Comparative genomics of nitrogen acquisition and assimilation gene-family copy-number architecture across chlorophytes.

Nitrogen acquisition and assimilation determine nitrogen availability for algal growth and metabolism; however, their gene-family organization across chlorophytes remains poorly resolved. We examined 10 gene families (NiR, NR, NRT, AMT, GS, GOGAT, GDH, ASN, CPS-L, and CPS-S) in 30 chlorophyte genome assemblies using family-specific sequence-based curation. The assemblies represented 26 biological species after taxonomic standardization and pseudoreplication control. One representative assembly per species defined the primary dataset (D1; n = 26), with BUSCO C ≥ 80% (D2; n = 22) and C ≥ 90% (D3; n = 18) subsets used for genome-completeness sensitivity analyses. A BUSCO-based species phylogeny was used to visualize copy-number patterns relative to relationships among sampled taxa. AMT and NRT showed the broadest copy-number distributions, whereas NiR and the CPS subunits were predominantly single-copy. After normalization for the unequal number of families in each module, nitrogen acquisition had a higher copy-number per family than intracellular assimilation in D1 (Wilcoxon W = 8, p = 4.93 × 10- 5), and this difference remained significant in D2 and D3. Of 45 pairwise gene-family associations in D1, none remained significant after Benjamini-Hochberg correction; isolated FDR-supported associations in D2 and D3 were not consistent across datasets. These results show heterogeneous nitrogen gene-family architecture across chlorophytes and indicate that the principal descriptive copy-number patterns were stable to genome-completeness filtering. The curated dataset, reproducibility package, and phylogenetic visualization framework provide a basis for further functional and gene-family evolutionary analyses.