Occasional paternal inheritance of the germline-restricted chromosome in songbirds.

Songbirds have one special accessory chromosome, the so-called germline-restricted chromosome (GRC), which is only present in germline cells and absent from all somatic tissues. Earlier work on the zebra finch (Taeniopygia guttata castanotis) showed that the GRC is inherited only through the female line-like the mitochondria-and is eliminated from the sperm during spermatogenesis. Here, we show that the GRC has the potential to be paternally inherited. Confocal microscopy using GRC-specific fluorescent in situ hybridization probes indicated that a considerable fraction of sperm heads (1 to 19%) in zebra finch ejaculates still contained the GRC. In line with these cytogenetic data, sequencing of ejaculates revealed that individual males from two families differed strongly and consistently in the number of GRCs in their ejaculates. Examining a captive-bred male hybrid of the two zebra finch subspecies (T. g. guttata and T. g. castanotis) revealed that the mitochondria originated from a castanotis mother, whereas the GRC came from a guttata father. Moreover, analyzing GRC haplotypes across nine castanotis matrilines, estimated to have diverged for up to 250,000 y, showed surprisingly little variability among GRCs. This suggests that a single GRC haplotype has spread relatively recently across all examined matrilines. A few diagnostic GRC mutations that arose since this inferred spreading suggest that the GRC has continued to jump across matriline boundaries. Our findings raise the possibility that certain GRC haplotypes could selfishly spread through the population via occasional paternal transmission, thereby outcompeting other GRC haplotypes that were limited to strict maternal inheritance, even if this was partly detrimental to organismal fitness.

Micro Germline-Restricted Chromosome in Blue Tits: Evidence for Meiotic Functions.

The germline-restricted chromosome (GRC) is likely present in all songbird species but differs widely in size and gene content. This extra chromosome has been described as either a microchromosome with only limited basic gene content or a macrochromosome with enriched gene functions related to female gonad and embryo development. Here, we assembled, annotated, and characterized the first micro-GRC in the blue tit (Cyanistes caeruleus) using high-fidelity long-read sequencing data. Although some genes on the blue tit GRC show signals of pseudogenization, others potentially have important functions, either currently or in the past. We highlight the GRC gene paralog BMP15, which is among the highest expressed GRC genes both in blue tits and in zebra finches (Taeniopygia guttata) and is known to play a role in oocyte and follicular maturation in other vertebrates. The GRC genes of the blue tit are further enriched for functions related to the synaptonemal complex. We found a similar functional enrichment when analyzing published data on GRC genes from two nightingale species (Luscinia spp.). We hypothesize that these genes play a role in maintaining standard maternal inheritance or in recombining maternal and paternal GRCs during potential episodes of biparental inheritance.

Mendelian nightmares: the germline-restricted chromosome of songbirds.

Germline-restricted chromosomes (GRCs) are accessory chromosomes that occur only in germ cells. They are eliminated from somatic cells through programmed DNA elimination during embryo development. GRCs have been observed in several unrelated animal taxa and show peculiar modes of non-Mendelian inheritance and within-individual elimination. Recent cytogenetic and phylogenomic evidence suggests that a GRC is present across the species-rich songbirds, but absent in non-passerine birds, implying that over half of all 10,500 bird species have extensive germline/soma genome differences. Here, we review recent insights gained from genomic, transcriptomic, and cytogenetic approaches with regard to the genetic content, phylogenetic distribution, and inheritance of the songbird GRC. While many questions remain unsolved in terms of GRC inheritance, elimination, and function, we discuss plausible scenarios and future directions for understanding this widespread form of programmed DNA elimination.

Satellitome comparison of two oedipodine grasshoppers highlights the contingent nature of satellite DNA evolution.

