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Proceedings of the Royal Society B: Biological Sciences logoLink to Proceedings of the Royal Society B: Biological Sciences
. 2008 Feb 12;275(1638):1029–1035. doi: 10.1098/rspb.2007.1622

Breakdown and delayed cospeciation in the arbuscular mycorrhizal mutualism

Vincent Merckx 1,*, Martin I Bidartondo 2,3
PMCID: PMC2600904  PMID: 18270159

Abstract

The ancient arbuscular mycorrhizal association between the vast majority of plants and the fungal phylum Glomeromycota is a dominant nutritional mutualism worldwide. In the mycorrhizal mutualism, plants exchange photosynthesized carbohydrates for mineral nutrients acquired by fungi from the soil. This widespread cooperative arrangement is broken by ‘cheater’ plant species that lack the ability to photosynthesize and thus become dependent upon three-partite linkages (cheater–fungus–photosynthetic plant). Using the first fine-level coevolutionary analysis of mycorrhizas, we show that extreme fidelity towards fungi has led cheater plants to lengthy evolutionary codiversification. Remarkably, the plants' evolutionary history closely mirrors that of their considerably older mycorrhizal fungi. This demonstrates that one of the most diffuse mutualistic networks is vulnerable to the emergence, persistence and speciation of highly specific cheaters.

Keywords: arbuscular mycorrhizal mutualism, myco-heterotrophy, parasitism, symbiosis

1. Introduction

Cooperation between species is one of the central problems in biology. Theoretically, it is widely assumed that mutualism is prone to break down into parasitism (Trivers 1971; Axelrod & Hamilton 1981; Doebeli & Knowlton 1998; Herre et al. 1999; Morris et al. 2003; Ferrière et al. 2007). However, mutualisms are ubiquitous in nature and there appear to be surprisingly few examples of breakdown, that is, there are few parasitic taxa nested within mutualistic clades (Sachs & Simms 2006).

Empirically, the overwhelming majority of information about shifts from mutualism to parasitism originates from two specialized reproductive mutualisms (i.e. yucca–yucca moth and fig–fig wasp). In both of these mutualisms the interaction between the partners is obligate: the insect oviposits into the ovaries of the plant and pollinates the flower. The insect larvae then feed on a subset of the developing seeds. A shift from mutualism to parasitism by one partner would lead to an interruption in the life cycle of the other partner and eventually to the extinction of both lineages. Yet there are yucca moths and fig wasps that lay their eggs into flowers without pollinating them. These parasites evolved when their ancestors colonized a novel plant host that already had a pollinator. In these plant–insect model systems, the critical barrier to mutualism breakdown and to the evolutionary stability of the resulting parasitic species is thus thought to be a difficult requirement for three-species coexistence: a parasitic insect specialized on a mutualistic plant specialized on a mutualistic insect (Pellmyr & Leebens-Mack 1999, 2000). In fact, there are only two parasitic species emanating from the yucca–yucca moth mutualism (Althoff et al. 2006) and a single one from the species-rich fig–fig wasp mutualism (Machado et al. 2001). The question of whether or not barriers to cheating are lowered in diffuse mutualisms, where multi-species coexistence is the rule, has received scant attention (Thompson 2005).

The most widespread diffuse mutualism is the mycorrhizal association between plants and fungi (Smith & Read 1997). In this mutualism, the plants exchange carbon for fungal nitrogen and phosphorus. Mycorrhizal plants can obtain approximately 75% of their nitrogen in exchange for approximately 15% of their carbon, and conversely, mycorrhizal fungi can obtain 100% of their C in exchange for approximately 40% of their N (Hobbie & Hobbie 2006). Sustaining this costly exchange is vital as it allows both partners to establish, grow and complete their life cycles. Mycorrhizas are diffuse symbioses because a mycorrhizal plant typically associates simultaneously with multiple fungi and a mycorrhizal fungus often associates simultaneously with multiple plants (Giovannetti et al. 2004; Lian et al. 2006).

