Tansley review. UMR8621, 91405, Orsay, France; 5 Museum National d Histoire Naturelle, 57 rue Cuvier, F-75231, Paris Cedex 05, France; 6 Section

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1 Review Cospeciation vs host-shift speciation: methods for, evidence from natural associations and relation to coevolution Author for correspondence: D. M. de Vienne Tel: Received: 22 November 2012 Accepted: 19 December 2012 D. M. de Vienne 1,2, G. Refregier 3,4,M.Lopez-Villavicencio 5, A. Tellier 6, M. E. Hood 7 and T. Giraud 8,9 1 Centre for Genomic Regulation (CRG), Dr. Aiguader 88, 08003, Barcelona, Spain; 2 Universitat Pompeu Fabra (UPF), 08003, Barcelona, Spain; 3 Universite Paris-Sud, Institut de Genetique et Microbiologie, UMR 8621, 91405, Orsay, France; 4 CNRS, UMR8621, 91405, Orsay, France; 5 Museum National d Histoire Naturelle, 57 rue Cuvier, F-75231, Paris Cedex 05, France; 6 Section of Population Genetics, Center of Life and Food Sciences Weihenstephan, Technische Universit at M unchen, D 85354, Freising, Germany; 7 Department of Biology, Amherst College, Amherst, MA, USA; 8 Universite Paris-Sud, Ecologie, Systematique et Evolution, UMR 8079, 91405, Orsay, France; 9 CNRS, UMR8079, 91405, Orsay, France Contents Summary 347 I. Introduction 348 II. Origin of the cospeciation concept 349 III. Theoretical framework and methods for for cospeciation 349 VI. Conclusion 381 Acknowledgements 381 References 381 Glossary 379 IV. Studies of natural associations reveal the prevalence of host shifts 355 V. Relationship between host symbiont coevolution and symbiont speciation 378 doi: /nph Key words: co-cladogenesis, cophylogenetic analysis, host-jump, host pathogen interaction, host-switch, PARAFIT, TREEFITTER, TREEMAP. Summary Hosts and their symbionts are involved in intimate physiological and ecological interactions. The impact of these interactions on the evolution of each partner depends on the time-scale considered. Short-term dynamics coevolution in the narrow sense has been reviewed elsewhere. We focus here on the long-term evolutionary dynamics of cospeciation and speciation following host shifts. Whether hosts and their symbionts speciate in parallel, by cospeciation, or through host shifts, is a key issue in host symbiont evolution. In this review, we first outline approaches to compare divergence between pairwise associated groups of species, their advantages and pitfalls. We then consider recent insights into the long-term evolution of host parasite and host mutualist associations by critically reviewing the literature. We show that convincing cases of cospeciation are rare (7%) and that cophylogenetic methods overestimate the occurrence of such events. Finally, we examine the relationships between short-term coevolutionary dynamics and long-term patterns of diversification in host symbiont associations. We review theoretical and experimental studies showing that short-term dynamics can foster parasite specialization, but that these events can occur following host shifts and do not necessarily involve cospeciation. Overall, there is now substantial evidence to suggest that coevolutionary dynamics of hosts and parasites do not favor long-term cospeciation. 347

2 348 Review Phytologist I. Introduction Interest in the reciprocal influences between hosts and symbionts has recently increased because of the need to control devastating diseases, to identify or develop biocontrol agents against invasive pests, to improve agricultural production and to decipher the processes of diversification in symbiosis as a widespread lifestyle (Poulin & Morand, 2004). Host symbiont interactions occur over short time-scales, from a single disease cycle in the case of opportunistic and transient infections by parasites, to very long time-scales persisting over multiple host speciation events. Short time-scales have been associated with reciprocal selection pressure between host and parasite, leading to changes in allele frequencies over successive generations (i.e. coevolution in the narrow sense, Clayton & Moore, 1997; see Box 1 for glossary of terms used in this review). By contrast, long time-scales may encompass several speciation events. The concomitant occurrence of speciation in hosts and their symbionts is referred to as cospeciation (Page, 2003). However, the speciation of symbionts may occur independently of host speciation, often through host shifts as the symbiont comes to occupy a new host environment in isolation from the ancestral lineage. Coevolution is used by some authors to describe long-term dynamics as a synonym for cospeciation but this usage may be misleading, as pointed out by some authors (Smith et al., 2008a). We will therefore use coevolution in the narrow sense here: reciprocal selection pressure and resulting micro-evolutionary changes. The often obligate and specialized interactions between hosts and symbionts suggest that any bifurcation of the host lineage is likely to result in the simultaneous isolation of its associated symbionts (Fig. 1a). Thus speciation in one lineage is then pegged to speciation in the other, and this process is referred to as cospeciation. Alternatively, new host symbiont combinations may arise owing to movement and specialization of the symbiont to a new host, on which the symbiont s immediate ancestor did not occur. Symbiont speciation subsequent to such movement is often referred to as host-shift speciation (Fig. 1b, Agosta et al., 2010; Giraud et al., 2010). In this review, we aim to: outline the origin of the concept of cospeciation; provide a description of the various methods developed for determining whether cospeciation has actually occurred, together with their advantages and pitfalls; critically review recent inferences on the history of host symbiont associations based on these methods; and examine the relationship between coevolution in its narrowest sense and symbiont speciation. We caution against the use of coevolution as a synonym for cospeciation because of the implication that short-term dynamics contributes directly to cospeciation in the long term, although the rationale underlying this idea and its potential implications have never been fully articulated. Indeed, recent studies comparing host (a) (b) (c) Fig. 1 Cophylogenetic patterns resulting from different types of parasite speciation. Black lines represent the host lineages; red and blue lines represent parasite lineages. (a) Cospeciation resulting in congruent. (b) Host-shift speciation resulting in congruent, but with shorter branches in the parasite lineages. (c) Host-shift speciations, resulting in incongruent. (d) Cospeciation occurring together with intrahost speciation (also known as duplication) and extinctions, resulting in incongruent without any host shift a host shift can thus be replaced in a reconciliation analysis by several independent events of intrahost speciation and extinctions.

3 Phytologist Review 349 and parasite and theoretical developments relating to parasite specialization and speciation seem to argue against cospeciation being the predominant mode of host and symbiont diversification, despite the occurrence of reciprocal selection over short time-scales. II. Origin of the cospeciation concept The idea of cospeciation was put forward in pioneering studies on avian parasites, such as those of Kellogg (1913) and Fahrenholz (1913), at the beginning of the 20th century. These authors noted that closely related avian parasites, with similar phenotypic features, were associated with closely related host species. They proposed the following hypothesis, known today as the Fahrenholz rule: parasite phylogeny mirrors that of its host (1913). A similar principle was proposed by Szidat some years later (1940): primitive hosts harbor primitive parasites. The idea was that similarity between the parasites of related hosts results from cospeciation (i.e. concurrent and interdependent bifurcation of host and parasite lineages), leading, in turn, to congruent host and parasite. The first studies referring to the Fahrenholz rule did not actually test cospeciation as a hypothesis. Without DNA sequencing being possible at the time it was therefore very important to obtain other forms of phylogenetic information. The narrow host distribution of many animal parasites led researchers to use parasites as characters for inferring phylogenetic relationships between host taxa (Hoberg et al., 1997). Similar hypotheses were proposed for plant parasites (Savile, 1979). Conversely, host taxa were often used as taxonomic criteria for the classification of parasites (see for example Downey, 1962). In both cases, the phylogeny of one partner was used to build the phylogeny of the other, so the two tended to be congruent. As congruence between host and parasite was the most widely accepted criterion for inferring cospeciation, this led to the widespread belief that cospeciation was common. However, this reasoning is clearly circular and the evidence put forward for cospeciation in host parasite associations was for many years inadequate. It was not until the late 1980s that robust, built independently for hosts and parasites, were used to test for cospeciation in a more specific manner (Hafner & Nadler, 1988). III. Theoretical framework and methods for for cospeciation Macro-evolutionary aspects of host parasite associations cannot be observed within the lifespan of a researcher. inferring the effects of interactions have thus been developed based on comparisons of the of the interacting species. These methods, which are described as cophylogenetic methods, are based on the idea that two interacting lineages will have completely congruent if they have diversified exclusively by cospeciation (Fig. 1a). However, it is important to note that congruent topologies can also be obtained after host shifts to closely related hosts under certain realistic conditions of time-span between host-switch and subsequent speciation (Fig. 1b, see de Vienne et al., 2007b for details). Events that reduce the congruence between host and symbiont include: (1) host-shift speciation (Fig. 1c), when a population of the symbiont species adapts to a new host followed by speciation (under certain conditions, see de Vienne et al., 2007b for details); (2) speciation of the symbiont without speciation of the host or host switching, also known as intrahost speciation or duplication; and (3) symbiont extinction (Fig. 1d). Cophylogenetic methods can be divided into two main classes (Table 1). The first class encompasses methods aiming to reconstruct the evolutionary history of the host and parasite lineages, to infer the nature and frequency of different evolutionary scenarios by comparing phylogenetic trees (event-based methods). Diversification by cospeciation is generally inferred if the number of cospeciation events is significantly greater than the number of cospeciation when randomizing the associations, although this merely indicates topological congruence and not necessarily cospeciation. Significant congruence can indeed be obtained after repeated host shifts, as noted above (Fig. 1b). The second class of methods tests the overall congruence between the host and parasite (i.e. topology or distance-based methods using the similarity and/or symmetry in the time of divergence between hosts and parasites) and it is generally considered that high levels of congruence provide evidence of frequent cospeciation although this conclusion may be similarly unwarranted (Fig. 1b). We will explain these two approaches in more detail in the following text and provide a brief overview of the existing cophylogenetic tools (summarized in Table 1). Finally, we will discuss some of the limitations of these methods in the light of recent results on the likelihood of host and parasite trees congruence in the absence of cospeciation. 1. Event-based methods The first event-based method developed was Brooks Parsimony Analysis (BPA; Brooks, 1981). It opened the way for such methods but considered parasites as character states of the hosts. The parasitic character states are assigned to each branch in the host phylogeny and the most parsimonious reconstruction, the one with smallest number of parasite presence vs absence state changes in the host phylogeny, is retained. If host and parasite are topologically congruent, then each internal branch in the host phylogeny is assigned one parasite state so that no state transition is required and cospeciation is inferred along the whole phylogeny. Although BPA was widely used in the 1980s and early 1990s, it received heavy criticism, particularly because of its requirement for a large number of a posteriori interpretations (Page, 1994). Another method, reconciliation analysis, proposed by Page (1990), considers parasites as evolutionary lineages rather than character states. Implemented in the COMPONENT program (Page, 1993), it estimates the minimum number of extinctions and intrahost speciations required to reconcile the separate host and parasite. Cospeciation is explicitly considered as the most parsimonious hypothesis. Page (1994) subsequently added host-shift speciation in the TREEMAP 1 program. This method tries to reconcile host and parasite by maximizing the

