Abstract
Phylostratigraphy, originally designed for gene age estimation by BLAST-based protein homology searches of sequenced genomes, has been widely used for studying patterns and inferring mechanisms of gene origination and evolution. We previously showed by computer simulation that phylostratigraphy underestimates gene age for a nonnegligible fraction of genes and that the underestimation is severer for genes with certain properties such as fast evolution and short protein sequences. Consequently, many previously reported age distributions of gene properties may have been methodological artifacts rather than biological realities. Domazet-Lošo and colleagues recently argued that our simulations were flawed and that phylostratigraphic bias does not impact inferences about gene emergence and evolution. Here we discuss conceptual difficulties of phylostratigraphy, identify numerous problems in Domazet-Lošo et al.'s argument, reconfirm phylostratigraphic error using simulations suggested by Domazet-Lošo and colleagues, and demonstrate that a phylostratigraphic trend claimed to be robust to error disappears when genes likely to be error-resistant are analyzed. We conclude that extreme caution is needed in interpreting phylostratigraphic results because of the inherent biases of the method and that reanalysis using genes exhibiting no error in realistic simulations may help reduce spurious findings.