# Ectomycorrhizal symbiosis evolved independently and by convergent gene duplication in rosid lineages

Fabian van Beveren, Yvet Boele, Camille Puginier, Matheus Bianconi, Cyril

Libourel, Maxime Bonhomme, Jean Keller, Pierre-marc Delaux

► To cite this version:

Fabian van Beveren, Yvet Boele, Camille Puginier, Matheus Bianconi, Cyril Libourel, et al.. Ectomy-corrhizal symbiosis evolved independently and by convergent gene duplication in rosid lineages. New Phytologist, 2025, 246 (4), pp.1432-1438. 10.1111/nph.70054. hal-05345646

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# Ectomycorrhizal symbiosis evolved independently and by convergent gene duplication in rosid lineages

Fabian van Beveren¹*§, Yvet Boele²,³*, Camille Puginier¹, Matheus Bianconi¹, Cyril Libourel¹, Maxime Bonhomme¹, Jean Keller¹, Pierre-Marc Delaux¹

1 Laboratoire de Recherche en Sciences Végétales (LRSV), Université de Toulouse, CNRS, UPS, INP, Toulouse, 31320, Castanet-Tolosan, France

2 Laboratory of Cell and Developmental Biology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, the Netherlands

3 Department of Terrestrial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Droevendaalsesteeg 10, 6708 PB Wageningen, the Netherlands

§ Current address: Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE Wageningen, the Netherlands

## Introduction

Many land plants rely on mutualistic symbiotic associations to thrive, starting with their common ancestor associating with arbuscular mycorrhizal (AM) fungi (Rich et al., 2021). Similar to AM symbiosis, multiple other intracellular symbiotic interactions have evolved in a variety of land plants, such as ericoid symbiosis in Ericaceae and root nodule symbiosis with nitrogen-fixing bacteria in the legumes and relatives (Strullu-Derrien et al., 2018; Radhakrishnan et al., 2020). Ectomycorrhizal (ECM) symbiosis contrasts with these relationships as there is no intracellular accommodation of the symbiont inside the plant cell, but only intercellular colonization (Brundrett & Tedersoo, 2018). This symbiotic relationship is common in a variety of seed plants, mostly trees and shrubs such as the gymnosperm pines, and dicot willows and oaks (Cairney, 2000; Wang & Qiu, 2006; Tedersoo & Brundrett, 2017). The fungal partners are primarily of the Ascomycete, Basidiomycete and sporadically Zygomycota clades, and evolved from a saprotrophic to symbiotic lifestyle (Cairney et al., 2000; Martin et al., 2016), with varying degrees of plant host specificity (Den Bakker et al., 2004; Plett et al., 2015; Lofgren et al., 2021).

Effort has been put to uncover the fungal side of this symbiotic relationship (Martin et al., 2008; Kohler et al., 2015; Liao et al., 2016; Martin et al., 2016; Zhang et al., 2018; Chowdhury et al., 2022; Zhang et al., 2022), but the plant side remains more elusive. Although it is known which plant lineages are involved in ECM symbiosis (Tedersoo & Brundrett, 2017), there has been little investigation into the evolutionary
origin of ECM symbiosis in these lineages. The current hypothesis is that plants
evolved the ability to engage in ECM repeatedly and independently as a response to
environmental change, but genetic evidence for this is lacking (Cairney, 2000; Wang
& Qiu, 2006; Brundrett & Tedersoo, 2018). Furthermore, the genetic innovations and
transcriptomic response related to this symbiosis have been studied largely in a genus
or species-specific context (Liao et al., 2016; Bouffaud et al., 2020; Chowdhury et al.,
2022; Hill et al., 2022), which hinders the study of their evolutionary origins. It has been
proposed that most ECM plants have evolved from AM plants, potentially repurposing
AM genes for ECM (Brundrett & Tedersoo, 2018; Li et al., 2024).

In this study, we reconstruct the origin of ECM in the rosid clade, showing at
least 16 independent origins, resulting in the 17 known extant ECM rosid lineages.
Moreover, comparative genomics of these lineages highlight genes involved in cell
wall remodeling which underwent duplications in a convergent manner across
ectomycorrhizal lineages.