4 350 Review Phytologist Table 1 Methods developed for the reconstruction or investigation of the history of the association between interacting host and parasite species (or other symbionts) Event-based methods Basic concept: consider cospeciation as the most parsimonious explanation for congruence between host and parasite trees Method Main feature Software/ method Estimation of the best reconstruction Advantages Disadvantages References Availability Brooks Parsimony analysis Reconciliation analysis Cost-based methods Considers parasites as character states of the hosts Mapping of the parasite phylogeny onto the host phylogeny. The best scenario may be that with the minimum number of or the least costly Cost associated with each event, no graphical representation BPA Minimum number of character state changes in the host phylogeny (parsimony) Component Minimization of the number of extinctions and intrahost speciations and maximization of the number of cospeciations TREEMAP 1* Minimization of the number of host shifts and maximization of the number of cospeciations TREEMAP 2* Minimization of the total cost of the reconstruction, a cost associated with each event Can handle more than just 1 : 1 correspondence between hosts and parasite tips Host shifts are taken into account Gives a graphical representation of the history of the host-parasite association Includes a test to assess whether the number of cospeciation events is higher than for random (thus also listed with topology-based methods) Cost is associated with each eventimplementation of the jungles method (Charleston, 1998), an algorithm allowing the rapid identification of the optimal reconstructions taking costs into account and ensuring the feasibility of each reconstruction TARZAN Possibility of defining the timing of nodes in the parasite phylogenyvery fast JANE Possibility of defining the timing of nodes in both the parasite and host Possibility of defining different hostswitch costs independentlyinteractive graphical interface Faster than TREEMAP 2 Possibility of defining the maximum permitted host-switch distance TREEFITTER Minimization of the total cost of the reconstruction, a cost being associated with each event Probability associated with each type of eventcosts of each event are set by the user Multiple equally parsimonious reconstructions for large and/or for multiple associations between host and parasitecospeciation considered the most parsimonious hypothesis No host shifts considered Cospeciation considered the most parsimonious hypothesis Needs 1 : 1 correspondence between hosts and parasites Cospeciation considered the most parsimonious hypothesis The number of parasites infecting ancestral host species can be unreasonably high Can give a very large number of reconstructions Does not guarantee that reconstructions involving more than one host shift are realistic (i.e. there may be timing incompatibilities) Needs one-to-one correspondence between host and parasite tips Cospeciation considered the most parsimonious hypothesis Very slow for large trees Does not guarantee that the solution is optimal Cannot always find a solution even when there is one Cospeciation considered the most parsimonious hypothesis Slower than TARZAN Cospeciation considered the most parsimonious hypothesis Cospeciation considered the most parsimonious hypothesis Cospeciations cannot be more costly than host-shift speciations Possible timing incompatibilities leading to potentially erroneous conclusions Brooks (1981); Brooks & McLennan (1991) To be implemented by the user. Refer to Brooks et al. (2001) for details Page (1993) zoology.gla.ac.uk/ rod/cpw.html Page (1994) zoology.gla.ac.uk/ rod/treemap.html Charleston (1998) au/engineering/it/ ~mcharles/ software/ treemap/ treemap.html Merkle & Middendorf (2005) informatik. uni-leipzig.de/ Download. html Conow et al. (2010) edu/~hadas/jane/ Jane1/index.html Ronquist (1995) net/projects/ treefitter/

5 Phytologist Review 351 Table 1 (Continued) Event-based methods Basic concept: consider cospeciation as the most parsimonious explanation for congruence between host and parasite trees Method Main feature Software/ method Estimation of the best reconstruction Advantages Disadvantages References Availability Bayesian methods Combination of two models, one estimating the probability of a given evolutionary scenario and one used to infer host and parasite Determines the most likely evolutionary scenario leading to the observed host and parasite DNA sequences, not their Does not consider the of the host and the parasites to be known Cospeciation considered the most parsimonious hypothesis Only considers host shift and cospeciation Works only for a 1 : 1 correspondence between host and parasite tips Huelsenbeck et al. (2000, 2003) Theoretically, upon request to author. But seems unavailable Topology and distance-based methods Basic concept: does not consider any event. These are simple tests of independence or similarity between trees or alignments Method Main features Software/method Input data Advantages Disadvantages References Availability Test of independence Looks at the probability of observing a certain level of congruence between two trees with respect to expectations if the trees were independent Icong index Trees. No branch lengths Methods based on Mantel test betweendistance matrices Sequence alignments (converted into distance matrices) PARAFIT Trees or alignments (converted into distance matrices) Method based on Pearson s correlation analysis between host distances and parasite distances Trees or alignments (converted into distance matrices) No random trees need to be generated for for higher levels of congruence than expected by chance Not restricted to 1:1 correspondence between host and parasitesallows of the contribution of each individual host-parasite link to the total congruence statistic (taking into account both topological congruence and branch lengths) Not restricted to 1:1 correspondence between host and parasitesapparently more accurate estimation of the contribution of each individual host parasite link to the total congruence than PARAFIT MRCAlink algorithm Trees Applicable to methods like PARAFIT: making it possible to take phylogenetic nonindependence into account Works only for a 1 : 1 correspondence between host and parasite tipsconsiders trees to be correct Does not account for phylogenetic nonindependence (Felsenstein, 1985) Does not account for phylogenetic nonindependence (Felsenstein, 1985) Considers trees to be correct (if trees used) Considers trees to be correct (if trees used) Considers trees to be correct TREEMAP 1 Trees Considers trees to be correct TREEMAPTREEMAP 2 Trees Based on the jungles method.several randomization test statistics available Considers trees to be correct de Vienne et al. (2007a) u-psud.fr/bases/ upresa/pages/ devienne/ Hafner et al. (1994) To be implemented by the user. Refer to Hafner et al. (1994) for details Legendre et al. (2002) Hommola et al. (2009) leeds.ac.uk/ ~kerstin/ and Hommola et al. (2009) Schardl et al. (2008) net/research.php Page (1994) zoology.gla.ac.uk/ rod/treemap.html Charleston (1998) au/engineering/it/ ~mcharles/ software/treemap/ treemap.html

6 352 Review Phytologist Table 1 (Continued) Topology and distance-based methods Basic concept: does not consider any event. These are simple tests of independence or similarity between trees or alignments Method Main features Software/method Input data Advantages Disadvantages References Availability Theoretically, upon request to author. But seems unavailable Huelsenbeck et al. (1997) Only topologies are considered, not branch lengths Sequence alignments Does not consider the trees to be known Maximum likelihood method Estimates the probability of observing the actual host and parasite DNA sequence variation assuming their are congruent Test of similarity or identity Theoretically, upon request to author. But seems unavailable Huelsenbeck et al. (1997) Only topologies are considered, not branch lengths Bayesian method Sequence alignments Does not consider the trees to be known Theoretically, upon request to author. But seems unavailable Huelsenbeck et al. (1997, 2003) Sequence alignments Does not consider the trees to be knowntests for temporal congruence, the null hypothesis being that the speciations occurred at the same time Second maximum Likelihood method *TREEMAPTREEMAP is also a topology-based program. All websites listed here have been verified at the date of submission of the paper. number of cospeciations and minimizing the number of host-shift speciations. There are no constraints on the numbers of intrahost speciations or extinctions or on numbers of parasites present on internal nodes, so the number of parasites infecting ancestral host species or number of intrahost speciations may be assumed to be unreasonably high (Refregier et al., 2008). A graphical representation of the history of the host parasite association is provided, although this representation is most often unlikely to be correct as exact costs for the events are impossible to assess a priori. TREEMAP 1 also determines whether the number of cospeciation events in the host and parasite trees compared is greater than that in random. This is the most useful part of the program, but it is often taken as a test for cospeciation, while in fact it is a test of topological congruence. Indeed, 100% of inferred events will be cospeciations in cases of complete congruence, while this can result from host-shift speciation (Fig. 1b, de Vienne et al., 2007b). Overall, reconciliation analyses overestimate cospeciation events because (1) they assume, a priori, that cospeciation is more likely than host-shift speciation or other events this assumption likely being unfounded (Ronquist, 1995) and (2) they interpret congruence as evidence for cospeciation, while this is not necessarily the case (Fig. 1b). The most recent version, TREEMAP 2, more rapidly identifies optimal phylogenetic reconstructions and takes into account the temporal feasibility of each reconstruction (host shifts only occurring between hosts present at the same time; for details on the method and its implementation in TREEMAP 2, see Charleston, 1998; Charleston & Perkins, 2003). Similar methods with faster computation have since been developed. TARZAN (Merkle & Middendorf, 2005) handles by allowing uncertainty on the age of parasite nodes (associating each node to a time zone) and selecting the cost of each event. JANE (Conow et al., 2010) takes into account uncertainty in time for the host phylogeny without substantially increasing the computation time. The first series of methods allowing the user to attribute a cost to each evolutionary event (cospeciation, host-shift speciation, intrahost speciation and extinction) was developed by Ronquist (1995). These cost-based methods find the most parsimonious scenario by minimizing the total cost. The most popular cost-based method is that implemented in TREEFITTER software (Ronquist, 1995). TREEFITTER estimates the number of events of each type that could explain the observed congruence between the two. It then associates each event with the probability that it arose by chance, calculated by permutations of the host and/or parasite leaves on the phylogeny. TREEFITTER finds the optimal numbers of each type of event by minimizing the total cost of the reconstruction, but it does not allow cospeciations to be more costly than hostshift speciation. All the methods presented consider the host and parasite to be known and fully resolved trees, and therefore they are sensitive to the selection of different optimal trees. The Bayesian method developed by Huelsenbeck et al. (2000, 2003) overcomes this problem. This method aims to determine the most likely evolutionary scenario leading to the observed host and parasite DNA sequences, rather than their. It is based on two simple stochastic models: one for host-shift speciations and the

