Germline-restricted chromosome (GRC) is widespread among songbirds

Results and Discussion

Using antibodies to the core proteinaceous structure of meiotic chromosomes, the synaptonemal complex (SC), we found that GRCs are present in all 16 songbird species examined (14 in this study and two in the previous studies). These species represent nine families of Passeri (Fig. 1). In 10 species, the GRCs were large acrocentric macrochromosomes (macro-GRCs) absent from bone marrow cells (Fig. 2 A and B and SI Appendix, Fig. S1). In oocytes, the macro-GRCs were usually present as a bivalent, containing one or two terminally located recombination sites visualized by antibodies to MLH1, a mismatch repair protein. In spermatocytes, the GRC usually occurred as a univalent lacking recombination sites, and was diffusely labeled with centromere antibodies (Fig. 2A and SI Appendix, Fig. S1). At the end of male meiotic prophase, this GRC was transformed into a dense round body and ejected from the nucleus (SI Appendix, Fig. S2). A similar meiotic behavior has been described for GRCs in zebra and Bengalese finches (3, 4).

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Fig. 1.

Topology of the bird species examined. Our sampling covers all major passerine groups (except for the basal oscines, suboscines, and acanthisittides) as well as Galloanserae, Columbaves, Apodiformes, Charadriiformes, Falconiformes, and Psittaciformes. Black circles indicate species with a macro-GRC, and white circles indicate species with a micro-GRC. Numbers after the species’ names indicate references for SC studies; asterisks indicate new data. A consensus topology of bird orders is based on the cladogram from Reddy et al. (10). Position of the common swift is defined according to Prum et al. (11). Topology of passerine birds is shown according to Roquet et al. (12). Positions of species within the Estrildidae lineage is established according to Hooper and Price (13).

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Fig. 2.

Discovery of GRCs in bird species. (A) SC spreads of four oscine species immunolabeled with antibodies against SYCP3, the main protein of the lateral element of SC (red), centromere proteins (blue) and MLH1, mismatch repair protein marking recombination sites (green). Arrowheads point to the largest chromosomes ordered according to their size ranks, ZZ (identified by its size and arm ratio), ZW (identified by heteromorphic SC and misaligned centromeres), and GRCs. Arrows in the Insets point to MLH1 foci in GRCs. Micro-GRC bivalents in female barn swallow and European pied flycatcher are indistinguishable from the microchromosomes of the somatic chromosome set. (B) DAPI-stained bone marrow cells. (C) Reverse and cross-species FISH of GRC DNA probes (green) derived from Bengalese finch (LST), zebra finch (TGU), Eurasian siskin (SSP), and pale martin (RDI) with SC spreads, immunolabeled with antibodies against SYCP3 (red). Centromeres are labeled with antibodies against centromere proteins (blue). Arrowheads point to GRCs and regions on the somatic chromosome set intensely painted with GRC probes in cross-species FISH. Insets show GRCs. The Bengalese finch GRC-specific DNA probe gives a strong signal on the Bengalese finch GRC and slightly paints some regions of the somatic chromosome set. The zebra finch GRC probe paints the distal area of the Bengalese finch GRC and a region of the short arm of SC3. The Eurasian siskin GRC probe paints a micro-GRC of European goldfinch, a region on the long arm of SC3 and some pericentromeric regions. The pale martin GRC probe gives a dispersed signal on the great tit GRC, the ZW bivalent and on SC5. Scale bar: 5 µm.

In male germline cells of six other species, we detected micro-GRCs, which appeared as a univalent surrounded by a cloud of centromere antibodies and lacking recombination sites, similar to the behavior of macro-GRCs in the 10 other species. In the oocytes of these species, the GRCs formed a bivalent indistinguishable from the standard microchromosomes. We did not observe any phylogenetic clustering for the GRCs by size. Both macro- and micro-GRCs were present within the families Fringillidae and Hirundinidae (Fig. 1).

Every examined primary spermatocyte of the 16 songbird species contained a GRC. This suggests that the GRC is an important component of the germline genome. However, no GRCs were observed [by reanalyzing our own data (14, 15) and published SC images (16⇓⇓–19)] in eight species of nonpasserine birds from seven separate lineages (Fig. 1). This implies a monophyletic origin of the GRC. The estimated time since songbird divergence from other avian lineages is 35 million years (20). However, basal oscines, suboscines, and Acanthisittidae species have not been examined yet, so we cannot exclude the possibility that GRCs formed in the common ancestor of all Passeriformes, about 60 million years ago (20).

