DNA barcoding and metabolite profiling reveal taxonomic and phytochemical diversity in Sonchus spp.

Código de barras de DNA e perfil de metabólitos revelam diversidade taxonômica e fitoquímica em Sonchus spp.

Abstract

Accurate species identification is fundamental to biodiversity studies, conservation, and the utilization of medicinal plants. The genusSonchus(Asteraceae) presents significant taxonomic challenges due to morphological plasticity and potential hybridization. DNA barcoding using the chloroplastrbcLand nuclear ribosomal ITS2 regions was used to delimit threeSonchusspecies (S. arvensis,S. oleraceus, andS. malayanus) collected from Bromo Mountain, Indonesia. To complement molecular data and explore chemotaxonomic markers, leaf metabolite profiles were analyzed using gas chromatography-mass spectrometry (GC-MS). TherbcLmarker showed high sequence conservation (99.58-100% identity) across species, confirming genus-level identification but failing to resolve species boundaries. The ITS2 region exhibited moderate variation (98.03-100% identity), with substantial overlap amongS. asper,S. oleraceus, andS. malayanus, suggesting recent divergence or possible conspecificity. Neighbor-joining phylogenetic analysis of both markers clustered accessions into broad clades corresponding to continental and island lineages, though low intraspecific divergence obscured taxonomic resolution. In contrast, GC-MS profiling revealed distinct species-specific chemotypes.Sonchus arvensiswas characterized by high abundances of neophytadiene (27.46%) and phytol (13.22%).Sonchus oleraceuscontained phytol (23.28%) as the major compound, with lower levels of neophytadiene (6.59%) and several methylated fatty acid esters.Sonchus malayanusdisplayed a distinct profile dominated by compounds with lower identification confidence, including (E)-4-trimethylsilyl-3-buten-2-ol (19.21%), alongside confidently identified guaiacol and trimethylamine. The discordance between molecular and chemical data underscores the limitations of single-locus barcoding in closely relatedSonchusspecies. This study demonstrates that integrative taxonomy, combining molecular, phytochemical, and morphological evidence, provides a more robust framework for species delimitation in this genus. The identified volatile compounds, particularly neophytadiene and phytol, represent promising chemotaxonomic markers and warrant further investigation for their ecological and pharmacological significance.

Keywords:
Asteraceae; chemotaxonomy; DNA barcoding; GC-MS; lowland; integrative taxonomy; Sonchus

Resumo

A identificação precisa de espécies é fundamental para estudos de biodiversidade, conservação e utilização de plantas medicinais. O gênero Sonchus (Asteraceae) apresenta desafios taxonômicos significativos devido à plasticidade morfológica e à possibilidade de hibridização. O uso de código de barras de DNA, com as regiões cloroplastídica rbcL e ribossômica nuclear ITS2, foi avaliado para delimitar três espécies de Sonchus (S. arvensis, S. oleraceus e S. malayanus) coletadas no Monte Bromo, na Indonésia. Para complementar os dados moleculares e explorar marcadores quimiotaxonômicos, os perfis de metabólitos foliares foram analisados ​​por meio de cromatografia gasosa acoplada à espectrometria de massas (GC-MS). O marcador rbcL apresentou alta conservação de sequência (99,58-100% de identidade) entre as espécies, confirmando a identificação em nível de gênero, mas não permitindo distinguir os limites entre as espécies. A região ITS2 exibiu variação moderada (98,03-100% de identidade), com sobreposição substancial entre S. asper, S. oleraceus e S. malayanus, sugerindo divergência recente ou possível coespecificidade. A análise filogenética por Neighbor-Joining de ambos os marcadores agrupou as amostras em grandes clados correspondentes a linhagens continentais e insulares, embora a baixa divergência intraespecífica tenha dificultado a resolução taxonômica. Em contrapartida, a análise dos perfis por GC-MS revelou quimiotipos distintos e específicos de cada espécie. Sonchus arvensis caracterizou-se por altas abundâncias de neofitadieno (27,46%) e fitol (13,22%). Sonchus oleraceus apresentou fitol (23,28%) como composto majoritário, com menor teor de neofitadieno (6,59%) e vários ésteres metílicos de ácidos graxos. Sonchus malayanus exibiu um perfil distinto, dominado por compostos com menor confiança de identificação, incluindo (E)-4-trimetilsilil-3-buten-2-ol (19,21%), juntamente com guaiacol e trimetilamina, identificados com maior segurança. A discordância entre os dados moleculares e químicos ressalta as limitações do uso de código de barras de locus único em espécies de Sonchus estreitamente relacionadas. Este estudo demonstra que a taxonomia integrativa, combinando evidências moleculares, fitoquímicas e morfológicas, fornece uma estrutura mais robusta para a delimitação de espécies nesse gênero. Os compostos voláteis identificados, particularmente o neofitadieno e o fitol, representam marcadores quimiotaxonômicos promissores e justificam investigações adicionais devido à sua relevância ecológica e farmacológica.

