Rapid gene content turnover on the germline-restricted chromosome in songbirds

Rapid gene content turnover on the germline-restricted chromosome in songbirds | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Rapid gene content turnover on the germline-restricted chromosome in songbirds Stephen Schlebusch, Jakub Rídl, Manon Poignet, Francisco Ruiz-Ruano, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1359388/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Jul, 2023 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The germline-restricted chromosome (GRC) of songbirds represents an extraordinary and taxonomically widespread example of programmed DNA elimination. Despite its apparent indispensability in songbirds, we still know very little about the GRC’s genetic composition, function, and evolutionary significance. Here we assembled the GRC in two closely related species, the common and thrush nightingale. We identified 585 and 406 genes on the GRC of each species, respectively, many of them present in multiple copies. Interestingly, the GRC gene content differed dramatically between the two species, with only 192 genes being shared despite only 1.8 million years of species divergence. The chromosome appears to be under little selective pressure, with most GRC genes being present in pseudogenized fragments. Only one gene, cpeb1 , had a complete coding region in all examined individuals of the two species and showed no copy number variation. The addition of this gene to the GRC corresponds with the earliest estimates of the GRC origin, making it a good candidate for the functional indispensability of the GRC in songbirds. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction In multicellular organisms, all cells of an individual normally contain the same genetic information. There are exceptions, however, where certain sequences are eliminated from all or some of the somatic cells during development, leaving the original genetic information to be maintained in the germ cells (Wang and Davis, 2014 ; Suh and Dion-Côté, 2021 ). An interesting example of this programmed DNA elimination has been described in songbirds, where a whole chromosome is lost from somatic cells early on in embryo development. The aptly named germline-restricted chromosome (GRC) was described for the first time in the zebra finch ( Taeniopygia guttata ) (Pigozzi and Solari 1998 ), with recent studies suggesting that it likely occurs in all songbirds (order Passeriformes, suborder Oscines) (Torgasheva, et al. 2019; Kinsella, et al. 2019). Songbirds diverged from the rest of the birds approximately 47 mya (Oliveros, et al. 2019) and comprise approximately 50% of all modern bird species, making them the largest taxonomic group with obligatory programmed DNA elimination. Despite the relatively wide distribution of the GRC, we still know very little about its genetic composition, evolutionary significance and function for birds. Besides its exclusive presence in the germline, there is little that is consistent about this chromosome. The GRC is normally maternally inherited, but paternal inheritance has been shown to be possible (Pei, et al. 2022). It occurs in a single copy in male germ cells, which is excluded from the nucleus during meiosis, and in two copies in female germ cells (Pigozzi and Solari 2005 ), although again, there are exceptions (Malinovskaya, et al. 2020; Torgasheva, et al. 2021 ). The chromosome size varies dramatically, from the largest macrochromosome in the cell (macro-GRC) to a small microchromosome (micro-GRC), with no apparent phylogenetic pattern (Torgasheva, et al. 2019). This lack of conservation is in clear contrast to the apparent ubiquity of the GRC in songbirds. Part of the reason why there is still so much unknown about the genetic composition of the GRC is that this chromosome is hard to sequence effectively. The GRC sequence is largely composed of recently diverged paralogous sequences from the other chromosomes in the cell (hereafter referred to as A chromosomes), and as such can be hard to differentiate in a sequencing library (Itoh et al. 2009 ; Biederman et al. 2018 ; Kinsella, et al. 2019; Asalone, et al. 2021 ). In addition, gonads are composed of both somatic and germ cells, so this chromosome is only found in a subset of testis cells and a minimal proportion of ovary cells (Kinsella, et al. 2019). Thus, GRC sequences are underrepresented in the sequencing libraries from these tissues. Assembled sequence information from the GRC is scarce and currently limited to T. guttata (Itoh et al. 2009 , Biedermann et al. 2018, Kinsella et al. 2019 and Pei, et al, 2022). Thus far, analyses of tissue-specific single-nucleotide polymporphisms (SNPs) and germline/somatic coverage differences have identified 269 putative genes as well as many high copy number regions on the T. guttata macro-GRC (Kinsella, et al. 2019; Asalone, et al. 2021 ). However, the total assembled length of GRC-linked sequences is 1.24 Mb (Kinsella, et al. 2019) plus 468 kb (Asalone, et al. 2021 ), which is approximately 1% of the expected 150 Mb T. guttata macro-GRC. In this paper, we sequenced and assembled the GRC in two closely related songbird species, the common nightingale ( Luscinia megarhynchos ) and thrush nightingale ( L. luscinia ), both of which possess a micro-GRC (Poignet et al., 2021 ). These species from the Muscicapidae family diverged approximately 1.8 mya (Storchová et al. 2010 ) and still hybridize in a secondary contact zone (Reifová, et al. 2011 ; Mořkovský, et al. 2018; Albrecht, et al. 2019). Using a novel method to identify GRC reads from germline sequencing libraries, we assembled the majority of the GRCs for both species. Our results show rapid gene content turnover with significant differences observed not only between species but even among individuals of the same species. The vast majority of genes on the GRC were only partially present and presumably non-functional. The gene cpeb1 was the only entire gene present in all individuals with no copy number variation. We show that this gene belongs to the oldest genes on the GRC, making it the standout candidate gene with an essential function on the GRC, which might be preventing the loss of the GRC in songbirds. Results GRC size estimation using meiotic spreads We visualized the pachytene chromosomes in testis cells using antibodies against the synaptonemal complex (anti-SYCP3) and centromere (CREST) (see Fig. 1 ). These antibodies enable the identification of the unpaired, univalent GRC (del Priore and Pigozzi 2014 ; Torgasheva, et al. 2019). In addition, we immunostained the eliminated GRC from the secondary spermatocytes in the form of a micronucleus (see Supplementary Fig. 1) using an antibody against histone H3 lysine 9 methylation (H3K9me) (del Priore and Pigozzi 2014 ). Both species had a GRC comparable in size with a microchromosome (i.e. a micro-GRC) as was described in Poignet et al. ( 2021 ). Consistent with this, the GRC micronucleus for both nightingale species was much smaller than in species with a macro-GRC (Supplementary Fig. 1; see del Priore and Pigozzi 2014 for visualization of the GRC micronucleus in T. guttata with a macro-GRC). The length of the GRC was estimated by measuring the size of the 22 largest chromosomes, as well as the GRC, in the pachytene cells and comparing the sizes with assembled chromosome lengths (in bp) in collared flycatcher ( Ficedula albicollis ), a songbird species that diverged from nightingales 15 mya (Jetz, et al. 2012 ). Given the conservation of bird karyotypes (Kawakami et al. 2014 ), we assumed that chromosome lengths would be similar between F. albicollis and nightingales (see Supplementary Fig. 2). Using this approach, we estimated the GRC size to be 9.6 Mbp for L. megarhynchos and 7.5 Mbp for L. luscinia . GRC assembly To identify GRC-derived sequences, we sequenced and compared somatic and germline genomes in three individuals of each species. One individual from each species was sequenced with 10x Chromium linked-read sequencing and two individuals with standard Illumina technology. The GRC was assembled using (i) 10x linked reads that aligned in a germline-specific way to the germline genome assembly, (ii) reads that contained germline-specific SNPs, (iii) reads containing germline-specific repetitive elements, which was only applicable for L. megarhynchos , and (iv) any 10x linked reads that shared their 10x barcodes with reads selected in previous steps (Supplementary Fig. 3). Approximately 23 thousand 10x barcodes were identified, resulting in 5.6 million read pairs to assemble the L. megarhynchos GRC. In comparison, only 13 thousand 10x barcodes were identified in L. luscinia , which resulted in 3 million read pairs. Despite having fewer reads, the L. luscinia GRC assembly was longer (5.6 Mbp) and of higher quality (see Table 1 ) than the L. megarhynchos assembly (3.5 Mbp). While the GRC assemblies were highly fragmented, their cumulative length suggests that a large proportion of each GRC was assembled (36–75% of the estimated size). This number is however probably an underestimate, as it does not take recent within-GRC duplicated sequences into account (see “Recent copy number variation within the GRC” below). Table 1 GRC assembly metrics for each nightingale species. L. megarhynchos L. luscinia Total Length 3.5 Mbp 5.6 Mbp Largest Scaffold 110 Kbp 370 Kbp Number of Scaffolds 1400 750 Scaffold N50 2.8 Kbp 46 Kbp Contig N50 2.5 Kbp 24 Kbp %N 3.3% 6.4% Recent copy number variation within the GRC The coverage across the GRC was calculated for the three individuals from each species. This was done in order to identify regions of the GRC that had been duplicated within the GRC but have not diverged sufficiently from their GRC paralogs to be differentiated by the assembly process. L. megarhynchos showed more near-identical duplications merged in the assembly process compared to L. luscinia . This was reflected by the higher average normalised GRC copy number in L. megarhynchos (3.0x) than in L. luscinia (1.6x). Importantly, this mostly accounts for recent within-GRC duplications and repetitive elements on the GRC, which the genome assembler was unable to differentiate. Older within-GRC duplications which have diverged in sequence are not captured in this metric and are expected to be assembled into separate paralogous sequences. The lower proportion of near-identical duplications in the L. luscinia GRC at least partially explains the higher assembly quality in this species. This is supported by the fact that the longest scaffolds from L. luscinia consistently had low copy number (see Fig. 2 ). It is also interesting to note that there is considerable variation in copy number among individuals. This is especially noticeable in L. megarhynchos but is also present in the L. luscinia scaffolds with higher copy numbers (see Fig. 2 ). This suggests that there is substantial variation in recent within-GRC duplications between the GRCs, even within species. The normalised copy number calculation allows for the estimation of the GRC size, accounting for near-identical duplicated sequences erroneously collapsed in the assembly, for each individual. Once these duplications are taken into account, the size of the GRC assembly is 1.3x-2.9x times larger than the original assembly suggested (see Fig. 3 ). The GRC assembly of the individuals sequenced by 10x linked reads was 10.2 Mbp long after this correction for L. megarhynchos and 7.0 Mbp for L. luscinia . These numbers are similar to the GRC size estimates from the meiotic spreads (9.6 Mbp and 7.5 Mbp, respectively), suggesting that we assembled the vast majority of the GRC in both species. And again, the variation among individuals within the same species is noticeable, with the L. megarhynchos GRC ranging in size from 8.8 Mbp to 12.5 Mbp and the L. luscinia GRC ranging from 7.0 Mbp to 11.6 Mbp. This variation is especially impressive considering that two of the three individuals in each species are not assembled, instead using the third individual as a reference, and will therefore underestimate any sequence that does not have a homologous region in the reference GRC. Genetic content of the GRC The variation in GRC size between the species and individuals may reflect highly different genetic content in the GRCs. To explore this possibility, we aligned the two GRC assemblies against each other as well as to the L. megarhynchos genome. Surprisingly, approximately only 1 Mbp of each GRC assembly aligned to the other GRC (before accounting for coverage; see Fig. 3 ), the rest being species-specific. Consistent with this, the A-chromosomal origins of each species’ GRC sequences are strikingly different (see Fig. 4 ). Curiously, the part of the GRC which is orthologous between the two species was disproportionately repetitive, with the average normalised copy number in these orthologous regions being 5.2x and 3.1x for L. megarhynchos and L. luscinia respectively, while the non-homologous regions had an average respective copy number of 2.1x and 1.4x. The L. luscinia GRC has a large proportion paralogous to Chromosome 2, which is absent in the L. megarhynchos GRC assembly. Most of this Chromosome 2 derived sequence comes from a single region of Chromosome 2. This paralogous region is visible in many of the largest scaffolds from the L. luscinia assembly (see Supplementary Fig. 4). Despite being present in large blocks, and likely originating from a single A-to-GRC duplication, it is no longer continuous, presumably as a result of internal rearrangements and within-GRC duplications or else later A-to-GRC duplications. For example, there are two large scaffolds with Chromosome 2 ancestry that are clearly within-GRC duplicates (Scaffolds 9 and 10, Supplementary Fig. 4), but because this duplication happened long enough ago, their sequences have sufficiently diverged for the assembly process to distinguish them (see Fig. 2 ). Divergence from A Chromosomes The proportion of mismatches between homologous regions of each GRC and the A chromosomes of each species was calculated to determine if the GRC sequences originated before or after the divergence of the two nightingale species (see Fig. 5 ). Interestingly, a large proportion of the L. megarhynchos GRC appears to have originated after speciation, with approximately 1 Mbp of the assembly aligning better to its A-chromosomal paralogs than to the L. luscinia genome. In comparison, virtually all of the L. luscinia GRC appears to predate the divergence of the species, with no noticeable difference in