Three amphioxus reference genomes reveal gene and chromosome evolution of chordates.

The slow-evolving invertebrate amphioxus has an irreplaceable role in advancing our understanding of the vertebrate origin and innovations. Here we resolve the nearly complete chromosomal genomes of three amphioxus species, one of which best recapitulates the 17 chordate ancestor linkage groups. We reconstruct the fusions, retention, or rearrangements between descendants of whole-genome duplications, which gave rise to the extant microchromosomes likely existed in the vertebrate ancestor. Similar to vertebrates, the amphioxus genome gradually establishes its three-dimensional chromatin architecture at the onset of zygotic activation and forms two topologically associated domains at the Hox gene cluster. We find that all three amphioxus species have ZW sex chromosomes with little sequence differentiation, and their putative sex-determining regions are nonhomologous to each other. Our results illuminate the unappreciated interspecific diversity and developmental dynamics of amphioxus genomes and provide high-quality references for understanding the mechanisms of chordate functional genome evolution.

Whole-Genome Analysis Reveals the Dynamic Evolution of Holocentric Chromosomes in Satyrine Butterflies.

Butterfly chromosomes are holocentric, i.e., lacking a localized centromere. Potentially, this can lead to rapid karyotypic evolution through chromosome fissions and fusions, since fragmented chromosomes retain kinetic activity, while fused chromosomes are not dicentric. However, the actual mechanisms of butterfly genome evolution are poorly understood. Here, we analyzed chromosome-scale genome assemblies to identify structural rearrangements between karyotypes of satyrine butterfly species. For the species pair Erebia ligea-Maniola jurtina, sharing the ancestral diploid karyotype 2n = 56 + ZW, we demonstrate a high level of chromosomal macrosynteny and nine inversions separating these species. We show that the formation of a karyotype with a low number of chromosomes (2n = 36 + ZW) in Erebia aethiops was based on ten fusions, including one autosome-sex chromosome fusion, resulting in a neo-Z chromosome. We also detected inversions on the Z sex chromosome that were differentially fixed between the species. We conclude that chromosomal evolution is dynamic in the satyrines, even in the lineage that preserves the ancestral chromosome number. We hypothesize that the exceptional role of Z chromosomes in speciation may be further enhanced by inversions and sex chromosome-autosome fusions. We argue that not only fusions/fissions but also inversions are drivers of the holocentromere-mediated mode of chromosomal speciation.

XY sex determination in a cnidarian.

BACKGROUND: Sex determination occurs across animal species, but most of our knowledge about its mechanisms comes from only a handful of bilaterian taxa. This limits our ability to infer the evolutionary history of sex determination within animals. RESULTS: In this study, we generated a linkage map of the genome of the colonial cnidarian Hydractinia symbiolongicarpus and used it to demonstrate that this species has an XX/XY sex determination system. We demonstrate that the X and Y chromosomes have pseudoautosomal and non-recombining regions. We then use the linkage map and a method based on the depth of sequencing coverage to identify genes encoded in the non-recombining region and show that many of them have male gonad-specific expression. In addition, we demonstrate that recombination rates are enhanced in the female genome and that the haploid chromosome number in Hydractinia is n = 15. CONCLUSIONS: These findings establish Hydractinia as a tractable non-bilaterian model system for the study of sex determination and the evolution of sex chromosomes.

Hi-C analysis of genomic contacts revealed karyotype abnormalities in chicken HD3 cell line.

