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Charadriiformes represent one of the largest orders of birds; members of this order are diverse in morphology, behavior and reproduction, making them an excellent model for studying evolution. It is accepted that the avian putative ancestral karyotype, with 2n = 80, remains conserved for about 100 million years. So far, only a few species of Charadriiformes have been studied using molecular cytogenetics. Here, we performed chromosome painting on metphase chromosomes of two species of Charadriidae, Charadrius collaris and Vanellus chilensis, with whole chromosome paint probes from Burhinus oedicnemus. Charadrius collaris has a diploid number of 76, with both sex chromosomes being submetacentric. In V. chilensi a diploid number of 78 was identified, and the Z chromosome is submetacentric. Chromosome painting suggests that chromosome conservation is a characteristic common to the family Charadriidae. The results allowed a comparative analysis between the three suborders of Charadriiformes and the order Gruiformes using chromosome rearrangements to understand phylogenetic relationships between species and karyotypic evolution. However, the comparative analysis between the Charadriiformes suborders so far has not revealed any shared rearrangements, indicating that each suborder follows an independent evolutionary path, as previously proposed. Likewise, although the orders Charadriiformes and Gruiformes are placed on sister branches, they do not share any signature chromosomal rearrangements.

Tandemly arranged and dispersed repetitive DNA sequences are important structural and functional elements that make up a significant portion of vertebrate genomes. Using high throughput, low coverage whole genome sequencing followed by bioinformatics analysis, we have identified seven major tandem repetitive DNAs and two fragments of LTR retrotransposons in the genome of the Nile crocodile (

X-autosome translocation (XY1Y2) has been reported in distinct groups of vertebrates suggesting that the rise of a multiple sex system within a species may act as a reproductive barrier and lead to speciation. The viability of this system has been linked with repetitive sequences located between sex and autosomal portions of the translocation. Herein, we investigate Oecomys auyantepui, using chromosome banding and Fluorescence In Situ Hybridization with telomeric and Hylaeamys megacephalus whole-chromosome probes, and phylogenetic reconstruction using mtDNA and nuDNA sequences. We describe an amended karyotype for O. auyantepui (2n = 64♀65♂/FNa = 84) and report for the first time a multiple sex system (XX/XY1Y2) in Oryzomyini rodents. Molecular data recovered O. auyantepui as a monophyletic taxon with high support and cytogenetic data indicate that O. auyantepui may exist in two lineages recognized by distinct sex systems. The Neo-X exhibits repetitive sequences located between sex and autosomal portions, which would act as a boundary between these two segments. The G-banding comparisons of the Neo-X chromosomes of other Sigmodontinae taxa revealed a similar banding pattern, suggesting that the autosomal segment in the Neo-X can be shared among the Sigmodontinae lineages with a XY1Y2 sex system.

X-autosome translocation (XY

Rhipidomys (Sigmodontinae, Thomasomyini) has 25 recognized species, with a wide distribution ranging from eastern Panama to northern Argentina. Cytogenetic data has been described for 13 species with 12 of them having 2n = 44 with a high level of autosomal fundamental number (FN) variation, ranging from 46 to 80, assigned to pericentric inversions. The species are grouped in groups with low FN (46–52) and high FN (72–80). In this work the karyotypes of Rhipidomys emiliae (2n = 44, FN = 50) and Rhipidomys mastacalis (2n = 44, FN = 74), were studied by classical cytogenetics and by fluorescence in situ hybridization using telomeric and whole chromosome probes (chromosome painting) of Hylaeamys megacephalus (HME). Chromosome painting revealed homology between 36 segments of REM and 37 of RMA. We tested the hypothesis that pericentric inversions are the predominant chromosomal rearrangements responsible for karyotypic divergence between these species, as proposed in literature. Our results show that the genomic diversification between the karyotypes of the two species resulted from translocations, centromeric repositioning and pericentric inversions. The chromosomal evolution in Rhipidomys was associated with karyotypical orthoselection. The HME probes revealed that seven syntenic probably ancestral blocks for Sigmodontinae are present in Rhipidomys. An additional syntenic block described here is suggested as part of the subfamily ancestral karyotype. We also define five synapomorphies that can be used as chromosomal signatures for Rhipidomys.

Whole-chromosome fusions play a major role in the karyotypic evolution of reptiles. It has been suggested that certain chromosomes tend to fuse with sex chromosomes more frequently than others. However, the comparative genomic synteny data are too scarce to draw strong conclusions. We obtained and sequenced chromosome-specific DNA pools of Sceloporus malachiticus, an iguanian species which has experienced many chromosome fusions. We found that four of seven lineage-specific fusions involved sex chromosomes, and that certain syntenic blocks which constitute the sex chromosomes, such as the homologues of the Anolis carolinensis chromosomes 11 and 16, are repeatedly involved in sex chromosome formation in different squamate species. To test the hypothesis that the karyotypic shift could be associated with changes in recombination patterns, we performed a synaptonemal complex analysis in this species and in Sceloporus variabilis (2n = 34). It revealed that the sex chromosomes in S. malachiticus had two distal pseudoautosomal regions and a medial differentiated region. We found that multiple fusions little affected the recombination rate in S. malachiticus. Our data confirm more frequent involvement of certain chromosomes in sex chromosome formation, but do not reveal a connection between the gonosome–autosome fusions and the evolution of recombination rate. This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)’.

Universidade Federal do Par?. Programa de P?s-Gradua??o em Biodiversidade e Biotecnologia da Rede Bionorte. Bel?m, PA, Brazil. Universidade Federal do Par?. Programa de P?s-Gradua??o em Gen?tica e Biologia Molecular. Bel?m, PA, Brazil / University of Cambridge. Cambridge Resource Centre for Comparative Genomics. Department of Veterinary Medicine. Cambridge, UK. Universidade Federal do Par?. Programa de P?s-Gradua??o em Gen?tica e Biologia Molecular. Bel?m, PA, Brazil. Universidade Federal do Par?. Programa de P?s-Gradua??o em Gen?tica e Biologia Molecular. Bel?m, PA, Brazil. Universidade Federal do Par?. Programa de P?s-Gradua??o em Neuroci?ncias e Biologia Celular. Bel?m, PA, Brazil. University of Cambridge. Cambridge Resource Centre for Comparative Genomics. Department of Veterinary Medicine. Cambridge, UK. University of Cambridge. Cambridge Resource Centre for Comparative Genomics. Department of Veterinary Medicine. Cambridge, UK. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Cultura de Tecidos e Citogen?tica. Ananindeua, PA, Brasil / Universidade Federal do Par?. Faculdade de Ci?ncias Naturais. Bel?m, PA, Brazil. Despite the variation observed in the diploid chromosome number of storks (Ciconiiformes, Ciconiidae), from 2n = 52 to 2n = 78, most reports have relied solely on analyses by conventional staining. As most species have similar macrochromosomes, some authors propose that karyotype evolution involves mainly fusions between microchromosomes, which are highly variable in species with different diploid numbers. In order to verify this hypothesis, in this study, the karyotypes of 2 species of storks from South America with different diploid numbers, the jabiru (Jabiru mycteria, 2n = 56) and the maguary stork (Ciconia maguary, 2n = 72), were analyzed by chromosome painting using whole chromosome probes from the macrochromosomes of Gallus gallus (GGA) and Leucopternis albicollis (LAL). The results revealed that J. mycteria and C. maguary share synteny within chromosome pairs 1-9 and Z. The syntenies to the macrochromosomes of G. gallus are conserved, except for GGA4, which is homologous to 2 different pairs, as in most species of birds. A fusion of GGA8 and GGA9 was observed in both species. Additionally, chromosomes corresponding to GGA4p and GGA6 are fused to other segments that did not hybridize to any of the macrochromosome probes used, suggesting that these segments correspond to microchromosomes. Hence, our data corroborate the proposed hypothesis that karyotype evolution is based on fusions involving microchromosomes. In view of the morphological constancy of the macrochromosome pairs in most Ciconiidae, we propose a putative ancestral karyotype for the family, including the GGA8/GGA9 fusion, and a diploid number of 2n = 78. The use of probes for microchromosome pairs should be the next step in identifying other synapomorphies that may help to clarify the phylogeny of this family.

