The sampling of the two nightingale species was carried out in allopatric regions (where only one of the two species occurs) to avoid possible sampling of interspecific hybrids. The common nightingale was sampled in South-western Poland by the Odra river, near the town Brzeg Dolny (N 51.2602°, E 16.7440°). The thrush nightingale was sampled in North-eastern Poland by the Narew river, near the town Łomża (N 53.1621°, E 22.1246°). In total, we sampled four common nightingales (one male, three females) and two thrush nightingales (one male, one female) for mitotic spreads and two males of each species for meiotic spreads. The birds were euthanized by a standard cervical dislocation and the tibia and testes were immediately dissected for the preparation of mitotic and meiotic chromosomal spreads, respectively. In addition, we collected a blood sample from the brachial vein from one female of each species. The blood sample was later used for DNA isolation and preparation of species-specific DNA probes for the interspecific comparative genomic hybridization (CGH) experiment. All individuals were sampled in May 2019, during the breeding season, and were captured using mist nets or collapsible traps. The work was approved by the General Directorate for Environmental Protection, Poland (permission no. DZP-WG.6401.03.123.2017.dl.3).

Bone marrow from the tibias of each bird was flushed out using a syringe needle with D-MEM medium (Sigma Aldrich) and cultivated in 5 ml of D-MEM medium (Sigma Aldrich) with 75 µl of colcemid solution (Roche) for 40 min at 37°C. After that, the cells were hypotonized in pre-warmed 0.075 M KCl solution for 25 min at 37°C. Finally, cells were washed four times with fixative solution (methanol:acetic acid, 3:1) and then stored at −20°C prior to use.

Chromosomal spreading was done using the air-drying technique followed by conventional Giemsa staining (5% Giemsa in 0.07 M phosphate buffer, pH 7.4). The C-banding method was applied for visualization of constitutive heterochromatin according to Sumner (1972). More specifically slides with chromosomal spreads were aged at 60°C for 1 h then successively soaked in 0.2 N HCl for 20 min at room temperature then in 5% Ba(OH)₂ solution for 4–5 min at 45°C and subsequently in 2× SSC for 1 h at 60°C, with intermediate washes in distilled water. Finally, metaphases were mounted with 4′,6-diamidino-2-phenylindole (DAPI) in mounting medium Vectashield (Vector laboratories).

Synaptonemal complex (SC) spreads were prepared from the testes of reproductively active males following Peters et al. (1997). Briefly, the left testis was cut into two pieces and placed in hypotonic solution (30 mM Tris, 50 mM sucrose, 17 mM trisodium citrate dehydrate and 5 mM EDTA; pH 8.2) for 50 min. The testis tissue was then disaggregated in 200 µl of 100 mM sucrose and the resulting cell suspension was applied in 40 µl drops and spread onto a slide previously treated with 1% PFA and 0.15% of Triton X100 (Sigma Aldrich). All slides were placed in a humid chamber for 90 min and washed for 2 min in 1× PBS. Slides were directly used for immunostaining.

Immunostaining was performed according to Moens et al. (1987) using the following primary antibodies: rabbit polyclonal anti-SYCP3 antibody (ab15093, Abcam) recognizing the lateral elements of the synaptonemal complex (dilution 1:200), and human anticentromere serum (CREST, 15-234, Antibodies Incorporated) binding kinetochores (dilution 1:50). The corresponding secondary antibodies were Alexa-594-conjugated goat anti-Rabbit IgG (H + L) (A32740, Invitrogen; dilution 1:200) and Alexa-488-conjugated goat anti-Human IgG (H + L) (A-11013, Invitrogen; dilution 1:200). Primary and secondary antibodies were diluted in PBT (3% BSA and 0.05% Tween 20 in 1× PBS) and incubated in a humid chamber for 90 min. Slides were washed three times in 1× PBS and dehydrated through an ethanol row (50, 70 and 96%, 3 min each). Finally, all slides were dried and stained with DAPI in mounting medium Vectashield (Vector laboratories).

Telomeric repeat probe (TTAGGG) _n was applied to meiotic and mitotic spreads using FISH. In both experiments, the telomeric repeat sequences were detected using a commercial kit probe directly labelled with Cy3 (DAKO). We followed the manufacturer’s instructions, with the hybridization extended to 1.5 h.

