Ultrafast Evolution and Loss of CRISPRs Following a Host Shift in a Novel Wildlife Pathogen,

Summary

Measureable rates of genome evolution are well documented in human pathogens but are less well understood in bacterial pathogens in the wild, particularly during and after host switches. Mycoplasma gallisepticum (MG) is a pathogenic bacterium that has evolved predominantly in poultry and recently jumped to wild house finches (Carpodacus mexicanus), a common North American songbird. For the first time we characterize the genome and measure rates of genome evolution in House Finch isolates of MG, as well as in poultry outgroups. Using whole-genome sequences of 12 House Finch isolates across a 13-year serial sample and an additional four newly sequenced poultry strains, we estimate a nucleotide diversity in House Finch isolates of only ∼2% of ancestral poultry strains and a nucleotide substitution rate of 0.8−1.2×10⁻⁵ per site per year both in poultry and in House Finches, an exceptionally fast rate rivaling some of the highest estimates reported thus far for bacteria. We also found high diversity and complete turnover of CRISPR arrays in poultry MG strains prior to the switch to the House Finch host, but after the invasion of House Finches there is progressive loss of CRISPR repeat diversity, and recruitment of novel CRISPR repeats ceases. Recent (2007) House Finch MG strains retain only ∼50% of the CRISPR repertoire founding (1994–95) strains and have lost the CRISPR–associated genes required for CRISPR function. Our results suggest that genome evolution in bacterial pathogens of wild birds can be extremely rapid and in this case is accompanied by apparent functional loss of CRISPRs.

Introduction

Populations of animals are under constant threat from bacterial pathogens, which can be particularly destructive following a switch to a new host or the evolution of novel virulence mechanisms. Understanding the rate and process of evolutionary change in pathogens is thus important to assessing the risks of pandemics and developing means to predict and avoid such catastrophic events. In 1994, a strain of Mycoplasma gallisepticum (MG) was identified as the causative agent of an emerging epizootic in House Finches, a wild songbird inhabiting Eastern North America [1]. This bacterial pathogen frequently causes disease in commercial chicken and turkey flocks, but it had never been reported in House Finches or any songbird, leading to the suggestion that the epidemic began when MG expanded its host range from poultry to this phylogenetically distant songbird. MG prevalence reached 60% in some areas, and killed an estimated 225 million finches in the first three years after detection [2]. The early detection of the epizootic allowed research and citizen-science teams to track its rapid spread throughout eastern North America in exceptional detail, making it one of the best documented wildlife pathogen outbreaks [3]–[7].

Although previous genome-wide studies have clarified rates of measurable evolution in viral pathogens [8], [9] and in bacterial populations evolving under laboratory conditions or as human pathogens [10]–[18], less is known about rates of genetic change in bacterial pathogens of non-mammalian vertebrates, particularly on short evolutionary time scales. Genome-wide and gene-specific estimates of point substitution in bacterial lineages measured over centuries [19] to millions of years [20] suggest maximum substitution rates on the order of 10⁻⁷ to 10⁻⁹ per site per year. Although recent work suggests the rate may be even faster for several bacterial species [12], [14], [19], the number of studies documenting whole-genome changes in bacteria during host switches is still small, particularly for wildlife pathogens [21], [22]. As part of ongoing surveillance, field isolates of MG obtained from infected finches were sampled at multiple time points from the start of the epidemic in 1994 to 2007, providing a genetic time series beginning immediately after the host switch, as well as an opportunity to directly measure the tempo and mode of evolution in a natural bacterial population whose genome is as yet uncharacterized.

To characterize patterns of genomic change during its host switch between distantly related avian species, we sequenced whole genomes of 12 House Finch MG isolates from this 13-year time series, with four samples each from the beginning (1994–1996), middle (2001) and recent (2007) periods (Table S1). In addition, to identify putative source strains as well to determine if differences between the House Finch MG strains and the ∼1 Mb published reference R_low strain from chicken [23] were ancestral or derived, we sequenced four additional strains from chicken and turkey based on phylogenetic analysis of a smaller multistrain data set (Figure S1). Our sequence, SNP filtering and between-platform cross-validation protocols yielded a high quality 756,552 bp alignment encompassing 612 genes (Tables S2, S3, S4, Text S1, Figure S2), and allowed us to monitor point substitutions, genomic indels, IS element insertions, and other changes across the entire genome (Figure 1), including the entire array of clustered regularly interspaced short palindromic repeats (CRISPR) of all 17 strains (finch and poultry isolates).