BACKGROUND: Karyotype abnormalities are frequent in immortalized continuous cell lines either transformed or derived from primary tumors. Chromosomal rearrangements can cause dramatic changes in gene expression and affect cellular phenotype and behavior during in vitro culture. Structural variations of chromosomes in many continuous mammalian cell lines are well documented, but chromosome aberrations in cell lines from other vertebrate models often remain understudied. The chicken LSCC-HD3 cell line (HD3), generated from erythroid precursors, was used as an avian model for erythroid differentiation and lineage-specific gene expression. However, karyotype abnormalities in the HD3 cell line were not assessed. In the present study, we applied high-throughput chromosome conformation capture to analyze 3D genome organization and to detect chromosome rearrangements in the HD3 cell line. RESULTS: We obtained Hi-C maps of genomic interactions for the HD3 cell line and compared A/B compartments and topologically associating domains between HD3 and several other cell types. By analysis of contact patterns in the Hi-C maps of HD3 cells, we identified more than 25 interchromosomal translocations of regions ≥ 200 kb on both micro- and macrochromosomes. We classified most of the observed translocations as unbalanced, leading to the formation of heteromorphic chromosomes. In many cases of microchromosome rearrangements, an entire microchromosome together with other macro- and microchromosomes participated in the emergence of a derivative chromosome, resembling "chromosomal fusions'' between acrocentric microchromosomes. Intrachromosomal inversions, deletions and duplications were also detected in HD3 cells. Several of the identified simple and complex chromosomal rearrangements, such as between GGA2 and GGA1qter; GGA5, GGA4p and GGA7p; GGA4q, GGA6 and GGA19; and duplication of the sex chromosome GGAW, were confirmed by FISH. CONCLUSIONS: In the erythroid progenitor HD3 cell line, in contrast to mature and immature erythrocytes, the genome is organized into distinct topologically associating domains. The HD3 cell line has a severely rearranged karyotype with most of the chromosomes engaged in translocations and can be used in studies of genome structure-function relationships. Hi-C proved to be a reliable tool for simultaneous assessment of the spatial genome organization and chromosomal aberrations in karyotypes of birds with a large number of microchromosomes.

Expansion and loss of sperm nuclear basic protein genes in Drosophila correspond with genetic conflicts between sex chromosomes.

Many animal species employ sperm nuclear basic proteins (SNBPs) or protamines to package sperm genomes tightly. SNBPs vary across animal lineages and evolve rapidly in mammals. We used a phylogenomic approach to investigate SNBP diversification in Drosophila species. We found that most SNBP genes in Drosophila melanogaster evolve under positive selection except for genes essential for male fertility. Unexpectedly, evolutionarily young SNBP genes are more likely to be critical for fertility than ancient, conserved SNBP genes. For example, CG30056 is dispensable for male fertility despite being one of three SNBP genes universally retained in Drosophila species. We found 19 independent SNBP gene amplification events that occurred preferentially on sex chromosomes. Conversely, the montium group of Drosophila species lost otherwise-conserved SNBP genes, coincident with an X-Y chromosomal fusion. Furthermore, SNBP genes that became linked to sex chromosomes via chromosomal fusions were more likely to degenerate or relocate back to autosomes. We hypothesize that autosomal SNBP genes suppress meiotic drive, whereas sex-chromosomal SNBP expansions lead to meiotic drive. X-Y fusions in the montium group render autosomal SNBPs dispensable by making X-versus-Y meiotic drive obsolete or costly. Thus, genetic conflicts between sex chromosomes may drive SNBP rapid evolution during spermatogenesis in Drosophila species.

In sperm, DNA is packaged more tightly than in other cells thanks to small proteins called 'sperm nuclear basic proteins' (SNBPs), also called protamines in mammals. SNBPs are important for sperm to develop properly and correctly perform their role during fertilization. Although the evolution of SNBPs has been studied in mammals, these proteins have not been as thoroughly examined in invertebrates. Chang et al. took advantage of the availability of high-quality sequences for the genomes of 78 species of Drosophila flies to investigate the evolution of the genes that code for SNBPs in these flies. The results showed that, just like in mammals, in Drosophila the protein sequences of SNBPs evolve rapidly. However, unlike mammals, Chang et al. also found that Drosophila species frequently gained and lost genes coding for SNBPs. Interestingly, the 'older' genes (genes that appeared earlier in evolution) that code for SNBPs are not essential for reproduction in the fruit fly Drosophila melanogaster. This is an unexpected finding because older genes usually have essential roles for survival and reproduction, which require them to be passed on to the next generation and remain in the genome. In contrast, younger SNBP genes that had appeared more recently and were not shared between different species of Drosophila were often essential for fertility. These results, combined with other observations about where SNBP genes are located in the genome, led Chang et al. to hypothesize that SNBPs present in sex chromosomes act as 'meiotic drivers' while those on other chromosomes (known as autosomes) suppress meiotic drive. In other words, SNBP genes present in the sex chromosomes may be responsible for killing sister sperm cells that do not carry those genes, while SNBP genes that are not located on sex chromosomes may suppress this activity. This is of particular interest because it indicates that SNBPs are involved in genetic conflicts between the two sex chromosomes: sperm that carry SNBPs on the X chromosome may kill sperm with a Y chromosome, and vice versa. The results of Chang et al. shed light on the mysterious evolution of SNBPs in Drosophila flies. Although previous hypotheses regarding the rapid evolution of SNBPs evolution have focused on their role in genome packaging, this new analysis suggests that much of the evolutionary change is likely driven by genetic conflicts between sex chromosomes.