Mycorrhizal promiscuity provides the potential for nutritional cheating when physiological continuity is established between a photosynthetic plant, its mycorrhizal fungi and a non-photosynthetic plant. The latter is referred to as a myco-heterotrophic (Leake 1994) epiparasite (Romell 1939; Björkman 1960). Myco-heterotrophy is an evolutionarily and ecologically widespread phenomenon that can provide escape from competitive exclusion in the shaded conditions of forest understorey habitats (Bidartondo et al. 2004); indeed, over 400 non-photosynthetic mycorrhizal plants have evolved within ten mycorrhizal plant families (Leake 1994).

Evolutionary trajectories have yet to be analysed in the cheaters of arbuscular mycorrhizas. In the relatively more specialized ectomycorrhizas, the ability to obtain carbon from neighbouring photosynthetic plants has led cheater plants to loose evolutionary codiversification (Bidartondo et al. 2004). Here we test whether evolutionary codiversification is tighter when plants cheat the more diffuse, much older and more pervasive arbuscular mycorrhizal mutualism. To accomplish this, we identified the mycorrhizal fungi in myco-heterotrophic plant species of Burmanniaceae and Triuridaceae from three regions in Cameroon. For five closely related Afrothismia species (Burmanniaceae), we compared a multi-gene plant phylogeny with a multi-gene phylogeny of their associated fungi. We used penalized likelihood (PL; Sanderson 2002) and Thorne & Kishino's (2002) method to estimate the divergence times in both Afrothismia and its arbuscular mycorrhizal symbionts. Finally, we propose a model for the evolution of non-photosynthetic mycorrhizal plants which explains the delayed cospeciation pattern observed in Afrothismia.

2. Material and methods

(a) Taxon sampling

The Burmanniaceae family has evolved non-photosynthetic lineages from within photosynthetic lineages at least five times (Merckx et al. 2006). Within the Burmanniaceae, the non-photosynthetic tribe Thismieae (approx. 40 species) represents one of those events and includes the genus Afrothismia. Populations of five Afrothismia species (electronic supplementary material, figure 1) and three co-occurring myco-heterotrophic plant species were sampled from three different regions in Southwest Cameroon: Mount Kupe, Diongo (western foothills of Mount Cameroon) and Korup National Park. These three regions are approximately 100 km apart from each other. Afrothismia hydra, Afrothismia winkleri and Afrothismia foertheriana plants were collected from two distinct locations approximately 1–2 km apart within each region. Sampled species, voucher information and sampling localities are listed in the electronic supplementary material, table 1. We extracted genomic DNA from 3 to 12 individual approximately 2 mm long sections of roots or whole enlarged bases of roots from each plant using methods described elsewhere (Gardes & Bruns 1993) with a purification step using GeneClean (QBioGene). We then amplified (PicoMaxx, Stratagene or JumpStart, Sigma), cloned (TOPO TA, Invitrogen) and sequenced (BigDye v. 3 on ABI3730 Genetic Analyzer, Applied Biosystems) the fungal 18s ribosomal RNA gene from NS1/ITS4 amplicons (White et al. 1990) for all plants, part of the fungal translation elongation factor 1α gene (EF1α) from EF1-526f/EF-1567r amplicons (S. Rehner 2001, unpublished data) for Afrothismia, and part of a homologue of the fungal TOR2 gene from GmTOR-1071f/GmTOR-1638r amplicons (Stuckenbrock & Rosendahl 2005) for Afrothismia. With the exception of TOR2, we used nearly universal eukaryotic primers; nonetheless, the only non-Glomus organism we detected was an unidentified stramenopile (EF1α from A. foertheriana). Stramenopiles do not have any mycorrhizal lineages and this is probably a pathogen. For Glomus, we examined variation in rDNA by DNA sequencing at least four clones from each plant and in non-rDNA two to eight clones from each plant. From plants, we amplified 18s rDNA with primers NS1, NS2, NS3, NS4, NS5 and NS8 (White et al. 1990), ITS with primers ITS1 and ITS4 (White et al. 1990), and atpA with primers ATPA-F1 and ATPA-B1 (Eyre-Walker & Gaut 1997). All PCR products were purified and sequenced on an ABI310 Genetic Analyzer (Applied Biosystems).

