A chromosome-level reference genome and pangenome for barn swallow population genomics.

Insights into the evolution of non-model organisms are limited by the lack of reference genomes of high accuracy, completeness, and contiguity. Here, we present a chromosome-level, karyotype-validated reference genome and pangenome for the barn swallow (Hirundo rustica). We complement these resources with a reference-free multialignment of the reference genome with other bird genomes and with the most comprehensive catalog of genetic markers for the barn swallow. We identify potentially conserved and accelerated genes using the multialignment and estimate genome-wide linkage disequilibrium using the catalog. We use the pangenome to infer core and accessory genes and to detect variants using it as a reference. Overall, these resources will foster population genomics studies in the barn swallow, enable detection of candidate genes in comparative genomics studies, and help reduce bias toward a single reference genome.

Replacement of owl monkey centromere satellite by a newly evolved variant was a recent and rapid process.

Alpha satellite DNA is a major DNA component of primate centromeres. We previously reported that Azara's owl monkey has two types of alpha satellite DNA, OwlAlp1 and OwlAlp2. OwlAlp2 (344 bp) exhibits a sequence similarity throughout its entire length with alpha satellite DNA of closely related species. OwlAlp1 (185 bp) corresponds to the part of OwlAlp2. Based on the observation that the CENP-A protein binds to OwlAlp1, we proposed that OwlAlp1 is a relatively new repetitive DNA that replaced OwlAlp2 as the centromeric satellite DNA. However, a detailed picture of the evolutionary process of this centromere DNA replacement remains largely unknown. Here, we performed a phylogenetic analysis of OwlAlp1 and OwlAlp2 sequences, and also compared our results to alpha satellite DNA sequences of other primate species. We found that: (i) OwlAlp1 exhibits a higher similarity to OwlAlp2 than to alpha satellite DNA of other species, (ii) OwlAlp1 has a single origin, and (iii) sequence variation is lower in OwlAlp1 than in OwlAlp2. We conclude that OwlAlp1 underwent a recent and rapid expansion in the owl monkey lineage. This centromere DNA replacement could have been facilitated by the heterochromatin reorganization that is associated with the adaptation of owl monkeys to a nocturnal lifestyle.

Not all yawns tell the same story: The case of Tonkean macaques.

Here we show for the first time that the plasticity in morphology and duration of yawning in Macaca tonkeana can be associated with different functional contexts. Macaca tonkeana is classified as a tolerant macaque species characterized by social interactions minimally constrained by dominance rank or kinship. Tonkean macaques, as other egalitarian species, rely on a complex facial communicative system. We found that the degree of mouth opening (ranging from covered to uncovered tooth yawns) and the duration of yawning were not strictly dependent. The shortest uncovered tooth yawns were associated with an intense locomotor/physical activity and peaked immediately after stressful social events thus indicating an increase in arousal. In contrast, longer yawns, independently from teeth exposure, were primarily associated with a relaxed state of the subject. In conclusion, our study suggests that to explore the potential different functions of yawning, it is necessary to focus on the variability of its expression both in terms of morphology and duration, because not all yawns tell the same story.

Bridging the Gap between Vertebrate Cytogenetics and Genomics with Single-Chromosome Sequencing (ChromSeq).

The study of vertebrate genome evolution is currently facing a revolution, brought about by next generation sequencing technologies that allow researchers to produce nearly complete and error-free genome assemblies. Novel approaches however do not always provide a direct link with information on vertebrate genome evolution gained from cytogenetic approaches. It is useful to preserve and link cytogenetic data with novel genomic discoveries. Sequencing of DNA from single isolated chromosomes (ChromSeq) is an elegant approach to determine the chromosome content and assign genome assemblies to chromosomes, thus bridging the gap between cytogenetics and genomics. The aim of this paper is to describe how ChromSeq can support the study of vertebrate genome evolution and how it can help link cytogenetic and genomic data. We show key examples of ChromSeq application in the refinement of vertebrate genome assemblies and in the study of vertebrate chromosome and karyotype evolution. We also provide a general overview of the approach and a concrete example of genome refinement using this method in the species Anolis carolinensis.

