Comparative chloroplast genomics of Cousinia (Asteraceae) based on nine newly sequenced Central Asian endemic species

Comparative chloroplast genomics of Cousinia (Asteraceae) based on nine newly sequenced Central Asian endemic species | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 11 February 2026 V1 Latest version Share on Comparative chloroplast genomics of Cousinia (Asteraceae) based on nine newly sequenced Central Asian endemic species Authors : Bobur Karimov 0009-0005-5874-1033 , Diyorjon Hamrayev , Husniddin Esanov , Alijon Eshonkulov , Oybek Omonov , Nodira Boboyeva , Damira Karimova , Abdullajon Umedov , Temur Asatulloev , Ziyoviddin Yusupov [email protected] , and Komiljon Tojibaev Authors Info & Affiliations https://doi.org/10.22541/au.177080775.52400647/v1 233 views 89 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Cousinia (Asteraceae, Cardueae) is one of the most diverse genera of the Irano–Turanian floristic region, but relationships among its species remain insufficiently resolved. In this study, nine complete chloroplast genomes of Cousinia species endemic to the Pamir–Alay mountain system were sequenced and analyzed together with previously published plastomes and nuclear ribosomal ITS data. The newly assembled plastomes were approximately 152 kb in length, exhibited a typical quadripartite structure, and had a GC content of 37.7%. Gene content was highly conserved, with 131 annotated genes in most species. Codon usage analysis of 16 plastomes showed a consistent preference for A/T-ending codons. Sliding-window analysis indicated low overall nucleotide diversity (Pi = 0–0.00918), with several variability hotspots located mainly in the large and small single-copy regions. A total of eleven types of simple sequence repeats were detected, of which A/T mononucleotide repeats were the most abundant. Phylogenetic analyses based on complete chloroplast genomes did not fully support the monophyly of morphologically defined sections, whereas ITS data provided better resolution of sectional boundaries. Morphological examination of anther appendages identified nine distinct structural groups and revealed patterns partly consistent with molecular results, suggesting the need for further taxonomic revision in some sections of Cousinia. Comparative chloroplast genomics of Cousinia (Asteraceae) based on nine newly sequenced Central Asian endemic species Bobur Karimov 1 , Diyorjon Hamrayev 1 , Husniddin Esanov 2 , Alijon Eshonkulov 3 , Oybek Omonov 4,5 , Nodira Boboyeva 6 , Damira Karimova 7 , Abdullajon Umedov 2 , Temur Asatulloev 1 , Ziyoviddin Yusupov 1* , Komiljon Sh. Tojibaev 1 1 Institute of Botany, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan 2 Bukhara State University, Bukhara, Uzbekistan 3 Bukhara State Medical Institute, Bukhara, Uzbekistan 4 Karshi State University, Karshi, Uzbekistan 5 Turan University, Karshi, Uzbekistan 6 Termez State University, Termez, Uzbekistan 7 Jizzakh State Pedagogical University, Jizzakh, Uzbekistan *Corresponding author: Mail: [email protected] ORCID: https://orcid.org/0000-0003-2278-542X Abstract . Cousinia (Asteraceae, Cardueae) is one of the most diverse genera of the Irano–Turanian floristic region, but relationships among its species remain insufficiently resolved. In this study, nine complete chloroplast genomes of Cousinia species endemic to the Pamir–Alay mountain system were sequenced and analyzed together with previously published chloroplast genomes and nuclear ribosomal ITS data. The newly assembled chloroplast genomes were approximately 152 kb in length, exhibited a typical quadripartite structure, and had a GC content of 37.7%. Gene content was highly conserved, with 131 annotated genes in most species. Codon usage analysis of 16 chloroplast genomes showed a consistent preference for A/T-ending codons. Sliding-window analysis indicated low overall nucleotide diversity (Pi = 0–0.00918), with several variability hotspots located mainly in the large and small single-copy regions. A total of eleven types of simple sequence repeats were detected, of which A/T mononucleotide repeats were the most abundant. Phylogenetic analyses based on complete chloroplast genomes did not fully support the monophyly of morphologically defined sections, whereas ITS data provided better resolution of sectional boundaries. Morphological examination of anther appendages identified nine distinct structural groups and revealed patterns partly consistent with molecular results, suggesting the need for further taxonomic revision in some sections of Cousinia . Keywords : anther appendage morphology; codon usage bias; nucleotide diversity; Pamir–Alay; phylogenetic incongruence; plastid genome evolution; simple sequence repeats Introduction The genus Cousinia Cass. (Asteraceae, Cardueae) represents one of the most diverse and taxonomically complex genera of flowering plants, comprising approximately 673 recognized species