(b) Datasets

To test for mycorrhizal specificity and for subsequent molecular dating analyses, a large 18s rDNA (109 taxa; 1721 bp) was compiled with all our fungal symbiont DNA sequences obtained from the roots of Afrothismia, Burmannia, Kupea, Sciaphila and Tacca as well as representative Glomeromycota 18s rDNA accessions from GenBank. Accessions of Ascomycota, Basidiomycota and Zygomycota were included for out-groups (table 4 in the electronic supplementary material).

A three-gene dataset was constructed for all fungi that were associated with Afrothismia, including Glomus intraradices, with Glomus mosseae as out-group. After excluding all ambiguously aligned positions, we obtained a total matrix of 2748 bp (18s rDNA: 1691 bp, EF1α: 464 bp, TOR2: 593 bp; see table 2 in the electronic supplementary material for GenBank accessions). In parallel, a three-gene dataset of Afrothismia was compiled with Thismia panamensis as out-group. Once combined, we obtained a dataset of 3113 bp (18s rDNA: 1566 bp, ITS: 501 bp, atpA: 1046 bp; see table 3 in the electronic supplementary material for GenBank accessions).

To allow inclusion of multiple calibration points for molecular dating analyses, the Afrothismia 18s rDNA and atpA data were combined with DNA sequences of all monocot orders and basal angiosperm groups with Amborella trichopoda as an out-group (table 5 in the electronic supplementary material). This resulted in a matrix of 2888 bp (18s rDNA: 1666 bp and atpA: 1222 bp). Individual alignments of all datasets were manually created with MacClade v. 4.06 (Maddison & Maddison 2001).

(c) Phylogenetic reconstruction

Both maximum parsimony (MP) and Bayesian (BI) analyses were performed with each of the above-mentioned datasets. MP analyses with PAUP* v. 4.0b10 (Swofford 2002) involved heuristic searches with 1000 replicates, tree bisection reconnection branch swapping and holding 100 trees in memory with the multrees option in effect. Clade support was assessed via non-parametric bootstrapping using 1000 replicates and the same search strategies. BI analyses were performed with the GTR+I+G model of DNA evolution, identified as the best-fitting model for each gene fragment under the Akaike information criterion implemented in Modeltest v. 3.06 (Posada & Crandall 1998). BI analyses were performed with MrBayes v. 3.1.2 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003). Multi-gene datasets were analysed under a mixed model approach partitioned over the different gene fragments. Each BI analysis consisted of two parallel Markov Chain Monte Carlo (MCMC) runs of four chains performed with a length of five million generations, a burn-in corresponding to the first 1.25 million (25%) and a sample frequency of one tree for every 1000 generations. In each analysis, the potential scale reduction factors for all model parameters (approx. 1.0) and the split frequency standard deviations (less than 0.01) confirmed convergence of runs. Convergence of the chains was also checked using Tracer v. 1.4 (Rambaut & Drummond 2007). For all analyses the effective sampling size of each parameter was found to exceed 100, suggesting acceptable mixing and sufficient sampling. During phylogenetic analyses of the 18s rDNA+atpA monocot dataset, the relationships between the orders were constrained to a seven-gene monocot topology (Chase 2004). All other datasets were analysed without constraints.

(d) Diversification time estimates

For both Glomeromycota and monocot datasets, a Χ2 likelihood ratio test rejected the hypothesis of evolution according to a strict molecular clock (for Glomeromycota Χ1092=276.22; p=3.12×10−16; for monocots Χ1352=2032.48; p=5.15×10−287). Therefore, we used two different relaxed molecular clock methods: PL as implemented in r8s (Sanderson 2002, 2003) and Thorne & Kishino's (TK) method (Thorne & Kishino 2002) with multidivtime (http://statgen.ncsu.edu/thorne/multidivtime.html). With the program Estbranches DNA, we calculated substitution branch lengths for each gene fragment using the F84+G model with parameters estimated using PAML (Yang 1997) and the 50% majority-rule topology from the Bayesian analysis (Rutschmann 2005). For each analysis, a single MCMC chain was run for 1.1 million generations with a burn-in of 0.1 million generations and a sampling frequency of one per 100 generations. To check for convergence of the MCMC chain, we ran analyses at least twice from different starting points. For the PL analyses, we used the 50% majority rule BI tree with branch lengths as input for r8s. Analyses were run with a Truncated-Newton optimization algorithm and optimal rate-smoothing parameter determined by the statistical cross-validation method in r8s (1.6e7 for Glomeromycota, 10.0 for monocots). We calculated credibility intervals and standard deviations for the PL age estimations of the main nodes by repeating the PL analysis on 1000 trees randomly sampled from the trees sampled during the BI analysis (Roelants et al. 2007). Details about priors and calibration points are given in the electronic supplementary material.