The taxonomy of the genus Calomyscus remains controversial. According to the latest systematics the genus includes eight species with great karyotypic variation. Here, we studied karyotypes of 14 Calomyscus individuals from different regions of Iran and Turkmenistan using a new set of chromosome painting probes from a Calomyscus sp. male (2n = 46, XY; Shahr-e-Kord-Soreshjan-Cheshme Maiak Province). We showed the retention of large syntenic blocks in karyotypes of individuals with identical chromosome numbers. The only rearrangement (fusion 2/21) differentiated Calomyscus elburzensis, Calomyscus mystax mystax, and Calomyscus sp. from Isfahan Province with 2n = 44 from karyotypes of C. bailwardi, Calomyscus sp. from Shahr-e-Kord, Chahar Mahal and Bakhtiari-Aloni, and Khuzestan-Izeh Provinces with 2n = 46. The individuals from Shahdad tunnel, Kerman Province with 2n = 51–52 demonstrated non-centric fissions of chromosomes 4, 5, and 6 of the 46-chromosomal form with the formation of separate small acrocentrics. A heteromorphic pair of chromosomes in a specimen with 2n = 51 resulted from a fusion of two autosomes. C-banding and chromomycin A3-DAPI staining after G-banding showed extensive heterochromatin variation between individuals.

Background The Scolopacidae family (Suborder Scolopaci, Charadriiformes) is composed of sandpipers and snipes; these birds are long-distance migrants that show great diversity in their behavior and habitat use. Cytogenetic studies in the Scolopacidae family show the highest diploid numbers for order Charadriiformes. This work analyzes for the first time the karyotype of Actitis macularius by classic cytogenetics and chromosome painting. Results The species has a diploid number of 92, composed mostly of telocentric pairs. This high 2n is greater than the proposed 80 for the avian ancestral putative karyotype (a common feature among Scolopaci), suggesting that fission rearrangements have formed smaller macrochromosomes and microchromosomes. Fluorescence in situ hybridization using Burhinus oedicnemus whole chromosome probes confirmed the fissions in pairs 1, 2, 3, 4 and 6 of macrochromosomes. Conclusion Comparative analysis with other species of Charadriiformes studied by chromosome painting together with the molecular phylogenies for the order allowed us to raise hypotheses about the chromosomal evolution in suborder Scolopaci. From this, we can establish a clear idea of how chromosomal evolution occurred in this suborder.

The unique evolutionary dynamics and complex structure make the Y chromosome the most diverse and least understood region in the mammalian genome, despite its undisputable role in sex determination, development, and male fertility. Here we present the first contig-level annotated draft assembly for the alpaca (Vicugna pacos) Y chromosome based on hybrid assembly of short- and long-read sequence data of flow-sorted Y. The latter was also used for cDNA selection providing Y-enriched testis transcriptome for annotation. The final assembly of 8.22 Mb comprised 4.5 Mb of male specific Y (MSY) and 3.7 Mb of the pseudoautosomal region. In MSY, we annotated 15 X-degenerate genes and two novel transcripts, but no transposed sequences. Two MSY genes, HSFY and RBMY, are multicopy. The pseudoautosomal boundary is located between SHROOM2 and HSFY. Comparative analysis shows that the small and cytogenetically distinct alpaca Y shares most of MSY sequences with the larger dromedary and Bactrian camel Y chromosomes. Most of alpaca X-degenerate genes are also shared with other mammalian MSYs, though WWC3Y is Y-specific only in alpaca/camels and the horse. The partial alpaca Y assembly is a starting point for further expansion and will have applications in the study of camelid populations and male biology.

The unique evolutionary dynamics and complex structure make the Y chromosome the most diverse and least understood region in the mammalian genome, despite its undisputable role in sex determination, development, and male fertility. Here we present the first contig-level annotated draft assembly for the alpaca (Vicugna pacos) Y chromosome based on hybrid assembly of short- and long-read sequence data of flow-sorted Y. The latter was also used for cDNA selection providing Y-enriched testis transcriptome for annotation. The final assembly of 8.22 Mb comprised 4.5 Mb of male specific Y (MSY) and 3.7 Mb of the pseudoautosomal region. In MSY, we annotated 15 X-degenerate genes and two novel transcripts, but no transposed sequences. Two MSY genes, HSFY and RBMY, are multicopy. The pseudoautosomal boundary is located between SHROOM2 and HSFY. Comparative analysis shows that the small and cytogenetically distinct alpaca Y shares most of MSY sequences with the larger dromedary and Bactrian camel Y chromosomes. Most of alpaca X-degenerate genes are also shared with other mammalian MSYs, though WWC3Y is Y-specific only in alpaca/camels and the horse. The partial alpaca Y assembly is a starting point for further expansion and will have applications in the study of camelid populations and male biology.

The mandarin vole, Lasiopodomys mandarinus, is one of the most intriguing species among mammals with non-XX/XY sex chromosome system. It combines polymorphism in diploid chromosome numbers, variation in the morphology of autosomes, heteromorphism of X chromosomes, and several sex chromosome systems the origin of which remains unexplained. Here we elucidate the sex determination system in Lasiopodomys mandarinus vinogradovi using extensive karyotyping, crossbreeding experiments, molecular cytogenetic methods, and single chromosome DNA sequencing. Among 205 karyotyped voles, one male and three female combinations of sex chromosomes were revealed. The chromosome segregation pattern and karyomorph-related reproductive performances suggested an aberrant sex determination with almost half of the females carrying neo-X/neo-Y combination. The comparative chromosome painting strongly supported this proposition and revealed the mandarin vole sex chromosome systems originated due to at least two de novo autosomal translocations onto the ancestral X chromosome. The polymorphism in autosome 2 was not related to sex chromosome variability and was proved to result from pericentric inversions. Sequencing of microdissection derived of sex chromosomes allowed the determination of the coordinates for syntenic regions but did not reveal any Y-specific sequences. Several possible sex determination mechanisms as well as interpopulation karyological differences are discussed.

The Columbidae species (Aves, Columbiformes) show considerable variation in their diploid numbers (2n = 68&ndash, 86), but there is limited understanding of the events that shaped the extant karyotypes. Hence, we performed whole chromosome painting (wcp) for paints GGA1-10 and bacterial artificial chromosome (BAC) probes for chromosomes GGA11-28 for Columbina passerina, Columbina talpacoti, Patagioenas cayennensis, Geotrygon violacea and Geotrygon montana. Streptopelia decaocto was only investigated with paints because BACs for GGA10-28 had been previously analyzed. We also performed phylogenetic analyses in order to trace the evolutionary history of this family in light of chromosomal changes using our wcp data with chicken probes and from Zenaida auriculata, Columbina picui, Columba livia and Leptotila verreauxi, previously published. G-banding was performed on all these species. Comparative chromosome paint and G-banding results suggested that at least one interchromosomal and many intrachromosomal rearrangements had occurred in the diversification of Columbidae species. On the other hand, a high degree of conservation of microchromosome organization was observed in these species. Our cladistic analysis, considering all the chromosome rearrangements detected, provided strong support for L. verreauxi and P. cayennensis, G. montana and G. violacea, C. passerina and C. talpacoti having sister taxa relationships, as well as for all Columbidae species analyzed herein. Additionally, the chromosome characters were mapped in a consensus phylogenetic topology previously proposed, revealing a pericentric inversion in the chromosome homologous to GGA4 in a chromosomal signature unique to small New World ground doves.

This work was supported by the Conselho Nacional de Desenvolvimento Cient??fico e Tecnolo?gico (CNPq) Grant number 307382/2019- 02 to EHCO and the Coordenac??o de Aperfeic?oamento de Pessoal de N??vel Superior (CAPES) Universidade Federal do Par?. Programa de P?s-gradua??o em Gen?tica e Biologia Molecular. Bel?m, PA, Brazil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Cultura de Tecidos e Citogen?tica. Ananindeua, PA, Brasil. Universidade Federal do Rio Grande do Sul. Programa de P?s-gradua??o em Gen?tica e Biologia Molecular. Porto Alegre, RS, Brazil. Universidade Federal do Par?. Programa de P?s-gradua??o em Gen?tica e Biologia Molecular. Bel?m, PA, Brazil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Cultura de Tecidos e Citogen?tica. Ananindeua, PA, Brasil. Universidade Federal do Pampa, Programa de P?s-gradua??o em Ci?ncias Biol?gicas. S?o Gabriel, RS, Brazil. Universidade Federal do Pampa, Programa de P?s-gradua??o em Ci?ncias Biol?gicas. S?o Gabriel, RS, Brazil. Universidade Federal de Minas Gerais. Instituto de Ci?ncias Biol?gicas. Departamento de Gen?tica, Ecologia e Evolu??o, Laborat?rio de Citogen?mica Evolutiva. Belo Horizonte, MG, Brazil. University of Cambridge. Cambridge Resource Centre for Comparative Genomics. Department of Veterinary Medicine. Cambridge, United Kingdom. University of Cambridge. Cambridge Resource Centre for Comparative Genomics. Department of Veterinary Medicine. Cambridge, United Kingdom. Universidade Federal do Par?. Programa de P?s-gradua??o em Gen?tica e Biologia Molecular. Bel?m, PA, Brazil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Cultura de Tecidos e Citogen?tica. Ananindeua, PA, Brasil / Universidade Federal do Par?. Instituto de Ci?ncias Exatas e Naturais. Bel?m, PA, Brazil. The Cuckoos have a long history of difficult classification. The species of this order have been the subject of several studies based on osteology, behavior, ecology, morphology and molecular data. Despite this, the relationship between Cuculiformes and species of other orders remains controversial. In this work, two species of Cuculidae, Guira guira (Gmelin, 1788) and Piaya cayana (Linnaeus, 1766), were analyzed by means of comparative chromosome painting in order to study the chromosome evolution of this group and to undertake the first chromosome mapping of these species. Our results demonstrate high chromosomal diversity, with 2n = 76 in G. guira, with fission and fusion events involving ancestral syntenies, while P. cayana presented only fissions, which were responsible for the high diploid number of 2n = 90. Interestingly, there were no chromosomal rearrangements in common between these species. Our results, based on Giemsa staining, were compared with previous data for other cuckoos and also with taxa proposed as sister-groups of Cuculiformes (Otidiformes, Musophagiformes and Opisthocomiformes). Cytogenetic comparisons demonstrated that cuckoo species can be divided into at least three major groups. In addition, we found no evidence to place Cuculiformes close to the groups proposed previously as sister-groups.

