Occasional paternal inheritance of the germline-restricted chromosome in songbirds

Abstract

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

Keywords: elimination efficiency; germline-restricted chromosome; paternal spillover; selfish DNA; zebra finch.

Fig. 1.

Cytogenetic evidence for the presence of the GRC in the nucleus of zebra finch Taeniopygia guttata castanotis sperm. The GRC-amplified probe dph6 (see Materials and Methods) indicates the presence of the GRC (pink) inside some sperm heads (white arrow in A–C) and in free-floating micronuclei (yellow arrow in A). Blue DAPI stain without red indicates sperm heads without GRC. Green autofluorescence shows the sperm flagellum in B. (A) 40× magnification. (B) 100× magnification. (C) Individual z-sections under a confocal microscope show the sequential appearance and disappearance of the dph6 signal along consecutive sections, indicating the location of the GRC within the nucleus of the spermatozoa. Time (in seconds) refers to Video S1. The video consisted of 24 sections representing a total of 6.0 μm in depth. (Scale bars, 20 μm.)

Fig. 2.

The elimination efficiency of the GRC differs between castanotis matrilines A and B. (A–C) Comparison of sequencing coverage of GRC-containing (testis, indicated in gray, and ejaculate, orange) and GRC-free tissue (liver) identifies sequences that are GRC linked in high copy number (4). Male identities are shown on the x-axis. The solid blue line refers to a log₂ germline-to-soma coverage ratio = 0 (i.e., no germline enrichment); the dashed blue line refers to a fourfold increase (Top) of coverage in germline compared to soma tissue. Pink dots highlight the 1-kb windows on dph6. (A) Violin box plots show coverage ratios of the selected windows with more than fourfold (log₂ > 2) enrichment in testes in comparison to soma in all nine castanotis males (A to I, of different matrilines). The thick horizontal lines show the median, and boxes indicate the 25th and 75th percentiles. (B) Coverage ratios of the selected windows in matriline A (three brothers A1 to A3 and uncle A0) show that ejaculates contain lower amounts of GRC-derived reads compared to testes (comparison of the median of eight ejaculates with two testis samples: b = −2.56, SE = 0.29, P < 0.001; Dataset S3) as expected from previous work (14, 15). (C) Coverage ratios of the selected windows in matriline B (three brothers B1 to B3) with a higher GRC content in ejaculates and hence a smaller difference with testis (comparison of the median of seven ejaculates with one testis sample: b = −1.21, SE = 0.39, P = 0.02; Dataset S3). (D–F) Illustration of the expected (D) and observed (E and F) number of sperm heads that are free of GRC (blue ovals) and that contain GRC (blue ovals with a pink circle inside; i.e., with a positive dph6 signal; Fig. 1) as well as the number of free-floating GRC-micronuclei (pink circles). (D) Error-free expulsion of GRC from spermatocytes (expected based on previous work) should result in up to 25 free-floating GRC micronuclei per 100 sperm heads in the ejaculate (1, 6, 13, 15). (E) Ejaculates from matriline A showed 1% of GRC-positive sperm heads (n = 677 scored sperm). (F) Ejaculates from matriline B showed 9% of GRC-positive sperm heads (n = 1,533 scored sperm; Dataset S1).

Fig. 3.