3. Results

(a) Mycorrhizal specificity

Bayesian and parsimony phylogenetic analysis of Glomeromycota 18s rDNA produced highly congruent estimates of phylogenetic relationships (figure 1; figure 2 in the electronic supplementary material). The topology is well resolved and shows high congruence with previous 18s rDNA-based hypotheses for Glomeromycota (Schwarzott et al. 2001). All fungal symbionts are placed within a maximally supported Glomus group A lineage and there is no fungal lineage overlap among the different myco-heterotrophic plant species. The mycorrhizal fungi obtained from the roots of Sciaphila ledermannii, Kupea martinetugei, Burmannia hexaptera, Afrothismia gesnerioides, A. hydra and Afrothismia korupensis are all placed in strongly supported monophyletic groups. The fungal symbionts of A. winkleri and A. foertheriana are part of paraphyletic groups due to the inclusion of the A. hydra and A. korupensis symbiont clades, respectively.

Figure 1.

Figure 1

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Glomus group A and B clades from the Bayesian phylogenetic analysis on 18s rDNA sequences of Glomeromycota (see figure 2 in the electronic supplementary material for the phylogeny with all taxa). Bayesian posterior probabilities of main clades are shown above branches, and non-parametric bootstrap percentages are placed below branches. The inset map shows the sites where collections were made. A Glomus group A symbiont 18s rDNA sequence from the roots of a Tacca species, the nearest extant photosynthetic relative of Afrothismia, is found in a clade with 18s rDNA sequences from Sciaphila ledermannii fungal symbionts.

(b) Multi-gene Afrothismia and fungal symbiont phylogenies

The three-gene phylogenies of Afrothismia and its fungal symbionts are both well resolved and strongly supported (figure 2). No incongruence was observed between parsimony and Bayesian analyses. The three-gene Glomus phylogeny shows improved resolution over the single 18s rDNA tree with fungal symbionts of each Afrothismia species in strongly supported clades. There is one topological difference between the plant and the fungus phylogenies: A. foertheriana is sister to A. hydra and A. winkleri while its fungal symbionts are sister to those of A. korupensis.

Figure 2.

Figure 2

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Comparison of multi-gene phylogenies for Afrothismia plants (right) and Glomus fungi (left) shows evidence of delayed cospeciation by the plants. Afrothismia species are extreme specialists that have tracked ancient fungal lineages over considerable evolutionary time. Thus, the plants' evolutionary history closely mirrors that of mycorrhizal fungi. Each photograph shows a flower of the plant species named above. Bayesian posterior probabilities (more than 95%) are shown above branches and non-parametric bootstrap percentages (more than 85%) are shown below branches. Branch lengths represent the number of substitutions per site. Divergence time estimates are indicated in boxes.

(c) Diversification time estimates

The chronograms obtained from the 18s rDNA of the fungi are shown in the electronic supplementary material, figure 3. Using the Heckman et al. (2001) interval, the stem node of the Glomeromycota is estimated at 1399±6 (PL) and 1304±44 Mya (TK). The crown node of this clade is estimated at 1307±30 (PL) and 1223±63 Mya (TK), much older than the oldest known Glomeromycota fossils (Redecker et al. 2000). Under the same priors, the split between Ascomycota and Basidiomycota is estimated at 1208±53 Mya (PL) and 1135±92 Mya (TK). This is congruent with prior estimates (Heckman et al. 2001; Padovan et al. 2005) when using the calibration date of 1576 Mya for the plant–animal–fungus split for this node. In contrast, when applying a 590–600 Mya interval constraint for calibration point I, we inferred an estimate of 600±38 (PL) and 595±32 Mya (TK) for the origin of the Glomeromycota. Table 6 in the electronic supplementary material lists the divergence time estimates obtained for the Afrothismia symbionts with both calibration strategies.