BACKGROUND: The full catalog of satellite DNA (satDNA) within a same genome constitutes the satellitome. The Library Hypothesis predicts that satDNA in relative species reflects that in their common ancestor, but the evolutionary mechanisms and pathways of satDNA evolution have never been analyzed for full satellitomes. We compare here the satellitomes of two Oedipodine grasshoppers (Locusta migratoria and Oedaleus decorus) which shared their most recent common ancestor about 22.8 Ma ago. RESULTS: We found that about one third of their satDNA families (near 60 in every species) showed sequence homology and were grouped into 12 orthologous superfamilies. The turnover rate of consensus sequences was extremely variable among the 20 orthologous family pairs analyzed in both species. The satDNAs shared by both species showed poor association with sequence signatures and motives frequently argued as functional, except for short inverted repeats allowing short dyad symmetries and non-B DNA conformations. Orthologous satDNAs frequently showed different FISH patterns at both intra- and interspecific levels. We defined indices of homogenization and degeneration and quantified the level of incomplete library sorting between species. CONCLUSIONS: Our analyses revealed that satDNA degenerates through point mutation and homogenizes through partial turnovers caused by massive tandem duplications (the so-called satDNA amplification). Remarkably, satDNA amplification increases homogenization, at intragenomic level, and diversification between species, thus constituting the basis for concerted evolution. We suggest a model of satDNA evolution by means of recursive cycles of amplification and degeneration, leading to mostly contingent evolutionary pathways where concerted evolution emerges promptly after lineages split.

Transposable element landscapes illuminate past evolutionary events in the endangered fern Vandenboschia speciosa.

Vandenboschia speciosa is an endangered tetraploid fern species with a large genome (10.5 Gb). Its geographical distribution is characterized by disjoined tertiary flora refuges, with relict populations that survived past climate crises. Here, we analyzed the transposable elements (TEs) and found that they comprise approximately 76% of the V. speciosa genome, thus being the most abundant type of DNA sequence in this gigantic genome. The V. speciosa genome is composed of 51% and 5.6% of Class I and Class II elements, respectively. LTR retrotransposons were the most abundant TEs in this species (at least 42% of the genome), followed by non-LTR retrotransposons, which constituted at least 8.7% of the genome of this species. We introduce an additional analysis to identify the nature of non-annotated elements (19% of the genome). A BLAST search of the non-annotated contigs against the V. speciosa TE database allowed for the identification of almost half of them, which were most likely diverged sequence variants of the annotated TEs. In general, the TE composition in V. speciosa resembles the TE composition in seed plants. In addition, repeat landscapes revealed three episodes of amplification for all TEs, most likely due to demographic changes associated with past climate crises.

Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish.

BACKGROUND: Eukaryote genomes frequently harbor supernumerary B chromosomes in addition to the "standard" A chromosome set. B chromosomes are thought to arise as byproducts of genome rearrangements and have mostly been considered intraspecific oddities. However, their evolutionary transcendence beyond species level has remained untested. RESULTS: Here we reveal that the large metacentric B chromosomes reported in several fish species of the genus Astyanax arose in a common ancestor at least 4 million years ago. We generated transcriptomes of A. scabripinnis and A. paranae 0B and 1B individuals and used these assemblies as a reference for mapping all gDNA and RNA libraries to quantify coverage differences between B-lacking and B-carrying genomes. We show that the B chromosomes of A. scabripinnis and A. paranae share 19 protein-coding genes, of which 14 and 11 were also present in the B chromosomes of A. bockmanni and A. fasciatus, respectively. Our search for B-specific single-nucleotide polymorphisms (SNPs) identified the presence of B-derived transcripts in B-carrying ovaries, 80% of which belonged to nobox, a gene involved in oogenesis regulation. Importantly, the B chromosome nobox paralog is expressed > 30× more than the A chromosome paralog. This indicates that the normal regulation of this gene is altered in B-carrying females, which could potentially facilitate B inheritance at higher rates than Mendelian law prediction. CONCLUSIONS: Taken together, our results demonstrate the long-term survival of B chromosomes despite their lack of regular pairing and segregation during meiosis and that they can endure episodes of population divergence leading to species formation.

Satellite DNA Is an Inseparable Fellow Traveler of B Chromosomes.

