Philippa Kaur

We retrieved 684 Lokiarchaeota and 31 Heimdallarchaeota near-full-length 16S rRNA sequences from sequence libraries generated from sediment sampled at 27 m water depth in 5 cm intervals between 0 and 40 cm below seafloor (cm.b.s.f) in Aarhus Bay (Supplementary Information). The maximum relative read abundance of Lokiarchaeota was 1.6% at 15–20 cm.b.s.f. and 0.1% for Heimdallarchaeota at 10–15 cm.b.s.f. (Fig. 1). The sequences were grouped into 58 Loki- and 3 Heimdallarchaeota operational taxonomic units (OTUs) using a 98% sequence identity threshold and formed three distinct Lokiarchaeota clades and one monophyletic Heimdallarchaeota cluster (Fig. 1). The primer-free sequencing of RNA extracts enabled us to broadly sample the Asgard archaeal diversity in Aarhus Bay sediments and provided a solid database to design oligonucleotide probes for their visualization.Fig. 1: Phylogenetic analysis and depth distribution of Loki- and Heimdallarchaeota 16S rRNA sequences from Aarhus Bay sediments.A Maximum likelihood phylogeny of Loki- and Heimdallarchaeota operational taxonomic units (OTUs) and related sequences selected from the SILVA database (v. 132). Specificities of FISH probes and the number of sequences constituting each OTU are also depicted. TACK archaea were selected as outgroup. Bar: 0.1 substitutions per nucleotide position. B Heatmap and relative abundances of Loki- and Heimdallarchaeota sequences at different sediment depths.Full size imageBased on the newly retrieved full-length sequences, we designed four novel oligonucleotide probes specifically targeting Loki- and Heimdallarchaeota 16S rRNA with high coverage (Fig. 1, Supplementary Table 1). Probe LOK1183 targets almost all sequences in Lokiarchaeota Clade A, which contains 92% of the retrieved Lokiarchaeota sequences from Aarhus Bay sediments, while probe LOK1378 targets 85% of the sequences in all three Lokiarchaeota clades. Probe HEIM329 and HEIM529 each target >97% of the retrieved Heimdallarchaeota sequences. All designed probes cover >89% sequences in their target groups in the SILVA database (v. 132). The two Lokiarchaeota probes match 5 and 10 different non-target sequences in the SILVA database (v. 132), respectively, while the Heimdallarchaeota probes have no match outside their target group. The broad coverage and high specificity suggest that our probes can also be used to detect Loki- and Heimdallarchaeota in other habitats. Furthermore, designing two probes for each phylum enabled us to identify Lokiarchaeota clade A and Heimdallarchaeota cells via double hybridizations with two distinct dyes and thus confidently distinguish true- and false-positive signals (Supplementary Fig. 1). The general archaeal probe ARC915 also targets Lokiarchaeota and thereby provided yet another control for specific hybridization of the two Lokiarchaeota-specific probes, while the non-sense probe NON338 served as the negative control. We also designed competitor probes to minimize the theoretical false-positive hybridizations with the most frequent one and two mismatches [11] in the SILVA database (v. 132) and helper probes to facilitate probe binding [12]. This comprehensive experimental design with appropriate controls enabled reliable detection of low-abundant Loki- and Heimdallarchaeota cells in Aarhus Bay sediments.We used both confocal laser scanning microscopy (CLSM) and three-dimensional super-resolution structured illumination (SR-SIM) microscopy for detailed imaging of dual-labeled Loki- and Heimdallarchaeota signals. Loki- and Heimdallarchaeota cells featured coccoid shapes and often formed clusters (Fig. 2) (Supplementary Fig. 2). Based on SR-SIM imaging, Lokiarchaeota cells (n = 18) were 1.27 ± 0.24 µm in diameter and 1.43 ± 0.25 µm in length, while the width and the length of Heimdallarchaeota cells (n = 11) were 1.30 ± 0.20 µm and 1.37 ± 0.21 µm, respectively (Supplementary Table 2). In addition, we observed a few large ( >3 µm) ovoid and filamentous cells, resembling some of the Lokiarchaeota morphotypes reported from lake sediment [9]; however, we never detected these cell types in double hybridizations with two probes (Supplementary Fig. 1P–R), and therefore consider them false-positives.Fig. 2: Visualization of Loki- and Heimdallarchaeota cells in Aarhus Bay sediments by CARD-FISH.Probe names and the dyes are indicated for each panel. Representative cell morphotypes were imaged in a super-resolution structured illumination microscope (SR-SIM; panels (A), (B), (D), (E)) and confocal laser scanning microscope (CLSM; panels (C) and (F)). For SR-SIM images, single slices from the center of the focal plane are shown. For CLSM images, three-dimensional (3D) surface reconstructions are depicted. All z-stack images taken in CLSM are included in Supplementary Fig. 2. 