This research was partially funded by a grant to EHCO from CNPq (307382/2019-2) and to MAFS from the Wellcome Trust in support of the Cambridge Resource Centre for comparative genomics Federal University of Par?. Post-Graduation Program in Genetics and Molecular Biology. Bel?m, PA, Brazil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Cultura de Tecido e Citogen?tica. Ananindeua, PA, Brasil / Cambridge Resource Centre for Comparative Genomics. Cambridge, UK. Cambridge Resource Centre for Comparative Genomics. Cambridge, UK / Federal University of Rio Grande do Sul. Pos-Graduation Program in Genetics and Molecular Biology. Porto Alegre, RS, Brazil. Cambridge Resource Centre for Comparative Genomics. Cambridge, UK. Cambridge Resource Centre for Comparative Genomics. Cambridge, UK. Cambridge Resource Centre for Comparative Genomics. Cambridge, UK. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Cultura de Tecido e Citogen?tica. Ananindeua, PA, Brasil / Federal University of Par?. Faculty of Natural Sciences. Institute of Exact and Natural Sciences. Bel?m, PA, Brazil. Abstract: Gruiformes is a group with phylogenetic issues. Recent studies based on mitochondrial and genomic DNA have proposed the existence of a core Gruiformes, consisting of five families: Heliornithidae, Aramidae, Gruidae, Psophiidae and Rallidae. Karyotype studies on these species are still scarce, either by conventional staining or molecular cytogenetics. Due to this, this study aimed to analyze the karyotype of two species (Aramides cajaneus and Psophia viridis) belonging to families Rallidae and Psopiidae, respectively, by comparative chromosome painting. The results show that some chromosome rearrangements in this group have different origins, such as the association of GGA5/GGA7 in A. cajaneus, as well as the fission of GGA4p and association GGA6/GGA7, which place P. viridis close to Fulica atra and Gallinula chloropus. In addition, we conclude that the common ancestor of the core Gruiformes maintained the original syntenic groups found in the putative avian ancestral karyotype

Background Thamnophilidae birds are the result of a monophyletic radiation of insectivorous Passeriformes. They are a diverse group of 225 species and 45 genera and occur in lowlands and lower montane forests of Neotropics. Despite the large degree of diversity seen in this family, just four species of Thamnophilidae have been karyotyped with a diploid number ranging from 76 to 82 chromosomes. The karyotypic relationships within and between Thamnophilidae and another Passeriformes therefore remain poorly understood. Recent studies have identified the occurrence of intrachromosomal rearrangements in Passeriformes using in silico data and molecular cytogenetic tools. These results demonstrate that intrachromosomal rearrangements are more common in birds than previously thought and are likely to contribute to speciation events. With this in mind, we investigate the apparently conserved karyotype of Willisornis vidua, the Xingu Scale-backed Antbird, using a combination of molecular cytogenetic techniques including chromosome painting with probes derived from Gallus gallus (chicken) and Burhinus oedicnemus (stone curlew), combined with Bacterial Artificial Chromosome (BAC) probes derived from the same species. The goal was to investigate the occurrence of rearrangements in an apparently conserved karyotype in order to understand the evolutionary history and taxonomy of this species. In total, 78 BAC probes from the Gallus gallus and Taeniopygia guttata (the Zebra Finch) BAC libraries were tested, of which 40 were derived from Gallus gallus macrochromosomes 1–8, and 38 from microchromosomes 9–28. Results The karyotype is similar to typical Passeriformes karyotypes, with a diploid number of 2n = 80. Our chromosome painting results show that most of the Gallus gallus chromosomes are conserved, except GGA-1, 2 and 4, with some rearrangements identified among macro- and microchromosomes. BAC mapping revealed many intrachromosomal rearrangements, mainly inversions, when comparing Willisornis vidua karyotype with Gallus gallus, and corroborates the fissions revealed by chromosome painting. Conclusions Willisornis vidua presents multiple chromosomal rearrangements despite having a supposed conservative karyotype, demonstrating that our approach using a combination of FISH tools provides a higher resolution than previously obtained by chromosome painting alone. We also show that populations of Willisornis vidua appear conserved from a cytogenetic perspective, despite significant phylogeographic structure.

Funder: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brazilian Federal Agency for the Support and Evaluation of Graduate Education); doi: https://doi.org/10.13039/501100002322, Funder: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Reproductive compatibility usually decreases according to increasing genetic difference and the time of divergence between species. However, the amount of modification required to influence hybridization may vary in different species. Thus, it is extremely important to conduct studies that seek to understand what and how variables influence the reproductive isolation of species. We have explored a system involving two species of subterranean rodents that present morphological, karyotypic, and evolutionary history differences and are capable of generating hybrids. To gain insight into the karyotype organization of genus Ctenomys, we examined the chromosome evolution by classical and molecular cytogenetics of both parental species and hybrids. Furthermore, we have used different approaches to analyze the differences between the parental species and the hybrids, and determined the origin of the hybrids. The results of our work demonstrate unequivocally that some species that present extensive differences in chromosome organization, phenotype, evolutionary history, sperm morphology and genetic, which are usually associated with reproductive isolation, can generate natural hybrids. The results also demonstrate that females of both species are able to generate hybrids with males of the other species. In addition, the chromosome-specific probes prepared from Ctenomys flamarioni provide an invaluable tool for comparative cytogenetics in closely related species.

Supernumerary elements of the genome are often called B chromosomes. They usually consist of various autosomal sequences and, because of low selective pressure, are mostly pseudogenized and contain many repeats. There are numerous reports on B chromosomes in mammals, fish, invertebrates, plants, and fungi, but only a few of them have been studied using sequencing techniques. However, reptilian supernumerary chromosomes have been detected only cytogenetically and never sequenced or analyzed at the molecular level. One model squamate species with available genome sequence is Anolis carolinensis. The scope of the present article is to describe the genetic content of A. carolinensis supernumerary chromosomes. In this article, we confirm the presence of B chromosomes in this species by reverse painting and synaptonemal complex analysis. We applied low-pass high-throughput sequencing to analyze flow-sorted B chromosomes. Anole B chromosomes exhibit similar traits to other supernumerary chromosomes from different taxons: they contain two genes related to cell division control (INCENP and SPIRE2), are enriched in specific repeats, and show a high degree of pseudogenization. Therefore, the present study confirms that reptilian B chromosomes resemble supernumerary chromosomes of other taxons.

Pigeons and doves (Columbiformes) are one of the oldest and most diverse extant lineages of birds. However, the karyotype evolution within Columbiformes remains unclear. To delineate the synteny-conserved segments and karyotypic differences among four Columbidae species, we used chromosome painting from Gallus gallus (GGA, 2n = 78) and Leucopternis albicollis (LAL, 2n = 68). Besides that, a set of painting probes for the eared dove, Zenaida auriculata (ZAU, 2n = 76), was generated from flow-sorted chromosomes. Chromosome painting with GGA and ZAU probes showed conservation of the first ten ancestral pairs in Z. auriculata, Columba livia, and Columbina picui, while in Leptotila verreauxi, fusion of the ancestral chromosomes 6 and 7 was observed. However, LAL probes revealed a complex reorganization of ancestral chromosome 1, involving paracentric and pericentric inversions. Because of the presence of similar intrachromosomal rearrangements in the chromosomes corresponding to GGA1q in the Columbidae and Passeriformes species but without a common origin, these results are consistent with the recent proposal of divergence within Neoaves (Passerea and Columbea). In addition, inversions in chromosome 2 were identified in C. picui and L. verreauxi. Thus, in four species of distinct genera of the Columbidae family, unique chromosomal rearrangements have occurred during karyotype evolution, confirming that despite conservation of the ancestral syntenic groups, these chromosomes have been modified by the occurrence of intrachromosomal rearrangements.

