Ecology

Generation timeDuring the infectious period, an infected individual may produce a secondary infection. However, the individual’s infectiousness is not constant during the infectious period, but it can be approximated by the probability distribution of the generation time (GT), which accounts for the time between the infection of a primary case and the infection of a secondary case. Unfortunately, such distribution is not as easy to estimate as that of the serial interval, which accounts for the time between the onset of symptoms in a primary case to the onset of symptoms of a secondary case. This is because the time of infection is more difficult to detect than the time of symptoms onset. Ganyani et al.27 developed a methodology to estimate the distribution of the GT from the distributions of the incubation period and the serial interval. Assuming an incubation period following a gamma distribution with a mean of 5.2 days and a standard deviation (SD) of 2.8 days, they estimated the serial interval from 91 and 135 pairs of documented infector-infectee in Singapore and Tianjin (China). Then, they found that the GT followed a gamma distribution with mean = 5.20 (95% CI = [3.78, 6.78]) days and SD = 1.72 (95% CI = [0.91, 3.93]) for Singapore (hereafter GT1), and with mean = 3.95 (95% CI = [3.01, 4.91]) days and SD = 1.51 (95% CI = [0.74, 2.97]) for Tianjin (hereafter GT2). Ng et al.28 applied the same methodology to 209 pairs of infector-infectee in Singapore and determined a gamma distribution with mean = 3.44 (95% CI = [2.79, 4.11]) days and SD 2.39 (95% CI = [1.27, 3.45]; hereafter GT3). Figure 3 shows the probability density functions (PDF) of such distributions, fGT. The differences between them are remarkable. For example, the 54.5%, 81.0%, and 80.7% of the contagions are produced in a pre-symptomatic stage (in the first 5.2 days after primary infection) assuming GT1, GT2, and GT3, respectively.Figure 3Probability density function of the generation time distribution, fGT(t), of GT1 (blue line; Singapore27), GT2 (yellow line; Tianjin27), GT3 (red line; Singapore28), and GTth (black line; theoretical distribution). Bars are the discretized version, (widetilde{{f_{GT} }}left( n right)), of the PDF of GTth.Full size imageTheoretically, assuming that the incubation periods of two individuals are independent and identically distributed, which is quite plausible, the expected/mean values of the GT and the serial interval should be equal29,30. The mean of the serial interval is easier to estimate than that of the GT. For that reason, we assume a mean serial interval as estimated from a meta-analysis of 13 studies involving a total of 964 pairs of infector-infectee, which is 4.99 days (95% CI = [4.17, 5.82])31, is more reliable than the aforementioned means of the GT. This value is within the error estimates of the means of GT1 and GT2, but not for GT3. Then, we construct a theoretical distribution for the GT that follows a gamma distribution (hereafter GTth) with mean = 4.99 days and SD = 1.88 days. This theoretical distribution can be seen in Fig. 3 and approximates the average PDF of three gamma distributions with mean = 4.99 and the SD of GT1, GT2, and GT3. We assume a conservative CI = [1.51, 2.39] for the theoretical SD, defined with the minimum and maximum SD values of GT1, GT2, and GT3. GTth shows 63.1% of pre-symptomatic contagions.
R

0

from r
In theory, the basic reproduction number R0 can be estimated as far as the intrinsic growth rate r, and the distributions of both the latent and infectious periods are known26,32,33,34. The latent period accounts for the period during which an infected individual cannot infect other individuals. It is observed in diseases for which the infectious period starts around the end of the incubation period, as happened with influenza35 and SARS36. However, from Fig. 3 it is inferred that COVID-19 is transmissible from the moment of infection, and we will assume a null latent period. Then, if the GT follows a gamma distribution, R0 can be estimated from the formulation of Anderson and Watson32, which was adapted to null latent periods by Yan26 as$$ R_{0} = frac{{mean_{GT} }}{{1 – left( {1 + mean_{GT} cdot r cdot frac{1}{{shape_{GT} }}} right)^{{ – shape_{GT} }} }} cdot r, $$
(4)
where meanGT is the mean GT and shapeGT is one of the two parameters defining the gamma distribution, which can be estimated as$$ shape_{GT} = frac{{left( {mean_{GT} } right)^{2} }}{{left( {SD_{GT} } right)^{2} }}. $$
(5)