360° rotation of 3D reconstructed images are also provided in Supplementary Video. Negative and positive controls are shown in Supplementary Fig. 1 together with large ovoid and filamentous false-positive signals. Images are representative of dual labeled Lokiarchaeota (n = 72) and Heimdallarchaeota (n = 70) cells in five individual experiments using two different sediment cores taken from the same sampling site. The scale bar is 1 µm.Full size imageThe DNA stain (4′,6-diamidino-2-phenylindole; DAPI) in the FISH-identified Loki- and Heimdallarchaeota cells was consistently confined to a single spherical central or lateral position (Fig. 2), corroborating the signal pattern suggested for some of the Asgard archaeal cells in lake sediments [9]. Using SR-SIM, we could image a clear gap, which separated the DNA from the ribosome-originated FISH signals with an average width of 0.18 ± 0.07 µm in Heimdallarchaeota and 0.16 ± 0.13 µm in Lokiarchaeota cells (Supplementary Table 2). The spatial separation of DNA and ribosomes in Loki- and Heimdallarchaeota cells represents an unusual observation since DAPI and FISH signals generally overlap partially or completely in prokaryotic cells [13]. Accordingly, SR-SIM imaging of benthic bacteria in Aarhus Bay sediments demonstrated the prevalence of this overlapping signal pattern (Supplementary Fig. 3). Also, the separated DNA signal observed in Loki- and Heimdallarchaeota cells appeared different from the condensed DNA formation previously described, for example, in Escherichia coli cells [14] and the Thaumarcheota Cenarcheum symbiosum [15] and Nitrosopumilus maritimus [16]. To corroborate this, we performed SR-SIM imaging of CARD-FISH-labeled E.coli and N. maritimus cells. Although their DNA was condensed in particular cellular locations, their FISH and DAPI signals always overlapped, indicating that their DNA and ribosomes are partially co-localized and not fully separated (Supplementary Fig. 4).To assess whether the gap between DAPI and FISH signals was indicative of an internal membrane, we tried various dyes to stain membranes of the CARD-FISH-labeled Asgard archaeal cells (Supplementary Information). However, none of these stainings was successful, not even for the outer cell membrane, most likely because cell membranes were disintegrated during the CARD-FISH protocol. We then used wheat germ agglutinin (WGA), a lectin primarily binding to N-acetyl-D-glucosamine but also other glycoconjugates and oligosaccharides [17] to at least be able to visualize the surfaces of Loki- and Heimdallarchaeota cells. WGA consistently decorated a cell surface that enclosed the proximal FISH and DAPI signals, suggesting that both signals originated from the same single cell (Supplementary Fig. 5). The WGA staining also demonstrated extracellular structures connected to some Heimdallarchaeota cells (Supplementary Fig. 5). These structures appear different than the membrane protrusions in the first cultured Lokiarchaeon “Ca. P. syntrophicum”, which has a considerably smaller cell size (550 nm in diameter) and does not possess the separated DNA and ribosome signals [5]. Our observations therefore indicate diverse cellular organizations and morphotypes within Asgard archaea superphylum.Our combined results suggest that genomic material is condensed and spatially distinct from the riboplasm within the detected Loki- and Heimdallarchaeota cells. Considering the anticipated role of Asgard archaea in eukaryogenesis, in particular the presence of ESPs potentially involved in dynamic cytoskeleton formation [18] and membrane remodeling [4, 19], the separation of DNA- and ribosome-derived signals might be indicative of cellular compartmentalization. Alternatively, the observed pattern could be the result of a membrane invagination to form a nucleoid region, similar to membrane organizations for example in Planctomycetes cells [20] or Atribacter laminatus [21].Our study demonstrates the first visualization of diverse Loki- and Heimdallarchaeota cells in the marine environment and provides a protocol for reliable in situ imaging of rare microorganisms in environmental samples. Future research should address the ultrastructure of Asgard archaeal cells using electron microscopy. This would help to elucidate the cell biology of Asgard archaea and provide insights into the emergence of subcellular complexity of the eukaryotic cell. More