Next-Generation Sequencing (NGS) has revealed that B chromosomes in several species are enriched in repetitive DNA, mostly satellite DNA (satDNA). This raises the question of whether satDNA is important to B chromosomes for functional reasons or else its abundance on Bs is simply a consequence of properties of B chromosomes such as their dispensability and late replication. Here we review current knowledge in this respect and contextualize it within the frame of practical difficulties to perform this kind of research, the most important being the absence of good full genome sequencing for B-carrying species, which is an essential requisite to ascertain the intragenomic origin of B chromosomes. Our review analysis on 16 species revealed that 38% of them showed B-specific satDNAs whereas only one of them (6%) carried an inter-specifically originated B chromosome. This shows that B-specific satDNA families can eventually evolve in intraspecifically arisen B chromosomes. Finally, the possibility of satDNA accumulation on B chromosomes for functional reasons is exemplified by B chromosomes in rye, as they contain B-specific satDNAs which are transcribed and occupy chromosome locations where they might facilitate the kind of drive shown by this B chromosome during pollen grain mitosis.

Out of patterns, the euchromatic B chromosome of the grasshopper Abracris flavolineata is not enriched in high-copy repeats.

In addition to the normal set of standard (A) chromosomes, some eukaryote species harbor supernumerary (B) chromosomes. In most cases, B chromosomes show differential condensation with respect to A chromosomes and display dark C-bands of heterochromatin, and some of them are highly enriched in repetitive DNA. Here we perform a comprehensive NGS (next-generation sequencing) analysis of the repeatome in the grasshopper Abracris flavolineata aimed at uncovering the molecular composition and origin of its B chromosome. Our results have revealed that this B chromosome shows a DNA repeat content highly similar to the DNA repeat content observed for euchromatic (non-C-banded) regions of A chromosomes. Moreover, this B chromosome shows little enrichment for high-copy repeats, with only a few elements showing overabundance in B-carrying individuals compared to the 0B individuals. Consequently, the few satellite DNAs (satDNAs) mapping on the B chromosome were mostly restricted to its centromeric and telomeric regions, and they displayed much smaller bands than those observed on the A chromosomes. Our data support the intraspecific origin of the B chromosome from the longest autosome by misdivision, isochromosome formation, and additional restructuring, with accumulation of specific repeats in one or both B chromosome arms, yielding a submetacentric B. Finally, the absence of B-specific satDNAs, which are frequent in other species, along with its euchromatic nature, suggest that this B chromosome arose recently and might still be starting a heterochromatinization process. On this basis, it could be a good model to investigate the initial steps of B chromosome evolution.

How dynamic could be the 45S rDNA cistron? An intriguing variability in a grasshopper species revealed by integration of chromosomal and genomic data.

To better understand the structure and variability of the 45S rDNA cistron and its evolutionary dynamics in grasshoppers, we performed a detailed analysis combining classical and molecular cytogenetic data with whole-genome sequencing in Abracris flavolienata, which shows extraordinary variability in the chromosomal distribution for this element. We found astonishing variability in the number and size of rDNA clusters at intra- and inter-population levels. Interestingly, FISH using distinct parts of 45S rDNA cistron (18S rDNA, 28S rDNA, and ITS1) as probes revealed a distinct number of clusters, suggesting independent mobility and amplification of the 45S rDNA components. This hypothesis is consistent with the higher genomic coverage of almost the entire cistron of 45S rDNA observed in A. flavolineata compared to other grasshoppers, besides coverage variability along the 45S rDNA cistron in the species. In addition, these differences in coverage for distinct components of the 45S rDNA cistron indicate emergence of pseudogenes evidenced by existence of truncated sequences, demonstrating the rDNA dynamics in the species. Although the chromosomal distribution of 18S rDNA was highly variable, the chromosomes 1, 3, 6, and 9 harbored rDNA clusters in all individuals with the occurrence of NOR activity in pair 9, suggesting ancestry or selective pressures to prevent pseudogenization of rDNA sequences in this chromosome pair. Additionally, small NORs and cryptic rDNA loci were observed. Finally, there was no evidence of enrichment and association of transposable elements, at least, inside or nearby rDNA cistron. These findings broaden our knowledge of rDNA dynamics, revealing an independent movement and amplification of segments of 45S rDNA cistron, which in A. flavolineata could be attributed to ectopic recombination.

