Mitochondrial DNA (mitogenomes) evolve independently from nuclear genomes, providing valuable insight into the evolutionary history of their host species (Seidl, 2024). Plant mitochondria can significantly influence various biological functions, including stress tolerance, growth vigor, and cytoplasmic male sterility (Guo et al., 2016; Kianian, 2016). To date, a total of 688 plant mitogenomes have been reported, the number less than the chloroplast genomes (n = 12,989) and plastid genomes (n = 1,718) (November 18, 2025, https://www.ncbi.nlm.nih.gov/datasets/organelle/). Plant mitochondria are recognized for their distinctive semi-autonomous genetic properties, including gene sequence transfer or loss, multiple RNA editing modifications, and multipartite genome arrangements (Handa, 2003; Broz, 2022a; Song et al., 2024). Among them, frequent recombination of repetitive sequences is a common feature in mitochondrial genomes, and extensive endosymbiotic gene transfer events regularly occur from chloroplasts to mitochondria (Keeling and Palmer, 2008; Bock, 2010; Broz, 2022a; Broz, 2022b). These processes drive plant mitogenome expansion and accelerated evolution (Prasad et al., 2022). Moreover, RNA editing events are prevalent in plant mitochondrial transcripts, and are essential for the generation of protein function and adaptive evolution (Ichinose and Sugita, 2017; Broz, 2022a). The frequency and type of these events are phylogenetically constrained among congeneric species, making them informative for elucidating evolutionary relationships (Zhang et al., 2024). In conclusion, plant mitogenomes have emerged as vital tool for species classification, evolutionary studies, and parentage tracing.

Artocarpus heterophyllus, commonly known as jackfruit, is an exotic species native to the Western Ghats of India (Prakash et al., 2009; Baliga et al., 2011; Almeida et al., 2022). It is widely cultivated the tropical regions, and serves as an important tropical commercial crop in Hainan Province of China (Kumar et al., 2022; Zhao et al., 2023b; Zhao et al., 2023a). Recent studies have demonstrated that Artocarpus species are valuable sources of enzyme inhibitors, antioxidants, resveratrol, cosmeceuticals, and other bioactive compounds (Borah et al., 2017; Liyanaarachchi et al., 2018; Liyanaararchchi et al., 2022). The genus Artocarpus (Moraceae) comprises more than 70 recognized species with taxonomically diverse members (Gardner et al., 2021). A. heterophyllus, A. heterophyllus (R) and A. integer are the three major varieties primarily cultivated in Hainan, with an annual output value of up to 2 billion. A. heterophyllus is a treasure trace of medicinal potential, as its fruits, bark, leaves, and roots can be used in the treatment of various conditions, such as anemia, asthma, dermatosis, diarrhea, and cough (Muyonga and Nansereko, 2021; Baliga et al., 2011; Kumar and Mishra, 2022). A. heterophyllus (R), a red-fleshed jackfruit, is a new dry-bulb cultivar selected from natural seedlings of Thai jackfruit with higher economic value than conventional varieties (Zehuai et al., 2012). In addition, chempedak (Artocarpus integer Merr), known as Champada in Thai, is indigenous to southeast Asia. The fruit is morphologically similar to A. heterophyllus, though smaller in size and characterized by a softer texture. Upon ripening, it exhibits a flavor profile reminiscent of durian, accompanied by a potent and distinctive aroma (Buttara et al., 2014). Given their substantial economic and medical significance, it is imperative to investigate their evolutionary history. Currently, comparative studies of A. heterophyllus focus solely on chloroplast (cp) genomics (Liu et al., 2018), and nuclear genomes (Lin X et al., 2022). However, there have been no reports on the mitochondrial genomics of Artocarpus species. Research demonstrates that plant mitochondrial genomes have significant potential for the development of molecular markers and taxonomic classification, serving as a key resource in advancing research on plant population genetics and evolutionary biology (Wang et al., 2024). Furthermore, comparative analysis of mitochondrial genomes across several species within the same genus enables a comprehensive understanding of their structural organization and genomic diversity, offering valuable insights into mitogenome evolution at the genus level (Soe et al., 2025).