Palavras-chave:
Asteraceae; quimiotaxonomia; código de barras de DNA; GC-MS; terras baixas; taxonomia integrativa; Sonchus

1. Introduction

The genusSonchusL. (Cichorieae: Asteraceae), commonly known as sow thistles, comprises approximately 50-60 recognized species of herbaceous plants distributed across temperate and tropical regions worldwide (Boulos, 1972;Mejías et al., 2018). Members of this genus exhibit remarkable ecological plasticity, colonizing disturbed habitats such as grasslands, roadsides, and agricultural areas (Boulos, 1972; Kim et al., 2007). In Southeast Asia, particularly Indonesia,Sonchus arvensisL.,Sonchus oleraceusL., andSonchus malayanusMiq. are widespread and often considered invasive (Padmanaba et al., 2013; Tjitrosoedirdjo et al., 2005).

Beyond their ecological significance, severalSonchusspecies hold considerable ethnobotanical importance (Wahyuni et al., 2023a). In Indonesian traditional medicine,S. arvensisis used to treat liver disorders, inflammation, and kidney stones (Suwartiny et al., 2022). Phytochemical investigations have revealed diverse secondary metabolites in this species, including flavonoids, saponins, alkaloids, phenolic acids, and terpenoids, which contribute to its reported anti-inflammatory, antioxidant, diuretic, and hepatoprotective properties (Khan, 2012; Suwartiny et al., 2022;Aldeeb et al., 2025). Similarly,S. oleraceusis consumed as a vegetable and used in traditional medicine for gastrointestinal ailments and wound healing, with bioactive compounds including luteolin, apigenin, chlorogenic acid, and β-sitosterol (Chen et al., 2021; Salim et al., 2023). In contrast,S. malayanusremains poorly studied phytochemically, though preliminary evidence suggests the presence of phenolics and terpenoids with potential antibacterial and hypolipidemic activities.

Accurate species identification is a prerequisite for understanding biodiversity, evolutionary relationships, and the medicinal potential of individual taxa. However, the morphological plasticity exhibited bySonchusspecies, coupled with possible hybridization and ecological overlap, often renders identification using traditional morphological keys challenging (Purnobasuki and Rakashiwi, 2022; Wahyuni et al., 2019). This challenge is particularly acute in dynamic landscapes such as Bromo Mountain in East Java, where volcanic disturbance and environmental heterogeneity create mosaic habitats that promote the coexistence and rapid spread of weedy taxa (Wahyuni et al., 2023b).

DNA barcoding has emerged as a powerful tool for species identification in taxonomically complex plant groups. The Consortium for the Barcode of Life (CBOL) has recommended the combination ofrbcLandmatKas core plant barcodes, with the nuclear ribosomal internal transcribed spacer (ITS) region serving as a supplementary marker (Hollingsworth et al., 2009, 2011). Within Asteraceae, plastid genes such asrbcLandmatKhave been widely employed due to their universality and phylogenetic signal (Hollingsworth et al., 2016; Wahyuni et al., 2019; Habila and Wahyuni, 2025). The ITS2 region often provides higher discriminatory power at the species level due to its faster evolutionary rate (Burgess et al., 2011; Hollingsworth et al., 2011, 2016).

Metabolite profiling using gas chromatography-mass spectrometry (GC-MS) offers a complementary approach to molecular data by revealing phytochemical diversity that may reflect underlying genetic differences or ecological adaptations (Tlili et al., 2011; Kessler and Kalske, 2018). When integrated with DNA barcoding, such chemotaxonomic data can enhance species characterization and provide insights into the functional significance of secondary metabolites (Aldeeb et al., 2025; Ilham et al., 2022).

The present study aims to: (1) evaluate the effectiveness ofrbcLand ITS2 DNA barcoding for delimiting three closely relatedSonchusspecies (S. arvensis,S. oleraceus, andS. malayanus) from Bromo Mountain, East Java; (2) characterize and compare the volatile metabolite profiles of these species using GC-MS; and (3) assess the congruence between molecular and chemical data to inform an integrative taxonomic approach for this genus.

2. Materials and Methods

2.1. Plant material

Leaf samples of threeSonchusspecies were collected from Bromo Mountain, East Java, Indonesia, in October 2025 (collection permits were obtained from the Bromo Tengger Semeru National Park Authority). The species were identified asSonchus arvensisL.,Sonchus oleraceusL., andSonchus malayanusMiq. based on morphological characteristics following Backer and Bakhuizen van den Brink (1965). Voucher specimens were prepared for each species and deposited at the Herbarium of the Laboratory of Plant Systematics, Department of Biology, Faculty of Science and Technology, Universitas Airlangga. Collection details are as follows:

1. Sonchus oleraceus: Payaman Andonosari, Tutur, Pasuruan (7°54’26.53” S, 112°50’9.06” E; 1258 m a.s.l.); voucher SO02102025

2. Sonchus malayanus: Sugro Andonosari, Tutur, Pasuruan (7°54’46.56” S, 112°50’17.26” E; 1327 m a.s.l.); voucher SM03102025

3. Sonchus arvensissubsp.arvensis: Payaman Andonosari, Tutur, Pasuruan (7°54’27.33” S, 112°50’8.16” E; 1258 m a.s.l.); voucher SA01102025

Species identification was confirmed by Mr. Dwi Narko, botanist at the Department of Biology, Universitas Airlangga.

2.2. DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from approximately 80 mg of fresh leaf tissue using the Plant Genomic DNA Kit (Tiangen, China) following the manufacturer's protocol. DNA quality and integrity were assessed by electrophoresis on 1% agarose gels (Promega, USA) stained with GelRed and visualized under UV transillumination.

