Reconocimiento social y vasopresina

Nature advance online publication 24 February 2010 | doi:10.1038/nature08826; Received 9 April 2009; Accepted 12 January 2010; Published online 24 February 2010

An intrinsic vasopressin system in the olfactory bulb is involved in social recognition

Vicky A. Tobin^1,7, Hirofumi Hashimoto^1,7, Douglas W. Wacker¹, Yuki Takayanagi², Kristina Langnaese³, Celine Caquineau¹, Julia Noack^3,4, Rainer Landgraf⁵, Tatsushi Onaka², Gareth Leng¹, Simone L. Meddle^1,6, Mario Engelmann³ & Mike Ludwig¹

1. Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, UK
2. Department of Physiology, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan
3. Institute of Biochemistry and Cell Biology,
4. Centre for Cellular Imaging and Innovative Disease Models, Otto von Guericke University Magdeburg, 39120 Magdeburg, Germany
5. Max Planck Institute of Psychiatry, 80804 Munich, Germany
6. The Roslin Institute & Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9PS, UK
7. These authors contributed equally to this work.

Correspondence to: Mike Ludwig¹ Correspondence and requests for materials should be addressed to M.L. (Email: mike.ludwig@ed.ac.uk).

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Abstract

Many peptides, when released as chemical messengers within the brain, have powerful influences on complex behaviours. Most strikingly, vasopressin and oxytocin, once thought of as circulating hormones whose actions were confined to peripheral organs, are now known to be released in the brain, where they have fundamentally important roles in social behaviours¹. In humans, disruptions of these peptide systems have been linked to several neurobehavioural disorders, including Prader–Willi syndrome, affective disorders and obsessive–compulsive disorder, and polymorphisms of V1a vasopressin receptor have been linked to autism^{2, 3}. Here we report that the rat olfactory bulb contains a large population of interneurons which express vasopressin, that blocking the actions of vasopressin in the olfactory bulb impairs the social recognition abilities of rats and that vasopressin agonists and antagonists can modulate the processing of information by olfactory bulb neurons. The findings indicate that social information is processed in part by a vasopressin system intrinsic to the olfactory system.

Complex social behaviour often depends on individual recognition, and most mammals distinguish individuals by their olfactory signatures. Some individuals are accorded a particular status, such as when a bond is formed between a mother and offspring, or between sexual partners in monogamous species. In these cases, an olfactory memory is forged in the olfactory bulb, partly as a result of the actions of peptides⁴. For example, oxytocin released in the mother’s brain during parturition helps to establish the olfactory signatures of the offspring as memorable⁵.

The converse of social attachment is rejection of, or aggression towards, individuals who are recognized as intruders or competitors⁶. For this, vasopressin, a peptide closely related to oxytocin, is important through its actions at V1 receptors, and mice without functional accessory olfactory systems show many of the same behavioural deficits as mice that lack V1 receptors. This suggests that vasopressin is involved in the processing and/or integration of olfactory stimuli, and that it couples socially relevant olfactory cues to an appropriate behavioural response⁷.

We have identified a hitherto unreported population of vasopressin neurons in the olfactory bulb (Fig. 1). We first saw these cells in a transgenic rat line in which enhanced green fluorescent protein (eGFP) was targeted to the vasopressin secretory pathway, resulting in its co-packaging with vasopressin in secretory vesicles⁸. The main olfactory bulb contains similar numbers of eGFP-expressing cells in males and females (99 ± 14 and 103 ± 10 cells per section, respectively; n = 16 in each case), giving an estimated 5,000–7,000 neurons per bulb; the accessory bulbs contained ~1,000 neurons. These large ovoid neurons (~15 μm in diameter) are mostly located in the external plexiform layer close to the glomeruli (the structures in the bulb that directly receive inputs from olfactory receptor cells). Each has several large dendrites, one of which penetrates a single glomerulus, where it gives rise to many small branches. This suggests that the neurons receive direct inputs from olfactory nerve afferents. Other dendrites travel laterally to the external zones around neighbouring glomeruli (Fig. 1a, b). Using immunocytochemistry, we showed that these cells indeed synthesize vasopressin (Fig. 1c, d), and we confirmed their presence in wild-type rats (Fig. 1e). We also confirmed that they express vasopressin messenger RNA, using in situ hybridization (Fig. 1f), and that vasopressin is released from olfactory bulb explants in vitro in response to depolarization using K⁺ in high concentration (release increased from 0.65 ± 0.19 to 4.88 ± 1.88 pg per sample; P < 0.01, n = 9). The total bulb vasopressin content was 42.9 ± 2.6 pg mg^-1 wet weight (n = 12; mixed sex).

