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

Comparative chloroplast genomics in Amorphophallus: Revealing codon usage patterns and phylogenetic clades across 16 species | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative chloroplast genomics in Amorphophallus: Revealing codon usage patterns and phylogenetic clades across 16 species Haoliang Shi, Qinghu Lu, Ying Qi, Min Yang, Meiwei Zhao, Penghua Gao, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9564592/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background 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. Results 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 strongly supported Amorphophallus as a monophyletic group (bootstrap support = 100%), which was further divided into three major clades: Continental Asia I, Continental Asia II, and Southeast Asia. Within Araceae, Amorphophallus occupies a crown group position and forms a sister group relationship with Syngonium , Xanthosoma , Zomicarpella , and Caladium , whereas Symplocarpus is resolved as a basal lineage. Conclusions 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. Amorphophallus chloroplast genome gene structure analysis codon usage bias analysis phylogenetic analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 7 Figure 8 Figure 11 Figure 12 Figure 13 1. Introduction Chloroplasts are organelles in plant cells responsible for photosynthesis and a range of metabolic processes. Beyond their central role in metabolism and energy conversion [ 1 , 2 ], chloroplasts are involved in the biosynthesis of starch, fatty acids, pigments, and amino acids. The chloroplast genome is considerably smaller than the nuclear genome. It exhibits several advantageous characteristics, including a moderate molecular weight, ease of sequencing, multiple copies, a relatively simple structure, moderate nucleotide substitution rates, pronounced differences in molecular evolutionary rates between coding and non-coding regions, and substantial synteny among chloroplast genomes across diverse taxa [ 1 , 2 ]. Chloroplasts contain a maternally inherited genome that operates independently of the nuclear genome. Owing to these features, chloroplast genomes have been widely applied in plant species identification, genetic diversity assessment, and phylogenetic reconstruction [ 3 ]. Their utility has become increasingly evident in plant phylogenetic studies integrating phylogenomic and comparative genomic analyses. Furthermore, a growing number of chloroplast genomes have been sequenced and assembled, and extensive genetic resources have been analyzed using various bioinformatics approaches, thereby offering novel insights into resolving taxonomically challenging plant groups [ 4 ]. The genetic code is degenerate, whereby most amino acids are encoded by multiple synonymous codons. Codon usage bias refers to the non-uniform utilization of synonymous codons and arises from the combined effects of mutation pressure, natural selection, and genetic drift [ 5 ]. This bias is widespread across all species and displays substantial variation among distantly related taxa. In applied contexts, exploitation of species-specific codon usage patterns can enhance the efficiency of transgene expression. Moreover, analysis of codon usage bias in chloroplast genomes provides crucial foundational information for elucidating phylogenetic relationships and supporting genetic engineering applications [ 6 ]. The genus Amorphophallus (Araceae) represents one of the most species-rich lineages globally, comprising approximately 250 species widely distributed across tropical and subtropical regions of Asia and Africa [ 7 ]. Members of this genus exhibit remarkable morphological and phytochemical diversity [ 8 , 9 ]. It includes glucomannan-rich resource species, such as A . konjac , A . muelleri , and A . albus [ 10 – 12 ], as well as A . paeoniifolius , which is used in ethnomedicine for treating gastrointestinal disorders [ 7 ]. Additionally, Amorphophallus includes species of considerable horticultural and ornamental value, including A . gigas , whose inflorescence can reach up to 3 meters in height [ 13 ], A . dunnii , which produces distinctive blue fruits [ 14 ], and A . atroviridis , characterized by conspicuous pink petioles and leaf veins [ 13 ]. Despite this diversity, taxonomic studies of Amorphophallus have long faced considerable challenges. Accurate species identification based solely on morphological characters is often hindered by pronounced morphological plasticity, hysteranthy (the temporal separation of leaf and flower emergence), and morphological convergence among species, leading to taxonomic ambiguity and misutilization of germplasm resources [ 13 , 15 ]. Consequently, the development of a high-resolution molecular phylogenetic framework is essential for elucidating the evolutionary history of the genus and supporting the accurate taxonomic application of its resources. Although chloroplast genomes of more than ten Amorphophallus species have recently been reported [ 7 , 16 – 26 ], sampling coverage remains critically insufficient given the genus's high species richness. Existing studies have mainly focused on genome assembly and basic annotation of individual species, with limited implementation of large-scale, multi-species comparative analyses based on complete chloroplast genomes. In particular, systematic synthesis remains lacking for key aspects, including patterns of structural variation in chloroplast genomes, the mechanisms underlying codon usage bias, and the evolutionary drivers associated with geographic isolation. Based on the currently limited number of sequenced species, a preliminary understanding of the basic features and initial variation of Amorphophallus chloroplast genomes has been established. Studies indicate that Amorphophallus species generally conform to the typical circular quadripartite structure characteristic of angiosperms, yet exhibit substantial interspecific variation in genome size. Specifically, chloroplast genome sizes range from 164,417 ( A . yunnanensis ) to 177,076 bp ( A . muelleri ), encoding 126–131 genes, with guanine–cytosine (GC) content ranging from 34.5% to 36.0%. Structural analyses further reveal species-specific variations in inverted repeat (IR) region length (26,225–35,204 bp) and in the distribution of key boundary genes, including rpl2 , trnH - GUG , and ycf1 . Moreover, mutational hotspot analysis has identified four highly variable regions, namely trnM - atpE (Pi = 0.16), atpB (Pi = 0.155), atpB - rbcL (Pi = 0.15), and ycf1 (Pi = 0.147) [ 7 , 16 – 26 ], underscoring their potential as molecular markers for species identification in Amorphophallus . However, these studies have largely focused on describing basic genomic characteristics, without fully elucidating the underlying evolutionary mechanisms. The relationships among interspecific genome size variation, IR boundary dynamics, and adaptive differentiation remain insufficiently understood and require broader taxon sampling and more systematic comparative genomic analyses. To address these research gaps, 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 high-throughput sequencing platform (Fig. 1 ). These datasets were then integrated with published chloroplast genome sequences of ten additional species to construct a comprehensive comparative genomic framework encompassing 16 representative species. The objective of this study were to (i) systematically compare structural features, including genome size, gene content, and IR boundary configurations, across the sampled species; (ii) elucidate the characteristics of codon usage bias in Amorphophallus and assess the relative contributions of driving factors (mutation pressure versus natural selection); (iii) resolve previously ambiguous phylogenetic relationships derived from fragment-based analyses, thereby establishing a robust framework for reconstructing the evolutionary history of the genus. Ultimately, this study provides a valuable reference for future breeding programs and the development of molecular markers in Amorphophallus . 2. Materials and Methods 2.1 Plant material sampling, DNA extraction, sequencing, and chloroplast genome assembly and annotation 2.1.1 Plant material sampling, DNA extraction, sequencing Six Amorphophallus species ( A . pendulus , A . glaucophyllus , A . fornicatus , A . macrophyllus , A . titanum , and A . hewittii ) were cultivated at Kunming University. Fresh leaves of each species were subsequently collected from the konjac germplasm nursery of Kunming University, Yunnan Province (latitude: 24.97406°N, longitude: 102.79605°E). Total genomic DNA was isolated from fresh leaf tissue using the DNeasy Plant Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. DNA purity and concentration were assessed using a NanoDrop 1000™ and a Qubit™ 4.0 system (Thermo Fisher Scientific). A 15 kb SMRTbell library was constructed with the SMRTbell Express Template Preparation Kit 2.0 (PacBio, Menlo Park, CA, USA), following a workflow that included DNA shearing, AMPure PB bead purification, ssDNA overhang removal, damage/end repair, hairpin adapter ligation, and library bead purification. After quality control, the library was sequenced on a PacBio Revio platform. Raw data were processed via CCS (v6.0.0; --minPasses 3, --minPredictedAccuracy 0.99, --maxLength 21,000) to yield high-accuracy HiFi reads. 2.1.2 Chloroplast genome assembly and annotation First, PacBio HiFi (high-fidelity) data (> 3 Gb, average fragment length > 13 kb) were aligned to the reference chloroplast genome using minimap2 (v2.15-r905) [ 27 ] to generate a PAF file. Filtered aligned reads (those with < 50× coverage were removed) were obtained using the PAF file. The filtered reads were assembled with Flye v2.6 (Kolmogorov et al. 2020) (reference: A . titanum NC_056329.1) [ 28 ] to generate a graph file. Redundant contigs were visualized and removed, and the assembly was edited into a circular sequence using Bandage v0.8.1 [ 29 ]; this circular sequence represents the complete chloroplast genome of the respective plant. The chloroplast genome assemblies of these six species were annotated using PGA [ 30 ], with manual correction of the results. Subsequently, each chloroplast genome was visualized as a circular diagram using OGDRAW [ 31 ]. 2.2 Comparative genomic analysis 2.2.1 Basic characteristics of chloroplast sequences of 16 species 2.2.2 Repeat sequence analysis Simple sequence repeats (SSRs) and long sequence repeats (LSRs) in the chloroplast genomes of the 16 Amorphophallus species—including forward (F), reverse (R), complement (C), and palindromic (P) repeat types—as well as their genomic positions, were identified using the SSR and LSR analysis modules of CPStools software with default parameters [ 32 ]. 2.2.3 IR region expansion and contraction analysis A comprehensive analytical profile illustrating the IR boundary coordinates, adjacent gene distribution, and structural features of the chloroplast genomes of the 16 Amorphophallus species was constructed using the online tool IRscope ( https://irscope.shinyapps.io/irapp/ ) [ 33 ]. 2.2.4 Nucleotide diversity (Pi) analysis The polymorphism analysis module of CPStools was employed to automatically extract shared genes and intergenic spacers (IGS) from the GenBank format files (.gb) of the 16 Amorphophallus chloroplast genomes. Multiple sequence alignments were performed, and nucleotide diversity (Pi) values were calculated. The Pi values for shared genes and IGS regions were subsequently visualized [ 32 ]. 2.2.5 Ka/Ks (non-synonymous/synonymous substitution rate) analysis Selection pressure analysis was conducted on 75 shared genes across the chloroplast genomes of the 16 Amorphophallus species using the Ka/Ks analysis module of CPStools (with A . konjac serving as the reference sequence). CDS nucleotide sequences and protein sequences were extracted for all samples, aligned, and converted to AXT format alignment files. Ka (non-synonymous substitution rate) and Ks (synonymous substitution rate) values were calculated, yielding a total of 620 valid Ka/Ks ratios. Ka/Ks > 1, Ka/Ks = 1, and Ka/Ks < 1 indicate positive selection, neutral selection, and purifying selection, respectively [ 34 ]. 2.2.6 Sequence variation analysis To assess the degree of sequence divergence among the chloroplast genomes of the 16 Amorphophallus species, a global comparative analysis was performed using the online tool VISTA ( https://genome.lbl.gov/vista/citeus.shtml ) with the A . konjac chloroplast genome serving as the reference. Structural variation plots were generated based on this analysis [ 24 ]. 2.2.7 Conservation of chloroplast RNA editing in Amorphophallus Potential RNA editing sites in the chloroplast genomes of the 16 Amorphophallus species were predicted using the online tool PREPACT 3.0 ( http://www.prepact.de/ ), with Spirodela polyrhiza (MN419335.1) employed as the reference. All other parameters were kept at default settings [ 35 ]. 2.3 Comparative analysis of codons in the chloroplast genomes of 16 Amorphophallus species 2.3.1 Analysis of codon composition and relative synonymous codon usage To ensure accuracy and comparability of results, CDS sequences shorter than 300 bp were removed from the chloroplast genomes of the 16 Amorphophallus species. The filtered CDS sequences were analyzed using CPStools software to obtain the following parameters for each individual: GC content at codon positions 1, 2, and 3 (GC1, GC2, GC3), overall GC content (GC_all), effective number of codons (ENC), and relative synonymous codon usage (RSCU) [ 36 ]. 2.3.2 ENC-plot analysis An ENC-plot was generated with ENC values plotted on the ordinate and GC3 values on the abscissa. The standard curve was calculated according to the formula ENC = 2 + GC3 + 29 / [GC3 2 + (1 – GC3) 2 ]. The extent of deviation of individual gene points from the standard curve was used to infer the influence of various factors on codon usage bias ([ 37 ]. 2.3.3 PR2-plot analysis Parity rule 2 (PR2) plot analysis was performed to assess the relative contributions of mutation pressure and selection pressure on codon usage. The contents of A, T, C, and G at the third codon position (designated A3, T3, C3, and G3, respectively) were calculated. PR2 plots were constructed with G3/(G3 + C3) as the abscissa and A3/(A3 + T3) as the ordinate. The direction and vector distance of each gene from the center point (where A = T and C = G, indicating unbiased codon usage) reflect the nature and magnitude of codon usage bias [ 38 ]. 2.3.4 Neutrality plot analysis Neutrality plot analysis was employed to evaluate the balance between mutational pressure and natural selection affecting codon usage, thereby elucidating the relationship between GC12 and GC3. A scatter plot was generated with the average GC content at the first and second codon positions (GC12) on the ordinate and GC3 on the abscissa, followed by regression analysis. In a neutrality plot, each point represents an individual gene, and the position of points relative to the diagonal line indicates whether codon usage patterns are predominantly shaped by mutation pressure or natural selection [ 39 ]. 2.3.5 Optimal codon analysis Optimal codons are defined as those used with the highest frequency within a genome. Based on the calculated ENC values, protein-coding genes from representative Amorphophallus species were ranked from lowest to highest ENC. The top 5 genes (lowest ENC, highest expression) and the bottom 5 genes (highest ENC, lowest expression) were selected to construct high-expression and low-expression datasets, respectively. Codons that simultaneously met the criteria of high frequency (RSCU > 1) and high expression (ΔRSCU ≥ 0.08) were designated as optimal codons [ 40 ]. 2.4 DNA barcode region identification for species differentiation based on multiple sequence alignment of 16 Amorphophallus species The Barcoding program within CPStools software was employed to perform DNA barcoding analysis on the 16 Amorphophallus species, using A . sp . as the reference (automatically recognized by the program). The analysis was based on multiple sequence alignment files, with a window size of 800 bp and a sliding step of 100 bp, generating five candidate windows. Constraints were set with a maximum of 20 diagnostic loci and a maximum gap proportion of 0.6. By analyzing variation characteristics across the 225,171 bp alignment—including SNPs, insertions, and deletions—the number of variable sites among the 16 species and the results of 120 pairwise species comparisons were tallied. The optimal DNA barcoding window capable of discriminating all species was identified, and diagnostic loci and species-specific fingerprints within the window were characterized, culminating in the selection of the highest-scoring effective window [ 32 , 41 ]. 2.5 Phylogenetic analysis To determine the phylogenetic placement of Amorphophallus within Araceae, a phylogenetic tree was constructed based on chloroplast genome data using the maximum likelihood (ML) method. The analysis included 61 taxa representing 51 genera of Araceae, with Zea mays designated as the outgroup (Table S7). All chloroplast genome sequences were aligned using MAFFT v7.526 [ 42 ]. The concatenated alignment was trimmed to remove poorly aligned regions using trimAl v1.5.0 [ 43 ]. Maximum likelihood analysis was performed with IQ-TREE v1.4.2, using 1,000 bootstrap replicates and the best-fit substitution model TVM + F+R7 [ 44 ]. The resulting phylogenetic tree was edited and visualized using FigTree v1.4.4 [ 45 ]. 3. Results and Analysis 3.1 Comparative genomic analysis This study investigated the chloroplast genomes of 16 Amorphophallus species, including six newly sequenced genomes (deposited in GenBank; accession numbers are provided in Table 1 ) and ten previously published genomes. The HiFi read sequencing datasets generated from the six newly sequenced samples ranged from 6,593,888,101 to 12,285,404,571 bp (Table S1 ), with average sequencing depths of 629.50× to 2,907.32× (Figure S1 ). All 16 chloroplast genomes exhibited the typical angiosperm quadripartite structure, comprising the large single-copy (LSC), small single-copy (SSC), IRa, and IRb regions. Genomic size variation was substantial among the 16 species, with A . macrophyllus exhibiting the largest chloroplast genome (185,810 bp) and A . fornicatus the shortest (152,492 bp), yielding an average genome size of 171,861 bp. Region-specific analyses revealed that the LSC region ranged from 90,524 to 98,561 bp, the SSC region from 10,983 to 36,366 bp, and IRa/IRb regions from 9,998 to 42,066 bp. GC content across the genomes ranged from 33.57% to 36.00%. Gene content analysis indicated 118–136 genes per genome, including 81–90 protein-coding genes, 33–39 transfer RNA (tRNA) genes, and 4–8 ribosomal RNA (rRNA) genes. After removal of duplicated genes, the chloroplast genomes contained 108–113 unique genes, comprising 76–79 protein-coding genes, 28–30 tRNA genes, and 4–8 rRNA genes (Fig. 1 ; Table 1 ). Table 1 Comparison of chloroplast genome features among 16 Amorphophallus species. Name Total length (bp) Total (unique) Protein coding genes (unique) rRNA (unique) tRNA (unique) LSC length (bp) IRb length (bp) SSC length (bp) IRa length (bp) Total GC (%) Accession number A . fornicatus 152,492 118 (111) 81 (78) 4 (4) 33 (29) 96,130 9,998 36,366 9,998 33.57 PZ226531 A . glaucophyllus 184,539 135 (110) 90 (78) 8 (4) 37 (28) 90,524 41,516 10,983 41,516 34.86 PZ243066 A . hewittii 171,707 128 (110) 83 (77) 8 (4) 37 (29) 93,724 31,505 14,973 31,505 34.85 PZ235491 A . macrophyllus 185,810 136 (110) 90 (78) 8 (4) 38 (29) 90,677 42,066 11,001 42,066 34.65 PZ250735 A . pendulus 176,384 130 (111) 85 (78) 8 (4) 37 (29) 95,372 32,546 15,920 32,546 34.53 PZ266516 A . titanium 170,976 130 (111) 87 (78) 8 (4) 37 (29) 95,500 29,695 16,086 29,695 34.87 PZ266517 A . albus 175,728 130 (113) 86 (79) 8 (4) 36 (30) 93,177 26,225 20,249 26,225 35.60 OP531918 A . coaetaneus 175,465 131 (111) 84 (78) 8 (4) 39 (29) 98,561 30,200 16,504 30,200 34.90 NC_072945.1 A . kachinensis 173,330 130 (110) 85 (78) 8 (4) 37 (28) 92,030 33,091 15,118 33,091 35.00 PP072244 A . kiusianus 166,269 129 (109) 84 (77) 8 (4) 37 (28) 90,701 31,383 14,802 31,383 36.00 PP072243 A . krausei 172,418 130 (110) 85 (78) 8 (4) 37 (28) 91,983 32,422 15,591 32,422 35.23 PP936071 A. muelleri 177,076 130 (111) 85 (78) 8 (4) 37 (29) 91,947 35,204 14,721 35,204 34.50 OR995733 A . tonkinensis 169,341 129 (110) 84 (77) 8 (4) 37 (29) 90,705 31,498 15,640 31,498 36.00 PP234804 A . yunnanensis 164,417 126 (108) 81 (76) 8 (4) 37 (28) 92,149 28,543 15,182 28,543 36.00 NC_082906.1 A . konjac 161,647 130 (113) 86 (79) 8 (4) 36 (30) 93,443 26,226 21,575 26,226 35.40 MK611803 A . sp 176,221 130 (111) 85 (78) 8 (4) 37 (29) 91,718 34,891 14,172 34,891 34.70 PP936070 Subsequent analysis indicated that genes associated with photosynthesis are highly conserved across Amorphophallus species, with only a few genes exhibiting copy-number variation among taxa. Specifically, ndhA , ndhH , and ndhI were present in duplicate copies in A . glaucophyllus and A . macrophyllus , whereas ndhB was a single-copy gene in A . fornicatus . rRNA genes ( rrn4 . 