For GTth, we get R0 = 1.50 (CI = [1.41, 1.61]) for REMEDID I(n) and R0 = 1.76 (CI = [1.60, 1.94]) for official I(n). For the other three GT distributions, R0 ranges from 1.39 (CI = [1.27, 1.58]) to 1.51 (CI = [1.34, 1.80]) for REMEDID I(n) and from 1.59 (CI = [1.40, 1.88]) to 1.78 (CI = [1.51, 2.23]) for official I(n) (Table 1). In all cases, R0 from GTth are within those from the three known GT distributions and indistinguishable from them within the error estimates. The lower (upper) bound of the CI is estimated as the minimum (maximum) R0 obtained from all the possible combinations of 100 evenly spaced values covering the CI of r, meanGT and SDGT. Then, following the Bonferroni correction, the reported CI present at least a 85% of confidence level for GT1, GT2, and GT3, but it cannot be assured for GTth since the CI of its SD is unknown. In general, all these R0 estimates are lower than those summarised by Park et al.20.Table 1 R0 and HIT values of the ancestral SARS-CoV-2 variant estimated from GT1, GT2, GT3, and GTth, and REMEDID and official infections. For date0, “Dec.” means December 2019, and “Jan.” means January 2020.Full size tableAlternatively, R0 can be estimated by applying the Euler–Lotka equation29,33,$$ R_{0} = frac{1}{{mathop smallint nolimits_{0}^{ + infty } e^{ – rt} cdot f_{GT} left( t right)dt}}. $$
(6)
In this case, we get values closer to previous estimates20. In particular, for GTth, we get R0 = 2.12 (CI = [1.81, 2.48]) for REMEDID I(n) and R0 = 2.92 (CI = [2.28, 3.75]) for official I(n). For the other three GT distributions, R0 ranges from 1.63 (CI = [1.43, 1.90]) to 2.21 (CI = [1.59, 2.95]) for REMEDID I(n) and from 1.97 (CI = [1.59, 2.54]) to 3.11 (CI = [1.84, 4.90]) for official I(n) (Table 1). The CI are estimated as in Eq. (4).R0 from a dynamical modelWe designed a dynamic model with Susceptible-Infected-Recovered (SIR) as stocks that accounts for the infectiousness of the infectors. Such a model is a generalisation of the Susceptible-Exposed-Infected-Recovered (SEIR) model37. Births, deaths, immigration and emigration are ignored, which seems reasonable since the timescale of the outbreak is too short to produce significant demographic changes. For the sake of simplicity, the recovered stock includes recoveries and fatalities, and it is denoted as R(t). A random mixing population is assumed, that is a population where contacts between any two people are equally probable. Time is discretized in days, so the real time variable t is replaced by the integer variable n. As a consequence, the derivatives in the differential equations defining the dynamic model explained below are discrete derivatives.The size of the population is fixed at N = 100,000, and then, for any day n we get$$ tilde{S}left( n right) + left( {mathop sum limits_{k = 0}^{20} tilde{I}left( n-k right)} right) + tilde{R}left( n right) = N, $$
(7)
where (tilde{S}left( n right)), (tilde{I}left( n right)), and (tilde{R}left( n right)) are the discretized versions of S(t), I(t), and R(t) and (tilde{I}) is assumed to be null for negative integers. The summation is a consequence of the infectiousness, which is approximated according to the GT, whose PDF is discretized as$$ widetilde{{f_{GT} }}left( n right) = mathop smallint limits_{n – 1}^{n} f_{GT} left( t right) dt, $$
(8)
from n = 1 to 20. Figure 3 shows (widetilde{{f_{GT} }}left( n right)) for GTth. Truncating at n = 20 accounts for 99.99% of the area below the PDF of all the GT. Then, an infected individual at day n0 is expected to produce on average$$ widetilde{{R_{e} }}left( {n_{0} + n} right) cdot widetilde{{f_{GT} }}left( n right) $$
(9)
infections n days later, where (widetilde{{R_{e} }}left( n right)) is the discretized version of Re(t). From this expression, it is obvious that values of (widetilde{{R_{e} }}left( n right) < 1) will produce a decline of infections. Conversely, infections at day n0 are produced by all individuals infected during the previous 20 days as$$ tilde{I}(n_{0} ) = tilde{R}_{e} left( {n_{0} } right) cdot left( {mathop sum limits_{n = 1}^{20} tilde{I}left( {n_{0} - n} right) cdot widetilde{{f_{GT} }}left( n right)} right), $$ (10) whose continuous version has been reported in previous studies29,38. The expression in brackets is called total infectiousness of infected individuals at day n039. According to Eq. (1), Eq. (10) can be expressed in terms of R0 as$$ tilde{I}(n_{0} ) = R_{0} cdot frac{{tilde{S}left( {n_{0} } right)}}{N} cdot left( {mathop sum limits_{n = 1}^{20} tilde{I}left( {n_{0} - n} right) cdot widetilde{{f_{GT} }}left( n right)} right). $$ (11) As we want a dynamic model capable of providing (tilde{I}left( {n_{0} } right)) from the stocks at time step n0 − 1, we replaced (tilde{S}left( {n_{0} } right)) by (tilde{S}left( {n_{0} - 1} right)) in Eq. (11). This assumption makes sense in a discrete domain since the infections at time n0 take place in the susceptible population at time n0 − 1. Then, assuming that all stocks are set to zero for negative