Although most birds show karyotypes with diploid number (2n) around 80, with few macrochromosomes and many microchromosomes pairs, some groups, such as the Accipitriformes, are characterized by a large karyotypic reorganization, which resulted in complements with low diploid numbers, and a smaller number of microchromosomal pairs when compared to other birds. Among Accipitriformes, the Accipitridae family is the most diverse and includes, among other subfamilies, the subfamily Aquilinae, composed of medium to large sized species. The Black-Hawk-Eagle (Spizaetus tyrannus-STY), found in South America, is a member of this subfamily. Available chromosome data for this species includes only conventional staining. Hence, in order to provide additional information on karyotype evolution process within this group, we performed comparative chromosome painting between S. tyrannus and Gallus gallus (GGA). Our results revealed that at least 29 fission-fusion events occurred in the STY karyotype, based on homology with GGA. Fissions occurred mainly in syntenic groups homologous to GGA1-GGA5. On the other hand, the majority of the microchromosomes were found fused to other chromosomal elements in STY, indicating these rearrangements played an important role in the reduction of the 2n to 68. Comparison with hybridization pattern of the Japanese-Mountain-Eagle (Nisaetus nipalensis orientalis), the only Aquilinae analyzed by comparative chromosome painting previously, did not reveal any synapomorphy that could represent a chromosome signature to this subfamily. Therefore, conclusions about karyotype evolution in Aquilinae require additional painting studies.

The taxonomy of the genus Calomyscus remains controversial. According to the latest systematics the genus includes eight species with great karyotypic variation. Here, we studied karyotypes of 14 Calomyscus individuals from different regions of Iran and Turkmenistan using a new set of chromosome painting probes from a Calomyscus sp. male (2n = 46, XY, Shahr-e-Kord-Soreshjan-Cheshme Maiak Province). We showed the retention of large syntenic blocks in karyotypes of individuals with identical chromosome numbers. The only rearrangement (fusion 2/21) differentiated Calomyscus elburzensis, Calomyscus mystax mystax, and Calomyscus sp. from Isfahan Province with 2n = 44 from karyotypes of C. bailwardi, Calomyscus sp. from Shahr-e-Kord, Chahar Mahal and Bakhtiari-Aloni, and Khuzestan-Izeh Provinces with 2n = 46. The individuals from Shahdad tunnel, Kerman Province with 2n = 51–52 demonstrated non-centric fissions of chromosomes 4, 5, and 6 of the 46-chromosomal form with the formation of separate small acrocentrics. A heteromorphic pair of chromosomes in a specimen with 2n = 51 resulted from a fusion of two autosomes. C-banding and chromomycin A3-DAPI staining after G-banding showed extensive heterochromatin variation between individuals.

Iguanians (Pleurodonta) are one of the reptile lineages that, like birds and mammals, have sex chromosomes of ancient origin. In most iguanians these are microchromosomes, making a distinction between the X and Y as well as between homeologous sex chromosomes in other species difficult. Meiotic chromosome analysis may be used to elucidate their differentiation, because meiotic prophase chromosomes are longer and less condensed than metaphase chromosomes, and the homologues are paired with each other, revealing minor heteromorphisms. Using electron and fluorescent microscopy of surface spread synaptonemal complexes (SCs) and immunolocalization of the proteins of the SC (SYCP3), the centromere, and recombination nodules (MLH1), we examined sex chromosome synapsis and recombination in 2 species of anoles (Dactyloidae), Anolis carolinensis and Deiroptyx coelestinus, in which the sex chromosomes represent the ancestral condition of iguanians. We detected clear differences in size between the anole X and Y microchromosomes and found an interspecies difference in the localization of the pseudoautosomal region. Our results show that the apparent homomorphy of certain reptile sex chromosome systems can hide a cryptic differentiation, which potentially may influence the evolution of sexual dimorphism and speciation.

Background The Neacomys genus is predominantly found in the Amazon region, and belongs to the most diverse tribe of the Sigmodontinae subfamily (Rodentia, Cricetidae, Oryzomyini). The systematics of this genus and questions about its diversity and range have been investigated by morphological, molecular (Cytb and COI sequences) and karyotype analysis (classic cytogenetics and chromosome painting), which have revealed candidate species and new distribution areas. Here we analyzed four species of Neacomys by chromosome painting with Hylaeamys megacephalus (HME) whole-chromosome probes, and compared the results with two previously studied Neacomys species and with other taxa from Oryzomyini and Akodontini tribes that have been hybridized with HME probes. Maximum Parsimony (MP) analyses were performed with the PAUP and T.N.T. software packages, using a non-additive (unordered) multi-state character matrix, based on chromosomal morphology, number and syntenic blocks. We also compared the chromosomal phylogeny obtained in this study with molecular topologies (Cytb and COI) that included eastern Amazonian species of Neacomys, to define the phylogenetic relationships of these taxa. Results The comparative chromosome painting analysis of the seven karyotypes of the six species of Neacomys shows that their diversity is due to 17 fusion/fission events and one translocation, pericentric inversions in four syntenic blocks, and constitutive heterochromatin (CH) amplification/deletion of six syntenic autosomal blocks plus the X chromosome. The chromosomal phylogeny is consistent with the molecular relationships of species of Neacomys. We describe new karyotypes and expand the distribution area for species from eastern Amazonia and detect complex rearrangements by chromosome painting among the karyotypes. Conclusions Our phylogeny reflects the molecular relationships of the Akodontini and Oryzomyini taxa and supports the monophyly of Neacomys. This work presents new insights about the chromosomal evolution of this group, and we conclude that the karyotypic divergence is in accord with phylogenetic relationships.

The involvement of chromosome changes in the initial steps of speciation is controversial. Here we examine diversification trends within the mole voles Ellobius, a group of subterranean rodents. The first description of their chromosome variability was published almost 40 years ago. Studying the G-band structure of chromosomes in numerous individuals revealed subsequent homologous, step-by-step, Robertsonian translocations, which changed diploid numbers from 54 to 30. Here we used a molecular cytogenetic strategy which demonstrates that chromosomal translocations are not always homologous, consequently, karyotypes with the same diploid number can carry different combinations of metacentrics. We further showed that at least three chromosomal forms with 2n = 34 and distinct metacentrics inhabit the Pamir-Alay mountains. Each of these forms independently hybridized with E. tancrei, 2n = 54, forming separate hybrid zones. The chromosomal variations correlate slightly with geographic barriers. Additionally, we confirmed that the emergence of partial or monobrachial homology appeared to be a strong barrier for hybridization in nature, in contradistinction to experiments which we reported earlier. We discuss the possibility of whole arm reciprocal translocations for mole voles. Our findings suggest that chromosomal translocations lead to diversification and speciation.

Chromosome homologies in reptiles have been investigated extensively by gene mapping and chromosome painting. Relative chromosome size can be estimated roughly from conventional karyotypes, but chromosome GC content cannot be evaluated by any of these approaches. However, GC content can be obtained by whole-genome sequencing, although complete data are available only for a limited number of reptilian species. Chromosomes can be characterized by size and GC content in bivariate flow karyotypes, in which the distribution of peaks represents the differences. We have analysed flow karyotypes from 9 representative squamate species and show chromosome profiles for each species based on the relationship between size and GC content. Our results reveal that the GC content of macrochromosomes is invariable in the 9 species. A higher GC content was found in microchromosomes, similar to profiles previously determined in crocodile, turtle, and chicken. The findings suggest that karyotype evolution in reptiles is characterized by unique features of chromosome GC content.

Multiple sex chromosome systems have been described for several mammalian orders, with different species from the same genus sharing the same system (e.g., X1X2Y or XY1Y2). This is important because the translocated autosome may be influenced by the evolution of the recipient sex chromosome, and this may be related to speciation. It is often thought that the translocation of an autosome to a sex chromosome may share a common origin among phylogenetically related species. However, the neo-X chromosomes of Proechimys goeldii (2n = 24♀, 25♂/NFa = 42) and Proechimys gr. goeldii (2n = 16♀, 17♂/NFa = 14) have distinct sizes and morphologies that have made it difficult to determine whether they have the same or different origins. This study investigates the origins of the XY1Y2 sex chromosome determination system in P. goeldii (PGO) and P. gr. goeldii (PGG) and elucidates the chromosomal rearrangements in this low-diploid-number group of Proechimys species. Toward this end, we produced whole-chromosome probes for P. roberti (PRO; 2n = 30♂/NFa = 54) and P. goeldii (2n = 25♂/NFa = 42) and used them in comparative chromosomal mapping. Our analysis reveals that multiple translocations and inversions are responsible for the karyotype diversity of these species, with only three whole-chromosomes conserved between PRO and PGO and eight between PGO and PGG. Our data indicate that multiple sex chromosome systems have originated twice in Proechimys. As small populations are prone to the fixation of chromosomal rearrangements, we speculate that biological features of Rodentia contribute to this fixation. We also highlight the potential of these rodents as a model for studying sex chromosome evolution.