mismatches between the GRC and the A-chromosomal paralogs from both species. Gene Annotation Genes were annotated on each of the GRC assemblies using F. albicollis protein-coding genes (FicAlb1.5), resulting in 585 identified genes in L. megarhynchos and 406 in L. luscinia . As might be expected given the largely different sequence origins of the two GRCs, the majority of genes were not shared between the two species, with only 192 genes being present in both species. Genes were assessed for their completeness (see Table 2 ) and copy number within the GRC (Supplementary Table 1). Notably, the vast majority of identified genes were both duplicated (with the average gene having a corrected copy number of 6.6x in L. megarhynchos and 3.7x in L. luscinia ) and only partially present (with only 23 genes in L. megarhynchos and 18 genes in L. luscinia having more than 95% of the coding region present in the assembly). This observed gene fragmentation was measured after correcting for a possible lack of sequence conservation between the nightingales and F. albicollis , which used the percentage of the gene that was found on the A chromosomes as a baseline of expected conservation (see Materials and Methods; Supplementary Table 1 contains gene-specific details). Ten of the genes with 95% of their coding sequence present were shared between the two nightingale species, from which six were previously reported on the T. guttata GRC (Kinsella, et al, 2019). Table 2 Number of GRC genes at varying levels of fragmentation identified in each nightingale species. A gene was counted as being shared between the two nightingales at a particular level of fragmentation if the proportion that was present was sufficient in both species. For genes shared between nightingales and T. guttata , the level of fragmentation had to be met for both nightingale species, and the gene had to be reported as putatively on the T. guttata GRC (Kinsella, et al. 2019). Proportion of coding region found Number of genes in L. megarhynchos Number of genes in L. luscinia Number of shared genes Number of genes shared between nightingales and T. guttata > 0% 585 406 192 25 > 25% 150 (26%) 99 (24%) 57 (30%) 13 (52%) > 50% 81 (14%) 63 (16%) 39 (20%) 11 (44%) > 75% 49 (8%) 36 (9%) 24 (13%) 9 (36%) > 95% 22 (4%) 17 (4%) 10 (5%) 6 (24%) While most genes on the GRC were species-specific, the genes that were shared had a similar percentage of their coding regions present in the two GRCs (see Fig. 6 ). The portion of these genes that was present on the two GRCs was also often the same portion (see Supplementary Fig. 5). This consistency suggests that the presence of these shared genes is the result of their presence on an ancestral GRC, rather than independent additions in each lineage. The consistency in the degree of fragmentation of these shared genes (see Fig. 6 ), as well as the parts of each gene which were missing (see Supplementary Fig. 5), also suggests that the observed gene fragmentation is largely a real phenomenon and not the result of incomplete and low quality GRC assemblies. Among the 10 genes which had more than 95% of their coding region present on both species’ GRCs, three were characterised as being homologs of endogenous retrovirus-derived proteins ( ervk genes in Fig. 7 ). This includes three of the six genes that were found to be shared with T. guttata. The genes also include three uncharacterized genes and two homologs of Hydrocephalus-inducing proteins ( hydin genes in Fig. 7 ). The two remaining genes are a zinc finger protein ( znf239 in Fig. 7 ) and a homolog of Cytoplasmic Polyadenylation Element Binding protein 1 ( cpeb1 ). With the notable exception of cpeb1 , these shared genes were often duplicated and differed in their copy number on the GRC, both between the species and between individuals of the same species (see Fig. 7 ). This means that while these genes are likely of ancient GRC linkage and potentially functional, they are still actively undergoing within-GRC duplication and deletion; and would be susceptible to all of the associated dosage changes. The cpeb1 homolog, on the other hand, maintained its single copy number and open reading frame despite having been present on the GRC for a long time, diverging from the A chromosomal version before the common ancestor of all oscines and suboscines, early in passerine evolution (see Fig. 8 ). Discussion The GRC is an unusual chromosome. On the one hand, the apparent universal presence among songbirds (Torgasheva, et al. 2019) suggests that the GRC is not just a parasitic supernumerary B chromosome, as has been previously suggested (Camacho et al. 2000 , Johnson Pokorná and Reifová 2021 ), but has some important function for these birds which prevents its loss. On the other hand, our data revealed the extremely dynamic nature of this chromosome, with a lack of conservation not only between closely related sister species that diverged merely 1.8 million years ago (Storchová et al. 2010 ), but even within species. Moreover, our results question the functionality of the majority of genes identified on the GRC. The GRC represents a challenge to assemble. The chromosome is only found in germ cells, which represent a small subset of cells, even when harvesting the testes or ovaries specifically. This, combined with the fact that the GRC only occurs as a single copy in males, means that sequencing coverage of regions not duplicated within the GRC is low (about 20% of the A chromosome coverage in our data). Additionally, the GRC sequence is often very similar or indistinguishable from the sequence of A chromosomes (Kinsella, et al. 2019). For these reasons, previous attempts to identify GRC sequences, which relied on using highly repetitive GRC regions (having high germline coverage compared to somatic coverage) and germline specific SNPs, were unable to assemble regions that have a low copy number and are not highly differentiated from the Achromosomal sequence. The method used in this paper to assemble the GRCs of the two nightingale species is able to assemble regions with low coverage and low divergence, as long as they have regions suitably nearby that are identifiable as GRC in origin, which can enable the classification of overlapping 10x linked barcodes. This is exemplified by the successful assembly of almost the whole GRC sequence in both nightingale species, despite the low copy number in L. luscinia . When near-identical duplications that are merged in the assembly are taken into account, our assemblies have a cumulative length that closely matches the chromosome sizes estimated from the cytogenetic visualisation, suggesting that we assembled the vast majority of the two nightingale GRCs. These assemblies thus represent the most complete and high quality GRC assemblies achieved to date. Using the coding genes of F. albicollis , we identified 799 different coding genes on the two GRCs combined. Interestingly, most of them were species-specific, with only 192 of them occurring in both nightingale species. This is striking, given the recent divergence of the two nightingale species (1.8 Mya; Storchová, et al. 2010 ) and low genetic divergence of the A-chromosomes (Storchová, et al. 2010 , Mořkovský, et al. 2018, Janoušek, et al. 2019). The comparison of the nightingale micro-GRC gene content with that of the T. guttata macro-GRC shows a similar lack of conservation. Of the 269 known GRC genes from T. guttata (Kinsella, et al. 2019), only 42 were found in either of the two nightingale species, with 25 being present in both. The GRCs of the two nightingale species are also surprisingly different in sequence origin, as well as the proportion of within-GRC duplicated sequences. One stark difference is the large paralogous region of chromosome 2 on the GRC of L. luscinia , making up approximately half of the L. luscinia GRC. Interestingly, analysis of the GRC and A chromosome divergence revealed that addition of this sequence to the GRC predates the divergence of the two species, suggesting the loss of the chromosome 2 paralogous region in L. megarhynchos rather than its recent addition in L. luscinia . On the other hand, we revealed that relatively large parts of the GRC in L. megarhynchos were added to the chromosome after the divergence of the two species. This suggests multiple frequent additions and deletions occurring on the GRC in a relatively short time span. Variation in copy number and the proportion of duplicated sequences was also high, even among individuals of the same species, suggesting that even within populations this chromosome is not well conserved. The striking divergence of the GRC sequence and gene content, even between such closely related species, allows for the intriguing possibility that this chromosome might be involved in speciation in songbirds. Songbirds have a higher diversification rate compared to other bird taxa and comprise more than half of all modern bird species, despite only being one of many present lineages (Prum, et al. 2015; Oliveros, et al. 2019). We identified 29 species-specific GRC genes with a complete coding region on the two GRCs, which might be theoretically involved in the reproductive isolation of the two nightingale species mediated by female-limited hybrid sterility and possibly divergence of sperm morphology (Reifová et al. 2011 ; Mořkovský et al. 2018 ; Albrecht et al. 2019 ). Most genes identified on the nightingale GRCs were only partially present, however, with approximately 75% of genes having less than a quarter of their coding region present and only 4% having their whole coding region present. This suggests that the vast majority of “genes” on the GRC are actually fragmented, non-functional pseudogenes. When this is combined with the observed rapid divergence of the GRC between species, it suggests that the GRC is largely non-functional and frequently acquires (and subsequently loses) sequences from the A-chromosomes. This, however, does not mean that at least a small proportion of the GRC is not important in function. Kinsella et al. ( 2019 ) found protein products for five genes on the T. guttata GRC, as well as signatures of selection on 10 GRC-linked genes, suggesting the functionality of at least some of the hundreds of GRC-linked genes. These genes could play important roles, for example in germline determination, oogenesis or spermatogenesis, although evolution of spermatogenesis functionality might be limited by the maternal inheritance of the GRC. In an attempt to identify conserved genes on the GRC that may be preventing its loss from the songbird germline genome, we searched for genes with a complete coding sequence that were present in both nightingale species and T. guttata . However, 5 of the 6 genes identified this way represent genes such as endogenous retroviral homologs and uncharacterized, or poorly characterized genes, which despite being present in all three species, also show high variation in copy number within species. It thus seems unlikely that they represent indispensable GRC sequences. The remaining complete gene that is present in both nightingale species as well as T. guttata is a paralog of cpeb1 , cytoplasmic polyadenylation element binding protein 1. In addition to the normal Achromosomal version, this gene has a single copy on the GRC present in all 3 individuals of both species and does not feature any stop codons along its entire length. We estimated cpeb1 to have diverged from the A-chromosomal version early on in passerine evolution (before the divergence of suboscines and oscines, but after the split of Acanthisittidae). This makes this gene one of the oldest genes identified on the GRC so far and suggests that the GRC might be present not only in all songbirds but all passerine birds except for a small group of Acanthisittidae. Previous analysis of this gene on the zebra finch GRC found that it is under long-term purifying selection, further supporting the functionality of this gene (Kinsella, et al. 2019). cpeb1 is known to play a role in transcript modification during oocyte maturation (Hake and Richter, 1994 ). Together, these findings make cpeb1 the best candidate for a functionally important gene which may be preventing the loss of the GRC from the songbird germline. The picture emerging of the GRC is that it is a tumultuous chromosome, where large stretches of DNA can be added and subtracted rapidly, seemingly without consequence. Once on the GRC, any sequence is liable to be duplicated on the chromosome multiple times. The pace and scale of these changes does not seem conducive to the fine scale refinement normally associated with natural selection. This, combined with the fact that most genes are fragmented, suggests that the vast majority of the chromosome is non-functional, with presumably a small ancestral region harbouring a gene or genes that are driving the continued existence of the chromosome in the songbird lineage. The seemingly contradictory picture of the GRC highlights how programmed DNA elimination can change the evolutionary landscape of genetic sequences. The fact that the GRC is eliminated from somatic cells means that there are much less pleiotropic constraints on this chromosome compared to A-chromosomes, which may lead to less selection pressure acting on this chromosome. As a consequence, many genetic changes, which would have large negative consequences for an individual if they occurred on an Achromosome, are effectively silenced on the GRC. Conclusion This work represents the first comparison of GRCs between closely related songbirds, demonstrating the speed with which the GRC undergoes change. It also represents the most complete, albeit fragmented, GRC assemblies produced to date. Our results emphasise how rapidly this chromosome evolves, with large variation being observed between the two species on almost every metric we measured, and moderate variation being observed even within each species. This contrasts starkly with the normally conserved bird karyotype and makes the GRC the fastest evolving chromosome in the genome. We also show that most genes that are present on the GRC are present in a fragmented, presumably non-functional, state. While the ubiquity of the GRC within the songbird clade does suggest an important role for the chromosome, it is still unclear what that role is. The chromosome appears to be under uniquely relaxed evolutionary pressure, presumably as a result of its elimination from somatic cells, and it seems likely that its main function is limited to a few consequential genes, one of which seems to be cpeb1 . Materials