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 Search Search ADVANCED SEARCH SCROLL Ecology and Evolution This is a preprint and has not been peer reviewed. Data may be preliminary. 24 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.177195994.44270542/v1 243 views 89 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Cousinia (Asteraceae: Cardueae) represents one of the most species-rich genera within the Irano–Turanian floristic region, yet interspecific relationships remain incompletely resolved. Here, nine chloroplast genomes from species endemic to the Pamir–Alay mountain system were newly sequenced and analyzed in combination with previously published plastome sequences and nuclear ribosomal ITS data. The assembled plastomes were highly uniform in size (approximately 152 kb), displayed the canonical quadripartite organization of angiosperm chloroplasts, and possessed a GC content of 37.7%. Gene composition was largely conserved, with 131 annotated genes identified in most taxa. Examination of synonymous codon usage across sixteen plastomes revealed a consistent bias toward A/T-ending codons. Sliding-window analysis demonstrated generally low nucleotide diversity (Pi = 0–0.00918), although several divergence hotspots were detected, primarily within the large and small single-copy regions. Eleven categories of simple sequence repeats were identified, with A/T-rich mononucleotide motifs predominating. Phylogenetic reconstruction based on complete plastome data did not consistently recover morphologically defined sections as monophyletic, whereas the ITS dataset provided improved resolution of sectional delimitations. Comparative analysis of anther appendage morphology recognized nine structural groups and showed partial congruence with molecular evidence. Together, these findings highlight incongruence between plastid and nuclear signals and indicate that certain infrageneric classifications within Cousinia warrant re-evaluation 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) represents one of the most species-rich genera within the Irano–Turanian floristic region, yet interspecific relationships remain incompletely resolved. Here, nine chloroplast genomes from species endemic to the Pamir–Alay mountain system were newly sequenced and analyzed in combination with previously published plastome sequences and nuclear ribosomal ITS data. The assembled plastomes were highly uniform in size (approximately 152 kb), displayed the canonical quadripartite organization of angiosperm chloroplasts, and possessed a GC content of 37.7%. Gene composition was largely conserved, with 131 annotated genes identified in most taxa. Examination of synonymous codon usage across sixteen plastomes revealed a consistent bias toward A/T-ending codons. Sliding-window analysis demonstrated generally low nucleotide diversity (Pi = 0–0.00918), although several divergence hotspots were detected, primarily within the large and small single-copy regions. Eleven categories of simple sequence repeats were identified, with A/T-rich mononucleotide motifs predominating. Phylogenetic reconstruction based on complete plastome data did not consistently recover morphologically defined sections as monophyletic, whereas the ITS dataset provided improved resolution of sectional delimitations. Comparative analysis of anther appendage morphology recognized nine structural groups and showed partial congruence with molecular evidence. Together, these findings highlight incongruence between plastid and nuclear signals and indicate that certain infrageneric classifications within Cousinia warrant re-evaluation. Keywords : anther appendage morphology; codon usage bias; nucleotide diversity; Pamir–Alay; phylogenetic incongruence; plastid genome evolution; plastome; simple sequence repeats Introduction Irano–Turanian floristic region represent one of the most important global centers of plant diversification, particularly for xerophytic and montane lineages within Asteraceae. Among these, the genus Cousinia Cass. stands out as an exceptional example of rapid radiation and taxonomic complexity. With approximately 673 currently accepted species (POWO, 2025), Cousinia constitutes one of the largest genera in the tribe Cardueae and is predominantly distributed across Central and Western Asia. The highest concentration of species richness occurs in the Pamir–Alay, Tianshan, and Iranian Plateau mountain systems, areas widely recognized for their high endemism and ecological heterogeneity (Knapp, 1987; Tscherneva, 1993). Understanding evolutionary relationships within such species-rich and geographically structured lineages requires robust genomic data capable of resolving fine-scale divergence patterns. One of the most effective methods for examining genome evolution and resolving intricate phylogenetic patterns is complete chloroplast (cp, plastome) genome sequencing. Plastome-scale data offer detailed information on nucleotide divergence patterns, codon usage bias, structural variation, gene content, genome organization, and simple sequence repeats (SSRs). Recent studies on plastomes in the Asteraceae have greatly improved phylogenetic resolution and helped to better understand evolutionary relationships among morphologically diverse genera (Yang et al., 2023; Zhong et al., 2023; Mahai et al., 2024; Nyamgerel et al., 2024; Karimov et al., 2025a; Rahmatulla et al., 2025; Xing et al., 2025). Despite these advancements, the plastome resources