5 , rrn5 , rrn16 , and rrn23 ) and several tRNA genes ( trnA - UGC and trnI - GAU ) were detected as single-copy genes exclusively in A . fornicatus . Moreover, trnM - CAU exhibited copy-number variation among taxa, occurring as either two or four copies. Among protein-coding genes, ycf1 was identified as a single copy in certain species (Table 2 ). These observed variations provide valuable molecular markers for phylogenetic studies and species identification within Amorphophallus . Table 2 Comparative chloroplast gene composition and interspecific differences in Amorphophallus species, with annotated variations. Category Gene Group Gene Photosynthesis Subunits of photosystem I psaA , psaB , psaC , psaI , psaJ Subunits of photosystem II psbA , psbB , psbC , psbD , psbE , psbF , psbH , psbI , psbJ , psbK , psbL , psbM , psbN , psbT , psbZ Subunits of NADH dehydrogenase ndhA (2) # A , ndhB (2) # B , ndhC, ndhD, ndhE, ndhF, ndhG, ndhH (2) C , ndhI (2) D , ndhJ, ndhK Subunits of cytochrome b/f complex petA , petB # , petD # , petG , petL , petN Large subunit of rubisco rbcL Subunits of ATP synthase atpA , atpB , atpE , atpF # , atpH , atpI Self-replication Proteins of large ribosomal subunit rpl2 (2) # , rpl14, rpl16 # E , rpl20, rpl22, rpl23 (2) # F , rpl32, rpl33, rpl36 Proteins of small ribosomal subunit rps2, rps3, rps4, rps7 (2) G , rps8, rps11, rps12 (2) ## H , rps14, rps15, rps16 # , rps18, rps19 Subunits of RNA polymerase rpoA , rpoB , rpoC1 # , rpoC2 Ribosomal RNAs rrn4 .5 (2) I , rrn5 (2) J , rrn16 (2) K , rrn23 (2) L Transfer RNAs trnA -UGC (2) # M , trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-GCC, trnG-UCC # , trnH, trnH-GUG N , trnI-GAU (2) # O , trnK-UUU # , trnL-CAA (2), trnL-UAA # , trnL-UAG, trnM, trnM-CAU (3) P , trnN-GUU, trnP-UGG, trnQ-UUG, trnR-ACG, trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC, trnV-UAC # , trnW-CCA, trnY-GUA Other genes Maturase matK Protease clpP## Envelope membrane protein cemA Acetyl-CoA carboxylase accD c-type cytochrome synthesis gene ccsA Conserved open reading frames ycf1 (2) Q , ycf2 (2), ycf3 ## , ycf4 , ycf68 Note: #: Intron number, (n): Gene copy number. A : In A . glaucophyllus and A . macrophyllus only, the ndhA gene is present in two copies. B : In A . fornicatus only, ndhB is a single-copy gene. C : In A . glaucophyllus and A . macrophyllus only, ndhH is present in two copies. D : In A . glaucophyllus and A . macrophyllus only, ndhI is present in two copies. E : In A . kachinensis and A . kiusianus , the rpl16 gene lacks an intron. F : In A . coaetaneus , rpl23 is a single-copy gene; it contains an intron only in A . kachinensis and A . krausei ; and it is absent in A . kiusianus , A . tonkinensis , and A . yunnanensis . G : In A . fornicatus only, rps7 is a single-copy gene. H : In A . fornicatus only, rps12 is a single-copy gene. I : In A . fornicatus only, rrn4 . 5 is a single-copy gene. J : In A . fornicatus only, rrn5 is a single-copy gene. K : In A . fornicatus only, rrn16 is a single-copy gene. L : In A . fornicatus only, rrn23 is a single-copy gene. M : In A . fornicatus only, trnA - UGC is a single-copy gene. N : In A . fornicatus , A . glaucophyllus , and A . macrophyllus , the gene is present in two copies. O : In A . fornicatus only, trnI - GAU is a single-copy gene. P : In A . albus , A . coaetaneus , and A . konjac , the copy number of trnM - CAU is two, whereas in A . kachinensis , A . krausei , and A . yunnanensis it is four. Q : In A . fornicatus , A . albus , A . yunnanensis , and A . konjac , ycf1 is a single-copy gene. 3.1.2 Repeat sequence analysis Repeat sequence analysis identified 87 (in A . konjac ) to 162 (in A . sp. ) simple sequence repeat (SSR) loci across the 16 species (Fig. 2 ). SSR distribution analysis revealed that SSRs were predominantly enriched in intergenic regions across the 16 Amorphophallus species, followed by intronic regions, whereas the fewest SSRs were observed in coding regions (Fig. 2 B). In terms of repeat motif classification, mononucleotide repeats were the most abundant (70–105 loci), followed by dinucleotide (11–55), trinucleotide (1–7), tetranucleotide (0–7), pentanucleotide (0–11), and hexanucleotide (0–2) repeats (Fig. 2 C). Across all repeat types, A (31–50)/T (33–56) motifs were the most prevalent, followed by AT (1–29)/TA (7–37) motifs, whereas other repeat patterns occurred at relatively low proportions (Fig. 2 ). Analysis of long sequence repeats (LSRs) in the chloroplast genomes of 16 Amorphophallus species revealed a generally comparable distribution pattern among species. The number of forward repeats ranged from 51 ( A . konjac ) to 1,209 ( A . muelleri ), palindromic repeats from 41 ( A . konjac ) to 345 ( A . hewittii ), complement repeats from 15 ( A . konjac ) to 313 ( A . titanum ), and reverse repeats from 44 ( A . konjac ) to 833 ( A . muelleri ) (Figs. 3 A and B). Overall, forward repeats constituted the predominant repeat type in 14 species, with the exception of A . hewittii and A . titanium , in which reverse repeats were more abundant (Fig. 3 A). Additional analysis indicated that LSRs in the six Amorphophallus species were highly concentrated within the 30–40 bp length interval, demonstrating a distribution pattern dominated by short repeat units. Moreover, positional distribution analysis revealed a strong regional bias, with the majority of LSRs located in intergenic regions, whereas substantially fewer were present in intronic and coding regions (Fig. 3 C). 3.1.3 IR region expansion and contraction analysis Analysis of IR boundary characteristics in the chloroplast genomes of 16 Amorphophallus species (Fig. 4 ) revealed pronounced variation in genome architecture. A . macrophyllus (185,810 bp) and A . glaucophyllus (184,539 bp) exhibited relatively large chloroplast genomes, both characterized by expanded IR regions (> 41,000 bp) and reduced SSC regions (< 12,000 bp), representing typical cases of IR expansion. In contrast, A . fornicatus displayed extreme IR contraction, with an IR region of only 9,998 bp and an expanded SSC region of 36,366 bp (the largest among all examined species). Based on gene position variation at LSC/IR/SSC boundaries, the 16 species were classified into eight structural types (I–VIII): Type I ( A . kiusianus , A . krausei , A . muelleri , A . tonkinensis , A . sp. , A . pendulus , A . titanum , and A . kachinensis ); Type II ( A . fornicates ); Type III ( A . yunnanensis ); Type IV ( A . konjac ); Type V ( A . glaucophyllus and A . macrophyllus ); Type VI ( A . hewittii ); Type VII ( A . albus ); Type VIII ( A . coaetaneus ). In Type I, LSC/IRb boundary was located between rps19 and rpl12 . The ndhF gene spanned IRb/SSC boundary in six species ( A . fornicatus , A . kiusianus , A . muelleri , A . yunnanensis , A . sp , and A . pendulus ). The rps15 gene spanned SSC/IRa boundary in A . muelleri , A . sp. , A . titanum , and A . kachinensis . In A . titanum , a 127 bp overlap was observed between rps15 and ycf1 . In Type II, LSC/IRb boundary was located between rps19 and trnH , IRb/SSC boundary was between trnL and ndhF , and SSC/IRa boundary was between ndhB and trnL . In Type III, a 136 bp overlap occurred between ycf1 and ndhF in A . yunnanensis , and SSC/IRa boundary was situated between rps15 and trnN . In Type IV, IRb/SSC boundary was located between trnN and ndhF , and SSC/IRa boundary was between ycf1 and trnN . In Type V, LSC/IRb boundary was between rps19 and psbA , IRb/SSC boundary was between ndhI and ndhF , and SSC/IRa boundary was between ndhG and ndhI . In Type VI, LSC/IRb boundary was between rps19 and rpl23 , and IRa/LSC boundary was between rpl23 and trnH . Type VII was generally similar to Type IV, except that IRb/SSC boundary was positioned between ycf1 and ndhF . In Type VIII, LSC/IRb boundary was located between rps12 and trnM . These structural variations provide valuable references for developing species-specific DNA barcodes for identification purposes. 3.1.4 Nucleotide diversity (Pi) analysis Pi analysis (Fig. 5 ) revealed substantial variation in polymorphism among shared genes and intergenic spacers across LSC, SSC, and IRa/b regions of the chloroplast genomes in 16 Amorphophallus species. In addition, the distribution of Pi values differed markedly among genes and spacers within the same genomic region. The LSC region exhibited the highest sequence variation, containing genes and intergenic regions with the greatest genome-wide Pi values. The gene accD (Pi = 0.1217) demonstrated the highest level of polymorphism across the entire genome, followed by clpP (Pi = 0.0951). Conversely, the SSC region displayed lower overall polymorphism than the LSC region, with gene Pi values ranging from 0.0037 to 0.0132. Within the SSC region, ndhF , ndhG , and ndhD exhibited relatively higher variability, whereas psaC exhibited the lowest. Intergenic spacer polymorphism was polarized, with petD _2– rpoA (Pi = 0.0743) exhibiting the highest Pi value among spacers, followed by ndhD – psaC (Pi = 0.0588). IRa/IRb regions exhibited the lowest overall polymorphism, with Pi values for most genes below 0.015. The gene ndhB (Pi = 0.0019) was identified as the most conserved across the genome. Only ycf1 (Pi = 0.0570) and rps15 (Pi = 0.0352) represented core variable loci within this region. In summary, hypervariable sites in the Amorphophallus chloroplast genome are predominantly concentrated in single-copy genes and intergenic spacers within the LSC region. In contrast, the SSC region is comparatively conserved with moderate localized variation. IRa/IRb regions remain highly conserved, with limited variability confined to ycf1 and rps15 . These findings provide a robust foundation for identifying hypervariable molecular markers and for phylogenetic analyses within the genus. 3.1.5 Ka/Ks analysis Using A . konjac as the reference species, Ka/Ks ratios were calculated for chloroplast genes across the remaining 15 Amorphophallus species (Fig. 6 ; Table S2 ). Ka/Ks ratios for the vast majority of chloroplast genes in all examined species ranged from 0 to 1, indicating that the Amorphophallus chloroplast genome has been predominantly subjected to purifying selection throughout its evolutionary history. This pattern is consistent with the generally highly conserved nature of plant chloroplast genomes. The highest Ka/Ks value, 12.274, was observed for the rps3 gene in the comparison between A . konjac and A . sp. , suggesting that this gene may have undergone rapid evolution or functional modification in A . sp. , potentially associated with species-specific environmental adaptation or physiological traits. Ka/Ks values of 1 were detected for the rpl16 gene in the A . konjac versus A . pendulus , the rps16 and rpoA genes in the A . konjac versus A . coaetaneus , and the rps16 and rpl16 genes in the A . konjac versus A . kiusianus . Subsequent analysis revealed that A . sp. exhibited the stronger signals of positive selection, with the highest number of genes under positive selection, including rps16 (1.007), rps2 (1.308), rpoC2 (0.725), rpl20 (1.011), clpP (2.072), rpoA (1.006), rps11 (1.165), rpl14 (3.818), rps3 (2.938), rps19 (1.698), ycf2 (1.554), ndhD (0.227), and ycf1 (2.332). These findings indicate that multiple functional genes in the chloroplast genome of A . sp. may have undergone adaptive evolution. 3.1.6 Sequence variation analysis Using the chloroplast genome of A . konjac as the reference, a comprehensive comparative analysis was conducted across the remaining 15 Amorphophallus species. This analysis revealed differential levels of sequence variation among exon regions, tRNAs, rRNAs, and conserved non-coding sequences (Fig. 7 ), indicating heterogeneous evolutionary dynamics across genomic regions. Notably, pronounced variation was observed in intergenic regions, such as tRNA - Ser (GCU)/ tRNA - Gly (UCC), tRNA - Glu (UUC)/ tRNA - Thr (GGU), tRNA - Thr (UGU)/ tRNA - Leu (UAA), and ndhC / tRNA - Val (UAC). Furthermore, notable sequence divergence was detected in genes, including ycf1 , ycf2 , rps16 , tRNA - Leu (CAA), and rps12 . 3.1.7 Conservation of chloroplast RNA editing in Amorphophallus To characterize chloroplast RNA editing in Amorphophallus , a systematic analysis of RNA editing patterns across the chloroplast genomes of 16 Amorphophallus species was conducted (Table S3 ). The results demonstrated that C→U base editing was the sole editing type detected in the chloroplasts of all species examined, whereas other base editing types, including U→C, G→A, and A→I, were absent. The number of C→U editing sites exhibited slight interspecific variation, ranging from 1,331 in A . fornicatus to 1,605 in A . kiusianus . Among the identified editing events, P→L (proline (Pro) → leucine (Leu)) and P→S (Pro → serine (Ser)) represented the two predominant editing types, constituting the core spectrum of amino acid substitutions mediated by C→U editing in Amorphophallus chloroplasts. In contrast, substitution types, such as A→V and H→Y, occurred at relatively rare frequencies. Furthermore, comparative analysis revealed that A . krausei possessed the highest number of nonsense mutations (Q→ 43 events, R→ 43 events; total 86 events), followed by A . albus (Q→ 39 events, R→ 39 events; total 78 events). Conversely, A . yunnanensis exhibited the lowest number of nonsense mutation events (Q→ 22 events, R→ 22 events; total 44 events). These species-specific RNA editing characteristics may serve as valuable molecular markers for species identification and provide insights into evolutionary differentiation within the genus. 3.2 Comparative analysis of codons in the chloroplast genomes of 16 Amorphophallus species 3.2.1 Basic characteristics of codon composition analysis Following quality screening, 50–52 coding DNA sequences (CDS) suitable for codon usage bias analysis were identified from the chloroplast genomes of 16 representative Amorphophallus species (Table S4 ). Across these species, the overall GC content of coding regions (GC_all) ranged from 38.00% to 38.57%. The base compositions at the three codon positions (GC1, GC2, and GC3) ranged from 46.21% to 47.25%, 39.37% to 40.12%, and 28.21% to 28.70%, respectively (Table S4 ), indicating a positional variation in nucleotide distribution. A consistent pattern of GC1 > GC2 > GC3 was observed across all examined species. Although GC content at different codon positions was relatively similar across Amorphophallus species, its distribution was uneven, with a bias toward the first two positions; notably, GC1, GC2, and GC_all were all below 50%. The mean effective number of codons (ENC) across the 16 chloroplast genomes was 46.91, and only the rps18 gene consistently exhibited ENC values below 40 in all chloroplast genomes, suggesting a generally weak codon usage bias in these species. Correlation analysis among parameters—including GC1, GC2, GC3, GC_all, ENC, and codon number—was performed using CDS from the chloroplast genomes of 16 representative Amorphophallus species. The results revealed that correlations among GC1, GC2, and GC3 were non-significant across all examined species (Fig. 8 ). In contrast, significant or highly significant positive correlations between GC1 and GC2 were detected in 13 species, with the exception of A . glaucophyllus (Fig. 8 B), A . macrophyllus (Fig. 8 D), and A . krausei (Fig. 8 K). This pattern indicates a comparable base composition at the first two codon positions across these 13 species and suggests that variation in chloroplast gene expression is primarily associated with GC content at the first codon position, with relatively minor associations at the second and third positions. Regarding codon usage bias, no significant correlation between ENC values and GC1 or GC2 was observed in 14 of the 16 representative Amorphophallus species, except for A . glaucophyllus (Fig. 8 B) and A . macrophyllus (Fig. 8 D). Conversely, significant or highly significant correlations between ENC values and GC3 were detected in all 16 species, suggesting that alterations in base composition at the third codon position are the primary determinant of codon usage bias in Amorphophallus . 3.2.2 Relative synonymous codon usage analysis After excluding stop codons and non-expressed codons, RSCU values across 16 representative Amorphophallus species were relatively comparable. Among all codons, UUA encoding Leu exhibited the highest RSCU value across all species (Figs. 9 and S2 ). In terms of high-frequency codon distribution, A . titanum , A . albus , A . kiusianus , and A . konjac each contained 31 high-frequency codons, whereas the remaining 12 species each harbored 30 high-frequency codons, accounting for 48.44% and 46.88% of the total codon sets, respectively. Furthermore, high-frequency codons in Amorphophallus species exhibited a bias toward A/T-ending codons, whereas low-frequency codons tended to terminate in G/C. 3.2.3 ENC-plot analysis ENC-plot analysis of chloroplast genomes from 16 Amorphophallus species revealed a strong association between codon usage patterns and gene function, with core functional genes exhibiting conserved codon usage across all examined species. Most genes were distributed below the standard curve, whereas only a few genes were located on or near the curve. Across most species, ycf3 represented the gene positioned farthest above the expected curve, with exceptions observed for clpP in A . albus , A . kachinensis , A . tonkinensis , and A . yunnanensis , and ycf68 in A . konjac . Additionally, most genes exhibited ENC values greater than 45. The frequency distribution of ENC ratios (Figs. 10 and S3 ) demonstrated that ratios ranged from − 0.05 to 0.35. Between 13 and 18 genes were concentrated within the interval near the standard curve (–0.05 to 0.05), indicating relatively small differences between observed and expected ENC values. Furthermore, 9 to 12 genes exhibited ENC ratios greater than 0.15 and were positioned below the standard curve, at a considerable distance from it. 3.2.4 Parity rule 2 (PR2)-plot analysis PR2 plot analysis was employed to assess the degree of nucleotide usage bias at the third codon position. In this analysis, the PR2 plot was centered at the coordinates (0.5, 0.5) based on A3/(A3 + T3) and G3/(G3 + C3), thereby dividing the plot into four quadrants. Protein-coding genes from the chloroplast genomes of 16 representative Amorphophallus species exhibited an uneven distribution across the four quadrants. Notably, the gene scatter points were highly concentrated in the fourth quadrant and did not cluster around the center point (Fig. 11 ). This distribution pattern indicates a pronounced bias in nucleotide usage at the third codon position in Amorphophallus species, characterized by a preference order of T > A and G > C. Collectively, these findings further support that natural selection is the primary factor influencing codon usage bias in this genus. From a functional gene perspective, scatter points corresponding to core chloroplast housekeeping genes—including those involved in photosystems, adenosine triphosphate synthase, nicotinamide adenine dinucleotide hydride (NADH) dehydrogenase, ribosomal proteins, and RNA polymerase—exhibited pronounced deviation from the central point. Notably, the deviation patterns for these gene categories were highly consistent across all 16 Amorphophallus species. This pattern indicates that nucleotide usage at the third codon position of core housekeeping genes is under strong directional natural selection, thereby promoting translational efficiency and accuracy of essential proteins and ensuring compatibility with the chloroplast translational machinery. 3.2.5 Neutrality plot analysis To assess the relationship between GC3 and GC12, neutrality plot analysis was conducted using the protein-coding genes from 16 representative Amorphophallus species. The results indicated that the majority of protein-coding genes were distributed above the diagonal line in the neutrality plot, whereas only a small proportion clustered along the diagonal. GC12 content ranged from 32.34% to 56.47%, while GC3 content ranged from 18.37% to 46.36%. The correlation coefficient (R²) between GC12 and GC3 ranged from 0.008 to 0.070 (Fig. 12 ), indicating that GC3 and GC12 exhibit distinct evolutionary trajectories among Amorphophallus genes. Both mutation pressure and natural selection contribute to codon usage bias in this genus, with natural selection representing the primary driving force. Subsequent analysis revealed that genes belonging to different functional categories occupy distinct positions in the neutrality plot. Core genes involved in transcription and translation, including those encoding ribosomal proteins and RNA polymerase, were substantially deviated from the diagonal, indicating that their evolutionary patterns are predominantly driven by natural selection pressure. In contrast, loci corresponding to NADH dehydrogenase-related genes were positioned between regions of high and moderate conservation, suggesting that their evolution is jointly influenced by natural selection and neutral mutation, with natural selection exerting a more pronounced effect. 3.2.6 Optimal codon analysis A total of 15 to 21 optimal codons were identified among the 16 representative Amorphophallus species (Table S4 ). Among them, A . coaetaneus , A . macrophyllus , and A . glaucophyllus possessed the highest number of optimal codons (21 each), whereas A . kiusianus exhibited the lowest number (15). Regarding base composition at the third codon position, these optimal codons displayed a pronounced bias toward A- or U-ending codons. Furthermore, comparative analysis identified six optimal codons shared by all 16 species: UUA, CGU, AAA, GUA, ACU, and AGU, which encode Leu, arginine (Arg), lysine (Lys), valine (Val), threonine (Thr), and Ser, respectively. Among these shared codons, three terminated in A and three in U. Furthermore, 15 differential optimal codons were identified, including ACA, ACC, AGA, AUU, CAU, CCC, GAA, GCU, GGA, GUU, UAG, UCC, UGU, UUG, and UUU. Within this set, four codons ended in A, six in U, three in C, and two in G. Notably, CCA (Pro) was unique to A . pendulus , whereas CUU (Leu), UCU (Ser), and CUA (Leu) were specific to A . konjac . 3.3 DNA barcode region identification Based on an 800 bp sliding window analysis, the optimal DNA barcoding region for the 16 Amorphophallus species was identified. This analysis revealed substantial genetic variation relative to the reference sequence ( A . sp. ), including 79,824 single-nucleotide polymorphisms (SNPs), 171,710 insertions, and 253,417 deletions, with deletions representing the predominant variant type (Table S5). Among the examined species, A . fornicatus exhibited the highest total number of variants (55,410), of which deletions constituted the majority (36,638), whereas A . muelleri displayed the lowest number of variants (1,125), comprising no SNPs, 990 insertions, and 135 deletions. In the remaining species, including A . glaucophyllus and A . macrophyllus , total variant numbers were distributed between 27,000 and 47,000, while SNP counts consistently ranged from 5,000 to 6,300. Furthermore, an 800 bp region spanning positions 80,401 to 81,200 bp was identified as the optimal DNA barcoding region, enabling discrimination among all 16 Amorphophallus species using five diagnostic loci (T|–|–|C|T; Table S6). 