A case of paternal inheritance of the GRC in a captive-bred hybrid castanotis × guttata population. (A) Reconstructed hypothetical breeding history during domestication of the recently wild-derived guttata subspecies. Presumably because females of wild-derived guttata birds do not easily reproduce in captivity, we hypothesized that guttata males were crossed with an already domesticated castanotis female (from Europe), and the resulting female hybrids were back crossed with pure guttata males for about five generations (P-BC5) until the population was phenotypically guttata-like. This reconstruction is based on the genotyping of one male (49) of the resulting hybrid population (yellow background, Bottom), which is characterized by a castanotis mother contributing the mtDNA (dark blue circle), the female-specific W chromosome (dark blue short rectangle; for details, see SI Appendix, Results), and 5% of the A-chromosomal DNA (blue fragments in the long rectangles) and guttata males contributing 95% of the A-chromosomal DNA (orange-brown long rectangles) and the GRC (orange-brown sigmoid symbol). Note that the paternal inheritance of the GRC must have happened sometime between generations P and BC5 (solid arrow with asterisk). (B and C) Raw read depth of selected genome regions of testis libraries [mapped to somatic reference taeGut1 (36)] for the castanotis × guttata hybrid (Top row of each panel, yellow background) and two representative castanotis individuals (Bottom two rows). GRC-specific windows are distinguishable from somatic reference-like windows by high read depth values and by testis-specific SNPs (cyan). Each dot represents a 1-kb window. Each cyan dot indicates a 1-kb window that contains ≥3 germline-specific SNPs. Each orange-brown dot shows a 1-kb window that contains private testis-specific SNPs. To visually emphasize highly diverged sequences, windows with private GRC SNPs are only shown (in orange brown) if there are at least three such SNPs within a 10-kb window. Note that only the hybrid has that many private testis-specific SNPs (B and C) as well as private GRC-linked sequences that are absent in castanotis individuals (C), suggesting that it carries a guttata GRC. See SI Appendix, Fig. S3 for genome-wide plots and more individuals. (D and E) Number of heterozygous sites (D) and number of fixed guttata-specific homozygous sites (E) in autosomal and Z-linked windows from somatic tissue of the castanotis × guttata hybrid compared to the pooled DNA from 100 wild-caught castanotis zebra finches (43). The horizontal blue line indicates the cutoff of 2,500 heterozygous sites (D) or 100 fixed guttata-specific homozygous sites per 500-kb window [i.e., each dot; (E)]. Dark blue dots show windows that contain an excess number of heterozygous sites (D), or a reduced number of guttata-specific homozygous sites (E), and indicate 10 introgressed castanotis segments. (F) Phylogenetic tree of the mitogenomes, showing that the hybrid’s mtDNA (“cas x gut,” orange-brown dot at the top of the tree) clusters with typical captive European castanotis zebra finches (dark blue) rather than with mitogenome assemblies of two published guttata datasets [SRA accession numbers SRR2299402 (47) and SRR3208120 (48), respectively; orange brown]. The scale bar shows the number of substitutions per site.

Fig. 4.

Tanglegram (A), haplotype sequences (B), and networks (C) showing the different phylogenies of the mtDNA haplotypes and the GRC-linked genes in the same castanotis zebra finch individuals. (A) Phylogenetic trees were built from gap-free alignments of all nine haplotypes (A through I) of the castanotis mitogenome (Left) and the nine associated GRC haplotypes (concatenated sequence of the nine single-copy GRC loci; Right). The mtDNA tree was rooted by the mitogenome assemblies of two published guttata datasets [SRA accession numbers SRR2299402 (47) and SRR3208120 (48) (not shown here, see Fig. 3F)], whereas the tree of GRC haplotypes was rooted at the midpoint of the two most distantly related haplotypes. Node support bootstrap values (in %) are shown if >60 (based on 1,000 bootstraps). The scale bar indicates 0.0004 substitutions per site. (B) The alignments of the mitogenomes and the nine single-copy loci on the GRC (4) from the nine individuals used in A. Gray indicates consensus among the nine haplotypes. Orange, red, blue, and dark blue indicate a mutation toward A, T, C, and G comparing to the consensus, respectively. (C) Haplotype networks built from gap-free alignments (used in A) of the castanotis mitogenomes (Left), pim3_GRC (Middle), and bicc1_GRC (Right) with their A-chromosomal paralogs (i.e., pim3_A and bicc1_A). A-chromosomal paralog haplotypes were constructed from all castanotis somatic libraries (Dataset S2). Colors represent the different mitogenome haplotypes. The size of each circle indicates the number of samples of each haplotype, and the length of the black lines corresponds to the number of mutational steps between haplotypes. Red numbers refer to the number of SNPs per kb for each cluster of haplotypes; black numbers refer to the number of mutations per kb between the GRC-linked and A-chromosomal paralogs. Also see SI Appendix, Fig. S8 for the haplotype networks of all germline samples and for the double-copy GRC paralog elavl4_GRC. Note the highly reduced genetic diversity (short branch length in A and little variation in B and C) in the GRC genes in comparison to the associated mitogenome (P < 0.0001). Further note that different mitogenome haplotypes from different clades may share the same GRC haplotype, indicating different evolutionary histories between mtDNA and GRC (Dataset S6).