Core genes can have higher recombination rates than accessory genes within global microbial populations

  1. Asher Preska Steinberg
  2. Mingzhi Lin
  3. Edo Kussell  Is a corresponding author
  1. Department of Biology, New York University, United States
  2. Department of Physics, New York University, United States

Abstract

Recombination is essential to microbial evolution, and is involved in the spread of antibiotic resistance, antigenic variation, and adaptation to the host niche. However, assessing the impact of homologous recombination on accessory genes which are only present in a subset of strains of a given species remains challenging due to their complex phylogenetic relationships. Quantifying homologous recombination for accessory genes (which are important for niche-specific adaptations) in comparison to core genes (which are present in all strains and have essential functions) is critical to understanding how selection acts on variation to shape species diversity and genome structures of bacteria. Here, we apply a computationally efficient, non-phylogenetic approach to measure homologous recombination rates in the core and accessory genome using >100,000 whole genome sequences from Streptococcus pneumoniae and several additional species. By analyzing diverse sets of sequence clusters, we show that core genes often have higher recombination rates than accessory genes, and for some bacterial species the associated effect sizes for these differences are pronounced. In a subset of species, we find that gene frequency and homologous recombination rate are positively correlated. For S. pneumoniae and several additional species, we find that while the recombination rate is higher for the core genome, the mutational divergence is lower, indicating that divergence-based homologous recombination barriers could contribute to differences in recombination rates between the core and accessory genome. Homologous recombination may therefore play a key role in increasing the efficiency of selection in the most conserved parts of the genome.

Editor's evaluation

Homologous recombination is an important process driving the evolution of bacterial genomes. This paper addresses how the rate of homologous recombination varies between core and accessory genes. The authors quantify recombination using an approach based on the decline of linkage between polymorphic sites over distance. The analysis indicates that core genes can be under higher rates of homologous recombination than accessory genes, which is an interesting observation that contrasts with the prevailing wisdom. Together, it suggests that homologous recombination may play a key role in increasing the efficiency of selection in conserved components of the genome.

https://doi.org/10.7554/eLife.78533.sa0

Introduction

Intrinsic to bacterial genome evolution is the process of recombination (Fraser et al., 2007; Hanage, 2016; Smith, 1991; Thomas and Nielsen, 2005), which can enhance the ability of microbes to adapt to their environment (Arnold et al., 2022; Maiden, 1998; van der Woude and Bäumler, 2004). Recombination in bacteria occurs in two forms. One is ‘homologous recombination’, in which fragments of DNA taken up from the environment are incorporated into homologous sites in the genome resulting in allele replacement and no gain or loss of chromosomal DNA (recently this has also been referred to as ‘allele transfer’ [Arnold et al., 2022]). The second is ‘nonhomologous recombination’ or ‘horizontal gene transfer’ (HGT), in which DNA fragments are inserted or removed from the genome, changing the amount of chromosomal DNA and potentially leading to gene gain or loss events (Arnold et al., 2022; Hanage, 2016). Both of these processes obfuscate clonal signals and confound phylogenetic analyses (Feil et al., 2001; Guttman and Dykhuizen, 1994; Spratt et al., 2001), and recombination is so ubiquitous in certain bacteria that whether or not bacterial genome evolution can truly be characterized as tree-like is the subject of much debate (Creevey et al., 2004; Doolittle, 1999; Sakoparnig et al., 2021; Spratt et al., 2001). Recombination rates vary greatly both between and within different bacterial species (Castillo-Ramírez et al., 2012; Chaguza et al., 2016; Chewapreecha et al., 2014; Croucher et al., 2013; Hanage, 2016; Hanage et al., 2009), and this variance is attributed to a complex interplay of selective pressure, molecular mechanisms, and ecological factors which have yet to be disentangled. Along the genome, recombination rates vary among specific genes (i.e., recombination ‘hotspots’) (Didelot et al., 2012; Everitt et al., 2014) and between gene classes such as ‘informational genes’ (those involved with transcription/translation) and ‘operational genes’ (those involved with biosynthesis, metabolism, and regulatory functions) (Jain et al., 1999; Novick and Doolittle, 2019).

This inter- and intragenomic variation in recombination rates has presented difficulties with using individual genes (e.g., 16S rRNA) to define genotypic clusters and species, and approaches such as multilocus sequence typing (MLST) have been developed to address this (Hanage et al., 2005; Hanage et al., 2006a; Maiden et al., 1998). Multilocus approaches attempt to resolve this issue by concatenating sequences from several housekeeping genes, thereby minimizing the chance that local inter- and intraspecific recombination at a single locus distorts our interpretation of clonal relationships. These housekeeping genes are part of the ‘core’ genome, which is found in nearly all strains of a given species and is thought to be less prone to recombination events (at least in the form of HGT) compared to the collections of genes found in only subsets of strains of a given species known as the ‘accessory’ genome (Daubin et al., 2002; Lan and Reeves, 2001; McInerney et al., 2017).

Quantifying the variation of homologous recombination rates across core and accessory genes is important for understanding of how selection and recombination interact to shape sequence diversity, as core genes are thought to be under strong, purifying selection (Bohlin et al., 2014; Bohlin et al., 2017; den Bakker et al., 2013; McInerney et al., 2017; Moulana et al., 2020) whereas many accessory genes are thought to be important for niche specialization and under positive or diversifying selection (Azarian et al., 2020; McInerney et al., 2017; Vernikos et al., 2015). Moreover, characterizing homologous recombination rates in niche-adaptive accessory genes will have far-reaching implications for understanding how bacteria adapt to stresses such as antibiotics, host immune responses, and nutrient conditions (Didelot et al., 2016; Povolo and Ackermann, 2019; Seifert, 1996; van der Woude and Bäumler, 2004). However, measuring homologous recombination rates across accessory genes has been stymied by the frequent gene loss and gain events that they experience, which cause difficulties in applying phylogeny-based approaches to infer homologous recombination rates (Arnold et al., 2018; Ansari and Didelot, 2014; Croucher et al., 2015; Didelot et al., 2010; Didelot et al., 2012; Didelot and Falush, 2007; Didelot and Wilson, 2015; Iranzo et al., 2019; Lefébure and Stanhope, 2007; Marttinen et al., 2012; Mostowy et al., 2017). For this reason, variation in recombination rates has primarily been studied across core genes (Didelot et al., 2012; Everitt et al., 2014; Jain et al., 1999). Whereas previous studies indicate that the core genome experiences fewer HGT events (Daubin et al., 2002; Haegeman and Weitz, 2012; Lan and Reeves, 2001; Lobkovsky et al., 2013; Wolf et al., 2016), direct comparisons of homologous recombination rates between the core and accessory genomes are currently lacking.

Here, we determine how homologous recombination rates differ between core and accessory genes. We focus on Streptococcus pneumoniae, which frequently engages in homologous recombination, has been extensively sequenced (Chaguza et al., 2016; Chewapreecha et al., 2014; Croucher et al., 2013; Hanage, 2016; Hanage et al., 2009), and is clinically relevant (O’Brien et al., 2009). For these reasons, S. pneumoniae provides a key case study for quantifying variation in homologous recombination rates across the core and accessory genome and, by analyzing additional species, we determine the generality of these trends across several bacterial species.

Results

Inferring recombination parameters for analyzed samples and unobserved gene pools using correlated substitutions

To infer the parameters of homologous recombination, we apply and extend the mcorr method (Lin and Kussell, 2017), an approach that avoids phylogenetic reconstruction by using a coalescent-based population genetics model with recombination to capture the statistics of large-scale sequencing data (see Lin and Kussell, 2019 for details). We define a ‘sample’ to be a set of lineages with mean coalescence time Tsample, which mutate and exchange homologous DNA fragments with a much larger, unobserved ‘pool’ of sequences having mean coalescence time Tpool (Figure 1A). By fitting the model to the data, we infer recombination parameters of both the sample (i.e., the specific set of sequences used for analysis) and its pool; sample construction and collection are discussed in the next section. We obtain the pool’s mutational divergence, θpool2μTpool, the pool’s recombinational divergence, ϕpool2γTpool, the sample’s recombination coverage, csample, and the mean recombined fragment size, f¯ (where μ and γ are the synonymous substitution rate and recombination rate, respectively). Together, θpool and ϕpool estimate the number of synonymous substitutions and recombination events that have occurred per site since coalescence of the pool, and csample estimates the fraction of the genome that has been replaced by homologous recombination with the pool since coalescence of the given sample. Unlike the synonymous substitutions, which we assume to be largely neutral, recombination events may be due to selective pressure or neutral drift.

Inferring parameters of homologous recombination from whole genome sequences (WGS).

(A) Schematic depicting exchange of homologous DNA fragments between the analyzed sequences (‘Sample’) and a larger, unobserved reservoir of bacterial genomes (‘Pool’). The coalescence times of the pool and sample are denoted as Tpool and Tsample , respectively. (B) Example correlation profiles of synonymous substitutions for the core genes in samples consisting of 568 S. pneumoniae and 2215 C. jejuni WGS (further description of these datasets are given in the following sections). (C) Recombination parameters inferred from fitting the profiles shown in panel C to a population genetics model (see Materials and methods). Left to right: the pool’s mutational divergence (θpool), the pool’s recombinational divergence (ϕpool), and the sample’s recombination coverage (csample). Error bars are 95% bootstrap confidence intervals (see Materials and methods). Colors correspond to panel B. θpool and ϕpool have units of bp-1 and csample is given as the percentage of genomic sites that have recombined.

The model predicts the conditional probability of a difference at genome site i+l given a difference at site i, which we refer to as the ‘correlation profile’, P(l), where l is the distance between sites in basepairs (bp). We take a set of whole genome sequences (WGS) to constitute a sample, and use alignments of protein coding (CDS) regions to measure profiles of synonymous substitutions for all possible sequence pairs, yielding an average profile for the sample. For each pair of sequences in the sample at genomic position i, a binary variable σi is assigned 1 for a difference or 0 for identity. The correlation profile is given by P(l)P(σi+l=1|σi=1), where i is restricted to third position sites of codons. While the profiles are computed using gene alignments, recombined fragments may span multiple genes, and the mean fragment size in the coalescent model can take values larger than the length of single genes. Fitting the model to these correlation profiles yields the parameters of homologous recombination described above (see Lin and Kussell, 2019 and Materials and methods for details of model and fitting); flat profiles indicate a lack of recombination, while in the presence of recombination P(l) has a monotonic decay with l, and declines more rapidly with increasing recombination (Lin and Kussell, 2017). Example profiles and inferred parameters are shown in Figure 1B, C using sets of S. pneumoniae and Campylobacter jejuni WGS; these sequences are part of the larger datasets used in the analysis that follows. Thus, the method infers parameters of both the sample and the pool of sequences with which it has recombined without using or inferring any phylogenetic relationships, offering a key advantage in determining homologous recombination rates across accessory genes.