Figure 1: Vasopressin neurons in the olfactory bulb.

a, Most vasopressin cells in eGFP transgenic rats (3,3′-diaminobenzidine staining) are in the periglomerular region throughout the main olfactory bulb. b, An apical dendrite ramifies into a glomerulus (blue staining, periglumerular cell-marker calbindin-D28k). c–e, Confirmation using antibodies against GFP (c) and vasopressin (d) in transgenic rats and vasopressin in wild-type rats (e). f, In situ hybridization for vasopressin mRNA. g–l, Vasopressin cells do not co-express calbindin, calretinin or GABA (red; g–i), but do contain glutamate (j–l). m–o, Fluorogold-labelling after injection into the anterior olfactory nucleus in mitral and periglomerular cells, but not vasopressin cells. p–r, V1a receptors are expressed on mitral cells and many periglomerular neurons (p), but not on vasopressin cells (q), whereas some vasopressin cells express V1b receptors (r). s, Patch-clamp recordings indicate firing patterns (spontaneous and depolarized) like those of external tufted cells¹⁰. GCL, granule cell layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. Scale bars, 20 μm.
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Unlike periglomerular cells and short axon cells (two other cell populations in the same region), the vasopressin cells are immunonegative for GABA (γ-aminobutyric acid), calretinin and calbindin-D28k (Fig. 1g–i), but like external tufted cells^{9, 10}, they are immunoreactive for glutamate (Fig. 1j–l). No cells were immunoreactive for oxytocin. Whole-cell patch-clamp recordings from olfactory bulb slices showed that the vasopressin cells have electrophysiological characteristics like those of external tufted cells¹⁰. They show spontaneous bursts of action potentials (1.5 bursts per second) arising at the start of a slow depolarizing potential envelope (6.5 ± 0.5 mV; n = 5) that grows from a resting membrane potential of -55 ± 2 mV, and have an input resistance of 189 ± 38 MΩ. This bursting is voltage dependent (Fig. 1s), and injection of depolarizing current converts bursts of action potentials to an irregular firing pattern.

Unlike most external tufted cells, most vasopressin cells do not project outside the olfactory bulb. Microinjections of the retrograde tracer Fluorogold into the cortical amygdala, the piriform cortex or the olfactory tubercle (major projection sites of olfactory bulb efferents¹¹) resulted in labelling of most mitral cells (the main output neurons) but no vasopressin cells (Fig. 1m–o), whereas injections into the anterior olfactory nucleus produced a very small number of labelled cells (data not shown). If the vasopressin cells do not project outside the olfactory bulb, any effects of the vasopressin that they release on olfactory information flow must be reflected by changes in the activity of other output cells. Vasopressin may be released from their axon terminals, but the dendrites may be a more important source as they are densely filled with vasopressin, and in the hypothalamus vasopressin is released from dendrites in both an activity-dependent manner and in an activity-independent way by agents that mobilize intracellular calcium¹².

Vasopressin receptors are widespread in the main and accessory olfactory bulbs¹³. We found no immunoreactivity for V1a receptors on vasopressin cells, but some for V1b receptors (Fig. 1p–r), so vasopressin may act as an autocrine regulator through this receptor subtype¹⁴. Many other cells in the periglomerular region were immunoreactive for both subtypes, as were mitral cells in the main and accessory bulbs.

We tested the hypothesis that olfactory bulb vasopressin is involved in social recognition. It has already been shown that infusion of vasopressin into the bulb can enhance social recognition in rats. In those experiments, a peptide V1 receptor antagonist had no significant effect¹⁵. Here we used a non-peptide V1 receptor antagonist (OPC-21268) that diffuses more readily, and which is effective in antagonizing the actions of dendritically released vasopressin in the hypothalamus¹⁶. We injected the antagonist bilaterally into the olfactory bulb and tested social discrimination¹⁷. In this test, a juvenile rat is placed in the home cage of an adult male and the time that the adult spends investigating it is measured. Later, the same juvenile and an unfamiliar juvenile are introduced into the cage. Normally the adult investigates the familiar juvenile only briefly, and pays most attention to the unfamiliar juvenile; this memory is short lasting (<40 min) and is based on olfactory characteristics. In these experiments, we gave the adults a microinjection of the antagonist just before the juvenile was first presented. When retested, the adults did not discriminate between the familiar and unfamiliar juveniles, indicating that no memory of the juvenile was retained (analysis of variance: F(3, 39) = 3.34, P = 0.026; Fig. 2a, b).