integers, our dynamic model can be expressed in terms of Eq. (7) and the following differential equations:$$ delta tilde{I}(n_{0} ) = R_{0} cdot frac{{tilde{S}left( {n_{0} - 1} right)}}{N} cdot left( {mathop sum limits_{n = 1}^{20} tilde{I}left( {n_{0} - n} right) cdot widetilde{{{text{f}}_{GT} }}left( n right)} right) - tilde{I}(n_{0} - 1), $$ (12) $$ delta tilde{S}left( {n_{0} } right) = {-}tilde{I}left( {n_{0} } right), $$ (13) $$ delta tilde{R}left( {n_{0} } right) = tilde{I}left( {n_{0} - 21} right), $$ (14) where (delta tilde{I}), (delta tilde{S}), and (delta tilde{R}) are the (discrete) derivatives of (tilde{I}), (tilde{S}), and (tilde{R}), respectively. Applying the initial conditions (tilde{S}left( 0 right) = N - 1), (tilde{I}left( 0 right) = 1), and (tilde{R}left( 0 right) = 0), it is assumed that the outbreak was produced by only one infector. The latter is not true in Spain, since several independent introductions of SARS-CoV-2 were detected40. However, for modelling purposes it is equivalent to introducing a single infection at day 0 or M infections produced by the single infection n days later. Then, the date of the initial time n = 0 is accounted as a parameter date0, which is optimised, as well as R0, to minimise the root-mean square of the residual between the model simulated (tilde{I}left( n right)) and the REMEDID and official I(n) for the period from date0 to n0.The model was implemented in Stella Architect software v2.1.1 (www.iseesystems.com) and exported to R software v4.1.1 with the help of deSolve (v1.28) and stats (v4.1.1) packages, and the Brent optimisation algorithm was implemented. For REMEDID I(n) and GTth, we obtained date0 = 13 December 2019 and R0 = 2.71 (CI = [2.33, 3.15]). Optimal solutions combine lower/higher R0 and earlier/later date0 (Fig. 4), which highlights the importance of providing an accurate first infection date to estimate R0. When the other three GT distributions were considered, we obtained similar date0, ranging from 12 to 17 December 2019, and R0 values ranging from 2.08 (CI = [1.86, 2.42]) to 2.85 (CI = [2.05, 3.25]; see Table 1). For official infections, date0 was set to 1 January 2020 for all cases, and R0 ranged from 1.81 (CI = [1.64, 2.07]) to 2.41 (CI = [1.80, 2.91]). The CI are estimated as in Eq. (4).Figure 4Root-mean square (RMS) of the residuals between infections from the model, which depends on date0 (x-axis) and R0 (y-axis), and REMEDID (from MoMo ED) and official infections. Parameters optimizing the model are highlighted in purple. RMS larger than 1275 (left panel) and 103 (right panel) are saturated in white.Full size image More

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Genome sequencing and assemblyGenome assemblies ranged in size from 799.9 Mbp in Melanocharis versteri to 1053.5 Mbp in Sericornis nouhuysi. The number of scaffolds ranged from 14,086 scaffolds in Melipotes ater to 87,957 scaffolds in Ficedula hyperythra and N50 ranged between ca. 40 Kbp to and 25 Mbp. Benchmarking Universal Single-Copy Orthologs (BUSCO) analyses of genome completeness ranged from a high proportion of complete BUSCOs in Melipotes ater, 86.8% to only 66.7% complete BUSCOs in Rhipidura albolimbata. For most species, the proportions of complete BUSCOs were 75–80%. Overall, the proportion of missing BUSCOs was low, ranging from 6.6% in Melipotes ater to 15.2% in Rhipidura albolimbata (see Supplementary Table 1 for all genome assembly statistics and Supplementary Fig. 1 for the number of SNP variants per species).Kinship analyses of individuals within populationsSampling of closely related individuals can dramatically bias estimates of population structure and demographics. Two Pachycephala schlegelii individuals (A117 and A118) showed a pairwise kinship coefficient of 0.144, indicative of being half-siblings. The two individuals were collected at the same locality on the same date. Similarly, two Ifrita kowaldi individuals (D116 and D117) showed a pairwise kinship coefficient of 0.135, also suggestive of being half-siblings. In this case, the individuals were collected on the same sampling locality on two consecutive days. To not bias downstream demographic analyses, one of the P. schlegelii (A118) and one of the I. kowaldi (D117) individuals were excluded from all subsequent analyses. For all other species, no closely related individuals were identified.Genetic differentiationEstimated levels of differentiation between populations were initially based on three approaches; (i) calculation of FST (the fixation index), which quantifies the degree of genetic differentiation between populations based on the variation in allele frequencies, ranging between 0 (complete mixing of individuals) and 1 (complete separation of populations) (Fig. 