Homologous recombination rates are higher in the core versus accessory genes for S. pneumoniae

To study recombination across the core and accessory genome of a given bacterial species, we analyze a wide range of samples from the global microbial population. In our model, each ‘sample’ is composed of collections of genomes from nature; they are statistical samples from a larger, unobserved bacterial population or gene pool. By examining a diverse set of samples from a given species, we are able to infer the distribution of recombination parameters for their unobserved gene pools. However, as many genomes are collected from specific geographic regions or individual hospitals (Chaguza et al., 2016; Chewapreecha et al., 2014; Pelton et al., 2007), a sample can be artificially overweighted by a single collection site accounting for many genomes. Therefore, to obtain a more meaningful set of samples for a given species, and ameliorate some of the compositional biases inherent in nonrandom sampling, we use sequence clusters obtained by clustering WGS solely based on sequence similarity using the average linkage algorithm (Wheeler and Kececioglu, 2007) with the pairwise synonymous diversity (ds) as the distance metric. Such approaches are widely used to define well-resolved genotypic clusters within bacterial populations without the inference of phylogenetic relationships (Hanage et al., 2006a). We then analyze single clusters or pairs of clusters to infer the distribution of pool parameters. For each cluster or cluster pair, we infer both the divergences of the unobserved gene pool with which they recombine (i.e., θpool and ϕpool), as well as the amount of recombination that has taken place within the set of analyzed sequences (i.e., the cluster or cluster pair) since coalescence (csample). For single clusters, we fit correlation profiles measured by averaging over all of a given cluster’s sequence pairs, which yields the parameters of the unobserved pool that the cluster interacts with. For cluster pairs, we fit correlation profiles measured by averaging exclusively over sequence pairs consisting of one sequence from each of the two clusters. By analyzing all possible pairs of clusters, we use diverse sets of sequences as our samples, yielding a parameter distribution that accounts for the full range of potential interactions between different sequences, regardless of their distance in sequence space and agnostic to population structure or sampling biases.

To sample the global population of S. pneumoniae, we used the PubMLST database (see Supplementary file 5) which includes WGS of strains from across the world. For each WGS, we aligned all sequencing reads to a reference genome to create an alignment of all CDS regions (see Materials and methods for details). We then measured ds across all genes (i.e., core and accessory) for each strain pair, clustered based on these distances to make a dendrogram, and split the sequences into flat clusters, where no two sequences within a cluster were more distant than the 10th percentile of pairwise distances, which corresponded to ds=0.015 (Figure 2A, Materials and methods for details; mean ds values within and between clusters for core and accessory genes are given in Supplementary file 3). This resulted in 44 major clusters (where a major cluster has >100 strains) which we used as our samples (946 total cluster pairs; Figure 2B shows an example correlation profile from a cluster), and we inferred recombination parameters for the core (defined here as present in >95% of strains) and accessory genes of each of the samples and the unobserved pools with which they recombine (Figure 2C–E).

Figure 2 with 4 supplements see all
Inference of recombination parameters for the core and accessory genome of S. pneumoniae.

(A) Dendrogram resulting from hierarchical clustering of 26,599 whole genome sequences (WGS) from the PubMLST genome collection for S. pneumoniae using the average linkage algorithm. The dendrogram was cut at the 10th percentile of all measured pairwise distances (ds ~ 0.015) yielding a discrete set of flat clusters. Here, we analyzed the 44 major clusters (those with >100 sequences) which resulted from this cut, encompassing 24,097 strains. In the dendrogram, alternating colors delineate adjacent clusters. (B) Correlation profiles measured across the core and accessory genes of a S. pneumoniae sequence cluster. Open-faced circle is the profile measured from sequencing data and solid line is the fit to the population genetics model. (C–E) Distributions of the pool’s mutational and recombinational divergence (θpool and ϕpool , shown in panels C and D, respectively) and the relative recombination rate of the pool ((γ/μ)pool , shown in panel E). For C–E, the bottom plot depicts empirical cumulative distribution functions (ECDFs) for each parameter and the top plots depict the medians of the bottom plot, where error bars are 95% bootstrap CIs created by sampling the distributions with replacement (n = 1000). Each step in the ECDF corresponds to a pool recombination parameter inferred from a correlation profile measured over a sequence cluster or pair of clusters like that shown in panel B. Core genes are defined as genes found in >95% of strains. We used model selection with the Akaike information criterion to ensure that each profile was well fit (see Materials and methods for details). θpool and ϕpool have units of bp-1 and (γ/μ)pool is unitless. Two-sided p-values were calculated using the Wilcoxon signed-rank test and were p = 1.9e−159, 3.9e−53, and 3.5e−99 for panels C–E, respectively.

Figure 2—source data 1

List of SRA accession numbers corresponding to the raw reads of genomes used from NCBI.

https://cdn.elifesciences.org/articles/78533/elife-78533-fig2-data1-v2.txt

We found that the core and accessory genes of S. pneumoniae have distinct distributions of θpool and ϕpool (Figure 2C, D), and the median of θpool is lower in core versus accessory genes, indicating that core genes are less mutationally diverged than accessory genes. Moreover, we observed that ϕpool is higher for the core genes, implying that core gene pools have experienced a greater number of recombination events per site compared to accessory genes (Figure 2D). We examined this further using the relation ϕpool/θpool=γ/μ, which removes the dependence on coalescence time to yield the relative recombination rates of the unobserved gene pools (from hereon we will append the subscript pool to indicate this, i.e., (γ/μ)pool). The inferred distribution of (γ/μ)pool shows that for S. pneumoniae, the core genome has a higher relative recombination rate than the accessory genome (Figure 2E), again indicating that core genes recombine more frequently. While the inferred distribution of recombination coverage for the samples initially suggests that the accessory genome may recombine more (Figure 2—figure supplement 1A), this can be reconciled by considering the mean size of the recombined fragments (f¯), which is higher for the accessory genes (Figure 2—figure supplement 1B). Taken together, we find that while accessory genes incorporate larger fragments, the core genes of the sampled genomes experience more recombination events on a per site basis (Figure 2—figure supplement 1C).

We tested the robustness of these results in several ways. As there are various thresholds used to define ‘core’ genes (Livingstone et al., 2018; McInerney et al., 2017; Page et al., 2015; Vernikos et al., 2015), we tested how the parameter distributions shifted with different thresholds and found similar trends (Figure 2—figure supplement 2). To determine whether the difference in the abundance of the core and accessory genes (i.e., how many strains a gene appears in) influences our inference of recombination parameters, for each cluster we artificially ‘thinned’ the core genome by replacing each accessory gene sequence with a randomly chosen core gene sequence. This yielded a thinned core genome where each core gene had the same number of sequence pairs as the accessory genome. We found that this thinned core genome showed similar recombination trends as the actual core genome (Figure 2—figure supplement 3).

Aligning raw reads to a reference genome to build consensus genomes allows for access to a much larger set of WGS, as there are comparably fewer whole genome assemblies for S. pneumoniae, which are typically a requirement for reference-free alignment (Ding et al., 2018; Lin and Kussell, 2019; Page et al., 2015) (as of the time of this analysis NCBI GenBank had 81 complete genome assemblies for S. pneumoniae versus the ~26,000 WGS accessed through PubMLST). However, a potential drawback of aligning reads to a single reference genome is that a subset of accessory genes will be missed. New approaches for aligning to reference graphs appear promising in this regard, yet remain computationally expensive for large numbers of reads (Colquhoun et al., 2021). We therefore sought to address this issue by testing how the parameters changed when more accessory genes were included by building a reference pangenome from all the genome assemblies in NCBI GenBank using Roary (Page et al., 2015) and aligned all the raw reads from PubMLST to this (see Materials and methods). Pangenome analysis suggests that this number of genomes should encompass the majority of genes in the S. pneumoniae pangenome (Donati et al., 2010). While the pangenome alignment included more genes (6200 versus 2018 genes), we found very similar trends for the inferred recombination parameters to those inferred using a single reference alignment (Figure 2—figure supplement 4).

To assess the generality of the trends in recombination parameters between the core and accessory genome, we analyzed 11 additional microbial species (encompassing >100,000 genomes including those already analyzed for S. pneumoniae). We used the same procedure described above to cluster sequences and measure correlation profiles for clusters and cluster pairs, and inferred distributions of recombination rates for the unobserved pools for these species. Pairing the inferred recombination rates of core and accessory genomes for each pool, we examined the effect sizes for each species using Cohen’s d statistic (Cohen, 1988; Nakagawa and Cuthill, 2007) to determine the magnitude and direction of the effect (Figure 3A; Supplementary file 1). We found that, in line with our observations in S. pneumoniae, certain species (e.g., Escherichia coli, Shigella flexneri, Neisseria meningitidis, and C. jejuni) showed markedly higher recombination rates for core genes, whereas others showed slightly higher recombination rates in the core genome. Helicobacter pylori had higher recombination rates in the accessory genome which were statistically significant, yet the magnitude of the effect was small (Cohen’s d = 0.18). Among the nine species for which Cohen’s d was nonzero (within 95% confidence intervals) all but H. pylori showed higher recombination rates (d < 0) in the core versus accessory genome. Similarly, of the 10 species with significantly different median recombination rates in core and accessory genomes (within 95% confidence intervals) (Supplementary file 1), all but H. pylori and N. gonorrhoeae showed higher median recombination rates in the core versus accessory genome. Different metrics thus yield largely consistent results regarding differences in recombination rates between core and accessory genomes across species. Lastly, to understand if core gene pools are less diverged than accessory pools as we had found for S. pneumoniae, we also assessed differences in θpool between the core and accessory genome for these same 12 species (Figure 3B; Supplementary file 2). We found that a majority of species had accessory gene pools which were markedly more diverged than core genes, and the few exceptions (e.g., N. gonorrhoeae and N. meningitidis) had small effect sizes.

Figure 3 with 1 supplement see all
Effect sizes (Cohen’s d) for recombination rates and mutational divergence for S. pneumoniae and 11 additional microbial species.