The following experimental workflow was implemented to address the main questions raised in our study (Fig. 1).Fig. 1: The scheme of experimental pipeline used in this study to examine the impact of lytic phage infection on the P. aeruginosa population and the development of phage-resistance.Experiments were conducted as follows: culture preparation (1); biofilm formation (2); phage infection with single or cocktail preparations (3); incubation (4); biofilm and planktonic populations sampling (5); culture plating on TSA agar and isolation of discrete colonies (6); phage typing determination (7); to select isolates with unique patterns (8) for further phenotypic (9) and genome sequencing analyses (10).Full size imageThe P. aeruginosa PAO1 reference strain and four other clinical representatives were infected with distinct lytic phages in a single or different cocktail combination. Randomly picked colonies from the surviving cultures were then tested in terms of susceptibility to inoculated phages as well as to the others from the Pseudomonas phages panel (Table 1). We were interested in exploring the broadest clonal variability developed in phage infected Pseudomonas population. Therefore, the first phase of the study was focused on examining the phenotypic heterogeneity of PAO1 reference mutants (phage typing) within planktonic and biofilm populations. Since the consequences of introducing lytic phages into the bacterial population are difficult to predict, a representative pool of bacterial clones that have survived infection was sampled. A total of 780 P. aeruginosa PAO1 clones were typed with phages (planktonic (320), biofilm populations (400) and 60 control clones). No resistance to phages was observed among the control clones taken from untreated biofilm or plankton. Therefore, three biofilm and three planktonic representatives and the wild-type PAO1 were selected for further genetic and fitness analyses (Table S1). Finally, a pool of 95 isolates has been filtered, representing seventeen different phage susceptibility patterns (Tables S1, 2). This selection was based on the maximum variety of phage-type profiles, without accounting for the origin of the isolate (biofilm/plankton), as the infected planktonic bacteria turned out to be less diverse and all phage types were also present in the biofilm population.Since we did not aim to analyse the differences of planktonic versus sessile cells response to phage infection but rather look for maximum population heterogeneity, we decided to focus the investigation on the biofilm population for the other clinical strains during the second stage of this research. Accordingly, 880 (30 clones from every condition plus 10 control clones for each strain) isolated colonies from A5803, AA43, CHA, and PA biofilm populations were first subjected to phage typing. No phage resistance was observed among clones taken from phage-untreated samples compared to the wild-type strain. Ultimately, 35 phage-treated colonies, three controls, and the wild-type from each strain were selected for further investigation, resulting in a pool of 156 clones in total (39 × 4 strains) representing over twenty different phage susceptibility patterns (Table S1 and S3).Do phages always select for cross-resistance to other phages recognising the same bacterial receptor?The application of monovalent phage against reference PAO1 population generally led to the selection of cross-resistance against phages that recognise the same receptor as the applied one (Table S2). This was observed for 12/17 and 23/24 PAO1 clones isolated after LPS- and T4P-dependent phages treatment, respectively. Similar relation (15/20) was only observed for other clinical cultures infected with phiKZ phage (T4P-dependent) (Table S3). The resistance to both groups of phages was less frequent in monovalent infections (14.5% in PAO1 and 32.5% for other clinical strains) compared to polyvalent infections (61.1%; 33/54) and 51.6% (31/60) for PAO1 and clinical strains, respectively. The use of a cocktail of two phages recognising LPS selected for PAO1 clones resistant only to LPS-dependent phages. In contrast, LPS-dependent phages application was mostly accompanied by the emergence of resistance to phages recognising alternative receptors in clinical strains (28/60 cases).The introduction of a particular phage into the population did not guarantee the isolation of clones resistant to this phage. This event was recorded in the case of single phages, as well as for polyvalent combinations (23 PAO1 mutants). However, the cross-resistance to other phages recognising the same or both receptors did also occur. Interestingly, LUZ7 and