Results

Phylogenomic diversity of House Finch and poultry MG

All House Finch MG samples were collected in the southeastern U.S. (Table S1), with an emphasis on the well studied population in Alabama [24], [25]. The population structure of Eastern House Finches before the epizootic was virtually panmictic [26], suggesting that there is likely to be little geographic structuring of MG in the east, a hypothesis that could be tested with additional data. The 12 House Finch strains from the three time periods spanned the known temporal and phylogenetic diversity of this lineage, and included strains that have been used to study host response to pathogen infection in House Finches [27]. To determine genetic diversity and phylogenetic identity of putative source populations of the House Finch MG strains, and to aid in sampling chicken and turkey strains for sequencing, we first analyzed a previously published data set [28]. Phylogenetic analysis of 1,363 bp obtained from four genomic regions for a large sample (n = 82) of MG strains suggests that turkeys rather than chickens were the source of House Finch MG and that the MG lineage colonizing House Finches first passed multiple times among chickens and turkeys (Figure S2). Although this analysis suggests frequent host switches between chickens and turkeys, which diverged 28–40 MYA [29], [30], it also suggests a single switch to the House Finch, a songbird species diverged from chickens by ∼80 MYA [31].

The whole genome alignment contained strong signals of a founder event as a result of colonization of House Finches. The total nucleotide diversity (π) in the House Finch strains for the four-gene region was only 3.1% of the diversity in circulating poultry strains prior to the epizootic, and only 2.3% of the poultry diversity when considering the entire House Finch MG genome [28] (Figure 2 and Table S5). In agreement with the four-gene analysis, our whole genome sequencing showed that the four sequenced poultry isolates were much more genetically diverse than the 12 House Finch isolates, possessing a total of 13,175 SNPs as compared to only 412 SNPs among the House Finch isolates (Table S2). The House Finch MG diversity corresponds to π = 0.00014, or roughly 1 SNP every 1,800 bp. Consistent with purifying selection acting over the longer time period encompassing the divergence of House Finch and poultry MG strains (as opposed to acting after the host-switch among House Finch strains alone), there was a stronger bias against non-synonymous substitutions among the more diverged poultry strains than among the recently diverged House Finch MG strains (Table S6). Across the entire genome, only 147 (35%) of the SNPs among the House Finch isolates were phylogenetically informative; the majority (265 or 64%) appeared as singletons.

To further quantify House Finch MG demography, we used a statistical model, the Bayesian skyline plot implemented with BEAST, that utilizes information on dates of sampling to estimate changes in genetic diversity through time [32], [33] (Text S2). The analysis is broadly consistent with field observations suggesting a mid-1990s origin followed by rapid population expansion, though it estimates that the House Finch MG lineages coalesced roughly in 1988, several years prior to the observation of sick birds in the field (estimated MRCA of the House Finch MG strains is 19.2 years prior to 2007 [95% HPD 16.9 – 21.7]; Figure 2d). Discrepancies between coalescence times and observed outbreaks in host populations have been observed for other pathogens, and could possibly be due to selective or demographic effects, or in our case low sample size [12]. Phylogenetic analysis suggests substantial turnover in the standing SNP variation between sampling intervals, with strong clustering of the 2007 strains, which are distinguished from other House Finch strains by 85 diagnostic SNPs (Figure 3). We found that one of the sequenced turkey strains, TK_2001, was highly similar in sequence to the House Finch strains and shares a number of genomic deletions and transposon insertions as well as duplications and losses of CRISPR spacers (see below) with the House Finch MG strains. This turkey strain may represent a poultry lineage close to the source lineage for House Finch MG (Figure 3).