Two barcode regions were amplified: the chloroplastrbcLgene and the nuclear ribosomal ITS region (ITS4-5). Primer sequences were:

1. rbcLforward: 5'-AAGTTCCTCCACCGAACTGTAG-3'

2. rbcLreverse: 5'-TACTGCGGGTACATGCGAAG-3'

3. ITS forward (ITS5): 5'-GGAAGTAAAAGTCGTAACAAGG-3'

4. ITS reverse (ITS4): 5'-TCCTCCGCTTATTGATATGC-3'

PCR amplifications were performed in 35 µL reaction volumes containing 17.5 µL GoTaq® Green Master Mix (Promega, USA), 0.5 µM of each primer, 50 ng DNA template, and nuclease-free water. Thermal cycling conditions were initial denaturation at 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 56 °C for 45 s, and 72 °C for 45 s; and final extension at 72 °C for 5 min. PCR products were verified by electrophoresis on 1.5% agarose gels in 0.5× TAE buffer. Successful amplicons (50 µL) were purified and sequenced bidirectionally by Macrogen Inc. (Seoul, Korea) using the same primers.

2.3. Sequence analysis and phylogenetic reconstruction

Raw sequences were assembled and edited using BioEdit version 7.2 (Hall, 1999). Consensus sequences were compared against the GenBank non-redundant nucleotide database using BLASTn (https://blast.ncbi.nlm.nih.gov) to identify the closest matches. For each sequence, the top five matches were retrieved based on maximum score and identity.

Sequences were aligned using ClustalW (Thompson et al., 1994) implemented in BioEdit. Phylogenetic analyses were conducted using the Neighbor-Joining (NJ) method with the Maximum Composite Likelihood model in MEGA version 11 (Kumar et al., 2016). Bootstrap support was assessed with 1000 replicates. Reference sequences from GenBank representing relatedSonchusspecies and outgroup taxa were included in the analyses.

2.4. Ethanol extraction of leaf material

Fresh leaves of eachSonchusspecies were cleaned, shade-dried at ambient temperature (25 ± 2 °C), and ground into fine powder. Ten grams of powdered leaf material from each species were macerated separately in 100 mL of absolute ethanol (Merck, Germany) for 24 h at room temperature with occasional shaking. The extraction was repeated three times. The combined extracts were filtered through Whatman No. 1 filter paper and concentrated under reduced pressure at 50 °C using a rotary evaporator. The resulting crude extracts were dried to constant weight at ambient temperature and stored at 4 °C in airtight containers until analysis.

2.5. Gas chromatography-mass spectrometry (GC-MS) analysis

GC-MS analysis was performed using an Agilent 7890B gas chromatograph coupled to an Agilent 5977A mass selective detector (Agilent Technologies, Germany). The extract was dissolved in ethanol to a final concentration of 0.2 g/mL, and 1 µL was injected in splitless mode. Chromatographic separation was achieved on an HP-5MS capillary column (30 m × 250 µm × 0.25 µm film thickness) with helium (99.9999% purity) as carrier gas at a constant flow rate of 1.5 mL/min. The oven temperature program was an initial hold at 150 °C for 2 minutes, a ramp to 250 °C at 20° C/minute, and a final hold at 250 °C for 5 minutes. The injector temperature was 300°C, and the transfer line was maintained at 280 °C.

Mass spectra were recorded in electron impact (EI) mode at 70 eV ionization energy, with a scan range of 50-550 m/z. The ion source temperature was 230 °C, and the quadrupole temperature was 150 °C.

Compound identification was performed by comparing mass spectra with the National Institute of Standards and Technology (NIST) Mass Spectral Library (version 14). Only compounds with match factors ≥80% (Quality score ≥80) were considered positively identified. Compounds with Quality scores <80 are reported with caution and noted as tentative identifications. For key compounds (neophytadiene, phytol, 9,12,15-octadecatrienoic acid methyl ester, and hexadecanoic acid methyl ester), identification was confirmed by co-injection with authentic standards (Sigma-Aldrich, USA) under the same analytical conditions. These compounds are marked with a red dagger (†) in the chromatograms. Relative peak areas (percentage of total ion current) were calculated for each identified compound.

2.6. Data analysis

Phylogenetic trees were visualized and annotated using MEGA 11. Heatmaps of BLAST identity percentages were generated using Microsoft Excel. Chemical profiles were compared qualitatively based on the presence/absence of compounds and their relative abundances. No statistical comparisons were made due to the absence of biological replicates (n=1 pooled sample per species).

3. Results

3.1. Morphology of Sonchus arvensis L., Sonchus oleraceus L., and Sonchus malayanus Miq.

3.1.1. Sonchus arvensis L.

Sonchus arvensis L. is an herb (Figure 1). Taproot strong. The stem in the upper part is often glandular, bristly, more than 1 m in height, fistulous with white, milky juice inside, and ribbed surface. Leaves alternate, amplexicaul, lower leaves approximate, higher ones remote, all oblong, spatulate-oblong or lyrate-pinnately lobed, with a large terminal lobe, a sagittate base and rounded basal auricles, mucronate at the apex, acutely shortly dentate, pale green beneath, length up to 25 cm, width up to 5 cm, upper ones smaller. Involucre 1,5-2 cm long; bracts linear-lanceolate, obtuse, outer ones densely clothed with coarse gland-bristles, 3-seriate; heads combined into widely branched corymbs; peduncles 2 cm long, covered with glandular-bristles; corolla ligulate, yellow, 1 cm long, often turning brownish-red with age, with a long-filiform tube and a narrow ligule, with a 5-dentate top. Achenes narrowly oblong, narrowed at both ends, laterally compressed, ribbed, glabrous, brown, length up to 4 mm, on both surfaces 5-ribbed, by the presence of fine tubercles on the ribs, transversely rugose. Pappus 1 cm long, numerous pappus hairs, thin, rough, white, connate at the base, often unequal.