Evolutionary History of the Poecilia picta Sex Chromosomes.

The degree of divergence between the sex chromosomes is not always proportional to their age. In poeciliids, four closely related species all exhibit a male heterogametic sex chromosome system on the same linkage group, yet show a remarkable diversity in X and Y divergence. In Poecilia reticulata and P. wingei, the sex chromosomes remain homomorphic, yet P. picta and P. parae have a highly degraded Y chromosome. To test alternative theories about the origin of their sex chromosomes, we used a combination of pedigrees and RNA-seq data from P. picta families in conjunction with DNA-seq data collected from P. reticulata, P. wingei, P. parae, and P. picta. Phylogenetic clustering analysis of X and Y orthologs, identified through segregation patterns, and their orthologous sequences in closely related species demonstrates a similar time of origin for both the P. picta and P. reticulata sex chromosomes. We next used k-mer analysis to identify shared ancestral Y sequence across all four species, suggesting a single origin to the sex chromosome system in this group. Together, our results provide key insights into the origin and evolution of the poeciliid Y chromosome and illustrate that the rate of sex chromosome divergence is often highly heterogenous, even over relatively short evolutionary time frames.

Newly identified sex chromosomes in the Sphagnum (peat moss) genome alter carbon sequestration and ecosystem dynamics.

Peatlands are crucial sinks for atmospheric carbon but are critically threatened due to warming climates. Sphagnum (peat moss) species are keystone members of peatland communities where they actively engineer hyperacidic conditions, which improves their competitive advantage and accelerates ecosystem-level carbon sequestration. To dissect the molecular and physiological sources of this unique biology, we generated chromosome-scale genomes of two Sphagnum species: S. divinum and S. angustifolium. Sphagnum genomes show no gene colinearity with any other reference genome to date, demonstrating that Sphagnum represents an unsampled lineage of land plant evolution. The genomes also revealed an average recombination rate an order of magnitude higher than vascular land plants and short putative U/V sex chromosomes. These newly described sex chromosomes interact with autosomal loci that significantly impact growth across diverse pH conditions. This discovery demonstrates that the ability of Sphagnum to sequester carbon in acidic peat bogs is mediated by interactions between sex, autosomes and environment.

Parallel Evolution of Sex-Linked Genes across XX/XY and ZZ/ZW Sex Chromosome Systems in the Frog Glandirana rugosa.

Genetic sex-determination features male (XX/XY) or female heterogamety (ZZ/ZW). To identify similarities and differences in the molecular evolution of sex-linked genes between these systems, we directly compared the sex chromosome systems existing in the frog Glandirana rugosa. The heteromorphic X/Y and Z/W sex chromosomes were derived from chromosomes 7 (2n = 26). RNA-Seq, de novo assembly, and BLASTP analyses identified 766 sex-linked genes. These genes were classified into three different clusters (XW/YZ, XY/ZW, and XZ/YW) based on sequence identities between the chromosomes, probably reflecting each step of the sex chromosome evolutionary history. The nucleotide substitution per site was significantly higher in the Y- and Z-genes than in the X- and W- genes, indicating male-driven mutation. The ratio of nonsynonymous to synonymous nucleotide substitution rates was higher in the X- and W-genes than in the Y- and Z-genes, with a female bias. Allelic expression in gonad, brain, and muscle was significantly higher in the Y- and W-genes than in the X- and Z-genes, favoring heterogametic sex. The same set of sex-linked genes showed parallel evolution across the two distinct systems. In contrast, the unique genomic region of the sex chromosomes demonstrated a difference between the two systems, with even and extremely high expression ratios of W/Z and Y/X, respectively.

Occurrence of Sex Chromosomes in Fish of the Genus Ancistrus with a New Description of Multiple Sex Chromosomes in the Ecuadorian Endemic Ancistrus clementinae (Loricariidae).