Pleurodont lizards are characterized by an ancient system of sex chromosomes. Along with stability of the central component of the system (homologous to the X chromosome of Anolis carolinensis [Dactyloidae], ACAX), in some genera the ancestral sex chromosomes are fused with microautosomes, forming neo-sex chromosomes. The genus Ctenonotus (Dactyloidae) is characterized by multiple X1X1X2X2/X1X2Y sex chromosomes. According to cytogenetic data, the large neo-Y chromosome is formed by fusion of the ancestral Y chromosome with 2 microautosomes (homologous to ACA10 or ACA11 and ACA12), the X1 chromosome is formed by fusion of the ancestral X chromosome with the autosome homologous to ACA10 or ACA11, and the X2 chromosome is homologous to autosome ACA12. To determine more precisely the content and evolution of the Ctenonotus sex chromosomes, we sequenced flow-sorted chromosomes (both sex chromosomes and microautosomes as control) of 2 species with a similar system: C. pogus and C. sabanus. Our results indicate that the translocated part of the X1 is homologous to ACA11, X2 is homologous to ACA12, and the Y contains segments homologous to both ACA11 and ACA12. Molecular divergence estimates suggest that the ancestral X-derived part has completely degenerated in the Y of Ctenonotus, similar to the degeneration of the Norops sagrei Y chromosome (Dactyloidae). The newly added regions show loss of DNA content, but without degeneration of the conserved regions. We hypothesize that the translocation of autosomal blocks onto sex chromosomes facilitated rapid degeneration of the pseudoautosomal region on the ancestral Y.

We developed new tools to build a high-quality chromosomal map of the Komodo dragon (Varanus komodoensis) available for cross-species phylogenomic analyses. First, we isolated chromosomes by flow sorting and determined the chromosome content of each flow karyotype peak by FISH. We then isolated additional Komodo dragon chromosomes by microdissection and amplified chromosome-specific DNA pools. The chromosome-specific DNA pools can be sequenced, assembled, and mapped by next-generation sequencing technology. The chromosome-specific paint probes can be used to investigate karyotype evolution through cross-species chromosome painting. Overall, the set of chromosome-specific DNA pools of V. komodoensis provides new tools for detailed phylogenomic analyses of Varanidae and squamates in general.

Glyphorynchus spirurus (GSP), also called the Wedge-billed Woodcreeper (Furnariidae) has an extensive distribution in the Americas, including the Atlantic coast of Brazil. Nevertheless, there is no information about its karyotype or genome organization. To contribute to the knowledge of chromosomal evolution in Passeriformes we analysed the karyotype of Glyphorynchus spirurus by classic and molecular cytogenetics methods. We show that Glyphorynchus spirurus has a 2n = 80 karyotype with a fundamental number (FN) of 84, similar to the avian putative ancestral karyotype (PAK). Glyphorynchus spirurus pair 1 was heteromorphic in the Tapajós population whereby the short arms varied in sizes, possibly due to a pericentric inversion, as described in other Furnariidae birds. FISH with the Histone H5 probe revealed a signal in the pericentromeric region of G. spirurus chromosome 5 and rDNA 18S showed interstitial signal in GSP-1. Chromosome painting with Gallus gallus (GGA) macrochromosomes 1–9 probes showed disruption of chromosome syntenies of GGA-1, 2 and 4 by fission in Glyphorynchus spirurus. Our results confirm that the GGA1 centric fission is a synapomorphic character for the phylogenetic branch composed of Strigiformes, Passeriformes, Columbiformes and Falconiformes. On the other hand, the GGA-2 fission is reported here for the first time in Passeriformes. Chromosome painting with BOE whole chromosome probes confirmed these rearrangements in Glyphorynchus spirurus revealed by Gallus gallus 1–9 probes, in addition to enabling the establishment of genome-wide homology map. Fil: Ribas, Talita Fernanda Augusto. Universidade Federal do Pará; Brasil Fil: Nagamachi, Cleusa Yoshiko. Universidade Federal do Pará; Brasil. Conselho Nacional de Desenvolvimiento Científico y Tecnológico; Brasil Fil: Aleixo, Alexandre. Museu Paraense Emílio Goeldi; Brasil Fil: Pinheiro, Melquizedec Luiz Silva. Universidade Federal do Pará; Brasil Fil: O'Brien, Patricia Caroline Mary. University of Cambridge; Reino Unido Fil: Ferguson Smith, Malcolm Andrew. University of Cambridge; Reino Unido Fil: Yang, Fengtang. Wellcome Sanger Institute; Reino Unido Fil: Suarez, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú; Argentina Fil: Pieczarka, Julio Cesar. Universidade Federal do Pará; Brasil. Conselho Nacional de Desenvolvimiento Científico y Tecnológico; Brasil

Chromocenters are interphase nuclear landmark structures of constitutive heterochromatin. The tandem repeat (TR)-enriched parts of different chromosomes cluster together in chromocenters. There has been progress in recent years in determining the protein content of chromocenters, although it is not clear which DNA sequences underly constitutive heterochromatin apart from the TRs. The aim of the current work was to find out which DNA sequences besides TRs are involved in chromocenters' formation. Biochemically isolated chromocenters and microdissected centromeric regions were amplified by DOP-PCR, then cloned and sequenced. Alignment to Repbase, the mouse reference genome and WGS databases separated the sequences from both libraries into three groups: (1) sequences with similarity to pericentromere mouse major satellite; (2) sequences without similarity to any repetitive sequences; (3) sequences with similarity to long interspersed nuclear elements (LINEs). LINE-related sequences have a disperse pattern distribution on chromosomes predicted in silico. Selected clones were used for fluorescent in situ hybridization (FISH). The 10 clones tested hybridized to chromocenters and centromeric regions of metaphase chromosomes. These clones were used for double FISH with four known cloned TRs (satDNA, satellite DNA) and a probe specific for the sex chromosomes. The probes bind various chromocenters' regions without overlapping; so, FISH results reveal a complex chromocenter composition. We mapped 18 LINE-derived clones to the RepBase L1 records. Most of them grouped in a ∼2-kb region at the end of the second ORF and 3' untranslated region (UTR). So, even the limited number of the clones allows us to determine the region of the L1 element that is specific for heterochromatic regions. Although the L1 full-length probe did not hybridize at detectable levels to the heterochromatic region on any chromosome, the 2-kb fragment found is definitely a part of these regions. The precise LINE ∼2-kb fragment is the component of mouse and human constitutive heterochromatin enriched with TRs. The method used for amplification of the probes from two sources of the heterochromatic material uncovered the enrichment of a precise fragment of LINE within chromocenters.

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.

A major goal of genomic and reproductive biology is to understand the evolution of sex determination and sex chromosomes. Species of the 2 genera of the Salamander family Proteidae - Necturus of eastern North America, and Proteus of Southern Europe - have similar-looking karyotypes with the same chromosome number (2n = 38), which differentiates them from all other salamanders. However, Necturus possesses strongly heteromorphic X and Y sex chromosomes that Proteus lacks. Since the heteromorphic sex chromosomes of Necturus were detectable only with C-banding, we hypothesized that we could use C-banding to find sex chromosomes in Proteus. We examined mitotic material from colchicine-treated intestinal epithelium, and meiotic material from testes in specimens of Proteus, representing 3 genetically distinct populations in Slovenia. We compared these results with those from Necturus. We performed FISH to visualize telomeric sequences in meiotic bivalents. Our results provide evidence that Proteus represents an example of sex chromosome turnover in which a Necturus-like Y-chromosome has become permanently translocated to another chromosome converting heteromorphic sex chromosomes to homomorphic sex chromosomes. These results may be key to understanding some unusual aspects of demographics and reproductive biology of Proteus, and are discussed in the context of models of the evolution of sex chromosomes in amphibians.