Figure 2: Effects of V1a receptor blockade and vasopressin cell destruction on social recognition.

a, A juvenile (J) is presented to an adult male (A) for 4 min. This juvenile is removed and after 30 or 180 min is re-presented together with a non-familiar juvenile, and the preference index (Methods Summary) is calculated. b, Administration of V1 receptor antagonist results in performance similar to that after extinction of short-term discrimination (after 180 min). c, V1a receptor siRNA similarly impairs discrimination after 4, 8 and 16 d of treatment. d, Selective destruction of vasopressin cells by means of diphtheria toxin injection results in a similar impairment of discrimination in transgenic rats, but not in wild-type rats. Data shown, mean + s.e.m; *P < 0.05 and **P < 0.001 versus control; for each group, n is shown at the bottom of the corresponding column.
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To test involvement of the V1a receptor subtype specifically, we used infusions of a small interference RNA (siRNA) targeted against V1a receptor mRNA (siRNA has previously been used to silence gene expression successfully, including silencing the V2 receptor in mouse kidney¹⁸); these infusions produced transfection in the olfactory bulb but not in the septum (Supplementary Fig. 1). The effects of siRNA treatment were similar to those obtained with antagonist (treatment: F(1, 16) = 17.86, P < 0.01; factor interaction: F(3, 48) = 4.37, P < 0.01; Fig. 2c). Control rats (but not siRNA-treated rats) could recognize juveniles by their complex individual olfactory fingerprint even in the presence of distracting monomolecular odours (Supplementary Fig. 2a). Treatment with siRNA impaired habituation/dishabituation to juvenile cues (control: F(4, 32) = 3.42, P < 0.02; siRNA: F(4, 32) = 0.47, P = 0.76), but not to volatile odours (control: F(4, 32) = 5.672, P < 0.01; siRNA: F(4, 32) = 4.09, P < 0.01) or object recognition (Fig. 3a–c), and did not affect open-field behaviour (Supplementary Fig. 2b, c).

Figure 3: Specificity of effects on social recognition.

a, Control rats and siRNA-treated rats show similar habituation and dishabituation to volatile scents. b, Control rats also show habituation and dishabituation to juveniles, whereas siRNA-treated rats show neither. c, Neither siRNA nor diphtheria toxin injection affects object recognition. Data shown, mean + s.e.m.; *P < 0.05 versus trial 4 and same treatment; n shown for each group in c.
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We also used a transgenic rat line in which the human diphtheria toxin receptor is inserted into the vasopressin promoter region. In these rats, infusion of diphtheria toxin^{19, 20} results in a local, selective destruction of vasopressin cells. Transgenic rats pretreated with toxin infusions into the olfactory bulb showed a similar impairment of juvenile recognition (treatment: F(1, 29) = 14.95, P < 0.01; factor interaction: F(1, 29) = 4.99, P = 0.033; Fig. 2d), again with no impairment of object recognition (Fig. 3c), locomotor activity or anxiety-related behaviours (Supplementary Fig. 2d–i).

Finally, we investigated vasopressin-dependent changes in olfactory bulb output. Current theories of glomerular function propose that olfactory nerve afferents activate external tufted cells, which activate short axon cells and periglomerular interneurons of the same glomerulus. This amplifies the olfactory nerve input and imposes on it a bursting pattern¹⁰, and this signal is transmitted to mitral cells. We recorded signals from mitral cells antidromically identified by electrical stimulation of the lateral olfactory tract in freely breathing, anaesthetized rats (Fig. 4a, b and Supplementary Fig. 3).