distributed mainly across the Irano-Turanian floristic region (POWO, 2025). The greatest concentration of diversity occurs in the mountain systems of Central and Western Asia, particularly the Pamir–Alay, Tianshan, and Iranian Plateau regions, which serve as major centers of endemism and speciation (Knapp, 1987; Tscherneva, 1993). Taxonomic studies of Cousinia have a long and intricate history. Following Cassini’s (1827) original description, early classification systems by de Candolle (1824) and Bunge (1865) divided the genus based on capitulum structure, receptacular bristles, and corolla morphology. Later revisions by Boissier (1875, 1888) and Winkler (1892, 1897) expanded the genus considerably, while regional floristic treatments such as Flora Iranica (Rechinger, 1972) and Flora of the USSR (Tscherneva, 1962, 1988) provided more refined sectional delimitations, recognizing over 350 species across approximately 50–58 sections. Subsequent morphological studies, notably by Sennikov (2010), redefined several sections, including Alpinae , Subappendiculatae , and Tianschanicae , based on detailed variations in involucre form, anther appendage shape, and basal leaf structure. Despite extensive morphological and taxonomic efforts, the evolutionary relationships within Cousinia remain difficult to resolve. The genus exhibits considerable morphological convergence and high intraspecific variability, often leading to taxonomic ambiguities. Molecular phylogenetic studies employing nuclear ITS and partial chloroplast regions (Atazadeh et al., 2021; Garcia-Jacas et al., 2002; Häffner & Hellwig, 1999; Kalouti et al., 2022; López-Vinyallonga et al., 2009, 2011; Mehregan & Kadereit, 2009; Susanna et al., 2003, 2006) have improved our understanding of the Arctium – Cousinia complex, revealing two primary clades, the Arctioid and the Cousinioid lineages. However, the limited number of genetic loci used and insufficient taxon sampling in these analyses have prevented full resolution of sectional boundaries and interspecific relationships. Complete chloroplast (cp) genome sequencing offers a powerful approach to resolve taxonomic and evolutionary gaps, and its application has rapidly expanded in the Asteraceae, one of the largest and most complex angiosperm families (Karimov et al., 2025a; Mahai et al., 2024; Nyamgerel et al., 2024; Rahmatulla et al., 2025; Xing et al., 2025; Yang et al., 2023; Zhong et al., 2023). Our recent study (Karimov et al., 2025b) marked the first major step toward presenting a comparative cp analysis for Cousinia species from Uzbekistan. However, broader taxon sampling and analysis remain necessary to evaluate structural diversity, codon usage bias, and phylogenetic congruence across taxonomic sections. In the present study, we expand upon this foundation by sequencing and analyzing nine additional chloroplast genomes of Cousinia species, all endemic to the Pamir–Alay mountain system. These newly generated cp genomes, combined with previously published sequences and nuclear ribosomal ITS data, provide a framework for examining the molecular evolution and phylogeny of the genus. The objectives of this study were to: (1) characterize the overall structure and codon usage bias of Cousinia chloroplast genomes; (2) identify nucleotide variability hotspots and SSR motifs as potential molecular markers; (3) assess genetic divergence among species and detect regions of elevated sequence variation; and (4) reconstruct phylogenetic relationships based on both cp and nuclear data to evaluate the correspondence between molecular and morphological classifications. Material and Methods Plant material Sampling was designed to capture the phylogenetic breadth of the genus by including species representing nine taxa belonging to eight taxonomic sections of Cousinia distributed across the Pamir–Alay region (Table 1, Figure 1). This strategy ensured broad lineage coverage for evaluating variation in chloroplast genome structure among distinct evolutionary groups. Fresh leaf material was collected from natural populations in Uzbekistan between 2023 and 2025 and immediately dried in silica gel. For species that could not be accessed during fieldwork, high-quality DNA was extracted from herbarium specimens housed at the National Herbarium of Uzbekistan (TASH) and the Herbarium of the Institute of Botany, Plant Physiology and Genetics, National Academy of Sciences of Tajikistan (TAD), with prior curator approval. DNA extraction and sequencing Genomic DNA was isolated from leaf tissue using the Tiangen DP305 Plant Genomic DNA Kit (Beijing, China). Libraries were prepared with the NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA; Cat. E7370L) following the manufacturer’s protocol, with index codes added during preparation. DNA was sonicated to ~350 bp, and fragments were end-repaired, A-tailed, and ligated to Illumina adapters, followed by PCR amplification. PCR products were