In addition to SNPs in House Finch MG we found five large genomic deletions that occurred by 2007 and amounted to ∼42, 245 bp and encompassing 34 genes relative to the chicken R_low strain (Figure 1 and Figure 3, Table S7). Three of these deletions are phylogenetically informative among the 17 MG strains (Table S7), but their conflicting phylogenetic distribution underscores the presence of recombination (see next section). Two deletions totaling 9,275 bp were shared among all strains except the reference. In addition, we detected six novel IS element insertions in the House Finch MG lineage (Text S3, Table S8) and three of the genomic deletions were likely mediated by illegitimate recombination between flanking IS elements (Table S7). In addition to the 34 genes deleted as part of genomic deletions, we found evidence for pseudogenization of 19 genes relative to the chicken MG reference (Text S3, Table S9). Two genes appear to have been disrupted by transposon insertions and 17 genes were pseudogenized by frameshift or nonsense mutations (Table S9). The substantial gene losses we detected, a total of 52 genes (∼8.6%) fixed in the House Finch MG lineage, presumably as a result of the bottleneck during host switch. By contrast, we failed to find a single novel gene in House Finch MG that was not also found in the poultry MG strains (Text S5). Comparative analysis with other Mycoplasma genomes showed that 15% of these lost genes also lacked a homologue in the other genomes surveyed whereas 13% had a homologue in every genome (Table S9).

Recombination and lateral gene flow

Despite the small amount of genetic variation segregating among our House Finch Mycoplasma samples (only 412 SNPs), it is not possible to construct a phylogenetic tree for these strains that is free of homoplasies. Although the four 2007 strains and all 2001 strains except AL_2001_17 clearly formed well defined clades based on 85 and 28 SNPs, respectively, establishing the phylogenetic relationships for the other 5 House Finch MG strains exclusively via SNPs was not possible (Text S6, Figure 3). Although a total of 16 SNPs were phylogenetically informative for the placement of these five strains, the largest cluster of SNPs that were phylogenetically consistent was seven, and overall, 13 different trees were supported by at least 3 SNPs each. Similarly, substantial homoplasy was found among the four newly sequenced poultry strains and the R_low reference. Although 6,152 SNPs were parsimony informative for these five strains, the unrooted tree with the best support was in conflict with 4,619 (75%) of these SNPs. These patterns are expected if sites are being shuffled by recombination or horizontal gene transfer (HGT) among isolates, and analysis of the entire data set found strong support for this (Text S4, Figures S3, S4, S5). Using the pairwise homoplasy index test [34] revealed a statistically significant signal of recombination (p<10⁻⁹). This signal comes predominantly from the four newly sequenced poultry strains because there is not enough genetic variation to make this test significant when only the House Finch strains are considered. However if we apply to the House Finch MG strains the homoplasy test by Maynard-Smith and Smith [35], which is found to perform well in situations of low nucleotide diversity [36], we again obtain a significant signal for recombination (p<10⁻⁶). We conclude that, despite a significant signal for recombination in both the poultry and House Finch strains, the House Finch MG cluster as a whole is a distinct and easily identifiable phylogenetic lineage with a long branch separating it from the poultry strains (Figure 3).

Substitution rate and robustness to model assumptions

Coalescent analysis [32] of the 12 House Finch isolates sampled at different dates suggested an extraordinary point substitution rate of 1.02×10⁻⁵ substitutions per site per year (95% HPD 7.95×10−6 to 1.23× 10−5 (Text S2), consistent with earlier suggestions that Mycoplasma may be among the fastest evolving bacteria [37]. This rate of point substitution is not restricted to House Finch MG strains but was also found in the poultry strains when analyzed separately (Text S2), suggesting that rapid evolution was characteristic of MG prior to the House Finch epizootic. We estimated a similar substitution rate when considering only the four-gene multistrain alignment use to identify poultry strains for sequencing (Text S2). We verified that our estimate of substitution rate is robust to different protocols for SNP identification, statistical models and data sets (Figure 4; Text S7). Altogether we estimated the substitution rate within a coalescent framework on 34 combinations of SNP calling and model assumptions and found consistent estimates throughout (Text S1, Figure 4, Figure S6). In addition, we achieved a similar estimate using a Poisson regression approach as well as a root-to-tip regression (Text S7 and Figure 4).