Figure 4: Vasopressin effects on mitral cells.

a, b, Effects of vasopressin (a) and V1 receptor antagonist (b) on firing rate (a) and instantaneous frequency in a single, representative mitral cell (a, b). The inset in a shows raw waveform traces of spike activity. c, The activity quotient decreases in six cells treated with 4 ng vasopressin and seven cells treated with 40 ng vasopressin, and increases in seven cells treated with the vasopressin antagonist. d, The hazard function overlay (light line, before vasopressin; heavy line, after vasopressin) shows the reduction in doublet firing in a typical cell. e, Change in the number of doublets (intervals <10 ms), quantified for all doublet cells. f, Top: spike activity in a mitral cell, showing activity modulated by respiratory rhythm 5 s before and 5 s after odour. Middle and bottom: cumulative spikes from four tests during bursts before (middle) and after (bottom) vasopressin. g, Number of spikes 5 s before (white) and 5 s after (black) odour for the tests in f. h, Responses of another cell to odour (arrows) before, during and after artificial cerebrospinal fluid (ACSF) and vasopressin. i, Magnified instantaneous frequency records of the cell shown in h. j, Mean odour response in 16 cells tested with vasopressin, seven of which were also tested with ACSF, and in ten of which recordings were maintained long enough to observe recovery. Data shown, mean + s.e.m.; *P < 0.05, **P < 0.01.
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Under urethane anaesthesia, most mitral cells displayed patterned discharge comprising prolonged intermittent bursts of action potentials²¹ (burst duration, 159 ± 10 s; interburst time, 97 ± 13 s; intraburst firing rate, 6.4 ± 0.5 Hz; n = 94; Supplementary Fig. 3). Within these bursts, the firing activity is modulated by the respiratory rhythm. In addition, many mitral cells (57 of 94) displayed a bimodal interspike interval distribution, reflecting the frequent occurrence of spike doublets within bursts. Thus, within bursts, these cells fired at two distinct instantaneous frequencies: at 100–250 Hz (doublets; mean modal interspike interval, 3.2 ± 0.4 ms) and at ~50 Hz (mean modal interspike interval, 18 ± 1 ms). The doublets are noteworthy as it is believed that only high-frequency firing episodes are back-propagated into the distal dendrites²². Topical administration of vasopressin or the V1 receptor antagonist onto the exposed bulb dorsal to the recording site modified the electrical activity of mitral cells. Vasopressin reduced the proportion of time they were active, and particularly reduced doublet firing, whereas the antagonist had the opposite effect (P < 0.05, paired t-tests; Fig. 4c–e).

In 16 experiments, each involving a long recording from an identified mitral cell, we identified an odour to which that cell was particularly responsive, established the repeatability of that response in basal conditions and then retested the response to stimulation after topical application of vasopressin. In every case, the response to the odour was suppressed after vasopressin, whereas topical application of artificial cerebrospinal fluid had no effect on the responses of seven cells tested (Fig. 4f–j).

These findings suggest that vasopressin is a retrograde signal that filters activation of the mitral cells. Its effects may involve presynaptic modulation of noradrenaline or acetylcholine release, both of which are increased by retrodialysis of vasopressin in the olfactory bulbs of ewes²³. Because this filtering is important for social recognition, it seems that the vasopressin release must depend on previous olfactory experience. In the hypothalamus, activity-dependent dendritic vasopressin release can be conditionally regulated (‘primed’) by recent experience^{12, 24}; such a mechanism in the olfactory bulb may therefore mediate conditional changes in olfactory recognition.

Thus, the olfactory bulb contains vasopressin cells that process olfactory signals relevant to social discrimination, and dendritic vasopressin release may be involved in filtering out familiar signals. Genetic variations in brain vasopressin signalling are associated with differences in social behaviours in humans^{25, 26} as well as in animal models. We are not suggesting that social recognition in humans depends on olfactory signals; vasopressin affects social behaviour at many other sites as well as at the olfactory bulb^{27, 28}, and in humans olfactory recognition probably has only a small role. However, these studies suggest a mechanism by which experience-dependent vasopressin release can facilitate social recognition, and this mechanism may be common to several sites of action.

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Methods Summary

Animal experimental procedures were conducted with regulatory approval and ethics committee approvals in the UK, Germany and Japan.

We processed brains immunocytochemically to detect cells expressing vasopressin, GFP, calbindin-D28k, calretinin, GABA, glutamate and V1a and V1b receptors, and by in situ hybridization to detect vasopressin mRNA and eGFP mRNA. Eleven eGFP rats were stereotaxically microinjected with the retrograde tracer Fluorogold at various sites to detect cells projecting from the olfactory bulb. Vasopressin content and potassium-stimulated release from olfactory bulb explants was measured by radioimmune assay.