of Cousinia remain limited when compared to its remarkable species diversity and geographic range. Although recent studies have begun to characterize chloroplast genome variation in specific Cousinia species, broader taxonomic sampling is required to better understand patterns of structural diversity and sequence variation across endemic lineages (Karimov et al., 2025b). The endemic taxa in the Pamir–Alay region, a major center of diversification, allow for the examination of genomic differentiation within the genus. The current study presents nine recently sequenced chloroplast genomes from Cousinia species that are endemic to the Pamir-Alay mountains in order to fill these gaps. By integrating these data with previously available plastome sequences and nuclear ribosomal ITS datasets, we aim to improve understanding of plastome structure, molecular evolution, and phylogenetic relationships within the genus. Specifically, we (1) analyze overall plastome architecture and codon usage patterns; (2) identify highly variable regions and characterize SSR loci as potential molecular markers; (3) evaluate interspecific genetic divergence; and (4) reconstruct phylogenetic relationships using combined chloroplast and nuclear data to assess congruence with morphology-based classifications. Material and Methods Plant material To reflect major evolutionary lineages, nine species representing eight infrageneric sections of Cousinia from the Pamir–Alay region were chosen (Table 1; Figure 1). During 2023–2025, fresh leaf material was gathered from Uzbekistan’s natural populations and dried in silica gel. DNA was extracted from herbarium specimens deposited in the National Herbarium of Uzbekistan (TASH) and the Herbarium of the Institute of Botany, Plant Physiology and Genetics of the National Academy of Sciences of Tajikistan (TAD), with prior curator authorization, when field sampling was not practical. DNA extraction and sequencing The Tiangen DP305 Plant Genomic DNA Kit (Beijing, China) was used to get genomic DNA from leaf tissue. While the libraries were being made with the NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA; Cat. E7370L), index codes were added according to the manufacturer’s instructions. After sonicating the DNA to about 350 bp, the pieces were A-tailed, fixed at the ends, and linked to Illumina adapters. After that, PCR amplification was done. We used AMPure XP beads from Beverly, USA, to clean up the PCR products. We used an Agilent 5400 system to check the quality of the library and qPCR (1.5 nM) to check the concentrations. Novogene (Beijing, China) used the PE150 method to combine and sequence qualified libraries on Illumina platforms. Genome assembly and annotation High-quality paired-end reads were assembled de novo with NOVOPlasty v4.3.5 (Dierckxsens et al., 2017), using the chloroplast genome of Cousinia rotundifolia (PQ240609) as a seed reference. This procedure produced a complete circular plastome for each sampled species. To validate assemblies, filtered reads were aligned against their corresponding plastomes using BWA-MEM v0.7.17 (Li, 2013). Resulting alignments were processed, sorted, and indexed with SAMtools v1.19.2 (Li et al., 2009), and sequencing depth across sites was calculated within the same package. Genome annotation was conducted through GeSeq (Tillich et al., 2017) with reference guidance from C. rotundifolia . Gene boundaries were subsequently verified and, where necessary, adjusted in Geneious v9.0.2 (Kearse et al., 2012), with particular attention to coding region start/stop positions and intron–exon structures. The physical representation of each plastome was visualized using Chloroplot (Zheng et al., 2020). Comparative genomic analyses Codon usage patterns and potential biases were investigated across sixteen complete Cousinia plastomes. Protein-coding regions were retrieved using Biopython v1.83 (Cock et al., 2009). To avoid redundancy, one copy of genes duplicated within the inverted repeat regions was retained. All sequences were verified for correct open reading frames and screened to exclude internal stop codons. Codon counts were derived from concatenated coding regions for each species. Termination codons (TAA, TAG, TGA), amino acids encoded by a single codon (Met and Trp), and the infrequently used cp arginine codons (AGA, AGG) were omitted from subsequent calculations. Relative Synonymous Codon Usage (RSCU) indices were then determined to assess synonymous codon preference. Levels of nucleotide diversity (Pi) across plastomes were evaluated in DnaSP v6.11 (Rozas et al., 2017) using a sliding window approach with an 800 bp window and 200 bp increment. Microsatellite loci were detected using MISA (Beier et al., 2017), applying minimum repeat thresholds of 10 for mononucleotides, 5 for dinucleotides, 4 for trinucleotides, and 3 for tetra-, penta-, and hexanucleotide motifs. For comparative analyses, SSR frequencies were compiled into a species-by-motif matrix. Compositional differences among taxa were quantified using Bray–Curtis distances (Bray and Curtis, 1957), followed by hierarchical clustering under the Ward D2 criterion (Ward, 1963; Murtagh and Legendre, 2014) to generate a phenetic dendrogram based on microsatellite profiles. Phylogenetic analysis Phylogenetic reconstruction was based on two independent molecular datasets: complete chloroplast genome sequences and nuclear ribosomal ITS regions. The plastome dataset included 19 taxa, comprising