Ancistrus Kner, 1854, is the most diverse genus among the Ancistrini (Loricariidae) with 70 valid species showing a wide geographic distribution and great taxonomic and systematic complexity. To date, about 40 Ancistrus taxa have been karyotyped, all from Brazil and Argentina, but the statistic is uncertain because 30 of these reports deal with samples that have not yet been identified at the species level. This study provides the first cytogenetic description of the bristlenose catfish, Ancistrus clementinae Rendahl, 1937, a species endemic to Ecuador, aiming to verify whether a sex chromosome system is identifiable in the species and, if so, which, and if its differentiation is associated with the presence of repetitive sequences reported for other species of the family. We associated the karyotype analysis with the COI molecular identification of the specimens. Karyotype analysis suggested the presence of a ♂ZZ/♀ZW1W2 sex chromosome system, never detected before in Ancistrus, with both W1W2 chromosomes enriched with heterochromatic blocks and 18S rDNA, in addition to GC-rich repeats (W2). No differences were observed between males and females in the distribution of 5S rDNA or telomeric repeats. Cytogenetic data here obtained confirm the huge karyotype diversity of Ancistrus, both in chromosome number and sex-determination systems.

Inclusion of sex chromosomes in noninvasive prenatal testing in Asia, Australia, Europe and the USA: A survey study.

OBJECTIVE: To examine the extent to which sex chromosomes are included in current noninvasive prenatal testing (NIPT) and the reporting practices with respect to fetal chromosomal sex and sex chromosome aberrations (SCAs), in addition to an update on the general implementation of NIPT. METHOD: A questionnaire addressing the research objectives was distributed by email to fetal medicine and clinical genetics experts in Asia, Australia, Europe and the USA. RESULTS: Guidelines on NIPT are available in the majority of the included countries. Not all existing guidelines address reporting of fetal chromosomal sex and SCAs. In most settings, NIPT frequently includes sex chromosomes (five Australian states, China, Hong Kong, Israel, Singapore, Thailand, USA and 23 of 31 European countries). This occurs most often by default or when parents wish to know fetal sex. In most settings, a potential SCA is reported by stating the risk hereof as "low" or "high" and/or by naming the SCA. Less than 50% of all pregnant women receive NIPT according to respondents from three Australian states, China, Israel, Singapore, Thailand and 24 of 31 European countries. However, this percentage, the genomic coverage of NIPT and its application as primary or secondary screening vary by setting. CONCLUSION: In most of the studied countries/states, NIPT commonly includes sex chromosomes. The reporting practices concerning fetal chromosomal sex and SCAs are diverse and most commonly not addressed by guidelines. In general, NIPT is variably implemented across countries/states.

Speech Sound Development in 18-Month-Old Children With Sex Chromosome Trisomies.

PURPOSE: This study aimed to describe speech sound development in a group of 18-month-old children with sex chromosome trisomies (SCTs), compared with a group of typically developing (TD) peers. Concurrent and longitudinal relationships between speech sound abilities and lexical development were examined. METHOD: A group of 76 children aged 18 months, 38 children prenatally diagnosed with SCTs (12 with XXY, 12 with XYY, and 14 with XXX) and 38 TD children, participated in the study. From video recordings of semistructured naturalistic parent-child play sessions, quantitative and qualitative measures of speech sound development were collected (e.g., the number of consonants, type and place of articulation, and syllable structures used), and group differences were observed. The relationships between the number of consonants produced and vocabulary size at 18 and 24 months were assessed. RESULTS: At 18 months, children with SCTs used a significantly lower number of consonants than TD children. Qualitatively, children with SCTs used significantly fewer articulatory complex consonants (fricative/affricates) and a more restricted inventory of syllable structures. The number of consonants used was significantly correlated with lexical development at 18 months. Moreover, in the SCTs group (but not in the TD group), the children with lower speech sound development at 18 months showed a significantly smaller vocabulary growth between 18 and 24 months than those with higher speech-sound development. CONCLUSIONS: Toddlers with SCTs showed a significantly delayed speech sound development pattern rather than an atypical one. Children with SCTs with low speech sound development also showed lower vocabulary growth between 18 and 24 months of age. These results can be clinically relevant for follow-up and treatment planning for children with SCTs.

Small RNAs and their protein partners in animal meiosis.