The resulting monocot topology based on 18s rDNA and atpA data is shown in the electronic supplementary material, figure 4. Our analysis shows a maximally supported placement for Afrothismia as sister clade to Tacca–Thismia–Haplothismia. The paraphyly of Burmanniaceae tribe Thismieae is unexpected, yet a previous study has indicated that in Burmanniaceae tribe Burmannieae many lineages lost their chlorophyll independently leading to convergent morphological evolution (Merckx et al. 2006). In general, many myco-heterotrophic plants, despite the very diverse range of families and genera, show remarkable convergence (Leake 1994). The stem and crown node age of the Dioscoreales are estimated at 120±6 Mya (TK) and 139±15 Mya (PL), and 113±8 Mya (TK) and 135±14 Mya (PL), respectively (figure 5 in the electronic supplementary material). These estimates bracket previous estimates for the stem (124 Mya) and the crown node (123 Mya) of Dioscoreales obtained with rbcL DNA sequence data (Jansen & Bremer 2004). Our analyses recover an estimate of 91±11 Mya (TK) and 120±11 Mya (PL) for the origin of Afrothismia (table 6 in the electronic supplementary material). Diversification of the genus, which should be treated as a minimum age since our sampling is incomplete, started between 50±13 Mya (TK) and 78±9 Mya (PL).

(d) Comparison of fungi and plant divergence time estimates

The Afrothismia chronogram is comparable with its Glomus host chronogram by three nodes (table 6 in the electronic supplementary material). No matter which calibration strategy or relaxed clock method we applied to obtain the divergence estimates, Glomus nodes are significantly older than corresponding nodes in Afrothismia (figure 6 in the electronic supplementary material).

4. Discussion

All mycorrhizal fungi in the roots of the sampled myco-heterotrophic plants belong to Glomus group A, a relatively species-rich group of arbuscular mycorrhizal fungi. This supports previous observations in myco-heterotrophic Burmanniaceae, Triuridaceae, Corsiaceae and Gentianaceae (Bidartondo et al. 2002; Franke et al. 2006). It is noteworthy that we did not observe any fungal overlap among the different plant species, not even between closely related Afrothismia species. Specificity towards narrow groups of arbuscular mycorrhizal fungi has been detected in one species of Corsiaceae in South America (Bidartondo et al. 2002) and our data show that African species of Burmanniaceae and Triuridaceae are also associated with extremely narrow subsets of arbuscular mycorrhizal fungi. Parasites are generally more specialized than mutualists (Thompson 2005) and accordingly breakdown of the arbuscular mycorrhizal mutualism has led to extreme specialization towards fungi by myco-heterotrophic plants.

A surprising result emerges when we focus on the specialization towards narrow fungal lineages in Afrothismia, where mycorrhizal overlap is lacking even between very closely related species growing in the same regions: the phylogenetic relationships between the Afrothismia species closely mirror those of its fungal associates (figure 2). However, the divergence time estimates obtained by us for fungal nodes are significantly older than for their corresponding nodes in the plant phylogeny, thus reflecting our current knowledge about the macroevolutionary diversification of fungi and plants. Fossil evidence shows arbuscular mycorrhizas to be at least as old as the earliest land plants (Remy et al. 1994; Redecker et al. 2000) and it has been suggested that colonization of land by plants was dependent upon symbiosis with arbuscular mycorrhizal fungi. The evolutionary barrier to cheating has indeed been lowered exceptionally in the arbuscular mycorrhizal mutualism. Not only have arbuscular mycorrhizal cheaters evolved independently multiple times and diversified as cheaters, but they have also achieved exceptional evolutionary stability (e.g. outliving cheaters in plant–insect mutualisms by 25–48 Mya; Pellmyr & Leebens-Mack 2000; Rønsted et al. 2005).