3.4 Phylogenetic analysis To determine the phylogenetic placement of A . fornicatus , A . glaucophyllus , A . hewittii , A . macrophyllus , A . pendulus , and A . titanum within Araceae, a phylogenetic tree was constructed based on chloroplast genome data using the maximum likelihood (ML) method. The dataset comprised 61 taxa representing 51 genera of Araceae, with Z ea mays designated as the outgroup (Table S8). The resulting phylogenetic topology (Fig. 14) indicated that Amorphophallus species form a distinct clade with 100% bootstrap support, constituting a strongly supported monophyletic lineage. Subsequent analysis resolved the Amorphophallus species into three distinct clades: Continental Asia II (CA-II), Continental Asia I (CA-I), and Southeast Asia (SEA). Within CA-II, A . glaucophyllus and A . macrophyllus clustered together with A . konjac , A . albus , A . krausei , and A . kachinensis (bootstrap support = 100%), indicating close phylogenetic relationships among these taxa. In the SEA clade, A . hewittii , A . titanum , A . fornicatus , and A . pendulus formed a closely related group (bootstrap support = 100%). Within the CA-I clade, A . coaetaneus , A . tonkinensis , A . yunnanensis , and A . muelleri were identified as the most closely related taxa (bootstrap support = 100%). Furthermore, in the broader phylogenetic tree of Araceae, Amorphophallus occupied a crown group position and formed a highly supported sister clade (bootstrap = 100%) with Syngonium , Xanthosoma , Zomicarpella , and Caladium . The base of the phylogenetic tree was represented by Symplocarpus , followed by the divergence of Wolffia . 4. Discussion The chloroplast genome is the smallest of the three primary genomes in plant cells, alongside the nuclear and mitochondrial genomes, and constitutes a relatively autonomous genetic system with limited dependence on nuclear genomic regulation [ 46 ]. Although generally conserved, chloroplast genome structure, size, and gene content exhibit variation across genera and even among species, thereby providing important reference data for studies in plant taxonomy, genetics, and ecological adaptation. In this study, complete chloroplast genomes from six Amorphophallus species were characterized and compared with the published chloroplast genomes from ten additional species within this genus, enabling a comprehensive assessment. The chloroplast genome size among the 16 Amorphophallus species varied substantially, ranging from 152,492 to 185,810 bp, while gene content ranged from 118 to 136 genes, suggesting species-specific differentiation potentially associated with metabolic pathways and ecological adaptation [ 46 , 47 ]. In contrast, the GC content of these genomes ranged from 33.57% to 36.00%, with a coefficient of variation of only 1.86%, indicating a strong evolutionary conservation of chloroplast genomes in Amorphophallus and the absence of substantial shifts in base composition. SSRs are widely used as molecular markers for assessing genetic polymorphism in plants [ 48 ]. Across the 16 species, the number of SSRs ranged from 74 to 113. Consistent with observations in most species, mononucleotide repeats constituted the predominant class, followed by dinucleotide, tetranucleotide, trinucleotide, pentanucleotide, and hexanucleotide repeats, the latter being the least frequent. Furthermore, A/T motifs were substantially more abundant than G/C motifs, consistent with previously reported patterns in Amorphophallus [ 17 , 25 ]. These results provide a valuable reference for future studies on population genetics and phylogeography within the genus. Analysis of characteristic genes at chloroplast genome boundary regions facilitates species identification and phylogenetic inference [ 49 ]. In this study, IR regions of chloroplast genomes from 16 Amorphophallus species were systematically examined. Although the overall genome sequences exhibited a high degree of similarity, minor differences were identified. Notably, A . macrophyllus (185,810 bp) and A . glaucophyllus (184,539 bp) represent typical IR expansion types, characterized by IR regions exceeding 41,000 bp in length and SSC regions shorter than 12,000 bp. In contrast, A . fornicatus represents an extreme case of IR contraction, with an IR region of only 9,998 bp and an SSC region extending to 36,366 bp. An inverse relationship between IR and SSC region lengths is evident, further supporting the role of IR dynamics as a central mechanism driving size variation and structural differentiation in angiosperm chloroplast genomes [ 50 ]. IR boundary shifts also resulted in species-specific patterns of gene distribution. The ndhF gene spanned the IRb/SSC boundary in six species, whereas rps19 was located in the LSC region in all species except A . coaetaneus , in which rpl2 was uniquely positioned in the LSC region. The ycf1 spanned the IRb/SSC boundary exclusively in A . yunnanensis and A . titanum . Additionally, gene overlaps of 136 bp between ycf1 and ndhF in A . yunnanensis , and 127 bp between rps15 and ycf1 in A . titanum , were identified; such structural configurations are extremely rare in chloroplast genomes [ 51 ]. The pronounced interspecific variation in IR boundary characteristics provides novel molecular targets for Amorphophallus species identification and may enhance resolution where traditional DNA barcoding approaches remain insufficient among closely related species. Integrating Pi and selection pressure (Ka/Ks) analyses revealed a highly consistent evolutionary pattern in the chloroplast genome of Amorphophallus , characterized by a coordinated distribution of hypervariable regions and signatures of positive selection. Pi analysis identified the LSC region as the primary reservoir of variation, with the accD gene and the petD _2– rpoA intergenic spacer exhibiting exceptionally high polymorphism, thereby representing promising targets for molecular marker development. In contrast, the SSC region remains generally conserved, as only ndhF and ndhD genes and the ndhD – psaC intergenic spacer demonstrate moderate levels of variation. Ka/Ks analysis further elucidated variation in selection pressure among genes, indicating that the vast majority of genes were under purifying selection, consistent with the highly conserved nature of chloroplast genomes [ 52 ]. Nevertheless, in comparisons involving A . sp. , several genes—including ycf1 , rps3 , clpP , and rpl14 —exhibited strong signals of positive selection (Ka/Ks > 1). Notably, ycf1 , which also constitutes a major variable locus within the IR region based on Pi analysis, may play a crucial role in the adaptive evolution of Amorphophallus across diverse environmental conditions [ 53 ]. The evolutionary pattern of the Amorphophallus chloroplast genome reflects a strong integration of structural conservation and localized variability. Similarity analysis demonstrates that overall gene order remains highly conserved, whereas ycf1 , ycf2 , and members of the ndh gene family constitute the principal variable loci. Despite their high variability, these genes are subject to purifying selection, with ycf1 and ycf2 exhibiting Ka/Ks ratios of 0.5–0.8, indicative of weak purifying selection, and ndh family genes displaying Ka/Ks ratios of 0.3–0.6, consistent with moderate purifying selection. Conversely, variable genes located in the LSC region are under stronger purifying selection (Ka/Ks = 0.2–0.4). Collectively, these findings suggest that, within an overall conserved genomic framework, the Amorphophallus chloroplast genome responds to environmental adaptation through weak purifying selection on specific genes, particularly ycf1 . This pattern provides precise targets for molecular marker development and phylogenetic studies within the genus [ 17 , 54 ]. During long-term evolution, plants have developed diverse codon usage patterns and base composition biases in response to different growth environments. In this study, chloroplast genome data from 16 Amorphophallus species were screened and characterized to determine base composition and codon usage patterns across 50–52 protein-coding genes. The results indicate that base composition is similar across Amorphophallus species, with a GC1 > GC2 > GC3 distribution pattern. In addition, the overall GC content and the GC content at all three codon positions remain below 50%, reflecting a bias toward A/U bases and a preference for A/U-ending codons within this genus. RSCU, defined as the ratio of observed to expected codon frequency, serves as a standard metric for evaluating codon usage bias across different organisms. Analysis of RSCU values demonstrates that high-frequency codons in all examined Amorphophallus species predominantly end in A/T, whereas low-frequency codons tend to terminate in G/C. This finding is consistent with previous reports on species within Monsteroideae and further supports the relatively conserved nature of codon usage patterns in the chloroplast genomes of Amorphophallus [ 55 ]. ENC reflects the degree of deviation from random codon usage and typically ranges from 20 to 61. An ENC value of 20 indicates exclusive usage of a single codon per amino acid, representing maximal codon bias. In contrast, an ENC value of 61 corresponds to random codon usage with no detectable bias [ 37 ]. Genes with ENC values ≤ 35 demonstrate strong codon usage bias, while those with higher values exhibit weak bias. Across the 16 Amorphophallus species, the average ENC values for CDS ranged from 46.3 to 47.4, all exceeding 45, indicating a generally weak codon usage bias. This pattern is consistent with previous reports on chloroplast genomes of numerous Araceae species [ 56 ]. Moreover, correlation analysis across the 16 Amorphophallus species revealed no significant correlation between GC3 and either GC1 or GC2, whereas GC3 exhibited a significant or highly significant positive correlation with ENC. These results indicate that base composition at the third codon position differs substantially from that at the first and second positions, and that codon usage bias is closely associated with base composition at the third codon position, which may influence functional gene expression. Previous studies have demonstrated that mutation pressure and natural selection are primary factors shaping species-specific codon usage bias, and ENC serves as an important metric for assessing the relative contributions of these two factors to the non-uniform usage of synonymous codons [ 37 ]. In this study, ENC-plot analyses of CDS across the 16 Amorphophallus species revealed similar patterns, with the vast majority of genes positioned below the standard curve. This pattern reflects a substantial deviation between observed and expected ENC values, indicating that codon usage bias in these species is predominantly driven by natural selection. Conversely, only a subset of genes is influenced by mutation pressure. PR2-plot analysis further revealed bias in base usage at the third codon position, characterized by an overall frequency pattern of T > A and G > C. These results indicate that codon usage bias in Amorphophallus is jointly shaped by natural selection and mutation pressure, with natural selection exerting a more pronounced influence [ 57 ]. Neutrality plot analysis revealed marked variation in the distribution of GC12 versus GC3 among genes with different functional categories across 16 Amorphophallus species. Core genes involved in transcription and translation, including those encoding ribosomal proteins and RNA polymerase, exhibited substantial deviation from the diagonal line, indicating that their codon usage bias is primarily driven by translational selection. This observation is consistent with established theoretical frameworks, which propose that highly expressed genes preferentially utilize optimized codons corresponding to high-abundance tRNAs to enhance translational efficiency and accuracy, and that ribosomal protein genes, as typical housekeeping genes, have undergone strong translational optimization during evolution [ 58 ]. In contrast, loci corresponding to NADH dehydrogenase-related genes were distributed near the diagonal, indicating an intermediate position between strict deviation and complete neutrality. This pattern suggests that their codon usage bias is more strongly influenced by mutation pressure, with a comparatively weaker contribution from translational selection. Such disparity may be associated with variation in gene expression levels and genomic localization [ 59 ]. Identifying optimal codons is essential for designing gene expression vectors and enhancing target gene expression. In this study, 15–21 optimal codons were identified across 16 Amorphophallus species. Among these, six optimal codons were shared by all representative species, namely UUA (Leu), CGU (Arg), AAA (Lys), GUA (Val), ACU (Thr), and AGU (Ser), and are highly conserved, underscoring their universal and functionally significant role in translational optimization. The evolution of species-specific optimal codons in certain taxa further highlights the combined influence of natural selection and habitat adaptation. For instance, in the widely cultivated A . konjac , unique optimal codons—including CUU and CUA (encoding Leu, which is abundant in transmembrane and receptor proteins) and UCU (encoding Ser, involved in photorespiration and cold-responsive signaling)—may reflect fine-tuned optimization of translational efficiency for relevant functional proteins during adaptation to complex habitats characterized by broad temperature fluctuations, as well as during specialized secondary metabolic processes such as substantial glucomannan synthesis [ 10 ]. In summary, the conservation and divergence of specific optimal codons result from the long-term coevolution between AT-biased mutational pressure and translational selection within distinct ecological niches, providing important molecular evidence for elucidating habitat-adaptation mechanisms in Amorphophallus . Based on an 800 bp sliding window analysis, the 80,401–81,200 bp interval of the Amorphophallus chloroplast genome was identified as the optimal DNA barcoding region. This region exhibits structural polymorphism dominated by deletion variants, combined with SNP patterns, enabling precise discrimination of all 16 examined species, including lineages characterized by extremely low and high genetic variation, using only five diagnostic loci. Consequently, it represents the highest-resolution molecular identification hotspot currently recognized within the genus [ 60 ]. Furthermore, the DNA barcoding region provides an effective target for developing low-cost, high-throughput species-specific markers, such as amplicon sequencing and quantitative polymerase chain reaction-based genotyping. When combined with the highly conserved nature of chloroplast RNA editing in Amorphophallus (limited exclusively to the C→U type), these findings further underscore a distinctive genomic architecture within the genus, wherein a conserved RNA editing mechanism coexists with a hypervariable DNA barcode region [ 61 ]. Phylogenetic analysis is widely used to elucidate evolutionary relationships among different species. To further resolve the evolutionary relationships among 16 Amorphophallus species and determine their phylogenetic placement within Araceae, a phylogenetic tree was constructed using 61 taxa based on the ML method, with Z . mays designated as the outgroup. The results indicated that the 16 Amorphophallus species form a well-supported monophyletic group (bootstrap = 100%). Subsequent analysis resolved the 16 species into 3 distinct clades: CA-II ( A . glaucophyllus , A . macrophyllus , A . konjac , A . albus , A . krausei , and A . kachinensis ); SEA ( A . hewittii , A . titanum , A . fornicatus , and A . pendulus ); CA-I ( A . coaetaneus , A . tonkinensis , A . yunnanensis , and A . muelleri ). These findings are consistent with previous studies. Within Araceae, Amorphophallus exhibits closer phylogenetic affinity with Caladium , Zomicarpella , Xanthosoma , and Syngonium than with other genera. 5. Conclusion In this study, chloroplast genomes from six rare Amorphophallu s species were sequenced. These data were integrated with previously reported genomes of ten Amorphophallus species to characterize genomic structural features, codon usage bias, and phylogenetic relationships. The results revealed that chloroplast genome sizes among the 16 species ranged from 164,417 to 177,076 bp, with gene numbers varying from 126 to 131. All genomes exhibit a conserved quadripartite structure comprising LSC, SSC, IRa, and IRb regions, although notable variation exists in the size of each region across species. The number of SSRs ranged from 87 to 162, with mononucleotide repeats representing the most abundant type and exhibiting a base composition biased toward A/T usage. Among LSR, reverse and forward repeats were the most prevalent types. IR boundary regions of the chloroplast genomes displayed evident contraction and expansion across the 16 species. Overall, chloroplast genome characteristics and codon usage patterns were similar among Amorphophallus species, although not identical. The GC content of all 16 species was below 50%. Six shared optimal codons, namely UUA, CGU, AAA, GUA, ACU, and AGU, were identified across the genus. Analyses including ENC-plot, neutrality plot, and PR2-plot indicated that codon usage bias across all species was more strongly influenced by natural selection than by mutation pressure. An 800 bp sliding window analysis identified the 80,401–81,200 bp interval as the optimal DNA barcoding region for the 16 species. Phylogenetic analysis further demonstrated that among the newly reported species, A . glaucophyllus and A . macrophyllus belong to the CA-II clade, whereas A . hewittii , A . titanum , A . fornicatus , and A . pendulus cluster within the SEA clade. These findings enrich the genetic resources available for Amorphophallus species and provide a valuable reference for species authentication and molecular-level determination of species origin. Declarations Acknowledgements Not applicable. Funding This research was supported by the Yunnan Provincial Science and Technology Department (grant no. 202449CE340009, 202503AP140005, 202401BA070001-001), Science and Technology Projects of Yunnan Universities Serving Key Industries (grant No. FWCY-QYCT2025017), NSFC (grant no. 32560733), Yunnan Province Yu Lei Expert Grassroots Research Workstation. Yunnan Province University Student Innovation and Entrepreneurship Training Program Project (S202511393064). Ethics declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Availability of data and materials The chloroplast genome sequences of A . fornicatus , A . glaucophyllus , A . hewittii , A . macrophyllus , A . pendulus , and A . titaniumi have been deposited in the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/nuccore/?term=) under the accession numbers PZ226531, PZ243066, PZ235491, PZ250735, PZ266516, and PZ266517, respectively. The raw sequence data reported in this paper have been deposited in the NCBI SRA (https://www.ncbi.nlm.nih.gov/sra/?term=) under BioSample accession numbers SAMN55469039, SAMN55971199, SAMN55975456, SAMN55872069, SAMN55958012, and SAMN55933366; BioProject accession numbers PRJNA1425632, PRJNA1427793, PRJNA1428900, PRJNA1426491, PRJNA1428559, and PRJNA1427781; and SRA accession numbers SRR37297627, SRR37374433, SRR37379527, SRR37351907, SRR37369025, and SRR37355529, respectively. Competing interests The authors declare no competing interests. Authors' contributions Haoliang Shi, Lifang Li, Lei Yu and Qinghu Lu conceived and designed the project. Ying Qi, and Min Yang performed the bioinformatics analysis. Meiwei Zhao, Penghua Gao, Feiyan Huang and Jianrong Zhao performed the experiments. Haoliang Shi wrote the manuscript. Lifang Li revised the manuscript. Lei Yu supervised the manuscript. All authors read and approved the manuscript. 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Genetic Diversity of Porang Populations ( Amorphophallus Muelleri Blume) In Central Java and West Java Based on LEAFY Second Intron Marker. J Trop Life Sci. 2016;6:23–7. Hao W, Liu G, Wang W, Shen W, Zhao Y, Sun J, Yang Q, Zhang Y, Fan W, Pei S, et al. RNA Editing and Its Roles in Plant Organelles. FRONT GENET. 2021;12:757109. Additional Declarations No competing interests reported. Supplementary Files TableS1S7.xlsx Table S1The basic information of 6 newly sequenced chloroplast genomes in Amorphophallus . Table S2Calculation of Ka/Ks ratios for chloroplast genes of 15 congeneric species using A . konjac as the reference species. Table S3Statistics of RNA base editing and amino acid changes in 16 Amorphophallus species. Table S4Summary of CDS GC content, effective number of codons and optimal codons. Table S5Genetic variation profiling of Amorphophallus taxa relative to A . sp reference sequence. Table S6Unique fingerprint sequences of Amorphophallus species. Table S7Chloroplast genome information for phylogenetic analysis. FigureS2.pdf Figure S2 Heatmap of relative synonymous codon usage (RSCU) in the chloroplast genomes of 16 Amorphophallus species. FigureS3.pdf Figure S3 Frequency distribution of ENC ratios for protein-coding genes across 16 Amorphophallus chloroplast genomes. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 15 May, 2026 Reviewers agreed at journal 10 May, 2026 Reviewers invited by journal 08 May, 2026 Editor assigned by journal 08 May, 2026 Editor invited by journal 08 May, 2026 Submission checks completed at journal 07 May, 2026 First submitted to journal 07 May, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9564592","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":640876228,"identity":"afd21b26-dbb9-4787-8429-26d42768367e","order_by":0,"name":"Haoliang 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10:41:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9564592/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9564592/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109799491,"identity":"ae95f5f5-7924-474b-8f13-a122e8468129","added_by":"auto","created_at":"2026-05-22 15:29:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":288554,"visible":true,"origin":"","legend":"\u003cp\u003eChloroplast genome maps of \u003cem\u003eAmorphophallus\u003c/em\u003e with annotated genes. 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Panels a, b, and c correspond to the annotations for A, B, and C, respectively.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9564592/v1/1c21a52c790404241edca1d6.png"},{"id":109906594,"identity":"97ac5b7d-ebe5-43ae-a22d-907615631439","added_by":"auto","created_at":"2026-05-25 06:40:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":93126,"visible":true,"origin":"","legend":"\u003cp\u003eLong sequence repea (LSRs) analysis. (A) Distribution of LSR types; (B) Length distribution of LSR; (C) Location type distribution.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9564592/v1/4be7ea90eaf779e37b850bd1.png"},{"id":109906410,"identity":"5c52b1f7-453f-4209-acce-b7de3ce9cdaf","added_by":"auto","created_at":"2026-05-25 06:40:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":581651,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the LSC, SSC, and IR boundaries among 16 chloroplast genomes.