Reassessment of coding-sequence constraint across the volvocine multicellularity gradient: correction of versions 1 and 2

Masato Tanigawa 

Versions 1 and 2 of this preprint interpreted lower dN/dS and shorter amino-acid terminal branches as evidence that coding-sequence constraint strengthens across the volvocine multicellularity gradient. Re-examination of the models and comparisons does not support that interpretation. The original rooted branch-model fits often reached boundary estimates. An unrooted specification, which is a different model for the original branch labels, gives lower foreground than background dN/dS in 71.5 % of 1,374 quality-controlled orthogroups, compared with the previously reported 88.6 % of 845; model specification and orthogroup selection both change. Separating the two lineages inferred in previous studies to represent independent origins of multicellularity yields different patterns: on a common set of 1,336 orthogroups, the fraction is 48.4 % for Tetrabaenaceae and 73.5 % for Goniaceae + Volvocaceae. At the latter origin, lower fitted dN/dS accompanies greater synonymous divergence relative to the Chlamydomonas reinhardtii branch (median path ratio 1.96), without a lower median nonsynonymous path (ratio 1.25). This arithmetic decomposition does not identify a cause or exclude stronger selection. On a separate public 263-gene dataset, the primary somatic-differentiation analysis retains only 2 usable contrasts. Numerically validated refits give a wide interval for the mean log path-length ratio (-0.33 to 0.45), including slower and faster evolution; applying both previously omitted filters is reported as a post hoc sensitivity check. Terminal branch length also confounds rate with time since the last sampled split, and no significant cell-number association was detected on the denser 57-OTU tree. The earlier comparisons therefore do not establish genome-wide strengthening of constraint or test disposable-soma theory. This corrected version identifies the unsupported interpretations, documents specification changes and numerical diagnostics, and provides the materials needed to reproduce its summaries.