1), (ii) Standardized pairwise FST used to conduct a Principal Component Analysis (PCA) in order to visualize population structure (Supplementary Fig. 1) and (iii) Admixture analysis as implemented in STRUCTURE (a clustering algorithm that infers the most likely number of groups [K]), in which individuals are grouped into clusters according to the proportion of their ancestry components (Supplementary Fig. 1). As a preliminary analysis, we calculated FST and constructed PCA plots for the four congeneric (incl. Sericornis/Aethomyias [until recently placed in the genus Sericornis]) species pairs in our study (Supplementary Fig. 2), which were aligned using the same reference genome. This was done to ascertain that no samples had been misidentified and to gauge levels of differentiation between distinct species. All species were genetically well separated and FST values ranged from 0.08 for the two Ptiloprora species to 0.20 for the two Ficedula species.For five out of six species from Buru/Seram, genetic differentiation (FST) was high between islands (Fig. 1), and comparable to differentiation between named congeneric species in this study (e.g. Ptiloprora and Melipotes); Ceyx lepidus (FST = 0.16), Thapsinillas affinis (FST = 0.15), Ficedula buruensis (FST = 0.13) and Pachycephala macrorhyncha (FST = 0.09). In contrast, differentiation in Ficedula hyperythra was consistent with population-level differentiation (FST = 0.04). In all cases, individuals from Buru and Seram were clearly differentiated in the PCA and STRUCTURE plots (Supplementary Fig. 1A). For Ceyx lepidus, Ficedula buruensis and Pachycephala macrorhyncha, samples were collected at multiple elevations and we therefore calculated genetic differentiation between elevations (Buru: 1097 m versus 1435 m and Seram: 1000 m versus 1300 m) to determine any potential parapatric differentiation along the gradients. In all possible comparisons, FST values did not differ significantly from 0. Moreover, PCA plots showed that samples did not cluster according to elevation (Supplementary Fig. 3A).Three of the thirteen New Guinean population pairs occurring in Mount Wilhelm and Huon showed relatively high genetic divergences: Melipotes fumigatus/ater (FST = 0.08), Paramythia montium (FST = 0.09) and Ifrita kowaldi (FST = 0.07) (Fig. 1) with populations clearly separated (Supplementary Fig. 1). By contrast, the two lowland species Toxorhamphus novaeguineae and Melilestes megarhynchus showed little genetic differentiation, FST = 0.00. For the remaining species, genetic differentiation between Mount Wilhelm and Huon ranged between FST = 0.01–0.05. Despite this moderate level of genetic differentiation, the populations of Mount Wilhelm and Huon could be clearly distinguished in the PCA plots. In all cases STRUCTURE suggested a scenario with K = 2 with some mixing of individuals, except for Rhipidura albolimbata, in which K = 1 was suggested.For five bird species we included an additional population from Mount Scratchley, which is also situated in the Central Range but ~400 km to the southeast of Mount Wilhelm. Genetic differentiation of this population from the other two populations was comparable with that between Mount Wilhelm and Huon. The highest genetic differentiation was found in Paramythia montium (FST = 0.10 both between Mount Wilhelm and Mount Scratchley and between Huon and Mount Scratchley). In the case of Peneothello sigillata, the Mount Scratchley population appeared genetically well-differentiated from both the populations of Mount Wilhelm (FST = 0.06) and Huon (FST = 0.07). In both cases, STRUCTURE suggested a scenario of K = 3, with individual assignments matching the three geographically circumscribed populations. For Pachycephala schlegelii, genetic differentiation was relatively high between Huon and Mount Scratchley (FST = 0.05), but low between Mount Wilhelm and Mount Scratchley (FST = 0.01). Accordingly, STRUCTURE suggested a scenario with K = 2 groups. For the remaining two species Sericornis nouhuysi showed some differentiation (FST = 0.03) between Mount Wilhelm and Huon and Aethomyias papuensis showed minor differentiation (FST = 0.02 between Mount Scratchley and Huon (Supplementary Table 2), but for both species, STRUCTURE suggested a scenario of K = 2 with considerable mixing of individuals between populations.Samples from Mount Wilhelm were collected at elevations ranging from 1700 to 3700 m, again allowing us to test for differences within populations on a single slope, a finding that would be consistent with incipient parapatric speciation. No species showed significant differences in FST when comparing individuals from different elevations, and concordantly there was little clustering of individuals by