Until now, only five mitochondrial genomes from species within the Moraceae family have been successfully assembled and deposited in the National Center for Biotechnology Information (NCBI) database (as of November 7, 2025), including those of Ficus carica, Morus notabilis, R. laevigata, R. hybrid cultivar, and R. chinensis. Notably, no Artocarpus mitochondrial genomes have been published accessible, that hampers our understanding of its phylogenetic classification as well as population identification. In this study, we first sequenced and assembled the complete mitogenomes of three Artocarpus species and further analyze their structural characteristics, including gene arrangement, repeat sequences, codon preferences, Ka/Ks value, gene loss, phylogenetic position, sequence transfer between chloroplast and mitochondrial DNA, and RNA editing sites. This research will enhance our understanding of the complex evolutionary features of Artocarpus species, and lay a theoretical foundation for their evolution.

Species within the Artocarpus genus, including A. integer, A. heterophyllus, and A. heterophyllus (R), possess significant edible and medicinal value (Gupta et al., 2023). These species are extensively cultivated on Hainan Island, contributing substantially to the local economy. The assembly and analysis of plastomes are essential for advancing plant breeding programs and other agronomic purposes (Kan et al., 2024). Plant mitochondrial DNA is crucial as organelle DNA exhibits a high degree of conservation and evolves at a distinct rate compared to nuclear genes, rendering it an invaluable molecular marker for studying evolution and molecular ecology (Zubaer et al., 2018). In the NCBI database, a limited number of mitochondrial DNA sequence have been documented in the Moraceae family (Liangliang, 2022; Wei et al., 2023). It is noteworthy that no reports currently exist on the mitochondrial genome of Artocarpus plants. Which has hindered our understanding of the mitochondrial genome structure with the Artocarpus genus and impeded research into its evolutionary relationships and molecular breeding.

Compared to animal mitochondrial DNA, plant mitochondrial DNA exhibit greater variability and demonstrates rich diversity in terms of size (ranging from 200 kb to 11 Mb), structure, and gene content across different species, and even within the same species (Barr et al., 2005; Davila et al., 2011; Sloan et al., 2012; Skippington et al., 2015). In this study, we successfully assembled the first complete mitochondrial of Artocarpus species using high-quality HiFi reads produced by PacBio technology. The three mitochondrial DNA (mtDNA) sequences contained six cotings and showed highly similarity in mitochondrial genomic size, with only minor base pair differences. Furthermore, all sequence exhibited a GC content of 44.9%, which aligned with that of certain species within the Moraceae family, such as F. carica (45.45%) (Wei et al., 2023); and M. alba (45.50%) (Liangliang, 2022). The Artocarpus species show notable similarity in mtDNA size and structure, in contrast to the greater variability observed in other closely related genera, such as Mangifera and Crataegus (Niu et al., 2022; Zhang et al., 2024), suggesting that Artocarpus species may exhibit relative conservation in organelle evolution. Additionally, we assembled three complete chloroplast DNA sequences (Supplementary Figures S5, Supplementary Figures S6), which also displayed high structural similarity (Supplementary Table S6). This finding is consistent with previous conclusions regarding the chloroplast genome of Artocarpus camansi (Souza et al., 2020). In conclusion, these results indicated that the organelle genomes of Artocarpus species remain highly conserved throughout breeding and evolutionary processes.