Effect sizes (Cohen’s d) for (A) the relative recombination rate of the pool ((γ/μ)p) and (B) the mutational divergence of the pool (θp) for core/accessory genome pairs for individual clusters and pairs of clusters for S. pneumoniae and 11 additional microbial species. Cohen’s d for paired samples was calculated as the mean paired difference (Xpacc-Xpcore, where X=(γ/μ)p or θp) divided by the standard deviation of paired difference (σ). Error bars are 95% bootstrap CIs, calculated by sampling the distributions with replacement 10,000 times. All effect sizes are listed in Supplementary files 1 and 2, as well as the medians of the distributions and the results of the Wilcoxon signed-rank test for each species. For A, the effect size and 95% bootstrap CI for S. flexneri are −25.5 and [−34.4,−21.1], respectively. When model selection was performed with Akaike information criterion (AIC) (described in Materials and methods) if part of a core/accessory pair was poorly fit, the paired sample was excluded. Full species names, number of strains, number of clusters, and mean synonymous diversity within and between clusters are reported for each species in Supplementary file 3. Legends of supplementary material.

Figure 3—source data 1

Lists of SRA accession numbers corresponding to the raw reads of genomes used from NCBI.

https://cdn.elifesciences.org/articles/78533/elife-78533-fig3-data1-v2.zip

Gene frequency as a recombination barrier in the core and accessory genome

One testable hypothesis consistent with our observation that core genes generally undergo more homologous recombination when compared with accessory genes is that the least frequent genes experience less recombination because they have fewer recombination partners. In other words, gene frequency could act as a recombination barrier. We tested this by binning the genes for the microbial species in Figure 3 into four gene frequency classes consistent with prior delineations of the pangenome as follows (q is frequency across strains) (Koonin and Wolf, 2012; Page et al., 2015): ‘cloud’ genes (q15%), ‘shell’ genes (divided into two segments, 15%<q55%,  55%<q95%), and core genes (q>95%). We then inferred the distributions of recombination parameters within each gene class for each of the 12 microbial species (Figure 3—figure supplement 1A, B). We found that certain species displayed trends consistent with a gene frequency barrier, particularly for the recombination coverage (Figure 3—figure supplement 1A). When considering the recombination rates (displayed as empirical cumulative distribution functions here because of disparate rates across and within species), C. jejuni, S. agalactiae, and P. aeruginosa showed trends most consistent with this hypothesis. For some of the exceptions in which cloud genes showed high recombination rates (e.g., N. gonorrhoeae, N. meningitidis, E. coli, and S. flexneri), this could be related to the high gene replacement rate experienced by rare genes such as ‘ORFans’ (Wolf et al., 2016), or diversifying selection experienced by these genes. Overall, we see a modest positive correlation with both recombination coverage and rates when considering the 50th and 25th percentiles of the matched parameter distributions for all species (Figure 3—figure supplement 1C, D).

Discussion

There is appreciable interest in understanding why microbes have pangenomes and how different parts of the genome have evolved (Lobkovsky et al., 2013; McInerney et al., 2017; Vernikos et al., 2015; Wolf et al., 2016). While it is known that homologous recombination plays a key role in shaping the genome (Fraser et al., 2007; González-Torres et al., 2019; Hanage, 2016; Iranzo et al., 2019; Thomas and Nielsen, 2005), large-scale analysis of this aspect of genome evolution has been limited. This was due to both computational bottlenecks and a reliance on phylogenetic methods (Arnold et al., 2018; Ansari and Didelot, 2014; Croucher et al., 2015; Didelot et al., 2010; Didelot and Falush, 2007; Didelot and Wilson, 2015; Marttinen et al., 2012; Mostowy et al., 2017), the latter of which hindered determination of recombination parameters for accessory genes, whose phylogenies are challenging to ascertain. Here, we expanded our non-phylogenetic, computationally efficient mcorr framework to overcome these obstacles, and present a detailed analysis of the variation in recombination rates between core and accessory genes for S. pneumoniae, along with several other microbial species.

We found that despite the high gene replacement rates and sequence variability experienced by some accessory genes (Iranzo et al., 2019; Kuenne et al., 2013; Kuhn et al., 2006; Wolf et al., 2016), core genes experience markedly higher recombination rates than the accessory genes in S. pneumoniae. In 8 out of 12 microbial species, we observe higher recombination rates in core genes relative to accessory genes. In a subset of microbial species, the magnitude of the effect is large (e.g., S. pneumoniae, E. coli, S. flexneri, N. meningitidis, and C. jejuni). While some work has previously shown housekeeping (Vos and Didelot, 2009) and core genes (Lin and Kussell, 2017; Park and Andam, 2020) can experience extensive recombination consistent with our results, our work offers a direct, quantitative comparison between the core and accessory genomes spanning multiple species and >100,000 sampled genomes. In certain species, mechanisms that give rise to higher recombination rates in the core genome are known; for example, some members of the Neisseriaceae (e.g., N. meningitidis and N. gonorrhoeae) and Pasteurellaceae families (e.g., H. influenzae) have DNA uptake sequences and uptake signal sequences accumulated in their core genomes which promote homologous recombination (Frye et al., 2013; Treangen et al., 2008).

We found that in S. pneumoniae, core gene pools had lower mutational divergence relative to accessory gene pools. Furthermore, we found that for the majority of the species we analyzed this was the case, and 9 out of 12 species analyzed had large effect sizes indicating core gene pools have lower mutational divergence (Cohen’s d = 0.92–2.8). Previous studies indicate that the core genome is under strong, purifying selection (Bohlin et al., 2014; Bohlin et al., 2017; den Bakker et al., 2013; McInerney et al., 2017; Moulana et al., 2020), and it is well known that purifying selection reduces mean coalescence times (Charlesworth et al., 1993; Nicolaisen and Desai, 2012). An additional consideration is that some accessory genes are used for niche specialization (Azarian et al., 2020; McInerney et al., 2017; Vernikos et al., 2015); if different alleles of the same accessory gene are adaptive to different niches, this could result in allelic diversity due to balancing selection, which would increase coalescence times of accessory genes. The lower value of θpool in core genes is therefore consistent with shorter coalescence times of core relative to accessory genes. While another possible explanation could involve codon usage biases (Plotkin and Kudla, 2011) causing differences in synonymous substitution rates between core and accessory genes, the selective coefficients for this process are tiny (s ~ 1/Ne Hershberg and Petrov, 2008); Ne=3×108 for S. pneumoniae (Bobay and Ochman, 2018) and thus effectively neutral over the timescales that we can observe in these data (i.e., coalescence times of S. pneumoniae samples and pools). Accordingly, evidence from long-term microbial evolution experiments has shown that on average the synonymous substitution rate is fairly homogeneous across the core and accessory genomes (Maddamsetti et al., 2015; Maddamsetti et al., 2017). Therefore, we attribute the difference in θpool between core and accessory genes to the difference in their coalescence times, which is consistent with core genes being under stronger purifying selection, and additionally with the possibility that a subset of accessory genes are under diversifying selection. We note that because higher levels of recombination in core genes are expected to reduce the effects of background selection (Charlesworth et al., 1993), the relative reduction in θpool observed in core versus accessory genes of S. pneumoniae and other species likely underestimates the difference in strength of purifying selection acting on these gene classes.

Experiments in numerous species show that homologous recombination rates decay with increasing sequence divergence (Fraser et al., 2007; Majewski, 2001; Majewski et al., 2000; Vulić et al., 1997; Zawadzki et al., 1995), and the role of this recombination barrier in bacterial speciation has been analyzed (Falush et al., 2006; Hanage et al., 2006b). This effect is primarily ascribed to the ubiquity of RecA and mismatch repair systems, both of which are essential for recombination and whose molecular mechanisms depend on sequence similarity. Consistent with these experimental and analytical results, the higher recombination rates and lower pool divergence we observe here for the core genome of S. pneumoniae and several other microbial species may suggest that the increased homology of the core genes reduces the barrier for homologous recombination in this part of the genome. This is also in line with modeling, which has suggested that homologous recombination slows sequence divergence in the core genome by homogenizing this part of the genome, whereas accessory genes do not have the opportunity to be homogenized as they are subject to continual gene gain and loss (Iranzo et al., 2019). Thus, we propose that an evolutionary feedback loop may operate in certain species of bacteria: purifying selection acting on core genes causes higher levels of homology at these loci, this increases their homologous recombination rates, which in turn enables more efficient purifying selection by breaking linkages between genes, effectively minimizing Hill–Robertson interference and hitchhiking effects (Barton, 1995; Barton, 2010; Felsenstein and Yokoyama, 1976) across the core genome. Further investigations are needed to examine whether such an evolutionary mechanism can act to fine-tune recombination rate variation across microbial genomes. Moreover, our work suggests that another recombination barrier for accessory genes may be related to their lower abundance in the population, as we found that the levels of homologous recombination are correlated with gene frequency in a subset of species.

In summary, we have presented a quantitative analysis of the variation in recombination rates between core and accessory genomes. As these two parts of the genome are under different forms of selective pressures, this work contributes broadly to our understanding of how selection and recombination act to shape diversity. The expansion of this approach to more specific gene classes could lead to a better understanding of how selective pressure and homologous recombination interplay to shape niche-adaptive genes such as those involved with drug resistance and antigen variation, for which allele shuffling due to homologous recombination can play a central role in their evolution (Bowler et al., 1994; Maiden, 1998; Seifert, 1996).

Materials and methods

Data and code availability

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  • Lists of SRA accession numbers corresponding to the raw reads used to build the multi-sequence alignments analyzed here are included as source data. All SRA files, reference genomes, and complete genome assemblies are available through NCBI. All sequence collections used are listed in Supplementary file 5. For the PubMLST sequence collections, PubMLST was used to identify WGS (by filtering for strains in the ‘Genome Collection’ of each species where the sequence length is at least that of the reference genome), then the raw reads were downloaded from NCBI using their SRA numbers. Accession numbers for reference genomes used for each microbial species are also listed in Supplementary file 5.

  • All original code has been deposited at GitHub and is publicly available (links provided throughout Methods).

Description of coalescent-based population genetics model with recombination

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The details of the model derivation and parameters are given in the ‘Supplementary Notes’ of Lin and Kussell, 2019. Here, we provide the model equations necessary for the fitting procedure used in this work.