elevation in the PCA plots. Even when individuals were collected as far as 2000 elevational meters apart (as in the case of Origma robusta), genetic differentiation was low (FST = 0.01). In Huon, all samples were collected at the same elevation, except for Ifrita kowaldi, for which genetic differentiation of FST = 0.03 was found between individuals collected at 2300 m and 2950 m (Supplementary Fig. 3B, Supplementary Table 2). These analyses however, suffer from very small sample sizes that hinder a thorough analysis of parapatric speciation events. Furthermore, we note that divergence with gene flow may not manifest as a genome-wide phenomenon (at least, not until the taxa are so differentiated that gene flow has ceased). Instead, it may proceed via selection acting to create small ‘islands of differentiation’ within the genome against a background of negligible differentiation22,23. Such analyses require large sample sizes and are therefore not possible herein.Correlations between genetic divergence and elevationIf lineages colonize mountains from the lowlands, followed by range contraction and differentiation in the highlands, we would expect a signature of larger genetic differentiation (FST) between populations inhabiting higher elevations. We found no relationship between genetic differentiation (FST) and the altitudinal floor (the lowest elevation at which a species/population occurs) for the five Moluccan species, but for all New Guinean taxa with the exception of Melipotes fumigatus/ater we found a significant positive correlation (r = 0.83, p  More

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General characterization of seven newly isolated HMO-2011-type phagesIn this study, we used four Roseobacter strains (FZCC0040, FZCC0042, FZCC0012, and FZCC0089) and one SAR11 strain (HTCC1062) to isolate phages. FZCC0040 and FZCC0042 belong to the Roseobacter RCA lineage [22], FZCC0012 shares 99.8% 16S rRNA gene identity with Roseobacter strain HIMB11 [57], and FZCC0089 belongs to a newly identified Roseobacter lineage located close to HIMB11 and SAG-019 lineages (Supplementary Fig. 1).A total of seven phages were newly isolated and analyzed in this study (Table 1). The complete phage genomes range in size from 52.7 to 54.9 kb, harbor 62 to 84 open reading frames (ORFs), and feature a G + C content ranging from 33.8 to 48.6%. Compared to other HMO-2011-type phages, pelagiphage HTVC033P has a relatively lower G + C content of 33.8%, similar to the G + C content of its host HTCC1062 (29.0%) and of other described pelagiphages [21, 26,27,28]. The G + C content of other six roseophages ranges from 42.2 to 48.6%, which is also similar to the G + C content of the hosts they infect (44.8 to 54.1%).Despite their distinct host origins, these phage genomes show considerable similarity in terms of gene content and genome architecture (Fig. 1). They all display clear similarity with the previously reported SAR116 phage HMO-2011 [20] and HMO-2011-type RCA phages [22]. Overall, these phages share 19.2 to 79.1% of their genes with previously reported HMO-2011-type phages and all contain homologues of HMO-2011-type DNA replication and metabolism genes, structural genes, and DNA packaging genes. Moreover, their overall genome structure is conserved with that of HMO-2011-type phages. Considering these observations, we tentatively classified these seven phages into the HMO-2011-type group. Of the 11 currently known HMO-2011-type isolates, one infects the SAR116 strain IMCC1322, one infects the SAR11 strain HTCC1062, and the remaining nine all infect Roseobacter strains; this suggest that HMO-2011-type phages infect diverse bacterial hosts. HTVC033P is the first pelagiphage identified to belong to the HMO-2011-type viral group. Our study has also increased the number of known types of pelagiphages. To date, pelagiphages belonging to a total of nine distinct viral groups have been isolated and analyzed [21, 26,27,28].Fig. 1: Alignment and comparison of genomes of HMO-2011-type isolates and representative HMO-2011-type MVGs from major subgroups.HMO-2011-type phage isolates are shown in red. Phages isolated in this study are indicated with red asterisks. Predicted open reading frames (ORFs) are represented by arrows, with the left or right arrow points indicating the direction of their transcription. The numbers inside the arrows indicate ORF numbers. ORFs annotated with known functions are marked using distinct colors according to their functions. HMO-2011-type core genes are indicated with blue asterisks. The color of the shading connecting homologous genes indicates the level of amino acid identity between the genes. To clearly present the genomic comparison, several MVGs were rearranged to start from