Repetitive sequences play a crucial role in promoting genetic recombination in the mitochondrial DNA of seed plants, as they can alter genome size and induce structural variations (Mackenzie, 1999; Chen et al., 2017). These sequences serve as valuable genetic markers in population analysis, thereby facilitating research on species evolution (Provan, 1996; Pfeifer et al., 2013; Li et al., 2022). In this study, we found that three species exhibit a high degree of similarity in their repetitive sequences. A total of 166 SSRs were identified in each mitogenome, which exceeds the number of SSRs found in the chloroplast genome (Souza et al., 2020). Furthermore, mononucleotide A/T repeats were the most frequent, consistent with findings in Punica granatum (Feng et al., 2023), and comparable to those in A. camansi and A. heterophyllus of plastid genomes (Souza et al., 2020). The mitochondrial genome of Artocarpus contains up to 308 dispersed repeats exceeding 30 base pairs, as well as a large repeat region greater than 8,000 base pairs. Extended repetitive sequences can promote genomic recombination, leading to structural modifications (Gualberto et al., 2014). Furthermore, 17 tandem repeats were identified, which differ from those found in F. carica and M. alba (Liangliang, 2022; Wei et al., 2023).

Homologous fragment transfer occurs among the nucleus, mitochondria, and chloroplasts. Frequent DNA transfers are estimated to have taken place in the common ancestor of gymnosperms and angiosperms approximately 300 million years ago (Timmis et al., 2004; Wang et al., 2007). Understanding this transfer relationship is essential for explaining the evolution of plant mitogenomes (Van Binh et al., 2020). In this study, the PCGs of psaA, PsaB, rps7 and ndhB were identified as completely transferred sequences between mtDNA and cpDNA of three Artocarpus species. The psaA, rps7 and ndhB genes were also transferred between organelle genome of Hibiscus cannabinus (Moghaddam et al., 2023).These genes play a crucial role in chloroplast functionality (Shikanai et al., 1998; Horvath et al., 2000; Grotjohann and Fromme, 2005).Their presence in the mitochondrial genome most likely represents chloroplast-derived insertions rather than functional mitochondrial genes.

Codon usage bias (CUB) is crucial for investigating the origins of species and their genetic differentiation (Athey et al., 2017). An RSCU value greater than 1 indicates a higher frequency of codon usage, while a value less than 1 suggests lower codon usage frequency (Parvathy et al., 2022). In the genomes of the three Artocarpus species, 4,978 codons exhibit an RSCU value above 1, with the third codon position predominantly being either A or U, that is a common feature in terrestrial plant mitochondrial DNA (Fang et al., 2022). Both neutrality plot and ENC-plot analyses indicate that the three Artocarpus species are influenced by both mutation and natural selection. However, the influence of natural selection is more dominant, similar to the findings in Angelica biserrata and Rhingia Scopoli (Wang et al., 2024; Zhao et al., 2024).

The Ka/Ks ratio is frequently employed to assess the selective pressure acting on PCGs, which determines the effects of environmental stress on the evolution of the mitochondrial genome (Qiao et al., 2022). Our results indicated that most PCGs have a Ka/Ks ratio of less than 1, suggesting that the majority of protein-coding genes have undergone purifying selection during evolution, leading to relatively stable protein functions (Zeng et al., 2024). Among these, four genes (ccmB, ccmC, ccmFc, and nad4L) exhibited positive selection in Artocarpus species. CcmB has frequently been identified as a positively selected gene in various plants, including F. carica, Ilex metabaptista, Astragalus membranaceus, Thuja sutchuenensis, Calophyllum soulattri and Phaseolus vulgaris (Bi et al., 2020; Cheng et al., 2021; Xia et al., 2023; Zhou et al., 2023; Cadorna et al., 2024; Zhang et al., 2024). Both ccmB and nad4L also function as positive selection genes in Hippophae tibetana and Diospyros kaki (Yang and Duan, 2024; Zeng et al., 2024). CcmB gene plays a significant role in helping plants resist stress and adapt to their environment (Xiong et al., 2017; Xu et al., 2023; Dai et al., 2024). Specially, genes ccmB, ccmC, and ccmFc are components of the mitochondrial cytochrome c maturation (CCM) complex (Brausemann et al., 2021), they plays a significant role in the repeated evolution of various species (Huang et al., 2024). These positive genes may play a crucial role in adaptation of Artocarpus species to tropical environments in Hainan province.