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9564592/v1/b90428fa01eecbb912d803cc.png"},{"id":109799482,"identity":"23df9f04-36f7-46ac-8775-5922a2480a2f","added_by":"auto","created_at":"2026-05-22 15:29:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":97480,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of nucleotide diversity (Pi) across the whole chloroplast genomes of \u003cem\u003eAmorphophallus\u003c/em\u003especies. (A) Nucleotide diversity of genes; (B) Nucleotide diversity of intergenic spacers (IGS).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9564592/v1/8ac8f5af5a6bff4bef3e630f.png"},{"id":109799483,"identity":"8a3583fd-0c2f-4fc9-aedb-7a19b9a5eed3","added_by":"auto","created_at":"2026-05-22 15:29:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":148458,"visible":true,"origin":"","legend":"\u003cp\u003eSequence alignment analysis of the chloroplast genomes of 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species generated by mVISTA.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-9564592/v1/6aa619927690a77b36b58364.png"},{"id":109906196,"identity":"c5e58bc9-d572-4e31-ad1a-6d4df9c0c600","added_by":"auto","created_at":"2026-05-25 06:39:30","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":299516,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis of basic codon parameters in the chloroplast genomes of 16 \u003cem\u003eAmorphophallus\u003c/em\u003especies. A-P correspond respectively to \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehewittii\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ependulus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ealbus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecoaetaneus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekachinensis\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekiusianus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekrausei\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emuelleri\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etonkinensis\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eyunnanensis\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esp\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-9564592/v1/d3003525fdf9de7ed8016f3b.png"},{"id":109799489,"identity":"40c64c43-fd37-4358-8acf-8ec1498fae34","added_by":"auto","created_at":"2026-05-22 15:29:45","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":283742,"visible":true,"origin":"","legend":"\u003cp\u003ePR2-plot of the chloroplast genomes of 16 species. The species corresponding to letters A-P are the same as in Figure 8.\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-9564592/v1/ebeafcdec4ee432b0c4e32eb.png"},{"id":109799490,"identity":"9c3a988e-3b5e-4e1b-8b12-2e3d1ab71739","added_by":"auto","created_at":"2026-05-22 15:29:45","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":318766,"visible":true,"origin":"","legend":"\u003cp\u003eNeutrality plot of chloroplast genomes of 16 species. The species corresponding to letters A-P are the same as in Figure 8.\u003c/p\u003e","description":"","filename":"Figure12.png","url":"https://assets-eu.researchsquare.com/files/rs-9564592/v1/0ea6b497fc1c53384ce00bb1.png"},{"id":109906377,"identity":"7748516b-80c8-4f36-8889-ddb93b53e212","added_by":"auto","created_at":"2026-05-25 06:40:02","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":269012,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic trees were constructed using the maximum likelihood (ML) based on the complete chloroplast genomes of 61 Araceae species. The numbers above the nodes indicate support values.\u003c/p\u003e","description":"","filename":"Figure13.png","url":"https://assets-eu.researchsquare.com/files/rs-9564592/v1/1ffae5bfa82f4f749b94afe6.png"},{"id":109465652,"identity":"ee4524ae-be20-4d43-b886-5322f8555cde","added_by":"auto","created_at":"2026-05-18 11:57:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":678139,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9564592/v1/f5ae007c-009d-4f62-8586-089382e1d83b.pdf"},{"id":109911885,"identity":"e2bae3e8-7748-4ee9-b0a0-faa9460fc8c8","added_by":"auto","created_at":"2026-05-25 07:25:17","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":186433,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1\u003c/strong\u003eThe basic information of 6 newly sequenced chloroplast genomes in \u003cem\u003eAmorphophallus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2\u003c/strong\u003eCalculation of Ka/Ks ratios for chloroplast genes of 15 congeneric species using \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac \u003c/em\u003eas the reference species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S3\u003c/strong\u003eStatistics of RNA base editing and amino acid changes in 16 \u003cem\u003eAmorphophallus \u003c/em\u003especies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S4\u003c/strong\u003eSummary of CDS GC content, effective number of codons and optimal codons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S5\u003c/strong\u003eGenetic variation profiling of \u003cem\u003eAmorphophallus \u003c/em\u003etaxa relative to \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esp \u003c/em\u003ereference sequence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S6\u003c/strong\u003eUnique fingerprint sequences of \u003cem\u003eAmorphophallus\u003c/em\u003e species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S7\u003c/strong\u003eChloroplast genome information for phylogenetic analysis.\u003c/p\u003e","description":"","filename":"TableS1S7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9564592/v1/167cc1c5c5297ff53b5d64aa.xlsx"},{"id":109906374,"identity":"7192534c-a4ac-4103-bf13-2513d7a9b93f","added_by":"auto","created_at":"2026-05-25 06:40:02","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":276328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S2 \u003c/strong\u003eHeatmap of relative synonymous codon usage (RSCU) in the chloroplast genomes of 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species.\u003c/p\u003e","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9564592/v1/d541b8a1b8c00e42d0ddf058.pdf"},{"id":109906602,"identity":"b6e4c826-601a-4c1b-9bd9-e0f4f2feaacf","added_by":"auto","created_at":"2026-05-25 06:40:32","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":84851,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S3 \u003c/strong\u003eFrequency distribution of ENC ratios for protein-coding genes across 16 \u003cem\u003eAmorphophallus\u003c/em\u003echloroplast genomes.\u003c/p\u003e","description":"","filename":"FigureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9564592/v1/fac662e89978bdc4e07253ec.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative chloroplast genomics in Amorphophallus: Revealing codon usage patterns and phylogenetic clades across 16 species","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChloroplasts are organelles in plant cells responsible for photosynthesis and a range of metabolic processes. Beyond their central role in metabolism and energy conversion [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], chloroplasts are involved in the biosynthesis of starch, fatty acids, pigments, and amino acids. The chloroplast genome is considerably smaller than the nuclear genome. It exhibits several advantageous characteristics, including a moderate molecular weight, ease of sequencing, multiple copies, a relatively simple structure, moderate nucleotide substitution rates, pronounced differences in molecular evolutionary rates between coding and non-coding regions, and substantial synteny among chloroplast genomes across diverse taxa [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Chloroplasts contain a maternally inherited genome that operates independently of the nuclear genome. Owing to these features, chloroplast genomes have been widely applied in plant species identification, genetic diversity assessment, and phylogenetic reconstruction [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Their utility has become increasingly evident in plant phylogenetic studies integrating phylogenomic and comparative genomic analyses. Furthermore, a growing number of chloroplast genomes have been sequenced and assembled, and extensive genetic resources have been analyzed using various bioinformatics approaches, thereby offering novel insights into resolving taxonomically challenging plant groups [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genetic code is degenerate, whereby most amino acids are encoded by multiple synonymous codons. Codon usage bias refers to the non-uniform utilization of synonymous codons and arises from the combined effects of mutation pressure, natural selection, and genetic drift [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This bias is widespread across all species and displays substantial variation among distantly related taxa. In applied contexts, exploitation of species-specific codon usage patterns can enhance the efficiency of transgene expression. Moreover, analysis of codon usage bias in chloroplast genomes provides crucial foundational information for elucidating phylogenetic relationships and supporting genetic engineering applications [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eAmorphophallus\u003c/em\u003e (Araceae) represents one of the most species-rich lineages globally, comprising approximately 250 species widely distributed across tropical and subtropical regions of Asia and Africa [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Members of this genus exhibit remarkable morphological and phytochemical diversity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It includes glucomannan-rich resource species, such as \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emuelleri\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ealbus\u003c/em\u003e [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], as well as \u003cem\u003eA\u003c/em\u003e. \u003cem\u003epaeoniifolius\u003c/em\u003e, which is used in ethnomedicine for treating gastrointestinal disorders [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, \u003cem\u003eAmorphophallus\u003c/em\u003e includes species of considerable horticultural and ornamental value, including \u003cem\u003eA\u003c/em\u003e. \u003cem\u003egigas\u003c/em\u003e, whose inflorescence can reach up to 3 meters in height [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], \u003cem\u003eA\u003c/em\u003e. \u003cem\u003edunnii\u003c/em\u003e, which produces distinctive blue fruits [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eatroviridis\u003c/em\u003e, characterized by conspicuous pink petioles and leaf veins [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Despite this diversity, taxonomic studies of \u003cem\u003eAmorphophallus\u003c/em\u003e have long faced considerable challenges. Accurate species identification based solely on morphological characters is often hindered by pronounced morphological plasticity, hysteranthy (the temporal separation of leaf and flower emergence), and morphological convergence among species, leading to taxonomic ambiguity and misutilization of germplasm resources [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Consequently, the development of a high-resolution molecular phylogenetic framework is essential for elucidating the evolutionary history of the genus and supporting the accurate taxonomic application of its resources.\u003c/p\u003e \u003cp\u003eAlthough chloroplast genomes of more than ten \u003cem\u003eAmorphophallus\u003c/em\u003e species have recently been reported [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20 CR21 CR22 CR23 CR24 CR25\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], sampling coverage remains critically insufficient given the genus's high species richness. Existing studies have mainly focused on genome assembly and basic annotation of individual species, with limited implementation of large-scale, multi-species comparative analyses based on complete chloroplast genomes. In particular, systematic synthesis remains lacking for key aspects, including patterns of structural variation in chloroplast genomes, the mechanisms underlying codon usage bias, and the evolutionary drivers associated with geographic isolation. Based on the currently limited number of sequenced species, a preliminary understanding of the basic features and initial variation of \u003cem\u003eAmorphophallus\u003c/em\u003e chloroplast genomes has been established. Studies indicate that \u003cem\u003eAmorphophallus\u003c/em\u003e species generally conform to the typical circular quadripartite structure characteristic of angiosperms, yet exhibit substantial interspecific variation in genome size. Specifically, chloroplast genome sizes range from 164,417 (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003eyunnanensis\u003c/em\u003e) to 177,076 bp (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003emuelleri\u003c/em\u003e), encoding 126\u0026ndash;131 genes, with guanine\u0026ndash;cytosine (GC) content ranging from 34.5% to 36.0%. Structural analyses further reveal species-specific variations in inverted repeat (IR) region length (26,225\u0026ndash;35,204 bp) and in the distribution of key boundary genes, including \u003cem\u003erpl2\u003c/em\u003e, \u003cem\u003etrnH\u003c/em\u003e-\u003cem\u003eGUG\u003c/em\u003e, and \u003cem\u003eycf1\u003c/em\u003e. Moreover, mutational hotspot analysis has identified four highly variable regions, namely \u003cem\u003etrnM\u003c/em\u003e-\u003cem\u003eatpE\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.16), \u003cem\u003eatpB\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.155), \u003cem\u003eatpB\u003c/em\u003e-\u003cem\u003erbcL\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.15), and \u003cem\u003eycf1\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.147) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20 CR21 CR22 CR23 CR24 CR25\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], underscoring their potential as molecular markers for species identification in \u003cem\u003eAmorphophallus\u003c/em\u003e. However, these studies have largely focused on describing basic genomic characteristics, without fully elucidating the underlying evolutionary mechanisms. The relationships among interspecific genome size variation, IR boundary dynamics, and adaptive differentiation remain insufficiently understood and require broader taxon sampling and more systematic comparative genomic analyses.\u003c/p\u003e \u003cp\u003eTo address these research gaps, chloroplast genomes of six \u003cem\u003eAmorphophallus\u003c/em\u003e species (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ependulus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehewittii\u003c/em\u003e) were sequenced, assembled, and annotated using the PacBio Revio high-throughput sequencing platform (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These datasets were then integrated with published chloroplast genome sequences of ten additional species to construct a comprehensive comparative genomic framework encompassing 16 representative species. The objective of this study were to (i) systematically compare structural features, including genome size, gene content, and IR boundary configurations, across the sampled species; (ii) elucidate the characteristics of codon usage bias in \u003cem\u003eAmorphophallus\u003c/em\u003e and assess the relative contributions of driving factors (mutation pressure versus natural selection); (iii) resolve previously ambiguous phylogenetic relationships derived from fragment-based analyses, thereby establishing a robust framework for reconstructing the evolutionary history of the genus. Ultimately, this study provides a valuable reference for future breeding programs and the development of molecular markers in \u003cem\u003eAmorphophallus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant material sampling, DNA extraction, sequencing, and chloroplast genome assembly and annotation\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 Plant material sampling, DNA extraction, sequencing\u003c/h2\u003e \u003cp\u003eSix \u003cem\u003eAmorphophallus\u003c/em\u003e species (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ependulus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehewittii\u003c/em\u003e) were cultivated at Kunming University. Fresh leaves of each species were subsequently collected from the konjac germplasm nursery of Kunming University, Yunnan Province (latitude: 24.97406\u0026deg;N, longitude: 102.79605\u0026deg;E). Total genomic DNA was isolated from fresh leaf tissue using the DNeasy Plant Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. DNA purity and concentration were assessed using a NanoDrop 1000\u0026trade; and a Qubit\u0026trade; 4.0 system (Thermo Fisher Scientific). A 15 kb SMRTbell library was constructed with the SMRTbell Express Template Preparation Kit 2.0 (PacBio, Menlo Park, CA, USA), following a workflow that included DNA shearing, AMPure PB bead purification, ssDNA overhang removal, damage/end repair, hairpin adapter ligation, and library bead purification. After quality control, the library was sequenced on a PacBio Revio platform. Raw data were processed via CCS (v6.0.0; --minPasses 3, --minPredictedAccuracy 0.99, --maxLength 21,000) to yield high-accuracy HiFi reads.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2 Chloroplast genome assembly and annotation\u003c/h2\u003e \u003cp\u003eFirst, PacBio HiFi (high-fidelity) data (\u0026gt;\u0026thinsp;3 Gb, average fragment length\u0026thinsp;\u0026gt;\u0026thinsp;13 kb) were aligned to the reference chloroplast genome using minimap2 (v2.15-r905) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] to generate a PAF file. Filtered aligned reads (those with \u0026lt;\u0026thinsp;50\u0026times; coverage were removed) were obtained using the PAF file. The filtered reads were assembled with Flye v2.6 (Kolmogorov et al. 2020) (reference: \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e NC_056329.1) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] to generate a graph file. Redundant contigs were visualized and removed, and the assembly was edited into a circular sequence using Bandage v0.8.1 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]; this circular sequence represents the complete chloroplast genome of the respective plant. The chloroplast genome assemblies of these six species were annotated using PGA [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], with manual correction of the results. Subsequently, each chloroplast genome was visualized as a circular diagram using OGDRAW [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Comparative genomic analysis\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Basic characteristics of chloroplast sequences of 16 species\u003c/h2\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Repeat sequence analysis\u003c/h2\u003e \u003cp\u003eSimple sequence repeats (SSRs) and long sequence repeats (LSRs) in the chloroplast genomes of the 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species\u0026mdash;including forward (F), reverse (R), complement (C), and palindromic (P) repeat types\u0026mdash;as well as their genomic positions, were identified using the SSR and LSR analysis modules of CPStools software with default parameters [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 IR region expansion and contraction analysis\u003c/h2\u003e \u003cp\u003eA comprehensive analytical profile illustrating the IR boundary coordinates, adjacent gene distribution, and structural features of the chloroplast genomes of the 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species was constructed using the online tool IRscope (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://irscope.shinyapps.io/irapp/\u003c/span\u003e\u003cspan address=\"https://irscope.shinyapps.io/irapp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4 Nucleotide diversity (Pi) analysis\u003c/h2\u003e \u003cp\u003eThe polymorphism analysis module of CPStools was employed to automatically extract shared genes and intergenic spacers (IGS) from the GenBank format files (.gb) of the 16 \u003cem\u003eAmorphophallus\u003c/em\u003e chloroplast genomes. Multiple sequence alignments were performed, and nucleotide diversity (Pi) values were calculated. The Pi values for shared genes and IGS regions were subsequently visualized [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5 Ka/Ks (non-synonymous/synonymous substitution rate) analysis\u003c/h2\u003e \u003cp\u003eSelection pressure analysis was conducted on 75 shared genes across the chloroplast genomes of the 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species using the Ka/Ks analysis module of CPStools (with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e serving as the reference sequence). CDS nucleotide sequences and protein sequences were extracted for all samples, aligned, and converted to AXT format alignment files. Ka (non-synonymous substitution rate) and Ks (synonymous substitution rate) values were calculated, yielding a total of 620 valid Ka/Ks ratios. Ka/Ks\u0026thinsp;\u0026gt;\u0026thinsp;1, Ka/Ks\u0026thinsp;=\u0026thinsp;1, and Ka/Ks\u0026thinsp;\u0026lt;\u0026thinsp;1 indicate positive selection, neutral selection, and purifying selection, respectively [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.2.6 Sequence variation analysis\u003c/h2\u003e \u003cp\u003eTo assess the degree of sequence divergence among the chloroplast genomes of the 16 Amorphophallus species, a global comparative analysis was performed using the online tool VISTA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://genome.lbl.gov/vista/citeus.shtml\u003c/span\u003e\u003cspan address=\"https://genome.lbl.gov/vista/citeus.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e chloroplast genome serving as the reference. Structural variation plots were generated based on this analysis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.2.7 Conservation of chloroplast RNA editing in \u003cem\u003eAmorphophallus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003ePotential RNA editing sites in the chloroplast genomes of the 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species were predicted using the online tool PREPACT 3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.prepact.de/\u003c/span\u003e\u003cspan address=\"http://www.prepact.