The measured synonymous diversity of the sample, ds, is a linear combination of the pool diversity, dp d(θp) (due to recombination), and the clonal diversity of the sample, d(θs), as follows:

(1) ds=csd(θp)+(1cs)d(θs),

where cs is the recombination coverage in the sample, and d(θ) is given by the classic population genetics expression for heterozygosity (Wakeley, 2009) (see table below). The predicted form of the correlation profile is

(2) P(l)=cs,0(l)d(2θs)+cs,1(l)dp+cs,2(l)Qp(l)/ds,

where the functional forms of cs,i(l) (i=0,1,2) and Qp(l) are given in the table below. As described in Lin and Kussell, 2019, we can reexpress Equation 2 in terms of the three parameters θs, ϕs, and f¯, determined by fitting the profiles. From these, the pool parameters are obtained using the following relations: θp=ds(1+ ϕswf¯+θsa~)θs(1dsa~)(ϕswf¯+θsa~)dsa~ and ϕp=θpϕs/θs ; see table below for values of constants a~ and w.

TermExpressionDescription
θs or θpMutational divergence of the sample or pool
ϕs or ϕpRecombinational divergence of the sample or pool
f¯Mean fragment size of homologous recombination
lDistance (bp) between two loci
The following constants are used below: a~=4/3 and w=2/3.
d(θ)θ1+θa~Synonymous diversity (heterozygosity) for divergence θ
csϕswf¯1+θsa~+ϕswf¯Recombination coverage of the sample
cs,1(l)2ϕswl1+2θsa~+ϕsw(f¯+l)Probability that the most recent event was a recombination affecting only one locus
cs,2(l)ϕsw(f¯l)1+2θsa~+ϕsw(f¯+l)Probability that the most recent event was a recombination affecting both loci
cs,0(l)1cs,1cs,2Probability that the most recent event at either locus was not a recombination
Qp(l)2(θp1+θpa~)2(1+θpa~+ϕpwl1+2θpa~+2ϕpwl)Probability of a difference at both loci in the pool

Calculation of correlation profiles

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For a given sample of aligned sequences, we measured the substitution profile, σi(k,g), for each pair of sequences k, at each position i along each gene g, where σi(k,g) = 1 for a difference and σi(k,g) = 0 for identity. In all calculations below, we only consider positions i that are third position sites of codons yielding synonymous substitutions. We computed the pairwise synonymous diversity of each gene g as:

(3) ds(g)=σi(k,g)¯

where the bar denotes averaging over all sequence pairs k and the bracket denotes averaging over all positions i. The joint probability of synonymous substitutions for two sites separated by a distance l was calculated for each gene as:

(4) Qs(l,g)=σi(k,g)σi+l(k,g)¯

We averaged these over all genes to obtain the sample diversity, ds=(1/ng)gds(g), and the joint probability of substitutions at two sites in the sample, Qs(l)=(1/ng)gQs(l,g), where ng is the total number of genes. The correlation profile is then given by P(l)=Qs(l)/ds.

For calculations of within-pool parameters, all possible sequence pairs within a cluster were considered. This was done with the original command-line (CLI) program mcorr-xmfa program in the mcorr package which takes as input a single extended multi-fasta (XMFA) file. For calculations of between-pool parameters using pairs of clusters, only sequence pairs where each sequence was from a different cluster were considered. This was done with the CLI program created for this paper called mcorr-xmfa-2clades, which uses two XMFA files (one from each sequence cluster) as inputs. These can be found in the mcorr GitHub repository: https://github.com/kussell-lab/mcorr.

Fitting of correlation profiles and model selection using Akaike information criterion

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The basic fitting procedure used was previously described in Lin and Kussell, 2019. We used the python package LMFIT (Newville et al., 2014) version 0.9.7 (https://lmfit.github.io/lmfit-py/) to fit P(l) to the analytical form given in Equation 2; in this work, we used the least-squares minimization with Trust Region Reflective method instead of the default Levenberg–Marquardt algorithm and increased the maximum number of function evaluations from 104 to 106. As described in the text, as an additional test of goodness of fit, we compared the fit of the data with Equation 2 where all parameters vary freely (which we refer to as the ‘full-recombination model’), to the ‘null-recombination model’ in which we set cs,1=cs,2=0, which yields P(l)=d(2θs)=2θs/(1+2θsa~), that is, a constant correlation profile which is fit by taking the average over l of the measured values, yielding a single parameter, θs. We perform model selection between the null- and full-recombination models by evaluating the Akaike information criterion (AIC) for each model, then computing the Akaike weight for each model, which can be roughly interpreted as the probability that a given model yields the best prediction of the data (Bois, 2020; Wagenmakers and Farrell, 2004). The AIC was computed using LMFIT using AIC=n ln(χ2/n)+2Nv , where n is the number of data points, Nv is the number of parameters being varied in the fitting, and χ2=i=1nResidi2 is the sum of the squared residuals for each data point i. The Akaike weight for model j{,r} can then be calculated as:

wj=e0.5(AICjAICmin)e0.5(AICAICmin)+e0.5(AICrAICmin) 

where AIC and AICr denote the AIC values for the null- and full-recombination models, respectively, and AICmin=min{AIC ,AICr}. The evidence ratio, which corresponds to the likelihood of one model being favored over the other in terms of minimizing Kullback–Leibler discrepancy (Wagenmakers and Farrell, 2004), can be computed as wr/w , where wr and w are the Akaike weights of the full- and null-recombination models, respectively. Fits to correlation profiles where wr / w < 100 were not included in the analyses presented in the main text. We also did not include fits to correlation profiles which yielded unphysical parameter values (θpool<0, ϕpool<0), or extreme values of recombination coverage where parameter fitting becomes less reliable due to too few recombination events (csample<1%) or the over-fitting of a flat profile (csample>99%). The CLI program mcorrFitCompare was built for this study to fit correlation profiles to both models and can be found in the mcorr GitHub repository: https://github.com/kussell-lab/mcorr.

Clustering using pairwise synonymous diversity

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In this study, we extended the mcorr method to measure the synonymous diversity separately for each sequence pair, denoted by dpair. This is the same as the calculation of ds described in ‘Calculation of correlation profiles’ without averaging over sequence pairs k. The CLI program mcorr-pair measures dpair for all possible sequence pairs from an XMFA file. We also built a CLI program called mcorr-pair-sync, which calculates a subset of all pairwise diversities from a given set of sequences. We primarily relied on the latter program, as it allows for parallelization of jobs on a high-performance computing cluster. The CLI programs mcorr-dm and mcorr-dm-chunks were built to collect outputs from mcorr-pair and mcorr-pair-sync, respectively, and write them to a square distance matrix for use with standard clustering algorithms. Lastly, we built a program (clusterSequences) relying on the python scipy package to cluster sequences using the unweighted pair group method with arithmetic mean or average linkage method with dpair as the distance metric. All necessary code to perform these calculations can be found in the github repository: https://github.com/kussell-lab/mcorr-clustering.

Generation of multisequence alignments for core and accessory genes using WGS

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For all collections of WGS (Supplementary file 5), with the exception for H. pylori (where the multisequence alignment was already assembled), we used reference-guided alignment to build consensus genomes from raw reads for each sequence, then extracted the CDS regions of each gene to make extended multi-fasta (XMFA) files. For the collections from the PubMLST database, we identified WGS by filtering for all sequences in the ‘Genome Collection’ for a given organism which had lengths greater than or equal to the length of the reference genome. We then exported tables which included the corresponding SRA numbers for each strain, and downloaded the reads from NCBI using the corresponding SRA. For the other sequence collections used, we used their associated NCBI Bioproject ID to obtain the SRA numbers for each strain, and downloaded reads in the same way. Reads were mapped against a reference genome (listed in Supplementary file 5 for each microbial species) using SMALT (version 0.7.6; https://www.sanger.ac.uk/tool/smalt-0/), and consensus genomes were created using SAMtools mpileup (Li et al., 2009). We then extracted the alignments of CDS regions from these consensus genomes using genomic coordinates provided from the general feature format (GFF) file of the reference genome to create an XMFA file of CDS regions for each gene. We filtered out any gene alignments where the gene sequence was 2% gaps. We then measured the percentage of the entire strain collection which had each gene (based on presence/absence of a gene sequence) to determine if a gene should be considered core or accessory. Code to perform this analysis is provided in the GitHub repository: https://github.com/kussell-lab/ReferenceAlignmentGenerator.

Generation of multisequence alignment to pangenome reference genome for S. pneumoniae

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We downloaded all complete genome assemblies from NCBI for S. pneumoniae as of December 18, 2020 (81 genomes), and used Prokka (Seemann, 2014) to reannotate the assemblies and generate general feature format version 3 (GFF3) files for use with Roary (Page et al., 2015). Roary was then used to generate a multi-FASTA for each gene CDS region in the pangenome (which we refer to as the ‘pangenome reference’). We aligned reads to this pangenome reference in the same manner described in ‘Generation of multisequence alignments for core and accessory genes using WGS’, then collected each gene alignment to create an XMFA file for the pangenome. We split the XMFA into files for the core and accessory genome by measuring the percentage of strains which had each gene. In this case, because alignment quality was not as high as when we aligned reads to a single reference genome (as described in the main text), we counted a gene as present if there was only a partially aligned sequence for the gene, and absent if there was no aligned sequence. Code to perform this analysis can be found in the following GitHub repository: https://github.com/kussell-lab/PangenomeAlignmentGenerator.

Generation of artificially ‘thinned’ core genome alignment for S. pneumoniae

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To create the artificially ‘thinned’ core genome alignment described in the main text, we took the core and accessory genome alignments for S. pneumoniae generated as described in ‘Generation of multisequence alignments for core and accessory genes using WGS’, and we replaced each of the accessory gene alignments for each sequence cluster with randomly selected core gene alignments from the same cluster until all accessory gene alignments were replaced. A CLI program called ReduceCoreGenome is used to generate these thinned core genome alignments (https://github.com/kussell-lab/ReferenceAlignmentGenerator).

Statistical analysis

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For the 95% bootstrap CIs appearing in Figure 1C, the same procedure was used as in Lin and Kussell, 2019. In brief, bootstrap replicates of the set of genes were created by resampling the list of all genes in the genome with replacement, then recalculating ds, Qs, and P(l) for each replicate. We then performed the same fitting procedure which was done on the actual dataset to infer recombination parameters on each of the bootstrap replicates to generate 95% confidence intervals. The resampling was done 1000 times.

For all other 95% bootstrap CIs, the procedure used for bootstrapping can be found in the figure legends of the corresponding figures. The process of model selection using AIC is described in ‘Fitting of correlation profiles and model selection using Akaike information criterion’. The number of major clusters and sequences used for each microbial species is given in Supplementary file 3.