The subfamily Arvicolinae consists of a great number of species with highly diversified karyotypes. In spite of the wide use of arvicolines in biological and medicine studies, the data on their karyotype structures are limited. Here, we made a set of painting probes from flow-sorted chromosomes of a male Palearctic collared lemming (Dicrostonyx torquatus, DTO). Together with the sets of painting probes made previously from the field vole (Microtus agrestis, MAG) and golden hamster (Mesocricetus auratus, MAU), we carried out a reciprocal chromosome painting between these three species. The three sets of probes were further hybridized onto the chromosomes of the Eurasian water vole (Arvicola amphibius) and northern red-backed vole (Myodes rutilus). We defined the diploid chromosome number in D. torquatus karyotype as 2n = 45 + Bs and showed that the system of sex chromosomes is X1X2Y1. The probes developed here provide a genomic tool-kit, which will help to investigate the evolutionary biology of the Arvicolinae rodents. Our results show that the syntenic association MAG1/17 is present not only in Arvicolinae but also in some species of Cricetinae; and thus, should not be considered as a cytogenetic signature for Arvicolinae. Although cytogenetic signature markers for the genera have not yet been found, our data provides insight into the likely ancestral karyotype of Arvicolinae. We conclude that the karyotypes of modern voles could have evolved from a common ancestral arvicoline karyotype (AAK) with 2n = 56 mainly by centric fusions and fissions.

Comparative chromosome-painting analysis among highly rearranged karyotypes of Sigmodontinae rodents (Rodentia, Cricetidae) detects conserved syntenic blocks, which are proposed as chromosomal signatures and can be used as phylogenetic markers. In the Akodontini tribe, the molecular topology (Cytb and/or IRBP) shows five low-supported clades (divisions: &ldquo, Akodon&rdquo, &ldquo, Bibimys&rdquo, Blarinomys&rdquo, Oxymycterus&rdquo, and &ldquo, Scapteromys&rdquo, ) within two high-supported major clades (clade A: &ldquo, clade B: &ldquo, ). Here, we examine the chromosomal signatures of the Akodontini tribe by using Hylaeamys megacephalus (HME) probes to study the karyotypes of Oxymycterus amazonicus (2n = 54, FN = 64) and Blarinomys breviceps (2n = 28, FN = 50), and compare these data with those from other taxa investigated using the same set of probes. We strategically employ the chromosomal signatures to elucidate phylogenetic relationships among the Akodontini. When we follow the evolution of chromosomal signature states, we find that the cytogenetic data corroborate the current molecular relationships in clade A nodes. We discuss the distinct events that caused karyotypic variability in the Oxymycterus and Blarinomys genera. In addition, we propose that Blarinomys may constitute a species complex, and that the taxonomy should be revised to better delimit the geographical boundaries and their taxonomic status.

Despite their long history with the basal split dating back to the Eocene, all species of monitor lizards (family Varanidae) studied so far share the same chromosome number of 2n = 40. However, there are differences in the morphology of the macrochromosome pairs 5–8. Further, sex determination, which revealed ZZ/ZW sex microchromosomes, was studied only in a few varanid species and only with techniques that did not test their homology. The aim of this study was to (i) test if cryptic interchromosomal rearrangements of larger chromosomal blocks occurred during the karyotype evolution of this group, (ii) contribute to the reconstruction of the varanid ancestral karyotype, and (iii) test homology of sex chromosomes among varanids. We investigated these issues by hybridizing flow sorted chromosome paints from Varanus komodoensis to metaphases of nine species of monitor lizards. The results show that differences in the morphology of the chromosome pairs 5–8 can be attributed to intrachromosomal rearrangements, which led to transitions between acrocentric and metacentric chromosomes in both directions. We also documented the first case of spontaneous triploidy among varanids in Varanus albigularis. The triploid individual was fully grown, which demonstrates that polyploidization is compatible with life in this lineage. We found that the W chromosome differs between species in size and heterochromatin content. The varanid Z chromosome is clearly conserved in all the analyzed species. Varanids, in addition to iguanas, caenophidian snakes, and lacertid lizards, are another squamate group with highly conserved sex chromosomes over a long evolutionary time.

The hoatzin (Opisthocomus hoazin Müller, 1776) is a folivorous bird, endemic to the Amazonian region. It presents some unique characteristics, including wing claws and foregut fermentation, which make its phylogenetic relationship to other birds difficult to determine. There have been various attempts to place it among the Galliformes, Gruiformes, Musophagiformes, Cuculiformes, and Charadriiformes, but phylogenetic analyses always show low supporting values. Nowadays, the hoatzin is included in the monotypic order Opisthocomiformes, but the relationship of this order to other groups of birds is still unclear. Although its karyotype resembles the typical avian model, fissions of the syntenic groups corresponding to chicken chromosomes 1 and 2 and 2 fusions were found. The presence of 18S rDNA clusters in 2 pairs of microchromosomes is another derived character. Hence, different rearrangements were detected in the karyotype of the hoatzin, indicating it has been derived from the putative ancestral karyotype by the occurrence of fissions and fusions. However, as these rearrangements are not exclusive to O. hoazin, they do not clarify the phylogenetic position of this enigmatic species.

Background The family Phyllostomidae (Chiroptera) shows wide morphological, molecular and cytogenetic variation; many disagreements regarding its phylogeny and taxonomy remains to be resolved. In this study, we use chromosome painting with whole chromosome probes from the Phyllostomidae Phyllostomus hastatus and Carollia brevicauda to determine the rearrangements among several genera of the Nullicauda group (subfamilies Gliphonycterinae, Carolliinae, Rhinophyllinae and Stenodermatinae). Results These data, when compared with previously published chromosome homology maps, allow the construction of a phylogeny comparable to those previously obtained by morphological and molecular analysis. Our phylogeny is largely in agreement with that proposed with molecular data, both on relationships between the subfamilies and among genera; it confirms, for instance, that Carollia and Rhinophylla, previously considered as part of the same subfamily are, in fact, distant genera. Conclusions The occurrence of the karyotype considered ancestral for this family in several different branches suggests that the diversification of Phyllostomidae into many subfamilies has occurred in a short period of time. Finally, the comparison with published maps using human whole chromosome probes allows us to track some syntenic associations prior to the emergence of this family. Electronic supplementary material The online version of this article (10.1186/s12862-018-1176-3) contains supplementary material, which is available to authorized users.

Universidade Federal do Par?. Faculdade de Ci?ncias Naturais. Instituto de Ci?ncias Exatas e Naturais. Programa de P?s-gradua??o em Gen?tica e Biologia Molecular. Bel?m, PA, Brazil / Universidade Federal do Par?. Faculdade de Ci?ncias Naturais. Instituto de Ci?ncias Exatas e Naturais. Bel?m, PA, Brazil. Universidade Federal do Rio Grande do Sul. Programa de P?s-gradua??o em Gen?tica e Biologia Molecular. Porto Alegre, RS, Brazil. Universidade Federal do Pampa, Campus S?o Gabriel. Programa de P?s-Gradua??o em Ci?ncias Biol?gicas. S?o Gabriel, RS, Brazil. Universidade Federal do Pampa, Campus S?o Gabriel. Programa de P?s-Gradua??o em Ci?ncias Biol?gicas. S?o Gabriel, RS, Brazil. Universidade Federal do Par?. Faculdade de Ci?ncias Naturais. Instituto de Ci?ncias Exatas e Naturais. Bel?m, PA, Brazil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. University of Cambridge. Cambridge Resource Centre for Comparative Genomics. Cambridge, UK. University of Cambridge. Cambridge Resource Centre for Comparative Genomics. Cambridge, UK. Tyrannidae is the largest family of Passeriformes in the Neotropical region. However, despite an interesting chromosomal diversity, there are only few cytogenetic studies of this family, and most of these are based on conventional cytogenetics. Hence, we analyzed here the chromosomal diversity and karyotypical evolution of this group by chromosome painting in 3 different species - Pitangus sulphuratus, Serpophaga subcristata, and Satrapa icterophrys - and make comparisons with previous data. In addition to chromosome painting with Gallus gallus (GGA) and Leucopternis albicollis (LAL) probes, karyotypes were analyzed by conventional staining, C-banding, and FISH with 18S rDNA and telomeric probes. Although this family is characterized by extensive chromosomal variation, we found similar karyotypes and diploid numbers ranging from 2n = 80 in P. sulphuratus to 2n = 82 in S. subcristata and S. icterophrys. Constitutive heterochromatin was located centromerically in all 3 species. Clusters of 18S rDNA were present in 1 pair of microchromosomes, except in S. subcristata, where 2 pairs of microchromosomes were labeled. No interstitial telomeric sequences were detected. GGA and LAL whole-chromosome probes revealed the occurrence of fissions and both paracentric and pericentric inversions commonly seen in other Passeriformes. In general terms, tyrants show the typical karyotype found in Passeriformes, suggesting that the observed rearrangements occurred before the division of the suborders Oscines and Suboscines.

