Comparative chloroplast genomics in Amorphophallus: revealing codon usage patterns and phylogenetic clades across 16 species

Amorphophallus is an important genus within Araceae, possessing economic value and considerable ornamental diversity. However, accurate taxonomic identification using traditional methods remains challenging due to pronounced morphological plasticity and the temporal separation of leaf and flower emergence. To date, large-scale comparative analyses of chloroplast genomes within this genus are lacking, and patterns of structural variation and phylogenetic relationships remain unclear. Accordingly, this study aims to elucidate the structural evolutionary characteristics and codon usage bias of Amorphophallus chloroplast genomes through comparative genomic analysis and to clarify their phylogenetic placement. The chloroplast genomes of six Amorphophallus species ( A . pendulus , A . glaucophyllus , A . fornicatus , A . macrophyllus , A . titanum , and A . hewittii ) were sequenced, assembled, and annotated using the PacBio Revio platform. These data were integrated with those from ten previously reported species and subjected to comparative genomic analysis, codon usage bias analysis, and phylogenetic reconstruction. The chloroplast genome sizes of the 16 Amorphophallus species ranged from 152,492 to 185,810 bp and exhibited a typical quadripartite structure. Inverted repeat (IR) region expansion was detected in A . macrophyllus and A . glaucophyllus , whereas IR contraction was identified in A . fornicatus . The gene accD (Pi = 0.1217) was identified as a highly variable region. Codon usage patterns exhibited a bias toward A- and U-ending codons, with natural selection identified as the predominant driving force. Six shared optimal codons were detected across all species. Only C → U base editing events were identified in the chloroplast genomes of the 16 Amorphophallus species. The genomic region spanning 80,401–81,200 bp was identified as an optimal DNA barcoding window, enabling discrimination of all 16 species using five diagnostic loci. Phylogenetic analysis revealed that all Amorphophallus species formed a monophyletic group, which was further divided into three major clades: Continental Asia I (CA- I), Continental Asia II (CA-II), and Southeast Asia (SEA). The SEA clade comprised four species: A . fornicatus , A. pendulus , A. hewittii , and A. titanium . The CA-II clade contained six species: A. glaucophyllus , A. macrophyllus , A. konjac , A. krausei , A. albus , and A. kachinensis . The CA-I clade included five species and one accession not identified to species: A. coaetaneus , A. tonkinensis , A. yunnanensis , A. kiusianus , A. muelleri , and Amorphophallus sp. Within Araceae, Symplocarpus is sister to the remaining sampled members of Araceae, and Amorphophallus was most closely related to Syngonium , Xanthosoma , Zomicarpella , and Caladium . This study systematically elucidates the patterns of structural variation and evolutionary features of Amorphophallus chloroplast genomes, clarifies the mechanisms underlying codon usage bias, and resolves the phylogenetic placement of the genus. These findings provide essential foundational data for species identification, germplasm conservation, and molecular breeding.