Gene expression changes elicited by a parasitic B chromosome in the grasshopper Eyprepocnemis plorans are consistent with its phenotypic effects.

Parasitism evokes adaptive physiological changes in the host, many of which take place through gene expression changes. This response can be more or less local, depending on the organ or tissue affected by the parasite, or else systemic when the parasite affects the entire host body. The most extreme of the latter cases is intragenomic parasitism, where the parasite is present in all host nuclei as any other genomic element. Here, we show the molecular crosstalk between a parasitic chromosome (also named B chromosome) and the host genome, manifested through gene expression changes. The transcriptome analysis of 0B and 1B females of the grasshopper Eyprepocnemis plorans, validated by a microarray experiment performed on four B-lacking and five B-carrying females, revealed changes in gene expression for 188 unigenes being consistent in both experiments. Once discarded B-derived transcripts, there were 46 differentially expressed genes (30 up- and 16 downregulated) related with the adaptation of the host genome to the presence of the parasitic chromosome. Interestingly, the functions of these genes could explain some of the most important effects of B chromosomes, such as nucleotypic effects derived from the additional DNA they represent, chemical defense and detoxification, protein modification and response to stress, ovary function, and regulation of gene expression. Collectively, these changes uncover an intimate host-parasite interaction between A and B chromosomes during crucial steps of gene expression and protein function.

Satellite DNA content of B chromosomes in the characid fish Characidium gomesi supports their origin from sex chromosomes.

The origin of supernumerary (B) chromosomes is clearly conditioned by their ancestry from the standard (A) chromosomes. Sequence similarity between A and B chromosomes is thus crucial to determine B chromosome origin. For this purpose, we compare here the DNA sequences from A and B chromosomes in the characid fish Characidium gomesi using two main approaches. First, we found 59 satellite DNA (satDNA) families constituting the satellitome of this species and performed FISH analysis for 18 of them. This showed the presence of six satDNAs on the B chromosome: one shared with sex chromosomes and autosomes, two shared with sex chromosomes, one shared with autosomes and two being B-specific. This indicated that B chromosomes most likely arose from the sex chromosomes. Our second approach consisted of the analysis of five repetitive DNA families: 18S and 5S ribosomal DNA (rDNA), the H3 histone gene, U2 snDNA and the most abundant satDNA (CgoSat01-184) on DNA obtained from microdissected B chromosomes and from B-lacking genomes. PCR and sequence analysis of these repetitive sequences was successful for three of them (5S rDNA, H3 histone gene and CgoSat01-184), and sequence comparison revealed that DNA sequences obtained from the B chromosomes displayed higher identity with C. gomesi genomic DNA than with those obtained from other Characidium species. Taken together, our results support the intraspecific origin of B chromosomes in C. gomesi and point to sex chromosomes as B chromosome ancestors, which raises interesting prospects for future joint research on the genetic content of sex and B chromosomes in this species.

High dynamism for neo-sex chromosomes: satellite DNAs reveal complex evolution in a grasshopper.

