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Ibrokhimjon Ergashov

https://orcid.org/0000-0002-0991-1076

Ibrokhimjon Ergashov

https://orcid.org/0000-0002-0991-1076

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Allium aktauense is a narrow endemic species from the Aktau Range in the Nurotau Mountains, Western Pamir-Alay, where it grows in rocky limestone crevices. Genomic resources for this species have not previously been available, limiting molecular understanding of its phylogenetic placement and conservation value. In this study, we sequenced, assembled and characterized the complete chloroplast genome of A. aktauense and evaluated its phylogenetic position within Allium using comparative plastome data. The plastome exhibited the typical circular quadripartite structure of angiosperms, with a total length of 152,443 bp, including a large single-copy region of 81,522 bp, a small single-copy region of 17,907 bp and two inverted repeat regions of 26,507 bp each. The overall genome organization, gene order and plastome structure were conserved, with no major rearrangements detected. The highest nucleotide diversity was observed in the ndhF–rpl32 region, suggesting its potential utility as a molecular marker for future studies. Phylogenetic analysis based on complete chloroplast genome sequences placed A. aktauense within subgenus Allium, close to A. ophiophyllum, with A. nikolaii and A. filidens forming a neighbouring lineage. These results provide the first plastome resource for A. aktauense and contribute to future phylogenetic, taxonomic and conservation-related studies of endemic Allium species in Central Asia.

The genus Allium L. is one of the largest and taxonomically most complex genera of monocotyledons, comprising more than 1000 species distributed mainly across temperate and seasonally dry regions of the Northern Hemisphere (Friesen et al., 2006, Li et al., 2010). The genus includes economically important vegetable crops, medicinal plants and ornamentals, and is traditionally recognized by bulbous or rhizomatous life forms, membranous to fibrous bulb tunics, free or nearly free tepals and a characteristic sulphur-containing odour. Southwestern and Central Asia, together with the Mediterranean region, represent one of the principal centers of diversity and diversification for Allium, whereas a second center occurs in western North America (Friesen et al., 2006; Nguyen et al., 2008; Li et al., 2010; Khassanov, 2018; Jang et al., 2023).

Central Asia is especially important for understanding the evolution of Allium, because the region harbours numerous narrowly distributed and locally endemic taxa associated with mountain systems, rocky slopes, gypsum- and limestone-rich substrates, and arid to semi-arid habitats (Khassanov and Yussupov 2022, Ergashov et al. 2025). Recent taxonomic, morphological and molecular studies have shown that species boundaries and infrageneric relationships in Allium are often difficult to resolve using morphology alone, owing to high morphological diversity, convergent ecological adaptations and incongruence between traditional sectional classifications and molecular phylogenies (Friesen et al., 2006; Li et al., 2010; Yusupov et al., 2022; Jang et al., 2023; Ergashov et al., 2025). In this context, complete plastome data provide an important complementary source of evidence for clarifying phylogenetic placement, identifying variable genomic regions and supporting taxonomic decisions.

Allium aktauense F.O.Khass. & Esankulov is a narrow endemic species described from the Aktau Range in the Western Pamir-Alay (Khassanov and Esankulov, 2015). The species was originally placed in Allium subgen. Allium sect. Minuta F.O.Khass., a small Middle Asian section characterized by diminutive plants, a compressed perianth and persistent spathe ( Figure 1) (Khassanov and Yussupov 2022). Its extremely restricted distribution and occurrence in rocky limestone crevices make it a valuable species for studies of local endemism, habitat specialization and conservation genetics in the Western Pamir-Alay (Beshko et al., 2025).