The arrangement of homologous regions has been widely utilized to elucidate the phylogenetic relationships among species (Mower et al., 2012; Zhang et al., 2023). In our study, the collinear blocks involving three Artocarpus species were observed to be the longest among all identified blocks. This suggests that more closely related species tend to exhibit elongated collinear regions. Concurrently, we noted distinct differences in the arrangement of these collinear blocks between Artocarpus and other species. This indicates that the mitochondrial genome of the three Artocarpus species have undergone extensive rearrangements compared to their close relatives, showcasing a high degree of structural variability.

In order to establish the phylogenetic position of Artocarpus species based on mt genome, we constructed a phylogenetic analysis utilizing 8 shared PCGs from 88 species. Results indicated that the three Artocarpus species are closely related, with the closest phylogenetic relationship observed between these species and M. notabilis (MK301435). This finding is consistent with previous classification results based on analyses of the ndhF gene, ITS sequences, and chloroplast DNA (Souza et al., 2020). Furthermore, the three Artocarpus species exhibit a close phylogenetic relationship with F. carica. However, this result is inconsistent with evolutionary relationship constructed using chloroplast genome (Liu et al., 2018; Souza et al., 2020). The limited availability of mitochondrial DNA sequences within the Moraceae family currently restricts the scope of our study. More mitochondrial DNA sequences from the Moraceae family should be research, particularly for the genus Artocarpus species.

Plant organelle gene expression correlates with a variety of post-transcriptional nucleic acid modifications, among which RNA editing is particularly significant (Castandet and Araya, 2011). Ancient RNA editing factors originated early in the evolutionary history of flowering plants, aiding researchers in tracing the evolutionary trajectory of plants (Hein and Knoop, 2018). In this research, three Artocarpus species exhibit a high degree of similarity in mitochondrial characteristics, their RNA editing analysis shows distinct differences. Notably, A. integer displays a markedly lower volume of RNA edits compared to the other two species. As plant evolution progresses, RNA editing events tend to diminish, with ancestral lineages typically exhibiting high editing rates in seed plants (Chen et al., 2011). Notably, a gradual reduction in cellular RNA editing rates also observed in evolution of angiosperms (Small et al., 2020). Therefore, in evolutionary terms, the lower number of RNA editing sites of A. integer may be a more derived position relative to A. heterophyllus and A. heterophyllus (R). In addition, C-T (U) types of RNA editing sites were dominant in single-base editing in this study, this finding that aligns with results from other plant mitochondrial genomes (Wang et al., 2024). The proportion of C-T (U) types varied among the three Artocarpus species, accounting for 79.73% in A. integer, 90.43% in A. heterophyllus (R), and 95.47% in A. heterophyllus. Correspondingly, A. integer, which had the fewest total editing sites, also exhibited the lowest proportion of C-to-U edits. In contrast, the number of reverse U(T)-to-C types in A. integer were significantly higher than other two Artocarpus species. On the other hand, RNA editing also has the capacity to alter amino acids, thereby modifying thus physical and chemical properties (Mower, 2009). Previously research indicated that a higher proportion of hydrophilic amino acids facilitates protein folding, whereas a reduced proportion contributes to enhanced protein stability (Wang et al., 2024). In our study, the majority of amino acids transitioned from hydrophilic to hydrophobic in three Artocarpus species similar to the patterns in F. carica, and M. alba (Liangliang, 2022; Wei et al., 2023). Additionally, A. integer exhibits a lower hydrophilic-to-hydrophobic ratio compared to the other two species, suggesting that its protein structure is more evolutionarily stable.

In this study, we successfully assembled and annotated the complete mitochondrial DNA of three economically significant tropical crops: A. integer, A. heterophyllus(R), and A. heterophyllus. This study represents the first report of the mitochondrial genome for the genus Artocarpus, further conducted a comprehensive investigation of various aspects based on the mitochondrial genome, including analysis of Ka/Ks ratios, repeat sequences, codon usage preference, RNA editing and evolutionary tree. That provide valuable insights for researchers to understand genetic characteristics, molecular differences, and taxonomic categorization of Artocarpus species, contribute to elucidating the evolutionary relationships for taxonomic studies within the Moraceae family.