nine newly sequenced species and ten accessions retrieved from GenBank. Arctium lappa , Jurinea auriculata , and Dolomiaea wardii were designated as outgroups. The ITS matrix consisted of 18 taxa (nine newly generated and nine obtained from GenBank), with Carduus acanthoides , Erigeron philadelphicus , and Atractylodes lancea selected for rooting purposes. The plastome alignment covered 154,308 bp, including 1,995 variable sites and 764 parsimony-informative positions. The ITS alignment spanned 5,844 bp, of which 195 sites were variable and 88 parsimony-informative. Alignments were manually checked, and insertion–deletion events were treated as missing data. Each dataset was analyzed independently. Maximum Likelihood analyses were implemented in RAxML v8.2.12 (Stamatakis, 2014). The plastome dataset was evaluated under the GTR+G substitution model with 1,000 bootstrap replicates. For the ITS matrix, jModelTest v2.1.10 (AICc criterion) selected GTR+I+G as the optimal model, which was subsequently applied in RAxML with 1,000 bootstrap replicates. Bayesian inference was performed using MrBayes v3.2.7 (Ronquist et al., 2012), running two independent MCMC analyses for 10 million generations with sampling every 1,000 generations. Convergence was confirmed when the average standard deviation of split frequencies fell below 0.01, and the initial 25% of sampled trees were discarded as burn-in. 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 Nine cp genomes were newly generated, representing species from eight taxonomic sections of Cousinia endemic to the Pamir–Alay mountain system. Plastome length was highly conserved across taxa, averaging approximately 152 kb, with an overall GC content of 37.7%. All genomes conformed to the conserved quadripartite organization typical of angiosperm chloroplasts, comprising a large single-copy (LSC) region, a small single-copy (SSC) region, and two homologous inverted repeat (IR) regions (Figure 2). Each of the nine plastomes encoded a total of 131 unique genes, comprising 87 protein-coding genes (PCGs), 36 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes, with no detectable variation in overall gene content among species. Eighteen loci were duplicated as a consequence of their position within the inverted repeat (IR) regions. These included seven tRNA genes ( trnA -UGC, trnI -CAU, trnI -GAU, trnL -CAA, trnN -GUU, trnR -ACG, trnV -GAC), four rRNA genes ( rrn16 , rrn23 , rrn4.5 , rrn5 ), and seven protein-coding genes ( ndhB , rpl2 , rpl23 , rps12 , ycf2 , ycf15 , rps7 ), consistent with the conserved organization of angiosperm plastomes. A total of sixteen genes contained introns, of which eleven were protein-coding ( atpF , rpoC1 , rpl2 , ndhB , ndhA , petB , petD , rps16 , rps12 , ycf3 , clpP ) and five were tRNA genes ( trnA -UGC, trnI -GAU, trnK -UUU, trnL -UAA, trnV -UAC). Among these, ycf3 and clpP harbored two introns each, whereas the remaining intron-bearing genes contained a single intron. Codon usage Analysis of RSCU across the chloroplast genomes of 16 Cousinia species showed highly conserved codon usage patterns, with only minor differences among species (Figure 3). Across all species, codon usage displayed a strong preference for A/T-ending codons, while G/C-ending codons were consistently underused. This AT bias was clear for nearly all amino acids and aligns with the AT-rich nucleotide makeup typical of cp genomes. Amino acids coded by two synonymous codons showed notable directional bias. For instance, AAT (Asn; RSCU ≈ 1.57) greatly outnumbered AAC (≈ 0.42), and GAT (Asp; ≈ 1.59) was clearly preferred over GAC (≈ 0.40). Similar trends were seen 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 coded by four or six synonymous codons also showed clear asymmetry in codon usage. For alanine, GCT was the most favored codon (RSCU ≈ 1.80), while GCC and GCG were underused (≈ 0.45–0.62). Glycine showed moderate preference, with GGT and GGA favored (≈ 1.33–1.53) and GGC underrepresented (≈ 0.49). Leucine showed one of the strongest preferences, with TTA (≈ 1.90) and TTG (≈ 1.22) being highly sought after, while CTG and CTC showed consistently low RSCU values (≈ 0.37–0.38). Similar A/T-ending codon preferences were evident 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 showed an uneven distribution of nucleotide diversity across the chloroplast genomes of the 16 Cousinia species studied. Overall Pi values were low, ranging from 0 to 0.00918. This indicates the highly conserved nature of cp genomes (Figure 4). However, several distinctly variable regions were found. Most variable nucleotides were concentrated in the LSC and SSC regions, while the IR regions displayed the highest stability and 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 peak of variability within the rbcL gene. In the SSC region, three notable hotspots were identified. These correspond 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 structural architecture of Cousinia plastomes, including their quadripartite configuration, approximate length (~152 kb), and overall GC proportion (37.7%)—aligns closely with patterns widely documented within Asteraceae (Mahai et al., 2024; Nyamgerel et al., 2024; Xing et al., 2025; Zhong et al., 2023). Both gene complement and intron distribution exhibit a high degree of uniformity across examined taxa, indicating strong structural conservation. Such stability is consistent with the evolutionary persistence typically reported for angiosperm