Plastome of Dehaasia pugerensis Koord. & Valeton: A Critically Endangered Lauraceae Species

Aulia Hasan Widjaya^1,2Andi Salamah^2*Tety Maryenti²Iyan Robiansyah¹Weibang SUN³Mahat Magandhi¹Irfan Martiansyah¹Muhammad Rifqi Hariri¹Aditya Nugroho¹

• ¹National Research and Innovation Agency (BRIN), Bogor, Indonesia
• ²Department of Biology, Faculty of Mathematics and Natural Sciences, University of Indonesia, Depok, West Java, Indonesia
• ³Kunming Institute of Botany, Chinese Academy of Sciences (CAS), Kunming, Yunnan, China

Dehaasia pugerensis Koord. & Valeton is an endemic Indonesian species restricted to Jember Regency, East Java, particularly in the Gunung Watangan area (Bijmoer et al., 2020). This species belongs to the Lauraceae family and has been classified as Critically Endangered (CR) on the IUCN Red List based on criteria B1ab (i, ii, iii, iv, v) and C2a (i) (Helmanto et al., 2022). Its distribution is highly limited, occurring at elevations between 61 and 391 meters above sea level, and its population is threatened by habitat degradation and overexploitation, particularly for charcoal production (Helmanto et al., 2022). These anthropogenic pressures have led to a significant population decline, highlighting the urgent need for science-based conservation efforts, including genomic studies as a foundation for sustainable management of rare species.One of the key approaches in plant genomics is the characterization of the chloroplast genome. The chloroplast genome in land plants typically exhibits a conserved circular structure, consisting of four major regions: the large single-copy (LSC), the small single-copy (SSC), and two inverted repeat (IR) regions. The total chloroplast genome size in land plants ranges from approximately 120 to 200 kb, with IR regions typically spanning 20-26 kb (Xiao-Ming et al., 2017). In recent decades, advances in sequencing technologies have enabled faster and more accurate assembly of chloroplast genomes. These developments have opened new opportunities to explore the structure, variation, and evolution of chloroplast genomes across plant species, contributing to phylogenetic studies and the conservation of rare genetic resources (An et al., 2022). Genomic DNA was extracted from plant leaves using the cetyltrimethylammonium bromide (CTAB) method as described by Doyle and Doyle (1987). The initial DNA concentration and purity were assessed using a Nanodrop 2000 spectrophotometer (Thermo Scientific, MA, USA). DNA integrity was visualized by agarose gel electrophoresis, and quantification was performed with the Qubit dsDNA HS Assay Kit (Thermo Scientific, MA, USA). Further evaluation of DNA integrity was conducted using the 4150 TapeStation system (Agilent Technologies, CA, USA). High-quality genomic DNA was then utilized for library preparation. Sequencing was carried out on the Illumina NextSeq 2000 platform (Genetika Science Lab, Tangerang, Indonesia) employing a paired-end 150 bp strategy, targeting a total data output of 10 Gb. The reads were assessed for quality using FASTQC software version 0.11.8 (Andrews 2010). Filtering and trimming were performed using Trimmomatic version 0.39 to remove low-quality bases (less than 30), adapters, nucleotide position bias at the 3' and 5' ends, and sequence contamination.The parameters applied included TruSeq3-PE.fa:2:30:10, SLIDINGWINDOW:4:28, LEADING:28, TRAILING:28, and MINLEN:20 (Bolger et al. 2014). The trimmed reads results were subsequently assembled utilizing GetOrganelle version 1.7.7.1 (Jin et al. 2020). The annotation of complete chloroplast genome of D. pugerensisreads was conducted utilizing CPGAVAS2 (http://47.96.249.172:16019/analyzer/annotate) (Shi et al. 2019;Lestari et al. 2024), with the cp genome of D. hainanensis (accession number: OP374101.1) serving as the reference. The annotation process faced challenges such as resolving ambiguous gene regions and validating gene boundaries, which were addressed via manual Formatted: Font: Italic verification using Unipro UGENEgene v. 45.1 (Okonechnikov et al. 2012) andNCBI Genomic Workbench v. 3.8.2 (Kuznetsov andBollin 2021). To ensure the cp genome sequence contained no N bases and had 21 amino acids, Unipro Ugene UGENEv. 45.1 was employed. Genes without a start codon were manually edited using the edit menu in NCBI Genomic Workbench v. 3.8.2. The circular genome visualization was performed using Organellar Genome DRAW (OGDRAW) accessed through MPI-MP Chlorobox (Greiner et al. 2019). Simple sequence repeats (SSRs) of D. pugerensis chloroplas genome were identified using the MIcroSAtellite (MISA) web tool (Beier et al. 2017). Search parameters were configured to detect perfect mono-, di-, tri-, tetra-, penta-, and hexa-nucleotide motifs, with minimum repeat thresholds of 810, 5, 35, 3, 3, and 3, respectively. Compound SSRs were allowed when two adjacent repeat motifs were separated by no more than 100 bp. Codon usage patterns and Relative Synonymous Codon Usage (RSCU) values were analyzed using MEGA X software (Kumar et al. 2018). Visualization of codon frequency distributions was subsequently performed using the "ggpubr" package in R version 4.2.3. The complete chloroplast genome of D. pugerensis spans 153,111 bp, exhibiting the typical quadripartite structure of angiosperms (Figure 1A), comprising a large single-copy (LSC) region of 93,852 bp, a small single-copy (SSC) region of 18,699 bp, and two inverted repeats (IRs) of 20,280 bp each. The GC content is 39.07%, consistent with other Lauraceae species. Genome annotation identified 128 functional genes, including 85 protein-coding genes, 8 rRNA genes, and 36 tRNA genes (Tables 1). Seven genes are duplicated in the IRs, and 16 genes contain introns, reflecting a level of structural and regulatory complexity typical of Lauraceae plastomes. genomic complexity and regulatory diversity.The