Gene loss is measurable, relaxed selection on the genes that remain is not: a retraction and recalibration of branch-specific selection tests in ectomycorrhizal fungi

The contraction of the plant-cell-wall-degrading enzyme (PCWDE) repertoire in ectomycorrhizal (ECM) fungi is among the most reproducible results in fungal comparative genomics, and it is routinely read as a loss of decay capacity - a statement about the genes that persist, which counts cannot make. We addressed that question in 183 fungal genomes at two levels and reached opposite conclusions. Gene loss is measurable. On a 182-tip species tree, ECM genomes carry 0.367 times the PCWDE complement of free-living decomposers (phylogenetic generalized least squares; 95% CI 0.295-0.457; P = 1.3 x 10^-15; Pagel's {lambda} = 0.92); the ECM coefficient is not absorbed into the phylogenetic covariance term. The selective regime of the copies that remain is not measurable with the standard branch-partitioned test, and this version retracts the relaxed-selection claims we made for GH6, GH7 and GH28 in version 1. Four independent controls support the retraction. (i) Calibration: applying the identical design to single-copy orthologues, which have no reason to experience a change of selective regime specific to ECM lineages, the relaxation test rejected the null for 35.8% of loci at = 0.05 (29 of 81; 43.9% of the 66 returning an interpretable statistic), against a nominal 5%. (ii) Non-convergence: in 46 of 410 fits the likelihood-ratio statistic was negative, an outcome that cannot occur if the optimisation has converged. (iii) Non-reproducibility: across 80 pairs of fits sharing alignment, tree and branch labels, none recovered its own log-likelihood to within 0.01 units, while the nested model fits computed inside the same runs agreed to within 1.99 units. (iv) Resolution: with 81 to 82 null replicates the smallest attainable empirical P value is about 0.012, against a Bonferroni threshold of 0.0125 for the four families tested here - a margin we had not computed, and one that any expansion of the family set removes. No family-level statement about selective regime is supported by these data. We emphasise the symmetry: we do not claim that these genes are maintained either. The pattern of gene loss described by previous work stands, and is strengthened by phylogenetic correction; what does not stand is the inference from that pattern to the functional state of the genes that remain.