And Methods Nightingale Sampling Three male individuals from each nightingale species were sampled for whole genome sequencing in allopatric regions (North-Eastern Poland for L. luscinia and South-Western Poland for L. megarhynchos ). From each individual, somatic tissue (kidney) and gonadal tissue (testis) were dissected and either used immediately for DNA isolation or frozen in liquid nitrogen and stored in 80°C prior to DNA isolation. The work was approved by the General Directorate for Environmental Protection, Poland (permission no. DZP-WG.6401.03.123.2017.dl.3). Preparation of meiotic spreads and estimation of GRC size Measurements were made of immunostained synaptonemal complexes of pachytene chromosomes from Poignet et al. ( 2021 ). Chromosomes were immunostained with anti-SYCP3 antibody recognizing the lateral elements of the synaptonemal complex, and human anticentromere serum (CREST, 15–234, Antibodies Incorporated) binding kinetochores (see Poignet et al. 2021 for details). The GRC can be recognized from other chromosomes on these slides by its relatively weaker staining by anti-SYCP3 antibody and the CREST signal which covers the whole chromosome, instead of just the centromere. The lengths of the 22 largest chromosomes and the GRCs were measured in high-quality cells for each species using ImageJ software (ImageJ 1.50i, Rueden et al. 2017 ). This resulted in 15 L. megarhynchos cells (12 from one individual and 3 from another) and 16 cells from L. luscinia (9 from one individual and 7 from the other) being used. The measured length of the GRC was divided by 1.5, due to a measurement discrepancy caused by its univalent nature (Malinovskaya et al. 2020 ), before the size was calculated using a linear regression (see Supplementary Fig. 2). This used the relationship between the logarithmic values of the 22 longest chromosomes lengths and the logarithmic size in base pairs of the 22 largest chromosomes from the F. albicollis genome, FicAlb1.5 (R 2 = 0.97 in L. megarhynchos and R 2 = 0.98 in L. luscinia ). The approximate size was checked against the size of the eliminated GRC micronucleus using rabbit monoclonal anti-H3K9me3 antibody (ab8898, Abcam) (dilution 1:200). Sequencing of somatic and germline genomes We sequenced DNA from somatic (kidney) and gonadal (testis) tissues from three individuals of each species. One individual from each species had DNA from both tissues sequenced using 10x linked (Zheng, et al. 2016 ) Illumina sequencing technology, while the other two individuals had DNA samples sequenced with standard paired-end Illumina sequencing. For 10x linked sequencing, high molecular weight DNA was extracted from frozen testis and liver samples using a phenol-chloroform methodology (Pajer, et al. 2006). The DNA was sent to SEQme (Dobris, Czech Republic) for 10x linked sequencing library construction and 2x150 bp paired-end sequencing using the NovaSeq 6000 (Illumina). For the standard Illumina sequencing, DNA was extracted from frozen tissue samples using MagAttract HMW DNA Kit (Qiagen) and sent to the Institute of Applied Biotechnologies (Prague, Czech Republic) where the sequencing libraries were prepared using NebNext Ultra II DNA Kit (New England Biolabs) and sequenced with the NovaSeq 6000 (Illuimna) using 2x150 bp paired-end mode. Testis samples were sequenced to higher depth (105-150x) than the kidney samples (45-120x) to ensure sufficient coverage over the GRC (see Supplementary Table 2). Identification and assembly of GRC reads Linked 10x reads that originated from the GRC were identified using the following methods before being assembled using Supernova (Weisenfeld, et al. 2017 ) and the “megabubbles” option (method visualised in Supplementary Fig. 3): 1) The testis samples were assembled using the 10x linked reads and Supernova (Weisenfeld, et al. 2017 ). The 10x reads from the testes and kidneys were then processed using Long Ranger v2.2.2, trimmed and checked for adapters using Trimmomatic v0.39 (Bolger, et al. 2014 ), before being aligned to their respective genome using bwa v0.7.17 (Li and Durbin 2010 ). Regions of the testis genome assembly were identified using Samtools v1.14 (Li, et al. 2009) which were fully covered by reads from the testis dataset while having no reads align to them from the somatic dataset and that were at least 500 bp long. Reads from testis samples that overlapped these regions by at least 10 bp were used to identify 10x barcodes and their associated reads as originating from the GRC. 2) Sequencing reads from 10x libraries were processed using Long Ranger v2.2.2 before all reads were trimmed and checked for adapters using Trimmomatic v0.39 (Bolger, et al. 2014 ). These reads were then aligned to draft somatic genomes from their respective species created using Oxford nanopore data and Illumina reads (Rídl, et al. unpublished). SNP variants were identified using GATK v4.1.7.0 (Poplin et al, 2017 ). If a variant was present in all three testis samples from a species, but in none of the kidney samples from that species, it was considered to be a GRC variant. These SNP variants were used to create 29 bp kmer sequences (i.e. each variant resulted in 29 kmers). Any kmer that was present in the 10x kidney reads was removed and the remaining kmers were used to identify reads from the 10x testis dataset that had a matching sequence. The barcodes from these reads were used to identify any associated reads. 3) A sample of 100 000 reads from each of the 10x datasets was used to identify repetitive elements that might be unique to the GRC of each species using RepeatExplorer (Novak, et al. 2010 ). While no such repeats were found in L. luscinia , a candidate repeat was found in the testis dataset of L. megarhynchos . This result was confirmed using all the 10x reads from L. megarhynchos and Blastn (Altschul, et al. 1990 ), with a word size of 8, an e-value of 1e-5, and a max hsps of 1. Any read that matched with at least 100 bp and greater than 90% identity to the repetitive element was selected. Once again, all reads with the associated barcodes were designated as having originated from the GRC. Gene Annotation The first frame of the F. albicollis transcriptome coding sequences (v FicAlb1.5) were aligned independently to the two assembled GRCs using Tblastx (Altschul, et al. 1990 ) and an e-value cut-off of 1e-6. Overlapping alignments on the same strand of the GRC were merged. The resultant regions were aligned back to the F. albicollis coding sequences and the top hit in the positive strand selected to identify which gene (and which portion of the gene) the exon represented. In order to account for a possible lack of conservation, when calculating what proportion of a gene was present on the GRC, the fraction of a gene that was found in the GRC was normalised by the fraction of the gene that was found in the L. megarhynchos genome, to a minimum of 0.75. In other words, if 80% of a gene was found in the genome, and 80% was found in the GRC, that gene was treated as being 100% present in the GRC. The cut-off of 0.75 fully corrects for 2/3rds of all genes identified on the genome. This correction resulted in an average increase in the measured proportion of the found gene of 8.5% in L. megarhynchos and 8.9% in L. luscinia . Coverage of GRC scaffolds In order to identify scaffolds that represent near-identical duplicates and/or erroneous sequences in the GRC assembly, the assembled GRC sequence for each species was combined with the corresponding draft somatic genome assembly (Rídl et al. unpublished). The sequencing reads from both tissue types for all three individuals were aligned to the combined genome and GRC assembly using bwa v0.7.17 (Li and Durbin 2010 ) for each species. For each individual, regions of the GRC which had zero read coverage from the kidney dataset were identified. The modal testis coverage value of these regions for each individual was used as an estimate of the expected coverage for single copy GRC regions. The ratio of the expected GRC coverage to the modal genomic coverage was used as a proxy for the “expected” GRC coverage in the kidney samples. These expected coverage values were used to normalise the observed coverage for each sample. The coverage of the kidney sample was then subtracted from the coverage of the testis sample to control for A chromosomal reads misaligning to the GRC sequence. Finally, the average GRC coverage was calculated across 1 kb windows along the two GRCs, with a minimum value of zero. GRC Scaffold Origin and Conservation Given that GRC sequences appear to originate from A chromosomes, the GRC scaffolds were aligned to the draft L. megarhynchos and L. luscinia somatic genomes (Rídl et al. unpublished) using Blastn and an e-value cutoff of 1e-6 (Altschul, et al. 1990 ). Additionally, the two GRCs were aligned to each other using Blastn. The A chromosomal origin of the GRC sequences were determined by the top hit in the L. megarhynchos genome, since it was the higher quality genome assembly. The genomic scaffold was then aligned to the F. albicolli genome assembly to determine the chromosome identity using Nucmer from the MUMmer package (Kurtz et al. 2004 ). cpeb1 Evolution The cpeb1 gene sequence was determined from the Tblastx results with the XP_005051706.1 transcript from the two GRCs and their respective genomes. The GRC sequence was used to identify homologous sequences in related species using the ‘nr’ database on NCBI and Tblastx. For each species, the top hit was selected. These species included: Acanthisitta chloris , Atrichornis clamosus , Calyptomena viridis , Corvus cornix cornix , Gallus gallus , Lonchura striata domestica , Sapayoa aenigma , Serilophus lunatus , Serinus canaria , Struthio camelus australis , Taeniopygia guttata and Tyrannus savana . The sequences were aligned using ClustalW (Thompson, et al. 1994 ) and a maximum likelihood tree drawn with MegaX (Kumar, et al. 2018 ). Declarations 800x600 Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 st1\:*{behavior:url(#ieooui) } Acknowledgements This research was funded by the Czech Science Foundation (grant 20-23794S to R.R. and T.A.), the Charles University grant PRIMUS/19/SCI/008 to R.R. and the Grant Agency of Charles University (grant 1169420 to M.P.). F.J.R.R. was supported by a postdoctoral fellowship from Sven och Lilly Lawskis fond and a Marie Curie Individual Fellowship (875732). 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Female heterogamety and speciation: reduced introgression of the z chromosome between two species of nightingales. Evolution 64 : 456–471. https://doi.org/10.1111/j.1558-5646.2009.00841.x Suh, A. and Dion-Côté, A-M. 2021. New Perspectives on the Evolution of Within-Individual Genome Variation and Germline/Soma Distinction. Genome Biol Evol 13 : 6 : evab095. https://doi.org/10.1093/gbe/evab095 Thompson, J. D., Higgins, D. G. and Gibson, T. J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22 : 22 : 4673–4680. https://doi.org/10.1093/nar/22.22.4673 Torgasheva A. A., et al. 2019. Germline-restricted chromosome (GRC) is widespread among songbirds. Proc Natl Acad Sci U S A 116 : 24 : 11845–11850. doi: 10.1073/pnas.1817373116 Torgasheva, A., Malinovskaya, L., Zadesenets, K., Shnaider, E., Rubtsov, N. and Borodin, P. 2021. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1359388","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":88978459,"identity":"8d722c87-5f1c-401e-af0f-7f3e38d9c93d","order_by":0,"name":"Stephen Schlebusch","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-2355-2652","institution":"Charles University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Stephen","middleName":"","lastName":"Schlebusch","suffix":""},{"id":88978460,"identity":"d424ca22-7b55-486c-90a0-0b70cf7ed571","order_by":1,"name":"Jakub Rídl","email":"","orcid":"","institution":"Czech Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jakub","middleName":"","lastName":"Rídl","suffix":""},{"id":88978461,"identity":"0b056b44-8b1f-4151-b2a6-ab1a6a680acd","order_by":2,"name":"Manon Poignet","email":"","orcid":"https://orcid.org/0000-0003-2445-830X","institution":"Charles University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Manon","middleName":"","lastName":"Poignet","suffix":""},{"id":88978462,"identity":"a8fc0e37-ac5c-4539-93aa-8ab4b5e97f2d","order_by":3,"name":"Francisco Ruiz-Ruano","email":"","orcid":"https://orcid.org/0000-0002-5391-301X","institution":"Uppsala University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"","lastName":"Ruiz-Ruano","suffix":""},{"id":88978463,"identity":"15998ecb-bba1-43f7-967b-dc090af429ed","order_by":4,"name":"Jiri Reif","email":"","orcid":"","institution":"Charles University in Prague; 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Arrowheads indicate the GRCs. The box in the top right corner shows the GRC in more detail (1.5x magnification) without the CREST signal. The scale bar represents 10 µm.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1359388/v1/0d7c7434dcc1bcfca5d86413.png"},{"id":18998526,"identity":"e2840fe3-adc7-42e2-be04-5da2de4703fd","added_by":"auto","created_at":"2022-03-08 20:01:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":57658,"visible":true,"origin":"","legend":"\u003cp\u003eAverage normalised copy number of the longest scaffolds in each nightingale individual.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"f2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1359388/v1/fc568c9ce381281062c2b3f0.jpg"},{"id":18998684,"identity":"14b7c4a4-d730-42c5-9e6a-6a353adc5a27","added_by":"auto","created_at":"2022-03-08 20:07:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31827,"visible":true,"origin":"","legend":"\u003cp\u003eGRC size estimates from genomic data for the three nightingale individuals of each species, taking near-identical duplications merged in the assembly into account. The proportion of the GRC that is shared between the species is shown in a darker colour, while the proportion of the GRC that is species-specific is in lighter colour. Assemblies are based on the individuals LM1 and LL1 respectively. The estimated GRC size for each species from meiotic spreads is shown by the horizontal dashed lines.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"f3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1359388/v1/6074e0b839cbbfff0c24085d.jpg"},{"id":18998842,"identity":"79441807-3579-4dd5-8ac9-6deac5bd98cb","added_by":"auto","created_at":"2022-03-08 20:10:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":48964,"visible":true,"origin":"","legend":"\u003cp\u003eOrigin of GRC sequence. A) Expected contribution from the A chromosomes to the GRC if each chromosome was contributing equally according to its size. B) Average observed contribution of A chromosomes to the \u003cem\u003eL. megarhynchos\u003c/em\u003e GRC. C) Average observed contribution of A Chromosomes to the \u003cem\u003eL.