Meiosis is characterized by highly regulated transitions in gene expression that require diverse mechanisms of gene regulation. For example, in male mammals, transcription undergoes a global shut-down in early prophase I of meiosis, followed by increasing transcriptional activity into pachynema. Later, as spermiogenesis proceeds, the histones bound to DNA are replaced with transition proteins, which are themselves replaced with protamines, resulting in a highly condensed nucleus with repressed transcriptional activity. In addition, two specialized gene silencing events take place during prophase I: meiotic silencing of unsynapsed chromatin (MSUC), and the sex chromatin specific mechanism, meiotic sex chromosome inactivation (MSCI). Notably, conserved roles for the RNA binding protein (RBP) machinery that functions with small non-coding RNAs have been described as participating in these meiosis-specific mechanisms, suggesting that RNA-mediated gene regulation is critical for fertility in many species. Here, we review roles of small RNAs and their associated RBPs in meiosis-related processes such as centromere function, silencing of unpaired chromatin and meiotic recombination. We will discuss the emerging evidence of non-canonical functions of these components in meiosis.

Madagascar Leaf-Tail Geckos (Uroplatus spp.) Share Independently Evolved Differentiated ZZ/ZW Sex Chromosomes.

Geckos are an excellent group to study the evolution of sex determination, as they possess a remarkable variability ranging from a complete absence of sex chromosomes to highly differentiated sex chromosomes. We explored sex determination in the Madagascar leaf-tail geckos of the genus Uroplatus. The cytogenetic analyses revealed highly heterochromatic W chromosomes in all three examined species (Uroplatus henkeli, U. alluaudi, U. sikorae). The comparative gene coverage analysis between sexes in U. henkeli uncovered an extensive Z-specific region, with a gene content shared with the chicken chromosomes 8, 20, 26 and 28. The genomic region homologous to chicken chromosome 28 has been independently co-opted for the role of sex chromosomes in several vertebrate lineages, including monitors, beaded lizards and monotremes, perhaps because it contains the amh gene, whose homologs were repeatedly recruited as a sex-determining locus. We demonstrate that all tested species of leaf-tail geckos share homologous sex chromosomes despite the differences in shape and size of their W chromosomes, which are not homologous to the sex chromosomes of other closely related genera. The rather old (at least 40 million years), highly differentiated sex chromosomes of Uroplatus geckos can serve as a great system to study the convergence of sex chromosomes evolved from the same genomic region.

In vitro spermatogenesis: Why meiotic checkpoints matter.

Successful in vitro spermatogenesis would generate functional haploid spermatids, and thus, form the basis for novel approaches to treat patients with impaired spermatogenesis or develop alternative strategies for male fertility preservation. Several culture strategies, including cell cultures using various stem cells and ex vivo cultures of testicular tissue, have been investigated to recapitulate spermatogenesis in vitro. Although some studies have described complete meiosis and subsequent generation of functional spermatids, key meiotic events, such as chromosome synapsis and homologous recombination required for successful meiosis and faithful in vitro-derived gametes, are often not reported. To guarantee the generation of in vitro-formed spermatids without persistent DNA double-strand breaks (DSBs) and chromosomal aberrations, criteria to evaluate whether all meiotic events are completely executed in vitro need to be established. In vivo, these meiotic events are strictly monitored by meiotic checkpoints that eliminate aberrant spermatocytes. To establish criteria to evaluate in vitro meiosis, we review the meiotic events and checkpoints that have been investigated by previous in vitro spermatogenesis studies. We found that, although major meiotic events such as initiation of DSBs and recombination, complete chromosome synapsis, and XY-body formation can be achieved in vitro, crossover formation, chiasmata frequency, and checkpoint mechanisms have been mostly ignored. In addition, complete spermiogenesis, during which round spermatids differentiate into elongated spermatids, has not been achieved in vitro by various cell culture strategies. Finally, we discuss the implications of meiotic checkpoints for in vitro spermatogenesis protocols and future clinical use.

Molecular characterization of eliminated chromosomes in Hessian fly (Mayetiola destructor (Say)).