To test effects on social discrimination, we bilaterally infused the V1 receptor antagonist or vehicle into the olfactory bulbs of adult rats¹⁵. A juvenile was introduced into the adult’s cage for 4 min and the duration of investigation by the adult was recorded; either 30 or 180 min later, the juvenile was reintroduced with another unfamiliar juvenile and the preference index ((time investigating unfamiliar juvenile)/(time investigating familiar juvenile + time investigating unfamiliar juvenile) × 100) was measured¹⁷. Olfactory habituation and dishabituation²⁹ was tested by exposing rats to four 1-min trials separated by 10 min. During a fifth dishabituation trial, the rats were exposed to a novel stimulus. In rats injected with siRNA directed against V1a receptors (or control vectors), behaviours were tested 4, 8 and 16 d after injection.

For conditional ablation of vasopressin neurons, we used transgenic rats with a mutated human heparin-binding epidermal growth factor-like growth factor³⁰ gene (HBEGF) under the control of vasopressin promoter. Diphtheria toxin was microinjected into the olfactory bulb in rats anaesthetized with tribromoethanol.

In urethane-anaesthetized rats, electrical activity of mitral cells was recorded before and after administration of vasopressin or V1 receptor antagonist onto a small exposure of the bulb. Cells were tested with odours applied for 2 s in an air stream directed at the nose. For in vitro electrophysiology, whole-cell current-clamp recordings were made from GFP-expressing cells in 300-μm horizontal olfactory bulb slices.

Full methods accompany this paper.

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Antagonistic coevolution accelerates molecular evolution

Nature advance online publication 24 February 2010 | doi:10.1038/nature08798; Received 8 September 2009; Accepted 23 December 2009; Published online 24 February 2010

Steve Paterson^1,5, Tom Vogwill^1,5, Angus Buckling², Rebecca Benmayor², Andrew J. Spiers³, Nicholas R. Thomson⁴, Mike Quail⁴, Frances Smith⁴, Danielle Walker⁴, Ben Libberton¹, Andrew Fenton¹, Neil Hall¹ & Michael A. Brockhurst^1,5

1. School of Biological Sciences, Biosciences Building, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK
2. Zoology Department, University of Oxford, South Parks Road, Oxford OX1 3PS, UK
3. SIMBIOS Centre, Level 5 Kydd Building, University of Abertay Dundee, Bell Street, Dundee DD1 1HG, UK
4. Pathogen Genomics, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
5. These authors contributed equally to this work.

Correspondence to: Michael A. Brockhurst^1,5 Correspondence and requests for materials should be addressed to M.A.B. (Email: michael.brockhurst@liverpool.ac.uk).

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Abstract

The Red Queen hypothesis proposes that coevolution of interacting species (such as hosts and parasites) should drive molecular evolution through continual natural selection for adaptation and counter-adaptation^{1, 2, 3}. Although the divergence observed at some host-resistance^{4, 5, 6} and parasite-infectivity^{7, 8, 9} genes is consistent with this, the long time periods typically required to study coevolution have so far prevented any direct empirical test. Here we show, using experimental populations of the bacterium Pseudomonas fluorescens SBW25 and its viral parasite, phage Φ2 (refs 10, 11), that the rate of molecular evolution in the phage was far higher when both bacterium and phage coevolved with each other than when phage evolved against a constant host genotype. Coevolution also resulted in far greater genetic divergence between replicate populations, which was correlated with the range of hosts that coevolved phage were able to infect. Consistent with this, the most rapidly evolving phage genes under coevolution were those involved in host infection. These results demonstrate, at both the genomic and phenotypic level, that antagonistic coevolution is a cause of rapid and divergent evolution, and is likely to be a major driver of evolutionary change within species.

According to the Red Queen hypothesis, biotic interactions are a fundamental driver of molecular evolution². The Red Queen hypothesis posits that for a given species, its effective environment is likely to be comprised of the other species in the ecosystem, such that an adaptation increasing the fitness of one species necessarily causes a decline in fitness of those species with which it interacts^{1, 3}. Such coevolutionary interactions give rise to continual natural selection for adaptation and counter-adaptation by ecologically interacting species^{1, 3}, thereby driving molecular evolution². Nowhere are such evolutionary dynamics thought to be so prevalent as in interactions between hosts and virulent parasites, in which selection is strongly antagonistic yet closely coupled¹². Comparative studies have found particularly high rates of molecular evolution in genes associated with infection^{7, 8, 9} or resistance to infection^{4, 5, 6}. However, there have been no direct empirical tests of whether antagonistic host–parasite coevolution accelerates molecular evolution in parasite genomes, and whether such evolution is particularly rapid at genes determining infectivity.