\u0026nbsp;luscinia\u003c/em\u003e GRC. The black and white outer circles show the proportion of each sequence that has a homologous sequence in the other species. The chromosome order is from largest to smallest. The proportions have been corrected for coverage, which accounts for near-identical duplications collapsed in the assembly as well as assembly errors.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"f4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1359388/v1/c07a17c2fa0110ea7f404bf3.jpg"},{"id":18998616,"identity":"76b35a24-b6c3-495a-bd52-295ac0bd54fa","added_by":"auto","created_at":"2022-03-08 20:04:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":51965,"visible":true,"origin":"","legend":"\u003cp\u003eDivergence of each GRC assembly from A-chromosomal sequences in \u003cem\u003eL.\u0026nbsp;megarhynchos \u003c/em\u003eand \u003cem\u003eL.\u0026nbsp;luscinia.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"f5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1359388/v1/b1546499149189164e8f122d.jpg"},{"id":18998534,"identity":"85aef3c4-b1c8-4ade-a494-76a929c6ca16","added_by":"auto","created_at":"2022-03-08 20:01:11","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":46428,"visible":true,"origin":"","legend":"\u003cp\u003eCompleteness of genes found on the GRC of both \u003cem\u003eL. megarhynchos\u003c/em\u003e and \u003cem\u003eL. luscinia\u003c/em\u003e. The grey dashed line represents a 1:1 ratio. The black line represents the linear fit of the data (R\u003csup\u003e2\u003c/sup\u003e = 0.76).\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"f6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1359388/v1/9c902595435f52fc0c0524cd.jpg"},{"id":18998532,"identity":"f6c6cdf0-e4e7-4d3f-8c92-a320a626d1a3","added_by":"auto","created_at":"2022-03-08 20:01:11","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":74431,"visible":true,"origin":"","legend":"\u003cp\u003eEstimated copy number of the 10 GRC genes with at least 95% of their coding region found in both nightingale species. Genes with bold names were also found in \u003cem\u003eT.\u0026nbsp;guttata\u003c/em\u003e. The copy number estimate for each individual is based off the average normalised coverage of each scaffold that the genes were present on.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"f7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1359388/v1/bb451aedd781f0b5f7574a15.jpg"},{"id":18998621,"identity":"686d512b-f368-4428-9393-9e9a60ca980d","added_by":"auto","created_at":"2022-03-08 20:04:11","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":62316,"visible":true,"origin":"","legend":"\u003cp\u003eDivergence of the \u003cem\u003ecpeb1\u003c/em\u003e GRC paralog from the A chromosomal version. The GRC paralogue diverges from the A-chromosomal version before the Oscine/Suboscine divergence. Branch values represent bootstrap support.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"f8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1359388/v1/e0663564ba6b2c1c221e1cda.jpg"},{"id":40772387,"identity":"ef16ef2b-2e32-4425-b44a-a74e2633e288","added_by":"auto","created_at":"2023-07-30 07:06:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":763903,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1359388/v1/830d29cc-60d6-4281-a4d6-1af82aee62dc.pdf"},{"id":18998529,"identity":"440694c5-f58f-4d27-9a81-554ffda46344","added_by":"auto","created_at":"2022-03-08 20:01:11","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":96234,"visible":true,"origin":"","legend":"Supplementary Tables","description":"","filename":"SupplementaryTablesv2.7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1359388/v1/fc6df1f0788b8499381be4a8.xlsx"},{"id":18998524,"identity":"043ea442-ab04-47e0-ae9a-fc005a1ecb48","added_by":"auto","created_at":"2022-03-08 20:01:11","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":705738,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationv2.7.docx","url":"https://assets-eu.researchsquare.com/files/rs-1359388/v1/ea3b4469aa163655cf28c6e2.docx"},{"id":18998619,"identity":"79b42582-61b7-4261-8ef4-e222dd85d879","added_by":"auto","created_at":"2022-03-08 20:04:11","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":366498,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"rs.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1359388/v1/d5e6aa53b6fb2703c53cc488.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Rapid gene content turnover on the germline-restricted chromosome in songbirds","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn multicellular organisms, all cells of an individual normally contain the same genetic information. There are exceptions, however, where certain sequences are eliminated from all or some of the somatic cells during development, leaving the original genetic information to be maintained in the germ cells (Wang and Davis, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Suh and Dion-C\u0026ocirc;t\u0026eacute;, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). An interesting example of this programmed DNA elimination has been described in songbirds, where a whole chromosome is lost from somatic cells early on in embryo development. The aptly named germline-restricted chromosome (GRC) was described for the first time in the zebra finch (\u003cem\u003eTaeniopygia guttata\u003c/em\u003e) (Pigozzi and Solari \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), with recent studies suggesting that it likely occurs in all songbirds (order Passeriformes, suborder Oscines) (Torgasheva, et al. 2019; Kinsella, et al. 2019). Songbirds diverged from the rest of the birds approximately 47 mya (Oliveros, et al. 2019) and comprise approximately 50% of all modern bird species, making them the largest taxonomic group with obligatory programmed DNA elimination. Despite the relatively wide distribution of the GRC, we still know very little about its genetic composition, evolutionary significance and function for birds.\u003c/p\u003e \u003cp\u003eBesides its exclusive presence in the germline, there is little that is consistent about this chromosome. The GRC is normally maternally inherited, but paternal inheritance has been shown to be possible (Pei, et al. 2022). It occurs in a single copy in male germ cells, which is excluded from the nucleus during meiosis, and in two copies in female germ cells (Pigozzi and Solari \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), although again, there are exceptions (Malinovskaya, et al. 2020; Torgasheva, et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The chromosome size varies dramatically, from the largest macrochromosome in the cell (macro-GRC) to a small microchromosome (micro-GRC), with no apparent phylogenetic pattern (Torgasheva, et al. 2019). This lack of conservation is in clear contrast to the apparent ubiquity of the GRC in songbirds.\u003c/p\u003e \u003cp\u003ePart of the reason why there is still so much unknown about the genetic composition of the GRC is that this chromosome is hard to sequence effectively. The GRC sequence is largely composed of recently diverged paralogous sequences from the other chromosomes in the cell (hereafter referred to as A chromosomes), and as such can be hard to differentiate in a sequencing library (Itoh et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Biederman et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kinsella, et al. 2019; Asalone, et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, gonads are composed of both somatic and germ cells, so this chromosome is only found in a subset of testis cells and a minimal proportion of ovary cells (Kinsella, et al. 2019). Thus, GRC sequences are underrepresented in the sequencing libraries from these tissues. Assembled sequence information from the GRC is scarce and currently limited to \u003cem\u003eT. guttata\u003c/em\u003e (Itoh et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Biedermann et al. 2018, Kinsella et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e and Pei, et al, 2022). Thus far, analyses of tissue-specific single-nucleotide polymporphisms (SNPs) and germline/somatic coverage differences have identified 269 putative genes as well as many high copy number regions on the \u003cem\u003eT. guttata\u003c/em\u003e macro-GRC (Kinsella, et al. 2019; Asalone, et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the total assembled length of GRC-linked sequences is 1.24 Mb (Kinsella, et al. 2019) plus 468 kb (Asalone, et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which is approximately 1% of the expected 150 Mb \u003cem\u003eT. guttata\u003c/em\u003e macro-GRC.\u003c/p\u003e \u003cp\u003eIn this paper, we sequenced and assembled the GRC in two closely related songbird species, the common nightingale (\u003cem\u003eLuscinia megarhynchos\u003c/em\u003e) and thrush nightingale (\u003cem\u003eL. luscinia\u003c/em\u003e), both of which possess a micro-GRC (Poignet et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These species from the Muscicapidae family diverged approximately 1.8 mya (Storchov\u0026aacute; et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and still hybridize in a secondary contact zone (Reifov\u0026aacute;, et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Mořkovsk\u0026yacute;, et al. 2018; Albrecht, et al. 2019). Using a novel method to identify GRC reads from germline sequencing libraries, we assembled the majority of the GRCs for both species. Our results show rapid gene content turnover with significant differences observed not only between species but even among individuals of the same species. The vast majority of genes on the GRC were only partially present and presumably non-functional. The gene \u003cem\u003ecpeb1\u003c/em\u003e was the only entire gene present in all individuals with no copy number variation. We show that this gene belongs to the oldest genes on the GRC, making it the standout candidate gene with an essential function on the GRC, which might be preventing the loss of the GRC in songbirds.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eGRC size estimation using meiotic spreads\u003c/p\u003e \u003cp\u003eWe visualized the pachytene chromosomes in testis cells using antibodies against the synaptonemal complex (anti-SYCP3) and centromere (CREST) (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These antibodies enable the identification of the unpaired, univalent GRC (del Priore and Pigozzi \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Torgasheva, et al. 2019). In addition, we immunostained the eliminated GRC from the secondary spermatocytes in the form of a micronucleus (see Supplementary Fig.\u0026nbsp;1) using an antibody against histone H3 lysine 9 methylation (H3K9me) (del Priore and Pigozzi \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBoth species had a GRC comparable in size with a microchromosome (i.e. a micro-GRC) as was described in Poignet et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consistent with this, the GRC micronucleus for both nightingale species was much smaller than in species with a macro-GRC (Supplementary Fig.\u0026nbsp;1; see del Priore and Pigozzi \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e for visualization of the GRC micronucleus in \u003cem\u003eT. guttata\u003c/em\u003e with a macro-GRC).\u003c/p\u003e \u003cp\u003eThe length of the GRC was estimated by measuring the size of the 22 largest chromosomes, as well as the GRC, in the pachytene cells and comparing the sizes with assembled chromosome lengths (in bp) in collared flycatcher (\u003cem\u003eFicedula albicollis\u003c/em\u003e), a songbird species that diverged from nightingales 15 mya (Jetz, et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Given the conservation of bird karyotypes (Kawakami et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), we assumed that chromosome lengths would be similar between \u003cem\u003eF. albicollis\u003c/em\u003e and nightingales (see Supplementary Fig.\u0026nbsp;2). Using this approach, we estimated the GRC size to be 9.6 Mbp for \u003cem\u003eL. megarhynchos\u003c/em\u003e and 7.5 Mbp for \u003cem\u003eL. luscinia\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eGRC assembly\u003c/p\u003e \u003cp\u003eTo identify GRC-derived sequences, we sequenced and compared somatic and germline genomes in three individuals of each species. One individual from each species was sequenced with 10x Chromium linked-read sequencing and two individuals with standard Illumina technology. The GRC was assembled using (i) 10x linked reads that aligned in a germline-specific way to the germline genome assembly, (ii) reads that contained germline-specific SNPs, (iii) reads containing germline-specific repetitive elements, which was only applicable for \u003cem\u003eL. megarhynchos\u003c/em\u003e, and (iv) any 10x linked reads that shared their 10x barcodes with reads selected in previous steps (Supplementary Fig.\u0026nbsp;3). Approximately 23 thousand 10x barcodes were identified, resulting in 5.6\u0026nbsp;million read pairs to assemble the \u003cem\u003eL. megarhynchos\u003c/em\u003e GRC. In comparison, only 13 thousand 10x barcodes were identified in \u003cem\u003eL. luscinia\u003c/em\u003e, which resulted in 3\u0026nbsp;million read pairs. Despite having fewer reads, the \u003cem\u003eL. luscinia\u003c/em\u003e GRC assembly was longer (5.6 Mbp) and of higher quality (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) than the \u003cem\u003eL. megarhynchos\u003c/em\u003e assembly (3.5 Mbp). While the GRC assemblies were highly fragmented, their cumulative length suggests that a large proportion of each GRC was assembled (36\u0026ndash;75% of the estimated size). This number is however probably an underestimate, as it does not take recent within-GRC duplicated sequences into account (see \u0026ldquo;Recent copy number variation within the GRC\u0026rdquo; below).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGRC assembly metrics for each nightingale species.