Figure 1
(A) Habit of Sonchus arvensis; (B) Leaves of Sonchus arvensis; (C) Flower of Sonchus arvensis. Scale bar: 10 cm.

3.1.2. Sonchus oleraceus L.

Sonchus oleraceus L. is an herb (Figure 2). Taproot strong. Stem obviously 5-ridged, purple, usually glabrous, at the top sometimes with a few gland-hairs, fistular with white milky juice inside, ribbed surface. Leaves alternate, length up to c. 20 cm, width up to c. 5 cm, higher ones sessile, amplexicaul, leaves oblong-obovate or lyrate, sagittate base and acute until rounded apex, very acute irregularly dentate, glaucous on the lower surface, lowermost radical, more or less narrowed into petiole, pinnatifid with irregular aciculate-dentate lobes; teeth of leaf-margins not pungent, basal auricles of leaves usually with a tooth obliquely pointing downwards. Involucre c. 2.5 cm long, without or with very few gland-bristles; bracts obtuse, glabrous or with very few gland-hairs; peduncle length up to c. 7 cm; corolla c. 2 cm long, light-yellow, sometimes tinged with purple, ligulate, with a long-filiform tube and a narrow ligule, with a 5-dentate top. Achenes narrowly oblong, narrowed at both ends, laterally compressed, ribbed, glabrous, brown, 2-3 mm long, on both surfaces 3-ribbed, transversely rugose by the presence of minute tubercles on the ribs; pappus 1 cm long, numerous pappus hairs, thin, rough, white, connate at the base, often unequal.

Figure 2
(A) Habit of Sonchus oleraceus; (B) Leaves of Sonchus oleraceus; (C) Flower of Sonchus oleraceus. Scale bar: 10 cm.

3.1.3. Sonchus malayanus Miq.

It is an herb (Figure 3) with a strong taproot. Stem in the upper part is often glandular-hairy, branches erect or ascending, fistular with white milky juice inside, ripped on the surface. Leaves alternate, higher ones sessile, amplexicaul, sagittate base and acute apex, with rounded or acute basal auricles, length up to 20 cm, width up to 7 cm, lower and medium leaves linear-lanceolate, upper ones lanceolate-sagittate, margin spinose-revolute. Involucre c.3 cm long; bracts with a distinct midrib, acute, glabrous or sometimes with some gland-hairs on the midrib, outer ones ovate; branched heads thinly corymbose, multiple branches like 4-7 branches or more; peduncle 3 cm long, sometimes with some gland-hairs, at first often white floccose; corolla ligulate, yellow, with a long-filiform tube and a narrow ligule, with a 5-dentate on top. Achenes narrowly oblong, narrowed at both ends, laterally compressed, ribbed, glabrous, brown, obscurely triquetrous, on each face 2-ribbed, by the presence of fine tubercles on the ribs, transversely rugose, numerous pappus hairs, thin, rough, white, connate at the base, often unequal.

Figure 3
(A) Habit of Sonchus malayanus; (B) Leaves of Sonchus malayanus; (C) Flower of Sonchus malayanus. Scale bar: 10 cm.

3.2. DNA barcoding and sequence similarity analysis

TherbcLand ITS2 regions were successfully amplified and sequenced from all threeSonchusspecies collected from Bromo Mountain. BLASTn analysis ofrbcLsequences (Table 1, Figure 4) revealed high similarity (99.58-100%) with multipleSonchusaccessions in GenBank, confirming genus-level identification but demonstrating limited resolution at the species level.Sonchus arvensisshowed 100% identity withS. asper,S. boulosii, andS. oleraceus, while exhibiting 99.58% similarity withS. canariensis. Similarly,S. malayanusdisplayed 100% identity withS. boulosiiand 99.80% identity withS. arvensis,S. asper,S. oleraceus, andS. wightianus. BothS. oleraceussamples ("kletak" and "sun kletak") showed 100% identity with multipleS. oleraceusandS. kirkiiaccessions, as well as 99.79% identity withS. arvensis.

Table 1
BLASTn analysis ofrbcLsequences fromSonchusspecies collected from Bromo Mountain, showing the closest GenBank matches.