7 Phytologist Review 353 other for DNA substitutions. The two models are mixed and subjected to Bayesian analysis. 2. Topology- and distance-based methods All the methods presented earlier and summarized at the top of Table 1 are based on the idea that host and parasite should be identical (congruent) in the absence of host-shift speciation, extinction and intrahost speciation. This is a logical conclusion of the principles first formulated by Fahrenholz (1913) and Szidat (1940) (see Section II, Origin of the cospeciation concept). Yet, host shifts can also lead to phylogenetic congruence under realistic conditions (Fig. 1b, see de Vienne et al., 2007b for details). Another set of methods is based on statistical tests for congruence between host and parasite. These methods do not directly consider high levels of congruence to constitute proof of cospeciation. Instead, they compare the probability of observing a certain level of congruence between two trees, with expectations based on the independence between trees. By linking the results obtained with such methods to the common history of the interacting lineages it is possible to obtain an a posteriori interpretation that is not integral to the test. This approach may thus be considered less biased than event-based or event- and costbased methods, such as those already presented. These methods can be assigned to different classes according to the null hypothesis tested (similarity or independence, Huelsenbeck et al., 2003) and the data used for the test (trees, distance matrices or raw sequence alignments; Light & Hafner, 2008). Tests of independence are based on comparisons of the topological or genetic distances of the focal host parasite association with a distribution of distances computed from a large number of randomly generated trees. If the distance of interest is significantly smaller than expected by chance, the association is considered to be significantly congruent. This principle is similar to that underlying the test implemented in TREEMAP 1. One of the weaknesses of these methods lies in the large number of random trees that must be generated de novo for each new comparison of trees. A test of tree independence has been proposed to overcome this problem, being based on the use of previously simulated associations (de Vienne et al., 2007a, 2009a; Kupczok & von Haeseler, 2009). Tests of independence have also been used to evaluate temporal congruence in the speciation histories of hosts and parasites. Repeated cospeciation events imply the simultaneous occurrence of speciation events (i.e. temporal congruence) and thus proportional branch length and identical dates for the nodes in the compared (Fig. 1a). One method (Hafner et al., 1994) tests whether the two species have accumulated similar numbers of genetic differences. Input data include host parasite species associations and the alignment of one specific locus (or several concatenated loci) for hosts and parasites. These alignments are used to calculate distance matrices. The significance of the correlation between the two matrices is then assessed using a Mantel test (Hafner et al., 1994). A second method compares matrices of branch lengths from host and parasite trees in the same way (Hafner et al., 1994; Page, 1996). If molecular clocks are available for both host and parasite it is possible to compare the estimated absolute ages of the nodes in the two trees. The determination of identical ages for each node is actually the only way to establish cospeciation with confidence. Indeed, identical relative divergence times, as deduced from proportional branch lengths, may exist in some host parasite associations in which speciation times are not identical. This can be the case when parasites jump preferentially onto closely related hosts and take a time to speciate that is proportional to the phylogenetic distance between initial and novel hosts (Charleston & Robertson, 2002). Furthermore, while Mantel tests account for statistical nonindependence in matrices, they do not account for phylogenetic nonindependence (Felsenstein, 1985; illustrated in Fig. 2), in that the data for divergence at ancient nodes include the same information as those for divergence at more recent nodes along the same branches (Felsenstein, 1985; Schardl et al., 2008). All the points used in the distance matrices are thus phylogenetically nonindependent, which should preclude the use of a Mantel test. PARAFIT (Legendre et al., 2002) tests the independence of host and symbiont genetic or patristic distances (patristic distances are calculated by summing the lengths of the branches in the estimated tree, joining each pair of taxa). This method is advantageous because it can (1) deal with cases in which multiple symbionts are associated with a single host, or where multiple hosts are associated with a single symbiont, and (2) be used to assess the contribution of each individual host symbiont link to the total congruence statistics. The host sequences and/or tree and the symbiont sequences and/or tree are transformed into distance matrices. A sum of the squared distances gives a value for the overall similarity between trees (ParafitGlobal), which is compared with a distribution of ParafitGlobal values obtained by permutations to assess significance. The contribution of each individual link to the overall congruence between trees is assessed by removing the links one by one. However, the problem of nonindependence of (Fig. 2, Felsenstein, 1985) also applies to this method. Hommola et al. (2009) recently introduced a new permutation method for evaluating the independence of host and parasite. This test is based on the calculation of Pearson s correlation coefficients between host distances and parasite Fig. 2 Illustration of the problem of phylogenetic nonindependence. Black lines represent the host lineages; color lines represent the parasite lineages. The phylogenetic distance between the taxa a and c is not independent of the phylogenetic distance between the taxa b and c, as a great proportion of these distances share an evolutionary history (in green). Similarly, the distances between c and d, and between c and e count as twice the distance between c and the common ancestor of d and e (in blue). Such a pseudoreplication can inflate the degree of congruence.

8 354 Review Phytologist distances, considering all pairs of interacting hosts and parasites. This correlation coefficient is then compared with that obtained after random permutations of the data, retaining the observed interaction links. This method is thus a generalization of the Mantel test, making it possible to test data in the absence of a one-to-one correspondence between hosts and parasites. This method seems to be more powerful than PARAFIT, with more accurate estimated P-values, although this superior performance may be attributable to the larger number of permutations performed ( , vs only 99 for PARAFIT). Finally, Schardl et al. (2008) proposed a modification for programs such as PARAFIT, taking into account the nonindependence of pairs of species from the same branch and using a method similar to the phylogenetic independent contrasts (PIC) method proposed by Felsenstein (1985). The algorithm, MRCAlink (MRCA for Most Recent Common Ancestor), identifies phylogenetically independent pairs between host and parasite trees and the reduced host and parasite matrices can then be compared. Fig. 3 Illustration of the recommended approach and pitfalls to avoid for inferring the history of host parasite associations. 3. Pitfalls in the theoretical framework when considering host parasite associations All the methods presented above and summarized in Table 1 have drawbacks (Nieberding et al., 2010). These problems include for congruence on the basis of estimated without taking into account uncertainty in the inference (TREEMAP, TREEFITTER or I cong, which require fully resolved trees), phylogenetic nonindependence (TREEMAP, TREEFITTER), tests considering only topologies and thus ignoring branch lengths (I cong, Huelsenbeck s methods) or underestimation of the potentially high probability of host-shift speciations (TREEMAP). A key issue that is rarely discussed (but see Hafner & Nadler, 1988; Hafner et al., 1994) is the common but potentially erroneous interpretation of these tests, specifically that congruence between host and parasite results from frequent cospeciations between host and parasite, whereas incongruence results from host-shift speciation, extinction, intrahost speciation and other evolutionary scenarios. A good illustration of the limitations of reconciliation methods was provided by Lanterbecq et al. (2010), who reviewed studies based on the use of TREEMAP to reconstruct the history of host parasite associations. Most of the examples in their Table 5 (Lanterbecq et al. 2010) refer to studies in which host shifts were eventually identified because of asynchronous splitting events as the main mode of parasite speciation, whereas the number of host shifts suggested by TREEMAP was smaller than numbers of cospeciation events. This was the case, for example, for legume-feeding insects and plants of the Genistae (Percy et al., 2004), for which 16 cospeciations and no host shifts were inferred and for algal and fungal mutualists (lichens, Piercey-Normore & DePriest, 2001), for which cospeciations and 3 5 host shifts were inferred. This example also illustrates one of the greatest pitfalls of eventbased methods (Fig. 3); the cospeciation events could only be inferred while assuming unreasonably large numbers of intrahost speciations and sorting events (29 intrahost speciations and 220 sorting events for the plant-insect interaction and 7 9 intrahost speciations and sorting events for lichens). Similarly unlikely inferences were also made in a cophylogenetic study between neobatrachian frogs and their parasitic platyhelminthes, for which 22 cospeciations were estimated for 26 species pairs, but with 10 intrahost speciations and 16 extinction events (Badets et al., 2011). The PARAFIT test was not significant and the tree node ages appeared to be inconsistent with cospeciations. The large number of cospeciation was thus clearly misleading. The default cost values for cospeciation, host-shift, intrahost speciation and sorting events in reconciliation methods thus bear little resemblance to the actual probabilities of these events (see Section IV Studies of natural associations reveal the prevalence of host shifts). For example, if parasite extinction occurs in a host lineage and this host lineage is then recolonized through host-shift speciation, reconstructions by event-based methods tend to suggest the occurrence of intrahost speciations in the distant past, followed by many extinction events (Fig. 1d). This tendency to avoid inferring host shift makes it necessary to include many more evolutionary steps to reconcile the two than reconstructions involving a host shift. Experimental and theoretical studies have shown that congruence between host and parasite can be achieved in the absence of cospeciation if there is a preferential host shift towards closely related hosts (Charleston & Robertson, 2002) and under certain conditions of time lag between the switch and the following speciation (Charleston & Robertson, 2002; de Vienne et al., 2007b; Fig. 1b). Preferential host shifts towards related hosts have been found using experimental cross-inoculations in many host parasite associations (Gilbert & Webb, 2007; de Vienne et al., 2009b) and the possibility of topological congruence without cospeciation highlights the importance of temporal congruence between host and parasite, as only such tests can validate the occurrence of cospeciation events (Charleston & Robertson, 2002; Hirose et al., 2005; Mikheyev et al., 2010). Another pitfall of cophylogenetic studies is the failure to delimit species correctly as this may lead several methods to artificially

9 Phytologist Review 355 inflate congruence when generalist species are found on closely related hosts (Refregier et al., 2008). Indeed, species delimitation in parasites is often difficult and generalist symbionts often infect closely related hosts; congruent intraspecific nodes then artificially increase the number of cospeciations inferred (Fig. 4). Multiple individuals per parasite species are often included in analyses, particularly when these species are generalists (Light & Hafner, 2007; Bruyndonckx et al., 2009), which can cause the same bias towards congruence. A last issue in cophylogenetic studies is the frequent use of mtdna. It is increasingly recognized that a single marker cannot reliably be used to reconstruct species, and this is particularly true for mtdna, which is more prone to introgression than nuclear DNA (Coyne & Orr, 2004) and can be subject to strong selective pressures and low recombination rates (Balloux, 2010). We present the approach we recommend to test for cospeciation in Fig. 3, to avoid as much as possible the different pitfalls discussed. IV. Studies of natural associations reveal the prevalence of host shifts The methods described earlier have been used in diverse host parasite associations to test cospeciation hypotheses. After > 50 yr of research, convincing examples of cospeciation between host and symbiont seem to be the exception rather than the rule. We have performed an extensive search in ISI Web of Knowledge, and we summarize in Table 2 and Fig. 5 the studies reporting cophylogeny analyses. We include the system and its type of symbiosis, the conclusion inferred by authors, the type of phylogenetic data, the results of cophylogenetic analyses, the results of the test for temporal congruence (when available) and our own conclusions. Convincing cospeciation between host and symbiont trees is seldom found except for a few mutualist associations, most often involving vertically transmitted symbionts. Host-shift speciation has been recognized for some time as the main mode of speciation in many systems involving plant viruses, plant fungi, plant Fig. 4 Illustration of the problem of sampling multiple individuals per (cryptic) species. Black lines represent the host lineages; red lines represent the parasite lineages. Host shifts are prevalent and result in incongruent. However, the intraspecific nodes increase the congruence without representing cospeciation, only intraspecific divergence. parasitoids and animal viruses (Table 2, Fig. 5). Host shifts are also frequent in phytophagous insects (for an extensive review see Nyman, 2010). In addition, we show here that even in associations where cospeciation has been claimed to occur together with other events, host shifts may be the only convincingly demonstrated mode of speciation. Indeed, in all these cases where absolute dates could be obtained, they indicated more recent speciation by symbionts, even when cophylogenetic analyses suggested cospeciation as the major mode of diversification. Furthermore, the number of duplications inferred is more often unrealistically high, casting doubt on the conclusion of cospeciation (Table 2, Fig. 5). Indeed, when host-shifts are considered costly, they will be replaced in most reconstructions by duplications and extinctions (Fig. 1d). Examples in the literature are also found illustrating that significant congruence between host and symbiont may occur without cospeciation, by the preferential occurrence of host shifts between closely related hosts under certain conditions of time lag between host shift and subsequent speciation. Indeed, most of the few studies in which absolute node dates were inferred have shown the dates of speciation to be incongruent for the interacting host and parasite species, despite the inference of cospeciation events by topology-based analyses (Charleston & Robertson, 2002; Sorenson et al., 2004; Huyse & Volckaert, 2005). Good illustrations are also found for our claim that mere correlations between branch lengths without absolute calibrations based on fossils are not sufficient to show temporal congruence. In a study analysing in a tritrophic association between Piper plants, Eois moths and their Parapanteles parasitoids (Wilson et al., 2012), the branch lengths of the were found to be significantly correlated, but dating analyses revealed that the correlation resulted from host conservationism (i.e. the moth radiated preferentially on closely related hosts after host shifts or closely related moths radiated on the same hosts) rather than. Another study has shown that correlations between branch lengths of the of Caryophyllaceous plants and their anther smut fungi most likely result from host shifts occurring preferentially between closely related hosts (Refregier et al., 2008). The well-known association between pocket gophers and their chewing lice (Hafner et al., 1994, 2003) remains the textbook example of cospeciation, and it played a central role in the development of the methods presented here. Interestingly, the high level of cospeciation in this system may be linked to the life history and ecology of these parasites and their hosts: pocket gophers (Rodentia: Geomyidae) are herbivorous rodents that spend most of their life in tunnels that they do not share with other individuals. Species of pocket gophers are mostly allopatric, decreasing the likelihood of their parasites shifting to other hosts. Moreover, the chewing lice (family Trichodectidae) are obligate parasites that spend their entire life on the host, with no dispersal stage (Reed & Hafner, 1997; Clayton et al., 2004). Experimental studies have shown that lice can colonize new gopher species, suggesting that limited dispersal is the main constraint preventing host shifts. The combination of the solitary and allopatric host lifestyle and the limited dispersal ability of the parasite may account for the rarity of host-shift speciation in this system (Clayton & Johnson, 2003;