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eL. megarhynchos\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. luscinia\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Length\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.5 Mbp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.6 Mbp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLargest Scaffold\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e110 Kbp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e370 Kbp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of Scaffolds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e750\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eScaffold N50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.8 Kbp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46 Kbp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eContig N50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5 Kbp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24 Kbp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e%N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.4%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eRecent copy number variation within the GRC\u003c/p\u003e \u003cp\u003eThe coverage across the GRC was calculated for the three individuals from each species. This was done in order to identify regions of the GRC that had been duplicated within the GRC but have not diverged sufficiently from their GRC paralogs to be differentiated by the assembly process. \u003cem\u003eL. megarhynchos\u003c/em\u003e showed more near-identical duplications merged in the assembly process compared to \u003cem\u003eL. luscinia\u003c/em\u003e. This was reflected by the higher average normalised GRC copy number in \u003cem\u003eL. megarhynchos\u003c/em\u003e (3.0x) than in \u003cem\u003eL. luscinia\u003c/em\u003e (1.6x). Importantly, this mostly accounts for recent within-GRC duplications and repetitive elements on the GRC, which the genome assembler was unable to differentiate. Older within-GRC duplications which have diverged in sequence are not captured in this metric and are expected to be assembled into separate paralogous sequences.\u003c/p\u003e \u003cp\u003eThe lower proportion of near-identical duplications in the \u003cem\u003eL. luscinia\u003c/em\u003e GRC at least partially explains the higher assembly quality in this species. This is supported by the fact that the longest scaffolds from \u003cem\u003eL. luscinia\u003c/em\u003e consistently had low copy number (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It is also interesting to note that there is considerable variation in copy number among individuals. This is especially noticeable in \u003cem\u003eL. megarhynchos\u003c/em\u003e but is also present in the \u003cem\u003eL. luscinia\u003c/em\u003e scaffolds with higher copy numbers (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This suggests that there is substantial variation in recent within-GRC duplications between the GRCs, even within species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe normalised copy number calculation allows for the estimation of the GRC size, accounting for near-identical duplicated sequences erroneously collapsed in the assembly, for each individual. Once these duplications are taken into account, the size of the GRC assembly is 1.3x-2.9x times larger than the original assembly suggested (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The GRC assembly of the individuals sequenced by 10x linked reads was 10.2 Mbp long after this correction for \u003cem\u003eL. megarhynchos\u003c/em\u003e and 7.0 Mbp for \u003cem\u003eL. luscinia\u003c/em\u003e. These numbers are similar to the GRC size estimates from the meiotic spreads (9.6 Mbp and 7.5 Mbp, respectively), suggesting that we assembled the vast majority of the GRC in both species. And again, the variation among individuals within the same species is noticeable, with the \u003cem\u003eL. megarhynchos\u003c/em\u003e GRC ranging in size from 8.8 Mbp to 12.5 Mbp and the \u003cem\u003eL. luscinia\u003c/em\u003e GRC ranging from 7.0 Mbp to 11.6 Mbp. This variation is especially impressive considering that two of the three individuals in each species are not assembled, instead using the third individual as a reference, and will therefore underestimate any sequence that does not have a homologous region in the reference GRC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGenetic content of the GRC\u003c/p\u003e \u003cp\u003eThe variation in GRC size between the species and individuals may reflect highly different genetic content in the GRCs. To explore this possibility, we aligned the two GRC assemblies against each other as well as to the \u003cem\u003eL. megarhynchos\u003c/em\u003e genome. Surprisingly, approximately only 1 Mbp of each GRC assembly aligned to the other GRC (before accounting for coverage; see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), the rest being species-specific. Consistent with this, the A-chromosomal origins of each species\u0026rsquo; GRC sequences are strikingly different (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Curiously, the part of the GRC which is orthologous between the two species was disproportionately repetitive, with the average normalised copy number in these orthologous regions being 5.2x and 3.1x for \u003cem\u003eL. megarhynchos\u003c/em\u003e and \u003cem\u003eL. luscinia\u003c/em\u003e respectively, while the non-homologous regions had an average respective copy number of 2.1x and 1.4x.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe \u003cem\u003eL. luscinia\u003c/em\u003e GRC has a large proportion paralogous to Chromosome 2, which is absent in the \u003cem\u003eL. megarhynchos\u003c/em\u003e GRC assembly. Most of this Chromosome 2 derived sequence comes from a single region of Chromosome 2. This paralogous region is visible in many of the largest scaffolds from the \u003cem\u003eL. luscinia\u003c/em\u003e assembly (see Supplementary Fig.\u0026nbsp;4). Despite being present in large blocks, and likely originating from a single A-to-GRC duplication, it is no longer continuous, presumably as a result of internal rearrangements and within-GRC duplications or else later A-to-GRC duplications. For example, there are two large scaffolds with Chromosome 2 ancestry that are clearly within-GRC duplicates (Scaffolds 9 and 10, Supplementary Fig.\u0026nbsp;4), but because this duplication happened long enough ago, their sequences have sufficiently diverged for the assembly process to distinguish them (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDivergence from A Chromosomes\u003c/p\u003e \u003cp\u003eThe proportion of mismatches between homologous regions of each GRC and the A chromosomes of each species was calculated to determine if the GRC sequences originated before or after the divergence of the two nightingale species (see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Interestingly, a large proportion of the \u003cem\u003eL. megarhynchos\u003c/em\u003e GRC appears to have originated after speciation, with approximately 1 Mbp of the assembly aligning better to its A-chromosomal paralogs than to the \u003cem\u003eL. luscinia\u003c/em\u003e genome. In comparison, virtually all of the \u003cem\u003eL. luscinia\u003c/em\u003e GRC appears to predate the divergence of the species, with no noticeable difference in mismatches between the GRC and the A-chromosomal paralogs from both species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGene Annotation\u003c/p\u003e \u003cp\u003eGenes were annotated on each of the GRC assemblies using \u003cem\u003eF. albicollis\u003c/em\u003e protein-coding genes (FicAlb1.5), resulting in 585 identified genes in \u003cem\u003eL. megarhynchos\u003c/em\u003e and 406 in \u003cem\u003eL. luscinia\u003c/em\u003e. As might be expected given the largely different sequence origins of the two GRCs, the majority of genes were not shared between the two species, with only 192 genes being present in both species. Genes were assessed for their completeness (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and copy number within the GRC (Supplementary Table\u0026nbsp;1). Notably, the vast majority of identified genes were both duplicated (with the average gene having a corrected copy number of 6.6x in \u003cem\u003eL. megarhynchos\u003c/em\u003e and 3.7x in \u003cem\u003eL. luscinia\u003c/em\u003e) and only partially present (with only 23 genes in \u003cem\u003eL. megarhynchos\u003c/em\u003e and 18 genes in \u003cem\u003eL. luscinia\u003c/em\u003e having more than 95% of the coding region present in the assembly). This observed gene fragmentation was measured after correcting for a possible lack of sequence conservation between the nightingales and \u003cem\u003eF. albicollis\u003c/em\u003e, which used the percentage of the gene that was found on the A chromosomes as a baseline of expected conservation (see Materials and Methods; Supplementary Table\u0026nbsp;1 contains gene-specific details). Ten of the genes with 95% of their coding sequence present were shared between the two nightingale species, from which six were previously reported on the \u003cem\u003eT. guttata\u003c/em\u003e GRC (Kinsella, et al, 2019).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNumber of GRC genes at varying levels of fragmentation identified in each nightingale species. A gene was counted as being shared between the two nightingales at a particular level of fragmentation if the proportion that was present was sufficient in both species. For genes shared between nightingales and \u003cem\u003eT. guttata\u003c/em\u003e, the level of fragmentation had to be met for both nightingale species, and the gene had to be reported as putatively on the \u003cem\u003eT. guttata\u003c/em\u003e GRC (Kinsella, et al. 2019).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProportion of coding region found\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of genes in\u003c/p\u003e \u003cp\u003e\u003cem\u003eL.\u0026nbsp;megarhynchos\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber of genes in\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. luscinia\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNumber of\u003c/p\u003e \u003cp\u003eshared genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNumber of genes\u003c/p\u003e \u003cp\u003eshared between nightingales and \u003cem\u003eT.\u0026nbsp;guttata\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e585\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e406\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;25%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e150 (26%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99 (24%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57 (30%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13 (52%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e81 (14%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e63 (16%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11 (44%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49 (8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36 (9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24 (13%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9 (36%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22 (4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17 (4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 (24%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhile most genes on the GRC were species-specific, the genes that were shared had a similar percentage of their coding regions present in the two GRCs (see Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The portion of these genes that was present on the two GRCs was also often the same portion (see Supplementary Fig.\u0026nbsp;5). This consistency suggests that the presence of these shared genes is the result of their presence on an ancestral GRC, rather than independent additions in each lineage. The consistency in the degree of fragmentation of these shared genes (see Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), as well as the parts of each gene which were missing (see Supplementary Fig.\u0026nbsp;5), also suggests that the observed gene fragmentation is largely a real phenomenon and not the result of incomplete and low quality GRC assemblies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong the 10 genes which had more than 95% of their coding region present on both species\u0026rsquo; GRCs, three were characterised as being homologs of endogenous retrovirus-derived proteins (\u003cem\u003eervk\u003c/em\u003e genes in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This includes three of the six genes that were found to be shared with \u003cem\u003eT. guttata.\u003c/em\u003e The genes also include three uncharacterized genes and two homologs of Hydrocephalus-inducing proteins (\u003cem\u003ehydin\u003c/em\u003e genes in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The two remaining genes are a zinc finger protein (\u003cem\u003eznf239\u003c/em\u003e in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) and a homolog of Cytoplasmic Polyadenylation Element Binding protein 1 (\u003cem\u003ecpeb1\u003c/em\u003e). With the notable exception of \u003cem\u003ecpeb1\u003c/em\u003e, these shared genes were often duplicated and differed in their copy number on the GRC, both between the species and between individuals of the same species (see Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This means that while these genes are likely of ancient GRC linkage and potentially functional, they are still actively undergoing within-GRC duplication and deletion; and would be susceptible to all of the associated dosage changes. The \u003cem\u003ecpeb1\u003c/em\u003e homolog, on the other hand, maintained its single copy number and open reading frame despite having been present on the GRC for a long time, diverging from the A chromosomal version before the common ancestor of all oscines and suboscines, early in passerine evolution (see Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe GRC is an unusual chromosome. On the one hand, the apparent universal presence among songbirds (Torgasheva, et al. 2019) suggests that the GRC is not just a parasitic supernumerary B chromosome, as has been previously suggested (Camacho et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Johnson Pokorn\u0026aacute; and Reifov\u0026aacute; \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), but has some important function for these birds which prevents its loss. On the other hand, our data revealed the extremely dynamic nature of this chromosome, with a lack of conservation not only between closely related sister species that diverged merely 1.8\u0026nbsp;million years ago (Storchov\u0026aacute; et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), but even within species. Moreover, our results question the functionality of the majority of genes identified on the GRC.