Publisher Correction: Characterization of Satellite DNAs in Squirrel Monkeys genus Saimiri (Cebidae, Platyrrhini).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Evolutionary rearrangements of X chromosomes in voles (Arvicolinae, Rodentia).

Euchromatic segments of the X chromosomes of placental mammals are the most conservative elements of the karyotype, only rarely subjected to either inter- or intrachromosomal rearrangements. Here, using microdissection-derived set of region-specific probes of Terricola savii we detailed the evolutionary rearrangements found in X chromosomes in 20 vole species (Arvicolinae, Rodentia). We show that the evolution of X chromosomes in this taxon was accompanied by multiple para- and pericentric inversions and centromere shifts. The contribution of intrachromosomal rearrangements to the karyotype evolution of Arvicolinae species was approximately equivalent in both the separate autosomal conserved segments and the X chromosomes. Intrachromosmal rearrangements and structural reorganization of the X chromosomes was likely accompanied by an accumulation, distribution, and evolution of repeated sequences.

Characterization of Satellite DNAs in Squirrel Monkeys genus Saimiri (Cebidae, Platyrrhini).

The genus Saimiri is a decades-long taxonomic and phylogenetic puzzle to which cytogenetics has contributed crucial data. All Saimiri species apparently have a diploid number of 2n = 44 but vary in the number of chromosome arms. Repetitive sequences such as satellite DNAs are potentially informative cytogenetic markers because they display high evolutionary rates. Our goal is to increase the pertinent karyological data by more fully characterizing satellite DNA sequences in the Saimiri genus. We were able to identify two abundant satellite DNAs, alpha (~340 bp) and CapA (~1,500 bp), from short-read clustering of sequencing datasets from S. boliviensis. The alpha sequences comprise about 1% and the CapA 2.2% of the S. boliviensis genome. We also mapped both satellite DNAs in S. boliviensis, S. sciureus, S. vanzolinii, and S. ustus. The alpha has high interspecific repeat homogeneity and was mapped to the centromeres of all analyzed species. CapA is associated with non-pericentromeric heterochromatin and its distribution varies among Saimiri species. We conclude that CapA genomic distribution and its pervasiveness across Platyrrhini makes it an attractive cytogenetic marker for Saimiri and other New World monkeys.

Eight million years of maintained heterozygosity in chromosome homologs of cercopithecine monkeys.

In the Cercopithecini ancestor two chromosomes, homologous to human chromosomes 20 and 21, fused to form the Cercopithecini specific 20/21 association. In some individuals from the genus Cercopithecus, this association was shown to be polymorphic for the position of the centromere, suggesting centromere repositioning events. We set out to test this hypothesis by defining the evolutionary history of the 20/21 association in four Cercopithecini species from three different genera. The marker order of the various 20/21 associations was established using molecular cytogenetic techniques, including an array of more than 100 BACs. We discovered that five different forms of the 20/21 association were present in the four studied Cercopithecini species. Remarkably, in the two Cercopithecus species, we found individuals in which one homolog conserved the ancestral condition, but the other homolog was highly rearranged. The phylogenetic analysis showed that the heterozygosity in these two species originated about 8 million years ago and was maintained for this entire arc of time, surviving multiple speciation events. Our report is a remarkable extension of Dobzhansky's pioneering observation in Drosophila concerning the maintenance of chromosomal heterozygosity due to selective advantage. Dobzhansky's hypothesis recently received strong support in a series of detailed reports on the fruit fly genome. Our findings are first extension to primates, indeed to Old World monkeys phylogenetically close to humans of an analogous situation. Our results have important implications for hypotheses on how chromosome rearrangements, selection, and speciation are related.

Isolation and phenotyping of potential stem cells from the umbilical cord of the bottlenose dolphin(Tursiops truncatus).