Our observations indicate a cospeciation pattern between Afrothismia and its mycorrhizal fungi, but not in a synchronous way as in some host–parasite interactions where speciation of the host triggers speciation in the parasite. Our analyses show that when the photosynthetic ancestor of Afrothismia began specializing on particular fungal lineages, initiating or following the shift from mutualism to parasitism, most extant Glomus lineages had already diverged. However, once adapted to a group of related arbuscular mycorrhizal fungi, subsequent specialization and a lack of host-switching forced the plants to track the Glomus phylogeny, resulting in a delayed cospeciation process (sensu Hafner et al. 2003; figure 3). Phylogenetic congruence between Afrothismia and Glomus is not perfect; this incongruence can be explained by host-switching over short phylogenetic distance in the lineage leading to A. foertheriana, or by ‘rough’ phylogenetic tracking where the ancestor of the A. foertheriana, A. hydra, A. winkleri clade was temporarily associated with a paraphyletic Glomus lineage.

Figure 3.

Figure 3

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A model for the evolution of non-photosynthetic mycorrhizal plants. (a) A community of generalist mycorrhizal mutualists. Black squares represent mycorrhizal plants in two distant lineages ((a,b),c) and (w,x). Circles represent mycorrhizal fungi in two distant lineages ((1,2),3) and ((9,8),7). Double-ended arrows show mutualistic links. For example, 1 and 2 are sister taxa and their closest relative is 3. All plants must be linked to fungi and all fungi must be linked to plants. Double-ended arrows show mutualistic mycorrhizal links (i.e. plants provide carbon and fungi provide mineral nutrients). (b) A mycorrhizal community where a plant species x (grey) has lost the ability to photosynthesize, so it cannot provide carbon to fungi, breaking down the mycorrhizal mutualism. Non-photosynthetic plants depend on fungi that link them to photosynthetic plants. One-ended dashed arrows show mycorrhizal links where fungi provide carbon to plants. Non-photosynthetic plants have a reduced mycorrhizal range and only associate with related fungi 1, 2 and 3. (c) Speciation of the non-photosynthetic plant lineage x into x′, and x′ leads to further specialization on fungal lineages; x′ depends on fungus 3 and x″ depends on fungi 1 and 2. The fungi and the photosynthetic plants remain generalist mycorrhizal mutualists. (d) Speciation of plant lineage x″ into y and z. Plant y specializes on fungal host 2 and plant z specializes on closely related fungal host 1. This form of phylogenetic tracking by non-photosynthetic mycorrhizal plants towards pre-existing fungal lineages produces an evolutionary pattern of delayed cospeciation.

While this study shows that the arbuscular mycorrhizal mutualism has given rise to coevolving extreme specialist cheaters, the selective forces behind the specialization process remain speculative. The pattern observed cannot be explained by an evolutionary arms race because then we would expect a synchronous, or stepwise, cospeciation process. Unlike ectomycorrhizal cheater plants, which show both narrow host shifting and host jumps to distant fungal lineages (Jackson 2004), the phylogenetic congruence we detected shows a lack of host jumping to distant fungal lineages that is probably due to the effects of interspecific interactions in which fungal detection (e.g. seed germination cues) and/or fungal metabolic usage by plants (e.g. carbon transporters) are species-specific.

5. Conclusions

The interaction between plants and arbuscular mycorrhizal fungi is probably the most important symbiotic association of terrestrial ecosystems. Its promiscuous character has created opportunities for diverse plant lineages to successfully exploit these mycorrhizal symbioses. The shift from a mutualistic plant to a non-photosynthetic cheater is accompanied by specialization towards narrow groups of arbuscular mycorrhizal fungi. This study shows that extreme specialization and a lack of host-switching in closely related species of Afrothismia have led to a pattern of conservative evolutionary tracking hitherto unknown in mycorrhizal symbioses and horizontally transmitted symbioses in general.

Acknowledgments

This work was supported by grants from IWT Vlaanderen, FWO Vlaanderen, K. U. Leuven (to V.M.) and NERC and the Royal Society (to M.I.B). M.I.B. thanks D. J. Read, N. Røusted and H. Döring for insightful discussions. V.M. thanks E. Smets, S. Huysmans, G. Chuyong, M. Sainge, J. Moonen, P. Schols and J. De Laet for their help.

Supplementary Material

Additioanl material

Supplementary methods, figures, and tables

rspb20071622s02.doc (2.2MB, doc)

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