Comparative plastome analysis and plastomic phylogeny of Moraceae with expanded species-level sampling

Moraceae is an economically and ecologically important angiosperm family. Although recent nuclear and plastid phylogenomic studies have greatly improved the systematic framework of Moraceae, structural evolution and lineage-specific variation of plastomes remain insufficiently evaluated under dense complete-plastome sampling. To address these issues, we conducted a plastome-based phylogenetic and comparative genomic analysis using 140 complete plastomes (49 newly sequenced and 91 publicly available), representing all seven tribes and 17 genera of Moraceae.

Moraceae plastomes were generally structurally conserved, although lineage-specific variation in inverted repeat (IR) boundaries was detected, particularly in Artocarpus and Ficus. Nucleotide diversity analyses identified several highly variable non-coding regions (including ndhC-trnV(UAC), ndhD-psaC, psbI-trnS(GCU), and trnL(UAG)-ccsA) and protein-coding genes (matK, rps11, ndhF, rps15, and ycf1), which may serve as candidate molecular markers for phylogenetic reconstruction and species identification in Moraceae. Analyses of repeat sequences and codon usage revealed a balance between structural conservation and sequence variability. Selection pressure analyses indicated that most plastid genes are under purifying selection, with a small number of genes showing signals consistent with positive selection in specific lineages, including rbcL in Morus, and clpP and rps19 in Ficus. Phylogenetic reconstruction based on complete plastome sequences strongly supported the monophyly of all seven recognized tribes of Moraceae, recovered the non-monophyly of Streblus, and placed Maclura within Chlorophoreae, consistent with recent nuclear and plastid phylogenomic frameworks.

Because plastomes represent a single, non-recombining organellar genome, our phylogenetic results should be interpreted as a plastome-based topology rather than a comprehensive species-tree reconstruction. This study provides an expanded species-level complete-plastome resource and phylogenetic framework for Moraceae, refines understanding of IR boundary evolution, and improves the robustness of candidate molecular markers for future systematic and species-identification studies.