The complete chloroplast genome sequence of Lophophora diffusa

Abstract

Lophophora diffusa is a psychoactive plant of Cactaceae, morphologically similar to the mescaline-rich Lophophora williamsii but with a distinct alkaloid profile, posing significant challenges for law enforcement. Here, we sequenced the complete chloroplast genome of L. diffusa using PacBio high-throughput sequencing technology. The chloroplast genome is 115,689 bp in length and exhibits a highly derived quadripartite structure, featuring a large single-copy (LSC) region of 79,403 bp, a small single-copy (SSC) region of 32,910 bp, and a pair of significantly reduced inverted repeat (IR) regions of 1,688 bp each. It encodes 100 unique genes and has a GC content of 36.52%. Phylogenetic analysis robustly resolved the position of L. diffusa, formed a monophyletic Lophophora clade that includes L. fricii and L. williamsii; under the current sampling, this clade was resolved as sister to a group containing Mammillaria species. This study provides a foundational genomic resource for future development of precise molecular tools to distinguish L. williamsii from its relatives, thereby supporting forensic botany and international narcotics control efforts.

Introduction

The genus Lophophora, belonging to Cactaceae, commonly known as peyote, was first formally described by John M. Coulter in 1894, who segregated it from the genus Anhalonium and designated Lophophora williamsii as the type species (Bruhn and Holmstedt, 1973; Coulter 1894). This small, spineless cactus is native to the arid regions of North America, primarily distributed from the Southwestern United States (Texas) into central Mexico, including the states of Coahuila, Nuevo León, and San Luis Potosí (Anderson 1969). Morphologically, L. williamsii is characterized by its blue-green stems with well-defined ribs and tufts of whitish hairs. The genus comprises another species, Lophophora diffusa (Croizat) Bravo 1978 (Bravo-Hollis 1978), which is distinguished by its yellow-green coloration, lack of prominent ribs, and restriction to a small area in the state of Querétaro, Mexico (Anderson 1969; Aragane et al. 2011). While phylogenetically closely related, the two species are chemically distinct (Aragane et al. 2011).

Lophophora williamsii is renowned for its psychoactive properties, attributed to the presence of mescaline (3,4,5-trimethoxyphenethylamine), a potent hallucinogenic phenethylamine alkaloid (Vamvakopoulou et al. 2023). This has led to its strict control under international drug laws, due to its high potential for abuse and associated public health risks (Johnson et al. 2019; UNODC 2022). In contrast, Lophophora diffusa contains predominantly phenolic tetrahydroisoquinoline alkaloids (mainly pellotine) and almost no mescaline, suggesting a significantly lower direct abuse liability (Aragane et al. 2011; Bruhn and Holmstedt, 1973). However, its morphological similarity to L. williamsii poses a substantial challenge for law enforcement and regulatory agencies tasked with visual identification.

The evolutionary relationships and precise biosynthetic pathways leading to the divergent alkaloid profiles within Lophophora remain active areas of research. Chloroplast genome sequencing has emerged as a powerful tool for phylogenetic studies, species identification, and understanding plant evolution due to its high conservation and sufficient sequence variability (Lee et al. 2020). Sequencing the complete chloroplast genome of L. diffusa is therefore critical. It will provide essential genetic data to clarify taxonomic boundaries, illuminate the evolutionary trajectory of alkaloid biosynthesis, and, most importantly, serve as the foundational resource for developing unambiguous molecular diagnostic tools for forensic botany. Such tools are urgently needed to accurately identify seized materials, distinguish controlled L. williamsii from its less-regulated relatives, and support narcotics control efforts.

In this study, the complete chloroplast genome of Lophophora diffusa was successfully assembled using PacBio high-throughput sequencing technology. The phylogenetic tree constructed within the genus Lophophora provides crucial insights for species identification, informs ecological and conservation studies, and furnishes a genetic basis for the development of precise detection methods. This work provides an indispensable genetic resource for forensic botany, aids international narcotics control by enabling the unambiguous identification of controlled substances, and contributes significantly to the scientific foundation of global efforts to prevent drug abuse.