To estimate the sequence homology between GRCs of different species and to get insight into their genetic content, we prepared DNA probes of macro-GRCs for four representatives of three families: Estrildidae (zebra and Bengalese finches), Fringillidae (Eurasian siskin), and Hirundinidae (pale martin). We microdissected the round dense bodies (SI Appendix, Fig. S2) containing the GRC from spermatocyte spreads and carried out whole genome amplification of the dissected material. The resulting probes were used for fluorescent in situ hybridization (FISH) and for sequencing.

Reverse FISH with these GRC probes produced strong specific signals on the GRCs of each species, indicating that the round dense bodies are indeed the ejected GRCs (SI Appendix, Figs. S2C and S3). In cross-species FISH experiments, the intensity of specific GRC signals was much lower. Interestingly, micro-GRCs were painted with DNA probes derived from macro-GRCs of closely related species, indicating that GRCs of related species share at least a part of their genetic content. In both reverse and cross species FISH, we also detected GRC signals on somatic chromosomes. Some signals remained visible after suppression of repeated sequences with Cot-1 DNA. This indicates that GRCs contain multiple copies of sequences homologous to genomic repeats, as well as sequences homologous to unique regions present in the somatic genomes.

To identify these sequences, we aligned the reads resulted from next-generation sequencing (NGS) of the GRCs to the repeat-masked zebra finch reference genome (Taeniopygia_guttata-3.2.4) using BLAT (21) with a 90% identity setting. Average genome coverage estimated in 10-kb windows was 0.15 ± (SD) 4.60, 0.12 ± 3.29, 0.03 ± 1.16, and 0.01 ± 0.25 for reads of zebra finch, Bengalese finch, Eurasian siskin, and pale martin GRC libraries, respectively. The coverage was highly uneven. GRCs of different species showed homology to different regions of the reference genome. Using the four GRC libraries, we characterized 27 regions longer than 10 kb, covered in at least 30% of their length and with an excess of two SD from the genome average (SI Appendix, Table S1). In some regions, where the GRC of one species showed a high coverage, GRCs of other species showed lower, but still above average, coverage. This may indicate that the unique sequences located in these regions have been copied from the ancestral somatic genome into the ancestral GRC and have then subsequently become diverged at the sequence level and/or in copy number.

The longest of such excessively covered genomic regions were also detected by FISH at the SCs of the corresponding species. Some regions partially overlapped sequences of zebra finch genes (22) or sequences homologous to nonzebra finch RefSeq genes (23) (SI Appendix, Table S1). For example, the zebra finch GRC probe gave a strong hybridization signal on the short arm of the zebra finch SC3 (corresponding to TGU1) and on one of the largest SCs of other species examined (Fig. 2C and SI Appendix, Fig. S3). In the corresponding region of TGU1, we found a 2.5-Mb long cluster of several regions with ∼70 fold coverage excess (SI Appendix, Table S1). This cluster overlapped with two genes: completely with ROBO1, a gene involved in vocal learning (24); and partially with GBE1, a gene encoding 1,4-alpha-glucan branching enzyme 1. The homology between the zebra finch GRC and a part of the genomic interval on TGU1 has been detected earlier by the random amplification of polymorphic DNA technique (9) and recently confirmed by Kinsella et al. (7).