\u003c/p\u003e \u003cp\u003eThe GRC represents a challenge to assemble. The chromosome is only found in germ cells, which represent a small subset of cells, even when harvesting the testes or ovaries specifically. This, combined with the fact that the GRC only occurs as a single copy in males, means that sequencing coverage of regions not duplicated within the GRC is low (about 20% of the A chromosome coverage in our data). Additionally, the GRC sequence is often very similar or indistinguishable from the sequence of A chromosomes (Kinsella, et al. 2019). For these reasons, previous attempts to identify GRC sequences, which relied on using highly repetitive GRC regions (having high germline coverage compared to somatic coverage) and germline specific SNPs, were unable to assemble regions that have a low copy number and are not highly differentiated from the Achromosomal sequence.\u003c/p\u003e \u003cp\u003eThe method used in this paper to assemble the GRCs of the two nightingale species is able to assemble regions with low coverage and low divergence, as long as they have regions suitably nearby that are identifiable as GRC in origin, which can enable the classification of overlapping 10x linked barcodes. This is exemplified by the successful assembly of almost the whole GRC sequence in both nightingale species, despite the low copy number in \u003cem\u003eL. luscinia\u003c/em\u003e. When near-identical duplications that are merged in the assembly are taken into account, our assemblies have a cumulative length that closely matches the chromosome sizes estimated from the cytogenetic visualisation, suggesting that we assembled the vast majority of the two nightingale GRCs. These assemblies thus represent the most complete and high quality GRC assemblies achieved to date.\u003c/p\u003e \u003cp\u003eUsing the coding genes of \u003cem\u003eF. albicollis\u003c/em\u003e, we identified 799 different coding genes on the two GRCs combined. Interestingly, most of them were species-specific, with only 192 of them occurring in both nightingale species. This is striking, given the recent divergence of the two nightingale species (1.8 Mya; Storchov\u0026aacute;, et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and low genetic divergence of the A-chromosomes (Storchov\u0026aacute;, et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Mořkovsk\u0026yacute;, et al. 2018, Janoušek, et al. 2019). The comparison of the nightingale micro-GRC gene content with that of the \u003cem\u003eT. guttata\u003c/em\u003e macro-GRC shows a similar lack of conservation. Of the 269 known GRC genes from \u003cem\u003eT. guttata\u003c/em\u003e (Kinsella, et al. 2019), only 42 were found in either of the two nightingale species, with 25 being present in both.\u003c/p\u003e \u003cp\u003eThe GRCs of the two nightingale species are also surprisingly different in sequence origin, as well as the proportion of within-GRC duplicated sequences. One stark difference is the large paralogous region of chromosome 2 on the GRC of \u003cem\u003eL. luscinia\u003c/em\u003e, making up approximately half of the \u003cem\u003eL. luscinia\u003c/em\u003e GRC. Interestingly, analysis of the GRC and A chromosome divergence revealed that addition of this sequence to the GRC predates the divergence of the two species, suggesting the loss of the chromosome 2 paralogous region in \u003cem\u003eL. megarhynchos\u003c/em\u003e rather than its recent addition in \u003cem\u003eL. luscinia\u003c/em\u003e. On the other hand, we revealed that relatively large parts of the GRC in \u003cem\u003eL. megarhynchos\u003c/em\u003e were added to the chromosome after the divergence of the two species. This suggests multiple frequent additions and deletions occurring on the GRC in a relatively short time span. Variation in copy number and the proportion of duplicated sequences was also high, even among individuals of the same species, suggesting that even within populations this chromosome is not well conserved.\u003c/p\u003e \u003cp\u003eThe striking divergence of the GRC sequence and gene content, even between such closely related species, allows for the intriguing possibility that this chromosome might be involved in speciation in songbirds. Songbirds have a higher diversification rate compared to other bird taxa and comprise more than half of all modern bird species, despite only being one of many present lineages (Prum, et al. 2015; Oliveros, et al. 2019). We identified 29 species-specific GRC genes with a complete coding region on the two GRCs, which might be theoretically involved in the reproductive isolation of the two nightingale species mediated by female-limited hybrid sterility and possibly divergence of sperm morphology (Reifov\u0026aacute; et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Mořkovsk\u0026yacute; et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Albrecht et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMost genes identified on the nightingale GRCs were only partially present, however, with approximately 75% of genes having less than a quarter of their coding region present and only 4% having their whole coding region present. This suggests that the vast majority of \u0026ldquo;genes\u0026rdquo; on the GRC are actually fragmented, non-functional pseudogenes. When this is combined with the observed rapid divergence of the GRC between species, it suggests that the GRC is largely non-functional and frequently acquires (and subsequently loses) sequences from the A-chromosomes. This, however, does not mean that at least a small proportion of the GRC is not important in function. Kinsella et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) found protein products for five genes on the \u003cem\u003eT. guttata\u003c/em\u003e GRC, as well as signatures of selection on 10 GRC-linked genes, suggesting the functionality of at least some of the hundreds of GRC-linked genes. These genes could play important roles, for example in germline determination, oogenesis or spermatogenesis, although evolution of spermatogenesis functionality might be limited by the maternal inheritance of the GRC.\u003c/p\u003e \u003cp\u003eIn an attempt to identify conserved genes on the GRC that may be preventing its loss from the songbird germline genome, we searched for genes with a complete coding sequence that were present in both nightingale species and \u003cem\u003eT. guttata\u003c/em\u003e. However, 5 of the 6 genes identified this way represent genes such as endogenous retroviral homologs and uncharacterized, or poorly characterized genes, which despite being present in all three species, also show high variation in copy number within species. It thus seems unlikely that they represent indispensable GRC sequences.\u003c/p\u003e \u003cp\u003eThe remaining complete gene that is present in both nightingale species as well as \u003cem\u003eT. guttata\u003c/em\u003e is a paralog of \u003cem\u003ecpeb1\u003c/em\u003e, cytoplasmic polyadenylation element binding protein 1. In addition to the normal Achromosomal version, this gene has a single copy on the GRC present in all 3 individuals of both species and does not feature any stop codons along its entire length. We estimated \u003cem\u003ecpeb1\u003c/em\u003e to have diverged from the A-chromosomal version early on in passerine evolution (before the divergence of suboscines and oscines, but after the split of Acanthisittidae). This makes this gene one of the oldest genes identified on the GRC so far and suggests that the GRC might be present not only in all songbirds but all passerine birds except for a small group of Acanthisittidae. Previous analysis of this gene on the zebra finch GRC found that it is under long-term purifying selection, further supporting the functionality of this gene (Kinsella, et al. 2019). \u003cem\u003ecpeb1\u003c/em\u003e is known to play a role in transcript modification during oocyte maturation (Hake and Richter, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Together, these findings make \u003cem\u003ecpeb1\u003c/em\u003e the best candidate for a functionally important gene which may be preventing the loss of the GRC from the songbird germline.\u003c/p\u003e \u003cp\u003eThe picture emerging of the GRC is that it is a tumultuous chromosome, where large stretches of DNA can be added and subtracted rapidly, seemingly without consequence. Once on the GRC, any sequence is liable to be duplicated on the chromosome multiple times. The pace and scale of these changes does not seem conducive to the fine scale refinement normally associated with natural selection. This, combined with the fact that most genes are fragmented, suggests that the vast majority of the chromosome is non-functional, with presumably a small ancestral region harbouring a gene or genes that are driving the continued existence of the chromosome in the songbird lineage.\u003c/p\u003e \u003cp\u003eThe seemingly contradictory picture of the GRC highlights how programmed DNA elimination can change the evolutionary landscape of genetic sequences. The fact that the GRC is eliminated from somatic cells means that there are much less pleiotropic constraints on this chromosome compared to A-chromosomes, which may lead to less selection pressure acting on this chromosome. As a consequence, many genetic changes, which would have large negative consequences for an individual if they occurred on an Achromosome, are effectively silenced on the GRC.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis work represents the first comparison of GRCs between closely related songbirds, demonstrating the speed with which the GRC undergoes change. It also represents the most complete, albeit fragmented, GRC assemblies produced to date. Our results emphasise how rapidly this chromosome evolves, with large variation being observed between the two species on almost every metric we measured, and moderate variation being observed even within each species. This contrasts starkly with the normally conserved bird karyotype and makes the GRC the fastest evolving chromosome in the genome. We also show that most genes that are present on the GRC are present in a fragmented, presumably non-functional, state. While the ubiquity of the GRC within the songbird clade does suggest an important role for the chromosome, it is still unclear what that role is. The chromosome appears to be under uniquely relaxed evolutionary pressure, presumably as a result of its elimination from somatic cells, and it seems likely that its main function is limited to a few consequential genes, one of which seems to be \u003cem\u003ecpeb1\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003eNightingale Sampling\u003c/p\u003e \u003cp\u003eThree male individuals from each nightingale species were sampled for whole genome sequencing in allopatric regions (North-Eastern Poland for \u003cem\u003eL. luscinia\u003c/em\u003e and South-Western Poland for \u003cem\u003eL. megarhynchos\u003c/em\u003e). From each individual, somatic tissue (kidney) and gonadal tissue (testis) were dissected and either used immediately for DNA isolation or frozen in liquid nitrogen and stored in 80\u0026deg;C prior to DNA isolation. The work was approved by the General Directorate for Environmental Protection, Poland (permission no. DZP-WG.6401.03.123.2017.dl.3).\u003c/p\u003e \u003cp\u003ePreparation of meiotic spreads and estimation of GRC size\u003c/p\u003e \u003cp\u003eMeasurements were made of immunostained synaptonemal complexes of pachytene chromosomes from Poignet et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Chromosomes were immunostained with anti-SYCP3 antibody recognizing the lateral elements of the synaptonemal complex, and human anticentromere serum (CREST, 15\u0026ndash;234, Antibodies Incorporated) binding kinetochores (see Poignet et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e for details). The GRC can be recognized from other chromosomes on these slides by its relatively weaker staining by anti-SYCP3 antibody and the CREST signal which covers the whole chromosome, instead of just the centromere.\u003c/p\u003e \u003cp\u003eThe lengths of the 22 largest chromosomes and the GRCs were measured in high-quality cells for each species using ImageJ software (ImageJ 1.50i, Rueden et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This resulted in 15 \u003cem\u003eL. megarhynchos\u003c/em\u003e cells (12 from one individual and 3 from another) and 16 cells from \u003cem\u003eL. luscinia\u003c/em\u003e (9 from one individual and 7 from the other) being used. The measured length of the GRC was divided by 1.5, due to a measurement discrepancy caused by its univalent nature (Malinovskaya et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), before the size was calculated using a linear regression (see Supplementary Fig.\u0026nbsp;2). This used the relationship between the logarithmic values of the 22 longest chromosomes lengths and the logarithmic size in base pairs of the 22 largest chromosomes from the \u003cem\u003eF. albicollis\u003c/em\u003e genome, FicAlb1.5 (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.97 in \u003cem\u003eL. megarhynchos\u003c/em\u003e and R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.98 in \u003cem\u003eL. luscinia\u003c/em\u003e). The approximate size was checked against the size of the eliminated GRC micronucleus using rabbit monoclonal anti-H3K9me3 antibody (ab8898, Abcam) (dilution 1:200).\u003c/p\u003e \u003cp\u003eSequencing of somatic and germline genomes\u003c/p\u003e \u003cp\u003eWe sequenced DNA from somatic (kidney) and gonadal (testis) tissues from three individuals of each species. One individual from each species had DNA from both tissues sequenced using 10x linked (Zheng, et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) Illumina sequencing technology, while the other two individuals had DNA samples sequenced with standard paired-end Illumina sequencing.\u003c/p\u003e \u003cp\u003eFor 10x linked sequencing, high molecular weight DNA was extracted from frozen testis and liver samples using a phenol-chloroform methodology (Pajer, et al. 2006). The DNA was sent to SEQme (Dobris, Czech Republic) for 10x linked sequencing library construction and 2x150 bp paired-end sequencing using the NovaSeq 6000 (Illumina). For the standard Illumina sequencing, DNA was extracted from frozen tissue samples using MagAttract HMW DNA Kit (Qiagen) and sent to the Institute of Applied Biotechnologies (Prague, Czech Republic) where the sequencing libraries were prepared using NebNext Ultra II DNA Kit (New England Biolabs) and sequenced with the NovaSeq 6000 (Illuimna) using 2x150 bp paired-end mode.