Materials and methods

Fresh stem epidermal tissue of Lophophora diffusa was collected from a specimen that was intercepted by China Customs and subsequently maintained in the laboratory of the Science and Technology Research Center of China Customs in Beijing, China (39.9°N, 116.3°E). The species is not native to China; the sampled plant originated from an intercepted shipment and was cultivated under laboratory conditions and the voucher specimens were deposited in the Science and Technology Research Center of China Customs (voucher number: WYY20240510-01; contact person: Yikai Wang; email: wangyikai_2001@163.com). Epidermal tissue from the stem was collected from L. diffusa plants. Total genomic DNA was extracted using an optimized cetyltrimethylammonium bromide (CTAB)-based protocol. Briefly, frozen, finely powdered tissue was incubated with pre-heated CTAB extraction buffer and Proteinase K at 65 °C for 3 h with gentle mixing. After centrifugation, the supernatant was treated with RNase A, followed by sequential extraction with chloroform-isoamyl alcohol. DNA was precipitated with isopropyl alcohol at −20 °C, washed with 80% ethanol, and finally re-suspended in elution buffer. DNA purity and quantity were evaluated using the NanoDrop 1000 and Qubit from Thermo Fisher Scientific. A 15-kb library was constructed using a SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, CA, USA). The construction included DNA shearing, AMPure PB bead purification, ssDNA overhang removal, damage repair, end repair, hairpin adapter ligation, and library bead purification. Following quality control, a SMRT bell library was obtained. The library was sequenced on the PacBio Revio platform (Pacific Biosciences, CA, USA). Raw data were processed using the CCS algorithm (version 6.0.0, parameters: ‐‐min Passes 3 ‐ min Predicted Accuracy 0.99 ‐‐max Length 21,000) to generate highly accurate HiFi reads. The data were assembled using the ptGAUL pipeline (Zhou et al. 2023). Initially, PacBio HiFi data (2.5 Gb, with an average read length of 14 kb) were aligned to the Lophophora diffusa chloroplast genome (GenBank accession: NC_064336.1) as the reference using minimap2 (version 2.24; Li 2018) to generate a PAF file. Based on the PAF file, aligned reads were filtered, and reads located in regions with sequencing coverage lower than 50× were discarded to ensure high-quality assembly. The filtered reads were then assembled using Flye (version 2.9; Kolmogorov et al. 2020) to produce an assembly graph. Subsequently, rigorous assembly graph filtering was performed: nodes with low coverage (<50×) and short contigs (<100 bp) were removed, while weakly supported edges and redundant connections were eliminated to retain only reliable paths. The graph was visualized with Bandage (version 0.8.1; Wick et al. 2015), and redundant contigs were removed and edited to form a circular sequence, which represented the complete chloroplast genome of L. diffusa. The minimum and average read mapping depths for the assembled genome were 983 × and 1379.35×(Figure S1), respectively. The assembled chloroplast genome was then annotated using the PGA (version 1.0; Qu et al. 2019), and the annotation results were manually corrected. The chloroplast genome map (Figure 2) and the diagrams of cis- and trans-spliced genes (Supplementary Figure S2 and S3) were generated using CPGView (Liu et al. 2023). Finally, the complete chloroplast sequence was submitted to GenBank (https://www.ncbi.nlm.nih.gov/) with the assigned accession PV742395.

Figure 2.

Schematic map of overall features of the chloroplast genome of Lophophora diffusa. In the chloroplast genome, the small single-copy (SSC) and large single-copy (LSC) regions are separated by inverted repeats (IRs: IRA and IRB). Genes inside the map are transcribed clockwise, and genes outside are transcribed counterclockwise. Genes with related functions are shown in the same color.

Figure 1.

Photograph of lophophora diffusa (the photograph was taken by author teng zhang in Beijing, China). The L. diffusa exhibits a spherical form with succulent, spineless tissue, characterized by smooth green epidermis covered in fine white trichomes. The ribs display a distinctive sinuous (S-shaped) pattern across the surface.