the same gene as in the HMO-2011-type phages. DNAP DNA polymerase, Endo endonuclease, RNR ribonucleoside-triphosphate reductase, PhoH phosphate starvation-inducible protein, MazG MazG nucleotide pyrophosphohydrolase domain protein, ThyX thymidylate synthase, GRX glutaredoxin, TerS terminase small subunit, TerL terminase large subunit.Full size imageIdentification and sequence analyses of HMO-2011-type MVGsTo identify HMO-2011-type MVGs, we performed a metagenomic mining and retrieved a total of 207 HMO-2011-type MVGs (≥50% genome completeness) from viromes in the worldwide ocean, from tropical to polar oceans (Supplementary Table 1). These MVGs range in size from 29.2 to 67.9 kb and their G + C content range from 31.3 to 52.4%. In addition, 45 HMO-2011-type MVGs were also identified from some non-marine habitats, suggesting that HMO-2011-type phages are widely distributed worldwide (Supplementary Table 1).Genomic analysis confirmed that all HMO-2011-type MVGs exhibit genomic synteny with HMO-2011-type phages (Fig. 1). Although some of these HMO-2011-type MVGs are highly similar to their cultivated relatives, most MVGs appear to have more genomic variations. To resolve the evolutionary relationship among the HMO-2011-type phages, a phylogenetic tree was constructed based on the concatenated sequences of five core genes. We found that HMO-2011-type phages are evolutionarily diverse and can be separated into at least 10 well-supported subgroups ( >2 members), with 140 MVGs clustering into previously identified HMO-2011-type groups (subgroups I and III in Fig. 2A) [22], and the remaining 67 MVGs forming new subgroups (Fig. 2A). Among these HMO-2011-type subgroups, three contain cultivated representatives (subgroups I, III, and IX). Subgroup I contains the greatest number of phages, including six cultivated representatives and 123 MVGs (Fig. 2A). The cultivated representatives in subgroup I include a phage that infect SAR116 strain and five phages that infect Roseobacter strains. Subgroup III contains four cultivated representatives that infect two Roseobacter strains, and 17 MVGs. Pelagiphage HTVC033P and nine MVGs form subgroup IX. Other subgroups have no cultivated representatives yet. The results of phylogenomic analysis showed that subgroups I to VI are closely related, whereas subgroups VII to X are located on a separate branch and are more distinct from the subgroups I to VI, which suggests that these subgroups are more evolutionarily distant. A phylogenomic-based approach with GL-UVAB workflow [53] was also performed to cluster these HMO-2011-type genomes, which showed similar grouping results (Supplementary Fig. 2).Fig. 2: Phylogenomic and shared-gene analyses of HMO-2011-type phages.A A maximum-likelihood tree was constructed using concatenated sequences of five hallmark genes. HMO-2011-type phages were grouped into 10 subgroups based on the phylogeny. Shading is used to indicate the subgroups. HMO-2011-type phage isolates are shown in red. Genomes containing an integrase gene are indicated by red triangles. The G + C content and completeness of the genomes are indicated. Scale bar indicates the number of amino acid substitutions per site. B Heatmap showing the percentage of shared genes between HMO-2011-type genomes. Phages in the same subgroup are boxed.Full size imageA previous study suggested the use of the percentage of shared proteins as a means of defining phage taxonomic ranks and proposed that phages with ≥20 and ≥40% orthologous proteins in common can be grouped at the taxonomic ranks of subfamily and genus, respectively [58]. Overall, most of the calculated percentages between HMO-2011-type genomes fall within the 20 to 100% range and most of the percentages between genomes within the same subgroup fall within the 40 to 100% range (Fig. 2B). Therefore, our results suggest that the HMO-2011-type is roughly a subfamily-level phage taxonomic group containing at least ten genus-level subgroups in the Podoviridae family.Conserved genomic structure and variation in HMO-2011-type phagesOf the 1235 orthologous protein groups (≥2 members) identified in HMO-2011-type genomes, only 254 proteins groups could be assigned putative biological functions (Supplementary Table 2). Comparative genomic analysis clearly revealed the conserved functional module structure of all HMO-2011-type genomes. All HMO-2011-type phage genomes can be roughly divided into the DNA metabolism and replication module, structural module and DNA packaging module (Fig. 1). Most of the homologous genes are scattered in similar loci of the HMO-2011-type genomes. Core genome analysis based on complete HMO-2011-type genomes revealed that HMO-2011-type genomes share a common set of ten core genes (Fig. 1). These core