\u003c/p\u003e \u003cp\u003eTestis samples were sequenced to higher depth (105-150x) than the kidney samples (45-120x) to ensure sufficient coverage over the GRC (see Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eIdentification and assembly of GRC reads\u003c/p\u003e \u003cp\u003eLinked 10x reads that originated from the GRC were identified using the following methods before being assembled using Supernova (Weisenfeld, et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and the \u0026ldquo;megabubbles\u0026rdquo; option (method visualised in Supplementary Fig.\u0026nbsp;3):\u003c/p\u003e \u003cp\u003e1) The testis samples were assembled using the 10x linked reads and Supernova (Weisenfeld, et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The 10x reads from the testes and kidneys were then processed using Long Ranger v2.2.2, trimmed and checked for adapters using Trimmomatic v0.39 (Bolger, et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), before being aligned to their respective genome using bwa v0.7.17 (Li and Durbin \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Regions of the testis genome assembly were identified using Samtools v1.14 (Li, et al. 2009) which were fully covered by reads from the testis dataset while having no reads align to them from the somatic dataset and that were at least 500 bp long. Reads from testis samples that overlapped these regions by at least 10 bp were used to identify 10x barcodes and their associated reads as originating from the GRC.\u003c/p\u003e \u003cp\u003e2) Sequencing reads from 10x libraries were processed using Long Ranger v2.2.2 before all reads were trimmed and checked for adapters using Trimmomatic v0.39 (Bolger, et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These reads were then aligned to draft somatic genomes from their respective species created using Oxford nanopore data and Illumina reads (R\u0026iacute;dl, et al. unpublished). SNP variants were identified using GATK v4.1.7.0 (Poplin et al, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). If a variant was present in all three testis samples from a species, but in none of the kidney samples from that species, it was considered to be a GRC variant. These SNP variants were used to create 29 bp kmer sequences (i.e. each variant resulted in 29 kmers). Any kmer that was present in the 10x kidney reads was removed and the remaining kmers were used to identify reads from the 10x testis dataset that had a matching sequence. The barcodes from these reads were used to identify any associated reads.\u003c/p\u003e \u003cp\u003e3) A sample of 100 000 reads from each of the 10x datasets was used to identify repetitive elements that might be unique to the GRC of each species using RepeatExplorer (Novak, et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). While no such repeats were found in \u003cem\u003eL. luscinia\u003c/em\u003e, a candidate repeat was found in the testis dataset of \u003cem\u003eL. megarhynchos\u003c/em\u003e. This result was confirmed using all the 10x reads from \u003cem\u003eL. megarhynchos\u003c/em\u003e and Blastn (Altschul, et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), with a word size of 8, an e-value of 1e-5, and a max hsps of 1. Any read that matched with at least 100 bp and greater than 90% identity to the repetitive element was selected. Once again, all reads with the associated barcodes were designated as having originated from the GRC.\u003c/p\u003e \u003cp\u003eGene Annotation\u003c/p\u003e \u003cp\u003eThe first frame of the \u003cem\u003eF. albicollis\u003c/em\u003e transcriptome coding sequences (v FicAlb1.5) were aligned independently to the two assembled GRCs using Tblastx (Altschul, et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) and an e-value cut-off of 1e-6. Overlapping alignments on the same strand of the GRC were merged. The resultant regions were aligned back to the \u003cem\u003eF. albicollis\u003c/em\u003e coding sequences and the top hit in the positive strand selected to identify which gene (and which portion of the gene) the exon represented.\u003c/p\u003e \u003cp\u003eIn order to account for a possible lack of conservation, when calculating what proportion of a gene was present on the GRC, the fraction of a gene that was found in the GRC was normalised by the fraction of the gene that was found in the \u003cem\u003eL. megarhynchos\u003c/em\u003e genome, to a minimum of 0.75. In other words, if 80% of a gene was found in the genome, and 80% was found in the GRC, that gene was treated as being 100% present in the GRC. The cut-off of 0.75 fully corrects for 2/3rds of all genes identified on the genome. This correction resulted in an average increase in the measured proportion of the found gene of 8.5% in \u003cem\u003eL. megarhynchos\u003c/em\u003e and 8.9% in \u003cem\u003eL. luscinia\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eCoverage of GRC scaffolds\u003c/p\u003e \u003cp\u003eIn order to identify scaffolds that represent near-identical duplicates and/or erroneous sequences in the GRC assembly, the assembled GRC sequence for each species was combined with the corresponding draft somatic genome assembly (R\u0026iacute;dl et al. unpublished). The sequencing reads from both tissue types for all three individuals were aligned to the combined genome and GRC assembly using bwa v0.7.17 (Li and Durbin \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) for each species. For each individual, regions of the GRC which had zero read coverage from the kidney dataset were identified. The modal testis coverage value of these regions for each individual was used as an estimate of the expected coverage for single copy GRC regions. The ratio of the expected GRC coverage to the modal genomic coverage was used as a proxy for the \u0026ldquo;expected\u0026rdquo; GRC coverage in the kidney samples. These expected coverage values were used to normalise the observed coverage for each sample. The coverage of the kidney sample was then subtracted from the coverage of the testis sample to control for A chromosomal reads misaligning to the GRC sequence. Finally, the average GRC coverage was calculated across 1 kb windows along the two GRCs, with a minimum value of zero.\u003c/p\u003e \u003cp\u003eGRC Scaffold Origin and Conservation\u003c/p\u003e \u003cp\u003eGiven that GRC sequences appear to originate from A chromosomes, the GRC scaffolds were aligned to the draft \u003cem\u003eL. megarhynchos\u003c/em\u003e and \u003cem\u003eL. luscinia\u003c/em\u003e somatic genomes (R\u0026iacute;dl et al. unpublished) using Blastn and an e-value cutoff of 1e-6 (Altschul, et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Additionally, the two GRCs were aligned to each other using Blastn. The A chromosomal origin of the GRC sequences were determined by the top hit in the \u003cem\u003eL. megarhynchos\u003c/em\u003e genome, since it was the higher quality genome assembly. The genomic scaffold was then aligned to the \u003cem\u003eF. albicolli\u003c/em\u003e genome assembly to determine the chromosome identity using Nucmer from the MUMmer package (Kurtz et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003ecpeb1\u003c/em\u003e Evolution\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ecpeb1\u003c/em\u003e gene sequence was determined from the Tblastx results with the XP_005051706.1 transcript from the two GRCs and their respective genomes. The GRC sequence was used to identify homologous sequences in related species using the \u0026lsquo;nr\u0026rsquo; database on NCBI and Tblastx. For each species, the top hit was selected. These species included: \u003cem\u003eAcanthisitta chloris\u003c/em\u003e, \u003cem\u003eAtrichornis clamosus\u003c/em\u003e, \u003cem\u003eCalyptomena viridis\u003c/em\u003e, \u003cem\u003eCorvus cornix cornix\u003c/em\u003e, \u003cem\u003eGallus gallus\u003c/em\u003e, \u003cem\u003eLonchura striata domestica\u003c/em\u003e, \u003cem\u003eSapayoa aenigma\u003c/em\u003e, \u003cem\u003eSerilophus lunatus\u003c/em\u003e, \u003cem\u003eSerinus canaria\u003c/em\u003e, \u003cem\u003eStruthio camelus australis\u003c/em\u003e, \u003cem\u003eTaeniopygia guttata\u003c/em\u003e and \u003cem\u003eTyrannus savana\u003c/em\u003e. The sequences were aligned using ClustalW (Thompson, et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) and a maximum likelihood tree drawn with MegaX (Kumar, et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003c!--[if gte mso 9]\u003e\u003cxml\u003e\n \u003co:OfficeDocumentSettings\u003e\n \u003co:TargetScreenSize\u003e800x600\u003c/o:TargetScreenSize\u003e\n \u003c/o:OfficeDocumentSettings\u003e\n\u003c/xml\u003e\u003c![endif]--\u003e\n\u003c!--[if gte mso 9]\u003e\u003cxml\u003e\n \u003cw:WordDocument\u003e\n \u003cw:View\u003eNormal\u003c/w:View\u003e\n \u003cw:Zoom\u003e0\u003c/w:Zoom\u003e\n \u003cw:TrackMoves/\u003e\n \u003cw:TrackFormatting/\u003e\n \u003cw:DoNotShowComments/\u003e\n \u003cw:PunctuationKerning/\u003e\n \u003cw:ValidateAgainstSchemas/\u003e\n \u003cw:SaveIfXMLInvalid\u003efalse\u003c/w:SaveIfXMLInvalid\u003e\n 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\u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"List Number 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"10\" QFormat=\"true\" Name=\"Title\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Closing\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Signature\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"1\" SemiHidden=\"true\"\n UnhideWhenUsed=\"true\" Name=\"Default Paragraph Font\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Body Text\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Body Text Indent\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"List Continue\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"List Continue 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"List Continue 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"List Continue 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"List Continue 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Message Header\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"11\" QFormat=\"true\" Name=\"Subtitle\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Salutation\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Date\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Body Text First Indent\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Body Text First Indent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Note Heading\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Body Text 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Body Text 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Body Text Indent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Body Text Indent 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Block Text\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Hyperlink\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"FollowedHyperlink\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"22\" QFormat=\"true\" Name=\"Strong\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"20\" QFormat=\"true\" Name=\"Emphasis\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Document Map\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Plain Text\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"E-mail Signature\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"HTML Top of Form\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"HTML Bottom of Form\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Normal (Web)\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"HTML Acronym\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"HTML Address\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"HTML Cite\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"HTML Code\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"HTML Definition\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"HTML Keyboard\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"HTML Preformatted\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"HTML Sample\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"HTML Typewriter\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"HTML Variable\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Normal Table\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"annotation subject\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"No List\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Outline List 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Outline List 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Outline List 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Simple 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Simple 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Simple 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Classic 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Classic 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Classic 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Classic 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Colorful 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Colorful 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Colorful 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Columns 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Columns 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Columns 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Columns 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Columns 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Grid 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Grid 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Grid 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Grid 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Grid 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Grid 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Grid 7\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Grid 8\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table List 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table List 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table List 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table List 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table