Here we use experimental evolution of populations of the bacterium Pseudomonas fluorescens SBW25 and its viral parasite, phage Φ2. We have previously demonstrated that P. fluorescens and Φ2 undergo a persistent coevolutionary ‘arms race’ with reciprocal selection for the evolution of new resistance and infectivity phenotypes through time in bacteria and phage, respectively^{10, 13}, but the link between this rapid phenotypic evolution and the underlying pattern of molecular evolution has not been resolved. Crucially, it is possible to separate bacteria and phage when transferring populations to fresh media¹⁴, which allows one partner to be held evolutionarily constant while the other partner is allowed to evolve^{15, 16, 17}. Initially isogenic, replicate populations of P. fluorescens and Φ2 were propagated by serial transfer under two conditions: (1) evolution, in which the bacterial genotype was held constant and only the phage was allowed to adapt, and (2) coevolution, in which both the bacterium and the phage were allowed to evolve adaptations and counter-adaptations. At the end of the selection experiment we obtained whole-genome sequences of phage populations by high coverage second-generation sequencing to determine the identity and frequency of mutations in each population. Mutations were partitioned into synonymous and non-synonymous changes; very few synonymous mutations were observed and only non-synonymous mutations were used in analyses (see Supplementary Information; note that each indel (that is, insertions or deletions) was counted as one mutation regardless of its length). From these data we calculated the number of sites that had acquired mutations in each population relative to the ancestral reference Φ2 sequence (obtained as part of this study; see Supplementary Information), and from allele frequencies, the genetic distance of each population from the ancestral Φ2 sequence, the genetic divergence among populations and the genetic diversity within each population.

Coevolved phage populations showed twice the genetic distance from the ancestor as that of the evolved populations (average pairwise genetic distances: coevolved, 22.7 ± 1.9 standard error (s.e.); evolved, 11.1 ± 0.4 s.e.; t = 6.64, d.f. = 4.46, P < 0.01), shown also by the increased branch lengths for coevolved populations in the phylogenetic tree in Fig. 1a. Similarly, coevolved populations had a greater number of sites exhibiting mutations than evolved populations (coevolved, mean 52.8, range 46–60; evolved, mean 37.5, range 29–42; likelihood-ratio test (LRT) = 14.3, P < 0.001). Furthermore, far greater genetic divergence was observed among replicate coevolved populations than was observed among replicate evolved populations (Φ_ST = 0.45 for coevolved populations versus Φ_ST = 0.06 for evolved populations (Supplementary Table 1), in which Φ_ST is a measure of the proportion of the total molecular variation attributable to differences among populations¹⁸). The tree in Fig. 1a also shows that replicate populations from the same treatment grouped together genotypically. This topology is not due to co-ancestry as all populations were split at the start of the experiment. Instead, the topology reflects parallel evolution: selection acting independently at the same sites among replicate populations. Thus, the tree reflects three key evolutionary patterns. Specifically, the extent to which replicates: (1) followed a similar trajectory away from the ancestral sequence, presumably as they adapted to laboratory conditions; (2) evolved similarly among replicates within a treatment but differently in response to consistent differences between treatments; and (3) showed independent evolution within each replicate, and at a far higher rate in the coevolved than the evolved treatment.

Figure 1: Genetic and phenotypic responses to selection.

a, Phylogenetic tree for evolved (E1–6) and coevolved (C1, C3–6) phage populations and ancestral reference genotype (ref) based on Euclidean distances calculated from the frequency and identity of mutations in each population. Scale bar indicates a Euclidean distance of one. b, The phage-infectivity range based on the ability of each coevolved population to infect 20 bacterial clones from each host population. Infection by phage is shown in red, and resistance by hosts is shown in grey. The dendrogram indicates phenotypic similarity between phage populations.
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Increased genetic divergence between parasite populations due to coevolution is likely to be driven primarily by divergent selection on infectivity traits. To address this prediction, we phenotypically characterized the infectivity profile of each phage population. Specifically, we used cross-infection experiments to test whether phage from each population were able to infect hosts from all coevolved populations. We found that coevolved phage populations varied in terms of both the range and identity of host genotypes that they were able to infect (Fig. 1b), but that phage from evolved populations failed to infect any coevolved hosts (data not shown). Indeed, phenotypic divergence of the infectivity profile of coevolved phage populations closely matched the genetic divergence of the phage genomes as demonstrated by the similar topologies of trees constructed using genetic or phenotypic traits (Fig. 1b).