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with \u003cem\u003eSpirodela polyrhiza\u003c/em\u003e (MN419335.1) employed as the reference. All other parameters were kept at default settings [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Comparative analysis of codons in the chloroplast genomes of 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Analysis of codon composition and relative synonymous codon usage\u003c/h2\u003e \u003cp\u003eTo ensure accuracy and comparability of results, CDS sequences shorter than 300 bp were removed from the chloroplast genomes of the 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species. The filtered CDS sequences were analyzed using CPStools software to obtain the following parameters for each individual: GC content at codon positions 1, 2, and 3 (GC1, GC2, GC3), overall GC content (GC_all), effective number of codons (ENC), and relative synonymous codon usage (RSCU) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 ENC-plot analysis\u003c/h2\u003e \u003cp\u003eAn ENC-plot was generated with ENC values plotted on the ordinate and GC3 values on the abscissa. The standard curve was calculated according to the formula ENC\u0026thinsp;=\u0026thinsp;2\u0026thinsp;+\u0026thinsp;GC3\u0026thinsp;+\u0026thinsp;29 / [GC3\u003csup\u003e2\u003c/sup\u003e + (1 \u0026ndash; GC3)\u003csup\u003e2\u003c/sup\u003e]. The extent of deviation of individual gene points from the standard curve was used to infer the influence of various factors on codon usage bias ([\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 PR2-plot analysis\u003c/h2\u003e \u003cp\u003eParity rule 2 (PR2) plot analysis was performed to assess the relative contributions of mutation pressure and selection pressure on codon usage. The contents of A, T, C, and G at the third codon position (designated A3, T3, C3, and G3, respectively) were calculated. PR2 plots were constructed with G3/(G3\u0026thinsp;+\u0026thinsp;C3) as the abscissa and A3/(A3\u0026thinsp;+\u0026thinsp;T3) as the ordinate. The direction and vector distance of each gene from the center point (where A\u0026thinsp;=\u0026thinsp;T and C\u0026thinsp;=\u0026thinsp;G, indicating unbiased codon usage) reflect the nature and magnitude of codon usage bias [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4 Neutrality plot analysis\u003c/h2\u003e \u003cp\u003eNeutrality plot analysis was employed to evaluate the balance between mutational pressure and natural selection affecting codon usage, thereby elucidating the relationship between GC12 and GC3. A scatter plot was generated with the average GC content at the first and second codon positions (GC12) on the ordinate and GC3 on the abscissa, followed by regression analysis. In a neutrality plot, each point represents an individual gene, and the position of points relative to the diagonal line indicates whether codon usage patterns are predominantly shaped by mutation pressure or natural selection [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e2.3.5 Optimal codon analysis\u003c/h2\u003e \u003cp\u003eOptimal codons are defined as those used with the highest frequency within a genome. Based on the calculated ENC values, protein-coding genes from representative \u003cem\u003eAmorphophallus\u003c/em\u003e species were ranked from lowest to highest ENC. The top 5 genes (lowest ENC, highest expression) and the bottom 5 genes (highest ENC, lowest expression) were selected to construct high-expression and low-expression datasets, respectively. Codons that simultaneously met the criteria of high frequency (RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1) and high expression (ΔRSCU\u0026thinsp;\u0026ge;\u0026thinsp;0.08) were designated as optimal codons [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.4 DNA barcode region identification for species differentiation based on multiple sequence alignment of 16\u003c/b\u003e \u003cb\u003eAmorphophallus\u003c/b\u003e \u003cb\u003especies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe Barcoding program within CPStools software was employed to perform DNA barcoding analysis on the 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species, using \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esp\u003c/em\u003e. as the reference (automatically recognized by the program). The analysis was based on multiple sequence alignment files, with a window size of 800 bp and a sliding step of 100 bp, generating five candidate windows. Constraints were set with a maximum of 20 diagnostic loci and a maximum gap proportion of 0.6. By analyzing variation characteristics across the 225,171 bp alignment\u0026mdash;including SNPs, insertions, and deletions\u0026mdash;the number of variable sites among the 16 species and the results of 120 pairwise species comparisons were tallied. The optimal DNA barcoding window capable of discriminating all species was identified, and diagnostic loci and species-specific fingerprints within the window were characterized, culminating in the selection of the highest-scoring effective window [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Phylogenetic analysis\u003c/h2\u003e \u003cp\u003eTo determine the phylogenetic placement of \u003cem\u003eAmorphophallus\u003c/em\u003e within Araceae, a phylogenetic tree was constructed based on chloroplast genome data using the maximum likelihood (ML) method. The analysis included 61 taxa representing 51 genera of Araceae, with \u003cem\u003eZea mays\u003c/em\u003e designated as the outgroup (Table S7). All chloroplast genome sequences were aligned using MAFFT v7.526 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The concatenated alignment was trimmed to remove poorly aligned regions using trimAl v1.5.0 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Maximum likelihood analysis was performed with IQ-TREE v1.4.2, using 1,000 bootstrap replicates and the best-fit substitution model TVM\u0026thinsp;+\u0026thinsp;F+R7 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The resulting phylogenetic tree was edited and visualized using FigTree v1.4.4 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Analysis","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Comparative genomic analysis\u003c/h2\u003e \u003cp\u003eThis study investigated the chloroplast genomes of 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species, including six newly sequenced genomes (deposited in GenBank; accession numbers are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and ten previously published genomes. The HiFi read sequencing datasets generated from the six newly sequenced samples ranged from 6,593,888,101 to 12,285,404,571 bp (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), with average sequencing depths of 629.50\u0026times; to 2,907.32\u0026times; (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). All 16 chloroplast genomes exhibited the typical angiosperm quadripartite structure, comprising the large single-copy (LSC), small single-copy (SSC), IRa, and IRb regions. Genomic size variation was substantial among the 16 species, with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e exhibiting the largest chloroplast genome (185,810 bp) and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e the shortest (152,492 bp), yielding an average genome size of 171,861 bp. Region-specific analyses revealed that the LSC region ranged from 90,524 to 98,561 bp, the SSC region from 10,983 to 36,366 bp, and IRa/IRb regions from 9,998 to 42,066 bp. GC content across the genomes ranged from 33.57% to 36.00%. Gene content analysis indicated 118\u0026ndash;136 genes per genome, including 81\u0026ndash;90 protein-coding genes, 33\u0026ndash;39 transfer RNA (tRNA) genes, and 4\u0026ndash;8 ribosomal RNA (rRNA) genes. After removal of duplicated genes, the chloroplast genomes contained 108\u0026ndash;113 unique genes, comprising 76\u0026ndash;79 protein-coding genes, 28\u0026ndash;30 tRNA genes, and 4\u0026ndash;8 rRNA genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of chloroplast genome features among 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal length (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal (unique)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProtein coding genes (unique)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003erRNA (unique)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003etRNA (unique)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLSC length (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIRb length (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eSSC length (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eIRa length (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eTotal GC (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eAccession number\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e152,492\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e118 (111)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81 (78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33 (29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e96,130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e9,998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e36,366\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e9,998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e33.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003ePZ226531\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e184,539\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e135 (110)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90 (78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37 (28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e90,524\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e41,516\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e10,983\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e41,516\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e34.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003ePZ243066\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehewittii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e171,707\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e128 (110)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83 (77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37 (29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e93,724\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e31,505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e14,973\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e31,505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e34.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003ePZ235491\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e185,810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e136 (110)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90 (78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e38 (29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e90,677\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e42,066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e11,001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e42,066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e34.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003ePZ250735\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ependulus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e176,384\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e130 (111)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85 (78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37 (29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e95,372\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e32,546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15,920\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e32,546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e34.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003ePZ266516\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e170,976\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e130 (111)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87 (78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37 (29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e95,500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e29,695\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e16,086\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e29,695\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e34.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003ePZ266517\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ealbus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e175,728\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e130 (113)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86 (79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e36 (30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e93,177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e26,225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e20,249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e26,225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e35.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eOP531918\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecoaetaneus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e175,465\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e131 (111)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e84 (78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e39 (29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e98,561\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e30,200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e16,504\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e30,200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e34.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eNC_072945.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekachinensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e173,330\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e130 (110)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85 (78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37 (28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e92,030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e33,091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15,118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e33,091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e35.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003ePP072244\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekiusianus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e166,269\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e129 (109)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e84 (77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37 (28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e90,701\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e31,383\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e14,802\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e31,383\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e36.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003ePP072243\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekrausei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e172,418\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e130 (110)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85 (78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37 (28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e91,983\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e32,422\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15,591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e32,422\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e35.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003ePP936071\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. muelleri\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e177,076\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e130 (111)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85 (78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37 (29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e91,947\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e35,204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e14,721\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e35,204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e34.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eOR995733\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003etonkinensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e169,341\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e129 (110)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e84 (77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37 (29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e90,705\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e31,498\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15,640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e31,498\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e36.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003ePP234804\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003eyunnanensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e164,417\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e126 (108)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81 (76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37 (28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e92,149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e28,543\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15,182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e28,543\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e36.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eNC_082906.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e161,647\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e130 (113)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86 (79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e36 (30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e93,443\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e26,226\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e21,575\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e26,226\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e35.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eMK611803\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003esp\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e176,221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e130 (111)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85 (78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37 (29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e91,718\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e34,891\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e14,172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e34,891\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e34.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003ePP936070\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSubsequent analysis indicated that genes associated with photosynthesis are highly conserved across \u003cem\u003eAmorphophallus\u003c/em\u003e species, with only a few genes exhibiting copy-number variation among taxa. Specifically, \u003cem\u003endhA\u003c/em\u003e, \u003cem\u003endhH\u003c/em\u003e, and \u003cem\u003endhI\u003c/em\u003e were present in duplicate copies in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e, whereas \u003cem\u003endhB\u003c/em\u003e was a single-copy gene in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e. rRNA genes (\u003cem\u003errn4\u003c/em\u003e.\u003cem\u003e5\u003c/em\u003e, \u003cem\u003errn5\u003c/em\u003e, \u003cem\u003errn16\u003c/em\u003e, and \u003cem\u003errn23\u003c/em\u003e) and several tRNA genes (\u003cem\u003etrnA\u003c/em\u003e-\u003cem\u003eUGC\u003c/em\u003e and \u003cem\u003etrnI\u003c/em\u003e-\u003cem\u003eGAU\u003c/em\u003e) were detected as single-copy genes exclusively in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e. Moreover, \u003cem\u003etrnM\u003c/em\u003e-\u003cem\u003eCAU\u003c/em\u003e exhibited copy-number variation among taxa, occurring as either two or four copies. Among protein-coding genes, \u003cem\u003eycf1\u003c/em\u003e was identified as a single copy in certain species (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These observed variations provide valuable molecular markers for phylogenetic studies and species identification within \u003cem\u003eAmorphophallus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparative chloroplast gene composition and interspecific differences in \u003cem\u003eAmorphophallus\u003c/em\u003e species, with annotated variations.