purified using AMPure XP beads (Beverly, USA). Library quality was assessed on an Agilent 5400 system, and concentrations were quantified by qPCR (1.5 nM). Qualified libraries were pooled and sequenced on Illumina platforms using the PE150 strategy at Novogene (Beijing, China). Genome assembly and annotation Clean paired-end reads were mapped de novo using NOVOPlasty v4.3.5 (Dierckxsens et al., 2017), with the Cousinia rotundifolia cp (PQ240609) employed as the reference. The assembly yielded a single circularized cp genome for each species. Clean paired-end reads were mapped to their respective chloroplast genomes using BWA-MEM v0.7.17 (Li, 2013). Alignments were processed, sorted, and indexed with SAMtools v1.19.2 (Li et al., 2009). Perbase coverage depth was calculated in SAMtools. Gene annotation was performed with GeSeq (Tillich et al., 2017), using the C. rotundifolia reference genome. Annotations were manually inspected and corrected in Geneious v9.0.2 (Kearse et al., 2012) to ensure the accuracy of start and stop codons as well as intron–exon boundaries of protein-coding genes. Furthermore, the cp map was generated using Chloroplot (https://irscope.shinyapps.io/chloroplot/) (Zheng et al., 2020) Comparative genomic analyses To characterize codon usage patterns and evaluate potential codon usage bias across Cousinia species, sixteen complete chloroplast genome sequences were analyzed. Coding sequences (CDS) were extracted using Biopython v1.83 (Cock et al., 2009), and duplicated genes located in the inverted repeat (IR) regions were removed to retain a single representative copy of each gene. All CDS were checked to ensure correct reading frames and to confirm the absence of internal stop codons. Codon frequencies were calculated from the concatenated CDS of each species. Stop codons (TAA, TAG, TGA), single-codon amino acids (ATG for Met and TGG for Trp), and the plastid-rare arginine codons (AGA, AGG) were excluded from downstream analyses. Relative Synonymous Codon Usage (RSCU) values were subsequently computed to characterize codon usage bias. Nucleotide variability (Pi) across the cp genomes was estimated using sliding window analysis implemented in DnaSP v.6.11 (Rozas et al., 2017), employing a window length of 800 bp and a step size of 200 bp. Simple Sequence Repeats (SSRs) were identified using MISA (Beier et al., 2017) with minimum repeat thresholds of 10, 5, 4, 3, 3, and 3 for mono-, di-, tri-, tetra-, penta-, and hexanucleotide motifs, respectively. For comparative assessment, SSR counts for each motif type were summarized into a species × motif matrix. Pairwise dissimilarities among species were quantified using the Bray–Curtis distance metric (Bray and Curtis, 1957). Hierarchical clustering was performed using the Ward D2 algorithm (Ward, 1963; Murtagh and Legendre, 2014) to construct a phenetic cladogram based on SSR motif composition. Phylogenetic analysis Two molecular datasets were used for phylogenetic reconstruction: (1) complete chloroplast genomes (19 species: 9 newly sequenced and 10 from GenBank, with Arctium lappa , Jurinea auriculata , and Dolomiaea wardii designated as outgroups) and (2) the nuclear ribosomal ITS region (18 species: 9 newly sequenced and 9 from GenBank, with Carduus acanthoides , Erigeron philadelphicus , and Atractylodes lancea used as outgroups). The chloroplast alignment comprised 154,308 bp (1,995 variable and 764 parsimony-informative sites), while the ITS alignment included 5,844 bp (195 variable and 88 parsimony-informative sites). All alignments were examined manually, and gaps were treated as missing data. Phylogenetic analyses were conducted separately for each dataset. The chloroplast dataset was analyzed using Maximum Likelihood (ML ) in RAxML v8.2.12 (Stamatakis, 2014) under the GTR+G substitution model with 1,000 bootstrap replicates . For the ITS dataset, model selection in jModelTest v2.1.10 (AICc) identified GTR+I+G as the best-fitting model. ML analysis of ITS sequences was performed in RAxML under this model with 1,000 bootstrap replicates. Bayesian inference was conducted in MrBayes v3.2.7 (Ronquist et al., 2012) using two independent MCMC runs of 10 million generations, sampling every 1,000 generations and discarding 25% as burn-in after confirming convergence (average standard deviation of split frequencies < 0.01). Final phylogenetic trees were visualized and annotated in iTOL v6 (Letunic and Bork, 2021). Sequencing and assembly The total number of reads per species ranged from 16,964,951 in C. coronata to 680,809,153 in C. campylaraphis (Table 2). The proportion of mapped reads ranged from 1.23% ( C. botschantzevii ) to 9.95% ( C. stellaris ). Properly paired reads showed a similar range, from 1.10% in C. botschantzevii to 9.74% in C. stellaris . Mean sequencing depth differed substantially among species, with the lowest depth observed in C. coronata (1,084.56×) and the highest in C. campylaraphis (30,784.2×). High coverage was also obtained for C. candicans (19,146.3×) and C. stellaris (13,586×), whereas moderate coverage levels were recorded for C. integrifolia (7,260.84×), C. spryginii (7,260.88×), C. speciosa (2,961.02×), C. laetevirens (2,341.92×), and C. botschantzevii (1,668.21×). Overall, all species achieved sufficient sequencing depth to support reliable chloroplast genome assembly. Chloroplast genome features In this study, nine chloroplast genomes were sequenced, representing eight sections of Cousinia , which are endemic to the Pamir–Alay mountain system. All cp genomes were ~152 kb in length with a GC content of 37.7%. The chloroplast genomes exhibited the typical quadripartite structure, consisting of a large single-copy (LSC) region, a small single-copy (SSC) region, and two identical inverted repeat (IR) regions (Figure 2). In all nine species, 131 genes were annotated, of which 87 were protein-coding genes (PCGs), 36 were transfer RNA (tRNA) genes, and 8 were ribosomal RNA (rRNA) genes. Notably, 18 genes within the cp genome are duplicated. This set of duplicated genes includes seven tRNAs ( trnA-UGC, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, trnV-GAC ), four rRNAs ( rrn16, rrn23, rrn4.5, rrn5 ), and seven PCGs ( ndhB, rpl2, rpl23, rps12, ycf2, ycf15, rps7 ). Sixteen genes possess introns, of which 11 are protein-coding genes ( atpF, rpoC1, rpl2, ndhB, ndhA, petB, petD, rps16, rps12, ycf3, and clpP ) and five are tRNA genes ( tRNA-UGC, trnI-GAU, trnK-UUU, trnL-UAA , and trnV-UAC ). Two genes, ycf3 and clpP have two introns, while the remaining 14 genes have only one. Codon usage Analysis of RSCU across the chloroplast genomes of 16 Cousinia species revealed highly conserved codon usage patterns, with only minor interspecific variation (Figure 3). Across all species, codon usage showed a strong bias toward A/T-ending codons, whereas G/C-ending codons were consistently underrepresented. This AT bias was evident for nearly all amino acids and is consistent with the AT-rich nucleotide composition typical of cp genomes. Amino acids encoded by two synonymous codons displayed pronounced directional bias. For example, AAT (Asn; RSCU ≈ 1.57) greatly exceeded AAC (≈ 0.42), and GAT (Asp; ≈ 1.59) was strongly favored over GAC (≈ 0.40). Similar patterns were observed for Cys (TGT > TGC), Gln (CAA > CAG), Glu (GAA > GAG), His (CAT > CAC), Phe (TTT > TTC), Tyr (TAT > TAC), and Lys (AAA ≈ 1.51 vs. AAG ≈ 0.49). Amino acids encoded by four or six synonymous codons also exhibited clear codon usage asymmetry. For alanine, GCT was the most preferred codon (RSCU ≈ 1.80), whereas GCC and GCG were underused (≈ 0.45–0.62). Glycine showed moderate bias, with GGT and GGA favored (≈ 1.33–1.53) and GGC underrepresented (≈ 0.49). Leucine displayed one of the strongest biases, with TTA (≈ 1.90) and TTG (≈ 1.22) being highly preferred, while CTG and CTC showed consistently low RSCU values (≈ 0.37–0.38). Similar A/T-ending codon preferences were observed for serine (TCT ≈ 1.77), proline (CCT ≈ 1.53), threonine (ACT ≈ 1.61), and valine (GTA and GTT ≈ 1.44–1.54). Nucleotide diversity Sliding-window analysis revealed an uneven distribution of nucleotide diversity across the chloroplast genomes of the 16 Cousinia species examined. Overall Pi values were low (0–0.00918), reflecting the highly conserved nature of cp genomes (Figure 4). Nevertheless, several distinctly variable regions were identified. Most variable nucleotides were concentrated in the LSC and SSC regions, whereas the IR regions showed the highest degree of stability and exhibited the lowest levels of diversity. Within the LSC region, five major peaks of variation were detected: trnK-UUU–rps16, trnE-UUC–rpoB, trnS-GGA–rps4, trnL-UAA–trnF-GAA–ndhJ , and a moderate variability peak within the rbcL gene. In the SSC region, three pronounced hotspots were identified, corresponding to the ndhF gene, the rpl32–trnL-UAG intergenic spacer, and the ycf1 gene. SSR markers Analysis of cp genomes revealed a total of 11 different SSR (simple sequence repeat) motifs, including 2 mononucleotide, 1 dinucleotide, 2 trinucleotide, 4 tetranucleotide, 1 pentanucleotide, and 1 hexanucleotide motifs (Figure 5). The most frequent motif type was the A/T mononucleotide repeat, which occurred with high frequency across all species, ranging from 20 repeats in C. coronata to 26 in C. spryginii and C. botschantzevii . In contrast, C/G-type mononucleotide SSRs were relatively rare, occurring only once in all examined species, and were completely absent in C. rotundifolia and C. orthacantha . The pentanucleotide motif AAATC–ATTTG was found exclusively in C. coronata in trnE-UUC – rpoB , while the hexanucleotide motif AATAGG–ATTCCT was unique to C. thomsonii in rpoC2 . Cluster analysis based on simple sequence repeat (SSR) profiles revealed distinct groupings among the Cousinia species. Notably, C. spryginii and C. botschantzevii