A common characteristic of sex chromosomes is the accumulation of repetitive DNA, which accounts for their diversification and degeneration. In grasshoppers, the X0 sex-determining system in males is considered ancestral. However, in some species, derived variants like neo-XY in males evolved several times independently by Robertsonian translocation. This is the case of Ronderosia bergii, in which further large pericentromeric inversion in the neo-Y also took place, making this species particularly interesting for investigating sex chromosome evolution. Here, we characterized the satellite DNAs (satDNAs) and transposable elements (TEs) of the species to investigate the quantitative differences in repeat composition between male and female genomes putatively associated with sex chromosomes. We found a total of 53 satDNA families and 56 families of TEs. The satDNAs were 13.5% more abundant in males than in females, while TEs were just 1.02% more abundant in females. These results imply differential amplification of satDNAs on neo-Y chromosome and a minor role of TEs in sex chromosome differentiation. We showed highly differentiated neo-XY sex chromosomes owing to major amplification of satDNAs in neo-Y. Furthermore, chromosomal mapping of satDNAs suggests high turnover of neo-sex chromosomes in R. bergii at the intrapopulation level, caused by multiple paracentric inversions, amplifications, and transpositions. Finally, the species is an example of the action of repetitive DNAs in the generation of variability for sex chromosomes after the suppression of recombination, and helps understand sex chromosome evolution at the intrapopulation level.

The complete mitochondrial genome of Talpa aquitania (Talpidae; Insectivora), a mole species endemic to northern Spain and southern France.

The complete mitogenome sequence of Talpa aquitania, a recently described Talpa species, was assembled using whole-genome sequencing data. It varies in length from 16,776 to 16,846 bp, contains 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, one origin of L-strand replication, and a control region. In the control region, which varied from 1320 to 1390 bp, we identified the extended termination-associated sequence (ETAS-1 and ETAS-2) and the conserved sequence blocks (CSB-1, 2, 3, B, C, D, E, F). In addition, this region includes a 10 bp tandem repeat DNA sequence, with a variable number of repeats that suggest the existence of heteroplasmy. Phylogeny reconstructions based on Maximum Likelihood, Neighbor-joining and Bayesian inference analyses yielded phylogenies with similar topologies demonstrating that T. aquitania and T. occidentalis are sister species.

Development and characterization of 15 novel polymorphic microsatellite loci for two important bot flies (Diptera, Oestridae) by next-generation sequencing.

Cephenemyia stimulator and Oestrus ovis are two important parasitic bot flies (Oestridae) species causing myiasis, with a potential negative impact on the welfare of the host. Using next-generation sequencing approach and bioinformatics tools, a large panel of possible microsatellites loci was obtained in both species. Primer pairs were designed for 15 selected microsatellite loci in C. stimulator and other 15 loci in O. ovis for PCR amplification. Loci amplification and analysis were performed in four populations of each species. The results demonstrated that all selected loci were polymorphic, with the number of alleles ranging from 2 to 6 per locus in C. stimulator and 3 to 13 per locus in O. ovis. This is the first time to describe these microsatellite loci for C. stimulator and O. ovis. These two sets of microsatellite markers could be further used for biogeographic and population genetics studies.

High-throughput analysis of satellite DNA in the grasshopper Pyrgomorpha conica reveals abundance of homologous and heterologous higher-order repeats.

Satellite DNA (satDNA) constitutes an important fraction of repetitive DNA in eukaryotic genomes, but it is barely known in most species. The high-throughput analysis of satDNA in the grasshopper Pyrgomorpha conica revealed 87 satDNA variants grouped into 76 different families, representing 9.4% of the genome. Fluorescent in situ hybridization (FISH) analysis of the 38 most abundant satDNA families revealed four different patterns of chromosome distribution. Homology search between the 76 satDNA families showed the existence of 15 superfamilies, each including two or more families, with the most abundant superfamily representing more than 80% of all satDNA found in this species. This also revealed the presence of two types of higher-order repeats (HORs), one showing internal homologous subrepeats, as conventional HORs, and an additional type showing non-homologous internal subrepeats, the latter arising by the combination of a given satDNA family with a non-annotated sequence, or with telomeric DNA. Interestingly, the heterologous subrepeats included in these HORs showed higher divergence within the HOR than outside it, suggesting that heterologous HORs show poor homogenization, in high contrast with conventional (homologous) HORs. Finally, heterologous HORs can show high differences in divergence between their constituent subrepeats, suggesting the possibility of regional homogenization.

Quantitative sequence characterization for repetitive DNA content in the supernumerary chromosome of the migratory locust.