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCategory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene Group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhotosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of photosystem I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epsaA\u003c/em\u003e, \u003cem\u003epsaB\u003c/em\u003e, \u003cem\u003epsaC\u003c/em\u003e, \u003cem\u003epsaI\u003c/em\u003e, \u003cem\u003epsaJ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of photosystem II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epsbA\u003c/em\u003e, \u003cem\u003epsbB\u003c/em\u003e, \u003cem\u003epsbC\u003c/em\u003e, \u003cem\u003epsbD\u003c/em\u003e, \u003cem\u003epsbE\u003c/em\u003e, \u003cem\u003epsbF\u003c/em\u003e, \u003cem\u003epsbH\u003c/em\u003e, \u003cem\u003epsbI\u003c/em\u003e, \u003cem\u003epsbJ\u003c/em\u003e, \u003cem\u003epsbK\u003c/em\u003e, \u003cem\u003epsbL\u003c/em\u003e, \u003cem\u003epsbM\u003c/em\u003e, \u003cem\u003epsbN\u003c/em\u003e, \u003cem\u003epsbT\u003c/em\u003e, \u003cem\u003epsbZ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of NADH dehydrogenase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003endhA (2)\u003cem\u003e#\u003c/em\u003e \u003cb\u003eA\u003c/b\u003e, ndhB (2)\u003cem\u003e#\u003c/em\u003e \u003cb\u003eB\u003c/b\u003e, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH (2) \u003cb\u003eC\u003c/b\u003e, ndhI (2) \u003cb\u003eD\u003c/b\u003e, ndhJ, ndhK\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of cytochrome b/f complex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epetA\u003c/em\u003e, \u003cem\u003epetB\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003epetD\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003epetG\u003c/em\u003e, \u003cem\u003epetL\u003c/em\u003e, \u003cem\u003epetN\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarge subunit of rubisco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erbcL\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of ATP synthase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eatpA\u003c/em\u003e, \u003cem\u003eatpB\u003c/em\u003e, \u003cem\u003eatpE\u003c/em\u003e, \u003cem\u003eatpF\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eatpH\u003c/em\u003e, \u003cem\u003eatpI\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSelf-replication\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteins of large ribosomal subunit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erpl2\u003c/em\u003e (2)\u003cem\u003e#\u003c/em\u003e, rpl14, rpl16\u003cem\u003e#\u003c/em\u003e \u003cb\u003eE\u003c/b\u003e, rpl20, rpl22, rpl23 (2)\u003cem\u003e#\u003c/em\u003e \u003cb\u003eF\u003c/b\u003e, rpl32, rpl33, rpl36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteins of small ribosomal subunit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003erps2, rps3, rps4, rps7 (2) \u003cb\u003eG\u003c/b\u003e, rps8, rps11, rps12 (2)\u003cem\u003e##\u003c/em\u003e \u003cb\u003eH\u003c/b\u003e, rps14, rps15, rps16\u003cem\u003e#\u003c/em\u003e, rps18, rps19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of RNA polymerase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erpoA\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003erpoC1\u003c/em\u003e\u003csup\u003e#\u003c/sup\u003e, \u003cem\u003erpoC2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRibosomal RNAs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003errn4\u003c/em\u003e.5 (2) \u003cb\u003eI\u003c/b\u003e, rrn5 (2) \u003cb\u003eJ\u003c/b\u003e, rrn16 (2) \u003cb\u003eK\u003c/b\u003e, rrn23 (2) \u003cb\u003eL\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTransfer RNAs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003etrnA\u003c/em\u003e-UGC (2)\u003cem\u003e#\u003c/em\u003e \u003cb\u003eM\u003c/b\u003e, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-GCC, trnG-UCC\u003cem\u003e#\u003c/em\u003e, trnH, trnH-GUG \u003cb\u003eN\u003c/b\u003e, trnI-GAU (2)\u003cem\u003e#\u003c/em\u003e \u003cb\u003eO\u003c/b\u003e, trnK-UUU\u003cem\u003e#\u003c/em\u003e, trnL-CAA (2), trnL-UAA\u003cem\u003e#\u003c/em\u003e, trnL-UAG, trnM, trnM-CAU (3) \u003csub\u003e\u003cb\u003eP\u003c/b\u003e\u003c/sub\u003e, trnN-GUU, trnP-UGG, trnQ-UUG, trnR-ACG, trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC, trnV-UAC\u003cem\u003e#\u003c/em\u003e, trnW-CCA, trnY-GUA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOther genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaturase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ematK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eclpP##\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnvelope membrane protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ecemA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcetyl-CoA carboxylase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eaccD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec-type cytochrome synthesis gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eccsA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConserved open reading frames\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eycf1\u003c/em\u003e (2) \u003cb\u003eQ\u003c/b\u003e, ycf2 (2), ycf3\u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eycf4\u003c/em\u003e, \u003cem\u003eycf68\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNote: #: Intron number, (n): Gene copy number.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e\u003cstrong\u003e\u003csub\u003eA\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e only, the \u003cem\u003endhA\u003c/em\u003e gene is present in two copies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eB\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e only, \u003cem\u003endhB\u003c/em\u003e is a single-copy gene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eC\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e only, \u003cem\u003endhH\u003c/em\u003e is present in two copies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eD\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e only, \u003cem\u003endhI\u003c/em\u003e is present in two copies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eE\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekachinensis\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekiusianus\u003c/em\u003e, the \u003cem\u003erpl16\u003c/em\u003e gene lacks an intron.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eF\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecoaetaneus\u003c/em\u003e, \u003cem\u003erpl23\u003c/em\u003e is a single-copy gene; it contains an intron only in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekachinensis\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekrausei\u003c/em\u003e; and it is absent in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekiusianus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etonkinensis\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eyunnanensis\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eG\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e only, \u003cem\u003erps7\u003c/em\u003e is a single-copy gene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e only, \u003cem\u003erps12\u003c/em\u003e is a single-copy gene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eI\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e only, \u003cem\u003errn4\u003c/em\u003e.\u003cem\u003e5\u003c/em\u003e is a single-copy gene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eJ\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e only, \u003cem\u003errn5\u003c/em\u003e is a single-copy gene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eK\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e only, \u003cem\u003errn16\u003c/em\u003e is a single-copy gene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eL\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e only, \u003cem\u003errn23\u003c/em\u003e is a single-copy gene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eM\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e only, \u003cem\u003etrnA\u003c/em\u003e-\u003cem\u003eUGC\u003c/em\u003e is a single-copy gene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eN\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e, the gene is present in two copies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eO\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e only, \u003cem\u003etrnI\u003c/em\u003e-\u003cem\u003eGAU\u003c/em\u003e is a single-copy gene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eP\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ealbus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecoaetaneus\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e, the copy number of \u003cem\u003etrnM\u003c/em\u003e-\u003cem\u003eCAU\u003c/em\u003e is two, whereas in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekachinensis\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekrausei\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eyunnanensis\u003c/em\u003e it is four.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003eQ\u003c/sub\u003e\u003c/strong\u003e: In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ealbus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eyunnanensis\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e is a single-copy gene.\u0026nbsp;\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Repeat sequence analysis\u003c/h2\u003e \u003cp\u003eRepeat sequence analysis identified 87 (in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e) to 162 (in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esp.\u003c/em\u003e) simple sequence repeat (SSR) loci across the 16 species (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). SSR distribution analysis revealed that SSRs were predominantly enriched in intergenic regions across the 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species, followed by intronic regions, whereas the fewest SSRs were observed in coding regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In terms of repeat motif classification, mononucleotide repeats were the most abundant (70\u0026ndash;105 loci), followed by dinucleotide (11\u0026ndash;55), trinucleotide (1\u0026ndash;7), tetranucleotide (0\u0026ndash;7), pentanucleotide (0\u0026ndash;11), and hexanucleotide (0\u0026ndash;2) repeats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Across all repeat types, A (31\u0026ndash;50)/T (33\u0026ndash;56) motifs were the most prevalent, followed by AT (1\u0026ndash;29)/TA (7\u0026ndash;37) motifs, whereas other repeat patterns occurred at relatively low proportions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of long sequence repeats (LSRs) in the chloroplast genomes of 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species revealed a generally comparable distribution pattern among species. The number of forward repeats ranged from 51 (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e) to 1,209 (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003emuelleri\u003c/em\u003e), palindromic repeats from 41 (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e) to 345 (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehewittii\u003c/em\u003e), complement repeats from 15 (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e) to 313 (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e), and reverse repeats from 44 (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e) to 833 (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003emuelleri\u003c/em\u003e) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). Overall, forward repeats constituted the predominant repeat type in 14 species, with the exception of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehewittii\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanium\u003c/em\u003e, in which reverse repeats were more abundant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Additional analysis indicated that LSRs in the six \u003cem\u003eAmorphophallus\u003c/em\u003e species were highly concentrated within the 30\u0026ndash;40 bp length interval, demonstrating a distribution pattern dominated by short repeat units. Moreover, positional distribution analysis revealed a strong regional bias, with the majority of LSRs located in intergenic regions, whereas substantially fewer were present in intronic and coding regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3 IR region expansion and contraction analysis\u003c/h2\u003e \u003cp\u003eAnalysis of IR boundary characteristics in the chloroplast genomes of 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e) revealed pronounced variation in genome architecture. \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e (185,810 bp) and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e (184,539 bp) exhibited relatively large chloroplast genomes, both characterized by expanded IR regions (\u0026gt;\u0026thinsp;41,000 bp) and reduced SSC regions (\u0026lt;\u0026thinsp;12,000 bp), representing typical cases of IR expansion. In contrast, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e displayed extreme IR contraction, with an IR region of only 9,998 bp and an expanded SSC region of 36,366 bp (the largest among all examined species). Based on gene position variation at LSC/IR/SSC boundaries, the 16 species were classified into eight structural types (I\u0026ndash;VIII): Type I (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekiusianus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekrausei\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emuelleri\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etonkinensis\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esp.\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ependulus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekachinensis\u003c/em\u003e); Type II (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicates\u003c/em\u003e); Type III (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003eyunnanensis\u003c/em\u003e); Type IV (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e); Type V (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e); Type VI (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehewittii\u003c/em\u003e); Type VII (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ealbus\u003c/em\u003e); Type VIII (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecoaetaneus\u003c/em\u003e). In Type I, LSC/IRb boundary was located between \u003cem\u003erps19\u003c/em\u003e and \u003cem\u003erpl12\u003c/em\u003e. The \u003cem\u003endhF\u003c/em\u003e gene spanned IRb/SSC boundary in six species (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekiusianus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emuelleri\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eyunnanensis\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esp\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ependulus\u003c/em\u003e). The \u003cem\u003erps15\u003c/em\u003e gene spanned SSC/IRa boundary in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emuelleri\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esp.\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekachinensis\u003c/em\u003e. In \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e, a 127 bp overlap was observed between \u003cem\u003erps15\u003c/em\u003e and \u003cem\u003eycf1\u003c/em\u003e. In Type II, LSC/IRb boundary was located between \u003cem\u003erps19\u003c/em\u003e and \u003cem\u003etrnH\u003c/em\u003e, IRb/SSC boundary was between \u003cem\u003etrnL\u003c/em\u003e and \u003cem\u003endhF\u003c/em\u003e, and SSC/IRa boundary was between \u003cem\u003endhB\u003c/em\u003e and \u003cem\u003etrnL\u003c/em\u003e. In Type III, a 136 bp overlap occurred between \u003cem\u003eycf1\u003c/em\u003e and \u003cem\u003endhF\u003c/em\u003e in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eyunnanensis\u003c/em\u003e, and SSC/IRa boundary was situated between \u003cem\u003erps15\u003c/em\u003e and \u003cem\u003etrnN\u003c/em\u003e. In Type IV, IRb/SSC boundary was located between \u003cem\u003etrnN\u003c/em\u003e and \u003cem\u003endhF\u003c/em\u003e, and SSC/IRa boundary was between \u003cem\u003eycf1\u003c/em\u003e and \u003cem\u003etrnN\u003c/em\u003e. In Type V, LSC/IRb boundary was between \u003cem\u003erps19\u003c/em\u003e and \u003cem\u003epsbA\u003c/em\u003e, IRb/SSC boundary was between \u003cem\u003endhI\u003c/em\u003e and \u003cem\u003endhF\u003c/em\u003e, and SSC/IRa boundary was between \u003cem\u003endhG\u003c/em\u003e and \u003cem\u003endhI\u003c/em\u003e. In Type VI, LSC/IRb boundary was between \u003cem\u003erps19\u003c/em\u003e and \u003cem\u003erpl23\u003c/em\u003e, and IRa/LSC boundary was between \u003cem\u003erpl23\u003c/em\u003e and \u003cem\u003etrnH\u003c/em\u003e. Type VII was generally similar to Type IV, except that IRb/SSC boundary was positioned between \u003cem\u003eycf1\u003c/em\u003e and \u003cem\u003endhF\u003c/em\u003e. In Type VIII, LSC/IRb boundary was located between \u003cem\u003erps12\u003c/em\u003e and \u003cem\u003etrnM\u003c/em\u003e. These structural variations provide valuable references for developing species-specific DNA barcodes for identification purposes.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.1.4 Nucleotide diversity (Pi) analysis\u003c/h2\u003e \u003cp\u003ePi analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e) revealed substantial variation in polymorphism among shared genes and intergenic spacers across LSC, SSC, and IRa/b regions of the chloroplast genomes in 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species. In addition, the distribution of Pi values differed markedly among genes and spacers within the same genomic region. The LSC region exhibited the highest sequence variation, containing genes and intergenic regions with the greatest genome-wide Pi values. The gene \u003cem\u003eaccD\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.1217) demonstrated the highest level of polymorphism across the entire genome, followed by \u003cem\u003eclpP\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.0951). Conversely, the SSC region displayed lower overall polymorphism than the LSC region, with gene Pi values ranging from 0.0037 to 0.0132. Within the SSC region, \u003cem\u003endhF\u003c/em\u003e, \u003cem\u003endhG\u003c/em\u003e, and \u003cem\u003endhD\u003c/em\u003e exhibited relatively higher variability, whereas \u003cem\u003epsaC\u003c/em\u003e exhibited the lowest. Intergenic spacer polymorphism was polarized, with \u003cem\u003epetD\u003c/em\u003e_2\u0026ndash;\u003cem\u003erpoA\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.0743) exhibiting the highest Pi value among spacers, followed by \u003cem\u003endhD\u003c/em\u003e\u0026ndash;\u003cem\u003epsaC\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.0588). IRa/IRb regions exhibited the lowest overall polymorphism, with Pi values for most genes below 0.015. The gene \u003cem\u003endhB\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.0019) was identified as the most conserved across the genome. Only \u003cem\u003eycf1\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.0570) and \u003cem\u003erps15\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.0352) represented core variable loci within this region. In summary, hypervariable sites in the \u003cem\u003eAmorphophallus\u003c/em\u003e chloroplast genome are predominantly concentrated in single-copy genes and intergenic spacers within the LSC region. In contrast, the SSC region is comparatively conserved with moderate localized variation. IRa/IRb regions remain highly conserved, with limited variability confined to \u003cem\u003eycf1\u003c/em\u003e and \u003cem\u003erps15\u003c/em\u003e. These findings provide a robust foundation for identifying hypervariable molecular markers and for phylogenetic analyses within the genus.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.1.5 Ka/Ks analysis\u003c/h2\u003e \u003cp\u003eUsing \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e as the reference species, Ka/Ks ratios were calculated for chloroplast genes across the remaining 15 \u003cem\u003eAmorphophallus\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Ka/Ks ratios for the vast majority of chloroplast genes in all examined species ranged from 0 to 1, indicating that the \u003cem\u003eAmorphophallus\u003c/em\u003e chloroplast genome has been predominantly subjected to purifying selection throughout its evolutionary history. This pattern is consistent with the generally highly conserved nature of plant chloroplast genomes. The highest Ka/Ks value, 12.274, was observed for the \u003cem\u003erps3\u003c/em\u003e gene in the comparison between \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esp.\u003c/em\u003e, suggesting that this gene may have undergone rapid evolution or functional modification in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esp.\u003c/em\u003e, potentially associated with species-specific environmental adaptation or physiological traits. Ka/Ks values of 1 were detected for the \u003cem\u003erpl16\u003c/em\u003e gene in the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e versus \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ependulus\u003c/em\u003e, the \u003cem\u003erps16\u003c/em\u003e and \u003cem\u003erpoA\u003c/em\u003e genes in the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e versus \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecoaetaneus\u003c/em\u003e, and the \u003cem\u003erps16\u003c/em\u003e and \u003cem\u003erpl16\u003c/em\u003e genes in the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e versus \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekiusianus\u003c/em\u003e. Subsequent analysis revealed that \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esp.\u003c/em\u003e exhibited the stronger signals of positive selection, with the highest number of genes under positive selection, including \u003cem\u003erps16\u003c/em\u003e (1.007), \u003cem\u003erps2\u003c/em\u003e (1.308), \u003cem\u003erpoC2\u003c/em\u003e (0.725), \u003cem\u003erpl20\u003c/em\u003e (1.011), \u003cem\u003eclpP\u003c/em\u003e (2.072), \u003cem\u003erpoA\u003c/em\u003e (1.006), \u003cem\u003erps11\u003c/em\u003e (1.165), \u003cem\u003erpl14\u003c/em\u003e (3.818), \u003cem\u003erps3\u003c/em\u003e (2.938), \u003cem\u003erps19\u003c/em\u003e (1.698), \u003cem\u003eycf2\u003c/em\u003e (1.554), \u003cem\u003endhD\u003c/em\u003e (0.227), and \u003cem\u003eycf1\u003c/em\u003e (2.332). These findings indicate that multiple functional genes in the chloroplast genome of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esp.\u003c/em\u003e may have undergone adaptive evolution.