Sample ID	Closest Match Species	GenBank Accession	% Identity	Max Score	Query Coverage	E-value
S. arvensis arvensis	S. asper	MF135322.1	100.00%	874	100%	0
	S. asper	NC_048510.1	100.00%	874	100%	0
	S. boulosii	MK016665.1	100.00%	872	100%	0
	S. canariensis	NC_042381.1	99.58%	863	100%	0
	S. oleraceus	MK371006.1	100.00%	872	100%	0
S. malayanus kletak	S. arvensis	MT435526.1	99.80%	898	100%	0
	S. asper	MK371016.1	99.80%	898	100%	0
	S. boulosii	NC_042244.1	100.00%	904	100%	0
	S. oleraceus	MN601475.1	99.80%	898	100%	0
	S. wightianus	JQ933486.1	99.80%	896	99%	0
S. oleraceus kletak	S. arvensis	JX848427.1	99.79%	867	100%	0
	S. kirkii	KT626829.1	100.00%	872	100%	0
	S. oleraceus	EU385018.1	100.00%	872	100%	0
	S. oleraceus	KM360989.1	100.00%	872	100%	0
	S. oleraceus	NC_048452.1	100.00%	872	100%	0
S. oleraceus sun kletak	S. acaulis	MK033507.1	99.59%	880	100%	0
	S. arvensis	MN601474.1	99.79%	885	100%	0
	S. asper	HM850372.1	100.00%	872	100%	0
	S. oleraceus	MH908962.1	100.00%	872	100%	0
	S. webii	NC_042383.1	99.59%	880	100%	0

• The parameters analyzed were percent identity, maximum alignment score, query coverage, and E-value.

Figure 4
Heatmap of BLASTn percentage identities forrbcLsequences ofSonchussamples from Bromo Mountain against GenBank reference sequences. Darker shades indicate higher similarity.

ITS2 sequences (Table 2, Figure 5) showed slightly lower overall similarity (98.03-100%) but similarly limited species-level resolution.Sonchus arvensisshowed 98.58% identity withS. arvensis(MG73059.1), 99.37% withS. oleraceus, and 98.03% with an unidentifiedSonchussp. Critically,S. malayanusexhibited 100% identity with multiple accessions of bothS. asperandS. oleraceus, indicating a complete lack of ITS2 differentiation between these taxa. BothS. oleraceussamples showed 99.40-100% identity withS. oleraceus,S. asper, andS. arvensisaccessions.

Table 2
BLASTn analysis of ITS2 sequences fromSonchusspecies collected from Bromo Mountain, showing closest GenBank matches.

Sample ID	Closest Match Species	GenBank Accession	% Identity	Max Score	Query Coverage	E-value
S. arvensisarvensis kletak	S. arvensis	DQ507957.1	98.21%	390	62%	3.00E-104
	S. arvensis	MG73059.1	98.58%	621	98%	9.00E-174
	S. arvensis	MH710813.1	95.60%	545	94%	5.00E-151
	S. oleraceus	MH050177.1	99.37%	573	88%	2.00E-159
	S. sp.	KF154363.1	98.03%	440	70%	3.00E-119
S. malayanus kletak	S. asper	KY700488.1	100.00%	652	98%	0
	S. asper	KY700494.1	100.00%	652	98%	0
	S. oleraceus	KX166543.1	100.00%	652	98%	0
	S. oleraceus	MG219232.1	100.00%	652	98%	0
	S. oleraceus	MK249725.1	100.00%	652	98%	0
S. oleraceus kletak	S. arvensis	MG256319.1	100.00%	547	81%	2.00E-151
	S. asper	MH808207.1	100.00%	558	83%	7.00E-155
	S. oleraceus	MG218471.1	100.00%	612	91%	5.00E-171
	S. oleraceus	MG219867.1	100.00%	612	91%	1.00E+00
	S. oleraceus	MH808210.1	100.00%	558	83%	7.00E-155
S. oleraceus sun kletak	S. arvensis	KU66679.1	99.56%	418	68%	1.00E-112
	S. arvensis	KU366680.1	99.56%	418	68%	1.00E+00
	S. asper	KY968828.1	99.70%	601	98%	1.00E-167
	S. oleraceus	AY862581.1	99.65%	518	84%	1.00E-142
	S. oleraceus	MK249725.1	99.40%	604	100%	8.00E-169

• The parameters analyzed were percent identity, maximum alignment score, query coverage, and E-value.

Figure 5
Heatmap of BLASTn percentage identities for ITS2 sequences ofSonchussamples from Bromo Mountain against GenBank reference sequences. Darker shades indicate higher similarity.

3.3. Phylogenetic relationships of Sonchus species

The neighbor-joining phylogenetic analysis ofrbcLsequences (Figure 6) resolved two major clades with moderate bootstrap support. The first clade comprised accessions ofS. asper,S. oleraceus,S. kirkii,S. boulosii, andS. wightianus. Within this group,S. asperandS. oleraceusformed distinct subclades (bootstrap 85-100%), indicating clear separation at the species level. The second major clade includedS. arvensis,S. webbii,S. acaulis, andS. canariensis, together with all three Bromo Mountain samples (S. arvensis,S. oleraceus, andS. malayanus). Branch lengths within this clade were short (<0.005 substitutions/site), indicating low sequence divergence among these accessions. The two major clades were separated by a branch length of 0.009 substitutions/site.

Figure 6
Neighbor-joining phylogenetic tree based onrbcLsequences ofSonchusspecies from Bromo Mountain and GenBank references. Bootstrap values (≥50%) from 1000 replicates are shown at nodes. The scale bar indicates the number of substitutions per site.

The ITS2-based phylogeny (Figure 7) similarly resolved two major clades but with generally lower bootstrap support. One clade containedS. oleraceus,S. asper,S. malayanus, and the BromoS. oleraceusaccessions, with minimal sequence divergence among them. Notably,S. malayanuswas embedded within theS. oleraceus/S. aspercluster with zero branch length, indicating identical ITS2 sequences. The second clade comprisedS. arvensisaccessions, including the BromoS. arvensissample, with branch lengths of 0.008-0.012 substitutions/site separating them from theS. oleraceusclade. One unidentifiedSonchussp. sequence (KF154363.1) grouped with theS. arvensisclade.