A possible mutator strains in House Finch MG

In addition to a high estimated substitution rate in MG, we found a mutation in the gene-encoding UvrB that could elevate this rate yet further. UvrB is an essential part of the nucleotide excision repair system, which has been posited to be the most important pathway for maintaining genomic integrity in Mycoplasma [38]. The mutation truncates the UvrB protein by three amino acids (Table S10) and raises the possibility of the origin of a mutator strain in House Finch MG [39] as the C-terminal of this protein is essential for its function [40]. Consistent with this idea, we found 14 instances of adjacent SNPs among the 12 House Finch isolates, a notable excess in an alignment with only 412 variable sites (Table S11). Moreover, 12 of these 14 are CC→TT double substitutions, which are normally repaired by the UVR system (Table S10). For 13 of the 14 doublets, both sites are inferred to have mutated on the same branch of the tree, suggesting single mutational events, and the proportion of doublet mutations involving the same base was drastically higher (92.8%) in lineages with the UvrB mutation as compared to those without (p<0.0001; Table S10). Nonetheless, these doublet mutations are not required to achieve the high rate of substitution that we measured. They account for less than 7% of the segregating variation and removal of these doublet sites does not affect the high estimated substitution rate. The UvrB mutation is found in all of our House Finch MG strains as well as the turkey strain TK_2001, but not in the ancestral chicken strains or the reference chicken strain. Thus, the mutation appears to have arisen on the lineage leading to the House Finch.

Degredation and apparent functional loss of CRISPR loci in House Finch MG

In some bacterial systems, CRISPRs have a well-recognized function in bacterial immunity and defense against phage, although they may possess additional functions, such as gene regulation [41]–[44]. We extensively catalogued CRISPR repeats in the House Finch and ancestral poultry strains (Figure 5, Text S8, Table S12). In so doing we observed drastic changes in the CRISPR system between House Finch and poultry strains (Figure 5) [45]–[48]. The House Finch MG strains from 1994–96 contain up to 50 unique spacers, none of which is shared with the four divergent poultry genomes, which each contained a unique set of 36 to 147 spacer regions consistent with a high rate of turnover for a population actively acquiring new spacer sequences. We found that less than 1% of the 302 unique spacer sequences had similarity to any sequences in the House Finch MG genomes and that none of the remaining spacers had any similarity to sequences in Genbank, indicating an external source for these sequences (Text S8). Surprisingly, no novel spacer elements are present in any of the House Finch MG samples or TK_2001, indicating that the CRISPR array ceased recruiting additional spacers around the time of host switch into the House Finch. In fact, over the 13-year period of the epizootic, the number of unique spacers present in the CRISPR array of the samples decreased to 28 (Figure 5). Further evidence for degradation of the CRISPR locus following the host switch is the complete loss of the four CRISPR-associated (i.e. “CAS”) genes in all of the 2007 isolates, a loss that likely renders the CRISPR system in House Finch MG non-functional [45].

Discussion

Rapid substitution rate

We conducted whole-genome sequencing on a unique 13-year serial sample of Mycoplasma strains circulating in wild House Finches to characterize genomic changes accompanying a host shift from poultry in the mid-1990s as well as to obtain a very high substitution rate for this avian pathogen. Previous estimates using serial samples and/or the known timing of events presumably tied to the divergence of bacterial strains have generally found much lower rates. An estimate of 2.0×10⁻⁶ was obtained for Staphyloccous aureus [12], 1.1×10⁻⁷ for Buchnera [19], 7.42×10⁻⁷ in Yersinia pestis and 1.4×10⁻⁶ in Heliobacter pylori [14]. Disentangling the effects of recombination and point substitution can be challenging and some previously published substitution rates are likely to be upper bounds rather than point estimates [12]. Our estimate appears to be among the highest reported for a bacterium, and is consistent with other reports of exceptionally high substitution rates in mycoplasmas [37].

Estimates of substitution rates can be influenced by the interval over which sequences are sampled, with estimates taken from short time intervals often exceeding those taken on biogeographic or geological time scales [49]. However the small number of SNPs that we detected segregating in House Finch MG populations suggest negligible effects of multiple hits on our estimate, and our use of a coalescent model suggests that effects of ancestral polymorphism on substitution rate estimates should be adequately accounted for [32], [50]. Additionally, our estimates of substitution rate were robust to many potential complicating factors, including SNP calling protocol and whether poultry or House Finches were used as the host for sampled sequences. Given the history and genetic isolation of the House Finch MG strains, the influence of recombination or lateral gene transfer on our estimate of substitution rate is likely also minimized (Text S7).