Repetitive DNA is a major component in most eukaryotic genomes but is ignored in most genome sequencing projects. Here, we report the quantitative composition in repetitive DNA for a supernumerary (B) chromosome, in the migratory locust (Locusta migratoria), by Illumina sequencing of genomic DNA from B-carrying and B-lacking individuals and DNA obtained from a microdissected B chromosome, as well as the physical mapping of some elements. B chromosome DNA of 94.9% was repetitive, in high contrast with the 64.1% of standard (A) chromosomes. B chromosomes are enriched in satellite DNA (satDNA) (65.2% of B-DNA), with a single satellite (LmiSat02-176) comprising 55% of the B. Six satDNAs were visualized by FISH on the B chromosome, and the only A chromosome carrying all these satellites was autosome 9, pointing to this chromosome, along with autosome 8 (which shares histone genes with the B) as putative ancestors of the B chromosome. We found several transposable elements (TEs) showing nucleotidic variation specific to B-carrying individuals, which was also present in B-carrying transcriptomes. Remarkably, an interstitial region of the B chromosome included a 17 kb chimera composed of 29 different TEs, suggesting reiterative TE insertion in this B chromosome region.

Satellite DNA content illuminates the ancestry of a supernumerary (B) chromosome.

B chromosomes are supernumerary genomic elements most likely derived from the standard (A) chromosomes, whose dispensability has freed their DNA sequences to evolve fast, thus making it difficult to uncover their ancestry. Here, we show the ancestry of a B chromosome in the grasshopper Eumigus monticola by means of the high-throughput analysis of the satellitome, i.e., the whole collection of satellite DNA (satDNA). The satellitome found in this species consists of 27 satDNA families, with monomer length between 5 and 325 nt and A + T content between 42.9 and 83.3 %. Two out of the 20 clustered satDNA families (EmoSat26-41 and EmoSat27-102) were observed only on the B chromosome. The A chromosome carrying the highest number of satDNA families was the megameric S8 (13 families), six of which were also present in the B chromosome, and three of these were exclusive of the S8 and B chromosomes. The absence in the B chromosome of the H3 histone gene cluster (located interstitially on S8) and three satDNA families (located distally on S8) allowed delimiting the possible origin of the B chromosome to the proximal third of the S8 autosome, through a breakpoint between EmoSat11-122 and the H3 cluster. Interestingly, bioinformatic analysis revealed the presence of seeds for the two B-specific satDNAs in the A chromosomes, suggesting their massive amplification in the B chromosome after its origin. Therefore, intraspecifically arisen B chromosomes can harbor DNA sequences apparently being B-specific.

Post-meiotic B chromosome expulsion, during spermiogenesis, in two grasshopper species.

Most supernumerary (B) chromosomes are parasitic elements carrying out an evolutionary arms race with the standard (A) chromosomes. A variety of weapons for attack and defense have evolved in both contending elements, the most conspicuous being B chromosome drive and A chromosome drive suppression. Here, we show for the first time that most microspermatids formed during spermiogenesis in two grasshopper species contain expulsed B chromosomes. By using DNA probes for B-specific satellite DNAs in Eumigus monticola and Eyprepocnemis plorans, and also 18S rDNA in the latter species, we were able to count the number of B chromosomes in standard spermatids submitted to fluorescence in situ hybridization, as well as visualizing B chromosomes inside most microspermatids. In E. plorans, the presence of B-carrying microspermatids in 1B males was associated with a significant decrease in the proportion of B-carrying standard spermatids. The fact that this decrease was apparent in elongating spermatids but not in round ones demonstrates that meiosis yields 1:1 proportions of 0B and 1B spermatids and hence that B elimination takes place post-meiotically, i.e., during spermiogenesis, implying a 5-25% decrease in B transmission rate. In E. monticola, the B chromosome is mitotically unstable and B number varies between cells within a same individual. A comparison of B frequency between round and elongating spermatids of a same individual revealed a significant 12.3% decrease. We conclude that B chromosome elimination during spermiogenesis is a defense weapon of the host genome to get rid of parasitic chromosomes.