10 356 Review Phytologist Table 2 Literature review of studies reporting cophylogeny analyses, with the type of association as we inferred it, the host symbiont system, the type of symbiont (parasite or mutualist), the number of taxa analysed, the methods used for topological incongruence, the main conclusion ( host shift), the percentage of cospeciation, data used for the host and symbiont, temporal congruence for the nodes of the two and reference 1a Devescovinid flagellates (Devescovina spp.) and Bacteroidales ectosymbionts 1a Trichonympha ter mite gut flagellates and Candidatus bacteria 1a Brachycaudus aphids and Buchnera aphidicola bacteria 1a Leafhoppers (Cicadellinae) and their two main symbionts: Sulcia (Bacteroidetes) and Baumannia (Prote obacteria) 1a Plataspidae Stinkbugs and c- Proteobacteria Codivergence Mutualistic termite gut flagellates and their bacterial symbionts Codivergence Mutualistic termite gut flagellates and their bacterial endosymbionts Codivergence Vertically transmitted mutualistic bacteria of aphids Codivergence Leafhoppers and two endosymbiont species providing nutrients Strict cospeciation Stinkbugs of the family Plataspidae, and their highly specific mutualistic gut endocellular c- Proteobacteria. Bacteria vertically transmitted 7 pairs of Devescovina flagellates and Bacteroidales ectosymbionts Flagellate and bacteria from 11 termite species 56 specimens of the host Brachycaudus, representing 27 species 29 leafhoppers species and their symbionts Three genera, seven species, and 12 populations of stinkbugs and their bacteria TREEMAPTREEMAP 100% SSU rrna for flagellates. For bacteria : 16S TREEMAPTREEMAP 7/11 cospeciation events TREEMAPTREEMAP and PARAFIT Parsimonybased ILD test, Shimodaira Hasegawa test and TREEFITTER 34 cospeciation events, 1 host shift; PARAFIT, also indicated significant The results of all tests suggest that the diversification of both endosymbionts was largely or entirely dependent on the phylogenetic history of their host leafhoppers TREEMAPTREEMAP Strict congruence (6 events) For both: SSU rrna genes For the bacteria: TrpB and two intergenic regions For the host: CytB, COI and ITS2 Host: COI, COII, 16S rdna and H3. For the symbionts: 16S rdna For the host: 16S rrna gene For the bacteria: 16S rrna gene Very good correlation of the host and symbiont coalescent times (r 2 = 0.98), but no absolute calibration Desai et al. (2010) Not tested Ikeda- Ohtsubo & Brune (2009) Strong correlation between the divergences in the two lineages, (R = ), the y-intercept was not significantly differ ent from 0 Likelihood-ratio test to assess whether the 16S rdna of Baumannia and Sulcia were evolving with a constant rate across different hostassociated lineages Jousselin et al. (2009) Takiya et al. (2006) Not tested Hosokawa et al. (2006)

11 Phytologist Review 357 1a Cockroaches (Polyphagidae, Cryptocercidae and Blattidae) and their Blattabacterium bacteria 1a Deep sea clams (Vesicomya, Calyptogena, and Ectenagena) and bacteria 1b Crematogaster ants and Macaranga plants 1b Camponotus Ants and their bacteria (Candidatus Blochmannia) Cospeciation Blattabacterium vertically transmitted intracellular mutualists (that presumably participate in the recycling of uric acid) that are located in specialized cells of cockroaches Four cockroach species and their Blattabacterium bacteria Cospeciation Vesicomyid clams depends entirely on their sulfur-oxidizing endosymbiotic bacteria Cospeciation Highly species specific mutualistic interaction between Crematogaster ants and Macaranga plants, but two ant species have multiple hosts 16 clam species and their associ ated bacteria Nine Macaranga plant species and four species of Crematogaster ants Cospeciation Mutualism between ants and their bacterial associates, that are located within bacteriocytes and are transmitted vertically although some horizontal transmission has been suggested 16 host species and their bacteria Component Lite, Templeton test and Shimodaira and Hasegawa test Kishino Hasegawa criteria Tree Mapping in Component Shimodaira Hasegawa test Host and symbiont topologies were found to be highly similar, and tests indicated that they were not statistically different The topologies are not significantly different The congruence of the two is statistically significant although there is a major disagreement No conflict on wellresolved nodes For the bacteria: 16S rdna. For the host: 18S rdna and COII, 12S rdna, and 16S rdna combined with morphological data already published Bacteria: 16S rdna; Clams: 16S and mtdna COI For the plant: phylogeny already published based on morphology and the nuclear ITS. For the ants: COI For the bacteria: 16S ribosomal DNA [rdna], groel, gida, and rpsb. For the host: the nuclear EF-1aF2 and COI and COII Congruence of divergence times Lo (2003) Congruent dates based on fossils Peek et al. (1998) Tertiary climate and the restriction of Macaranga to sea sonal forests sug gest that this plant clade diversified in the late Tertiary, which corresponds to the diversification period of the ants Correlated rates of synonymous substitution (ds) in the two Itino et al. (2001) Degnan et al. (2004)

12 358 Review Phytologist 2 Tephritinae fruit flies and bacteria (Candidatus spp.) 2 Makialgine mites (Acari, Psoroptidae, Makialginae) and Galagalges primates 2 Crinoids (Echinodermata) and myzostomids (Myzostomida, Annelida) 2 Rodents (Muridae: Sigmodontinae) and their hoplopleurid sucking lice (Phthiraptera: Anoplura) 62.5% of nodes with inferred Mutualistic relationships between fruit flies and their extracellular bacterial symbionts (some vertically transmitted) 33 Tephritinae flies species in 17 different genera Mainly cospeciations and duplications Permanent and highly specialized ectoparasite mites For the parasite : 9 taxa Mainly cospeciation and losses Cospeciation but with prevalent host switching Obligate and highly specific commensal marine worms Generalist parasitic sucking lice of rodents 16 species of crinoids (belonging to 6 different families) and their 16 associated myzostomids (belonging to 15 species) 15 distinct louse species and 19 rodent species TREEMAP, PARAFIT and Shimodaira Hasegawa likelihoodbased test TREEFITTER and TREEMAP TREEMAP, PARAFIT and KH and SH tests TREEMAP and TREEFITTER A maximum of 20 events (= 10 cospeciations), from 6 to 17 losses, 1 to 6 switches and 12 to 14 duplication events 4/5 cospeciation, but at least as many duplication events as cospeciation events 8 or 9 cospeciations, but 7 10 losses and 3 4 host shifts TREEMAP:12 20 s, duplications, extinctions, 3 4 host switchings. TREEFITTER:6 9 s, 0 duplications, 0 3 extinctions, 6 10 host switchings For the host: 16S rdna and COItRNALeu-COII; for the symbiont: 16S rdna Morphological traits For crinoids: 18S rdna, and COI; for myzostomid: 18S rdna, 16S rdna, and COI CO I and EF1a Not tested Mazzon et al. (2010) Not tested Bochkov et al. (2011) Not tested Lanterbecq et al. (2010) Not tested Smith et al. (2008b)

13 Phytologist Review Fig trees (Moraceae, Ficus) and fig wasps 2 Geomydoecus lice on Cratogeomys pocket gophers 2 Figs (Ficus spp., Moraceae) and wasps (Hymenop tera, Agaonidae, Chalcidoidea) Significant cospeciation but with host shifts and duplications Pollinating and nonpollinating fig wasps and Ficus 23 fig species TREEMAP and PARAFIT Codivergence Chewing parasite lice and their pocket gopher hosts 41 specimens of chewing lice from seven species. Gophers: 16 individuals from 3 species TREEMAP, PARAFIT, KH and SH tests, Diffuse coevolution Host specific mutualistic pollinator and nonpollinator wasps of figs 411 individuals from 69 pollinating and nonpollinating fig wasp species, 17 species of Urostigma figs TREEMAP and PARAFIT Pollinators: no significant cospeciation in the tree with all species, but significant cospeciation in the combined tree with fewer species. Non pollinators: significant cospeciation, but with almost as many duplications needed as cospeciation events TREEMAP: significant cophylogeny between host and parasites, 16 events, 6 8 duplications, 3 4 extinctions, 3 4 host switches Significant congruence. Hostswitching and multiple wasp species per host are however ubiquitous; 1 6 cospeciations, 1 10 duplications, 4 68 sorting events, 0 1 host switch Figs: two nuclear DNA fragments (ETS and ITS). Wasps: 28S and ITS2 Louse: COI and EF-1a For the host, COI Wasp phylogeny based on COI Significant correlation of MRCA, with intercept at 0 but slope < 1 Jousselin et al. (2008) Regression analyses of estimated branch lengths in gophers and lice showed intercepts that were not significantly different from zero Light & Hafner (2007) Not tested Marussich & Machado (2007)