We have investigated the complex karyotype of a living zebra-donkey hybrid for the first time using chromosome-specific painting probes produced from flow-sorted chromosomes from a zebra (Equus burchelli) and horse (Equus caballus). As the chromosomes proved difficult to distinguish from one another, a successful new strategy was devised to resolve the difficulty and characterize each chromosome. This was based on selecting five panels of whole chromosome painting probes that could differentiate zebra and donkey chromosomes by labelling the probes with either FITC or Cy3 fluorochromes. Each panel was hybridized sequentially to the same G-Q-banded metaphases and the results combined so that every zebra and donkey chromosome in each suitable metaphase could be identified. A diploid number of 2n = 53, XY was found, containing haploid sets of 22 chromosomes from the zebra and 31 chromosomes from the donkey, without evidence of chromosome rearrangement. This new strategy, developed for the first time, may have several applications in the resolution of other complex hybrid karyotypes and chromosomal aberrations.

The Neacomys genus (Rodentia, Sigmodontinae) is distributed in the Amazon region, with some species limited to a single endemic area, while others may occur more widely. The number of species within the genus and their geographical boundaries are not known accurately, due to their high genetic diversity and difficulties in taxonomic identification. In this work we collected Neacomys specimens from both banks of the Tapajós River in eastern Amazon, and studied them using chromosome painting with whole chromosome probes of Hylaeamys megacephalus (HME; Rodentia, Sigmodontinae), and molecular analysis using haplotypes of mitochondrial genes COI and Cytb. Chromosome painting shows that Neacomys sp. A (NSP-A, 2n = 58/FN = 68) and Neacomys sp. B (NSP-B, 2n = 54/FN = 66) differ by 11 fusion/fission events, one translocation, four pericentric inversions and four heterochromatin amplification events. Using haplotypes of the concatenated mitochondrial genes COI and Cyt b, Neacomys sp. (2n = 58/FN = 64 and 70) shows a mean divergence of 6.2% for Neacomys sp. A and 9.1% for Neacomys sp. B, while Neacomys sp. A and Neacomys sp. B presents a medium nucleotide divergence of 7.4%. Comparisons were made with other published Neacomys data. The Tapajós and Xingu Rivers act as geographic barriers that define the distribution of these Neacomys species. Furthermore, our HME probes reveal four synapomorphies for the Neacomys genus (associations HME 20/[13,22]/4, 6a/21, [9,10]/7b/[9,10] and 12/[16,17]) and demonstrate ancestral traits of the Oryzomyini tribe (HME 8a and 8b, 18 and 25) and Sigmodontinae subfamily (HME 15 and 24), which can be used as taxonomic markers for these groups.

Trogons are forest birds with a wide distribution, being found in Africa, Asia, and America, and are included in the order Trogoniformes, family Trogonidae. Phylogenetic studies using molecular data have not been able to determine the phylogenetic relationship among the different genera of trogons. So far, no cytogenetic data for these birds exist. Hence, the aim of this study was to characterize the karyotype of Trogon surrucura surrucura by means of classical and molecular cytogenetics. We found a diploid chromosome number of 2n = 82, similar to most birds, with several derived features compared to chicken and the putative ancestral avian karyotype. T. s. surrucura showed 3 pairs of microchromosomes bearing 18S rDNA clusters. The Z and W sex chromosomes were of similar size but could readily be identified by morphological differences. Using chromosome painting with whole chromosome probes from Gallus gallus and Leucopternis albicollis, we found that the chromosomes homologous to chicken chromosomes 2 and 5 correspond to 2 different pairs in T. s. surrucura and L. albicollis, due to the occurrence of centric fissions. Paracentric inversions were detected in the segment homologous to chicken chromosome 1q, and we confirmed the recurrence of breakpoints when our results were compared to other species of birds already analyzed by FISH or by in silico genome assembly.

Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Programa de P?s-Gradua??o em Gen?tica e Biologia Molecular. Bel?m, PA, Brazil. Universidade Federal do Rio Grande do Sul. Programa de P?s-Gradua??o em Gen?tica e Biologia Molecular. Porto Alegre, RS, Brazil. Max Planck Institute for Ornithology. Department of Behavioral Neurobiology. Seewiesen, Germany. Max Planck Institute for Ornithology. Department of Behavioral Neurobiology. Seewiesen, Germany. Max Planck Institute for Ornithology. Department of Behavioral Neurobiology. Seewiesen, Germany. University of Cambridge. Department of Veterinary Medicine. Cambridge Resource Centre for Comparative Genomics. Cambridge, United Kingdom. University of Cambridge. Department of Veterinary Medicine. Cambridge Resource Centre for Comparative Genomics. Cambridge, United Kingdom. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Cultura de Tecidos e Citogen?tica. Ananindeua, PA, Brasil / Universidade Federal do Par?. Instituto de Ci?ncias Exatas e Naturais. Faculdade de Ci?ncias Naturais. Bel?m, PA, Brazil. Songbird species (order Passeriformes, suborder Oscines) are important models in various experimental fields spanning behavioural genomics to neurobiology. Although the genomes of some songbird species were sequenced recently, the chromosomal organization of these species is mostly unknown. Here we focused on the two most studied songbird species in neuroscience, the zebra finch (Taeniopygia guttata) and the canary (Serinus canaria). In order to clarify these issues and also to integrate chromosome data with their assembled genomes, we used classical and molecular cytogenetics in both zebra finch and canary to define their chromosomal homology, localization of heterochromatic blocks and distribution of rDNA clusters. We confirmed the same diploid number (2n = 80) in both species, as previously reported. FISH experiments confirmed the occurrence of multiple paracentric and pericentric inversions previously found in other species of Passeriformes, providing a cytogenetic signature for this order, and corroborating data from in silico analyses. Additionally, compared to other Passeriformes, we detected differences in the zebra finch karyotype concerning the morphology of some chromosomes, in the distribution of 5S rDNA clusters, and an inversion in chromosome 1.

Among the Oryzomyini (Sigmodontinae), Oecomys is the most speciose, with 17 species. This genus presents high karyotypic diversity (2n = 54 to 2n = 86) and many taxonomic issues at the species level because of the presence of cryptic species and the overlap of morphological characters. For these reasons the real number of species of Oecomys may be underestimated. With the aim of verifying if the taxon Oecomys catherinae is composed of more than one species, we made comparative studies on two populations from two regions of Brazil, one from the Amazon and another from the Atlantic Forest using both classical cytogenetics (G- and C-banding) and comparative genomic mapping with whole chromosome probes of Hylaeamys megacephalus (HME), molecular data (cytochrome b mitochondrial DNA) and morphology. Our results confirm that Oecomys catherinae occurs in the southeast Amazon, and reveal a new karyotype for the species (2n = 62, FNa = 62). The comparative genomic analysis with HME probes identified chromosomal homeologies between both populations and rearrangements that are responsible for the different karyotypes. We compared our results in Sigmodontinae genera with other studies that also used HME probes. These chromosomal differences together with the absence of consistent differentiation between the two populations on morphological and molecular analyses suggest that these populations may represent cryptic species.

Here, for the first time, we describe the karyotype of Myiopsitta monachus (Psittacidae, Arini). We found 2n = 48, corresponding to the lowest diploid number observed in Neotropical Psittaciformes so far, with an uncommonly large W chromosome homomorphic to the Z. In order to better understand the evolution of the sex chromosomes in this species, we applied several molecular cytogenetic approaches, including C-banding, FISH mapping of repetitive DNAs (several microsatellite repeats), and whole-chromosome painting on metaphases of M. monachus. For comparison, another species belonging to the same tribe but with a smaller W chromosome (A. aestiva) was also analyzed. The results show that the constitutive heterochromatin has a very diverse distribution pattern in these species revealing heterochromatic blocks in the centromeric region of all chromosomes and in most of the length of the W chromosome in A. aestiva, while in M. monachus they were found in interstitial and telomeric regions. Concerning the microsatellites, only the sequence (CG)n produced signals on the W chromosome of A. aestiva, in the distal region of both arms. However, in M. monachus, (CAA)n, (CAG)n, and (CG)n probes were accumulated on the W chromosome, and, in addition, the sequence (CAG)n also hybridized to heterochromatic regions in macrochromosomes, as well as in microchromosomes. Based on these results, we suggest that the increase in length of the W chromosome in M. monachus is due to the amplification of repetitive elements, which highlights their significant role in the evolutionary process of sex chromosome differentiation.