KTN6 phages could still infect surviving clinical populations with a frequency of 23/60 and 44/80, respectively. Indicating that the resistance to LPS-dependent phages in clinical strains was more difficult to develop compared to those impaired by giant viruses, with 11/60 and 1/20 still sensitive to phiKZ and PA5oct phages, respectively. Almost all PAO1 (80/95) and clinical (127/140) clones treated with phages developed resistance to phage PA5oct, whereas the resistance to the entire phage panel emerged regardless of the single or cocktails application.To conclude, the selection of cross-resistance to other phages recognising the same bacterial receptor was mostly valid in the PAO1 model, whereas the other clinical strains primarily developed the cross-resistance to T4P-dependent phages.Do phages from different taxonomy groups recognising the same receptor cause the emergence of the same type of resistant mutants? Are the defence response and genome changes correlated with the receptor specificity of infecting phage?To assess the genetic basis of the resistance selected by phages, we performed single nucleotide polymorphisms (SNPs) and mapping analyses of 102 reference PAO1 clones and 156 clones derived from clinical strains (Figs. 2, 3, Table S2–S4). The wild-type P. aeruginosa strains were also re-sequenced with Illumina and PacBio technologies to ascertain their complete genomic background. Missense, nonsense, and frameshift mutation variants were taken into account in the analyses. Mutations that also occurred in control isolates were excluded from further consideration. The remaining mutations were divided into six groups: LPS-related genes, mucoidity-associated genes (EPS production, biofilm formation), T4P-related genes, global regulatory genes, and others (hypothetical or undefined function genes). The comparative analysis showed the presence of point mutations in 64 out of 95 examined PAO1 mutants. The frequency of mutations in PAO1 clones isolated after treatment with single or multiple phages was similar (73% vs 61%, respectively). In most of those isolates, only one gene mutation event was recorded (43%). However, in 23 cases SNPs occurred in two or three genes belonging to different metabolic gene groups. Five PAO1 isolates showed the presence of mutations in two genes from one gene group. The 33 cases of SNPs related to LPS synthesis were found in 29 mutants selected with single LPS-dependent phage preparations or in polyvalent combinations. Among these, the most frequent mutation (21/33 cases) was observed within the wzy gene, encoding the B-band O-antigen polymerase [30]. These frequent mutations in the LPS-biosynthesis cluster confirmed the phage resistance results emerging after LUZ7, KT28, and KTN6 phages propagation. In some cases, the LPS gene modification was accompanied by changes in EPS-related genes, leading to a mucoid phenotype. The T4P-dependent phage treatment also led to the selection of specific mutations in genes responsible for T4P expression, but also alterations in flagella-related genes (flgH, fliN, fliP, flhA). The mutations in global regulatory genes (most frequent yqjG and vfr) and “others” gene groups did not show any correlation to the type of phages used.Fig. 2: Graphical presentation of genetic changes occurring in the population of P. aeruginosa as a result of the infection by selected phages.The colour dots refer to particular gene groups where the point mutations (accumulated results) were recorded within the genomes of examined mutant clones. The lower line contains information on the maximum and minimum size of large deletions (grey bands) and the presence of intact prophages (light blue bands). * means mutation in promoter region of the gene.Full size imageFig. 3: The frequency of genetic changes per clone detected in P. aeruginosa strains.Panel (A) represents the PAO1 clones, and panel (B) represents the clinical strains populations. Populations were selected by specific phages targeting LPS (red dots) or T4P (blue dots) as a single treatment or in cocktails. The colour bars refer to particular gene groups where the point mutations were recorded within the genomes of examined mutant clones. N means the number of analysed clones for each strain.Full size imageApart from point mutations, 23% of phage-resistant PAO1 isolates contained large genomic deletions (23,983 bp–544,729 bp) appeared regardless of the phage-type and cocktail composition used as selective pressure agents. All deletions were located in the same region and despite different starting/ending points, they hold a core element of 19,038 bp. This core