14 360 Review Phytologist 2 Pelecaniform birds and Pectinopygus lice 2 Wing lice of the genus Anaticola (Is chnocera) and sev eral genera of fla mingoes and ducks 2 Polyomaviridae (polyomaviruses) and vertebrates (avian and mammals) Significant congruence but with host shifts Host-specific parasitic lice that infect a single order of birds (Pelecaniform) 17 Pectinopygus species and their pelecaniform host Cospeciations and host shifts Parasitic lice infecting flamingoes and ducks 43 genera of avian lice Codivergence Parasitic doublestranded DNA viruses, which are widely distributed among vertebrates; avian viruses infect a broader host range than the highly specific mammalian polyomaviruses 72 full genomes: nine mammalian (67 strains) and two avian (5 strains) polyomavirus TREEFITTER, TREEMAP, ILD, and PARAFIT Significant overall congruence. However, without invoking any host switching, TREEMAP had to introduce cospeciation events, 5 6 duplications, and sorting events. Allowing host shifts: cospeciations, 5 6 duplications, 3 19 losses, and 0 6 switches TREEMAP Codivergences = 4 5, duplications = 5 6, losses = 1 32, host switches = 0 6 TREEMAP Codivergences = 12, duplications = 8, losses = 2 13, host switches = S rrna, 16S rrna, COI, and nuclear wingless and EFl-a gene. For the host: 12S rrna, COI, and ATPases 8 and 6 genes nuclear EF-1a, 12S and cytochrome oxidase I (COI). Avian phylogeny already published For the virus: the main five genes of the genome (VP1, VP2, VP3, large T antigen, and small T antigen) Significant correlation between coalescence times (r = 0.94). The intercept of the slope is positive but not significantly dif ferent from zero Hughes et al. (2007) Not tested Johnson et al. (2006) Not tested Perez-Losada et al. (2006)

15 Phytologist Review Mealybugs Hemiptera (Subfamily Pseudococcinae) and endosymbiont bacteria 2 Plants (Fabaceae, Asteraceae, Rosa ceae, Cyperaceae) and gall-forming nematodes (Tylenchida: Anguinidae) 2 Doves and pigeons (Aves: Columbiformes) and feather lice in the genus Columbicola (In secta: Phthiraptera) 2 Feather mites (Subfamily Avenzoariinae) and birds (Charadriiformes, Procellariiformes, Pelecaniformes, Ciconiiformes, and Falconiformes) Codivergence and sorting events Hemipterans, mealybugs and their obligate intracellular bacterial symbionts, thought to be strictly vertically inherited Cospeciation Gall-forming nematodes, obligate specialized parasites of plants Cospeciation, but also significant level of incongruence and host switches Vertically transmitted parasitic lice of pigeons and doves. Some species are host specific, other are found on multiple host species Cospeciation Mostly commensal and some parasitic mites of birds from the Subfamily Avenzoariinae 21 host mealybug taxa and their bacterial symbionts 58 nematode samples from 53 populations 27 host species and their associated 15 lice species TREEMAP and SOWH test TREEMAP:14 s, 0 3 duplications, 7 12 sorting events and 2 5 host shifts. Significantly congruent TREEMAP 12 cospeciations, 4 6 duplications, 1 4 host switches. The level of cospeciation was estimated as 60% TREEMAP and TREEFITTER 9 cospeciation events, 11 duplications and 61 sorting events. Up to 3 host switches under certain costs. Number of cospeciation events higher than expected by chance 26 mite species TREEMAP cospeciation events, 6 7 duplications, 2 host shifts, and sorting events For the mealybugs: EF-1a, 28S and 18S. For the endosymbionts: 16S and 23S rdna For the parasitic nematode: ITS1, 5.8S and ITS2. For the plant: ITS1 and ITS2 COI and the nuclear EF-1a. For the host: cyt b, COI and the nuclear FIB7 Mite phylogeny based on 41 morphological characters and mtdna. For birds, phylogeny constructed from several published based on morphological and molecular data Strong correlation between branch lengths in host and symbiont trees (r = 0.785, P < 0.001) Downie & Gullan (2005) Not tested Subbotin et al. (2004) Not tested Johnson et al. (2003) Not tested Dabert (2001)

16 362 Review Phytologist 2 Seabirds (Procellariiformes and Sphenisciformes) and lice (Phthiraptera) 3 Three trophic levels: geometrid moths (Eois), braconid parasitoids (Para panteles) and plants in the genus Piper 3 Neobatrachian anurans (frogs and toads) and Platyhelminthes (Monogenea) 3 Chewing lice (Pappogeomys) and Geomydoecus pocket gophers Cospeciation Seabirds and their parasitic lice Host shifts and host conservatism (shifts to closely related hosts) in Eois Herbivore moths, specialist moth parasitoid wasp 11 species of seabirds from the sphenisciform genera and 14 species of lice from six genera N = 94 (> 13 spp.) for Eois, N = 38 (> 10 spp.) for Parapanteles N = 52 for Piper Host shifts Parasitic relationship: flatworm and anurian 26 parasite species, 23 anuran species Prevalent cospeciation Highly host-specific parasitic chewing lice on pocket gophers occurring on a single pocket gopher species or subspecies 57 individuals from the Geomydoecus bulleri species group TREEMAP One host-switching, 9 cospeciation, 3 4 intrahost speciation, and sorting events Permutation test of Hommola (nonrandom association of matrices) NASignificant correlation between the branch lengths, but due to host conservationism TREEMAP, PARAFIT, DIVA analysis TREEMAP and PARAFIT 4 host shifts, 22 s, 10 duplications, and 16 extinction events; Parafit test nonsignificant 12 cospeciation events, 4 duplications, 1 loss, and 2 host switches 12S rrna. For the hosts: 12S ribosomal RNA, isoenzyme, and behavioral data COI and Ef1-a for Eois; ITS1 and ITS2 for Piper; COI and two nuclear genes for Parapanteles 18S and 28S. For the host: Rhodopsin and (12S and 16S) COI for chewing lice. Phylogeny of the host previously published based on mtdna Cytb and CoI and 1 nuclear gene (b-fib) Not tested Paterson et al. (2000) Fossil calibration for the Piper and Eois trees, molecular clock estimate for the Parapanteles tree: lack of temporal congruence No : datations inconsistent with Wilson et al. (2012) Badets et al. (2011) Absolute time congruence not tested, but the estimated molecular substitution rate is fourfold higher in lice than in hosts under assumed Demastes et al. (2012)

17 Phytologist Review Cyttaria fungi on southern beech trees (Nothofagus) 3 Nosema (Microspori dia: Nosematidae) and bees (Hyme noptera: Apidae) 3 Wheat, barley and oat (Poaceae) and Wheat dwarf viruses (WDV) (Mastrevirus) 3 Heteromyid Rodents (Rodentia: Heteromyidae) and Fahrenholzia suck ing lice (Phthirap tera: Anoplura) Codivergence, but also host shifts and extinction events Obligate Ascomycete fungi parasites of trees 12 species of Cyttaria and their hosts Cospeciation and host shifts Microsporidian parasites in bees 4 host species and 4 parasite species Codivergence for some viruses but not for others Parasitic DNA viruses Full genomes of 46 isolates of Wheat dwarf virus Codivergence Rodents and their permanent and obligate ectoparasitic sucking lice 43 heteromyid specimens and their lice PARAFIT Significant cophylogenetic structure with Parafit; reconstruction of the history by hand with 7 8, 1 2 duplications, 1 2 host shifts TREEMAP and TREEFITTER 0 1 cospeciation, 1 2 host shifts TREEMAP 6 s and 2 host jumps PARAFIT, TREEMAP PARAFIT: 39 of the 44 host-parasite pairs were significant. TREEMAP:26 s, 14 duplications, 23 extinctions, 1 host switching Cyttaria already published. For Nothofagus: cpdna, rbcl, nucits, rrna, cpdna atpbrbcl intergenic spacer and morphological data LS and SS rrna. For the host: cytochrome b For viruses: Phylogenetic trees constructed using full genomes. Host: rbcl Host and parasite : COI BEAST calibrated with fossils inferred a more ancient divergence of the fungus than Nothofagus Peterson et al. (2010) Not tested Shafer et al. (2009) Correlation between host lineage and WDV divergence estimates. However, assuming, the inferred rate of substitutions implied stronger constraints against change than by other methods Correlation between branch lengths, but r is weak (r = 0.7) and the slope is 2.8, interpreted as different rates of substitutions in lice; intercept significantly < 0, indicating delayed divergence in lice relative to host divergence Wu et al. (2008) Light & Hafner (2008)

18 364 Review Phytologist 3 Lice (Pediculus, Pedicinus, Pthirus) and primates (Homo, Pan, Gorilla) 3 Simian foamy viruses and primates (Hominoidea and Cercopithecoidea) 3 Gyrodactylus flat worms and Pomatoschistus Gobies fishes Significant cospeciation, but also parasite duplication, extinction, and host switching Highly specialized and permanent obligate ectoparasites of primates 5 species of lice from primates and one species from rodents as outgroup Cospeciation Non-pathogenic RNA retroviruses infecting mammals 55 primate species and viruses isolated from 44 primate species Host switches Two types of platyhelminth parasites: a monophyletic group of host-specific species, mainly infecting gills and a second group with lower specificity, dominantly found on fin and skin 15 Gyrodactylus taxa TREEMAP TREEMAP:5 cospeciation events and one host switch 1 duplication and 2 losses. Significantly greater similarity between the host and parasite trees than expected by chance TREEMAP Significant support for overall cospeciation (22 events/44), with some obvious cases of some instances of cross-species infections TREEFITTER, TREEMAP and PARAFIT The overall fit between trees was significant according to TREEMAP and TREEFITTER, but not according to the timed analysis in TREEMAP or to the PARAFIT analysis For the lice: Cox1 and elongation factor 1 alpha (EF-1a) gene For the virus: polymerase gene (pol). For the host: (mtdna) cytochrome oxidase subunit II (COII) the V4 region of the 18S rrna and the complete ITS rdna region. For the host: the 12S and 16S mtdna fragments Divergence date estimates show that the nodes in the host and parasite trees are not contemporaneous Reed et al. (2007) Significant linear relationship (r = ) between branch lengths. However, the molecular clock calibrations under cospeciation hypothesis infers an extremely low rate of SFV evolution, that would make it the slowestevolving RNA virus documented so far An absolute timing of speciation events in host and parasite ruled out the possibility of synchronous speciation for the gill parasites Switzer et al. (2005) Huyse et al. (2005)

19 Phytologist Review Primate lentiviruses (PLV) and primates 3 Brood parasitic finches (Vidua spp.) and their finch hosts (Estrildidae) 3 Malaria parasites (Plasmodium and Haemoproteus) and Haemoproteus birds Host switches Parasitic retroviruses that have been cited as evidence for 12 primate taxa (including outgroup) and their lentiviruses: 11 events Host shifts inferred from dates while cophylogeny tests pointed to cospeciations Host specific African brood parasitic finches (Vidua spp.) that mimic the songs and nestling mouth markings of their finch hosts (family Estrildidae) 74 estrildids, 21 parasitic finches, and nine ploceid finches as the outgroup Cospeciation Plasmodium parasites and Haemoproteus birds. Individual parasite species are thought to be restricted to host taxonomic families 68 lineages of Plasmodium/ Haemoproteus recovered from 79 species of birds in 20 avian families TREEMAP 8 s events of a possible 11 events for perfectly matched trees, but simulated based on the hypothesis of preferential shifts between closely related hosts were mostly congruent, and cospeciation was inferred TREEMAP and PARAFIT Basal divergences among Vidua spe cies are more recent than those among host species, allow ing cospeciation to be rejected, while tests for cospeciation indicated significant congru ence between host and parasite tree topologies TREEFITTER Significantly more cospeciation events (9 16) than in randomized trees; however, they required up to 52 switching events or 366 extinction events Host and parasite based on a number of published studies For host and parasites: most of the analyses were done using mtdna data set, although some nuclear sequences were also used in some clades Cytochrome b. For the host: already pub lished based on the DNA DNA hybridization studies Divergence time incompatible Charleston & Robertson (2002) More recent divergence of parasites than hosts Sorenson et al. (2004) Assuming, the DNA nucleotide substitution appears to occur about three times faster in hosts than in parasites Ricklefs & Fallon (2002)