**Results and Discussion**

***Ancestral state reconstruction supports multiple independent gains of ECM in angiosperms.***

The many different plant ECM lineages have been proposed to have originated
independently (Brundrett, 2009; Tedersoo & Smith, 2013). In rosids, the main ECM
lineages are Fagales, Salicaceae (Malpighiales) and Myrtoideae (Myrtales), but also
include less species-diverse clades in the orders Fabales, Rosales, Malpighiales and
Malvales (Tedersoo & Brundrett, 2017). We reconstructed a phylogenetic tree of
rosids based on sequences of two plastid-encoded proteins: the large subunit of
ribulose-bisphosphate carboxylase (RbcL) and Maturase K (MatK) (Supplementary
table S1), to model the evolution of the ECM character. Using the best-performing
model (ARD+kappa), we found a largely independent origin of ECM lineages, in
agreement with the literature (Fig. 1; Supplementary methods S1, Supplementary
table S2). The rate of non-ECM to ECM was modeled to be higher than vice-versa
(0.21; 0.063), suggesting ECM symbiosis is more frequently gained than lost. The
kappa value in our model is low (~0.07), which results in little weight being put on the
branch lengths for the trait evolution. A clear single origin in the common ancestor of
Fagales can be detected, with a loss in the Myricaceae, a group that instead
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associates with nitrogen-fixing bacteria of the genus *Frankia* (Huguet *et al.*, 2005).

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ECM symbiosis in Myrtoideae has a clear single origin with a putative single loss in

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the common ancestor of *Pimenta* and *Myrtus*, both members of Myrteae. This single

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loss agrees with the literature (Tedersoo & Brundrett, 2017), although additional losses

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have been suggested that can only be recovered with a denser taxon sampling of this

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clade (Thornhill *et al.*, 2015; Tedersoo & Brundrett, 2017).

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Tedersoo and Brundrett (2017) separate four ECM lineages within the

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Detarioideae (Fabales; Fig. 1), two of these we estimate to have originated

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independently: the Afzelia group (*Afzelia* and *Intsia*) and the Berlinia group (here

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represented by *Brachystegia*, *Gilbertiodendron* and *Julbernardia*). We detect a single

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origin in the common ancestor of the other two groups (*Dicymbe* and

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*Cryptosepalum*+*Paramacrolobium*), followed by a loss in *Amherstia* (Fig. 1). However,

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the relationships among lineages of Detarioideae remain uncertain, and more data

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besides *RbcL* and *MatK* markers might be necessary to resolve their phylogeny (de la

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Estrella *et al.*, 2017; 2018). This is especially the case for the tribe Amherstiae, where

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for example the species *Polystemonanthus dinklagei* may be important as it is sister

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to the genus *Dicymbe*, but its ECM status is unknown (Tedersoo and Brundrett, 2017).

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All other rosid ECM lineages are shown to have evolved the symbiosis independently

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(Fig. 1). Overall, our reconstruction of ancestral states within the rosids shows that the

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identified ECM lineages largely evolved this symbiotic association independently.

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As ancestral state reconstruction is limited by phylogenetic signal, taxon

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sampling and limited models for character evolution, we decided to also look for

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genetic signal that may indicate an ancestral origin of ECM in rosids. With a database

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of 63 genomes, including 25 ECM and 19 non-ECM rosids (Supplementary table S3),

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we reconstructed orthologous groups associated to the common ancestral node of

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rosids, and mined for orthogroups being largely absent in the non-ECM rosids

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(Supplementary Methods S1). If there would be a common ECM ancestor, convergent

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gene losses would be observed in the non-ECM lineages following the loss of ECM, a

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phenomenon observed following the loss of other symbioses (Delaux *et al.*, 2014;

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Bravo *et al.*, 2016; Griesmann *et al.*, 2018). After manual curation, we identified six

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genes that share an origin in the common ancestor of rosids, but have been retained