The increased rate of molecular evolution observed in the coevolved populations was not distributed uniformly across the phage genome (Fig. 2a). Four genes showed significantly increased molecular evolution in coevolved versus evolved phage genomes. SBWP25_0036 (EMBL accession FN594518), which encodes a tail-fibre protein (gp49), had a greater number of sites with mutations in the coevolved versus evolved treatment, and, based on the allele frequencies of mutations at these sites, a substantially higher divergence from the ancestral genotype (number of mutational sites: coevolved, mean 17.6, range 15–20; evolved, mean 12.7, range 11–14; LRT = 4.43, P < 0.05; pairwise genetic distance: coevolved, 10.86 ± 0.90 s.e.; evolved, 4.51 ± 0.19 s.e.; t = 7.69, d.f. = 4.38, P < 0.01). SBWP25_0027, which encodes a structural protein (gp40), also had a greater number of sites with mutations and a higher divergence from the ancestor in the coevolved than the evolved populations (number of mutational sites: coevolved, mean 5.8, range 4–7; evolved mean 1.0, range 0–2; LRT = 20.9, P < 0.001; pairwise genetic distance: coevolved, 1.79 ± 0.23 s.e.; evolved, 0.17 ± 0.05 s.e.; t = 7.69, d.f. = 4.38, P < 0.01). SBWP25_0034 and SBWP25_0035, which encode internal virion structural proteins gp47 and gp48, respectively, also showed higher rates of molecular evolution in coevolved populations, although to a lesser extent than SBWP25_0027 and SBWP25_0036 (pairwise genetic distance: SBWP25_0034 coevolved, 1.21 ± 0.13 s.e.; evolved, 0.34 ± 0.06 s.e.; t = 6.79, P < 0.01; SBWP25_0035 coevolved, 1.23 ± 0.22 s.e.; evolved, 0.48 ± 0.06 s.e.; t = 3.61, P < 0.05). SBWP25_0027 and SBWP25_0036 had a similar density of mutations (Fig. 2c) as each other, but, because of its smaller size, SBWP25_0027 contributed less to the overall divergence of the coevolved genomes from the ancestor than SBWP25_0036. Consistent with the observed evolution of the phage-infectivity range (Fig. 1b), all four of these proteins are predicted to be involved in host attachment¹⁹. In tailed bacteriophages, attachment is a two-step process consisting of an initial reversible adsorption by tail fibres, followed by irreversible adhesion by structural proteins²⁰. The average size of deletions in the tail fibre gene (SBWP25_0036) was positively correlated to the number of bacterial genotypes the phage populations could infect (infectivity range) (Supplementary Fig. 1), suggesting that tail fibres are under strong directional selection for reduced protein length during coevolution but that the precise genetic changes varied between populations. Shortened tail fibres also evolved in the evolution treatment, although to a lesser degree than under coevolution and without a concomitant increase in the infectivity range (Supplementary Fig. 1), suggesting that to some extent shorter tail fibres may also be a general adaptation to laboratory conditions, perhaps through increasing adsorption efficiency. SBWP25_0032, encoding a tail tubular protein (gp45), showed divergence from the ancestor in both evolved and coevolved treatments, but at different sites in each treatment. SBWP25_0027 (gp40) and SBWP25_0036 (gp49) accounted for most of the divergence between replicate, coevolved phage populations (Supplementary Fig. 2).