An unrooted phylogenetic tree was constructed using the available complete chloroplast genome sequence in GenBank to explore the evolutionary relationships between Lophophora diffusa and the other 23 species in Cactaceae and 3 outgroups with PhyloSuite (Zhang et al. 2020). To avoid alignment difficulties caused by structural complexity and gene content variation across plastomes, common single-copy genes were extracted from each genome using PhyloSuite and concatenated into a supergene matrix. Multiple chloroplast sequences were aligned using MAFFT with default settings (Katoh and Standley, 2013). Then, the alignment was optimized using Gblocks to remove poorly aligned positions and divergent regions. The phylogenetic tree was then constructed using IQ-TREE (Nguyen et al. 2015) with the best-fit model TVM+F + I + G4, which was chosen according to the Bayesian Information Criterion (BIC) (Susko and Roger, 2020). Node support was assessed using ultrafast bootstrap approximation (UFBS) with 1,000 replicates (Hoang et al. 2018). Bootstrap values below 60% are not shown in the tree. Finally, the resulting tree was visualized using the Interactive Tree of Life (iTOL) platform (Letunic and Bork 2019), with branch lengths drawn to scale and bootstrap support values indicated at the nodes.

Results

We have obtained a fully sequenced chloroplast genome of Lophophora diffusa that is 115,689 bp in sequence length and displays a highly derived circled quadripartite structure containing a 79,403 bp large single-copy (LSC) region, a 32,910 bp small single-copy (SSC) region while two inverted repeat regions (IRA and IRB) showed the same length of 1688 bp, which exhibits a highly reduced inverted repeat (IR) region (Figure 2). This significant size reduction suggests substantial degradation and contraction of the IR regions, a common evolutionary phenomenon in some plant lineages. The GC content in the chloroplast genome was 36.52%. Comparison with the previously reported L. diffusa plastome (MW046207.1) revealed high consistency between the two assemblies. Both genomes share an identical inverted repeat length of 1,688 bp and nearly identical GC content (36.52% vs. 36.5%). The total genome lengths differ by only 93 bp (115,689 bp vs. 115,596 bp), with minor discrepancies in the LSC and SSC boundary regions that may reflect natural intraspecific variation or differences in sequencing platforms (PacBio HiFi vs. Illumina) and assembly strategies. These results confirm the reliability of our assembled genome. The L. diffusa chloroplast genome was annotated with 100 unique genes, including 67 protein-coding genes, 29 tRNA genes, and 4 rRNA genes. Of these, 5 genes (atpF, rpl16, petD, petB, and ycf3) are cis-spliced, and rps12 is a trans-spliced gene (Figure S2). The phylogenetic analysis robustly resolved the phylogenetic position of L. diffusa within Cactaceae. L. diffusa formed a well-supported clade with its congeners, L. fricii and L. williamsii (including the variety L. williamsii var. super spialis), confirming their close phylogenetic affinity and the monophyly of the genus Lophophora. Under the current taxon sampling, the Lophophora clade was recovered as sister to a clade comprising several Mammillaria species (Mammillaria bocasana, Mammillaria erythrosperma, and Mammillaria elongata). The phylogenetic tree resolved major cactus lineages, with Opuntioideae (Nopalea cochenillifera, Opuntia sulphurea, Tephrocactus geometricus) and Pereskioideae (Pereskia aculeata) forming distinct clades, while outgroups (Portulaca oleracea, Colobanthus subulatus, and Dianthus caryophyllus) were positioned basal to the cactus lineage, supporting the phylogenetic structure revealed in this study (Figure 3).

Figure 3.

Phylogenetic relationships of the Lophophora diffusa and other cactaceae species, with Portulaca oleracea, Colobanthus subulatus and Dianthus caryophyllus serve as outgroup. Branch lengths are proportional to the number of substitutions per site (scale bar: 0.01 substitutions per site). Numbers above branches indicate ultrafast bootstrap support values (%; 1,000 replicates). The complete chloroplast genome sequences and accession ID were used as follows: Carnegiea gigantea NC 027618 (Sanderson et al. 2015), Lophocereus schottii NC 041727, Polaskia chende OR738629, Neoraimondia herzogiana OQ458703, Cleistocactus hyalacanthus OQ458704, Echinopsis candicans OR577372, Melocactus glaucescens NC 081993, Rhipsalis teres NC 057526 (Morais da Silva et al. 2021), Echinocactus grusonii MW553048, Ferocactus latispinus MW553072, Ferocactus setispinus MW553071, Leuchtenbergia principis MW553057, Lophophora fricii PV872148, Lophophora williamsii PV904153, Lophophora williamsii var. super spialis PV904154, Mammillaria bocasana OR863748 (Ortiz-Brunel et al. 2024), Mammillaria erythrosperma OR863749 (Ortiz-Brunel et al. 2024), Mammillaria elongata MW553058 (Ni et al. 2023), Sclerocactus unguispinus NC 086707, Nopalea cochenillifera NC 087798 (J. Liu et al. 2024), Opuntia sulphurea PV464448 (Chen et al. 2025), Tephrocactus geometricus NC 064335, Pereskia aculeata NC 062888, Portulaca oleracea NC 036236 (X. Liu et al. 2018), Colobanthus subulatus NC 053723 (Androsiuk et al. 2020), Dianthus caryophyllus OP136027 (Lin et al. 2022).