The distribution of 18S rDNA genes was analyzed on mitotic spreads using FISH. The 18S rDNA probe was generated by PCR amplification and nick-translation labelling according to the protocol of Cioffi et al. (2009). The template genomic DNA originated from a reptile species, slow-worm (Anguis fragilis), and the PCR product was 1,456 bp in length (sequence is provided in Supplementary Table 1). The probe showed high sequence similarity with the rDNA of several bird species, Gallus gallus (97.73%), Hirundo rustica and Motacilla alba species (both 97.11%), and thus was considered similar enough to detect the distribution of 18S rDNA clusters in the nightingale species. Slides were aged for 1 h at 60°C, then treated with RNase for 1 h at 37°C and washed three times for 5 min in 2× SSC. Chromosomes were treated with pepsin for 3 min at 37°C and then fixed for 10 min in 1% formaldehyde solution. Slides were dehydrated in an ethanol row (70, 85 and 96%, 3 min each) and air-dried. The chromosomes were denatured in 75% formamide/2× SSC at 76°C for 3 min followed by dehydration in an ethanol row. Meanwhile, the probe was denatured at 80°C for 6 min and chilled on ice for 10 min prior to the hybridization. The probe-chromosome hybridization was performed overnight at 37°C. Post-hybridization washes were performed three times for 5 min in 50% formamide/2× SSC at 37°C and washed twice for 5 min in 2× SSC and finally for 5 min in 4× SSC/0.05% Tween 20 (Sigma Aldrich). Slides were first incubated for 30 min at 37°C with 4× SSC/5% blocking reagent (Roche), then the probe signal was detected by 4× SSC/5% blocking reagent mixed with fluorescein conjugated avidin (Vector laboratories) for 30 min at 37°C, followed by three washes in 4× SSC/0.05% Tween 20 for 5 min. Slides were then incubated with biotinylated anti-avidin (Vector laboratories) for 30 min at 37°C, followed by a second round of fluorescein conjugated avidin treatment for signal amplification. Slides were finally washed in 4× SSC/0.05% Tween 20 twice for 5 min, dehydrated through an ethanol row and air-dried. Chromosomes were stained with DAPI in mounting medium, Vectashield (Vector laboratories).

The CGH experiment was performed with (i) common nightingale and (ii) thrush nightingale metaphase chromosomes. In both cases, equal concentrations of DNA probes from the common nightingale and the thrush nightingale were hybridized to the chromosomes (Figure 1) following the procedure described in Symonová et al. (2015) with slight modification. The DNA probe was labelled by biotin (detected by streptoavidin-FITC, green) in the common nightingale and digoxigenin (detected by antidigoxigenine-rhodopsine, red) in the thrush nightingale. In both experimental designs, the green signal suggests a higher copy number of a particular repetitive sequence in the common nightingale, while the red signal indicates a higher copy number in the thrush nightingale. An intermediate yellow/orange signal suggests the same copy number in both species. Finally, a green signal in one design, while red in the other, indicates the presence of species-specific sequence (Figure 1).

Genomic DNA for the preparation of probes was extracted from blood samples using DNeasy Blood and Tissue Kit (Qiagen). The probes were prepared by nick translation (Abbott Laboratories) according to the manufacturer’s protocol and labelled with biotin-dUTP (Roche) and digoxigenin-dUTP (Roche). The nick translation took place at 15°C for 2 h. From each sample, 1 µg of DNA was co-precipitated overnight at −20°C with an additional 5 µl of salmon sperm DNA (10 mg/ml, Sigma Aldrich), 3 µl of 3 M sodium acetate (pH 5.2) and 2.5 volume of 96% ethanol. After precipitation, the dry pellets were resuspended in 11 µl of hybridization buffer for each slide (50% formamide in 2× SSC, 10% dextran sulfate, 10% sodium dodecyl sulfate and 1× Denhardt’s buffer, pH 7.0), denatured at 86°C for 6 min and then chilled on ice for 10 min prior to hybridization.

