Relaxed selection and convergent loss erode the retained plant-cell-wall-degrading enzymes of ectomycorrhizal fungi

<jats:p>The repeated evolution of the ectomycorrhizal (ECM) symbiosis from saprotrophic ancestors is accompanied by a contraction of the plant-cell-wall-degrading enzyme (PCWDE) repertoire, a pattern established from gene counts. Whether the PCWDE that ECM fungi retain remain functional, however, cannot be read from counts alone. Using 182 fungal genomes spanning ECM fungi and free-living decomposers, we separate two processes that gene counts conflate: the loss of genes and the relaxation of selection on the genes that persist. Catalytic-residue scoring shows that retained ECM PCWDE are 83-96% intact, yet this is true across all lifestyles and therefore uninformative about ECM specifically. We therefore tested selective regime directly with HyPhy RELAX and gene reduction with phylogenetic generalized least squares (PGLS). In the dedicated cellulose cellobiohydrolases GH6 and GH7 and the pectinase GH28, selection is significantly relaxed on ECM branches (relaxation parameter K = 0.46-0.87; P &lt;= 2.7 x 10^-5), whereas the broad, multifunctional family GH5 shows no relaxation (K = 1.00). Independently, PGLS shows that the PCWDE reduction in ECM fungi remains highly significant after phylogenetic correction (ECM carry ~0.41x the raw and ~0.55x the catalytically intact PCWDE of free-living decomposers; P &lt;= 9.8 x 10^-10; Pagel's lambda ~ 0.93-1.0), i.e. the reduction is convergent across the numerous independent ECM origins (spanning 11 fungal orders in our panel) rather than phylogenetic inertia. Comparing the two analyses family by family shows that gene loss (H3) and relaxed selection (H1) are separable: count reduction affects every family, including GH5, but relaxed selection is restricted to the dedicated PCWDE, which are also nearly absent from ECM genomes (present in 11-13% of ECM species) and under relaxed constraint where they do persist. The dedicated lignocellulolytic enzymes of ECM fungi are therefore both lost and, where retained, released from selective constraint, whereas the multifunctional GH5 is reduced in copy number but kept under purifying selection. Gene count, and even catalytic-residue integrity, overstate the lignocellulolytic capacity of ectomycorrhizal fungi.</jats:p>