Discussion and conclusions

This study successfully deciphered the complete chloroplast genome of Lophophora diffusa using PacBio sequencing, providing a crucial genomic foundation for this taxonomically and forensically significant species. The assembled genome reveals a highly reduced inverted repeat (IR) region, a structural feature consistent with significant IR contraction observed in other Cactaceae lineages (Sanderson et al. 2015; Köhler et al. 2020), suggesting an accelerated evolutionary rate in the Lophophora lineage. Such structural variations provide valuable insights into the dynamic evolutionary history of plastomes within Cactaceae and highlight the genus as a model for studying chloroplast genome reduction (Jansen and Ruhlman, 2012; Köhler et al. 2020). Phylogenetic analysis based on this complete plastome robustly resolves the evolutionary position of L. diffusa, confirming the monophyly of the genus Lophophora and was resolved as sister to a clade of Mammillaria species under the current sampling scheme. It should be noted that a more comprehensive sampling of Cactaceae genera would be necessary to fully resolve the sister-group relationships of Lophophora. The high support values at key nodes underscore the utility of plastome-scale data for clarifying relationships among closely related taxa. Overall, the genomic resource presented here not only serves as a definitive reference for taxonomy but also contributes to understanding broader patterns of plastome evolution within the family.

The complete chloroplast genome sequence provides a foundational genomic resource for developing unambiguous molecular diagnostic tools. Previous studies have successfully employed specific chloroplast regions—such as the polymorphic trnL/trnF intergenic spacer or novel intronic VNTRs—for distinguishing Lophophora williamsii from L. diffusa (Ng et al. 2016; Hwang et al. 2025). However, reliance on a limited set of markers presents challenges, including potential amplification failure when dealing with degraded, processed, or trace-evidence samples where DNA quality is poor. The complete plastome presented here serves as a comprehensive genomic repository, harboring a vast reservoir of potentially informative sites beyond any single locus. While the present study focuses on the assembly and characterization of the L. diffusa chloroplast genome, comparative analyses with chloroplast genomes of other Lophophora species will enable the identification of species-specific variable regions. Such analyses will facilitate the development of robust molecular markers for accurate species identification, thereby overcoming the limitations of individual barcodes and providing scientifically reliable evidence for forensic applications.

The species-specific molecular markers, developed from foundational resources like the complete chloroplast genome, directly enhanced capabilities in forensic botany and narcotics regulation. The morphological similarity between the controlled, mescaline-containing Lophophora williamsii and its less-regulated relative L. diffusa poses a significant challenge for law enforcement (Aragane et al. 2011). Robust DNA-based assays allow for the accurate identification and individualization of Lophophora specimens, even from trace or processed material, providing scientifically defensible and legally admissible evidence. This strengthens the monitoring of illicit plant trade and supports judicial authentication, contributing directly to effective narcotics control.

In summary, the complete chloroplast genome of Lophophora diffusa serves as a pivotal genetic resource bridging evolutionary biology and applied forensic science. Future research should leverage this genome to design rapid, field-deployable detection methods and to perform comparative genomic analyses across the Lophophora genus to identify robust, species-specific markers. Ultimately, this work contributes to global efforts in preventing drug abuse by enabling accurate plant identification, supporting law enforcement, and fostering a deeper scientific understanding of the evolution of psychoactive compounds in the Cactaceae family.

Funding Statement

This work was supported by the National Key Research and Development Program of China under Grant No. [2022YFF1202203].

Ethical approval

Research on plant organelle genome sequencing does not affect the population and does not require ethical approval. The analyzed species is widely distributed in China, and the material was not obtained from nature reserves.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The genome sequence data that support the findings of this study are openly available at NCBI (https://www.ncbi.nlm.nih.gov/) under the accession no. PV742395. The associated BioProject, BioSample, and SRA/DRA numbers are PRJNA1268711, SAMN48761952, and SRR33724456, respectively.