Data availability

Lists of SRA accession numbers corresponding to the raw reads used to build the multisequence alignments analyzed in this manuscript are included as Figure 2—source data 1 and Figure 3—source data 1. All SRA files, reference genomes, and complete genome assemblies are available through NCBI. All sequence collections used are listed in Supplementary file 5. For the PubMLST sequence collections, PubMLST was used to identify whole genome sequences (by filtering for strains in the 'Genome Collection' of each species where the sequence length is at least that of the reference genome), then the raw reads were downloaded from NCBI using their SRA numbers. Accession numbers for reference genomes used for each microbial species are also listed in Supplementary file 5. All original code has been deposited at GitHub and is publicly available at https://github.com/kussell-lab (copies archived at swh:1:rev:4adea90557f1e592123e073b46a859d879143a2a, swh:1:rev:1d34860183a5dd55332e08edf9503c4ce6daebdf, swh:1:rev:49bf399a9d1ceae722c0c8c4aeb8376f2644d40f, and swh:1:rev:2228e8ee2df7339d28ef8b9107381d2e4767ac11).

The following previously published data sets were used
    1. Thorell K
    (2018) Dryad Digital Repository
    Data from: Rapid evolution of distinct Helicobacter pylori subpopulations in the Americas.
    https://doi.org/10.5061/dryad.8qp4n

References

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    3. Hanage WP
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    https://doi.org/10.1007/978-3-030-38281-0
    1. Barton NH
    (2010) Mutation and the evolution of recombination
    Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 365:1281–1294.
    https://doi.org/10.1098/rstb.2009.0320
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    1. Hanage WP
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    3. Spratt BG
    (2006a) Sequences, sequence clusters and bacterial species
    Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 361:1917–1927.
    https://doi.org/10.1098/rstb.2006.1917
    1. Hanage WP
    2. Spratt BG
    3. Turner KME
    4. Fraser C
    (2006b) Modelling bacterial speciation
    Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 361:2039–2044.
    https://doi.org/10.1098/rstb.2006.1926
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Decision letter

  1. Paul B Rainey
    Reviewing Editor; Max Planck Institute for Evolutionary Biology, Germany
  2. Gisela Storz
    Senior Editor; National Institute of Child Health and Human Development, United States
  3. Edward J Feil
    Reviewer; University of Bath, United Kingdom
  4. Daniel Falush
    Reviewer; Institute Pasteur Shanghai, Shanghai, China

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting the paper "Core genes can have higher recombination rates than accessory genes within global microbial populations" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Daniel Falush (Reviewer #3).

Comments to the Authors:

We are sorry to say that, after consultation with the reviewers, we have decided that this work will not be considered further, at least in this version, for publication by eLife. This said, as you will see from the reviewers' comments, all recognise the importance of the subject and the need for new approaches to understand evolutionary forces shaping pan and core genomes. Your work is very much a step in the right direction. If you find it possible to deal with the requests made by the referees, we would be willing to consider a fresh submission.

Specifically, on the problematic side, is seeming discord between the major claims of the paper and the data supporting these claims. This may in part stem from lack of clarity concerning methodological aspects, but as both referees point out, there are reasons for concern. And, importantly, for this work to appeal to a broad readership, it is necessary for the work to be generally accessible. Additionally, the work would benefit greatly from a more nuanced placement of the subject within context of an extensive and relevant literature. This would particularly aid motivation for the study.

Reviewer #2:

Homologous recombination is a key process driving the evolution of bacterial genomes, and the rate at which this occurs varies widely between different species. Whilst this variation is still not completely understood it is certainly due to a complex combination of mechanistic and ecological drivers. In this paper Steinberg and Kussel ask a slightly different question by the addressing the variation in intra-genomic rate of homologous recombination, and specifically between core and accessory genes. The use of core genes for phylogenetic and population-level inference (for example in multi-locus sequence typing) is predicated on the view that the genes encoding essential 'housekeeping' functions are under high levels of purifying selection, and are therefore assumed not to be impacted by high levels of recombination from diverged sources that might act to accelerate diversification. In contrast, accessory genes, which are more likely to encode niche-specific traits involving interactions with the external environment, may become more selectively favoured from homologous recombination leading to increased diversification. It is important to critically re-evaluate this model as evidence accumulates, both for understanding how selection shapes the genome and populations, but also for understanding the utility of different parts of the genome for phylogenetic and population genetics inference.

Even before the concept of the pan and core genome was established, varying rates of recombination between different classes of genes was a subject of active research. Jain et al. (Proc Natl Acad Sci U S A 1999 Mar 30;96(7):3801-6. doi: 10.1073/pnas.96.7.3801) speculated over 20 years ago, that 'operational' (metabolic) genes experience higher rates of transfer than 'informational' genes (those involved in DNA processing) because the latter were more likely to be involved in complex protein-protein interactions (the so-called 'complexity hypothesis'). Together with the underlying rational of MLST, and the prior use of 16s rRNA as a universal marker, there remains a common wisdom that the most conserved, ubiquitous and essential genes are also those least likely to undergo recombination. For a recent discussion of the implications of the complexity hypothesis see Novick, A., Doolittle, W.F.. Biol Philos 35, 2 (2020). https://doi.org/10.1007/s10539-019-9727-6

Whilst it is unfortunate that none of this broad perspective is elaborated in the paper, Steinberg and Kussel address this question on a large-scale (10 species, >100,000 genomes), and note that in many (but not all) species accessory genes tend to undergo lower rates of homologous recombination than core genes, although this trend is far from universal. To measure recombination they use an approach called mcorr which gauges recombination on the basis of the steepness of the decline in linkage between polymorphic sites as a function of genome distance. Their model allows them to distinguish between mutational and recombinational divergence, as well as estimating the percentage of each gene sequence that has been impacted by recombination. Their results are a mixed bag, with variation between species and between the different parameters within single species. However, overall the suggestion is that accessory genes may experience less homologous recombination than core genes. This trend is consistent with arguments (some of which are expressed by the authors) relating to the homogenising effect of recombination between close parental sequences, and how, in this case, recombination can reinforce purifying rather than directional selection.

While the question is an important one, and the use of large genome sequences to address it is laudable, I sense that there are two many factors at play over this broad scale to draw any firm conclusions about general trends. My main concerns with the analyses are two-fold – first it is carried out on single gene alignments which severely limits the range over which recombination can be detected and quantified (this would not be so critical if all homologous recombination was by localised transformation, but transduction and conjugation also play major roles). My second concern lies in the use of a single reference genome for each species. For species with very large pan-genomes, such as E. coli, this effectively means that the majority of accessory genes (those that happen to be absent in the reference) will not be included. The authors do acknowledge this, and present an analysis based on a composite reference, but they reject this approach due to the poor alignments that result. This is the perennial problem with working with accessory genes – but a reference free method for detecting SNPs in accessory genes has recently been (eg Colquhoun, R.M., Hall, M.B., Lima, L. et al. Pandora: nucleotide-resolution bacterial pan-genomics with reference graphs. Genome Biol 22, 267 (2021)). There are additional potential complications relating to how the different datasets have been sampled that have not been acknowledged. In sum, the analysis should be of interest to the field as it does demonstrate the potential for addressing this fundamental question using large WGS datasets, but my sense is that the authors would have been better served to analyse one or two species in detail, taking into account all possible confounders, rather than spread themselves too thinly across such diverse datasets.

General: the paper would be vastly improved by a broader review of the literature and a more clear motivation for the study. The introduction is frankly a bit waffly.

Abstract:

The observation that some species consist of overlapping gene pools (or even one single gene pool) to a degree simply reflects inconsistencies in our demarcation of species (for example S. flexneri is really just a clone of E. coli, and N. gonorrhoeae is really just a clone of N. meningitidis).

Line 25 the role of population structure in genome evolution – I'm not sure about the causal direction here – it seems to make more sense the other way around to me.

Line 29 – it has been established for a long time that core genes are under strong purifying selection. Claiming this as news somehow detracts from the aspects of the paper that are really novel.

Line 43 – the ability to quantify rates in different parts of the genome hasn't been limited, but has been going on for a long time. For an example, see Nat Commun 2014 May 23;5:3956. doi: 10.1038/ncomms4956.

As I understand the model, it is based on the decay of linkage over distance (Figure 1C,D) but I have struggled a bit with how you move from this to the data you present in the other figures – it would be reassuring to us non-modellers if you could show some more examples of this decay to understand more clearly what the results you present are really based on.

Are recombination events, like synonymous SNPs, also assumed to be neutral?

Line 120 – explain w/n.

Line 136 – meaning 'no two sequences within a cluster'?

Figure 2B – why not separate points for within and between clusters?

Figure S2 – why exclude the marginal plots?

Line 194 – There is certainly an effect of codon bias on substitution rate – the fact the experimental evolution data hasn't picked this up is more a comment on these experiments.

Line 264 – the P values presented in S table 1 are two-signed – which ones indicate higher rates of recombination in the core genes?

Figure 4 – 60% of the core gene sequences in S. enterica have experienced recombination? This seems a lot -how does this figure (and other species specific data) compare with what is in the literature? Could the authors comment on the fact that in E. coli (and S. flexneri) the strict core genes show less recombination than the cloud core.

Line 322 'complicates' not 'complexifies'.

Line 332 – 340 this model isn't really novel.

Reviewer #3:

This paper presents interesting results comparing core and accessory genome evolution.

The headline result, if correct, is both important and reasonably easy to understand; namely that in Pneumococci and other bacterial species, on average homologous recombination rates (or more precisely the effect of recombination in reassorting sequence diversity) are higher in core genome elements, while diversity is higher in accessary genome elements.

This is an interesting result and is buttressed by showing some dependence on gene frequencies. It is not entirely surprising, since core genes have more opportunities for recombination due to being present in every strain, while accessory genome elements have more opportunity for being imported from other species. Nevertheless, a quantitative result is important enough to be newsworthy, even though it does not do an enormous amount to elucidate the mechanisms responsible for the difference. This would require more explicitly phylogenetic methods or a more in-depth analysis of the difference between species, which is currently superficial.

However, I am not sure I am persuaded by the second part of the result, since looking at supplementary figure 4, it is not obvious to me that there is a clear trend for recombination parameters, core and accessory genomes seem to give very inconsistent results in different species, for which no real explanations are given.