exhibited nearly identical SSR profiles, while C. stellaris displayed a highly similar profile, suggesting close genetic affinity among these taxa. Species from the sections Alpinae and Tianschanicae — C. rhodantha , C. speciosa , and C. pseudodshizakensis —formed a strongly supported cluster, differing only at a single mono- and dinucleotide locus. Likewise, C. laetevirens and C. campylaraphis shared fully concordant SSR profiles across all mono-, di-, tri-, and tetranucleotide loci, with the sole distinction being an additional A–T repeat in C. campylaraphis . Similarly, C. integrifolia and C. rotundifolia exhibited highly comparable SSR patterns, differing primarily in the C–G motif, which was absent in C. rotundifolia . Phylogenetic analysis Analyses based on the cp genome indicated no support for the monophyly of the morphologically defined sections within Cousinia (Figure 6A). Species attributed to section Homalochaete did not form an exclusive group; instead, four of them were recovered in a well-supported clade (BS = 100) together with taxa representing sections Acanthotoma , Dichotomae , and Coronophora . All members of this clade are confined to the Pamir–Alay region. The closest relative of this assemblage was Cousinia thomsonii , a species whose native range extends from Afghanistan eastward to Nepal and southern Tibet. A similar pattern emerged for section Alpinae . Four species traditionally placed in this section grouped with representatives of sections Tianschanicae and Olgaeanthe , forming a second, distinct clade composed entirely of Pamir–Alay endemics. This clade was inferred to be sister to a lineage comprising Cousinia botschantzevii (section Regelianae ) and Cousinia spryginii (section Heliantheae ), both of which likewise exhibit strict endemism to the Pamir–Alay region. In contrast, the ITS-based phylogeny better supported the monophyly of the morphologically defined sections of Cousinia , although most nodes showed lower bootstrap values compared with the chloroplast tree (Figure 6B). The four species assigned to section Homalochaete formed a coherent and well-defined clade. The single representatives included from sections Acanthotoma, Dichotomae, Coronophora, Olgaeanthe , and Heliantheae each occupied isolated lineages without intermixing with other sections, fully consistent with their morphological delimitation. Only two exceptions were observed: C. speciosa (section Tianschanicae) nested within the Alpinae clade (ML = 87), and C. stellaris (section Alpinae) clustered with C. botschantzevii of section Regelianae , forming a moderately supported monophyletic lineage (ML = 67). The form of the anther appendages We examined the anther-appendage morphology of more than forty species of Cousinia (Supplementary 1) and distinguished nine discrete structural groups (Figure 7). Group I, consisting of irregularly dentate appendages, was recorded only in C. botschantzevii Juz. ex Tscherneva and C. regelii C. Winkl. of section Regelianae . Group II, characterized by narrowly elongate and smooth appendages, comprised five species: C. spryginii Kult. (section Helianthae ), C. ferghanensis Bornm. and C. simulatrix C. Winkl. (section Subappendiculatae ), C. divaricata C. Winkl. (section Leiacanthos ), and C. princeps Franch. (section Alpinae ). Group III, defined by narrowly elongate appendages with a toothed apex, included twelve species— C. proxima , C. corymbosa , C. podophylla , C. coerulea , C. campylaraphis , C. subcandicans , and C. litwinowiana from section Homalochaete ; C. verticillaris , C. laetevirens , C. sarawschanica , and C. splendida from section Acanthotoma ; and C. magnifica from section Racemosae . Group IV, possessing short, smoothly rounded lobes forming a dome-shaped or arched apex, was restricted to four species of section Alpinae : Cousinia stellaris Bornm., C. pseudodshizakensis Tschern. & Vved., C. rotundifolia C. Winkl., and C. grigoriewii . Group V, consisting of narrowly elongate appendages without an arched outline, was observed in C. rosea Kult., C. alpina Bunge, and C. calva Juz. (section Alpinae ), C. speciosa C. Winkl. (section Tianschanicae ), and C. dubia Popov and C. submutica Franch. (section Jurineopsis ). Group VI, defined by long, acute appendages with an arched outline, included C. integrifolia Franch. (section Olgaeanthe ), C. coronata Franch. and C. radians Bunge (section Coronophora ), and C. outichaschensis Franch., C. buphthalmoides Regel (together with its synonym C. auriculata Hook.f.), and C. rava C. Winkl. of section Alpinae . Group VII, a crown-shaped apex bearing teeth, was restricted to C. psammophila Kult. of section Chrysoptera . Group VIII, having a short, entire, and rounded apex, was observed in C. pseudolanata Popov ex Tscherneva and C. lanata C. Winkl. of section Racemosae . Group IX, defined by elongate appendages with a minute or absent apical tooth, characterized four species of section Dichotomae : C. tedshenica Tscherneva, C. sylvicola Bunge, C. patentispina Tscherneva, and C. orthacantha Tscherneva. Discussion The present study represents chloroplast genome analysis of Cousinia to date, expanding our understanding of cp genome structural variation, codon usage evolution, and phylogenetic relationships in this taxonomically challenging genus. The overall organization of Cousinia chloroplast genomes, quadripartite structure, size (~152 kb), and GC content (37.7%)—is consistent with typical patterns observed across Asteraceae (Mahai et al., 2024; Nyamgerel et al., 2024; Xing et al., 2025; Zhong et al., 2023). Gene content and intron composition are remarkably conserved among species, suggesting that the cp genome structure in Cousinia has remained largely stable through evolutionary history (Jansen and Ruhlman, 2012) The codon usage analysis across cp genomes of 16 Cousinia species revealed highly conserved and AT-biased patterns, typical of angiosperm cp genomes (Parvathy et al., 2022). Codons ending in A or T were consistently preferred, while G/C-ending codons were underrepresented, reflecting the AT-rich nature of these genomes. This bias was most evident in amino acids like leucine, lysine, and alanine, suggesting a neutral effect of mutational pressure as the main driver (Zhang et al., 2007). The uniformity of RSCU profiles among Cousinia species indicates this conservation is likely driven by functional constraints and natural selection favoring codons that enhance translation efficiency and accuracy (Li et al., 2025) Sliding window analysis indicated that Cousinia cp genomes are largely conserved, with overall nucleotide diversity (Pi = 0–0.00918) comparable to that reported in other thistle-like genera such as Saussurea (He et al., 2023 ). The observed variability hotspots ( trnK-UUU–rps16, trnE-UUC–rpoB, trnS-GGA–rps4, trnL-UAA–trnF-GAA–ndhJ, rbcL, ndhF, rpl32–trnL-UAG and ycf1 ) correspond well to regions known to evolve rapidly in Asteraceae and serve as useful molecular markers for species delimitation (Shen et al., 2020; Kim et al., 2024). These molecular markers provide a foundation for future genomic barcoding and biogeographic reconstruction within Cousinia. Analyses of the cp genomes demonstrated a total of eleven SSR motif types in Cousinia species, among which A/T mononucleotide repeats were the most abundant. In contrast, C/G-type motifs were exceedingly rare and were entirely absent in some species. This predominance of A/T-rich repeats is considered a widespread feature of chloroplast genomes in higher plants, and it is generally attributed to codon-usage bias, as preferred codons tend to terminate in A or U. Previous studies have further suggested that this pattern may be influenced by natural selection and underlying mutational processes (Zhang et al., 2023). The chloroplast genome–based phylogeny did not fully support the monophyly of the morphologically defined Cousinia sections, suggesting possible chloroplast capture, introgressive hybridization, or incomplete lineage sorting. Because chloroplast DNA is typically maternally inherited, its phylogenetic signal may not fully represent organismal history, particularly in lineages with hybridization or reticulate evolution (Rieseberg and Soltis, 1991; Soltis and Kuzoff, 1995; Soltis & Soltis, 1998; Tsitrone et al., 2003). Hybridization in Cousinia occurs between species of the same clade and of different clades (Mehregan and Kadereit, 2009). Conversely, the ITS-based phylogeny better reflected morphological classifications, delineating clearer sectional boundaries despite lower statistical support. Only C. stellaris from section Alpinae showed exceptions, forming mixed clades with species of section Regelianae . This pattern indicates that C. stellaris does not belong to the Alpinae section, a conclusion supported by both cp and ITS data. This congruence implies that nuclear markers, being biparentally inherited, may better represent evolutionary relationships (Álvarez and Wendel, 2003; Feliner and Rosselló, 2007). The observed diversity of anther appendage morphology within Cousinia demonstrates pronounced structural differentiation among sections. Certain appendage types were confined to particular lineages ( Regelianae ), whereas others were distributed across unrelated sections. In Flora of the USSR (Tscherneva 1962), C. pseudolanata, C. lanata , and C. magnifica were originally assigned to section Acanthotoma (formerly Alpinae ). Later, in Conspectus florae Asiae Mediae (Tscherneva 1993), these three species were transferred to the newly established section Racemosae . The transfer of C. pseudolanata and C. lanata is supported by their similar anther appendage morphology, characterized by short, entire, and rounded apices. However, C. magnifica possesses narrowly elongate appendages with a toothed apex, aligning morphologically with species of Acanthotom a. Consequently, we recommend