element carries the galU gene (responsible for LPS biosynthesis), as well as the hmgA gene, which causes the accumulation of brown pigment in bacterial cells when absent. Besides, the cumulated deletion range contained a total of 706,374 bp, including many key genes involved in the bacterial metabolism.Mutations detected in other clinical phage-resistant clones were classified according to the same criteria as in PAO1 (Figs. 2, 3, Table S3, S4). The genome-driven response to phage infection of A5803 was primarily located in global (71%, cpdA) and other genes (34%, PA2911); of AA43 in other genes (31%, PA2911); of CHA in T4P (34%) and global genes (34%, morA); and of PAK in T4P (25%) and other genes (23%, PA2911). Most of the mutations selected by LPS-dependent phage exposition were found in the global regulatory genes (9–11–25–54%) or “other” genes (17–23–31%), rather than in the LPS biosynthesis locus (0–3–6–17%) depending on the impacted strain (Table S3). That confirmed the phage-typing results where LUZ7 and KTN6 phages remained lytic towards surviving clones. In contrast, the application of phiKZ selected for the cross-resistance to T4P-dependent phages as well as for the genetic modifications in pili-related genes. Mutations in global regulatory and “others” genes show no correlation to the receptor specificity of phages used. Interestingly, a portion of phenotypically phage-resistant clones in each clinical P. aeruginosa population (5-9/35 clones) did not reveal any distinguishable genetic modifications. Consistent with PAO1, large genomic deletions were observed in A5803, AA43, and PAK strains ranging between 92,207 bp and 383,693 bp in size, encompassing the galU region. The MEME analysis of the regions flanking the deletions did not reveal specific motifs that would indicate recombination events. Interestingly, the unique large deletion found in CHA strain (15,126 bp) turned out to be the induced ssDNA filamentous Pf1-like phage.Summarising the analyses one might say that phages from different taxonomy groups recognising the same receptor generally cause the emergence of a similar type of resistance within a particular strain. However, the defence response and genome changes correlated with the receptor specificity of infecting phage differ in a strain-dependent manner.Do different strains of P. aeruginosa react similarly to a specific phage infection?The next step aimed to assess the impact of gaining phage resistance in terms of population growth efficiency as an indicator for bacterial fitness. Three of the examined wild-type strains (PAO1, A5803, and CHA) have a naturally rapid growth rate, while the other two (AA43 and PAK) display moderate growth rates. For this reason, the final results are expressed as the cumulated OD600 (Fig. 4, Table S2, S3). Overall, the majority of PAO1 mutants (61/95; 64%, p  0.001) for the clones resistant to 6–7 phages but only in the PAO1 group. Moreover, only the selection done by phage cocktails gave a statistically significant reduction of bacterial growth (p  > 0.001), while no differences were observed regarding groups treated with single LPS- or T4P- dependent phages. In contrast to the PAO1 reference strain, the statistical analyses conducted in the A5803, AA43, CHA, and PAK strains did not show any differences in terms of phage-typing profile nor phage-type selection pressure versus the population fitness reduction (growth rate).Fig. 4: The population growth efficiency as an indicator for bacterial fitness expressed as the cumulative OD600 values of 18 h culture at 37 °C measured at 20-minute intervals.Dots represent the growth of particular clones: the wild-type and control clones (green dots); mutants selected by LPS-dependent phage (red dots); mutants selected by T4P-dependent phage (blue dots); mutants selected by LPS/T4P-dependent PA5oct phage (orange dots); mutants selected by phage cocktail (black dots). * statistically different cumulative OD value compared to phage-untreated pool (p  More