Rapid evolution and degradation of CRISPRs

The CRISPR dynamics we observed in House Finch MG differ from that seen in other pathogen and bacterial populations. A recent study of Y. pestis CRISPR arrays from 131 strains [51] indicated a slower pace of CRISPR evolution than observed in MG and pattern of evolution in which acquisition of novel sequences does not play a prominent role. This study found that in Y. pestis the first part of the CRISPR arrays were conserved and that over 76% of all spacer sequences derived from within the Y. pestis genome. Similarly, a recent study of E. coli and Salmonella genomes found that strains within 0.02% divergence typically have identical CRISPR loci [52] and that spacer sequences were often matched to elements of the E. coli genome. Additionally, some spacer sequences were shared between strains within a species exhibiting over 1% sequence divergence. These observations and an estimated substitution rate on the order of 10⁻¹⁰ per site per year suggested that E. coli strains that had diverged for 1,000 years sometimes shared identical CRISPR loci, suggesting patterns of evolution different from that expected for a rapidly changing adaptive immune system primed to combat phages, a conclusion that was supported by later work [53].

By contrast to the pattern seen in these γ-proteobacteria, none of the House Finch MG strains in this study have the same CRISPR locus despite differing at only 0.01–0.02% of sites and likely having last shared a common ancestor less than 20 years ago. Our serial sampling suggests that the loss of spacer sequences and the CRISPR system itself can take place on very short time scales in Mycoplasma. Unlike the patterns seen in E. coli, Y. pestis, and Salmonella, the poultry MG strains in our study did not share any spacer sequences, even though they differed by ∼1%. These strains had very large CRISPR arrays and 99% of all spacer sequences did not match any known sequence in their genome or in the databases. Therefore the MG CRISPR loci studied here differ from the those observed in some γ-proteobacteria, a group for which CRISPR dynamics can appear functionally unrelated to ecology or immunity [53]–[55].

Instead, our finding of rapid evolution and degradation of the CRISPR loci more closely resembles patterns found in other bacterial groups, particularly those in which CRISPR is involved in phage defense [56]. CRISPRs are found in only 40% of sequenced bacteria investigated thus far, and often have major roles in bacterial immunity in several lineages investigated in detail [45]. We were surprised to find a gradual degradation and ultimate apparent functional loss of the CRISPR system in House Finch MG after the host switch and a shift in CRISPR dynamics appears to be a major correlate of host switch in this system. One possible explanation for this pattern is that MG experienced release from its ancestral phage parasite community (or other mobile genetic elements such as plasmids) following introduction into the House Finch. Loss of traits upon removal of the agent of selection is a common evolutionary response, as are population expansions of animals and plants when introduced into novel habitats unaccompanied by their parasites [57].

Despite the large amount of ecological research focusing on this host-pathogen system [3]–[7], at present nothing is known about phages that infect MG or their role in its evolutionary dynamics. Therefore the hypothesis of parasite release as a driver of CRISPR loss is purely speculative. We know of no phage known to infect the Pneumoniae phylogenetic group of mycoplasmas and the few phages known to infect Mycoplasma have proven difficult to characterize [58]. We might expect Mycoplasma bacteriophages to be host-specific given that they seem to be unusual in their ability to bind to a bacterium with no cell wall and a diverse assortment of surface proteins [58]. However, we are not aware of even basic data on the degree to which Mycoplasma might be susceptible to the many bacteriophages that they presumably encounter in their environment. Although phage represent one possible source for these novel ∼30 bp sequences, another possible explanation for the source of the spacer sequences is that they derive from plasmids. Although unprecedented (we know of no examples of a naturally occurring plasmid in the Pneumoniae mycoplasmas), such a scenario could raise the possibility of easier genetic manipulations in MG where development of such tools has been challenging [59]. Of the many other possibilities that could explain the observed degradation of the CRISPR loci, we can at least rule out self-interference as an explanation in derived MG strains, given that there is only a single CRISPR cluster in House Finch MG [54]. Measurement of costs, possible advantages and consequences of CRISPR loss, as well as functional and evolutionary assays and surveys of phage diversity will help determine if the rapid and deadly spread of Mycoplasma following their expansion into the House Finch was facilitated by a lack of phage predation, a short-term advantage of CRISPR degradation or some other, possibly neutral, mechanism. Although our sequence data is suggestive, explicit functional studies will also be required to demonstrate CRISPR functionality or lack thereof in poultry and House Finch MG and its role, if any, in phage defense.