20 366 Review Phytologist 3 Frankia bacteria and angiosperm plants (Actinorhizae) 4 Sigma viruses (Rhabdoviruses) and Drosophila fruit flies 4 Papillomavirus and mammals 4 Gammaretroviruses and bats (Chiroptera) 4 Lymphocystis viruses and fishes (Paralich thyidae) 4 Maculinea butterfly and Myrmica ants Significant tree congruence but incongruent dates Actinorhizae, mutualistic relation between angiosperm roots and nitrogen fixing Frankia bacteria Host shifts Parasite vertically transmitted RNA virus Host shifts Parasitic doublestranded DNA viruses Host shifts Exogenous parasitic retroviruses transmitted horizontally Independent divergence Independent divergence Parasitic DNA viruses causing lymphocystis disease in fish Parasitic relationship: caterpillars need to be adopted and nursed by ants 19 Actinorhizal angiosperms 4 species of Diptera TREEMAP, Component Shimodaira Hasegawa test and Robinson Foulds distance 207 PV genomes TREEMAP, TREEFITTER, and PARAFIT 8 events of and 9 duplication events. The probability of eight coevolutionary events occurring by chance was about 0.23 when 1000 host and symbiont trees were randomly associated For the bacteria: nifh and 16S rdna. For actinorhizal plants: rbcl 4/7 RNA polymerase gene for viruses 1/3 3 genes for Papilloma; 68- genes for the hosts 11 bat species TREEMAP 2/7 Viruses: Gag and 25 virus isolates, 8 fish species 32 Maculinea specimens (8 species includ ing outgroup), 14 species of Myrmica TREEMAP 3 s, 11 duplications and 19 sorting events PARAFIT, TREEFITTER Random association between the host and the parasite Pol proteins. Host tree from the tree of life Cytochrome b for the fishes, mcp gene for Lymphocystis COI, trna-leu, trnl, COII and Elongation Factor for Maculinea. For Myrmica: COI, Cytb, 28S ArgK, EF 1 alpha and LwRh Estimated divergence times among Frankia and plant clades indicated that Frankia clades diverged more recently than plant clades Jeong et al. (1999) Not tested Longdon et al. (2011) Not tested Gottschling et al. (2011) Not tested Cui et al. (2012) Not tested Yan et al. (2011) Not tested Jansen et al. (2011)

21 Phytologist Review Tobamovirus and plants (monocoty ledonous and dicot yledonous) 4 Fig trees (Ficus) and fig wasps (Elisab ethiella, Courtella, Alfonsiella) 4 Steinernema nema todes and c-proteo bacteria (Xenorhabdus) 4 Picornaviruses and animals (Aves and mammals: Primates, Rodentia, Carnivora, Perissodactyla, Certatiodactyla) 4 Malaria (Plasmodium) and primates 4 Hantavirus and Rodents (Arvicolinae, Murinae, and Sigmodontinae subfamilies) Independent evolution Parasitic relationship: plant RNA viruses 31 species of Tobamovirus Host shifts Mutualistic relation between Ficus and extreme host specific African fig wasps 42 wasp taxa and 26 Ficus species Host shifts Mutualistic relationship between nematodes and their associated c-proteobacteria 30 host species and their associated bacteria Host shifts Parasitic RNA viruses causing a broad spectrum of diseases in several orders of birds and mammals 752 complete genome sequences of piconaviruses Independent evolution Parasitic Plasmodium and their primate hosts 18 Plasmodium species Mainly host shifts Parasitic singlestranded RNA viruses 65 taxa. For the host: 95 sequences TREEMAP Lack of congruence between the host and the parasite Genes for the virus: CP (ORF4). For the plants: rbcl PARAFIT, TREEFITTER Nonsignificant Parafit test; A least twice as many host shifts as cospeciation events, even with high costs to host shifts Tarzan 12 cospeciation events, 17 hostswitches and 7 occurrences of sorting EF-1a and Cytb for Ficus and CO1 for wasps For the nematode: 28S, 12S, and COI. For the bacteria: 16S, RecA and SerC genes PARAFIT Lack of congruence 2C, 3Cpro, and 3Dpol TREEFITTER and PARAFIT 0 5 cospeciations, but assuming either up to 93 sorting events or up to 12 duplications or up to 11 host shifts TREEMAP 13 14, host shifts, 5 7 duplications and 4 10 sorting events; Parafit test nonsignificant For Plasmodium: 18S rrna, b- tubulin, cell divi sion cycle 2, EF, cyt b, merozoite surface; Host phylogeny pre viously pub lished For the virus: S, M, and L segments. For the host: cyt b More recent divergence of viruses than of their hosts (BEAST estimations for viruses) Host shifts occurred later than host diversification events, although overall confidence intervals overlap Pagan et al. (2010) Mcleish & Noort (2012) Not tested Lee & Stock (2010) Not tested Lewis-Rogers & Crandall (2010) Not tested Garamszegi (2009) Overlap of the mean node ages Ramsden et al. (2008)

22 368 Review Phytologist 4 Candidatus endobugula bacte ria and their Bugula bryozoan host 4 Grasses (Pooideae) and Epichlo e fungal endophytes 4 Mussels (Mytilidae: Bathymodiolinae) and endosymbiotic bacteria 4 Fig trees (Ficus) and their associated fig wasps No support for a history of strict cospeciation Mutualistic vertically transmitted bacteria of bryozoan Five host species and their associated symbionts Overall nonsignificant congruence, but early suggested Symbiont (from mutualist to parasites) fungal Endophytes in grasses, mostly vertically transmitted 26 grass species- Epichlo e species Incongruence Bathymodiolin mussels and their associated thiotrophic (sulfuroxidizing) bacterial endosymbiont Incongruence Figs and their mutualistic pollinators For the host, 25 OTU For the host: 18 neotropical fig species TREEMAP 3 cospeciation events and 1 host switch, but this was not significantly more congruent than expected by chance PARAFIT and MRCALink Analysis of the 26 associations did not reject random association. When five obvious host jumps were removed, the analysis significantly rejected random association and supported grass endophyte PARAFIT Host and symbiont tree topologies were not congruent TREEMAP No significant. Reconciliation of inferred 3 5 cospeciations. If switching events are excluded, reconciliation required losses Host: 16S LSU rrna and COI; Symbiont: 16S SSU rrna For the plant: a trnl intron and two intergenic spacers (trnttrnl, trnl-trnf) from cpdna. For the fungus: tubb (formerly tub2) and tefa (formerly tef1) For the host: ND4, COI and 28S. For the parasite: 16S rrna For the host: g3pdh, tpi and the ITS. For the pollinator: Phy logeny based on data already published Not tested Lim-Fong et al. (2008) No correlation between MRCA ages in the 26 species tree Schardl et al. (2008) time-depths of the gene trees were inconsistent (Mantel s test) Won et al. (2008) Not tested Jackson et al. (2008)

23 Phytologist Review Chaetodactylid mites and long-tongued bees (Apidae and Megachilidae) 4 Polyomavirus in human populations (Homo sapiens sapiens) 4 Penguins (Sphenisciformes) and chewing lice (Phthiraptera: Philopteridae) 4 Urophora insects (Diptera: Tephriti dae) and plants (Centaureinae) Infrequent host shifts at a higher taxonomic level, and frequent shifts at a lower level No evidence for Mites of bees including mutualists (feeding on nest waste), parasitoids (killing the bee egg or larvae), commensals or cleptoparasites Double-stranded DNA viruses transmitted in a quasi-vertical manner (from parent to child postnatally) 230 mite species from 1500 museum specimens of long-tongued bees 333 viral genomes and 158 human sequences Incongruence interpreted as caused by failure to speciate (parasites not speciating in response to their hosts speciating) Multihost parasites, all species of chewing lice are parasites of an entire host order 15 species of chewing lice parasitizing all 17 species of penguins No evidence for overall congruence Herbivorous insects fruit fly genus 11 European Urophora taxa PARAFIT, DistPCoA, TREEFITTER 0 3 cospeciation, 5 8 duplications, 0 6 host shifts, 0 35 extinctions TREEMAP < 10 events TREEFITTER, TREEMAP and PARAFIT No evidence of extensive cospeciation but support for significant congruence between the interpreted as possible failure to speciate events TREEMAP The number of cospeciation events (3 and 4) did not differ from random expectation Mite phylogeny: 51 morphological characters. Host already published Viral genomes and human sequences For the parasitic lice: 12S and COI regions. Host phylogeny based on 70 integumentary and breeding characters For the herbivore: allozyme frequency data from 20 loci. Host phylogeny already published based on allozymes Not tested Klimov et al. (2007) The analysis suggests that this virus may evolve nearly two orders of magnitude faster than predicted under the hypothesis Shackelton et al. (2006) Not tested Banks et al. (2006) divergence times indicated that the split of insect taxa lagged behind the split of their hosts Br andle et al. (2005)

24 370 Review Phytologist 4 Anther smut fungi (Microbotryum) and their host plants (Caryophyllaceae) 4 Achrysocharoides parasitoid wasps, Lepidoptera insects and plants (Rosales, Sapindales and Fabaceae) 4 Glochidion trees and Epicephala moths Host shifts between relatively closely related species Microbotryum com plex: Parasitic sexually transmitted and species-specific fungi of the Caryophyllaceae 21 host plants and their fungal parasites Incongruence between the three Achrysocharoides parasitoid wasps, highly host-specific and attack leafmining Lepidoptera and the plant host of Lepidoptera larvae 15 Achrysocharoides species No perfect congruence Obligate speciesspecific pollination mutualism between plants and their seedparasitic pollinators 18 Glochidion species. For the pollinator a sin gle individual from each of the 18 morphologi cally delimited species TREEMAP, TREEFITTER, Maximum Agreement Subtrees (Icong index), PARAFIT TREEMAP to compare the three pairwise TREEFITTER, TREEMAP and PARAFIT Overall, results suggest that cospeciation is not the rule in the Microbotryum Caryophyllaceae system, that host shifts were perva sive, but that fungal species could not shift to too distant host species No evidence that the were more congruent than expected by chance Greater congruence between the than expected in a random association. Perfect congruence between is not found, which likely resulted from host shift by the moths For the host plant: ITS and cpdna (trnl and trnf). b-tubulin, c- tubulin and Elongation factor 1 a For the parasitoid: cyt b sequences and 28S. For the Lepidoptera and the plant host, already published For the plant: the entire ITS-1, 5.8S rdna, and ITS-2 regions and the entire intergenic spacer region between 28S and 18S rdna including ETS For the moth: CO1, ArgK and EF-lox Not tested Refregier et al. (2008) Not tested Lopez- Vaamonde et al. (2005) Not tested Kawakita (2010)