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Whole-genome sequencing of Zhoushan cattle and Wenling cattle populationsWe collected seven individuals of Zhoushan cattle (Fig. 1a, upper panel). We also collected nine individuals of Wenling cattle (Fig. 1a, lower panel). Wenling cattle have a prominent hump on the back, dewlap, and larger ears, suggesting that its genetic background is largely B. indicus (Fig. 1a, lower panel). We performed whole-genome sequencing of these samples. To resolve their phylogenetic positions and interrelationships within domesticated cattle, we combined our data of 16 cattle individuals with publicly-available whole-genome sequencing data of five individuals from the Angus breed, a typical B. taurus in Europe, and 33 individuals from nine breeds with genetic backgrounds similar to B. indicus3, giving a total of 54 individuals (Fig. 1b, c; Table S1). We performed read trimming and aligned the trimmed reads to the UOA_Brahman_1 assembly of the cattle genome11. This assembly represents the maternal haplotype of an F1 hybrid of Brahman cattle (dam) and Angus (sire)11. After variant calling and filtering, we identified 32,970,327 single-nucleotide polymorphisms (SNPs) and 3,331,322 small indels. Based on this genomic variant information, we conducted the population genomic analyses.Figure 1Phylogenetic analysis of Zhoushan cattle and other cattle breeds. (a) Gross appearance of Zhoushan (upper panel) and Wenling cattle (lower panel). Note that Zhoushan cattle have a dark black coat colour. The arrow indicates the curving horn of Zhoushan cattle. (b) Geographic map indicating the origins of Zhoushan (green dot) and Wenling (orange dot) cattle analysed in this study. We also examined other Chinese cattle (red dots) whose genome sequencing data were available. (c) Regional map around the Zhoushan islands. Wenling, Wannan, and Guangfeng are mainland regions close to the Zhoushan islands. (d) Neighbour-joining tree of the 54 domesticated cattle. The scale bar represents pairwise distances between different individuals. The maps were constructed by R38 and R packages of maps v3.3.0 (https://cran.r-project.org/web/packages/maps) and mapdata v2.3.0 (https://cran.r-project.org/web/packages/mapdata).Full size imageGenetic relationship between Zhoushan cattle and other domesticated cattleTo reveal the phylogenetic positions and interrelationships of Zhoushan and other domesticated cattle, we performed population genomic analyses on 54 cattle individuals. First, we calculated the pairwise evolutionary distance between individuals and generated a neighbour-joining (NJ) tree to reconstruct the phylogenetic relationships between individuals of Zhoushan and other domesticated cattle (Fig. 1d). In the NJ tree, cattle clustered consistently with their geographical location (Fig. 1d). Angus individuals formed a sister group to all other individuals, including Zhoushan cattle, Wenling cattle, and other B. indicus (Fig. 1d). The individuals of Zhoushan and Wenling cattle formed monophyletic groups and were sisters to each other (Fig. 1d). The cattle in Guangfeng formed another monophyletic group and were sisters to both Zhoushan and Wenling cattle (Fig. 1d). Cattle in Wannan, Ji’an, and Leiqiong formed a single group, sister to the cattle of Zhoushan, Wenling, and Guangfeng (Fig. 1d). Zhoushan, Wenling, Guangfeng, Wannan, and Ji’an are geographically close to each other (Fig. 1b, c). The cattle of Dianzhong and Wenshan, which are in the south part of China, were distant from them (Fig. 1d). Cattle in Pakistan and India were located near the root of the phylogenetic tree (Fig. 1d). The branch lengths of Zhoushan cattle were shorter than other B. indicus cattle, suggesting the reduced genetic diversity of Zhoushan cattle (Fig. 1d).To estimate the relatedness between Zhoushan and other domesticated cattle, we performed unsupervised clustering analysis with ADMIXTURE v1.3.0 software (https://dalexander.github.io/admixture/index.html)12. At K = 2, Angus cattle were distinct from all other cattle (Fig. 2a). At K = 3, Zhoushan and Wenling cattle were newly segregated from other cattle, suggesting that these two cattle breeds are genetically close to each other (Fig. 2a). The cattle of Guangfeng, Wannan, Ji’an, Leiqiong, and Wenshan had intermediate genetic structures between Zhoushan cattle and Dianzhong cattle (Fig. 2a). At K = 4, Zhoushan cattle and Wenling cattle were separated from each other (Fig. 2a).Figure 2Admixture and principal component analysis of Zhoushan cattle and other cattle breeds. (a) Admixture plot (K = 2, 3, 4) for the 54 cattle individuals. Each individual is shown as a vertical bar divided into K colours. (b) PCA plot showing the genetic structure of the 54 cattle individuals. The degree of explained variance is given in parentheses. Colours reflect the geographic regions of sampling in Fig. 1d. The cluster composed of cattle in Wenling, Guangfeng, Wannan, Ji’an, and Leiqiong is highlighted in the black dotted ellipse. (c) Estimate of the effective population sizes of Zhoushan (green) and Wenling (orange) cattle over the past 100 generations.Full size imageTo infer the population structure of cattle individuals analysed in this study, we conducted