our previous study, large-scale GGA autosome syntenies have been conserved in turtle and crocodile despite hundreds of millions of years divergence time. Based on phylogenetic hypotheses that crocodiles diverged after the divergence of birds and turtles, our results in CNI suggest that GGA1-2 and TSC1-2 represent the ancestral state and that chromosome fissions followed by fusions have been the mechanisms responsible for the reduction of chromosome number in crocodiles.

Karyotype evolution of eulipotyphla (insectivora): the genome homology of seven sorex species revealed by comparative chromosome painting and banding data.

The genus Sorex is one of the most successful genera of Eulipotyphla. Species of this genus are characterized by a striking chromosome variability including XY1Y2 sex chromosome systems and exceptional chromosomal polymorphisms within and between populations. To study chromosomal evolution of the genus in detail, we performed cross-species chromosome painting of 7 Sorex species with S. granarius and S. araneus whole-chromosome probes and found that the tundra shrew S. tundrensis has the most rearranged karyotype among these. We reconstructed robust phylogeny of the genus Sorex based on revealed conserved chromosomal segments and syntenic associations. About 16 rearrangements led to formation of 2 major Palearctic groups after their divergence from the common ancestor: the S. araneus group (10 fusions and 1 fission) and the S. minutus group (5 fusions). Further chromosomal evolution of the 12 species inside the groups, including 5 previously investigated species, was accompanied by multiple reshuffling events: 39 fusions, 20 centromere shifts and 10 fissions. The rate of chromosomal exchanges upon formation of the genus was close to the average rate for eutherians, but increased during recent (about 6-3 million years ago) speciation within Sorex. We propose that a plausible ancestral Sorex karyotype consists of 56 elements. It underwent 20 chromosome rearrangements from the boreoeutherian ancestor, with 14 chromosomes retaining the conserved state. The set of genus-specific chromosome signatures was drawn from the human (HSA)-shrew comparative map (HSA3/12/22, 8/19/3/21, 2/13, 3/18, 11/17, 12/15 and 1/12/22). The syntenic association HSA4/20, that was previously proposed as a common trait of all Eulipotyphla species, is shown here to be an apomorphic trait of S. araneus.

Chromosome mapping of H1 histone and 5S rRNA gene clusters in three species of Astyanax (Teleostei, Characiformes).

We report here on the physical mapping of the H1 histone genes (hisDNA) and the 5S ribosomal DNA (rDNA) in 3 Neotropical fish species of the genus Astyanax(A. altiparanae, A. bockmanni and A. fasciatus) and the comparative analysis of the chromosomes bearing these genes. Nucleotide analyses by sequencing of both genes were also performed. The distribution of the H1 histone genes was more conserved than that of the rRNA genes, since these were always located in the pericentromeric regions of 2 chromosome pairs. 5S rDNA was found on one of the pairs that presented an H1 histone cluster; this seems to be a conserved chromosomal feature of the genus Astyanax. In addition, individuals of A. bockmanni and A. fasciatus showed clusters of 5S rDNA on 1 pair of acrocentric chromosomes, not found in A. altiparanae. The results obtained by chromosome mapping as well as by sequencing of both genes showed that A.bockmanni is more closely related to A. fasciatus than to A. altiparanae. The results allow the characterization of cytogenetic markers for improved elucidation of the processes involved in karyotype differentiation of fish genomes.

Chromosomal homologies among vampire bats revealed by chromosome painting (phyllostomidae, chiroptera).

Substantial effort has been made to elucidate karyotypic evolution of phyllostomid bats, mostly through comparisons of G-banding patterns. However, due to the limited number of G-bands in respective karyotypes and to the similarity of non-homologous bands, an accurate evolutionary history of chromosome segments remains questionable. This is the case for vampire bats (Desmodontinae). Despite several proposed homologies, banding data have not yet provided a detailed understanding of the chromosomal changes within vampire genera. We examined karyotype differentiation of the 3 species within this subfamily using whole chromosomal probes from Phyllostomus hastatus (Phyllostominae) and Carollia brevicauda (Carolliinae). Painting probes of P. hastatus respectively detected 22, 21 and 23 conserved segments in Diphylla ecaudata, Diaemus youngi, and Desmodus rotundus karyotypes, whereas 27, 27 and 28 were respectively detectedwith C. brevicauda paints. Based on the evolutionary relationships proposed by morphological and molecular data, we present probable chromosomal synapomorphies for vampire bats and propose chromosomes that were present in the common ancestor of the 5 genera analyzed. Karyotype comparisons allowed us to relate a number of conserved chromosomal segments among the 5 species, providing a broader database for understanding karyotype evolution in the family.

Geographic patterns of inversion polymorphisms in a wild African rodent, Mastomys erythroleucus.