Figure 7
Neighbor-joining phylogenetic tree based on ITS2 sequences ofSonchusspecies from Bromo Mountain and GenBank references. Bootstrap values (≥50%) from 1000 replicates are shown at nodes. The scale bar indicates the number of substitutions per site.

3.4. GC-MS metabolite profiling in Sonchus arvensis, S. malayanus, and S. oleraceus leaf extracts

GC-MS analysis revealed distinct volatile metabolite profiles for eachSonchusspecies (Table 3, Figure 8). A total of 12, 31, and 32 peaks were detected inS. arvensis,S. malayanus, andS. oleraceus, respectively. Only compounds with NIST library match factors ≥80% (Quality score ≥80) are reported as confidently identified in Table 3; lower-confidence identifications are noted separately.

Table 3
Major volatile compounds identified by GC-MS in ethanol extracts ofSonchusleaves. Only compounds with NIST library match factor ≥80% (Quality ≥80) are included.

RT (min)	Compound	CAS Number	S. arvensisArea %	S. malayanusArea %	S. oleraceusArea %	Quality Score
1.51	Trimethylamine	75-50-3	-	4.12	-	86
7.75	Phenol, 2-methoxy- (Guaiacol)	90-05-1	-	1.52	-	93
11.11	2-Methoxy-4-vinylphenol	7786-61-0	-	-	4.59	93
13.34	Cycloheptasiloxane, tetradecamethyl-	107-50-6	-	3.39	-	90
17.04	2-Cyclohexen-1-one	930-68-7	-	4.12	-	80
17.47	Neophytadiene	504-96-1	27.46	-	6.59	97-99
17.57	2-Hexadecene, 3,7,11,15-tetramethyl-	14237-73-1	10.13	-	-	94
17.57	3,7,11,15-Tetramethylhexadec-2-ene	55162-49-3	-	-	5.41	89
17.84	Neophytadiene (isomer)	504-96-1	12.58	-	1.73	84-86
18.12	3-Methylene-7,11-dimethyl-1-dodecene	55162-48-2	9.11	-	-	86
18.83	Hexadecanoic acid, methyl ester	112-39-0	-	-	3.92	98
18.96	Velleral	37841-91-3	1.81	-	-	80
22.76	8,11-Octadecadienoic acid, methyl ester	56554-60-4	-	-	1.44	93
22.96	9,12,15-Octadecatrienoic acid, methyl ester	301-00-8	4.22	-	4.6	97-99
23.32	Phytol	150-86-7	13.22	-	23.28	90-91

• Note: Several additional peaks were detected inS. malayanuswith higher relative abundances (e.g., RT 14.91 min, 17.96%; RT 15.10 min, 19.21%; RT 15.36 min, 6.39%) but had Quality scores <80 and are therefore reported as tentative identifications only (see Supplementary File 1).

Figure 8
GC-MS chromatograms of ethanol leaf extracts from (A)Sonchus arvensis, (B)Sonchus malayanus, and (C)Sonchus oleraceus. Major peaks corresponding to compounds in Table 3 are labeled. The x-axis shows retention time (min); the y-axis shows total ion current (arbitrary units). Retention times corresponding to major peaks are displayed above each peak. The red dagger (†) denotes compounds confirmed by co-injection with authentic standards. All other identifications are based on NIST library matching with a match factor ≥80. (A) S. arvensis — major peaks: A = 2-Hexadecene, B = Neophytadiene, C = 2-Hexadecene, D = Neophytadiene, E = 3-Methylene-7, F = Velleral, G = 9,12,15-Octadecatrienoic acid, and H = Phytol; (B) S. malayanus — major peaks: A = Trimethylamine, B = Phenol, C = Cycloheptasiloxane, and D = 2-Cyclohexen-1-one; (C) S. oleraceus — major peaks: A = 2-Methoxy-4-vinylphenol, B = Neophytadiene, C = 3,7,11,15-Tetramethylhexadec-2-ene, D = Neophytadiene, E = Neophytadiene, F = Hexadecanoic acid, G = 8,11-Octadecadienoic acid, H = 9,12,15-Octadecatrienoic acid, and I = Phytol.

Sonchus arvensis(Figure 8A) was characterized by high abundances of diterpenes. The predominant compound was neophytadiene (retention time [RT] 17.47 min), comprising 27.46% of the total chromatogram area (Quality score 97). Phytol (RT 23.33 min) was the second most abundant compound (13.22%, Quality 90). Other confidently identified compounds included a neophytadiene isomer (RT 17.84 min, 12.58%, Quality 84), 2-hexadecene, 3,7,11,15-tetramethyl- (RT 17.57 min, 10.13%, Quality 94), 3-methylene-7,11-dimethyl-1-dodecene (RT 18.12 min, 9.11%, Quality 86), and 9,12,15-octadecatrienoic acid methyl ester (RT 22.96 min, 4.22%, Quality 97). Velleral (RT 18.96 min, 1.81%, Quality 80) was also detected.