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.1.6 Sequence variation analysis\u003c/h2\u003e \u003cp\u003eUsing the chloroplast genome of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e as the reference, a comprehensive comparative analysis was conducted across the remaining 15 \u003cem\u003eAmorphophallus\u003c/em\u003e species. This analysis revealed differential levels of sequence variation among exon regions, tRNAs, rRNAs, and conserved non-coding sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003e), indicating heterogeneous evolutionary dynamics across genomic regions. Notably, pronounced variation was observed in intergenic regions, such as \u003cem\u003etRNA\u003c/em\u003e-\u003cem\u003eSer\u003c/em\u003e(GCU)/\u003cem\u003etRNA\u003c/em\u003e-\u003cem\u003eGly\u003c/em\u003e(UCC), \u003cem\u003etRNA\u003c/em\u003e-\u003cem\u003eGlu\u003c/em\u003e(UUC)/\u003cem\u003etRNA\u003c/em\u003e-\u003cem\u003eThr\u003c/em\u003e(GGU), \u003cem\u003etRNA\u003c/em\u003e-\u003cem\u003eThr\u003c/em\u003e(UGU)/\u003cem\u003etRNA\u003c/em\u003e-\u003cem\u003eLeu\u003c/em\u003e(UAA), and \u003cem\u003endhC\u003c/em\u003e/\u003cem\u003etRNA\u003c/em\u003e-\u003cem\u003eVal\u003c/em\u003e(UAC). Furthermore, notable sequence divergence was detected in genes, including \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003eycf2\u003c/em\u003e, \u003cem\u003erps16\u003c/em\u003e, \u003cem\u003etRNA\u003c/em\u003e-\u003cem\u003eLeu\u003c/em\u003e(CAA), and \u003cem\u003erps12\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e3.1.7 Conservation of chloroplast RNA editing in \u003cem\u003eAmorphophallus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo characterize chloroplast RNA editing in \u003cem\u003eAmorphophallus\u003c/em\u003e, a systematic analysis of RNA editing patterns across the chloroplast genomes of 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species was conducted (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The results demonstrated that C\u0026rarr;U base editing was the sole editing type detected in the chloroplasts of all species examined, whereas other base editing types, including U\u0026rarr;C, G\u0026rarr;A, and A\u0026rarr;I, were absent. The number of C\u0026rarr;U editing sites exhibited slight interspecific variation, ranging from 1,331 in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e to 1,605 in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekiusianus\u003c/em\u003e. Among the identified editing events, P\u0026rarr;L (proline (Pro) \u0026rarr; leucine (Leu)) and P\u0026rarr;S (Pro \u0026rarr; serine (Ser)) represented the two predominant editing types, constituting the core spectrum of amino acid substitutions mediated by C\u0026rarr;U editing in \u003cem\u003eAmorphophallus\u003c/em\u003e chloroplasts. In contrast, substitution types, such as A\u0026rarr;V and H\u0026rarr;Y, occurred at relatively rare frequencies. Furthermore, comparative analysis revealed that \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekrausei\u003c/em\u003e possessed the highest number of nonsense mutations (Q\u0026rarr; 43 events, R\u0026rarr; 43 events; total 86 events), followed by \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ealbus\u003c/em\u003e (Q\u0026rarr; 39 events, R\u0026rarr; 39 events; total 78 events). Conversely, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eyunnanensis\u003c/em\u003e exhibited the lowest number of nonsense mutation events (Q\u0026rarr; 22 events, R\u0026rarr; 22 events; total 44 events). These species-specific RNA editing characteristics may serve as valuable molecular markers for species identification and provide insights into evolutionary differentiation within the genus.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Comparative analysis of codons in the chloroplast genomes of 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species\u003c/h2\u003e \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Basic characteristics of codon composition analysis\u003c/h2\u003e \u003cp\u003eFollowing quality screening, 50\u0026ndash;52 coding DNA sequences (CDS) suitable for codon usage bias analysis were identified from the chloroplast genomes of 16 representative \u003cem\u003eAmorphophallus\u003c/em\u003e species (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Across these species, the overall GC content of coding regions (GC_all) ranged from 38.00% to 38.57%. The base compositions at the three codon positions (GC1, GC2, and GC3) ranged from 46.21% to 47.25%, 39.37% to 40.12%, and 28.21% to 28.70%, respectively (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), indicating a positional variation in nucleotide distribution. A consistent pattern of GC1\u0026thinsp;\u0026gt;\u0026thinsp;GC2\u0026thinsp;\u0026gt;\u0026thinsp;GC3 was observed across all examined species. Although GC content at different codon positions was relatively similar across \u003cem\u003eAmorphophallus\u003c/em\u003e species, its distribution was uneven, with a bias toward the first two positions; notably, GC1, GC2, and GC_all were all below 50%. The mean effective number of codons (ENC) across the 16 chloroplast genomes was 46.91, and only the \u003cem\u003erps18\u003c/em\u003e gene consistently exhibited ENC values below 40 in all chloroplast genomes, suggesting a generally weak codon usage bias in these species.\u003c/p\u003e \u003cp\u003eCorrelation analysis among parameters\u0026mdash;including GC1, GC2, GC3, GC_all, ENC, and codon number\u0026mdash;was performed using CDS from the chloroplast genomes of 16 representative \u003cem\u003eAmorphophallus\u003c/em\u003e species. The results revealed that correlations among GC1, GC2, and GC3 were non-significant across all examined species (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003e). In contrast, significant or highly significant positive correlations between GC1 and GC2 were detected in 13 species, with the exception of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekrausei\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eK). This pattern indicates a comparable base composition at the first two codon positions across these 13 species and suggests that variation in chloroplast gene expression is primarily associated with GC content at the first codon position, with relatively minor associations at the second and third positions. Regarding codon usage bias, no significant correlation between ENC values and GC1 or GC2 was observed in 14 of the 16 representative \u003cem\u003eAmorphophallus\u003c/em\u003e species, except for \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eB) and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Conversely, significant or highly significant correlations between ENC values and GC3 were detected in all 16 species, suggesting that alterations in base composition at the third codon position are the primary determinant of codon usage bias in \u003cem\u003eAmorphophallus\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Relative synonymous codon usage analysis\u003c/h2\u003e \u003cp\u003eAfter excluding stop codons and non-expressed codons, RSCU values across 16 representative \u003cem\u003eAmorphophallus\u003c/em\u003e species were relatively comparable. Among all codons, UUA encoding Leu exhibited the highest RSCU value across all species (Figs.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003e and \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In terms of high-frequency codon distribution, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ealbus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekiusianus\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e each contained 31 high-frequency codons, whereas the remaining 12 species each harbored 30 high-frequency codons, accounting for 48.44% and 46.88% of the total codon sets, respectively. Furthermore, high-frequency codons in \u003cem\u003eAmorphophallus\u003c/em\u003e species exhibited a bias toward A/T-ending codons, whereas low-frequency codons tended to terminate in G/C.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3 ENC-plot analysis\u003c/h2\u003e \u003cp\u003eENC-plot analysis of chloroplast genomes from 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species revealed a strong association between codon usage patterns and gene function, with core functional genes exhibiting conserved codon usage across all examined species. Most genes were distributed below the standard curve, whereas only a few genes were located on or near the curve. Across most species, \u003cem\u003eycf3\u003c/em\u003e represented the gene positioned farthest above the expected curve, with exceptions observed for \u003cem\u003eclpP\u003c/em\u003e in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ealbus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekachinensis\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etonkinensis\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eyunnanensis\u003c/em\u003e, and \u003cem\u003eycf68\u003c/em\u003e in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e. Additionally, most genes exhibited ENC values greater than 45. The frequency distribution of ENC ratios (Figs.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e10\u003c/span\u003e and \u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) demonstrated that ratios ranged from \u0026minus;\u0026thinsp;0.05 to 0.35. Between 13 and 18 genes were concentrated within the interval near the standard curve (\u0026ndash;0.05 to 0.05), indicating relatively small differences between observed and expected ENC values. Furthermore, 9 to 12 genes exhibited ENC ratios greater than 0.15 and were positioned below the standard curve, at a considerable distance from it.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003e3.2.4 Parity rule 2 (PR2)-plot analysis\u003c/h2\u003e \u003cp\u003ePR2 plot analysis was employed to assess the degree of nucleotide usage bias at the third codon position. In this analysis, the PR2 plot was centered at the coordinates (0.5, 0.5) based on A3/(A3\u0026thinsp;+\u0026thinsp;T3) and G3/(G3\u0026thinsp;+\u0026thinsp;C3), thereby dividing the plot into four quadrants. Protein-coding genes from the chloroplast genomes of 16 representative \u003cem\u003eAmorphophallus\u003c/em\u003e species exhibited an uneven distribution across the four quadrants. Notably, the gene scatter points were highly concentrated in the fourth quadrant and did not cluster around the center point (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e11\u003c/span\u003e). This distribution pattern indicates a pronounced bias in nucleotide usage at the third codon position in \u003cem\u003eAmorphophallus\u003c/em\u003e species, characterized by a preference order of T\u0026thinsp;\u0026gt;\u0026thinsp;A and G\u0026thinsp;\u0026gt;\u0026thinsp;C. Collectively, these findings further support that natural selection is the primary factor influencing codon usage bias in this genus.\u003c/p\u003e\u003cp\u003eFrom a functional gene perspective, scatter points corresponding to core chloroplast housekeeping genes\u0026mdash;including those involved in photosystems, adenosine triphosphate synthase, nicotinamide adenine dinucleotide hydride (NADH) dehydrogenase, ribosomal proteins, and RNA polymerase\u0026mdash;exhibited pronounced deviation from the central point. Notably, the deviation patterns for these gene categories were highly consistent across all 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species. This pattern indicates that nucleotide usage at the third codon position of core housekeeping genes is under strong directional natural selection, thereby promoting translational efficiency and accuracy of essential proteins and ensuring compatibility with the chloroplast translational machinery.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003e3.2.5 Neutrality plot analysis\u003c/h2\u003e \u003cp\u003eTo assess the relationship between GC3 and GC12, neutrality plot analysis was conducted using the protein-coding genes from 16 representative \u003cem\u003eAmorphophallus\u003c/em\u003e species. The results indicated that the majority of protein-coding genes were distributed above the diagonal line in the neutrality plot, whereas only a small proportion clustered along the diagonal. GC12 content ranged from 32.34% to 56.47%, while GC3 content ranged from 18.37% to 46.36%. The correlation coefficient (R\u0026sup2;) between GC12 and GC3 ranged from 0.008 to 0.070 (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e12\u003c/span\u003e), indicating that GC3 and GC12 exhibit distinct evolutionary trajectories among \u003cem\u003eAmorphophallus\u003c/em\u003e genes. Both mutation pressure and natural selection contribute to codon usage bias in this genus, with natural selection representing the primary driving force.\u003c/p\u003e\u003cp\u003eSubsequent analysis revealed that genes belonging to different functional categories occupy distinct positions in the neutrality plot. Core genes involved in transcription and translation, including those encoding ribosomal proteins and RNA polymerase, were substantially deviated from the diagonal, indicating that their evolutionary patterns are predominantly driven by natural selection pressure. In contrast, loci corresponding to NADH dehydrogenase-related genes were positioned between regions of high and moderate conservation, suggesting that their evolution is jointly influenced by natural selection and neutral mutation, with natural selection exerting a more pronounced effect.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section3\"\u003e \u003ch2\u003e3.2.6 Optimal codon analysis\u003c/h2\u003e \u003cp\u003eA total of 15 to 21 optimal codons were identified among the 16 representative \u003cem\u003eAmorphophallus\u003c/em\u003e species (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Among them, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecoaetaneus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e possessed the highest number of optimal codons (21 each), whereas \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekiusianus\u003c/em\u003e exhibited the lowest number (15). Regarding base composition at the third codon position, these optimal codons displayed a pronounced bias toward A- or U-ending codons. Furthermore, comparative analysis identified six optimal codons shared by all 16 species: UUA, CGU, AAA, GUA, ACU, and AGU, which encode Leu, arginine (Arg), lysine (Lys), valine (Val), threonine (Thr), and Ser, respectively. Among these shared codons, three terminated in A and three in U. Furthermore, 15 differential optimal codons were identified, including ACA, ACC, AGA, AUU, CAU, CCC, GAA, GCU, GGA, GUU, UAG, UCC, UGU, UUG, and UUU. Within this set, four codons ended in A, six in U, three in C, and two in G. Notably, CCA (Pro) was unique to \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ependulus\u003c/em\u003e, whereas CUU (Leu), UCU (Ser), and CUA (Leu) were specific to \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec36\" class=\"Section2\"\u003e \u003ch2\u003e3.3 DNA barcode region identification\u003c/h2\u003e \u003cp\u003eBased on an 800 bp sliding window analysis, the optimal DNA barcoding region for the 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species was identified. This analysis revealed substantial genetic variation relative to the reference sequence (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003esp.\u003c/em\u003e), including 79,824 single-nucleotide polymorphisms (SNPs), 171,710 insertions, and 253,417 deletions, with deletions representing the predominant variant type (Table S5). Among the examined species, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e exhibited the highest total number of variants (55,410), of which deletions constituted the majority (36,638), whereas \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emuelleri\u003c/em\u003e displayed the lowest number of variants (1,125), comprising no SNPs, 990 insertions, and 135 deletions. In the remaining species, including \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e, total variant numbers were distributed between 27,000 and 47,000, while SNP counts consistently ranged from 5,000 to 6,300. Furthermore, an 800 bp region spanning positions 80,401 to 81,200 bp was identified as the optimal DNA barcoding region, enabling discrimination among all 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species using five diagnostic loci (T|\u0026ndash;|\u0026ndash;|C|T; Table S6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Phylogenetic analysis\u003c/h2\u003e \u003cp\u003eTo determine the phylogenetic placement of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehewittii\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ependulus\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e within Araceae, a phylogenetic tree was constructed based on chloroplast genome data using the maximum likelihood (ML) method. The dataset comprised 61 taxa representing 51 genera of Araceae, with \u003cem\u003eZ\u003c/em\u003eea \u003cem\u003emays\u003c/em\u003e designated as the outgroup (Table S8). The resulting phylogenetic topology (Fig.\u0026nbsp;14) indicated that \u003cem\u003eAmorphophallus\u003c/em\u003e species form a distinct clade with 100% bootstrap support, constituting a strongly supported monophyletic lineage. Subsequent analysis resolved the \u003cem\u003eAmorphophallus\u003c/em\u003e species into three distinct clades: Continental Asia II (CA-II), Continental Asia I (CA-I), and Southeast Asia (SEA). Within CA-II, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e clustered together with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ealbus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekrausei\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekachinensis\u003c/em\u003e (bootstrap support\u0026thinsp;=\u0026thinsp;100%), indicating close phylogenetic relationships among these taxa. In the SEA clade, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehewittii\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ependulus\u003c/em\u003e formed a closely related group (bootstrap support\u0026thinsp;=\u0026thinsp;100%). Within the CA-I clade, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecoaetaneus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etonkinensis\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eyunnanensis\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emuelleri\u003c/em\u003e were identified as the most closely related taxa (bootstrap support\u0026thinsp;=\u0026thinsp;100%). Furthermore, in the broader phylogenetic tree of Araceae, \u003cem\u003eAmorphophallus\u003c/em\u003e occupied a crown group position and formed a highly supported sister clade (bootstrap\u0026thinsp;=\u0026thinsp;100%) with \u003cem\u003eSyngonium\u003c/em\u003e, \u003cem\u003eXanthosoma\u003c/em\u003e, \u003cem\u003eZomicarpella\u003c/em\u003e, and \u003cem\u003eCaladium\u003c/em\u003e. The base of the phylogenetic tree was represented by \u003cem\u003eSymplocarpus\u003c/em\u003e, followed by the divergence of \u003cem\u003eWolffia\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe chloroplast genome is the smallest of the three primary genomes in plant cells, alongside the nuclear and mitochondrial genomes, and constitutes a relatively autonomous genetic system with limited dependence on nuclear genomic regulation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Although generally conserved, chloroplast genome structure, size, and gene content exhibit variation across genera and even among species, thereby providing important reference data for studies in plant taxonomy, genetics, and ecological adaptation. In this study, complete chloroplast genomes from six \u003cem\u003eAmorphophallus\u003c/em\u003e species were characterized and compared with the published chloroplast genomes from ten additional species within this genus, enabling a comprehensive assessment. The chloroplast genome size among the 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species varied substantially, ranging from 152,492 to 185,810 bp, while gene content ranged from 118 to 136 genes, suggesting species-specific differentiation potentially associated with metabolic pathways and ecological adaptation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In contrast, the GC content of these genomes ranged from 33.57% to 36.00%, with a coefficient of variation of only 1.86%, indicating a strong evolutionary conservation of chloroplast genomes in \u003cem\u003eAmorphophallus\u003c/em\u003e and the absence of substantial shifts in base composition.\u003c/p\u003e \u003cp\u003eSSRs are widely used as molecular markers for assessing genetic polymorphism in plants [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Across the 16 species, the number of SSRs ranged from 74 to 113. Consistent with observations in most species, mononucleotide repeats constituted the predominant class, followed by dinucleotide, tetranucleotide, trinucleotide, pentanucleotide, and hexanucleotide repeats, the latter being the least frequent. Furthermore, A/T motifs were substantially more abundant than G/C motifs, consistent with previously reported patterns in \u003cem\u003eAmorphophallus\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These results provide a valuable reference for future studies on population genetics and phylogeography within the genus.\u003c/p\u003e \u003cp\u003eAnalysis of characteristic genes at chloroplast genome boundary regions facilitates species identification and phylogenetic inference [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In this study, IR regions of chloroplast genomes from 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species were systematically examined. Although the overall genome sequences exhibited a high degree of similarity, minor differences were identified. Notably, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e (185,810 bp) and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e (184,539 bp) represent typical IR expansion types, characterized by IR regions exceeding 41,000 bp in length and SSC regions shorter than 12,000 bp. In contrast, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e represents an extreme case of IR contraction, with an IR region of only 9,998 bp and an SSC region extending to 36,366 bp. An inverse relationship between IR and SSC region lengths is evident, further supporting the role of IR dynamics as a central mechanism driving size variation and structural differentiation in angiosperm chloroplast genomes [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. IR boundary shifts also resulted in species-specific patterns of gene distribution. The \u003cem\u003endhF\u003c/em\u003e gene spanned the IRb/SSC boundary in six species, whereas \u003cem\u003erps19\u003c/em\u003e was located in the LSC region in all species except \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecoaetaneus\u003c/em\u003e, in which \u003cem\u003erpl2\u003c/em\u003e was uniquely positioned in the LSC region. The \u003cem\u003eycf1\u003c/em\u003e spanned the IRb/SSC boundary exclusively in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eyunnanensis\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e. Additionally, gene overlaps of 136 bp between \u003cem\u003eycf1\u003c/em\u003e and \u003cem\u003endhF\u003c/em\u003e in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eyunnanensis\u003c/em\u003e, and 127 bp between \u003cem\u003erps15\u003c/em\u003e and \u003cem\u003eycf1\u003c/em\u003e in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e, were identified; such structural configurations are extremely rare in chloroplast genomes [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The pronounced interspecific variation in IR boundary characteristics provides novel molecular targets for \u003cem\u003eAmorphophallus\u003c/em\u003e species identification and may enhance resolution where traditional DNA barcoding approaches remain insufficient among closely related species.