There is one important technical check that should be carried out, which is that core genes should be artificially thinned to match the frequency of accessory gene elements and then the analysis repeated, to see whether this has any effect on in the inference. Indeed, because the alignment method is shown to have a considerable effect on the inference, especially of recombination parameters (supplementary figure 2) this thinning would be ideally done before alignment, although I recognize that this would be time-consuming to implement.

Figure 3 is presented as the results from 12 species but it’s really hard to judge what this figure actually tells us. Agglomorating different species into single graphs seems meaningless and makes it impossible to see what is actually going on with individual species.

Aside from questions about to what extent the data really supports the headline result, my main problem with the paper is that the analysis underlying the result and in particular the meaning of the figures is difficult to understand. As it stands, I do not think it is suitable for a broad readership. The concept about "sample" and "pool" are not well explained and seem in fact to be conceptually misleading.

Sample is a misnomer. The samples that are actually used in the data are sequence clusters. The species is clusters into groups of strains with high sequence similarity as is done in many other analyses of bacterial genomes (for example using the software POPPUNK). Each of these clusters is a putative clonal complex, i.e. a set of strains that share a recent common ancestor, relative to the rest of the sample. This is seen for example in figure 2A, where the clusters are shown in alternate colours.

Sample is a misleading name because sample refers to the process of collection of bacteria, which is not directly related to their evolutionary relationships. A sample of strains is collected over a time period or at a particular hospital or from a particular set of patients or with a particular growth media. It’s not determined post-hoc based on sequence similarity.

Pool is presented about being an idea about gene pools. But the actual gene pool for any bacteria is unknown and indeed may not have a discreate boundary. As I understand it, the entities that are in fact used as pools in this analysis are actually just designated species, which are essentially designated by a combination of traditional phenotypic criteria and (in the genomics era) sequence similarity. It is not based on any measurement of gene flow. Therefore, implying that the measurement is taken over pools is misleading.

The manuscript mentions that pools may be sets of strains isolated from particular geographic locations, but this is in fact a sample. A pool, as in a set of organisms that constitute a gene pool, is not just a collection of genomes, it is a set of organisms in nature.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Core genes can have higher recombination rates than accessory genes within global microbial populations" for further consideration by eLife. Your revised article has been evaluated by Gisela Storz (Senior Editor) and a Reviewing Editor.

The manuscript has been significantly improved with all reviewers commenting on the considerable effort that you and co-authors have made. However, there remains one issue that needs attention as outlined below in the request from Reviewer 1.

Reviewer #1 (Recommendations for the authors):

The correlations in figure 3 seem very strong but I am doubtful of their interpretation. It seems to me that a more direct approach than to compare different samples (which I continue to think should be called clusters) is to compare different genes. I.e. do genes with higher divergence show lower recombination? This could also help to answer the extent to which lower recombination in the accessory genome was largely a function of higher divergence levels, for example. In any case I think the method should be applied to data simulated from a recombining population with homogeneous bacterial recombination. If the method is really picking up homology dependent recombination rates, then it should give no correlation when applied to such data. Generally, simulations are very valuable for this kind of paper because they force the authors to clarify what it is they are trying to measure and what the null expectation is in the absence of any such effect.

The abstract is light on results and has too much background in it.

Reviewer #2 (Recommendations for the authors):

This is a greatly improved version of the paper - my main concerns regarding placing the work within a broader context of the literature have been addressed very well. My specific questions relating to the approach have bee addressed / clarified in the rebuttal and the text modified appropriately.

https://doi.org/10.7554/eLife.78533.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Comments to the Authors:

We are sorry to say that, after consultation with the reviewers, we have decided that this work will not be considered further, at least in this version, for publication by eLife. This said, as you will see from the reviewers' comments, all recognise the importance of the subject and the need for new approaches to understand evolutionary forces shaping pan and core genomes. Your work is very much a step in the right direction. If you find it possible to deal with the requests made by the referees, we would be willing to consider a fresh submission.

Specifically, on the problematic side, is seeming discord between the major claims of the paper and the data supporting these claims. This may in part stem from lack of clarity concerning methodological aspects, but as both referees point out, there are reasons for concern. And, importantly, for this work to appeal to a broad readership, it is necessary for the work to be generally accessible. Additionally, the work would benefit greatly from a more nuanced placement of the subject within context of an extensive and relevant literature. This would particularly aid motivation for the study.

Reviewer #2:

Homologous recombination is a key process driving the evolution of bacterial genomes, and the rate at which this occurs varies widely between different species. Whilst this variation is still not completely understood it is certainly due to a complex combination of mechanistic and ecological drivers. In this paper Steinberg and Kussel ask a slightly different question by the addressing the variation in intra-genomic rate of homologous recombination, and specifically between core and accessory genes. The use of core genes for phylogenetic and population-level inference (for example in multi-locus sequence typing) is predicated on the view that the genes encoding essential 'housekeeping' functions are under high levels of purifying selection, and are therefore assumed not to be impacted by high levels of recombination from diverged sources that might act to accelerate diversification. In contrast, accessory genes, which are more likely to encode niche-specific traits involving interactions with the external environment, may become more selectively favoured from homologous recombination leading to increased diversification. It is important to critically re-evaluate this model as evidence accumulates, both for understanding how selection shapes the genome and populations, but also for understanding the utility of different parts of the genome for phylogenetic and population genetics inference.

Even before the concept of the pan and core genome was established, varying rates of recombination between different classes of genes was a subject of active research. Jain et al. (Proc Natl Acad Sci U S A 1999 Mar 30;96(7):3801-6. doi: 10.1073/pnas.96.7.3801) speculated over 20 years ago, that 'operational' (metabolic) genes experience higher rates of transfer than 'informational' genes (those involved in DNA processing) because the latter were more likely to be involved in complex protein-protein interactions (the so-called 'complexity hypothesis'). Together with the underlying rational of MLST, and the prior use of 16s rRNA as a universal marker, there remains a common wisdom that the most conserved, ubiquitous and essential genes are also those least likely to undergo recombination. For a recent discussion of the implications of the complexity hypothesis see Novick, A., Doolittle, W.F.. Biol Philos 35, 2 (2020). https://doi.org/10.1007/s10539-019-9727-6

Whilst it is unfortunate that none of this broad perspective is elaborated in the paper, Steinberg and Kussel address this question on a large-scale (10 species, >100,000 genomes), and note that in many (but not all) species accessory genes tend to undergo lower rates of homologous recombination than core genes, although this trend is far from universal. To measure recombination they use an approach called mcorr which gauges recombination on the basis of the steepness of the decline in linkage between polymorphic sites as a function of genome distance. Their model allows them to distinguish between mutational and recombinational divergence, as well as estimating the percentage of each gene sequence that has been impacted by recombination. Their results are a mixed bag, with variation between species and between the different parameters within single species. However, overall the suggestion is that accessory genes may experience less homologous recombination than core genes. This trend is consistent with arguments (some of which are expressed by the authors) relating to the homogenising effect of recombination between close parental sequences, and how, in this case, recombination can reinforce purifying rather than directional selection.

While the question is an important one, and the use of large genome sequences to address it is laudable, I sense that there are two many factors at play over this broad scale to draw any firm conclusions about general trends. My main concerns with the analyses are two-fold – first it is carried out on single gene alignments which severely limits the range over which recombination can be detected and quantified (this would not be so critical if all homologous recombination was by localised transformation, but transduction and conjugation also play major roles). My second concern lies in the use of a single reference genome for each species. For species with very large pan-genomes, such as E. coli, this effectively means that the majority of accessory genes (those that happen to be absent in the reference) will not be included. The authors do acknowledge this, and present an analysis based on a composite reference, but they reject this approach due to the poor alignments that result. This is the perennial problem with working with accessory genes – but a reference free method for detecting SNPs in accessory genes has recently been (eg Colquhoun, R.M., Hall, M.B., Lima, L. et al. Pandora: nucleotide-resolution bacterial pan-genomics with reference graphs. Genome Biol 22, 267 (2021)). There are additional potential complications relating to how the different datasets have been sampled that have not been acknowledged. In sum, the analysis should be of interest to the field as it does demonstrate the potential for addressing this fundamental question using large WGS datasets, but my sense is that the authors would have been better served to analyse one or two species in detail, taking into account all possible confounders, rather than spread themselves too thinly across such diverse datasets.

We thank the Reviewer for their comprehensive feedback on the manuscript, and we greatly appreciate their efforts in helping us improve the manuscript.

Regarding the first main concern, which is that our use of single gene alignments truncates the range over which recombination can be quantified, we have now clarified this point in the text (Lines 143-145). While our method does rely on single gene alignments, our coalescent model with recombination does not limit the size of the recombined fragment to the length of single genes. Each recombined fragment affects pairs of loci at distances l that are less than the fragment size, thus recombined fragments that span multiple genes contribute to the measured correlation profiles across all the affected genes. The coalescent model infers the mean fragment size by fitting the correlation profiles, and we find that mean fragment sizes may range from

~102 to ~105 bp. We have included some distributions of the fragment sizes predicted for the core and accessory genomes of Streptococcus pneumoniae to help clarify this in Figure S1.

To address the second main concern, regarding the use of a single reference genome, we have completed an analysis using both an alignment to a single reference genome and to a reference pangenome created from 81 complete genome assemblies (which encompasses the majority of the S. pneumoniae pangenome according to previous analysis of its pangenome). We found very similar results with both methods. In the original manuscript, we had included this comparison in Figure S2. To better facilitate this comparison, we have reformatted these data as empirical cumulative distribution functions in Figure S4. The trends for the inferred recombination parameters are very similar, which gives us confidence that the recombination rate trends for

S. pneumoniae hold for both alignment methods. We have re-examined the alignments, and while the alignment quality is slightly lower for pangenome alignments (as we reported originally), our results in Figure S4 show that this does not impact the results regarding recombination parameters. Furthermore, while the Reviewer’s suggestion for using a new method for reference free alignment of raw reads does indeed look promising, it also appears to be very computationally expensive; in the manuscript the Reviewer refers to, the Authors report that aligning raw reads from just 20 isolates took ~9 hours. This method would therefore be prohibitive for our analysis of ~26,000 isolates for S. pneumoniae alone. We revised the Results section to emphasize these points (Lines 260-275).

To focus our analysis on a detailed study of a single species, as recommended by the reviewer, we have rewritten the manuscript to highlight primarily our analysis of Streptococcus pneumoniae. We have removed Figure 3 from the original manuscript which contained the multi-species analysis of recombination rates and instead provide a supplemental figure (Figure S5) and a supplemental table (Table S1) briefly summarizing recombination rate trends in different species. We also moved Figure 4 (which contained another multi-species analysis) to the supplement as Figure S7. Both Figure S5 and Table S1 now simply highlight that other species either show higher or equal recombination rates in core versus accessory genes, except for Helicobacter pylori, for which the effect size statistic is small. We revised the text to reflect these points (Lines 276-295).