Chloroplast genomes have become widely used in plant systematics because of their generally conserved quadripartite structure, uniparental inheritance, moderate evolutionary rate and usefulness for reconstructing phylogenetic relationships at interspecific and infrageneric levels. In Allium, plastome-scale studies have improved understanding of phylogenetic relationships, divergence patterns, adaptive evolution and the utility of hypervariable regions as candidate molecular markers (Xie et al., 2020; Chen et al., 2022; Yang et al., 2023). Despite increasing plastome sampling across Allium, genomic information for many narrowly distributed Central Asian endemics remains limited. This is particularly true for species of small, geographically restricted sections such as sect. Minuta, for which molecular data are still insufficient to test morphology-based classifications and to evaluate relationships with other lineages of subg. Allium. Generating the complete chloroplast genome of A. aktauense is therefore important not only for documenting the genomic resources of a narrow endemic species, but also for improving comparative plastome sampling in Central Asian Allium.

In the present study, we sequenced, assembled and annotated the complete chloroplast genome of A. aktauense collected from its type region in the Aktau Range, Western Pamir-Alay, Uzbekistan. We aimed to: (1) characterize the overall structure, gene content and organization of the chloroplast genome; (2) identify highly variable regions that may be useful as molecular markers; (3) infer its phylogenetic position within the genus using complete chloroplast genome data. This study provides the first plastome resource for A. aktauense and contributes to ongoing efforts to clarify the evolution, taxonomy and conservation value of endemic Allium species of Central Asia.

Fresh leaf material of Allium aktauense was sampled in May 2025 from a natural population at the type locality on the northern slopes of Lyangar Pass, Aktau Range, Nurotau Mountains, Western Pamir-Alay, Uzbekistan (40°20′37.08″ N, 66°00′34.69″ E; Fig. 1). Species identification was carried out by Ibrokhimjon Ergashov at the National Herbarium of Uzbekistan, and representative voucher specimens were deposited in the National Herbarium of Uzbekistan (TASH) under voucher accession number TASH0043. Leaf samples collected for molecular work were rapidly desiccated in silica gel in the field and subsequently kept at room temperature until DNA isolation.

Genomic DNA was isolated from the dried leaf tissue using the Tiangen plant genomic DNA extraction kit according to the supplier’s instructions. The overall plastome sequencing and bioinformatic workflow followed standard procedures used in recent chloroplast genome studies, with minor modifications appropriate for Allium material (Ergashov et al., 2026a). Illumina sequencing libraries were constructed using a NEB library preparation kit. Genomic DNA was sheared to an average insert size of approximately 350 bp, followed by end repair, adapter ligation, and PCR enrichment. Library integrity and fragment-size distribution were evaluated using an Agilent 5400 system, and the final library concentration was quantified before sequencing. Qualified libraries were sequenced on an Illumina platform by Novogene Bioinformatics Technology Co.

Raw sequencing data were processed to obtain high-quality clean reads, which were then used for chloroplast genome assembly with NOVOPlasty (Dierckxsens et al., 2017). Genome annotation was carried out in Geneious Prime v2025.1.2, using the chloroplast genome of Allium nikolaii F.O.Khass. & Achilova (NC_068793) as a reference. The preliminary annotation was inspected manually, and gene boundaries were corrected where necessary, including verification of start and stop codons as well as exon–intron junctions in protein-coding genes (Kearse et al., 2012). The graphical map of the circular chloroplast genome was produced with OGDRAW (Greiner et al., 2019; Fig. 2).

For comparative plastome analyses, complete chloroplast genome sequences of representative Allium species were downloaded from NCBI GenBank ( Table 1). The plastome sequences were aligned using MAFFT v7.5 (Katoh and Standley, 2013). Nucleotide diversity (Pi) was estimated in DnaSP v6.12.03 (Rozas et al., 2017). Highly variable regions were detected using a sliding-window approach with a window size of 800 bp and a step size of 200 bp.

Phylogenetic relationships among the sampled Allium species were reconstructed using complete chloroplast genome sequences. The aligned plastome matrix generated in MAFFT v7.5 was analyzed under a maximum likelihood (ML) framework in RAxML v8.2.12 (Stamatakis, 2014), applying the GTRGAMMA substitution model. Branch support was assessed with 1,000 bootstrap replicates. The resulting tree was rooted with Agapanthus coddii F.M.Leight. and Narcissus poeticus L., representing related genera within Asparagales, to provide an external comparative framework for assessing the phylogenetic placement of A. aktauense within Allium.