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mostly by ECM species, although they remain present in several genomes of non-

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ECM species (Fig. 2, Supplementary Figs. S1-S6). Part of the reason may be that the

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pressure to lose them is strengthened in non-ECM lineages, but not enough to result
in a complete loss in these lineages. It may also be that the loss of ECM is recent in some of these lineages. For example, a specific short-chain dehydrogenase/reductase (SDR) gene was retained in non-ECM Rosales, but Rosales contains several ECM-lineages, thus the presence of this gene could be explained by the recent loss of ECM (Fig. 2). It is however difficult to tell how many genes are expected to follow such a pattern by chance, with retention only in a small subset of lineages. In addition, none of these genes are conserved in all the ECM lineages covered by our analysis.

Overall, the retention of these genes does not clearly support an ancestral origin of this symbiosis, and in light of the ancestral state reconstruction the occurrence of ECM in rosids is still best explained by multiple independent origins.

**ECM evolution is linked with convergent gene duplications**

The independent emergence of traits may result either from completely different or from convergent genetic pathways. Convergent evolution occurs through the co-option of existing genes or following gene-duplication and may result in similar molecular responses (Satterlee *et al.*, 2024).

First, we reasoned that convergence towards the ability to engage in ECM symbiosis should be detected at the transcriptomic level, thus we looked for shared transcriptomic responses to ECM in three host species: *Quercus robur*, *Populus tremula* × *P. tremuloides* and *Eucalyptus grandis* (Bouffaud *et al.*, 2020; Chowdhury *et al.*, 2022; Hill *et al.*, 2022). Comparing the overlap in deregulation of genes during ECM, we find a set of 537 orthogroups that are deregulated in all three species (Supplementary Methods S1, Supplementary Fig. S7, Supplementary table S4). This number of shared differentially expressed orthogroups is higher than would be expected by chance (mean: 359, p-value: 0.00001), which is especially notable considering different symbionts and time points tested in the three studies included in this analysis. Exclusion of orthogroups that are also deregulated in response to chitin in *M. truncatula* and *A. thaliana* (Feng *et al.*, 2019; Bjornson *et al.*, 2021), datasets that mimic a generic response to fungi, still results in a set of larger than expected (observed: 447, sampled mean: 327, p-value: 0.00001). This shows that at least from a transcriptomic perspective, there is a similar response to the presence of ECM fungi.

Then, we investigated the potential contribution of gene duplication to the convergent evolution of ECM. To do so, we looked into gene duplication events that
have occurred independently in the ancestral nodes of the five ECM rosid lineages that were covered in our genome analysis: Fagales, Salicaceae, Myrtoideae, Dipterocarpaceae-Cistacea and *Acacia* (Supplementary Methods S1). Using OrthoFinder, we detected 61 orthogroups with duplication events in the groups represented by multiple genera: the common ancestors of the Salicaceae and the one of the Fagales. Among these 61 orthogroups, a number are overall frequently duplicated across the whole phylogeny. To further identify relevant candidates in this list, we selected the ones that matched one of the following three additional criteria: (1) at least one of the duplicated genes is deregulated in the three ECM species with available RNAseq data (Supplementary table S4), (2) a high proportion (> 1/7) of duplication co-occur with the gain of ECM, or (3) more than half of the duplication occurred in any ECM node. This filter resulted in the shortlisting of 27 orthogroups, which after manual curation through single-gene phylogenetic analyses resulted in a final selection of nine genes (Supplementary Figs. S8-S16). These genes are duplicated in a minimum of three ECM origin nodes and not frequently duplicated in a set of non-ECM nodes (Fig. 2; supplementary table S5).