Besides functional genes, GRCs also contain multiple repeated sequences. We estimated their representation in the GRC reads and in the somatic genome of zebra finch using RepeatMasker (25) with the RepBase avian library (26) (SI Appendix, Table S2). This revealed both simple and low complexity repeats. The fraction of transposable elements (TEs) in the GRCs was typical for avian genomes (27). The majority were long terminal repeats (LTRs) and long interspersed nuclear elements (LINEs), while short interspersed nuclear elements (SINEs) and DNA TEs were represented in lower fractions than in the somatic genome. Overall abundance of LTRs and LINEs and their ratio varied between different species’ GRCs, reflecting their different evolutionary trajectories. It has been shown that although activity of TEs in avian genomes was rather low and ancient (especially for SINEs), avian species differed for the timing of TE family activities. Interestingly, the zebra finch genome shows a peak of LTR activity from 5 to 20 million years ago (27). This is a likely reason why LTRs are more abundant in zebra finch GRC than in other GRCs. On the other hand, SINEs are rare in avian genomes and they did not show any activity during last 30 million years, yet they are present in the GRCs of all four examined species, likely being inherited from the GRC ancestor. This provides further evidence for the formation of GRCs in the songbird genome rather than in older avian ancestors, because GRCs had a chance to accumulate at higher rate LTRs active in songbirds but not older SINEs. Therefore, a few SINEs found in the GRCs likely represent copies transferred from the somatic genome and amplified in the GRCs rather than those inserted during the actual activity of SINEs.

To examine the general pattern of GRC transcription in oogenesis, we analyzed lampbrush GRCs isolated from zebra finch oocytes at the previtellogenic growth phase. The lampbrush GRC exhibited a typical chromomere-loop pattern, with several pairs of transcriptionally active lateral loops extending from all chromomeres except for those located in a prominent DAPI-positive region. Antibodies against RNA-polymerase II labeled the whole GRC except for this region (SI Appendix, Fig. S4). Thus, lampbrush GRCs display a pattern of transcription typical for somatic chromosomes (28).

Indeed, recent studies demonstrated that many GRC-linked genes are transcribed (7, 8) and at least some of them are translated in the zebra finch germline (7). The evolutionary history of some of these genes points to the songbird origin of the proto-GRC. This is an excellent complementary confirmation of our own findings which drove us to the same conclusion based on a direct (cytogenetic) observation and indirect (transposable element composition) analysis of our sequenced GRC libraries. Our study, however, also points to the existence of both micro- and macrochromosome versions of GRCs in avian lineages, suggesting that this chromosome is highly dynamic in songbird evolution.

Thus, GRCs are present in all of the songbirds studied, but are absent from germlines of birds from other orders. These chromosomes vary drastically in size and show a low sequence similarity between different species. GRCs contain various highly duplicated regions represented in the somatic genome by both unique and repetitive sequences. The spectrum of transposable elements found in our sequenced GRC libraries suggests that the GRC was more likely formed in the ancestral songbird lineage followed by an extensive sequence divergence in the descendent species genomes rather than to appear in the avian ancestor and then being lost in the nonsongbirds.

Therefore, we propose that the GRC has formed as an additional “parasitic B-like” microchromosome in the ancestral songbird genome likely due to a whole-chromosome duplication (Fig. 3). If this proto-GRC already contained some copies of somatic genes contributing to reproductive and developmental processes, it could become beneficial due to providing a higher dosage of these genes and therefore escape purifying selection pressure in the germline. Its presence in the germline only could also relax selection for the functional integrity of the GRC’s genetic content.

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Fig. 3.

Scenario of GRC origin and evolution. A proto-GRC forms due to duplication (Ctl-D) of a microchromosome likely containing genes involved in germ cell development. Copies of unique somatic cell sequences and repetitive elements invade the GRC (Ctl-C+Ctl-V). Divergence of GRCs in different songbird lineages occurs due to amplification and deletion (Del) of their sequences.

This in turn could make the GRC a target for selfish genetic elements active during its evolutionary history. Additional copies of unique sequences (e.g., genes) from the somatic genome could also populate the GRC through nonallelic recombination process, using its own and somatic genome transposable elements as templates. Suppression of recombination along GRCs (except for their termini in female meiosis) could facilitate their divergence and the degradation of their original genetic content via Muller’s ratchet (29). This could lead to a rapid and massive loss of homology between various species’ GRCs.

However, as the contemporary GRCs contain expressed and transcribed genes and persist in the germline of all songbirds studied, it likely has changed its original “parasitic” state to a more “symbiotic” one, providing evolutionary benefits to the representatives of the most speciose group of birds. We believe that a detailed comparison of micro- and macro-GRCs, phylogenetic studies of shared and lineage-specific GRC sequences, and detailed analysis of their stratification within each GRC will shed further light on the origin and evolution of this highly dynamic and surprising chromosome.