Although more than thirty mammalian genomes have been sequenced to draft quality, very few of these include the Y chromosome. This has limited our understanding of the evolutionary dynamics of gene persistence and loss, our ability to identify conserved regulatory elements, as well our knowledge of the extent to which different types of selection act to maintain genes within this unique genomic environment. Here, we present the first MSY (male-specific region of the Y chromosome) sequences from two carnivores, the domestic dog and cat. By combining these with other available MSY data, our multiordinal comparison allows for the first accounting of levels of selection constraining the evolution of eutherian Y chromosomes. Despite gene gain and loss across the phylogeny, we show the eutherian ancestor retained a core set of 17 MSY genes, most being constrained by negative selection for nearly 100 million years. The X-degenerate and ampliconic gene classes are partitioned into distinct chromosomal domains in most mammals, but were radically restructured on the human lineage. We identified multiple conserved noncoding elements that potentially regulate eutherian MSY genes. The acquisition of novel ampliconic gene families was accompanied by signatures of positive selection and has differentially impacted the degeneration and expansion of MSY gene repertoires in different species.

Background B chromosomes are dispensable and variable karyotypic elements found in some species of animals, plants and fungi. They often originate from duplications and translocations of host genomic regions or result from hybridization. In most species, little is known about their DNA content. Here we perform high-throughput sequencing and analysis of B chromosomes of roe deer and brocket deer, the only representatives of Cetartiodactyla known to have B chromosomes. Results In this study we developed an approach to identify genomic regions present on chromosomes by high-throughput sequencing of DNA generated from flow-sorted chromosomes using degenerate-oligonucleotide-primed PCR. Application of this method on small cattle autosomes revealed a previously described KIT gene region translocation associated with colour sidedness. Implementing this approach to B chromosomes from two cervid species, Siberian roe deer (Capreolus pygargus) and grey brocket deer (Mazama gouazoubira), revealed dramatically different genetic content: roe deer B chromosomes consisted of two duplicated genomic regions (a total of 1.42-1.98 Mbp) involving three genes, while grey brocket deer B chromosomes contained 26 duplicated regions (a total of 8.28-9.31 Mbp) with 34 complete and 21 partial genes, including KIT and RET protooncogenes, previously found on supernumerary chromosomes in canids. Sequence variation analysis of roe deer B chromosomes revealed a high frequency of mutations and increased heterozygosity due to either amplification within B chromosomes or divergence between different Bs. In contrast, grey brocket deer B chromosomes were found to be more homogeneous and resembled autosomes in patterns of sequence variation. Similar tendencies were observed in repetitive DNA composition. Conclusions Our data demonstrate independent origins of B chromosomes in the grey brocket and roe deer. We hypothesize that the B chromosomes of these two cervid species represent different stages of B chromosome sequences evolution: probably nascent and similar to autosomal copies in brocket deer, highly derived in roe deer. Based on the presence of the same orthologous protooncogenes in canids and brocket deer Bs we argue that genomic regions involved in B chromosome formation are not random. In addition, our approach is also applicable to the characterization of other evolutionary and clinical rearrangements. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2933-6) contains supplementary material, which is available to authorized users.

Squamate reptiles show a striking diversity in modes of sex determination, including both genetic (XY or ZW) and temperature-dependent sex determination systems. The genomes of only a handful of species have been sequenced, analyzed and assembled including the genome of Anolis carolinensis. Despite a high genome coverage, only macrochromosomes of A. carolinensis were assembled whereas the content of most microchromosomes remained unclear. Most of the Anolis species have homomorphic XY sex chromosome system. However, some species have large heteromorphic XY chromosomes (e.g., A. sagrei) and even multiple sex chromosomes systems (e.g. A. pogus), that were shown to be derived from fusions of the ancestral XY with microautosomes. We applied next generation sequencing of flow sorting-derived chromosome-specific DNA pools to characterize the content and composition of microchromosomes in A. carolinensis and A. sagrei. Comparative analysis of sequenced chromosome-specific DNA pools revealed that the A. sagrei XY sex chromosomes contain regions homologous to several microautosomes of A. carolinensis. We suggest that the sex chromosomes of A. sagrei are derived by fusions of the ancestral sex chromosome with three microautosomes and subsequent loss of some genetic content on the Y chromosome.

The sunbittern (Eurypyga helias) is a South American Gruiformes, the only member of Family Eurypigidae. In most phylogenetic proposals, it is placed in a more distant position than other families of the so-called "core Gruiformes". Different studies based on molecular, morphological and biogeographical data suggest that the Eurypigidae is closely related to the kagu (Rhynochetos jubatus), the only species in Rynochetidae, another family not included in the core Gruiformes. Here, the karyotype of the sunbittern is described for the first time, by classical and molecular cytogenetics, using whole chromosome probes derived from Gallus gallus and Leucopternis albicollis. We found a diploid number of 80, with only one pair of biarmed autosomal macrochromosomes, similar to that observed in the kagu. Chromosome painting revealed that most syntenies found in the avian putative ancestral karyotype (PAK) were conserved in the sunbittern. However, PAK1, PAK2, and PAK5 corresponded to two chromosome pairs each. Probes derived from L. albicollis confirm that fissions in PAK1 and PAK2 were centric, whereas in PAK5 the fission is interstitial. In addition, there is fusion of segments homologous to PAK2q and PAK5. From a phylogenetic point of view, comparisons of our results with two other Gruiformes belonging to family Rallidae suggest that the PAK5q fission might be a synapomorphy for Gruiformes. Fissions in PAK1 and PAK2 are found only in Eurypigidae, and might also occur in Rynochetidae, in view of the similar chromosomal morphology between the sunbittern and the kagu. This suggests a close phylogenetic relationship between Eurypigidae and Rynochetidae, whose common ancestor was separated by the Gondwana vicariancy in South America and New Caledonia, respectively.

The genome size in turtles and crocodiles is thought to be much larger than the 1.2 Gb of the chicken ( Gallus gallus domesticus , GGA), according to the animal genome size database. However, GGA macrochromosomes show extensive homology in the karyotypes of the red eared slider ( Trachemys scripta elegans , TSC) and the Nile crocodile ( Crocodylus niloticus , CNI), and bird and reptile genomes have been highly conserved during evolution. In this study, size and GC content of all chromosomes are measured from the flow karyotypes of GGA, TSC and CNI. Genome sizes estimated from the total chromosome size demonstrate that TSC and CNI are 1.21 Gb and 1.29 Gb, respectively. This refines previous overestimations and reveals similar genome sizes in chicken, turtle and crocodile. Analysis of chromosome GC content in each of these three species shows a higher GC content in smaller chromosomes than in larger chromosomes. This contrasts with mammals and squamates in which GC content does not correlate with chromosome size. These data suggest that a common ancestor of birds, turtles and crocodiles had a small genome size and a chromosomal size-dependent GC bias, distinct from the squamate lineage.

Background Previous cross-species painting studies with probes from chicken (Gallus gallus) chromosomes 1–10 and a paint pool of nineteen microchromosomes have revealed that the drastic karyotypic reorganization in Accipitridae is due to extensive synteny disruptions and associations. However, the number of synteny association events and identities of microchromosomes involved in such synteny associations remain undefined, due to the lack of paint probes derived from individual chicken microchromosomes. Moreover, no genome-wide homology map between Accipitridae species and other avian species with atypical karyotype organization has been reported till now, and the karyotype evolution within Accipitriformes remains unclear. Results To delineate the synteny-conserved segments in Accipitridae, a set of painting probes for the griffon vulture, Gyps fulvus (2n = 66) was generated from flow-sorted chromosomes. Together with previous generated probes from the stone curlew, Burhinus oedicnemus (2n = 42), a Charadriiformes species with atypical karyotype organization, we conducted multidirectional chromosome painting, including reciprocal chromosome painting between B. oedicnemus and G. fulvus and cross-species chromosome painting between B. oedicnemus and two accipitrid species (the Himalayan griffon, G. himalayensis 2n = 66, and the common buzzard, Buteo buteo, 2n = 68). In doing so, genome-wide homology maps between B. oedicnemus and three Accipitridae species were established. From there, a cladistic analysis using chromosomal characters and mapping of chromosomal changes on a consensus molecular phylogeny were conducted in order to search for cytogenetic signatures for different lineages within Accipitriformes. Conclusion Our study confirmed that the genomes of the diurnal birds of prey, especially the genomes of species in Accipitriformes excluding Cathartidae, have been extensively reshuffled when compared to other bird lineages. The chromosomal rearrangements involved include both fusions and fissions. Our chromosome painting data indicated that the Palearctic common buzzard (BBU) shared several common chromosomal rearrangements with some Old World vultures, and was found to be more closely related to other Accipitridae than to Neotropical buteonine raptors from the karyotypic perspective. Using both a chromosome-based cladistic analysis as well as by mapping of chromosomal differences onto a molecular-based phylogenetic tree, we revealed a number of potential cytogenetic signatures that support the clade of Pandionidae (PHA) + Accipitridae. In addition, our cladistic analysis using chromosomal characters appears to support the placement of osprey (PHA) in Accipitridae.