The complete chloroplast genome of Allium aktauense showed a typical quadripartite structure, including one large single-copy region, one small single-copy region and two inverted repeat regions ( Figure 2). The chloroplast genome was 152,443 bp in length, including an LSC region of 81,522 bp, an SSC region of 17,907 bp, and two IR regions of 26,507 bp each. Genome annotation identified 131 genes, including 85 protein-coding sequences (CDS), 38 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. This genome organization is consistent with most angiosperm plastomes and agrees with previously published chloroplast genomes of Allium species (Munavvarov et al. 2022; Ergashov et al. 2025a,b, 2026a,b). The plastome size of A. aktauense (152,443 bp) is also close to those reported for other members of the genus, such as A. wallichii (152,496 bp), suggesting that the overall chloroplast genome structure is highly conserved within Allium (Zhao et al., 2023).

Sliding-window analysis of nucleotide diversity (Pi) revealed a heterogeneous distribution of sequence variation across the chloroplast genome alignment ( Figure 3). Several highly variable regions were identified, mainly in intergenic spacers and selected coding regions. The most variable regions included rps16, trnD-GUC–trnE-UUC , psbJ–psbE, ndhF–rpl32, and ycf1. Among these, the ndhF–rpl32 region showed the highest nucleotide diversity, with Pi reaching approximately 0.044. The ycf1 region also showed elevated variation, with Pi values approaching 0.030. These regions may be useful as candidate molecular markers for species identification, phylogenetic reconstruction and future population-level studies. Among these loci, ycf1 is particularly notable because it has frequently been reported as one of the most variable and phylogenetically informative plastid regions in angiosperms and in several monocot lineages (Dong et al. 2012, 2015). In the present study, ycf1 also showed high nucleotide diversity, supporting its potential value for resolving relationships among closely related Allium species ( Figure 3). The ndhF–rpl32 intergenic region showed the highest variation, suggesting that non-coding plastid regions may contain stronger species-level signals than many conserved coding regions (Gielly and Taberlet, 1994; Shaw et al. 2007).

The chloroplast genome phylogeny placed A. aktauense within subgenus Allium, supporting its current broad taxonomic placement ( Figure 4). The maximum likelihood tree recovered several major clades corresponding to recognized Allium lineages, including Cepa, Polyprason, Reticulatobulbosa, Amerallium, Rhizirideum, and subgenus Allium. Bootstrap support values indicated that complete chloroplast genome sequences provided a strong phylogenetic signal for resolving relationships among the sampled taxa. In the plastome tree, A. aktauense was recovered close to A. ophiophyllum, while A. nikolaii and A. filidens formed a neighbouring lineage. However, this result should be interpreted cautiously. The close position of A. aktauense to A. ophiophyllum in the plastome tree does not necessarily mean that these species are closest relatives in a complete evolutionary sense. Chloroplast genomes represent a maternally inherited or uniparentally inherited history in many angiosperms and may differ from nuclear phylogenetic patterns, especially in groups affected by hybridization, incomplete lineage sorting or rapid radiation (Folk et al. 2017; Ye et al. 2021). Therefore, the plastome tree provides strong evidence for the placement of A. aktauense within subgenus Allium, but broader sampling of sect. Minuta and related Middle Asian sections, together with nuclear markers, will be necessary to clarify its precise sectional relationships.

Overall, the present study provides the first complete chloroplast genome of A. aktauense and adds an important genomic resource for Central Asian Allium. The conserved plastome structure, identification of highly variable loci and phylogenetic placement within subgenus Allium contribute to a better understanding of the taxonomy, evolution and conservation value of this narrow endemic species.

No custom code was used in this study.

Software used in the analysis included NOVOPlasty, Geneious Prime, OGDRAW, DnaSP v6.12.03, MAFFT, RAxML, and jModelTest v2.1.4, as cited in the Methods section.

Grant information

The author(s) declared that no grants were involved in supporting this work.

Copyright

© 2026 Ergashov I et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Version 1

VERSION 1 PUBLISHED 05 Aug 2026

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