Sonchus malayanus(Figure 8B) exhibited a distinct profile dominated by several compounds with low identification confidence. The most abundant peak (RT 15.10 min, 19.21%) corresponded to (E)-4-trimethylsilyl-3-buten-2-ol, but its low-Quality score (25) renders this identification tentative. Similarly, peaks at RT 14.91 min (17.96%, Quality 35) and RT 15.36 min (6.39%, Quality 45) could not be confidently identified. Four compounds were confidently identified (Quality ≥80): trimethylamine (RT 1.51 min, 4.12%, Quality 86), guaiacol (2-methoxyphenol; RT 7.75 min, 1.52%, Quality 93), cycloheptasiloxane tetradecamethyl- (RT 13.34 min, 3.39%, Quality 90), and 2-cyclohexen-1-one (RT 17.04 min, 4.12%, Quality 80).

Sonchus oleraceus(Figure 8C) showed a profile dominated by phytol (RT 23.32 min, 23.28%, Quality 91), with lower abundances of neophytadiene (RT 17.47 min, 6.59%, Quality 99) and related compounds. Other confidently identified compounds included 9,12,15-octadecatrienoic acid methyl ester (RT 22.96 min, 4.60%, Quality 99), hexadecanoic acid methyl ester (RT 18.83 min, 3.92%, Quality 98), 2-methoxy-4-vinylphenol (RT 11.11 min, 4.59%, Quality 93), 8,11-octadecadienoic acid methyl ester (RT 22.76 min, 1.44%, Quality 93), and 3,7,11,15-tetramethylhexadec-2-ene (RT 17.57 min, 5.41%, Quality 89).

4. Discussion

This study employed an integrative approach combining DNA barcoding and GC-MS metabolomic profiling to assess taxonomic boundaries and phytochemical diversity among threeSonchusspecies from Bromo Mountain, Indonesia. Our results reveal a striking discordance between molecular and chemical data, highlighting both the limitations of single-locus barcoding in recently diverged lineages and the potential of chemotaxonomic markers for species discrimination.

The high sequence conservation observed in therbcLgene (99.58-100% identity across species) confirms its utility for genus-level identification but demonstrates its inadequacy for resolving closely relatedSonchusspecies, consistent with previous studies in Asteraceae (Mejías et al., 2018; Wahyuni et al., 2019, 2020; Habila and Wahyuni, 2025). The complete identity betweenS. arvensisand multiple otherSonchusspecies (S. asper,S. boulosii,S. oleraceus) underscores the limited discriminatory power of this plastid marker.

The ITS2 region, despite its faster evolutionary rate, showed similarly limited resolution, withS. malayanusexhibiting 100% sequence identity to bothS. asperandS. oleraceus. This finding suggests either very recent divergence among these taxa, ongoing gene flow, or potential conspecificity. The phylogenetic trees further support this interpretation, withS. malayanusembedded within theS. oleraceus/S. aspercluster with zero branch length in the ITS2 analysis. Similar patterns of incomplete lineage sorting or hybridization have been reported in other Asteraceae genera (Hodac̆ et al., 2016; Kim et al., 2007).

The two major clades resolved by both markers, one containingS. asper, S. oleraceus, S. malayanus, and the other containing S. arvensis with island endemics, may reflect broader biogeographic patterns, possibly corresponding to continental versus island lineages as suggested by Mejías et al. (2018). However, the low bootstrap support for many nodes and short branch lengths within clades indicate that additional markers (e.g., plastid genomes, nuclear single-copy genes) are needed to fully resolve evolutionary relationships in this genus.

In contrast to the molecular data, GC-MS profiling revealed distinct species-specific chemical profiles that may serve as chemotaxonomic markers.Sonchus arvensiswas characterized by high levels of neophytadiene (27.46%) and phytol (13.22%), consistent with previous reports of diterpene accumulation in this species (Khan, 2012). Neophytadiene, a diterpene hydrocarbon, has been reported to be a major volatile constituent in several Asteraceae species and may play roles in plant defense and pollinator attraction (Tlili et al., 2011).

Sonchus oleraceusexhibited a profile dominated by phytol (23.28%) with lower neophytadiene (6.59%) and a greater diversity of methylated fatty acid esters (e.g., hexadecanoic acid methyl ester, 9,12,15-octadecatrienoic acid methyl ester). Phytol, a diterpene alcohol and chlorophyll breakdown product, has demonstrated antioxidant and anti-inflammatory activities that may contribute to the medicinal properties ofS. oleraceus(Salim et al., 2023; Chen et al., 2021). The presence of 2-methoxy-4-vinylphenol, a phenolic compound with antimicrobial properties, further distinguishesS. oleraceusfrom the other species.

Sonchus malayanuspresented the most distinctive profile, though interpretation is complicated by low-confidence identifications for its major peaks. The confidently identified compounds—trimethylamine, guaiacol, cycloheptasiloxane, and 2-cyclohexen-1-one—were unique to this species. Guaiacol (2-methoxyphenol) is a phenolic compound with antimicrobial and antioxidant properties, while trimethylamine is a simple amine that may contribute to the plant's volatile signature. The absence of neophytadiene and phytol, which dominated the other two species, suggests fundamental differences in terpenoid metabolism, possibly reflecting distinct ecological adaptations or phylogenetic placement (Kessler and Kalske, 2018).