that C. magnifica be retained within section Acanthotoma . The morphological heterogeneity of section Alpinae is evident from the diversity of anther appendage forms represented among its species. Members of this section occur in several morphological groups: C. princeps in Group II (narrowly elongate, smooth appendages); C. stellaris , C. pseudodshizakensis, C. rotundifolia , and C. grigoriewii in Group IV (short, dome-shaped, arched in outline); C. rosea, C. alpina , and C. calva in Group V (narrowly elongate, not arched in outline); and C. outichaschensis, C. buphthalmoides, and C. rava in Group VI (long, arched appendages with acute apices). These findings suggest that the current circumscription of Alpinae may not accurately reflect evolutionary relationships and that both morphological and molecular characters should be considered in future taxonomic revisions of Cousinia. Conclusion The findings of this study highlight that resolving relationships within Cousinia requires a multilayered approach rather than reliance on a single data source. Plastid genomes, although structurally stable and highly conserved, do not reflect the morphological sectional system traditionally used in the genus. Their discordance with both ITS phylogeny and anther-appendage morphology shows that cp genome inheritance captures only part of the evolutionary history, likely influenced by historical hybridization events and lineage sorting. Importantly, the morphological assessment demonstrates that anther appendages carry a strong phylogenetic signal and can reveal hidden structure within sections that appear morphologically heterogeneous. Rather than offering a classification, this work provides a framework to guide future taxonomic decisions. The cp genome markers identified here, together with nuclear loci and targeted morphological traits, create new opportunities for fine-scale species delimitation, testing sectional monophyly, and reconstructing the diversification of Cousinia across the Pamir–Alay. Broader genomic sampling and expanded nuclear datasets will be essential next steps to clarify the evolutionary processes shaping this complex Irano–Turanian lineage Acknowledgments. This research was carried out within the framework of the projects “Digital Nature: Development of a digital platform for the flora of Central Uzbekistan” (2025–2029), and “Assessing climate change adaptation in endangered plants of Uzbekistan: A DNA barcoding approach” (AL-9224104464), implemented by the Institute of Botany, Academy of Sciences of the Republic of Uzbekistan. Conflict of interest statement. The authors declare that there is no conflict of interest regarding the publication of this paper. Data availability statement. The raw sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1124612 (Cousinia genome sequencing and assembly, Tree of Life Uzbekistan; multispecies). All other data generated or analyzed during this study are included in this published article and its supplementary information files. References Álvarez, I. J. F. W., & Wendel, J. F. (2003). Ribosomal ITS sequences and plant phylogenetic inference. Molecular phylogenetics and evolution, 29(3), 417-434. https://doi.org/10.1016/S1055-7903(03)00208-2 Atazadeh, N., Sheidai, M., Attar, F., and Koohdar, F. (2021). Molecular phylogeny and morphometric analyses in the genus Cousinia Cass. (Family Asteraceae), sections Cynaroideae Bunge and Platyacanthae Rech. f. 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Physiology and Molecular Biology of Plants, 29(3), 409-420. https://doi.org/10.1007/s12298-023-01292-x Table 1. Species, sectional placement, distribution, voucher data, and NCBI accession numbers for Cousinia cp genome samples sequenced in this study. C. botschantzevii Juz. ex Tscherneva Sect. Regelianae (Juz.) Tschern.; endemic to the Pamir–Alay (Nuratau Mountains) Uzbekistan, Navoiy Region, Nuratau Mountains. 11 May 2025. Leg. Karimov B. Observation link: https://www.inaturalist.org/observations/280210636 PX230056 PX632335 C. campylaraphis Tschern. Sect. Homalochaete C. Winkl.; endemic to the Pamir–Alay (Southwestern Hissar) Uzbekistan, Kashkadarya Region, road to Vuar village. 7 June 2023. Leg. Turginov et al., (TASH) PX230055 PX632341 C. candicans Juz. Sect. Homalochaete C. Winkl.; endemic to the Pamir–Alay (Babatag Range) Uzbekistan, Surkhondarya Region, Babatag Mountains. 25 May 2019. Leg. Beshko et al., (TASH) PX230051 PX632343 C. coronata Franch. Sect. Coronophora (Juz.) Rech. f.; endemic to the Pamir–Alay Uzbekistan, Surkhondarya Region, Topalang River basin, vicinity of Bakhcha village. 