We have successfully isolated cells with stem-like properties from bottlenose dolphin (Tursiops truncatus) umbilical cord. Our results show that this cetacean species has embryonic fetal and adult stem cells as do humans and other studied mammals. This accomplishment allows to eventually investigate whether dolphins, due to their unique adaptations to aquatic environments, have special stem cell lineages or distinctive mechanisms of cell programming. Further characterization of their potency to differentiate into multiple cell lineages would fulfill numerous applicative purposes. We characterized, developed and refined a new protocol for obtaining potential stem cells from umbilical cord tissues of the bottlenose dolphin. Tissue samples were taken from umbilical cords of successful deliveries immediately after placenta ejection and collection from the water. Umbilical cord samples (2-3 cm3) were excised and subjected to enzymatic digestion and mechanical dissociation. Viable cells from specimens resident in the Oceanografic Valencia were cultured and subsequently isolated and tested for pluripotent characteristics (cell morphology, phenotype and expression of surface markers). Cell viability was confirmed also after freezing/thawing. The established protocol is suitable for collection/isolation/culture of dolphin potential mesenchymal stem cells from dolphin umbilical cord, which can be deposited in cell banks for future research needs.

Insights into Emydid Turtle Cytogenetics: The European Pond Turtle as a Model Species.

Our knowledge of Testudines evolution is limited by the lack of modern cytogenetic data. Compared to other reptiles, there is little information even on chromosome banding, let alone molecular cytogenetic data. Here, we provide detailed information on the karyotype of the European pond turtle Emys orbicularis, a model Emydidae, employing both chromosome banding and molecular cytogenetics. We provide a high-resolution G-banded karyotype and a map of rDNA genes and telomeric sequences using fluorescence in situ hybridization. We test hypotheses of sex-determining mechanisms in Emys by comparative genomic hybridization to determine if Emys has a cryptic sex-specific region. Our results provide valuable data to guide future efforts on genome sequencing and anchoring in Emydidae and for understanding karyotype evolution in Testudines.

Conserved sex chromosomes and karyotype evolution in monitor lizards (Varanidae).

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.

Isolating Chromosomes of the Komodo Dragon: New Tools for Comparative Mapping and Sequence Assembly.

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.

Multiple intrasyntenic rearrangements and rapid speciation in voles.

Remarkably stable genomic chromosome elements (evolutionary conserved segments or syntenies) are the basis of large-scale chromosome architecture in vertebrate species. However, these syntenic elements harbour evolutionary important changes through intrachromosomal rearrangements such as inversions and centromere repositioning. Here, using FISH with a set of 20 region-specific probes on a wide array of 28 species, we analyzed evolution of three conserved syntenic regions of the Arvicolinae ancestral karyotype. Inside these syntenies we uncovered multiple, previously cryptic intrachromosomal rearrangements. Although in each of the three conserved blocks we found inversions and centromere repositions, the blocks experienced different types of rearrangements. In two syntenies centromere repositioning predominated, while in the third region, paracentric inversions were more frequent, whereas pericentric inversions were not detected. We found that some of the intrachromosomal rearrangements, mainly paracentric inversions, were synapomorphic for whole arvicoline genera or tribes: genera Alexandromys and Microtus, tribes Ellobini and Myodini. We hypothesize that intrachromosomal rearrangements within conserved syntenic blocks are a major evolutionary force modulating genome architecture in species-rich and rapidly-evolving rodent taxa. Inversions and centromere repositioning may impact speciation and provide a potential link between genome evolution, speciation, and biogeography.

Chromosome painting of the red-handed tamarin (Saguinus midas) compared to other Callitrichinae monkeys.

Here we present, for the first time, the complete chromosome painting map of Saguinus midas, the red-handed tamarin. Chromosome banding and painting with human chromosome-specific probes were used to compare the karyotype of this species with those of four other Neotropical primates of the subfamily Callitrichinae: Leontopithecus rosalia, Callithrix geoffroyi, C. penicillata, and Mico argentatus. The chromosome painting map of S. midas was identical to that of L. rosalia and other previously studied tamarin species (genera Saguinus and Leontopithecus). The three marmoset species studied (genera Callithrix and Mico) differed in the painting pattern of four human probes (chromosomes 1, 2, 10, and 16). These paints identified the presence or absence of chromosome associations HSA 1/10 and 2/16 in these taxa. By integrating our data with those from the literature, we were able to propose an ancestral Callitrichinae karyotype. The genera Saguinus and Leontopithecus (tamarins) conserve the ancestral Callitrichinae karyotype, while Mico and Callithrix (marmosets) show more derived karyotypes due to chromosome translocations and fissions that occurred during the evolution of these taxa.