Metaphase slides were prepared by treatment with RNase and pepsin before being fixed with 1% formaldehyde, dehydrated through an ethanol row (70, 85 and 96%, 3 min each) and air-dried. Chromosomes were then denatured in 75% formamide/2× SSC at 76°C for 3 min and dehydrated again in an ice cold ethanol raw (70, 80 and 96%, 3 min each). Finally, 11 µl of the probe mix was applied to each slide and hybridization took place at 37°C for 48 h. The same probe mix was applied to metaphases of both nightingale species in the two CGH designs.

Post-hybridization washes were performed two and three times in 50% formamide/2× SSC and 1× SSC at 44°C, respectively. Each slide was incubated with 100 μl of 4× SSC/5% blocking reagent (Roche) at 37°C for 30 min and then with 100 μl of detection mix containing 4× SSC/5% blocking reagent, 2 μl of streptavidin-FITC (Vector Laboratories) and 1 μl of anti-digoxigenin-rhodamine (Roche) at 37°C for 1 h. The slides were subsequently washed in 4× SSC/0.01% Tween 20 (Sigma Aldrich) at 44°C, dehydrated through an ethanol row (70, 85 and 96%, 3 min each) and air-dried. Finally, the chromosomes were counterstained with DAPI in mounting medium Vectashield (Vector laboratories).

Mitotic spreads were captured with an Axio Imager Z2 microscope (Zeiss) equipped with the automatic Metafer-MSearch scanning platform and a CoolCube 1 b/w digital camera (MetaSystems). Meiotic spreads were analyzed using an Olympus BX53 fluorescence microscope (Olympus) equipped with a DP30BW digital camera (Olympus). Ikaros karyotyping software (Metasystems) was used to remove the background from the metaphase images and to arrange the karyotypes. The colors of the C-banded metaphase images were inverted. All color images were captured in black and white, and later pseudocolored and superimposed using Adobe Photoshop software (version CC 2017).

A total of 18 and 17 metaphases from bone marrow were analyzed for the common nightingale and the thrush nightingale, respectively. The W chromosome was detected by C-banding due to its heterochromatic character. The Z chromosome was identified by comparing the female (ZW) and male (ZZ) metaphases. The size of each bivalent and its arm ratio was measured using the LEVAN plugin in the program ImageJ (Schindelin et al., 2012). Depending on the position of the centromere, we distinguished for each macrochromosome whether it was telocentric, acrocentric, submetacentric or metacentric chromosomes (Levan, 1964). For microchromosomes, the telocentric/acrocentric categories and submetacentric/metacentric categories were merged as they were difficult to distinguish clearly. The Z chromosome was identified among bivalents based on its relative size to the other macrochromosomes identified on the mitotic spreads. Chromosomes were measured in 15 pachytene cells in each species.

The metaphase chromosomes with applied CGH were analyzed using Photoshop (version CC 2017). For each CGH design, three cells were analyzed and compared. The centromeric red and green signals of the nine largest macrochromosomes and the sex chromosomes were measured using the histogram color tools. Each metaphase was measured three times to reduce the technical error associated with the signal measurement. The color ratio was calculated using the median value of both colors, after their normalization using the total red and green signal color.

Both species showed a more or less continuous decrease in chromosome size without a clear distinction between macrochromosomes and microchromosomes (Figure 2). We categorized the 10 largest chromosome pairs including the sex chromosomes to be macrochromosomes with the remaining chromosomes considered to be microchromosomes. Because the mitotic chromosomes were not always well spread, it was difficult to estimate the diploid chromosome number from metaphase spreads. This was especially true with respect to the number of microchromosomes. We thus calculated the diploid chromosome number for both species based on immunostained meiotic spreads (Hale et al., 1988; del Priore and Pigozzi, 2020). Both the common nightingale and the thrush nightingale consistently displayed 42 bivalents, establishing a diploid chromosome number of 84 for each species (Figure 3). In addition to these bivalents, both species displayed an extra univalent chromosome in male germ cells, corresponding to the GRC (Figure 3). The GRC was stained weaker by anti-SYCP3 antibody and showed a CREST signal not only in the centromere, but along the whole chromosome, as has been described previously in other passerine species (Torgasheva et al., 2019).

