Parajuli, S.; Adhikari, B.; Fennell, A.; Nepal, M. P.

Genome-wide identification of plant gene families is essential for functional and evolutionary studies but often requires the use of multiple independent tools for homolog detection, domain validation, orthology assignment, and phylogenetic analysis. This fragmented approach involves extensive manual scripting, complicates reproducibility and parameter tracking, and may require additional steps to remove redundant protein isoforms. To address these challenges, we developed PhytoFam, a Nextflow-based workflow that automates gene family identification from proteome input through phylogenetic reconstruction. The pipeline integrates HMMER for candidate sequence identification, isoform-aware deduplication, InterProScan for domain confirmation, BLAST reciprocal best hit (RBH) analysis for orthology assignment, MUSCLE for multiple sequence alignment with optional outgroup incorporation, TrimAl for alignment trimming, and IQ-TREE3 for phylogenetic reconstruction. PhytoFam is portable across local workstations and high-performance computing environments and supports deployment through Conda, Docker, and Singularity. We validated the workflow using the Morus alba MADS-box gene family, where the complete analysis finished in 1 h 10 min (9 CPU h). IQ-TREE3 accounted for most of the execution time, whereas InterProScan showed the highest memory requirement with a peak resident set size of 4.5 GB. PhytoFam provides a reproducible, automated, and scalable solution for plant gene family identification and phylogenetic analysis. The pipeline is freely available at https://github.com/sanamparajuli/PhytoFam.