Figure 2: Patterns of molecular evolution in the Φ2 genome.

a, b, Pairwise genetic distance between each phage population and the ancestral genotype (a), and genetic diversity within each phage population (b). Symbols denote means ± s.e.m. of replicate populations within the coevolved (magenta; n = 5) and the evolved (blue; n = 6) treatments. The locations of mutations within each population are shown as bars underneath each coding sequence, with the colour of each bar indicating the frequency of each mutation within each population (white, rare; red, common). c, Magnified view of identity and frequency of mutations in each population for SBWP25_0027 (gp40) and SBWP25_0036 (gp49). bp, base pairs.
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Coevolved populations also showed higher genetic diversity within populations than did evolved populations (Supplementary Table 1). This was predominantly due to variation in SBWP25_0027 (gp40), as SBWP25_0036 (gp49) displayed within-population diversity in both treatments (Fig. 2b). This high genetic diversity at SBWP25_0036 is surprising given the apparent directional selection for reduced tail-fibre protein length during coevolution. This suggests that SBWP25_0036 polymorphisms may be transient and the result of recurrent continuing selective sweeps, and/or clonal interference. Alternatively, both SBWP25_0036 and SBWP25_0027 may be subject to diversifying or fluctuating selection within populations. Together these genes (SBWP25_0036 and SBWP25_0027) are believed to control host adhesion¹⁹; thus, it is possible that diversity at these genes may determine fine-scale host-specificity differences between phage genotypes. Such phenotypic differences between individual phage clones from the same population have been observed in a previous study in this system¹⁶.

Overall, our results are consistent with accelerated evolution in the coevolution treatment that is driven by selective effects, rather than purely demographic differences between treatments. Demographic effects, such as reduced generation time or population size, or reduced fidelity of DNA replication, would have led to a genome-wide increase in divergence and diversity, which was not observed. By contrast, genetic divergence and diversity for most phage genes were roughly similar in the two treatments (Fig. 2), indicating selection under coevolution on specific infectivity genes/traits, such as that for tail-fibre protein length or infectivity range (Supplementary Fig. 1). Furthermore, whereas greater genetic divergence among coevolved populations (Fig. 1a) could be explicable simply if coevolved populations are also smaller, and hence more susceptible to genetic drift, this explanation is incompatible with the higher genetic diversity observed in SBWP25_0027 in coevolved populations (Fig. 2b). In line with this, there was no significant difference in phage population size between treatments over the course of the experiment (log₁₀(plaque-forming units (p.f.u.) ml^-1) averaged through time: coevolved, 7.39 ± 0.14 s.e.; evolved, 7.51 ± 0.08 s.e.; t = 0.71, d.f. = 9, P = 0.5).

Our results highlight coevolution as a fundamental driver of molecular evolution, and emphasize the utility of genome re-sequencing for quantifying evolutionary dynamics in experimental evolution²¹. We directly demonstrate that antagonistic coevolution accelerates molecular evolution and can generate genetic divergence both between and within populations. By contrast, populations adapting to a fixed host genotype showed a remarkable degree of parallel evolution, indicating genetic constraints on the evolutionary trajectories taken by replicate populations^{22, 23}. Coevolutionary interactions between species are likely therefore to be responsible for rapid evolutionary change within species, potentially causing sufficient between-population genetic divergence to drive speciation itself²⁴.

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Methods Summary

Twelve replicate microcosms (30 ml glass universals containing 6 ml of King’s B (KB) broth) were inoculated with 10⁷ isogenic cells of P. fluorescens SBW25 and 10⁴ isogenic particles of phage Φ2. Cultures were propagated by 12 serial transfers in a static incubator at 28 °C. Transfers for the six coevolving populations involved transferring 60 μl (1%) of culture to a fresh KB microcosm every 48 h. Transfers for the six evolving populations involved isolating phage populations using 0.1 vol. chloroform and centrifuging at 14,000g for 2 min, and then inoculating fresh microcosms with 60 μl (1%) of the phage population plus 10⁷ ancestral SBW25 cells every 48 h. Every two transfers we estimated phage population density by plating dilutions of each phage population onto KB agar plates with a semi-solid overlay bacterial lawn. At the end of the experiment phage DNA was isolated from each population²⁵ and sequenced on a Roche 454 Titanium pyrosequencer. Reads were mapped to the Φ2 reference sequence and mutations were identified and their frequencies calculated using the Roche Newbler mapping tool. We used all non-synonymous changes to construct a phylogenetic tree using Euclidean genetic distance (the square root of pairwise differences), which is suggested as an appropriate metric for molecular variation data¹⁸. To determine phage population infectivity profiles, 20 independent bacterial colonies were isolated from each of the five coevolved populations by plating on KB agar, and these were streaked against a perpendicular line of each phage population that had been previously applied to a KB agar plate¹⁰. A bacterial colony was deemed susceptible if it showed any inhibition of growth after encountering the line of phage.

Full methods accompany this paper.

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