Strikingly, three out of the nine candidate genes have functions associated with cell-wall remodeling: a Pectinesterase inhibitor, a Pectin methylesterase and a Cellulase (Fig. 2). Modification of the plant cell wall is a common feature in intra- and intercellular plant - fungi mutualistic symbioses (Su, 2023). In particular, extensive pectin modifications have been observed during the interaction between ECM fungi and their hosts, and part of these modifications are mediated by the fungal partner (Chowdhury et al., 2022). However, comparative phylogenomics of the fungal symbionts have revealed that ECM fungal lineages have a reduced set of plant cell wall degrading enzymes, a genomic reduction that occurred in a convergent manner (Kohler et al., 2015). The convergent expansions of gene families encoding for cell-wall modifying enzymes in plant lineages that evolved the ability to engage in ECM symbiosis might reflect a shift in the distribution of this functionality from the symbiont to the host which may represent a common principle for the evolution of ECM symbiosis.

**Convergent co-option of the common symbiosis pathway for ECM may have occurred in rosids**
Besides gene duplication, convergent evolution originates from co-option of existing genes and pathways. In the context of ECM, it has been proposed that its evolution in Fagales was facilitated by the co-option of the common signaling pathway (CSP; Li et al. 2024), a module of several proteins essential for the establishment of intracellular symbiosis (Parniske, 2008). This hypothesis, supported by reverse genetic analyses in poplar (Cope et al., 2019), is based on the conservation of those genes in certain plant species forming ECM which have, in parallel, lost the ability to do any form of intracellular symbioses. Indeed, the loss of this ability has been correlated in many plant lineages with the loss of associated genes by relaxed selection (Delaux et al., 2014; Bravo et al., 2016; Radhakrishnan et al., 2020). Thus, the retention of these genes may reflect the evolution of another selection pressure, possibly by ECM (Li et al., 2024). Similarly, genes have been found to be specifically dedicated to the ability to engage in arbuscular mycorrhizal symbiosis (AMS). The retention of one of such genes in ECM Fagales, the Glycerol 3-Phosphate Acyltransferase RAM2, an enzyme essential for the functioning of the AMS (Wang et al., 2012), also indicates that it might have been co-opted for ECM in that lineage (Li et al., 2024).

To determine whether the convergent evolution of ECM relied more broadly on the co-option of these genes, we conducted targeted phylogenies on the CSP genes *SYMRK*, *CCaMK* and *CYCLOPS*, and on the AMS-specific genes *RAD1*, *STR* and *STR2* (Supplementary Methods S1, Supplementary Figs. S17-S22). Only three of the included genomes are lacking *RAD1*, *STR1* and *STR2*. Two of these are *Arabidopsis thaliana* and *Lupinus albus*, both known to lack mycorrhizal (both AM and ECM) symbioses. The other is *Morella rubra*, which has multiple observations of AM listed on the FungalRoot database (Soudzilovskaia et al., 2020, 2024). Moreover, other members of the genus *Myrica/Morella* are considered to be both ECM and AMS species (Urgiles et al., 2014; Teste et al., 2020). It is possible that the loss of AMS is species-specific, although we cannot rule out that the genes were simply not recovered in the genome assembly (BUSCO completeness 92.2%). More interestingly, the AMS-specific genes were mostly retained in the ECM species that lost AMS: *C. fangiana* and *B. pendula* retained all three genes, whereas *Q. robur* and *F. sylvatica* retained two of the tree genes (Fig. 2). Similarly, among the CSP genes, *SYMRK* and *CCaMK* were found in all ECM species, including those that lost AMS (Fig. 2). The same pattern was observed for *CYCLOPS* with the notable exception of
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Fagaceae and the genus *Morella*, in which the gene was either not detected or only partially recovered. Although this may be attributable to low quality genomes, the observation that these potential losses are restricted to a single clade might deserve further investigation. For the retained genes, we also investigated the expression level during ECM symbiosis. CSP and AMS-specific genes were generally not deregulated, except for one copy of *CYCLOPS* in *P. tremula* × *P. tremuloides* and the *STR* gene of *E. grandis* (Supplementary table S4). Altogether, this suggests there may be co-option of CSP and AMS-specific genes to independent instances of ECM, or that the losses of AMS in ECM-only lineages are relatively recent.