principal component analysis (PCA). The top three principal components accounted for 21.1% of the total variance (Fig. 2b). In the first component of PCA, Angus individuals were separated from all other cattle (Fig. 2b). Additionally, cattle of Wenling, Guangfeng, Wannan, Ji’an, and Leiqiong formed a cluster (dotted ellipse in Fig. 2b). In the second component of PCA, individuals of Zhoushan cattle were separated from all other cattle (Fig. 2b). In the third principal component, Wenling cattle individuals were separated from all other cattle (Fig. 2b).We estimated the trends of the effective population size of Zhoushan and Wenling cattle over the past 100 generations (Fig. 2c). Both populations showed decreasing trends of effective population sizes (Fig. 2c). The effective population size of Zhoushan cattle was estimated to be smaller than that of Wenling cattle, suggesting the effect of island isolation on the genetic diversity of Zhoushan cattle (Fig. 2c).Detection of candidate genes associated with dark black coat colour of Zhoushan cattleTo identify putative genes associated with the dark black coat colour of Zhoushan cattle, we searched genomic regions where the same mutations were shared between Zhoushan cattle and Angus cattle. To achieve this, we calculated the average fixation index (Fst) values in 40 kb windows with 10 kb steps (Fig. 3a). We identified four peaks of Fst at chromosomes 2, 4, 8, and 18 (Fig. 3a). Among these peaks, the highest peak of Fst was identified in the region from 51.05 to 51.35 Mbp on chromosome 18 (Fig. 3a, b). This region contains 18 genes (Fig. 3c). We searched for genes that have mutations altering the amino acid sequence and have been reported to be involved in the regulation of coat colour. Among these 18 genes, only the gene of melanocyte-stimulating hormone receptor (MC1R) is known to involved in the regulation of coat colour13,14,15. Therefore, we regarded MC1R as a strong candidate gene associated with the dark black coat colour of Zhoushan and Angus cattle (Fig. 3c). This gene is located in the region between 51,094,227 bp and 51,095,177 bp on chromosome 18. MC1R is expressed in the skin melanocyte and plays a crucial role in regulating animal coat colour formation16. Mutations of MC1R have been reported to be associated with black coat colour in some animals, such as cattle17, sheep16, pigs18, reindeer19, and geese20. In the protein-coding region of MC1R, we identified one missense mutation (c.583T  > C, p.F195L) and one synonymous mutation (c.663C  > T) (Figs. 3d, 4a). The missense mutation is located in the fifth transmembrane region of MC1R (Fig. 4b). All seven Zhoushan cattle were homozygous for the missense mutation (Figs. 3d, 4a). Four of five Angus individuals were homozygous for the missense mutation, and the remaining one was heterozygous for the missense mutation (Figs. 3d, 4a). Conversely, only 19% (8/42) and 33% (14/42) of B. indicus individuals were homozygous or heterozygous, respectively, for the missense mutation (Figs. 3d, 4a). The remaining 48% (20/42) of individuals of B. indicus were homozygous for the wild-type allele (Figs. 3d, 4a). We also found that the p.F195L mutation is also present in MC1R of Black Angus (accession number: ABX83563.1) in the NCBI Protein database (Fig. S1). Furthermore, we identified 15 upstream variants and three downstream variants in the intergenic regions between neighbouring genes (Table S2).Figure 3Genomic regions associated with dark black coat colour of Zhoushan cattle. (a) Manhattan plot for average Fst values in 40 kb windows with 10 kb steps between Zhoushan cattle plus Angus and other B. indicus. A region with an average Fst of more than 0.6 is coloured in green. The arrow indicates the highest peak. The x-axis represents chromosomal positions, and the y-axis represents the average Fst values. (b) Manhattan plot on chromosome 18 for average Fst values in 40 kb windows with 10 kb steps between Zhoushan cattle, Angus, and other B. indicus. (c) Regional plot around the MC1R gene. The genotype of each individual at each variant site is shown. The genotype homozygous for the reference allele is coloured grey. Heterozygous variants are coloured blue. The homozygous genotype for alternative alleles is coloured light blue. Note that homozygous genotypes for alternative alleles are enriched in Zhoushan and Angus cattle in this region. (d) Regional plot showing the mutations around MC1R gene.Full size imageFigure 4Secondary structure of MC1R and protein sequence alignment of MC1R orthologs. (a) Regional highlight of the c.583 T  > C mutation of MC1R. The genomic region from 