genes are mostly genes related to essential function in phage replication and development, including genes encoding DNA helicase, DNA primase, DNA polymerase (DNAP), portal protein, capsid protein, and terminase small and large subunits (TerL and TerS) as well as several genes with no known function, suggesting that phages in this group employ similar overall infection and propagation processes (Fig. 1).Most members in subgroups I and III and one member in subgroup II possess a tyrosine integrase gene (int) located upstream of the DNA replication and metabolism module, whereas all subgroup IV to X genomes contain no identifiable lysogeny-related genes. This result suggests that members of subgroups IV to X might be obligate lytic phages. Integrase genes typically occur in the genomes of temperate phages and are responsible for site-specific recombination between phage and host bacterial genomes [59, 60]. In subgroup III, RCA phage CRP-3 has been experimentally demonstrated to be capable of integrating into the host genome [22]. Thus, certain int-containing HMO-2011-type phages are also likely to be temperate phages.In the DNA metabolism and replication modules, genes encoding DNA primase, DNA helicase, DNAP, ribonucleotide reductase (RNR), and endonuclease can be identified; and DNA helicase, DNA primase, and DNAP are core to all HMO-2011-type phages. All reported HMO-2011-type phages contain an atypical DNAP, in which a partial DnaJ central domain is located between the exonuclease domain and the DNA polymerase domain [20, 22]. The Escherichia coli DnaJ protein, a co-chaperone [61], has been shown to be involved in diverse functions [62] and to be critical for the replication of phage Lambda [63,64,65]. The sequence analysis revealed that DNAP sequences of these seven new HMO-2011-type phages and 207 MVGs also present this unusual domain structure and contain two repeats of the CXXCXGXG motifs involved in zinc binding [66] in the partial DnaJ domain (Supplementary Fig. 3). RNR gene is frequently detected in subgroups I, II, III, IV, V, and X genomes but not in the other subgroup genomes. RNRs, which are widely distributed in diverse phage genomes, are involved in catalyzing the reduction of ribonucleotides to deoxyribonucleotides, and thus play a crucial role in providing deoxyribonucleoside triphosphates for phage DNA biosynthesis and repair [67,68,69]. RNR genes clustered with the RNR gene in phage HMO-2011 were previously reported to dominate the class II viral RNRs in examined marine viromes [69]. In the remaining two modules, genes involved in phage structure (e.g., genes encoding capsid and portal proteins), packaging of DNA (TerL and TerS genes), and cell lysis were detected. The proteins encoded by these genes play key roles in phage morphogenesis and virion release.Examination of the distribution of the orthologous groups among the subgroups revealed clear pan-genome differences in various subgroups (Fig. 3). Most subgroups harbor subgroup-specific genes not identified in other subgroups, although  no function has yet been assigned to most of these genes. Notably, the phages in subgroups VII, VIII, and IX possess genomic features that differentiate them from phages in other subgroups, specifically with regard to the G + C content and gene content. The members of these three subgroups are closely related to each other in the phylogenetic tree and harbor several subgroup-specific genes. The G + C content of the phage genomes in these subgroups ranges from 31.9 to 35.4%, significantly smaller than other subgroups but similar to the G + C content of SAR11 bacteria and other known pelagiphages. HTVC033P is the only cultivated representative of subgroup IX. The aforementioned results suggest that the phages in subgroup VII, VIII, and IX might have related bacterial hosts and are highly likely to be pelagiphages. The host prediction using RaFAH tool also assigned Pelagibacter as their potential hosts (Supplementary Table 1). Subgroup X is located near these three subgroups in the phylogenetic tree, and the G + C content of the phages in this subgroup ranges from 34.4 to 39.0%. The host prediction assigned Roseobacter as their potential hosts. The hosts of this subgroup still remain to be experimentally investigated.Fig. 3: Distribution and functional classification of orthologous protein groups across HMO-2011-type genomes.Only orthogroups containing >10 members or showing subgroup-specific features are shown. Subgroup-specific genes are boxed in red. Genes that are absent in a specific subgroup are boxed in orange.Full size imageMetabolic capabilities of HMO-2011-type phagesAll HMO-2011-type phage genomes harbor several host-derived auxiliary metabolic genes (AMGs) potentially