By suppressing recombination and reducing gene flow, chromosome inversions favor the capture and protection of advantageous allelic combinations, leading to adaptive polymorphisms. However, studies in non-model species remain scarce. Here we investigate the distribution of inversion polymorphisms in the multimammate rat Mastomys erythroleucus in West Africa. More than 270 individuals from 52 localities were karyotyped using G-bands and showed widespread polymorphisms involving four chromosome pairs. No significant deviations from Hardy-Weinberg equilibrium were observed either through space or time, nor were differences retrieved in viability or sex contribution between cytotypes. The distribution of chromosomal variation, however, showed perfect congruence with that of mtDNA-based phylogeographic clades. Thus, inversion diversity patterns in M. erythroleucus appeared more related to historical and/or demographic processes than to climate-based adaptive features. Using cross-species chromosome painting and G-banding analyses to identify homologous chromosomes in related out-group species, we proposed a phylogenetic scenario that involves ancestral-shared polymorphisms and subsequent lineage sorting during expansion/contraction of West African savannas. Our data suggest that long-standing inversion polymorphisms may act as regions in which adaptation genes may accumulate (nucleation model).

The unique sex chromosome system in platypus and echidna.

A striking example of the power of chromosome painting has been the resolution of the male platypus karyotype and the pairing relationships of the chain often sex chromosomes. We have extended our analysis to the nine sex chromosomes of the male echidna. Cross-species painting with platypus shows that the first five chromosomes in the chain are identical in both, but the order of the remainder are different and, in each species, a different autosome replaces one of the five X chromosomes. As the therian X is homologous mainly to platypus autosome 6 and echidna 16, and as SRY is absent in both, the sex determination mechanism in monotremes is currently unknown. Several of the X and Y chromosomes contain genes orthologous to those in the avian Z but the significance of this is also unknown. It seems likely that a novel testis determinant is carried by a Y chromosome common to platypus and echidna. We have searched for candidates for this determinant among the many genes known to be involved in vertebrate sex differentiation. So far fourteen such genes have been mapped, eleven are autosomal in platypus, two map to the differential regions of X chromosomes, and one maps to a pairing segment and is likewise excluded. Search for the platypus testis-determining gene continues, and the extension of comparative mapping between platypus and birds and reptiles may shed light on the ancestral origin of monotreme sex chromosomes.

Neo-XY body: an analysis of XY1Y2 meiotic behavior in Carollia (Chiroptera, Phyllostomidae) by chromosome painting.

Classical and molecular cytogenetic analyses of mitotic and meiotic cells were performed on two species of Carollia from the family Phyllostomidae (Chiroptera), which have an XX/XY(1)Y(2) sex determination system. Our results show that the species Carollia perspicillata and Carollia brevicauda have the same Xq-autosome translocation (neo-X). Using multicolor FISH we observed different levels of condensation of the original X and Y chromosomes when compared to the translocated autosomal segment, a likely consequence of the nucleolar organizer region blocking spreading of inactivation to the autosomal region of the neo-X. The use of chromosome painting showed the behavior of the sex chromosome trivalent--here called the 'neo-XY body'--in meiosis. We compared the variation between the condensation of the original X and Y and the autosome-sex chromosome axis and described the pairing between the original X-Y segments (pseudoautosomal region) and the XY(2) homologous segments, suggesting genetic activity of the latter during meiosis.

Skinks (Reptilia: Scincidae) have highly conserved karyotypes as revealed by chromosome painting.

Skinks represent the most diversified squamate reptiles with a great variation in body size and form, and are found worldwide in a variety of habitats. Their remarkable diversification has been accompanied by only a few chromosome rearrangements, resulting in highly-conservative chromosomal complements of these lizards. In this study cross-species chromosome painting using Scincus scincus (2n = 32) as the source genome, was used to detect the chromosomal rearrangements and homologies between the following skinks: Chalcides chalcides (2n = 28), C. ocellatus (2n = 28), Eumeces schneideri (2n = 32), Lepidothyris fernandi (2n = 30), Mabuya quinquetaeniata (2n = 32). The results of this study confirmed a high degree of chromosome conservation between these species. The main rearrangements in the studied skinks involve chromosomes 3, 5, 6 and 7 of S. scincus. These subtelocentric chromosomes are homologous to the p and q arms of metacentric pair 3 and 4 in C. chalcides, C. ocellatus, L. fernandi, and M. quinquetaeniata, while they are entirely conserved in E. schneideri. Other rearrangements involve S. scincus 11 in L. fernandi and M. quinquetaeniata, supporting the monophyly of Lygosominae, and one of the chromosomes S. scincus 12-16, in M. quinquetaeniata. In conclusion, our data support the monophyly of Scincidae and confirm that Scincus-Eumeces plus Chalcides do not form a monophyletic clade, suggesting that the Scincus-Eumeces clade is basal to other members of this family. This study represents the first time the whole genome of any reptile species has been used for cross-species chromosome painting to assess chromosomal evolution in this group of vertebrates.