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The combination of ancestral state reconstruction, comparative phylogenomics and transcriptomics, and single-gene phylogeny conducted here do not support an ancestral gain of ECM in rosids, but rather the independent evolution of this symbiotic ability. These independent events, involving different fungal lineages (Kohler *et al.*, 2015), occurred in a convergent manner by gene co-option and duplications leading to the emergence of a similar transcriptional response.

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## Acknowledgements

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The authors thank the Genotoul bioinformatics platform Toulouse Occitanie (Bioinfo Genotoul, https://doi.org/10.15454/1.5572369328961167E12) for providing computing resources. P-M.D was supported by the project Engineering Nitrogen Symbiosis for Africa (ENSA) currently funded through a grant to the University of Cambridge by the Bill and Melinda Gates Foundation (OPP1172165) and the UK Foreign, Commonwealth and Development Office as Engineering Nitrogen Symbiosis for Africa (OPP1172165). This project received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement no. 101001675 - ORIGINS) to P.-M.D. This project was supported by the Laboratoires d'Excellence (LABEX)' TULIP (ANR-10-LABX-41) and the 'École Universitaire de Recherche (EUR)' TULIP-GS (ANR-18-EURE-0019). MBi has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 101105838 as SYMBIOLOSS.

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237 **Author contributions**

238 FvB, YB, CP, MBi, CL, MBo and JK conducted analyses. FvB, YB, PMD and JK
239 planned and designed the research. PMD coordinated the project. FvB, YB and PMD
240 wrote the manuscript. FvB and YB contributed equally.

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242 **Data availability**

243 Code used for this work is available on GitLab
244 (https://gitlab.com/fabianvanbeveren/ecm/) and phylogenies and alignments can be
245 found on Figshare (https://figshare.com/s/21bc629367a4c979ae70). Genome data
246 used is listed in table S1 and sources for the RbcL and MatK sequences in table S3.

247
<figure><img src="image_2.png" /><figcaption>**Fig. 1:** Ancestral reconstruction of ectomycorrhizal symbiosis in plant lineages of rosids. Phylogenetic tree was reconstructed based on plastid protein sequences of RbcL and MatK, reconstructed under the model Q.plant+C60+F0+R5. Ancestral reconstruction is based on the ARD+kappa model. Likelihood of the ECM (red) or non-</figcaption></figure>
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ECM (white) status is shown proportionally for each node as a pie diagram, whereas status of included rosid species is shown next to the species name. The 17 rosid ECM clades identified by Tedersoo and Brundrett (2017) are shown with thick black lines and their names and the 16 yellow stars represent independent origins of ECM symbiosis. The two clades represented by multiple species that lost the ability for ECM symbiosis are shown with dashed lines. Asterisks denote species also included in the OrthoFinder analysis and the species name is placed between parentheses if a different species was used. Scale bar represents estimated substitutions per site. Cu. = Cucurbitales, Crypt. gr. = Cryptosepalum group, Myr. = Myrteae, Myrtoi. = Myrtoideae, Dipt.-Cist. = Dipterocarpaceae-Cistacea, Br. = Brassicales, Sp. = Sapindales.
<figure><img src="image_3.png" /><figcaption>**Fig. 2:** Cladogram of rosid species included in the orthogroup analysis, depicting ECM-specific gene duplications and retainment of genes with a known or putative role in symbiosis. Phylogenetic relationships are based on Figure 1. Duplications are shown for 11 taxonomic groups. A single faint circle means no duplications occurred, whereas two or multiple circles represent one or more duplications, and the circles are colored by the ECM (red) or non-ECM (gray) status of that node. Presence (colored) or absence (white) are shown for genes part of the common signaling pathway (CSP), specific to arbuscular mycorrhizal symbiosis (AMS) and/or root nodule symbiosis (RNS) and those retained in ECM lineages. When the AMS state of a species is uncertain the circle is half-colored. Genes partially present are shown by a half-colored square. Duplicated genes related to cell wall remodeling are shown in bold. Abbreviations: compl. = completeness, HAD = haloacid dehalogenase-like, SDR = short-chain dehydrogenase/reductase, O-methylfransf. = O-methyltransferase, TF = transcription factor.</figcaption></figure>

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