chloroplast genomes (Jansen and Ruhlman, 2012), suggesting limited large-scale genomic restructuring within the genus. Assessment of synonymous codon usage across the chloroplast genomes of sixteen Cousinia species demonstrated a strongly conserved pattern characterized by a pronounced preference for A/T-terminating codons. Such compositional asymmetry is a common feature of angiosperm plastomes and corresponds to their overall AT-rich nucleotide composition (Parvathy et al., 2022). Codons ending in G or C were comparatively infrequent, indicating that base composition exerts a substantial influence on codon selection. This tendency was particularly apparent in amino acids such as leucine, lysine, and alanine, where A/T-ending codons predominated, a pattern consistent with mutational bias operating under predominantly neutral evolutionary processes (Zhang et al., 2007). At the same time, the near-uniformity of RSCU profiles across sampled taxa suggests the presence of evolutionary constraints maintaining translational stability. Such conservation may reflect selective pressures that favor codons contributing to efficient and accurate protein synthesis within the cp translational system (Li et al., 2025). Sliding-window evaluation of nucleotide diversity demonstrated that Cousinia plastomes exhibit a high degree of sequence conservation, with Pi values ranging from 0 to 0.00918. These estimates fall within the range reported for other Cardueae genera, including Saussurea (He et al., 2023), indicating comparable levels of plastome stability within thistle-like lineages. Regions exhibiting elevated variability— trnK -UUU– rps16 , trnE -UUC– rpoB , trnS -GGA– rps4 , trnL -UAA– trnF -GAA– ndhJ , rbcL , ndhF , rpl32 – trnL -UAG, and ycf1 —coincide with loci frequently identified as rapidly evolving in Asteraceae (Shen et al., 2020; Kim et al., 2024). The recurrence of these divergence hotspots across related genera highlights their potential utility as informative markers for species discrimination and phylogenetic inference. Accordingly, these regions represent promising candidates for future plastid-based barcoding initiatives and for reconstructing the historical biogeography of Cousinia . Microsatellite screening of the plastomes identified eleven distinct SSR motif categories across the examined Cousinia species. Mononucleotide repeats composed of A/T bases represented the dominant class, whereas C/G-rich motifs occurred infrequently and were completely absent in several taxa. Such enrichment of A/T repeats is a well-documented characteristic of angiosperm chloroplast genomes and reflects their intrinsic nucleotide composition bias. The scarcity of C/G-type repeats is consistent with broader patterns of cp genome evolution, where codon preference and base composition collectively shape sequence architecture. The predominance of A/T-rich motifs may therefore arise from the combined effects of codon usage tendencies—favoring A- or U-ending codons—and underlying mutational biases (Zhang et al., 2023). Additionally, selective constraints acting on genome stability may further reinforce the retention of A/T-enriched repeat structures within cp DNA. Phylogenetic reconstruction based on cp genomes did not recover all morphologically circumscribed Cousinia sections as monophyletic, indicating discordance between cp-derived topologies and traditional sectional classifications. Such incongruence may reflect processes including chloroplast capture, historical introgression, or incomplete lineage sorting. Given the predominantly maternal inheritance of cp DNA, chloroplast-based phylogenies may capture only a partial representation of species evolutionary history, particularly in groups where hybridization and reticulate evolution are prevalent (Rieseberg and Soltis, 1991; Soltis and Kuzoff, 1995; Soltis & Soltis, 1998; Tsitrone et al., 2003). Evidence of interspecific hybridization within Cousinia , occurring both within and between major clades, further supports this interpretation (Mehregan and Kadereit, 2009). In contrast, the nuclear ITS dataset yielded a topology more consistent with morphology-based sectional delimitations, although nodal support values were generally lower. An exception was Cousinia stellaris (section Alpinae ), which grouped with representatives of section Regelianae in both cp and nuclear reconstructions. This consistent placement across datasets suggests that C. stellaris may be misassigned within the current sectional framework. The closer correspondence between ITS phylogeny and morphological classification likely reflects the biparental inheritance of nuclear markers, which can provide a more comprehensive representation of organismal evolutionary history (Álvarez and Wendel, 2003; Feliner and Rosselló, 2007). Together, these findings underscore the importance of integrating cp and nuclear evidence when reconstructing phylogenetic relationships in taxonomically complex and potentially reticulate plant lineages. 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). 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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 24 February 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Ecology and Evolution 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 243 views 89 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Bobur Karimov, Diyorjon Hamrayev, Husniddin Esanov, et al. 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