14 May 2023. Leg. Turdiboev et al., (TASH) PX230054 PX632330 C. laetevirens C. Winkl. Sect. Acanthotoma Juz.; endemic to the Pamir–Alay Tajikistan, Vanch Range, southern approach to the Gushkhon Pass, stony light-loam slope, ca. 3500 m. July 1929. No. 1121 (TASH) PX230053 PX632334 C. integrifolia Franch. Sect. Olgaeanthe Tschern.; endemic to the Pamir–Alay Uzbekistan, Samarkand Region, vicinity of Aman-Kutan Pass. 2 Jun 2024. Leg. Karimov B. Observation link: https://www.inaturalist.org/observations/219948664 PX230050 PX632340 C. speciosa C. Winkl. Sect. Tianschanicae Sennikov; endemic to the Pamir–Alay (Alay and Zaalayskiy Ranges) Kyrgyzstan, Northern slope of the Alay Range, foot of the Taldyk Pass, near Ak-Bashi pasture, 2850–2940 m. 7 September 1952. No. 1173, Leg. Ovchinnikov et al., (TAD) PX230049 PX632331 C. stellaris Bornm. Sect. Alpinae Bunge; endemic to the Pamir–Alay (Alay Range) Kyrgyzstan, Northern slope of the Alay Range, left bank of the Shakhimardan River, Okhna village, rocky slopes, 1400–1450 m. 17 June 1959. No. 357, Leg. Ismatova et al., (TAD) PX230048 PX632332 C. spryginii Kult. Sect. Helianthae Bge.; endemic to the Pamir–Alay (Southwestern Hissar) Uzbekistan, Kashkadarya Region, Dehkanabad District, near roadside. 24 May 2024. Leg. Karimov B. Observation link: https://www.inaturalist.org/observations/217885461 PX230047 PX632333 Table 2. Summary of read mapping statistics for cp genome sequencing of Cousinia species. C. integrifolia 148,619,123 5.20 4.98 7260.84 C. spryginii 157,978,956 4.83 4.64 7260.88 C. botschantzevii 153,580,807 1.23 1.10 1668.21 C. laetevirens 152,724,222 1.77 1.63 2341.92 C. stellaris 142,085,434 9.95 9.74 13,586 C. speciosa 146,445,132 2.30 2.18 2961.02 C. candicans 628,412,024 3.26 3.09 19,146.3 C. campylaraphis 680,809,153 4.70 4.53 30,784.2 C. coronata 16, 964, 951 6.72 6.41 1084.56 Figure 1 . Cousinia species sampled for DNA extraction and sequencing in this study. A. Cousinia botschantzevii, B. Cousinia integrifolia, C. Cousinia coronata, D. Cousinia spryginii, E. Cousinia speciosa, F. Cousinia stellaris, G. Cousinia campylaraphis, H. Cousinia candicans, I. Cousinia laetevirens Figure 2 . The circular map of the chloroplast genomes of Cousinia was drawn using the Chloroplot to show the genes present in each region (LSC, SSC, and IRs). The transcription directions for the inner and outer genes are clockwise and anticlockwise, respectively, and each functional group of genes is distinctively color-marked. In the inner circle, the darker gray shades represent the GC content, and the lighter gray shades signify the AT content. Figure 3 . Relative synonymous codon usage (RSCU) patterns across the chloroplast genomes of 16 Cousinia species. Figure 4. Variation in nucleotide diversity (π) across LSC, IR, and SSC regions of Cousinia chloroplast genomes. Figure 5 . Heatmap of simple sequence repeat (SSR) motif abundance in Cousinia chloroplast genomes with hierarchical clustering. Figure 6. Comparison of phylogenetic trees of Cousinia species inferred from complete chloroplast genomes (A) and nuclear ribosomal ITS sequences (B). Figure 7. Variation in anther appendage morphology among Cousinia species. A. Irregularly dentate appendages, B. Narrowly elongate, smooth appendages, C. Narrowly elongate appendages with a toothed apex, D. Short, smoothly rounded lobes forming a dome-shaped or arched apex, E. Narrowly elongate, not arched in outline, F. Narrowly elongate, arched in outline, G. Crown-shaped apex with a toothed apex, H. Apex short, entire, and rounded, I. Elongate appendages with a very small or absent apical tooth Supplementary 1. Observed anther appendage morphology Information & Authors Information Version history V1 Version 1 11 February 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords comparative genetics molecular genetics plants sequencing terrestrial Authors Affiliations Bobur Karimov 0009-0005-5874-1033 Institute of Botany of the Academy of Sciences of the Republic of Uzbekistan View all articles by this author Diyorjon Hamrayev Institute of Botany of the Academy of Sciences of the Republic of Uzbekistan View all articles by this author Husniddin Esanov Bukhara State University View all articles by this author Alijon Eshonkulov Bukhara State Medical Institute View all articles by this author Oybek Omonov Karshi State University View all articles by this author Nodira Boboyeva Termez State University View all articles by this author Damira Karimova Jizzakh State Pedagogical Institute named after Abdullah Kadiri View all articles by this author Abdullajon Umedov Bukhara State University View all articles by this author Temur Asatulloev Institute of Botany of the Academy of Sciences of the Republic of Uzbekistan View all articles by this author Ziyoviddin Yusupov [email protected] Institute of Botany of the Academy of Sciences of the Republic of Uzbekistan View all articles by this author Komiljon Tojibaev Institute of Botany of the Academy of Sciences of the Republic of Uzbekistan View all articles by this author Metrics & Citations Metrics Article Usage 233 views 89 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Bobur Karimov, Diyorjon Hamrayev, Husniddin Esanov, et al. 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