To address the concern that the “results are a mixed bag, with variation between species and between the different parameters within single species” we sought to determine whether general relationships hold across species with regard to divergence-based recombination barriers, which have been described in many bacteria. We hypothesized that due to the highly conserved recA-mediated homologous recombination pathway, sequence divergence should play a key role in determining recombination rates within species, and a signal of this recombination barrier should be present in the inferred recombination parameters. In the new Figure 3, we present the analysis which shows that, in all species that we analyzed, the difference in recombination coverage between accessory and core genes declines with the difference in their mutational divergences. This general relation demonstrates that, despite variation in parameters within and between species, we detect the existence of genome-wide, within-species recombination barriers in many different bacteria. We describe these results in a new section (Lines 297-327).

Lastly, to address the request that the manuscript acknowledge potential complications with how datasets were sampled, we have revised the text to explicitly discuss issues related to sampling and sample construction on Lines 172-197 and to point out how our method addresses these. We now call the reader’s attention to this discussion earlier on at Lines 126-127.

General: the paper would be vastly improved by a broader review of the literature and a more clear motivation for the study (see public response for some pointers). The introduction is frankly a bit waffly.

We thank the Reviewer for this very helpful feedback on the structure of the introduction. We have almost entirely re-written the Introduction to the paper (Lines 35-113) to better encompass literature findings and clearly motivate the study. We also appreciate the useful pointers the Reviewer gave in their public response, which helped guide this revision. We hope we have covered much of the key related literature, and if the reviewer is aware of additional papers we may have missed, we would be glad for those references as well.

Abstract:

The observation that some species consist of overlapping gene pools (or even one single gene pool) to a degree simply reflects inconsistencies in our demarcation of species (for example S. flexneri is really just a clone of E. coli, and N. gonorrhoeae is really just a clone of N. meningitidis).

We agree with the Reviewer’s comment, and because this point was in fact tangential to our main findings, we have decided to remove the analysis of overlapping gene pool structure from the manuscript. This decision is also responsive to comments by Reviewer 3, and enables us to maintain focus throughout the text on the major results regarding the differences in recombination rates between the core and accessory genome.

Line 25 the role of population structure in genome evolution – I'm not sure about the causal direction here – it seems to make more sense the other way around to me.

We removed Line 25 as it was no longer relevant without the discussion of pool structure.

Line 29 – it has been established for a long time that core genes are under strong purifying selection. Claiming this as news somehow detracts from the aspects of the paper that are really novel.

We removed Line 29 from the Abstract as we agree that it detracts from the main points of the paper.

Line 43 – the ability to quantify rates in different parts of the genome hasn't been limited, but has been going on for a long time. For an example, see Nat Commun 2014 May 23;5:3956. doi: 10.1038/ncomms4956.

We have edited the text of the Introduction (Lines 53-57) to reference this and other work quantifying variation in recombination across different genes.

As I understand the model, it is based on the decay of linkage over distance (Figure 1C,D) but I have struggled a bit with how you move from this to the data you present in the other figures – it would be reassuring to us non-modellers if you could show some more examples of this decay to understand more clearly what the results you present are really based on.

We have added additional examples of correlation profiles in Figure 2B (line 207), and on lines 190-197 we discuss how clusters are used to measure the decaying correlation profiles, yielding the parameters distributions shown in Figure 2C-E.

Are recombination events, like synonymous SNPs, also assumed to be neutral?

There is no implicit assumption of neutrality for recombination events in the model. We have added text to clarify this (Lines 134-135), and thank the Reviewer for this question as we now realize this key point may not have been clear.

Line 120 – explain w/n.

As we have removed the analysis of pool structure from the manuscript, we no longer use the terms ‘w/n’ and ‘btwn’ in the manuscript.

Line 136 – meaning 'no two sequences within a cluster'?

That is correct, and we have amended this line in the text correspondingly (presently on Lines 192-194).

Figure 2B – why not separate points for within and between clusters?

Figure S2 – why exclude the marginal plots?

We have changed Figure 2 to just show empirical cumulative distribution functions (ECDFs) of the recombinational and mutational divergence (Figure 2C-D in the revised manuscript). We chose to not separate the points for within and between clusters in the revised manuscript because we have removed the analysis of pool structure, for which the distinction of within and between clusters was important. Figure S2 has been replaced with Figure S4, which now just shows ECDFs of recombination parameters for the different alignment methods side-by-side, which we hope allows for a clearer comparison between the results using an alignment to a single reference genome versus a pangenome reference genome.

Line 194 – There is certainly an effect of codon bias on substitution rate – the fact the experimental evolution data hasn't picked this up is more a comment on these experiments.

We agree with the Reviewer’s comment, and have revised the text to estimate the magnitude of the effect in S. pneumoniae and relate it to our analysis and to the prior results in experimental evolution. We moved this section to the Discussion (Lines 403-411).

Line 264 – the P values presented in S table 1 are two-signed – which ones indicate higher rates of recombination in the core genes?

We have amended Table S1 so that it also includes the median recombination rates and effect size statistics (Cohen’s d) quantifying the difference in rates between the core/accessory genes. Values of d < 0 indicate higher rates of recombination in core genes.

Figure 4 – 60% of the core gene sequences in S. enterica have experienced recombination? This seems a lot -how does this figure (and other species specific data) compare with what is in the literature? Could the authors comment on the fact that in E. coli (and S. flexneri) the strict core genes show less recombination than the cloud core.

We checked the literature with regard to S. enterica, and note that another recent study using a smaller dataset shows comparable levels of recombination in the core genes (see Park and Andam, mSystems 5:e00515-19, 2020; doi:10.1128/mSystems.00515-19). While other studies have examined recombination across housekeeping and core genes, there is scant existing data for accessory genes. In particular, no studies to our knowledge have presented direct comparisons between core and accessory genes. We have revised the text to address these points (Lines 385-388). We have also added discussion commenting on potential reasons why the cloud genes for E. coli and S. flexneri show higher recombination rates than core genes (Lines 358361). We would also like to note that Figure 4 has been moved to the supplement as Figure S7.

Line 322 'complicates' not 'complexifies'.

Line 322 has been removed as it refers to the analysis of pool structure, which is not included in the revised manuscript.

Line 332 – 340 this model isn't really novel.

We have amended the text preceding this statement to reference research which has shown that genes with lower sequence divergence have a reduced barrier to homologous recombination and modelling results which have suggested that core genes experience more homologous recombination leading to homogenization (Lines 420-429).

Reviewer #3:

This paper presents interesting results comparing core and accessory genome evolution.

The headline result, if correct, is both important and reasonably easy to understand; namely that in Pneumococci and other bacterial species, on average homologous recombination rates (or more precisely the effect of recombination in reassorting sequence diversity) are higher in core genome elements, while diversity is higher in accessary genome elements.

This is an interesting result and is buttressed by showing some dependence on gene frequencies. It is not entirely surprising, since core genes have more opportunities for recombination due to being present in every strain, while accessory genome elements have more opportunity for being imported from other species. Nevertheless, a quantitative result is important enough to be newsworthy, even though it does not do an enormous amount to elucidate the mechanisms responsible for the difference. This would require more explicitly phylogenetic methods or a more in-depth analysis of the difference between species, which is currently superficial.

We thank the Reviewer for their comprehensive feedback on the manuscript, and for highlighting the broad interest of this work to the community. We greatly appreciate their efforts in helping us improve the manuscript.

To address the Reviewer’s comment regarding connecting the quantitative result on the difference in recombination rates between core and accessory genes to underlying mechanisms, we have performed additional analysis which is presented in a new Figure 3 and in Results (Lines 298-328). Specifically, we tested whether divergence-based recombination barriers are related to differences between core and accessory genes’ rates of recombination, across clusters within each species. We find pronounced negative correlations in the paired differences of sequence divergence and recombination coverage for accessory and core genes in S. pneumoniae (Figure 3A), and in the other bacteria that we have analyzed (Figure 3B,C). We include this general finding as a first major step toward elucidating the mechanisms underlying the quantitative differences we have presented.

However, I am not sure I am persuaded by the second part of the result, since looking at supplementary figure 4, it is not obvious to me that there is a clear trend for recombination parameters, core and accessory genomes seem to give very inconsistent results in different species, for which no real explanations are given.

To address the Reviewer’s concern regarding the generalizability of our results to multiple species, we have performed two statistical analyses on the relative recombination rates, shown in Figure S4 and Table S1.

First, we computed Cohen’s d statistic showing the effect size for the difference in relative recombination rates (Figure S4, Table S1). This shows a non-zero effect size in 9 out of 12 species, and in all but one of these (H. pylori), we find that core genes have higher recombination rates than accessory genes (d < 0). Second, we list the median relative recombination rates for core and accessory genes in each species (Table S1). We find that in 10 out of 12 species the medians are significantly different (within 95% confidence intervals), and among these in all but H. pylori and N. gonorrhoeae the core genes have higher recombination rates than accessory genes. Thus, the general trend according to these two independent metrics is quite consistent. Additionally, in the exceptional cases of H. pylori and N. gonorrhoeae, the d statistic is small in magnitude, indicating the overall effect size is small.

In response to both Reviewers’ feedback we have decided to focus most of the results on the analysis of Streptococcus pneumoniae, which serves as the main case study of the paper. We then present a very focused set of results on the additional bacterial species in order to test the generality of the results that we found in S. pneumoniae. In addition to Figure S4 and Table S1, the new Figure 3 (discussed in the previous comment) addresses the reviewer’s concern regarding explanation of the observed trend in recombination parameters between core and accessory genes.

There is one important technical check that should be carried out, which is that core genes should be artificially thinned to match the frequency of accessory gene elements and then the analysis repeated, to see whether this has any effect on in the inference. Indeed, because the alignment method is shown to have a considerable effect on the inference, especially of recombination parameters (supplementary figure 2) this thinning would be ideally done before alignment, although I recognize that this would be time-consuming to implement.