conserved structure of the plastome provides a robust molecular framework for phylogenetic resolution within Lauraceae, where morphological convergence often obscures species boundaries (Liu et al. 2017;Tian et al. 2021). Duplicated genes and intron-rich regions offer potential molecular markers for evaluating genetic diversity, population structure, and gene flow (Han et al. 2022). This is critical for D. pugerensis, a narrowly endemic and critically endangered species, as chloroplast genomic data inform historical biogeography, demographic shifts, and conservation prioritization (Crawford & Stuessy 2016).The chloroplast genome of D.ehaasia pugerensis harbors 11 genes with cis-splicing introns, where exons and introns reside on the same transcript (Figure 1B). These include rps16, atpF, rpoC1, ycf3, clpP, petB, petD, rpl2, ndhA, and ndhB (the latter two located in IRs and thus duplicated). Gene structures, including exons (black) and introns (white), are annotated with genomic coordinates in the corresponding figure. Most genes, such as atpF and rpoC1, contain one intron, while ycf3 and clpP have two, indicating complex splicing regulation. Genes located in the IR regions, such as rpl2 and ndhB, appear twice in the genome.The presence of introns in genes like clpP and ycf3 is conserved among angiosperms and may reflect regulatory or evolutionary functions (Rogalski et al. 2015). The cis-splicing profile in D. pugerensis mirrors that of other Lauraceae species, indicating a conserved regulatory mechanism across the family (Song et al. 2017).The chloroplast genome of D.ehaasia pugerensis contains a single trans-splicing gene, rps12 (Figure 1C). Unlike cis-splicing, trans-splicing joins exons located in separate genomic regions. In D. pugerensis, rps12 is split into three exons: exon 1 resides in the LSC region, while exons 2 and 3 are duplicated in the IRs. These exons are spliced post-transcriptionally to form a functional mRNA. This complex splicing pattern is highly conserved in land plants and is essential for proper chloroplast gene expression. rps12 encodes a component of the small ribosomal subunit, critical for translation of chloroplast-encoded proteins. Accurate transsplicing is thus fundamental to chloroplast function and plant development, underscoring the evolutionary significance of maintaining structural and functional integrity in organelle genomes (Oldenburg & Bendich 2015). The stability of this arrangement also makes rps12 a reliable phylogenetic marker, offering taxonomic utility in resolving species relationships within Lauraceae. (Jacobs et al. 2010;Horiuchi & Aigaki, 2006) The chloroplast genome of D.ehaasia pugerensis contains 149 simple sequence repeats (SSRs), comprising 53 mononucleotide, 11 dinucleotide, 72 trinucleotide, 10 tetranucleotide, one pentanucleotide, and two hexanucleotide repeats. Trinucleotide repeats-particularly TTA and TAA-are most abundant, followed by A/T-rich mononucleotide repeats (Figure 1CD). In contrast, the plastome of D. hainanensis (NC_068504) is dominated by mononucleotide SSRs with relatively few trinucleotide repeats (n=10), reflecting interspecific variation in SSR profiles (Gao et al. 2018). Such patterns may indicate lineage-specific mutation rates or demographic processes such as historical bottlenecks or prolonged population isolation (Dobrogojski et al., 2020). The elevated proportion of trinucleotide SSRs in D. pugerensis may therefore signal unique evolutionary pressures acting on its small, fragmented populations, making these markers valuable for future population-genetic and conservation studies.Synonymous codon usage analysis in the D. pugerensis chloroplast genome provides insights into translational dynamics and selective pressures shaping plastome evolution. Relative Synonymous Codon Usage (RSCU) analysis revealed a distinct codon bias among proteincoding genes (Figure 4). AGA (arginine) showed the highest RSCU value (1.79), while CGG (arginine) had the lowest (0.35), indicating a strong preference among synonymous codons. Leucine was the most abundant amino acid, and tryptophan the least (Figure 1DE).A marked bias toward codons ending in A or U was observed, consistent with the AT-rich nature of plastid genomes. Most A/U-ending codons had RSCU > 1, enhancing translational efficiency, while codons such as CGA, GGU, and AGC deviated from this trend. AUG (methionine) and UGG (tryptophan) had neutral RSCU values (1.0), reflecting their lack of synonymous alternatives.Genomic analyses demonstrate that D. pugerensis shares strong plastome conservation with D. hainanensis, supporting the need for coordinated conservation strategies across their overlapping native range in East Java. Despite this similarity, distinct SSR profiles and codon usage signatures provide powerful genomic markers for species identification and for evaluating adaptive potential. Combined with ongoing conservation research-including population surveys, DNA barcoding using rbcL, matK, trnH-psbA, and ITS (Widjaya et al. 2025), SSR and ISSR-based genetic diversity assessments, and vegetative propagation-the plastome data Recent conservation efforts have resulted in the collection of 23 seeds and 15 seedlings of D. pugerensis, now cultivated at the Bogor Botanic Gardens. These ex situ collections serve as essential material for research, propagation trials, and public education (Williams et al. 2015;Westwood et al. 2021). They also support in situ recovery efforts by supplying seeds or planting stock for population reinforcement (Heywood 2017;Abeli et al. 2020). Field assessments by Helmanto et al. (2022) confirm the species' extremely restricted distribution and high vulnerability, underscoring the need to incorporate genomic evidence into conservation management to enhance long-term survival. psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