The identification of several high-abundance peaks inS. malayanuswith low Quality scores (<80) represents a significant limitation of this study. These compounds could not be confidently identified using the NIST library and may represent novel or uncommon metabolites in this understudied species. Future studies employing high-resolution mass spectrometry or nuclear magnetic resonance spectroscopy would be valuable for structural elucidation.

The discordance between molecular similarity (high ITS2 identity betweenS. malayanusandS. oleraceus) and chemical dissimilarity (distinct volatile profiles) raises important questions about the forces shaping genetic and metabolic diversity inSonchus. Several explanations are possible:

First, the ITS2 region may simply lack sufficient variation to distinguish recently diverged species, despite their accumulation of chemical differences. Metabolic pathways, particularly those involved in secondary metabolism, can evolve rapidly in response to ecological selection pressures without concomitant changes in neutral genetic markers (Bohlmann et al., 1998; Agrawal and Fishbein, 2006).

Second, the observed chemical differences may reflect phenotypic plasticity in response to local environmental conditions rather than fixed genetic differences. Although all samples were collected from Bromo Mountain, microhabitat variation could influence metabolite profiles (Tlhapi et al., 2024). Replicate sampling across multiple populations and environments would be necessary to distinguish fixed chemotaxonomic markers from environmentally induced variation.

Third, the possibility of hybridization or introgression cannot be excluded. IfS. malayanusandS. oleraceushybridize in sympatry, nuclear markers like ITS2 might show limited differentiation despite distinct parental chemotypes. Analysis of additional unlinked nuclear markers and population-level sampling would be required to test this hypothesis.

Our findings reinforce the growing consensus that single-locus DNA barcoding is insufficient for species delimitation in taxonomically complex genera likeSonchus(Pante et al., 2015; Hollingsworth et al., 2016). The integration of multiple data types, molecular, chemical, morphological, and ecological, provides a more robust framework for understanding species boundaries and evolutionary relationships.

The species-specific chemical profiles identified here, particularly the contrasting abundances of neophytadiene and phytol, represent promising chemotaxonomic markers that could supplement molecular data in species identification. However, validation in larger samples and across multiple populations is essential before these markers can be used diagnostically.

From a conservation and utilization perspective, the distinct phytochemical profiles of these threeSonchusspecies suggest they may possess different pharmacological properties, warranting species-specific investigation of bioactivities. The traditional medicinal uses attributed toS. arvensisandS. oleraceusshould not be assumed forS. malayanuswithout independent verification.

This study has several limitations that should be acknowledged. First, sampling was limited to single populations of each species from one geographic location, precluding assessment of intraspecific variation. Second, the absence of biological replicates for GC-MS analysis prevented statistical comparisons of chemical profiles. Third, low-confidence identifications for major compounds inS. malayanuslimit interpretation of its chemical profile. Fourth, using only two DNA markers provides limited phylogenetic resolution.

Future studies should address these limitations through: (1) expanded sampling across the geographic ranges of these species; (2) inclusion of multiple individuals per population with biological replicates for metabolomic analysis; (3) application of high-resolution mass spectrometry and NMR for structural elucidation of unknown compounds; (4) genome-wide markers (e.g., RAD-seq, whole plastomes) for improved phylogenetic resolution; and (5) bioactivity-guided fractionation to link specific compounds to traditional medicinal uses.

5. Conclusion

This integrative study demonstrates that while single-locus DNA barcoding usingrbcLand ITS2 provides reliable genus-level identification forSonchus, it fails to resolve closely related species, particularlyS. malayanusandS. oleraceus, which share identical ITS2 sequences. In contrast, GC-MS metabolite profiling revealed distinct species-specific chemotypes:S. arvensischaracterized by high neophytadiene and phytol content;S. oleraceusdominated by phytol with diverse fatty acid esters; andS. malayanusdistinguished by guaiacol, trimethylamine, and several unidentified compounds.

The discordance between molecular and chemical data highlights the need for integrative taxonomic approaches that combine multiple lines of evidence. Neophytadiene and phytol emerge as promising chemotaxonomic markers for discriminating Sonchus species, though validation across broader geographic and genetic samples is required. The distinct chemical profiles also suggest species-specific pharmacological potential, warranting further investigation of bioactivities.

This study contributes to the growing body of evidence that DNA barcoding alone is insufficient for species delimitation in recently radiated or hybridizing plant groups, and that integration with metabolomic data provides a more comprehensive understanding of biodiversity.

Supplementary Material

Supplementary material accompanies this paper.

Supplementary File 1.

GC-MS tentative identifications (NIST Quality score <80) for compounds detected in Sonchus leaf extracts that were not included in Table 3. The table lists retention time (min), compound name (as matched in the NIST library), CAS number (where available), relative peak area (% of total ion current) for each species, and the corresponding Quality score.

This material is available as part of the online article from https://doi.org/10.1590/1519-6984.306506

Data Availability Statement

The entire data set supporting the results of this study was published in the article itself.

Acknowledgements

The authors thank the Bromo Tengger Semeru National Park Authority for granting collection permits. We are grateful to Mr. Dwi Narko for assistance with species identification and in the field. This research was supported by Universitas Airlangga (Research Group Program with contract number 7543/B/UN3.FST/PT.01.03/2025).

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*

e-mail: [e-mail]; [e-mail]

Editor:

Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  12 Oct 2026
• Date of issue
  2026

History

• Received
  15 Mar 2026
• Accepted
  10 Aug 2026