involved in diverse metabolic processes. Some AMGs in HMO-2011-type phages have been discussed previously [20, 22].Subgroups VII, VIII, IX, and X possess distinct AMGs as compared with the other subgroups. For example, the genes encoding FAD-dependent thymidylate synthase (ThyX, PF02511) and MazG pyrophosphohydrolase domains are absent in all subgroups VII, VIII, IX, and X genomes but frequently detected in other subgroup genomes. ThyX protein is essential for the conversion of dUMP to dTMP mediated by an FAD coenzyme and is therefore a key enzyme involved in DNA synthesis [70, 71]. The thyX gene is commonly found in microbial genomes and phage genomes. Phage-encoded ThyX has been suggested to compensate for the loss of host-encoded ThyA and thus play crucial roles in phage nucleic acid synthesis and metabolism during infection [72]. Except in the case of subgroups VII, VIII, IX, and X genomes, the mazG gene, which encodes a nucleoside triphosphate pyrophosphohydrolase is sporadically distributed in HMO-2011-type genomes. MazG protein is predicted to be a regulator of nutrient stress and programmed cell death [73] and has been hypothesized to promote phage survival by keeping the host alive during phage propagation [74]. The Escherichia coli MazG can interfere with the function of the MazEF toxin–antitoxin system by decreasing the cellular level of (p)ppGpp [73]. However, a recent study showed that a cyanophage MazG has no binding or hydrolysis activity against alarmone (p)ppGpp but has high hydrolytic activity toward dGTP and dCTP, and it was speculated to play a role in hydrolyzing high G + C host genome for phage replication [75]. Whether the MazG proteins encoded by HMO-2011-type phages play a similar role in phage propagation remained to be investigated.Five MVGs in subgroup I contain a gene encoding a DraG-like family ADP-ribosyl hydrolase (ARH). In cellular ADP-ribosylation systems, ARH catalyzes the cleavage of the ADP-ribose moiety, and thereby counteract the effects of ADP-ribosyl transferases [76]. It has been reported that ARH in Rhodospirillum rubrum regulates the nitrogen fixation [77]. However, the function of this phage-encoded ARH in the phage propagation process remains unclear.We also observed that several MVGs possess genes involved in iron–sulfur (Fe–S) cluster biosynthesis, including an Fe–S cluster assembly scaffold gene (iscU) that involved in Fe–S cluster assembly and transfer [78] and an Fe–S cluster insertion protein gene (erpA). Fe–S cluster participates in a wide variety of cellular biological processes [79]. The discovery of these genes suggests that these phages may play important roles in Fe–S cluster biogenesis and function.The gene encoding sodium-dependent phosphate transport protein (PF02690) has been identified in eight subgroup I genomes. The Na/Pi cotransporter family protein is responsible for high-affinity, sodium-dependent Pi uptake, and thus the protein plays a critical role in maintaining phosphate homeostasis [80]. This gene might function in the transport of phosphate into cells during phage infection. The presence of Na/Pi cotransporter genes suggests that some HMO-2011-type phages may have the potential to regulate host phosphate uptake in phosphate-limited ocean environments in order to benefit phage replication and propagation.Identification and phylogenetic analysis of HMO-2011-type DNAPsThe genetic diversity and geographically distribution of HMO-2011-type phages in marine environments was further inferred from DNAP gene analyses. A total of 2433 HMO-2011-type DNAP sequences with sequence sizes ranging from 540 to 779 amino acids were identified and subjected to phylogenetic analysis (Supplementary Table 3).Among the identified HMO-2011-type DNAPs, 2030 sequences were retrieved from the GOV 2.0 Tara expedition upper-ocean viral populations (0–1000 m), from tropical to polar regions. HMO-2011-type DNAP genes were identified from all analyzed upper-ocean viromes, suggesting the global prevalence of HMO-2011-type phages in upper oceans.A previous study revealed that marine viromes contain various types of tailed phage genomes that encode a family A DNAP gene [81]. To estimate the importance of HMO-2011-type phages, we calculated the proportion of HMO-2011-type DNAPs based on the number of HMO-2011-type DNAP sequences and the total number of family A DNAP sequences ( >470 aa) in each GOV 2.0 viral population dataset. This analysis revealed that HMO-2011-type DNAPs accounted for up to 19.7% of all family A DNAPs in each GOV 2.0 dataset (Supplementary Table 4). We found that the HMO-2011-type DNAP sequences appear to be more dominant in epipelagic viromes than in mesopelagic viromes (p  More

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