The authors are to be commended for the breadth and coherence of their multi-platform experimental design. The integration of PacBio HiFi genome assemblies, Iso-seq long-read transcriptomics, Illumina RNA-seq, and mass spectrometry proteomics across multiple species and tissues produces a robustly triangulated dataset that is difficult to dismiss. The use of independent lines of evidence to support each major claim; confirming venom gland expression of the hemostatic F10 paralog by both long-read isoform sequencing and short-read mapping, and then further validating protein presence by mass spectrometry,reflects commendable scientific rigor.

The comparative genomic framework is also well-constructed. By selecting outgroup species at carefully chosen phylogenetic distances; a non-venomous colubrid, an Asian elapid, an Australian elapid lacking vF5, and multiple sea snake species; the authors establish a clear evolutionary backdrop against which each genomic event can be contextualized. The synteny-based approach to tracing segmental duplications is particularly elegant, allowing the authors to reconstruct the order and boundaries of duplication events with high confidence.

Nevertheless, the study presents several methodological and inferential limitations that temper the strength of its conclusions. First, the argument for the temporal ordering of co-option and duplication rests on a most parsimonious basis rather than direct evidence. The authors propose that co-option preceded duplication for both F10 and F5 because residual expression of the liver-expressed paralogs is detectable in the venom gland today. However, this residual expression could equally reflect incomplete subfunctionalization after duplication or other post-duplication phenomena. The study cannot definitively exclude these alternatives, meaning the proposed sequential model remains a well-supported inference rather than a proven pathway.

Second, the study lacks functional experimentation for several of its key toxin candidates. This is most evident in the case of the procoagulant PLA₂ in O. microlepidotus, which the authors themselves describe as a "candidate" whose biochemical activity "has yet to be functionally validated". Similarly, the entire case for coagulation factor VII as a venom toxin is built exclusively on expression data and mass spectrometry protein detection, with no enzymatic assay provided to confirm its biological role in prey envenomation.

Third, the study draws broad conclusions about the evolutionary history of the entire Australian elapid radiation while only generating new genome assemblies for two in-group species: Pseudonaja textilis and Oxyuranus microlepidotus. Given the considerable diversity of the Hydrophiinae, deeper taxonomic sampling would be necessary to robustly support the proposed stepwise co-option model across the clade.

Fourth, the comparative analysis relies partly on a N. scutatus genome reassembled using ONT long-read sequencing, which is known to have accuracy limitations in homopolymeric regions. The authors acknowledge having to manually correct exonic indels where the F10 locus was initially absent, introducing a degree of subjectivity into a key outgroup comparison.

Finally, despite the introduction explicitly recognizing the importance of cis-regulatory mutations as the primary mechanism driving gene co-option in animal evolution, the study never characterizes how F10 and F5 came to be expressed in the venom gland at the molecular level. This omission represents a significant open question in an otherwise mechanistically detailed study.