Like other cecidomyiid Diptera, Hessian fly has stable S chromosomes and dispensable E chromosomes that are retained only in the germ line. Amplified fragment length polymorphisms (AFLP), suppressive subtractive hybridization (SSH), fluorescent in-situ hybridization (FISH), and sequencing were used to investigate similarities and differences between S and E chromosomes. More than 99.9% of AFLP bands were identical between separated ovary and somatic tissue, but one band was unique to ovary and resembled Worf, a non-LTR retrotransposon. Arrayed clones, derived by SSH of somatic from ovarian DNA, showed no clones that were unique to ovary. FISH with BAC clones revealed a diagnostic banding pattern of BAC positions on both autosomes and both sex chromosomes, and each E chromosome shared a pattern with one of the S chromosomes. Sequencing analysis showed that E chromosomes are nearly identical to S chromosomes, since no sequence could be confirmed to belong only to E chromosomes. There were a few questionably E-specific sequences that are candidates for further investigation. Thus, the E chromosomes appear to be derived from S chromosomes by the acquisition or conversion of sequences that produce the negatively heteropycnotic region around the centromere.

Why Are X Autosome Rearrangements so Frequent in Beetles? A Study of 50 Cases.

Amongst the 460 karyotypes of Polyphagan Coleoptera that we studied, 50 (10.8%) were carriers of an X autosome rearrangement. In addition to mitotic metaphase analysis, the correct diagnosis was performed on meiotic cells, principally at the pachytene stage. The percentages of these inter-chromosomal rearrangements, principally fusions, varied in relation to the total diploid number of chromosomes: high (51%) below 19, null at 19, low (2.7%) at 20 (the ancestral and modal number), and slightly increasing from 7.1% to 16.7% from 22 to above 30. The involvement of the X in chromosome fusions appears to be more than seven-fold higher than expected for the average of the autosomes. Examples of karyotypes with X autosome rearrangements are shown, including insertion of the whole X in the autosome (ins(A;X)), which has never been reported before in animals. End-to-end fusions (Robertsonian translocations, terminal rearrangements, and pseudo-dicentrics) are the most frequent types of X autosome rearrangements. As in the 34 species with a 19,X formula, there was no trace of the Y chromosome in the 50 karyotypes with an X autosome rearrangement, which demonstrates the dispensability of this chromosome. In most instances, C-banded heterochromatin was present at the X autosome junction, which suggests that it insulates the gonosome from the autosome portions, whose genes are subjected to different levels of expression. Finally, it is proposed that the very preferential involvement of the X in inter-chromosome rearrangements is explained by: (1) the frequent acrocentric morphology of the X, thus the terminal position of constitutive heterochromatin, which can insulate the attached gonosomal and autosomal components; (2) the dispensability of the Y chromosome, which considerably minimizes the deleterious consequences of the heterozygous status in male meiosis, (3) following the rapid loss of the useless Y chromosome, the correct segregation of the X autosome-autosome trivalent, which ipso facto is ensured by a chiasma in its autosomal portion.

High-density linkage maps and chromosome level genome assemblies unveil direction and frequency of extensive structural rearrangements in wood white butterflies (Leptidea spp.).

Karyotypes are generally conserved between closely related species and large chromosome rearrangements typically have negative fitness consequences in heterozygotes, potentially driving speciation. In the order Lepidoptera, most investigated species have the ancestral karyotype and gene synteny is often conserved across deep divergence, although examples of extensive genome reshuffling have recently been demonstrated. The genus Leptidea has an unusual level of chromosome variation and rearranged sex chromosomes, but the extent of restructuring across the rest of the genome is so far unknown. To explore the genomes of the wood white (Leptidea) species complex, we generated eight genome assemblies using a combination of 10X linked reads and HiC data, and improved them using linkage maps for two populations of the common wood white (L. sinapis) with distinct karyotypes. Synteny analysis revealed an extensive amount of rearrangements, both compared to the ancestral karyotype and between the Leptidea species, where only one of the three Z chromosomes was conserved across all comparisons. Most restructuring was explained by fissions and fusions, while translocations appear relatively rare. We further detected several examples of segregating rearrangement polymorphisms supporting a highly dynamic genome evolution in this clade. Fusion breakpoints were enriched for LINEs and LTR elements, which suggests that ectopic recombination might be an important driver in the formation of new chromosomes. Our results show that chromosome count alone may conceal the extent of genome restructuring and we propose that the amount of genome evolution in Lepidoptera might still be underestimated due to lack of taxonomic sampling.