Calming an aggressor through spontaneous post-conflict triadic contacts: Appeasement in Macaca tonkeana.

Peaceful third-party interventions usually occur after an aggressive encounter and can be directed toward the victim or the aggressor. Macaca tonkeana, a cercopithecine species characterized by high levels of tolerance, frequently engage in consolatory contacts, which both calm the victim and reduce the probability of further attacks against him/her. Other post-conflict affiliative interventions such as reconciliation and quadratic affiliation are also common in this species. However, little attention has been given to contacts directed toward the aggressor. Here, we explore the role of bystander affiliative interventions toward the aggressor in influencing the affective state of the aggressor and the consequences of triadic interventions at group level. We found that triadic post-conflict affiliation occurred independently from the intensity of the conflict and that it was more frequent in absence of the conciliatory contact between the opponents (reconciliation). Bystanders showed a higher amount of post-conflict affiliation toward low ranking aggressors. Post-conflict triadic affiliation functioned as a tension reduction mechanism by lowering the arousal of the aggressor, which less frequently engaged in renewed aggression. All these findings suggest that post-conflict triadic contacts in Tonkean macaques can be considered as a strategic mechanism to calm the aggressor and reduce the risk of retaliatory aggression.

Rapid emergence of independent "chromosomal lineages" in silvered-leaf monkey triggered by Y/autosome translocation.

Sex/autosome translocations are rare events. The only known example in catarrhines is in the silvered-leaf monkey. Here the Y chromosome was reciprocally translocated with chromosome 1. The rearrangement produced an X1X2Y1Y2 sex chromosome system. At least three chromosomal variants of the intact chromosome 1 are known to exist. We characterized in high resolution the translocation products (Y1 and Y2) and the polymorphic forms of the intact chromosome 1 with a panel of more than 150 human BAC clones. We showed that the translocation products were extremely rearranged, in contrast to the high level of marker order conservation of the other silvered-leaf monkey chromosomes. Surprisingly, each translocation product appeared to form independent "chromosome lineages"; each having a myriad of distinct rearrangements. We reconstructed the evolutionary history of the translocation products by comparing the homologous chromosomes of two other colobine species: the African mantled guereza and the Indian langur. The results showed a massive reuse of breakpoints: only 12, out of the 40 breaks occurred in domains never reused in other rearrangements, while, strikingly, some domains were used up to four times. Such frequent breakpoint reuse if proved to be a general phenomenon has profound implications for mechanisms of chromosome evolution.

Evolutionary Origin of OwlRep, a Megasatellite DNA Associated with Adaptation of Owl Monkeys to Nocturnal Lifestyle.

Rod cells of many nocturnal mammals have a "non-standard" nuclear architecture, which is called the inverted nuclear architecture. Heterochromatin localizes to the central region of the nucleus. This leads to an efficient light transmission to the outer segments of photoreceptors. Rod cells of diurnal mammals have the conventional nuclear architecture. Owl monkeys (genus Aotus) are the only taxon of simian primates that has a nocturnal or cathemeral lifestyle, and this adaptation is widely thought to be secondary. Their rod cells were shown to exhibit an intermediate chromatin distribution: a spherical heterochromatin block was found in the central region of the nucleus although it was less complete than that of typical nocturnal mammals. We recently demonstrated that the primary DNA component of this heterochromatin block was OwlRep, a megasatellite DNA consisting of 187-bp-long repeat units. However, the origin of OwlRep was not known. Here we show that OwlRep was derived from HSAT6, a simple repeat sequence found in the centromere regions of human chromosomes. HSAT6 occurs widely in primates, suggesting that it was already present in the last common ancestor of extant primates. Notably, Strepsirrhini and Tarsiformes apparently carry a single HSAT6 copy, whereas many species of Simiiformes contain multiple copies. Comparison of nucleotide sequences of these copies revealed the entire process of the OwlRep formation. HSAT6, with or without flanking sequences, was segmentally duplicated in New World monkeys. Then, in the owl monkey linage after its divergence from other New World monkeys, a copy of HSAT6 was tandemly amplified, eventually forming a megasatellite DNA.

Identification and characterization of a subtelomeric satellite DNA in Callitrichini monkeys.