List 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table List 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table List 7\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table List 8\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table 3D effects 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table 3D effects 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table 3D effects 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Contemporary\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Elegant\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Professional\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Subtle 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Subtle 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Web 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Web 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Web 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Balloon Text\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"39\" Name=\"Table Grid\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" UnhideWhenUsed=\"true\"\n Name=\"Table Theme\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" Name=\"Placeholder Text\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"1\" QFormat=\"true\" Name=\"No Spacing\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"60\" Name=\"Light Shading\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"61\" Name=\"Light List\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"62\" Name=\"Light Grid\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"63\" Name=\"Medium Shading 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"64\" Name=\"Medium Shading 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"65\" Name=\"Medium List 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"66\" Name=\"Medium List 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"67\" Name=\"Medium Grid 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"68\" Name=\"Medium Grid 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"69\" Name=\"Medium Grid 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"70\" Name=\"Dark List\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"71\" Name=\"Colorful Shading\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"72\" Name=\"Colorful List\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"73\" Name=\"Colorful Grid\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"60\" Name=\"Light Shading Accent 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"61\" Name=\"Light List Accent 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"62\" Name=\"Light Grid Accent 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"63\" Name=\"Medium Shading 1 Accent 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"64\" Name=\"Medium Shading 2 Accent 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"65\" Name=\"Medium List 1 Accent 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" SemiHidden=\"true\" Name=\"Revision\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"34\" QFormat=\"true\"\n Name=\"List Paragraph\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"29\" QFormat=\"true\" Name=\"Quote\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"30\" QFormat=\"true\"\n Name=\"Intense Quote\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"66\" Name=\"Medium List 2 Accent 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"67\" Name=\"Medium Grid 1 Accent 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"68\" Name=\"Medium Grid 2 Accent 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"69\" Name=\"Medium Grid 3 Accent 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"70\" Name=\"Dark List Accent 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"71\" Name=\"Colorful Shading Accent 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"72\" Name=\"Colorful List Accent 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"73\" Name=\"Colorful Grid Accent 1\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"60\" Name=\"Light Shading Accent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"61\" Name=\"Light List Accent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"62\" Name=\"Light Grid Accent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"63\" Name=\"Medium Shading 1 Accent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"64\" Name=\"Medium Shading 2 Accent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"65\" Name=\"Medium List 1 Accent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"66\" Name=\"Medium List 2 Accent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"67\" Name=\"Medium Grid 1 Accent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"68\" Name=\"Medium Grid 2 Accent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"69\" Name=\"Medium Grid 3 Accent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"70\" Name=\"Dark List Accent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"71\" Name=\"Colorful Shading Accent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"72\" Name=\"Colorful List Accent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"73\" Name=\"Colorful Grid Accent 2\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"60\" Name=\"Light Shading Accent 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"61\" Name=\"Light List Accent 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"62\" Name=\"Light Grid Accent 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"63\" Name=\"Medium Shading 1 Accent 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"64\" Name=\"Medium Shading 2 Accent 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"65\" Name=\"Medium List 1 Accent 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"66\" Name=\"Medium List 2 Accent 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"67\" Name=\"Medium Grid 1 Accent 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"68\" Name=\"Medium Grid 2 Accent 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"69\" Name=\"Medium Grid 3 Accent 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"70\" Name=\"Dark List Accent 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"71\" Name=\"Colorful Shading Accent 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"72\" Name=\"Colorful List Accent 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"73\" Name=\"Colorful Grid Accent 3\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"60\" Name=\"Light Shading Accent 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"61\" Name=\"Light List Accent 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"62\" Name=\"Light Grid Accent 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"63\" Name=\"Medium Shading 1 Accent 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"64\" Name=\"Medium Shading 2 Accent 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"65\" Name=\"Medium List 1 Accent 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"66\" Name=\"Medium List 2 Accent 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"67\" Name=\"Medium Grid 1 Accent 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"68\" Name=\"Medium Grid 2 Accent 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"69\" Name=\"Medium Grid 3 Accent 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"70\" Name=\"Dark List Accent 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"71\" Name=\"Colorful Shading Accent 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"72\" Name=\"Colorful List Accent 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"73\" Name=\"Colorful Grid Accent 4\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"60\" Name=\"Light Shading Accent 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"61\" Name=\"Light List Accent 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"62\" Name=\"Light Grid Accent 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"63\" Name=\"Medium Shading 1 Accent 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"64\" Name=\"Medium Shading 2 Accent 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"65\" Name=\"Medium List 1 Accent 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"66\" Name=\"Medium List 2 Accent 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"67\" Name=\"Medium Grid 1 Accent 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"68\" Name=\"Medium Grid 2 Accent 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"69\" Name=\"Medium Grid 3 Accent 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"70\" Name=\"Dark List Accent 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"71\" Name=\"Colorful Shading Accent 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"72\" Name=\"Colorful List Accent 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"73\" Name=\"Colorful Grid Accent 5\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"60\" Name=\"Light Shading Accent 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"61\" Name=\"Light List Accent 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"62\" Name=\"Light Grid Accent 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"63\" Name=\"Medium Shading 1 Accent 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"64\" Name=\"Medium Shading 2 Accent 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"65\" Name=\"Medium List 1 Accent 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"66\" Name=\"Medium List 2 Accent 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"67\" Name=\"Medium Grid 1 Accent 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"68\" Name=\"Medium Grid 2 Accent 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"69\" Name=\"Medium Grid 3 Accent 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"70\" Name=\"Dark List Accent 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"71\" Name=\"Colorful Shading Accent 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"72\" Name=\"Colorful List Accent 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"73\" Name=\"Colorful Grid Accent 6\"/\u003e\n \u003cw:LsdException Locked=\"false\" Priority=\"19\" QFormat=\"true\"\n Name=\"Subtle 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\t{size:8.5in 11.0in; \tmargin:1.0in 1.0in 1.0in 1.0in; \tmso-header-margin:.5in; \tmso-footer-margin:.5in; \tmso-paper-source:0;} div.WordSection1 \t{page:WordSection1;} \n --\u003e\n\u003c/style\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Czech Science Foundation (grant 20-23794S to R.R. and T.A.), the Charles University grant PRIMUS/19/SCI/008 to R.R. and the Grant Agency of Charles University (grant 1169420 to M.P.). F.J.R.R. was supported by a postdoctoral fellowship from Sven och Lilly Lawskis fond and a Marie Curie Individual Fellowship (875732). Computational analysis was mostly done using the Institute of Molecular Genetics (Czech Academy of Sciences, Prague, Czech Republic) computers. Additional computational resources were provided by the ELIXIR-CZ project (LM2018131), part of the international ELIXIR infrastructure.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eContributions\u003c/p\u003e\n\u003cp\u003eThe project was conceptualized by R.R., S.A.S., A.S., T.A. and J.P.; Samples were collected by J.Re., T.A., R.R., and M.P.; Cytogenetic analysis was performed by M.P.; DNA extraction was done by J.R\u0026iacute;. Bioinformatic analyses were done by S.A.S.; Manuscript was written by S.A.S. and R.R. and all authors contributed to text editing. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlbrecht, T., et al. 2019. Sperm divergence in a passerine contact zone: Indication of reinforcement at the gametic level. Evolution \u003cb\u003e73\u003c/b\u003e: 202\u0026ndash;213. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/evo.13677\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. 1990. Basic local alignment search tool. J Mol Biol \u003cb\u003e215\u003c/b\u003e: 403\u0026ndash;410\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsalone, K. C., Takkar, A. K., Saldanha, C. J. and Bracht, J. R. 2021. 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Nat Biotechnol 34: 303\u0026ndash;311. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nbt.3432\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-1359388/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1359388/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe germline-restricted chromosome (GRC) of songbirds represents an extraordinary and taxonomically widespread example of programmed DNA elimination. Despite its apparent indispensability in songbirds, we still know very little about the GRC\u0026rsquo;s genetic composition, function, and evolutionary significance. Here we assembled the GRC in two closely related species, the common and thrush nightingale. We identified 585 and 406 genes on the GRC of each species, respectively, many of them present in multiple copies. Interestingly, the GRC gene content differed dramatically between the two species, with only 192 genes being shared despite only 1.8\u0026nbsp;million years of species divergence. The chromosome appears to be under little selective pressure, with most GRC genes being present in pseudogenized fragments. Only one gene, \u003cem\u003ecpeb1\u003c/em\u003e, had a complete coding region in all examined individuals of the two species and showed no copy number variation. The addition of this gene to the GRC corresponds with the earliest estimates of the GRC origin, making it a good candidate for the functional indispensability of the GRC in songbirds.\u003c/p\u003e","manuscriptTitle":"Rapid gene content turnover on the germline-restricted chromosome in songbirds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-03-08 20:01:09","doi":"10.21203/rs.3.rs-1359388/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7db32cc5-b20d-479a-ab07-566b8086c77f","owner":[],"postedDate":"March 8th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-07-30T07:06:15+00:00","versionOfRecord":{"articleIdentity":"rs-1359388","link":"https://doi.org/10.1038/s41467-023-40308-8","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2023-07-29 04:00:00","publishedOnDateReadable":"July 29th, 2023"},"versionCreatedAt":"2022-03-08 20:01:09","video":"","vorDoi":"10.1038/s41467-023-40308-8","vorDoiUrl":"https://doi.org/10.1038/s41467-023-40308-8","workflowStages":[]},"version":"v1","identity":"rs-1359388","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1359388","identity":"rs-1359388","version":["v1"]},"buildId":"ehx78VzkSd0WSzXnipQa-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}