We have carried out the technical check suggested by the Reviewer – artificially thinning the core genome – and found similar results for the “thinned” core genome compared to the actual core genome, suggesting that the difference in sequence pairs between core and accessory genome does not affect parameter inference enough to change our interpretations. We provide these data as Figure S3, and have amended the text (Lines 253-259). We note that we performed this control post-alignment because, as we have discussed in our response to Reviewer #2, the distributions of recombination parameters inferred from the different alignment methods are similar, and we have provided a new supplemental figure (Figure S4, described in the response to Reviewer #2’s question) which we hope better highlights the similarity in parameter inference from different alignment methods.

Figure 3 is presented as the results from 12 species but it’s really hard to judge what this figure actually tells us. Agglomorating different species into single graphs seems meaningless and makes it impossible to see what is actually going on with individual species.

We agree with the Reviewer’s comment on the difficulty of comparing and assessing multiple species on a single graph. In response to this comment, as well as the comments of Reviewer #2, we focus most of our analysis throughout the main text on a detailed study of Streptococcus pneumoniae. We have removed Figure 3 from the original manuscript which contained the multi-species analysis of recombination rates and instead provide a supplemental figure (Figure S5) and a supplemental table (Table S1) briefly summarizing recombination rate trends in different species. We also moved Figure 4 (which contained another multi-species analysis) to the supplement as Figure S7. Both Figure S5 and Table S1 now simply highlight that other species either show higher or equal recombination rates in core versus accessory genes, except for Helicobacter pylori, for which the effect size statistic is small. We revised the text to reflect these points (Lines 276-295).

We hope that with these changes the interpretation of the main results is much clearer.

Aside from questions about to what extent the data really supports the headline result, my main problem with the paper is that the analysis underlying the result and in particular the meaning of the figures is difficult to understand. As it stands, I do not think it is suitable for a broad readership. The concept about "sample" and "pool" are not well explained and seem in fact to be conceptually misleading.

We thank the Reviewer for bringing these points regarding clarity and suitability for a broad readership to our attention. To address these, we have simplified Figure 1 so that it no longer includes the analysis of pool structure, which was tangential to our central results. We now focus the figure entirely on inferring recombination parameters from correlation profiles and explaining the model. We have added additional example correlation profiles of synonymous substitutions into Figure 2 to better illustrate how we go from sequencing data to recombination parameters. We also removed the analysis of pool structure from the text of the manuscript and from Figures 1-2 (as well as Figures S1 and S3).

We have re-written the Introduction almost entirely in order to better place the paper into context for a broad readership, and to introduce the major concepts and methods more clearly for this audience. We have also revised the Results section to clearly explain the issues involved in the concept of “sample” and “pool”, (Lines 122-127) and particularly in terms of sample construction (Lines 172-197), discussed further in the next comment.

We hope that these changes have substantially improved the readability and suitability of the manuscript for a broad readership.

Sample is a misnomer. The samples that are actually used in the data are sequence clusters. The species is clusters into groups of strains with high sequence similarity as is done in many other analyses of bacterial genomes (for example using the software POPPUNK). Each of these clusters is a putative clonal complex, i.e. a set of strains that share a recent common ancestor, relative to the rest of the sample. This is seen for example in figure 2A, where the clusters are shown in alternate colours.

Sample is a misleading name because sample refers to the process of collection of bacteria, which is not directly related to their evolutionary relationships. A sample of strains is collected over a time period or at a particular hospital or from a particular set of patients or with a particular growth media. It’s not determined post-hoc based on sequence similarity.

We thank the Reviewer for bringing this point to our attention, which we hope we have now been able to fully clarify. We understand that the term “sample” as it was previously described in the manuscript may have been misleading. By “sample” we do not mean to refer to the process of collection itself, but instead we are using the term in its statistical sense, referring to the process of statistically sampling from a larger distribution; in this case, sampling from the bulk gene pool. In our analysis, the sequence clusters we use “sample” from these bulk gene pools. We have made this point explicit in the text (Lines 173-175), and we have added additional text about the role of clustering in our sample construction (Lines 175-197).

Pool is presented about being an idea about gene pools. But the actual gene pool for any bacteria is unknown and indeed may not have a discreate boundary. As I understand it, the entities that are in fact used as pools in this analysis are actually just designated species, which are essentially designated by a combination of traditional phenotypic criteria and (in the genomics era) sequence similarity. It is not based on any measurement of gene flow. Therefore, implying that the measurement is taken over pools is misleading.

The manuscript mentions that pools may be sets of strains isolated from particular geographic locations, but this is in fact a sample. A pool, as in a set of organisms that constitute a gene pool, is not just a collection of genomes, it is a set of organisms in nature.

The Reviewer makes an excellent point, and we have sought to clarify this important aspect of the paper. We agree with the Reviewer that the gene pool is unknown and may not have discrete bounds. We recognize that the original manuscript may not have adequately described what our method assigns to be the pool. In fact, our method does not use or designate any entity as the pool, but instead we use the analyzed sequences (i.e., the “sample”) to infer recombination parameters of the greater, unobserved gene pool with which the sample recombines. The pool parameters are distinct from those of the sample (the sample being the set of sequenced genomes used to compute each correlation profile, e.g., a single sequence cluster). We revised the text to clarify these points (Lines 122-127; 172-177).

We also noticed that Line 108 in the original manuscript may have been confusing, as it read: “Samples may have access to different sequence pools, e.g., due to geographic constraints or differences in host niches.” We recognize that the way this was written may have been unintentionally misleading; we meant that samples could be constrained biogeographically, which may lead to access to different gene pools, not that the gene pools are constrained. We have removed this line from the manuscript, and amended the text to further clarify this point (Lines 177-197).

[Editors’ note: what follows is the authors’ response to the second round of review.]

The manuscript has been significantly improved with all reviewers commenting on the considerable effort that you and co-authors have made. However, there remains one issue that needs attention as outlined below in the request from Reviewer 1.

Reviewer #1 (Recommendations for the authors):

The correlations in figure 3 seem very strong but I am doubtful of their interpretation. It seems to me that a more direct approach than to compare different samples (which I continue to think should be called clusters) is to compare different genes. I.e. do genes with higher divergence show lower recombination? This could also help to answer the extent to which lower recombination in the accessory genome was largely a function of higher divergence levels, for example. In any case I think the method should be applied to data simulated from a recombining population with homogeneous bacterial recombination. If the method is really picking up homology dependent recombination rates, then it should give no correlation when applied to such data. Generally, simulations are very valuable for this kind of paper because they force the authors to clarify what it is they are trying to measure and what the null expectation is in the absence of any such effect.

We agree with the Reviewer that multiple effects could determine the homology-based recombination barriers examined in Figure 3 of the previous version of the manuscript, and that simulations may play an important role in revealing these dependencies.

As the modeling work needed to fully address this point is extensive, given the complex population structure underlying these datasets, we have removed the original Figure 3 from the manuscript and corresponding text (Lines 27-32, 90-101, 108-110, 299-327, and 423-426 in the previous version).

The revised manuscript is thus focused on (i) the major result showing that core genes can recombine more frequently than accessory genes, and the related findings (ii) that mutational divergence is lower in core genes, and (iii) that gene frequency correlates with recombination rate.

We moved the previous Figure S5 to be the new Figure 3, showing the key statistical analysis supporting the differences in the recombination rates of core and accessory genes. We have also added a part B to the new Figure 3, which displays effect sizes for the pool’s mutational divergence, and we include additional statistics to support these results in Supplementary File 2. In the revised manuscript, we have added a description of the pool’s mutational divergence results on lines 242-247 and include a brief discussion of their potential relationship to the observed recombination rate differences on lines 328-337 of the revised manuscript.

The results presented in the new Figure 3 are consistent with the Reviewer’s comment above regarding divergence levels and recombination rates. To confirm this, we conducted the additional control that the reviewer suggested, i.e., to test if genes with higher divergence show lower recombination. Using the S. pneumoniae dataset shown in Figure 2, we binned genes by their synonymous diversity into low or high diversity bins (i.e., lower 50%, upper 50%), and inferred recombination rates separately in each bin for all clusters and cluster pairs. The results are shown in the bar plot in Author response image 1: genes with higher diversity (and, correspondingly, divergence) show lower recombination rates (the y-axis shows the median pool recombination rates, and the error bars are 95% bootstrap confidence intervals).

Author response image 1

We leave further detailed analysis on the role of homology barriers to future work.

The abstract is light on results and has too much background in it.

We agree with the Reviewer’s comments, and have added additional text to the Abstract describing key results on Lines 24-31 of the revised manuscript. To remove some of the background, we took out Lines 17-19 of the original Abstract.

We would like to thank the Reviewer for taking the time to carefully read the manuscript, and for their insightful feedback which has further improved the manuscript.

https://doi.org/10.7554/eLife.78533.sa2

Article and author information

Author details

  1. Asher Preska Steinberg

    Department of Biology, New York University, New York, United States
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8694-7224
  2. Mingzhi Lin

    Department of Biology, New York University, New York, United States
    Contribution
    Conceptualization, Software, Investigation
    Competing interests
    No competing interests declared
  3. Edo Kussell

    1. Department of Biology, New York University, New York, United States
    2. Department of Physics, New York University, New York, United States
    Contribution
    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    edo.kussell@nyu.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0590-4036

Funding

National Institutes of Health (R01-GM097356)

  • Edo Kussell

Simons Foundation (Simons Foundation Awardee of the Life Sciences Research Foundation)

  • Asher Preska Steinberg

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported in part by NIH grant R01-GM-097356. Asher Preska Steinberg is a Simons Foundation Awardee of the Life Sciences Research Foundation. We gratefully acknowledge Nobuto Takeuchi for useful discussions and feedback on the manuscript; the New York University (NYU) high-performance computing cluster for resources, and its staff for technical support.

Senior Editor

  1. Gisela Storz, National Institute of Child Health and Human Development, United States

Reviewing Editor

  1. Paul B Rainey, Max Planck Institute for Evolutionary Biology, Germany

Reviewers

  1. Edward J Feil, University of Bath, United Kingdom
  2. Daniel Falush, Institute Pasteur Shanghai, Shanghai, China

Publication history

  1. Preprint posted: September 14, 2021 (view preprint)
  2. Received: March 10, 2022
  3. Accepted: June 30, 2022
  4. Accepted Manuscript published: July 8, 2022 (version 1)
  5. Version of Record published: September 5, 2022 (version 2)

Copyright

© 2022, Preska Steinberg et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Asher Preska Steinberg
  2. Mingzhi Lin
  3. Edo Kussell
(2022)
Core genes can have higher recombination rates than accessory genes within global microbial populations
eLife 11:e78533.
https://doi.org/10.7554/eLife.78533

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