Repetitive DNAs are abundant fast-evolving components of eukaryotic genomes, which often possess important structural and functional roles. Despite their ubiquity, repetitive DNAs are poorly studied when compared with the genic fraction of genomes. Here, we took advantage of the availability of the sequenced genome of the common marmoset Callithrix jacchus to assess its satellite DNAs (satDNAs) and their distribution in Callitrichini. After clustering analysis of all reads and comparisons by similarity, we identified a satDNA composed by 171 bp motifs, named MarmoSAT, which composes 1.09% of the C. jacchus genome. Fluorescent in situ hybridization on chromosomes of species from the genera Callithrix, Mico and Callimico showed that MarmoSAT had a subtelomeric location. In addition to the common monomeric, we found that MarmoSAT was also organized in higher-order repeats of 338 bp in Callimico goeldii. Our phylogenetic analyses showed that MarmoSAT repeats from C. jacchus lack chromosome-specific features, suggesting exchange events among subterminal regions of non-homologous chromosomes. MarmoSAT is transcribed in several tissues of C. jacchus, with the highest transcription levels in spleen, thymus and heart. The transcription profile and subtelomeric location suggest that MarmoSAT may be involved in the regulation of telomerase and modulation of telomeric chromatin.

Intrachromosomal Rearrangements in Rodents from the Perspective of Comparative Region-Specific Painting.

It has long been hypothesized that chromosomal rearrangements play a central role in different evolutionary processes, particularly in speciation and adaptation. Interchromosomal rearrangements have been extensively mapped using chromosome painting. However, intrachromosomal rearrangements have only been described using molecular cytogenetics in a limited number of mammals, including a few rodent species. This situation is unfortunate because intrachromosomal rearrangements are more abundant than interchromosomal rearrangements and probably contain essential phylogenomic information. Significant progress in the detection of intrachromosomal rearrangement is now possible, due to recent advances in molecular biology and bioinformatics. We investigated the level of intrachromosomal rearrangement in the Arvicolinae subfamily, a species-rich taxon characterized by very high rate of karyotype evolution. We made a set of region specific probes by microdissection for a single syntenic region represented by the p-arm of chromosome 1 of Alexandromys oeconomus, and hybridized the probes onto the chromosomes of four arvicolines (Microtus agrestis, Microtus arvalis, Myodes rutilus, and Dicrostonyx torquatus). These experiments allowed us to show the intrachromosomal rearrangements in the subfamily at a significantly higher level of resolution than previously described. We found a number of paracentric inversions in the karyotypes of M. agrestis and M. rutilus, as well as multiple inversions and a centromere shift in the karyotype of M. arvalis. We propose that during karyotype evolution, arvicolines underwent a significant number of complex intrachromosomal rearrangements that were not previously detected.

Co-Opted Megasatellite DNA Drives Evolution of Secondary Night Vision in Azara's Owl Monkey.

Owl monkeys (genus Aotus) are the only taxon in simian primates that consists of nocturnal or otherwise cathemeral species. Their night vision is superior to that of other monkeys, apes, and humans but not as good as that of typical nocturnal mammals. This incomplete night vision has been used to conclude that these monkeys only secondarily adapted to a nocturnal lifestyle, or to their cathemeral lifestyle that involves high night-time activity. It is known that the rod cells of many nocturnal mammals possess a unique nuclear architecture in which heterochromatin is centrally located. This "inverted nuclear architecture", in contrast with "conventional nuclear architecture", provides elevated night vision by passing light efficiently to the outer segments of photoreceptors. Owl monkey rod cells exhibit an intermediate chromatin distribution, which may provide them with less efficient night vision than other nocturnal mammals. Recently, we identified three megasatellite DNAs in the genome of Azara's owl monkey (Aotus azarae). In the present study, we show that one of the three megasatellite DNAs, OwlRep, serves as the primary component of the heterochromatin block located in the central space of the rod nucleus in A. azarae. This satellite DNA is likely to have emerged in the Aotus lineage after its divergence from those of other platyrrhini taxa and underwent a rapid expansion in the genome. Our results indicate that the heterochromatin core in the A. azarae rod nucleus was newly formed in A. azarae or its recent ancestor, and supports the hypothesis that A. azarae, and with all probability other Aotus species, secondarily acquired night vision.