\u003c/p\u003e \u003cp\u003eIntegrating Pi and selection pressure (Ka/Ks) analyses revealed a highly consistent evolutionary pattern in the chloroplast genome of \u003cem\u003eAmorphophallus\u003c/em\u003e, characterized by a coordinated distribution of hypervariable regions and signatures of positive selection. Pi analysis identified the LSC region as the primary reservoir of variation, with the \u003cem\u003eaccD\u003c/em\u003e gene and the \u003cem\u003epetD\u003c/em\u003e_2\u0026ndash;\u003cem\u003erpoA\u003c/em\u003e intergenic spacer exhibiting exceptionally high polymorphism, thereby representing promising targets for molecular marker development. In contrast, the SSC region remains generally conserved, as only \u003cem\u003endhF\u003c/em\u003e and \u003cem\u003endhD\u003c/em\u003e genes and the \u003cem\u003endhD\u003c/em\u003e\u0026ndash;\u003cem\u003epsaC\u003c/em\u003e intergenic spacer demonstrate moderate levels of variation. Ka/Ks analysis further elucidated variation in selection pressure among genes, indicating that the vast majority of genes were under purifying selection, consistent with the highly conserved nature of chloroplast genomes [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Nevertheless, in comparisons involving \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esp.\u003c/em\u003e, several genes\u0026mdash;including \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003erps3\u003c/em\u003e, \u003cem\u003eclpP\u003c/em\u003e, and \u003cem\u003erpl14\u003c/em\u003e\u0026mdash;exhibited strong signals of positive selection (Ka/Ks\u0026thinsp;\u0026gt;\u0026thinsp;1). Notably, \u003cem\u003eycf1\u003c/em\u003e, which also constitutes a major variable locus within the IR region based on Pi analysis, may play a crucial role in the adaptive evolution of \u003cem\u003eAmorphophallus\u003c/em\u003e across diverse environmental conditions [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe evolutionary pattern of the \u003cem\u003eAmorphophallus\u003c/em\u003e chloroplast genome reflects a strong integration of structural conservation and localized variability. Similarity analysis demonstrates that overall gene order remains highly conserved, whereas \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003eycf2\u003c/em\u003e, and members of the \u003cem\u003endh\u003c/em\u003e gene family constitute the principal variable loci. Despite their high variability, these genes are subject to purifying selection, with \u003cem\u003eycf1\u003c/em\u003e and \u003cem\u003eycf2\u003c/em\u003e exhibiting Ka/Ks ratios of 0.5\u0026ndash;0.8, indicative of weak purifying selection, and ndh family genes displaying Ka/Ks ratios of 0.3\u0026ndash;0.6, consistent with moderate purifying selection. Conversely, variable genes located in the LSC region are under stronger purifying selection (Ka/Ks\u0026thinsp;=\u0026thinsp;0.2\u0026ndash;0.4). Collectively, these findings suggest that, within an overall conserved genomic framework, the \u003cem\u003eAmorphophallus\u003c/em\u003e chloroplast genome responds to environmental adaptation through weak purifying selection on specific genes, particularly \u003cem\u003eycf1\u003c/em\u003e. This pattern provides precise targets for molecular marker development and phylogenetic studies within the genus [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDuring long-term evolution, plants have developed diverse codon usage patterns and base composition biases in response to different growth environments. In this study, chloroplast genome data from 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species were screened and characterized to determine base composition and codon usage patterns across 50\u0026ndash;52 protein-coding genes. The results indicate that base composition is similar across \u003cem\u003eAmorphophallus\u003c/em\u003e species, with a GC1\u0026thinsp;\u0026gt;\u0026thinsp;GC2\u0026thinsp;\u0026gt;\u0026thinsp;GC3 distribution pattern. In addition, the overall GC content and the GC content at all three codon positions remain below 50%, reflecting a bias toward A/U bases and a preference for A/U-ending codons within this genus. RSCU, defined as the ratio of observed to expected codon frequency, serves as a standard metric for evaluating codon usage bias across different organisms. Analysis of RSCU values demonstrates that high-frequency codons in all examined \u003cem\u003eAmorphophallus\u003c/em\u003e species predominantly end in A/T, whereas low-frequency codons tend to terminate in G/C. This finding is consistent with previous reports on species within Monsteroideae and further supports the relatively conserved nature of codon usage patterns in the chloroplast genomes of \u003cem\u003eAmorphophallus\u003c/em\u003e [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eENC reflects the degree of deviation from random codon usage and typically ranges from 20 to 61. An ENC value of 20 indicates exclusive usage of a single codon per amino acid, representing maximal codon bias. In contrast, an ENC value of 61 corresponds to random codon usage with no detectable bias [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Genes with ENC values\u0026thinsp;\u0026le;\u0026thinsp;35 demonstrate strong codon usage bias, while those with higher values exhibit weak bias. Across the 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species, the average ENC values for CDS ranged from 46.3 to 47.4, all exceeding 45, indicating a generally weak codon usage bias. This pattern is consistent with previous reports on chloroplast genomes of numerous Araceae species [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Moreover, correlation analysis across the 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species revealed no significant correlation between GC3 and either GC1 or GC2, whereas GC3 exhibited a significant or highly significant positive correlation with ENC. These results indicate that base composition at the third codon position differs substantially from that at the first and second positions, and that codon usage bias is closely associated with base composition at the third codon position, which may influence functional gene expression.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that mutation pressure and natural selection are primary factors shaping species-specific codon usage bias, and ENC serves as an important metric for assessing the relative contributions of these two factors to the non-uniform usage of synonymous codons [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this study, ENC-plot analyses of CDS across the 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species revealed similar patterns, with the vast majority of genes positioned below the standard curve. This pattern reflects a substantial deviation between observed and expected ENC values, indicating that codon usage bias in these species is predominantly driven by natural selection. Conversely, only a subset of genes is influenced by mutation pressure. PR2-plot analysis further revealed bias in base usage at the third codon position, characterized by an overall frequency pattern of T\u0026thinsp;\u0026gt;\u0026thinsp;A and G\u0026thinsp;\u0026gt;\u0026thinsp;C. These results indicate that codon usage bias in \u003cem\u003eAmorphophallus\u003c/em\u003e is jointly shaped by natural selection and mutation pressure, with natural selection exerting a more pronounced influence [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNeutrality plot analysis revealed marked variation in the distribution of GC12 versus GC3 among genes with different functional categories across 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species. Core genes involved in transcription and translation, including those encoding ribosomal proteins and RNA polymerase, exhibited substantial deviation from the diagonal line, indicating that their codon usage bias is primarily driven by translational selection. This observation is consistent with established theoretical frameworks, which propose that highly expressed genes preferentially utilize optimized codons corresponding to high-abundance tRNAs to enhance translational efficiency and accuracy, and that ribosomal protein genes, as typical housekeeping genes, have undergone strong translational optimization during evolution [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. In contrast, loci corresponding to NADH dehydrogenase-related genes were distributed near the diagonal, indicating an intermediate position between strict deviation and complete neutrality. This pattern suggests that their codon usage bias is more strongly influenced by mutation pressure, with a comparatively weaker contribution from translational selection. Such disparity may be associated with variation in gene expression levels and genomic localization [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIdentifying optimal codons is essential for designing gene expression vectors and enhancing target gene expression. In this study, 15\u0026ndash;21 optimal codons were identified across 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species. Among these, six optimal codons were shared by all representative species, namely UUA (Leu), CGU (Arg), AAA (Lys), GUA (Val), ACU (Thr), and AGU (Ser), and are highly conserved, underscoring their universal and functionally significant role in translational optimization. The evolution of species-specific optimal codons in certain taxa further highlights the combined influence of natural selection and habitat adaptation. For instance, in the widely cultivated \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e, unique optimal codons\u0026mdash;including CUU and CUA (encoding Leu, which is abundant in transmembrane and receptor proteins) and UCU (encoding Ser, involved in photorespiration and cold-responsive signaling)\u0026mdash;may reflect fine-tuned optimization of translational efficiency for relevant functional proteins during adaptation to complex habitats characterized by broad temperature fluctuations, as well as during specialized secondary metabolic processes such as substantial glucomannan synthesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In summary, the conservation and divergence of specific optimal codons result from the long-term coevolution between AT-biased mutational pressure and translational selection within distinct ecological niches, providing important molecular evidence for elucidating habitat-adaptation mechanisms in \u003cem\u003eAmorphophallus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eBased on an 800 bp sliding window analysis, the 80,401\u0026ndash;81,200 bp interval of the \u003cem\u003eAmorphophallus\u003c/em\u003e chloroplast genome was identified as the optimal DNA barcoding region. This region exhibits structural polymorphism dominated by deletion variants, combined with SNP patterns, enabling precise discrimination of all 16 examined species, including lineages characterized by extremely low and high genetic variation, using only five diagnostic loci. Consequently, it represents the highest-resolution molecular identification hotspot currently recognized within the genus [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Furthermore, the DNA barcoding region provides an effective target for developing low-cost, high-throughput species-specific markers, such as amplicon sequencing and quantitative polymerase chain reaction-based genotyping. When combined with the highly conserved nature of chloroplast RNA editing in \u003cem\u003eAmorphophallus\u003c/em\u003e (limited exclusively to the C\u0026rarr;U type), these findings further underscore a distinctive genomic architecture within the genus, wherein a conserved RNA editing mechanism coexists with a hypervariable DNA barcode region [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePhylogenetic analysis is widely used to elucidate evolutionary relationships among different species. To further resolve the evolutionary relationships among 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species and determine their phylogenetic placement within Araceae, a phylogenetic tree was constructed using 61 taxa based on the ML method, with \u003cem\u003eZ\u003c/em\u003e. \u003cem\u003emays\u003c/em\u003e designated as the outgroup. The results indicated that the 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species form a well-supported monophyletic group (bootstrap\u0026thinsp;=\u0026thinsp;100%). Subsequent analysis resolved the 16 species into 3 distinct clades: CA-II (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ealbus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekrausei\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekachinensis\u003c/em\u003e); SEA (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehewittii\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ependulus\u003c/em\u003e); CA-I (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecoaetaneus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etonkinensis\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eyunnanensis\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emuelleri\u003c/em\u003e). These findings are consistent with previous studies. Within Araceae, \u003cem\u003eAmorphophallus\u003c/em\u003e exhibits closer phylogenetic affinity with \u003cem\u003eCaladium\u003c/em\u003e, \u003cem\u003eZomicarpella\u003c/em\u003e, \u003cem\u003eXanthosoma\u003c/em\u003e, and \u003cem\u003eSyngonium\u003c/em\u003e than with other genera.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, chloroplast genomes from six rare \u003cem\u003eAmorphophallu\u003c/em\u003es species were sequenced. These data were integrated with previously reported genomes of ten \u003cem\u003eAmorphophallus\u003c/em\u003e species to characterize genomic structural features, codon usage bias, and phylogenetic relationships. The results revealed that chloroplast genome sizes among the 16 species ranged from 164,417 to 177,076 bp, with gene numbers varying from 126 to 131. All genomes exhibit a conserved quadripartite structure comprising LSC, SSC, IRa, and IRb regions, although notable variation exists in the size of each region across species. The number of SSRs ranged from 87 to 162, with mononucleotide repeats representing the most abundant type and exhibiting a base composition biased toward A/T usage. Among LSR, reverse and forward repeats were the most prevalent types. IR boundary regions of the chloroplast genomes displayed evident contraction and expansion across the 16 species. Overall, chloroplast genome characteristics and codon usage patterns were similar among \u003cem\u003eAmorphophallus\u003c/em\u003e species, although not identical. The GC content of all 16 species was below 50%. Six shared optimal codons, namely UUA, CGU, AAA, GUA, ACU, and AGU, were identified across the genus. Analyses including ENC-plot, neutrality plot, and PR2-plot indicated that codon usage bias across all species was more strongly influenced by natural selection than by mutation pressure. An 800 bp sliding window analysis identified the 80,401\u0026ndash;81,200 bp interval as the optimal DNA barcoding region for the 16 species. Phylogenetic analysis further demonstrated that among the newly reported species, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e belong to the CA-II clade, whereas \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehewittii\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ependulus\u003c/em\u003e cluster within the SEA clade. These findings enrich the genetic resources available for \u003cem\u003eAmorphophallus\u003c/em\u003e species and provide a valuable reference for species authentication and molecular-level determination of species origin.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Yunnan Provincial Science and Technology Department (grant no. 202449CE340009, 202503AP140005, 202401BA070001-001), Science and Technology Projects of Yunnan Universities Serving Key Industries (grant No.\u0026nbsp;FWCY-QYCT2025017),\u0026nbsp;NSFC (grant no. 32560733), Yunnan Province Yu Lei Expert Grassroots Research Workstation.\u0026nbsp;Yunnan Province University Student Innovation and Entrepreneurship Training Program Project (S202511393064).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chloroplast genome sequences of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehewittii\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ependulus\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitaniumi\u0026nbsp;\u003c/em\u003ehave been deposited in the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/nuccore/?term=) under the accession numbers PZ226531, PZ243066, PZ235491, PZ250735, PZ266516, and PZ266517, respectively. The raw sequence data reported in this paper have been deposited in the NCBI SRA (https://www.ncbi.nlm.nih.gov/sra/?term=) under BioSample accession numbers SAMN55469039, SAMN55971199, SAMN55975456, SAMN55872069, SAMN55958012, and SAMN55933366; BioProject accession numbers PRJNA1425632, PRJNA1427793, PRJNA1428900, PRJNA1426491, PRJNA1428559, and PRJNA1427781; and SRA accession numbers SRR37297627, SRR37374433, SRR37379527, SRR37351907, SRR37369025, and SRR37355529, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaoliang Shi, Lifang Li, Lei Yu and Qinghu Lu conceived and designed the project. Ying Qi, and Min Yang performed the bioinformatics analysis. Meiwei Zhao, Penghua Gao, Feiyan Huang and Jianrong Zhao performed the experiments. Haoliang Shi wrote the manuscript. Lifang Li revised the manuscript. Lei Yu supervised the manuscript. All authors read and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGolenberg EM, Clegg MT, Durbin ML, Doebley J, Ma DP. Evolution of a noncoding region of the chloroplast genome. MOL PHYLOGENET EVOL. 1993;2(1):52\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartin W, Rujan T, Richly E, Hansen A, Cornelsen S, Lins T, Leister D, Stoebe B, Hasegawa M, Penny D. Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. 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FRONT GENET. 2021;12:757109.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Amorphophallus, chloroplast genome, gene structure analysis, codon usage bias analysis, phylogenetic analysis","lastPublishedDoi":"10.21203/rs.3.rs-9564592/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9564592/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAmorphophallus\u003c/em\u003e 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 \u003cem\u003eAmorphophallus\u003c/em\u003e chloroplast genomes through comparative genomic analysis and to clarify their phylogenetic placement.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe chloroplast genomes of six \u003cem\u003eAmorphophallus\u003c/em\u003e species (\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ependulus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etitanum\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ehewittii\u003c/em\u003e) 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 \u003cem\u003eAmorphophallus\u003c/em\u003e species ranged from 152,492 to 185,810 bp and exhibited a typical quadripartite structure. Inverted repeat (IR) region expansion was detected in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003emacrophyllus\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eglaucophyllus\u003c/em\u003e, whereas IR contraction was identified in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003efornicatus\u003c/em\u003e. The gene \u003cem\u003eaccD\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;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\u0026rarr;U base editing events were identified in the chloroplast genomes of the 16 \u003cem\u003eAmorphophallus\u003c/em\u003e species. The genomic region spanning 80,401\u0026ndash;81,200 bp was identified as an optimal DNA barcoding window, enabling discrimination of all 16 species using five diagnostic loci. Phylogenetic analysis strongly supported \u003cem\u003eAmorphophallus\u003c/em\u003e as a monophyletic group (bootstrap support\u0026thinsp;=\u0026thinsp;100%), which was further divided into three major clades: Continental Asia I, Continental Asia II, and Southeast Asia. Within Araceae, \u003cem\u003eAmorphophallus\u003c/em\u003e occupies a crown group position and forms a sister group relationship with \u003cem\u003eSyngonium\u003c/em\u003e, \u003cem\u003eXanthosoma\u003c/em\u003e, \u003cem\u003eZomicarpella\u003c/em\u003e, and \u003cem\u003eCaladium\u003c/em\u003e, whereas \u003cem\u003eSymplocarpus\u003c/em\u003e is resolved as a basal lineage.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study systematically elucidates the patterns of structural variation and evolutionary features of \u003cem\u003eAmorphophallus\u003c/em\u003e 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.\u003c/p\u003e","manuscriptTitle":"Comparative chloroplast genomics in Amorphophallus: Revealing codon usage patterns and phylogenetic clades across 16 species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-18 11:57:51","doi":"10.21203/rs.3.rs-9564592/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"234868350587022537765224673948456023532","date":"2026-05-15T13:01:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"243078736417524217710191560954478096086","date":"2026-05-10T10:57:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-08T07:50:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-08T07:45:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-05-08T07:27:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-07T16:25:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-05-07T13:25:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"de2c0fd4-75d8-4682-b08d-3f4befd34f91","owner":[],"postedDate":"May 18th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"234868350587022537765224673948456023532","date":"2026-05-15T13:01:52+00:00","index":35,"fulltext":""},{"type":"reviewerAgreed","content":"243078736417524217710191560954478096086","date":"2026-05-10T10:57:37+00:00","index":33,"fulltext":""},{"type":"reviewersInvited","content":"14","date":"2026-05-08T07:50:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-08T07:45:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-05-08T07:27:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-07T16:25:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-05-07T13:25:12+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T11:57:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-18 11:57:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9564592","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9564592","identity":"rs-9564592","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}