Staining of centromeres in meiotic chromosomes by the CREST antibody allowed us to estimate the arm ratio for each chromosome and compare chromosome morphology between species in a more precise way than was possible with mitotic chromosomes. The ten largest chromosomes had the same morphology between the species, suggesting that no chromosomal rearrangements that would have changed the position of the centromere occurred on these chromosomes. In both species, the largest chromosome, SC1, was identified as acrocentric, SC2 to SC4 as telocentric, SC5 as submetacentric, SC6 as metacentric and SC7 to SC9 as telocentric. However, SC10 was telocentric in the common nightingale, but acrocentric in the thrush nightingale (Figure 3; Supplementary Table 2), indicating that some rearrangements might have occurred on this chromosome.

Based on the comparison of male and female mitotic spreads, we identified the Z chromosome as the fourth largest chromosome in both species and in both species it was telocentric. The W chromosome was also telocentric, with a size between the 10th and 11th chromosome in the common nightingale and between the nineth and 10th chromosome in the thrush nightingale (Figure 4).

The morphology of the microchromosomes slightly differed between the two species with 17 acrocentric/telocentric and 15 submetacentric/metacentric microchromosomes in the common nightingale and 19 acrocentric/telocentric and 13 submetacentric/metacentric microchromosomes in the thrush nightingale (Figure 3; Supplementary Table 2). The GRC was present in both nightingale species, with its size corresponding to a microchromosome.

The distribution of the constitutive heterochromatin revealed by C-banding displayed the same pattern in the two nightingale species. C-banding signals mainly occurred in the centromeric regions of macrochromosomes and microchromosomes, but sometimes the signal covered the entire microchromosome. The W chromosome displayed a large C-banding signal in both species, but the signal was slightly larger in the thrush nightingale than in the common nightingale (Figure 4). In both species, the Z chromosome had a small heterochromatic band in the centromeric region (Figure 4).

18S rDNA clusters were consistently located on 10 microchomosomes in both species (Figure 4). The same number and distribution of the 18S rDNA clusters suggests that no rearrangements that would include rDNA genes had occurred between the two species.

The telomeric motif (TTAGGG) _n was detected at the terminal regions of all chromosomes. No interstitial telomeric signal was detected (Figure 4; Supplementary Figure 1). This can be seen in both the mitotic (Figure 4) and meiotic spreads (Supplementary Figure 1).

In both interspecific CGH experimental designs (i.e., with the common nightingale and the thrush nightingale metaphases, Figure 1), the distribution pattern of the probe signal was similar to that of the heterochromatin from the C-banding experiment, meaning that the probe signal was brightest in the centromeric regions of the macrochromosomes and microchromosomes (Figures 4, 5). In some microchromosomes, the whole chromosome appeared to be generating signal, however, due to the small size of chromosomes and the signal strength of the probes, this might still only represent centromeric binding.

Interestingly, the centromeric regions of the nine largest autosomes were mostly green (common nightingale probe) in the CGH with common nightingale metaphases and red (thrush nightingale probe) in the CGH with thrush nightingale metaphases, suggesting sequence divergence of repetitive elements in the centromeric regions. The exceptions were the first and fifth chromosome pairs, which showed an increased red signal in both CGH designs, indicating a higher copy number of centromeric repetitive elements in the thrush nightingale genome. The fourth pair produced variable signals across the three metaphases, making the results difficult to interpret (Figures 5, 6; Supplementary Table 3).

The whole W chromosome displayed a higher red signal in both interspecific CGH designs, indicating a higher number of repetitive elements in the thrush nightingale genome. Contrastingly, the probe signal in the centromeric region of the Z chromosome was greener in both the common nightingale and the thrush nightingale experimental designs (Figures 5, 6; Supplementary Table 3). Thus, the Z chromosome seems to have a higher copy number of centromeric repetitive elements in the common nightingale compared to the thrush nightingale.

The centromeric regions of microchromosomes were mostly green in the CGH with the common nightingale metaphases and red in the CGH with the thrush nightingale metaphases, suggesting sequence divergence of centromeric repeats on most microchromosomes (Figure 5).