Pseudogenization and possible mutator strains

Genome evolution of MG during its host-switch from poultry to House Finches adds to a growing list of host-switches that are successful in the complete absence of novel genes [21], [60], [61] and bacterial lineages exhibiting high rates of point substitution [14]. Mycoplasmas are some of the fastest evolving organisms on earth [62] having lost many of the repair mechanisms present in other bacteria [38] and this high mutation rate could help introduce deleterious mutations and contribute to the substantial level of pseudogenization that was observed in this study. The high basal substitution rate in MG may well be elevated yet further by UvrB mutation that we detected, a mutation that could have consequences for the long term genomic integrity of this MG lineage, particularly if it remains genetically distinct from and unable to exchange genes with the poultry MG lineages with a functional UvrB. Alternatively, given the short (3 amino acid) truncation of this gene in the House Finch strains, another explanation for the greatly increased number of doublet mutations in the lineage carrying the UvrB truncation is that selection has not had enough time to remove them as it has for poultry strains without this mutation. Although mutator strains are known to have a selective advantage in rapidly evolving laboratory and natural populations [39], [63], additional functional and experimental work will be required to determine the selective and functional effect of the mutation we have detected in UvrB, and over what time scales such selective effects might persist. For this and other endeavors, serial sampling of additional bacterial populations in nature will further clarify the rate at which genomes are remolded during host switches in the wild.

Materials and Methods

Sampling of House Finch and poultry MG strain diversity

DNA sequence data for 4 gene fragments collected from 74 strains in Ferguson et. al. [28], was combined with data from 8 strains newly sequenced in this study to yield a Large Sample Multiple Sequence Alignment (LS-MSA) 1,363 bp in length (Figure S2). We estimated nucleotide diversity and the standard deviation of this estimate within and among subgroups of these sequences using DNAsp version 4.10.9 [64] (Table S5). In estimating diversity of MG strains sampled from chickens and turkeys, we restricted analysis to those strains sampled during 1994–1996 for comparison with our earliest House Finch strains sampled in a similar time interval.

Strain selection and genome sequencing

Twelve strains of MG isolated from House Finches in the Southeastern US were sequenced with the Roche 454 Gene Sequencer. The average coverage level was 9.4X (Table S1). Additionally, four MG strains isolated from poultry hosts and selected based on their positions in the multistrain phylogenetic tree were sequenced with the Illumina sequencing platform to an average coverage of ∼410 X (Tables S2, S3, S4, Text S1, Figure S2).

Inference of substitutions rates, times to common ancestry, and population dynamics

Using a coalescent model and a Bayesian framework as implement in BEAST v1.52 [32] we estimated the mutation rate and times to common ancestry from a 13-taxon alignment composed of the reference MG genome and all of the House Finch MG strains whose genomes were sequenced in this study (Text S2). We also ensured that the conclusions from this inference were not sensitive to the SNP calling procedures or the choice of substitution models (Text S2, S7, Figure S6). In order to compare the mutation rate between the poultry and House Finch MG populations, these quantities were similarly estimated from the 82 taxon LS-MSA after removing nine laboratory strains from the alignment that likely experienced different population dynamics than the wild strains and had unknown sampling dates. A Poisson regression model was also used to estimate substitution rates by counting mutations along a single lineage assumed to span the dates of sampling for each strain (Text S7).

Transposon movements, recombination, and lateral gene flow

We catalogued IS elements using BLAST and the ISFinder database [65, Text S4]. We tested for evidence of genetic recombination between MG strains using the genome sequences from our 4 poultry and 2 House Finch strains using the pairwise homoplasy index test [34] as implement in splitstree4 [66], and the homoplasy test by Maynard-Smith and Smith [35]. Further evidence for the presence of recombination and the number of nonrecombining blocks was provided by other methods (Text S6, Figures S3, S4, S5).

Supporting Information

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