51,094,590 to 51,094,598 bp on chromosome 18 is shown. Note that MC1R is located on the reverse strand. (b) Secondary structure of MC1R. MC1R is a seven-transmembrane receptor. The p.F195L mutation is located in the 5th transmembrane region and enclosed by the red circle. This figure is generated by using the Protter server application39. (c) Multiple sequence alignment of MC1R orthologs. The black rectangle highlights the 195th phenylalanine residues. The red rectangle encloses the p.F195L mutation in Zhoushan cattle. The cladogram of the species is shown to the left of the species name. The cladogram topology is derived from a previous study40.Full size imageTo characterise the missense mutation of MC1R (c.583T  > C, p.F195L) found in Zhoushan and Angus cattle, we estimated the degree of evolutionary conservation of the 195th phenylalanine of MC1R. We obtained various MC1R orthologs of vertebrates from eight eutherian mammals, two marsupial mammals, four reptiles, two birds, two amphibians, one lobe-finned fish, one polypterus fish, four teleost fish, and two cartilaginous fish (Table S3). We aligned these 26 sequences with MC1R of Zhoushan cattle and B. indicus (Fig. 4c). This analysis revealed that the 195th phenylalanine of MC1R is highly conserved among vertebrates (Fig. 4c).Furthermore, we verified whether any larger structural variants are spanning the MC1R region (chr18:51,058,185–51,148,307 bp) of Zhoushan cattle and Angus. If there are large structural variants in this region for these breeds, we should see regions where the read depth distributions are different among the groups. We assessed the integrated read depth distributions of Wenling cattle (n = 9), Zhoushan cattle (n = 7) and Angus (n = 5) (Fig. 5a). The read depth distribution was very similar among the three groups suggesting that there are not large structural variants spanning the MC1R region in these breeds (Fig. 5a). We also collected the sequence reads mapped to this region, and performed BreakDancer to detect structural variants21. However, no structural variants were detected in this region in any breeds. Moreover, we compared the reference genome sequence in MC1R region of the UOA_Brahman_1 assembly and that of the UOA_Angus_1 assembly11. The UOA_Brahman_1 assembly represents the maternal haplotype of an F1 hybrid of Brahman cattle (dam) and Angus (sire), and the UOA_Angus_1 assembly represents its paternal haplotype11. The results showed that the genome sequence in the MC1R region are highly preserved between these two assemblies (Fig. 5b).Figure 5Read depth distribution, genome alignment and admixture analysis of the MC1R region. (a) Read depth distributions in the MC1R region. The left panel shows the read depth distributions in the region from 51,058,185 to 51,148,307 bp on chromosome 18. The right panel shows the read depth distributions in the region from 51,090,618 to 51,099,796 bp on chromosome 18. For each breed, the sequencing reads were integrated. The first track represents read depth distribution in each breed, and the second track represents read alignments to the reference genome. For a given base position, if the base call in the sequencing read and the corresponding base in the reference genome are different, adenine is shown in green, thymine in red, guanine in orange, and cytosine in blue. (b) Dot plots showing the genome alignments of the MC1R regions of the UOA_Angus_1 assembly (chr18:49,477,288–49,566,766 bp) and the UOA_Brahman_1 assembly (chr18:51,058,185–51,148,307 bp). The left panel shows the genome alignment by minimap2 aligner and the right one shows the genome alignment by LASTZ aligner. The region corresponding to the MC1R gene body is highlighted in red. (c) Admixture analysis of the MC1R region. The SNPs located in the MC1R region (chr18:51,058,185–51,148,307 bp) were collected and subjected to admixture analysis. The order of the samples is the same as in Fig. 2a.Full size imageFinally, we deduced the origin of the MC1R haplotype in Zhoushan cattle. We collected the SNPs located in the MC1R region (chr18:51,058,185–51,148,307 bp) from all individuals and performed admixture analysis using these SNPs. The result showed that Zhoushan cattle and Angus shared highly similar genetic components (Fig. 5c). However, the other individuals of B. indicus showed genetic components that differed from both Zhoushan cattle and Angus (Fig. 5c). These results suggest that the MC1R haplotype in Zhoushan cattle is derived from B. taurus, even though the genome of Zhoushan cattle as a whole is that of B. indicus. More

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