Comparative chromosome painting between chicken and spectacled owl (Pulsatrix perspicillata): implications for chromosomal evolution in the Strigidae (Aves, Strigiformes).

The spectacled owl (Pulsatrix perspicillata), a species found in the Neotropical region, has 76 chromosomes, with a high number of biarmed chromosomes. In order to define homologies between Gallus gallus and Pulsatrixperspicillata (Strigiformes, Strigidae), we used chromosome painting with chicken DNA probes of chromosomes 1-10 and Z and telomeric sequences. This approach allowed a comparison between Pulsatrixperspicillata and other species of Strigidae already analyzed by chromosome painting (Strix nebulosa and Bubo bubo, both with 2n = 80). The results show that centric fusions and fissions have occurred in different chromosomal pairs and are responsible for the karyotypic variation observed in this group. No interstitial telomeric sequences were found. Although the largest pair of chromosomes in P. perspicillata and Bubo bubo are submetacentric, they are homologous to different chicken chromosomes: GGA1/GGA2 in P. perspicillata and GGA2/GGA4 in B. bubo.

Cytogenetic studies and karyotype nomenclature of three wild canid species: maned wolf (Chrysocyon brachyurus), bat-eared fox (Otocyon megalotis) and fennec fox (Fennecus zerda).

We have analysed the chromosomes of three wild and endangered canid species: the maned wolf (Chrysocyon brachyurus), the bat-eared fox (Otocyon megalotis) and the fennec fox (Fennecuszerda) using classical and molecular cytogenetic methods. For the first time detailed and encompassing descriptions of the chromosomes are presented including the chromosomal assignment of nucleolar organizer regions and the 5S rRNA gene cluster. We propose a karyotype nomenclature with ideograms including more than 300 bands per haploid set for each of these three species which will form the basis for further research. In addition, we propose four basic different patterns of karyotype organization in the family Canidae. A comparison of these patterns with the most recent molecular phylogeny of Canidae revealed that the karyotype evolution of a species is not always strongly connected with its phylogenetic position. Our findings underline the need and justification for basic cytogenetic work in rare and exotic species.

Non-homologous sex chromosomes in two species of the genus Eigenmannia (Teleostei: Gymnotiformes).

The Neotropical genus Eigenmannia is a fish group with unknown species diversity where representatives possess a broad range of chromosomal sex determining systems namely XY/XX, X(1)X(2)Y/X(1)X(1)X(2)X(2), ZZ/ZW as well as homomorphic sex chromosomes. To test the homology of two heteromorphic XY sex chromosome systems present in two sympatric populations, reciprocal cross-species FISH experiments were performed using probes derived by microdissection of X and Y chromosomes present in analyzed specimens of Eigenmannia virescens and Eigenmannia sp.2, respectively. While X and Y paint probes hybridized to species-specific sex chromosomes, in reciprocal cross-FISH both probes hybridized exclusively to autosomes. The result suggests multiple independent origins of the XY systems in the analyzed populations.

Chromosomal evolution in tenrecs (Microgale and Oryzorictes, Tenrecidae) from the Central Highlands of Madagascar.

Tenrecs (Tenrecidae) are a widely diversified assemblage of small eutherian mammals that occur in Madagascar and Western and Central Africa. With the exception of a few early karyotypic descriptions based on conventional staining, nothing is known about the chromosomal evolution of this family. We present a detailed analysis of G-banded and molecularly defined chromosomes based on fluorescence in situ hybridization (FISH) that allows a comprehensive comparison between the karyotypes of 11 species of two closely related Malagasy genera, Microgale (10 species) and Oryzorictes (one species), of the subfamily Oryzorictinae. The karyotypes of Microgale taiva and M. parvula (2n = 32) were found to be identical to that of O. hova (2n = 32) most likely reflecting the ancestral karyotypes of both genera, as well as that of the Oryzorictinae. Parsimony analysis of chromosomal rearrangements that could have arisen following Whole Arm Reciprocal Translocations (WARTs) showed, however, that these are more likely to be the result of Robertsonian translocations. A single most parsimonious tree was obtained that provides strong support for three species associations within Microgale, all of which are consistent with previous molecular and morphological investigations. By expanding on a recently published molecular clock for the Tenrecidae we were able to place our findings in a temporal framework that shows strong chromosomal rate heterogeneity within the Oryzorictinae. We use these data to critically examine the possible role of chromosomal rearrangements in speciation within Microgale.