25 Phytologist Review World arenaviruses (NWA) and rodents (subfamilies Sigmodontinae and Neotominae) 4 Seabirds (Procellariidae) and lice (Phthiraptera: Ischnocera) 4 Decacrema ants and Macaranga trees 4 Avian malaria parasites (Plasmodium) and birds (Aves) Host switches Single-stranded parasitic RNA viruses. One-quarter of them infect multiple hosts and one-third of the host species can be infected by more than one NWA virus Codivergence and host switches Parasitic lice from seabirds (petrels, albatrosses, and their relatives) with a high degree of lineage specificity 21 host taxa and 22 viral taxa 39 lice species from diverse hosts. The louse tree was broken into four subtrees and analysed separately Lack of overall phylogenetic congruence Highly specific mutualistic ants that inhabits and defends trees in Southeast Asia Decacrema ants from 262 trees corresponding to 22 Macaranga species Host shifts Bird parasites vectortransmitted parasites from the genus Plasmodium and Haemoproteus 65 parasite lineages, 44 host species, and 121 host parasite links Parafit 22 of 31 host virus associations were not significantly congruent TREEMAP Mixture of cospeciation and host switching, with some clades of lice showing close fidelity to their hosts (high ) and other clades showing higher levels of host switching TREEMAP and PARAFIT The Parafit analysis suggests only partial congruence between ants and plants. No cospeciation events were inferred by TREEMAP Component, TREEFITTER and PARAFIT Lack of significant congruence For the virus: complete coding region sequences of GP, NP, L and Z proteins. For the host: cytochrome b For the parasite 12S rrna and COI. Previously published elongation factor 1a. For the host, phylogeny constructed using a published dataset based on cytochrome b For the ant phylogeny based on COI. Macaranga phy logeny based on morphological characters and nuclear ITS already pub lished For the parasite and the host: cytochrome b Not tested Irwin et al. (2012) Correlation between sequence divergences Page et al. (2004) Not tested Quek et al. (2004) Not tested Ricklefs et al. (2004)

26 372 Review Phytologist 4 Austrophilopterus chewing lice and Ramphastos toucans 4 Drosophila fruit flies and Howardula nematodes 4 Deep sea vestimentiferan tubeworms and bacteria 4 Fishes (Sparidae) and monogenean parasites Lamellodiscus Host switches Chewing lice, parasites of toucans, considered to be host specific 26 Austrophilopterus lice col lected from 10 Ramphastos toucans and 7 Pteroglossus toucans Host shifts Howardula nematodes, horizontally transmit ted parasites of Drosophila Almost all known Drosophila hosts of Howardula No evidence for cospeciation Associations considered to be due more to ecological factors than to cospeciation Vestimentiferan tubeworm relying on intracellular sulfideoxidizing bacteria located in specialized tissues Fish hosts (Sparidae) and their highly host specific monogenean parasites (Lamellodiscus) 15 Vestimentiferan taxa and their symbionts 20 described Lamellodiscus species and 16 Sparidae TREEMAP and TREEFITTER Overall, TREEMAP indicated lack of cospeciation. Analyses identified one potential cospeciation event but then required 3 duplications and sorting events. TREEFITTER: 0 2 cospeciation events and 6 host switches TREEMAP Host and parasite are not congruent. The reconstruction with the fewest steps yielded 3 cospeciation events, 5 host switches, 0 duplication events and 25 sorting events TREEMAP No evidence for cospeciation TREEFITTER, TREEMAP and PARAFIT All methods agreed on the absence of widespread cospeciation if the cost of a host switch is not assumed to be very high For parasitic lice: COI and EF-1a. For the toucans: phylogeny already published based on different sequences such as COI and Cyt b rdna: 18S, ITS1 and COI. For the host: COI, COII, COIII For the symbiont: 16S ribosomal gene. For the host: COI 18S rdna. For the host: cyt b and previously published 16S mtdna sequences Not tested Weckstein (2004) Not tested Perlman et al. (2003) Not tested McMullin et al. (2003) Not tested Desdevises et al. (2002)

27 Phytologist Review Wolbachia and fig wasps (Hymenoptera) 4 Brueelia lice and birds (Passeriformes, Trogoniformes, Piciformes, Coracii formes, Psittacifor mes, Caprimulgifor mes, Charadriifor mes and Columbif ormes) 4 Fig trees (Malvanthera) and fig wasps (Pleistodontes, Sycoscapter) Incongruent Mainly vertically (and pervasive horizontally) transmitted Wolbachia bacteria in fig wasps 70 individuals representing 22 wasp species and their 23 species of associated Wolbachia Inconruent Brueelia parasitic lice considered to be highly host-specific, infecting birds 15 species of Brueelia col lected from 21 host species Partial ; Host plant switching less constrained in parasites than in pollinators Figs, obligated mutualistic pollinating Pleistodontes wasps and parasitic nonpolli nating Sycoscapter wasps. Each Ficus species is typically host to one pollinating and many different non pollinating wasp species 20 species of Pleistodontes and 16 species of Sycoscapter associated with Ficus species in the section Malvanthera TREEMAP The total number of matches between the two cladograms (7 cospeciation events) was not signicantly different from random expectation TREEMAP 7 cospeciation events not beyond that expected by chance TREEMAP,SH tests, ILD The level of cospeciation is significantly greater than that expected by chance. However, the maximum level of cospeciation was only 50 64% of nodes wsp gene. For the host: phy logeny already published based on partial COI and COII sequences nuclear EF-1a and COI. For the host: already published based on the DNA- DNA hybridization studies For the mutualistic wasp Pleistodontes: cyt b, 28S, and ITS2. For the parasitic Sycoscapter:cyt b and 28S Not tested Dewayne Shoemaker et al. (2002) Not tested Johnson et al. (2002) The greater genetic distances between Sycoscapter species than between their associated pollinators suggest that Sycoscapter may have the higher rate of molecular evolution. Another possi bility is that Sycoscapter species are older Lopez- Vaamonde et al. (2001)

28 374 Review Phytologist 4 Lichens (Trebouxia): algae and fungi 4 Primates and Oxyuridae nematodes 4 Puccinia rust fungi and Brassicaceae plants 4 Ascomycete mycangial (Ophiostomataceae) fungi and Dendroctonus bark beetles Switching of algal genotypes occurred repeatedly among these symbiotic lichen associations Hostswitching and Long-term mutualism between of photosynthetic algae or cyanobacteria and heterotrophic fungi. Low algal specificity Enterobiinae oxyurid, nematodes parasites of primates. In most of the cases, one parasite species per host species 33 natural lichen associations: 46 fungal species are associated with only 36 genotypes, representing four or fewer species of algae 48 species of Enterobiinae analysed (46 species of the subfamily and 2 outgroup species) and their hosts Host shifts more common than No widespread Crucifers and their flower-mimicking fungal pathogens Mutualist and specific relationship: beetles carry mycangia, tegument invagination for fungal dissemination 17 Brassicaceae species and 3 rust species (multiple individuals of each) 11 fungal species and 6 beetle species TREEMAP cospeciations. However, this required 7 9 duplications, 3 5 switches and sorting events TREEMAP 6 8 cospeciation events, 1 duplication, 1 3 host switching, 1 4 sorting events Partition homogeneity test Incongruent TREEMAP 4 cospeciations, 3 duplications, 4 sorting events and 1 host shift; more cospeciations than expected by chance For both symbionts: ITS Not tested Piercey- Normore & DePriest (2001) 45 morphological characters from various organ systems. For the host, modified from a previously published phylogeny For the host: cp trnl-f and ITS; for the fungi: ITS and 5.8S Not tested Hugot (1999) Not tested Roy (2001) Isoenzymes Not tested Six & Paine (1999)

29 Phytologist Review (8 cases) and 5 7 cases) 15 Plant fungal symbioses 5 Fungal Pneumocystis and mammals 5 Spinturnix mites and bats (Rhinolophus, Myotis, Nyctalus, Plecotus, Miniopterus and Barbastellus) 5 a-proteobacteria and Ishikawaella stink bugs A continuum of cophylogenetic patterns ranging from mostly to mostly switching Different plant-fungal associations, ranging from parasitism to mutualism Symbioses from 5 Orders and 10 families Codivergence Parasitic fungus 19 species of mammals Cospeciation and host shifts European bats and their ectoparasitic mites 78 Spinturnix mites (11 mor phospecies) from 20 Euro pean bat species Mainly Vertically transmitted gut mutualistic bacteria of stinkbugs 14 host species and their symbiotic bacteria POpt and TREEMAP Seven associations showed significant congruence while eight were incongruent. Even the association inferred as significantly congruent exhibited a number of losses or duplication and/ or host shifts TREEMAP 14 cospeciation out of 18 events (number of other not indicated) PARAFIT, MESQUITE Significant cophylogenetic structure, but at least five host switch events TREEMAP and TREEFITTER events, 2 3 host shifts, 2 3 duplications, 2 3 sorting events Phylogenies already published and bases on different molecules depending on the symbiosis. In general, for the fungal symbiont: ITS or nuclear rrna. Different molecules used for the host phylogeny mtlsu rdna, mtssu rdna and DHPS. Phylogeny of the mammals previously published For mites: two genes (16S COI). For bats, published plus cyt b For the bacteria: 16S rrna and groel t. For the host, COI Not tested Jackson (2004) Not tested Chabe et al. (2012) Not tested Bruyndonckx et al. (2009) Not tested Kikuchi et al. (2009)

30 376 Review Phytologist 5 Fungal Pneumocystis and Primates 5 Cryptocercus cock roaches and their bacteria Blattabacterium cuenoti 5 Uroleucon aphids and endosymbiotic Buchnera bacteria 4 and 2 Viruses (Partitiviridae), plants (Viridiplantae) and fungi (Ascomycetes and Basidiomycetes) Cospeciation Highly specific fungal parasites 20 primate species Cospeciation Cryptocercus subsocial, xylophagous cock roaches and their endosymbiotic and vertically transmitted bacteria Blattabacterium cuenoti Cospeciation Aphids and their mutualistic vertically transmitted endobacteria, required for host reproduction Six out of the seven Cryptocercus species and their endosymbionts 14 representative species of Uroleucon and their bacteria Two virus families with inferred and two families without Parasitic relationship: Vertically and horizontally transmitted RNA virus 175 viral genomes TREEMAP 61 77% of the nodes interpreted as resulting from events, but the numbers of other events then required are not reported COMPONENT Lite Significant similarity between TREEMAP, Kishino Hasegawa test, likelihoodratio test Highly significant levels of similarity between the trees: 8 9 cospeciation out of 14 possible PARAFIT as implemented in AXPARAFIT, TREEFITTER, TREEMAP Many duplication and switching even for the families where is suggested DHPS, mtssurrna, and mtlsu-rrna. For the host: already published based on several and nuclear sequences and morphological characters For the bacteria: 16S rrna and 23S rrna. For the host: portions of the 28S rrna and 5.8S rrna genes and the entire ITS2 For the mutualist: partial sequences of trpb. For the host: tree based on and nuclear sequences already published Complete genomes for viruses Not tested Hugot et al. (2003) Not tested Clark et al. (2001) Not tested Clark et al. (2000) Not tested G oker et al. (2011)

31 Phytologist Review and 5 Doves (Aves: Columbiformes) and lice (Columbicola and Physconelloides) 4 and 5 Fig trees (Sycomorus) and fig wasps (Ceratosolen and Apocryptophagus) 4 and 5 Chondracanthid copepods and fishes (Ophidiiformes, Pleuronectiformes, Scorpaeniformes, Zeiformes and Gadiformes) Cospeciation in body lice but not in wing lice Cospeciation for mutualists and host shift for parasites Cospeciation in one fish order but not in the second Dove body lice (Physconelloides) and dove wing lice (Columbicola) parasitiz ing pigeons and doves, dove body lice being more host-specific than dove wing lice Different types of symbionts of figs: Mutualist pollinator Ceratosolen wasps and parasite Apocryptophagus wasps Chondracanthid copepods parasitic on fish considered to be host specific although this has been debated 13 species of doves and their associated wing and body lice 19 species of Sycomorus figs. 19 Ceratosolen species and 18 species of Apocryptophagus 26 Chondracanthus spp. and their teleost host genera from five orders TREEMAP For dove wing lice: 4/ 12 cospeciation events, which is not more than expected by chance. For body lice: 8/12 cospeciation events, congruence being inferred as significant, but the numbers of other events assumed are not reported TREEMAP 9 10 cospeciation (significant) for mutualists and 7 8 for the parasites (not significant) TREEMAP Support for cospeciation of copepods and their fish hosts in the orders Ophidiiformes, Pleuronectiformes, Scorpaeniformes and Zeiformes, but no support for cospeciation in the Gadiformes COI and 12S rrna and the nuclear EF-1a For the host: cyt b and the nuclear FIB7 For the symbiotic wasps: COI. For the host fig: ITS Phylogenies already published Not tested Clayton & Johnson (2003) Not tested Weiblen & Bush (2002) Not tested Paterson & Poulin (1999) 1 Type: 1: convincing cases of cospeciation (i.e. with comparison of divergence times): 1a, mutualists, vertically inherited; 1b, mutualists; 1c, endoparasites; 1d, parasites. 2: cospeciation inferred by authors, but host shifts possibly more likely given the high number of other (i.e. unlikely high number of intrahost speciation and ancestral numbers of parasites); absolute time congruence not tested. 3: cospeciation inferred (i.e. significant topological congruence, high number of cospeciation events) but contradicted by time inference (either absolute or relative); this is indicative of host shifts occurring preferentially between closely related hosts (host conservationism). 4: frequent host shifts inferred by authors because of lack of phylogenetic congruence. 5: unclear (e.g. congruence without absolute time inferred or other number of events than cospeciation not provided). ITS, internal transcribed spacer.

32 378 Review Phytologist Clayton et al., 2003). Other ecological factors that may influence the probability of include the abundance of the main host, the community of parasites, the degree of specialization, the population sizes and generation times of hosts and symbionts (Whiteman et al., 2007; Gibson et al., 2010; Nieberding et al., 2010). Notwithstanding the exemplary nature of the case of pocket gophers and their chewing lice, analyses of their association have assumed multiple host shifts and intrahost speciation events to reconcile, even with the great costs assumed for these events (Light & Hafner, 2007). Lice species other than those of the pocket gopher have been investigated for. The heteromyid gophers, which are more social than pocket gophers, display lower levels of tree congruence with their sucking lice (Light & Hafner, 2008). Furthermore, the intercept of the regression line between the gopher and lice divergence times was significantly < 0, indicating that lice divergence occurred after host divergence (Light & Hafner, 2008). Similarly, the estimated dates of divergence between lice and primates shows that the nodes in the host and parasite trees did not coincide temporally (Reed et al., 2007). Nevertheless, eventbased methods analyses misleadingly inferred significant cospeciation (Page, 1996). The most convincing examples of cospeciation appear to concern mutualist associations in which the symbiont is transmitted vertically (Table 2, Fig. 5), as could be expected (Nieberding & Olivieri, 2007). A few host shifts have, nevertheless, been detected in associations of mutualists with vertical transmission (Table 2, cases Fig. 1b). Important conclusions from this literature review and theoretical considerations are that symbiont speciation by host shift appears to be more common than cospeciation even more than is currently recognized (Fig. 5, convincing examples of cospeciation represent only 7% of the cases) and that the results of cophylogenetic tests are often overinterpreted to suggest cospeciation. A key question thus concerns the short-term ecological and genetic mechanisms promoting host-shift speciation rather than cospeciation. Nieberding et al. (2010) put forward a list of ecological traits that might influence the degree of cospeciation. In the next section, we consider the evolutionary mechanisms affecting the likelihood of symbiont specialization and speciation in relation to short-term coevolution with hosts. V. Relationship between host symbiont coevolution and symbiont speciation We aim here to review the processes by which coevolutionary mechanisms can promote symbiont diversification. For this to occur, coevolution must first foster the specialization of symbionts, which could then lead to speciation. We thus review studies (1) showing how coevolution can promote symbiont specialization and (2) providing experimental and theoretical evidence for symbiont specialization leading to speciation. We argue that divergence as a result of specialization may occur, but that it occurs more frequently through host-shift speciation than cospeciation. 1. Coevolution: short-term host parasite interaction Host parasite coevolution is a process of prolonged reciprocal selection, for better recognition of the parasite by its host, and for greater infectious ability of the parasites and the prevision of parasitism by the host. In the simplest systems, this selection involves a single locus in each partner. Two outcomes for the dynamics of host and pathogen allele frequencies are commonly distinguished under frequency-dependent selection (Holub, 2001; Woolhouse et al., 2002). The arms race model describes allele frequency dynamics where advantageous new variants go to fixation. By contrast, the trench warfare model depicts allele frequencies in oscillating dynamically over time or converging to equilibrium frequencies, resulting in the maintenance of several host and pathogen alleles (Brown & Tellier, 2011). Another classification considers the dynamics of phenotype shifts caused by selection. When the phenotype values always shift in the same direction, as in predator prey systems with densitydependent selection, the interaction has been termed phenotype difference (Dawkins & Krebs, 1979), whereas when the system oscillates depending on the phenotypic value of the interacting species, as in most self/nonself recognition systems with frequencydependent selection, the interaction has been called phenotype matching (Lahti, 2005). Such dynamical systems led Van Valen (1973) to refer to the coevolutionary processes between hosts and parasites as Red Queen dynamics, in reference to Lewis Carroll s tale Through the Looking Glass (the Red Queen character explains to Alice that in her world that it takes all the running you can do, to keep in the same place ). His paper was the first to connect short-term coevolutionary dynamics with macroevolution, including the long-term persistence of species in particular. The question here is whether coevolution, regardless of the prevailing mechanism (arms race, trench warfare, etc.), can actually directly promote parasite specialization. 2. From coevolution to specialization, models and observations A priori, we might expect all species to be selected for the exploitation of broad ecological niches. Becoming a generalist decreases the spatial and temporal risks and efforts required for food collection and ensures survival in conditions in which the availability of particular resources may reveal unreliable. Generalism is common in plant viruses (Garcia-Arenal et al., 2003) and in animal viruses (Pedersen et al., 2005). However, specialization seems to be far more common than generalism in various parasite species ranging from phytophagous insects (Dres & Mallet, 2002; Nyman, 2010) to fungi (Giraud et al., 2008) and avian parasites (Proctor & Owens, 2000). The relative paucity of generalist parasites may result from tradeoffs between the ability to infect a broad range of host species and optimized rates of exploitation for any particular host type. Such trade-offs have been observed in serial passage experiments, in which propagating a microorganism on a host species different from its original host species consistently leads to a decrease in fitness on the

33 Phytologist Review 379 Fig. 5 Illustration of the literature survey in Table 2, with number of cases representing either convincing cases of cospeciation (in red), cases of host shifts inferred from incongruent topologies, discordant times of divergence or likely given the high number of duplication and extinction inferred (in blue), or finally unclear cases (in green). Box 1 Glossary Codivergence Coevolution (to be distinguished from cospeciation) Congruence Cospeciation Generalist Host Host-shift speciation Intrahost speciation (called duplication in some papers and cophylogeny software) Mutualist Parasite Specialist Symbiont Process whereby a symbiont population or species splits at the same time as that of its host population or species. This is a pattern and does not assume causal relationships. Process of never-ending reciprocal selection for improvements in parasite recognition in the host, and for improvements in recognition escape mechanisms in the parasite. Phylogenetic trees are said to be congruent when their topologies are highly similar; temporal congruence also implies that the corresponding nodes are of similar ages in the two. Process whereby a symbiont speciates at the same time as another species (this may result from vicarious events or from narrow host specificity). This is a pattern and does not assume causal relationships. Symbiont able to take resources from different host species. Organism from which another smaller organism (the symbiont), from another species, takes resources; the symbiont may be either a parasite or a mutualist. Mutualists also provide the host with resources. Speciation of the symbiont by specialization of a daughter species on a new host. Speciation of the symbiont without speciation of the host or host shift: both daughter symbiont species continue to parasitize the same host species. This may be because of vicarious events affecting only the symbiont or specialization on different organs of the host. Organism both taking and resources from and providing resources to another larger organism (the host), from another species, resulting in an overall increase in host fitness. Organism taking resources from another larger organism (the host), from another species, decreasing host fitness. Symbiont able to take resources from a single host species. Organism taking resources from another larger organism (the host) from another species. The symbiont is either a parasite or a mutualist. Mutualists also provide the host with resources. original host (Ebert, 1998). By contrast, the instability of host abundance proposed as a factor explaining the evolution of generalists in natural systems (Jaenike, 1990; Norton & Carpenter, 1998) has received some experimental support (Soler et al., 2009). A combination of these selection pressures may occur, as both specialists and generalists have emerged in several experimental evolution studies (Little et al., 2006; Poullain et al., 2008). Factors favoring specialization even in the absence of fitness trade-offs and in the presence of stable host populations have been investigated in theoretical studies. In particular, parasite specialization may also evolve because of the more rapid adaptation of specialists than generalists to each host species (Whitlock, 1996; Kawecki, 1998) as assumed in the Red Queen dynamics theory (Whitlock, 1996). According to the model developed by Kawecki (1998), if recurrent selection for new alleles at the loci controlling infectivity occurs because of coevolution, then specialization will be selected for because specialist parasites adapt more rapidly than generalists. Indeed, selection for a greater ability to infect a given host operates at every generation in specialized parasites, but only occasionally in generalists distributed between several host species.