Lysine and cadaverine differentially regulate huperzine biosynthesis in axenic gametophyte cultures of Huperzia selago (L.) Bernh. ex Schrank et Mart.

Abstract

Background

Specialized metabolites represent a major component of plant adaptive and chemical diversity and constitute an important source of pharmacologically relevant compounds. Among them, Lycopodiaceae alkaloids form a distinctive group of lysine-derived metabolites characteristic of club mosses (Lycopodiaceae), a relict lineage of early-diverging lycophytes sister to the remaining extant vascular plants. Huperzia selago produces huperzine A (HupA) and huperzine B (HupB), potent acetylcholinesterase inhibitors, yet the regulation of their biosynthesis remains poorly understood, particularly in the gametophyte generation. Because lysine and its decarboxylation product cadaverine represent key entry metabolites into the Lycopodiaceae alkaloid network, precursor supplementation provides a tractable approach for probing regulatory nodes of this pathway under controlled conditions.

Results

Axenic gametophyte cultures of H. selago were maintained for 24 weeks on Whittier–Storchová medium supplemented with L-lysine (Lys), cadaverine (Cad), or combined Lys + Cad (5 + 5, 10 + 10, or 20 + 20 mg L⁻¹) under plant-growth-regulator-free conditions or in the presence of indole-3-butyric acid (IBA) + kinetin. Two-way ANOVA revealed significant effects of culture time, medium composition, and their interaction on both alkaloids. HupA showed its strongest response during the early to intermediate culture phase, reaching a maximum at week 12 in Lys + Cad-10 + 10 (5.70 ± 0.01 mg g⁻¹ DW), whereas the highest late-stage HupA levels were observed under cadaverine supplementation at week 24 (6.79 ± 0.10 mg g⁻¹ DW). In contrast, HupB exhibited delayed accumulation, with peak levels at week 24 in Lys + Cad-5 + 5 (0.97 ± 0.05 mg g⁻¹ DW). Plant growth regulators modified the feeding response in a time- and variant-dependent manner rather than acting as a uniform stimulator. LC–MS/IT–TOF profiling detected 27 signals corresponding to Lycopodiaceae alkaloids, with HupA and HupB confirmed using authentic standards.

Conclusions

Precursor supplementation with lysine and cadaverine effectively modulates huperzine biosynthesis in H. selago gametophytes and reveals distinct temporal regulation patterns for HupA and HupB. The results support the view that the lysine-derived entry node represents an important regulatory node within the Lycopodiaceae alkaloid network. Axenic gametophyte cultures therefore provide a tractable experimental system for investigating specialized metabolism in early-diverging vascular plants and for refining strategies for the controlled production of pharmaceutically relevant alkaloids.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12870-026-09868-x.

Background

Plant specialized (secondary) metabolites are among the most diverse and functionally important products of plant metabolism. Far from being mere biochemical “by-products”, they mediate interactions with both biotic and abiotic environments, co-shape developmental plasticity, and contribute to plant adaptation through roles in herbivore defense, stress tolerance, and ecological signaling and communication [1]. At the same time, they represent a major pool of compounds with high application value (including pharmaceuticals and functional ingredients), which justifies research aimed both at understanding the mechanisms governing their biosynthesis and at developing methods for predictable control of metabolic fluxes in controlled systems. In this context, plant biotechnology—including the “plant cell factories” concept—remains a timely paradigm that integrates fundamental questions with translational potential [1].

Among specialized metabolites, alkaloids occupy a distinctive position as nitrogen-containing compounds that link primary nitrogen metabolism with dedicated pathways producing molecules of complex architecture and often strong bioactivity. Alkaloids fulfill important ecological functions (including defensive and signaling roles), and from a human perspective they constitute a persistent source of pharmacologically relevant substances [2, 3]. In practice, however, their acquisition is frequently challenging: chemical synthesis of many alkaloids is costly or impractical, while natural resources may be limited, seasonal, and variable in quality [1, 3].

Within spore-bearing vascular plants, club mosses of the family Lycopodiaceae P. Beauv. in Mirb. (homosporous lycophytes; Lycopodiales DC. ex Bercht. & J. Presl) are particularly notable as an ancient, relict, and evolutionarily distinctive lineage of vascular plants that traces back to early Devonian ancestors from the time of terrestrial colonization [4]. Owing to their early divergence from the lineages that later evolved toward seed plants, lycophytes combine ancestral and highly specialized traits; as a sister group to the remaining vascular plants, they also exhibit features that in some cases likely reflect convergent evolutionary solutions. Consequently, Lycopodiaceae display biological attributes uncommon in other plant groups, and their specialized metabolism has relevance beyond descriptive phytochemistry: Lycopodiaceae alkaloids constitute one of the most characteristic components of the metabolome of this lineage and can be treated as a model system for considering the evolution and regulation of specialized biosynthetic pathways in vascular plants (Fig. 1) [5–8]. In the relatively few Lycopodiaceae species studied to date (out of ~ 388 species according to recent estimates), these alkaloids may accumulate in sporophytes from fractions to several mg·g⁻¹ dry weight, while both pool size and qualitative profiles are strongly dependent on species, organ/tissue, and environmental conditions [5–10].

Fig. 1.

Study system and biosynthetic rationale for lysine/cadaverine supplementation in Huperzia selago cultures. A Huperzia selago sporophytes in their natural habitat (main image) and a representative axenic in vitro culture of H. selago gametophyte (inset) used as a controlled platform for metabolite production. B Simplified entry of L-lysine (Lys) into Lycopodiaceae alkaloid biosynthesis: lysine decarboxylation releases CO₂ to yield cadaverine (Cad), which is converted (via Δ¹-piperideine; not shown) to pelletierine, a key intermediate that can feed into multiple Lycopodiaceae alkaloid scaffold types (illustrated as lycopodine-, lycodine- and fawcettimine-type skeletons). Additional downstream steps and enzymes are omitted for clarity. C Representative Lycopodiaceae alkaloids illustrating scaffold diversity; huperzine A (HupA) and huperzine B (HupB) are highlighted as the quantified targets in this study (HPLC), whereas the remaining examples provide chemical context consistent with the broader alkaloid profile annotated by LC–MS/IT-TOF (Table 1). Structures are shown in simplified form (not to scale)

Structurally, Lycopodiaceae alkaloids comprise multiple skeleton classes, dominated by the lycopodine, fawcettimine, phlegmarine, and lycodine-types (including huperzines and related derivatives), alongside rarer structures that are often phylogenetically restricted [5–8]. They are typically built from 16 to 18 carbon atoms and one or two nitrogen atoms. The C₁₈ alkaloids are most often acetylated derivatives of C₁₆ alkaloids, although compounds with higher carbon counts have also been reported [7]. This diversity—combined with the high biological activity of many members—makes club moss alkaloids an important target for both fundamental research (pathway regulation and evolution) and applied studies (pharmacological relevance) [5–8]. A further interpretive layer is provided by chemotaxonomy: certain alkaloid skeleton types show non-random, clade-structured distributions within Lycopodiaceae, consistent with phylogenetically conserved variants of biosynthetic networks [5–8]. In particular, huperzine-derived alkaloids (selagines), mainly huperzine A (HupA) and huperzine B (HupB) belonging to the lycodine-type alkaloids, exhibit a distribution strongly concentrated within the subfamily Huperzioideae W. H. Wagner & Beitel ex B. Øllg. Available evidence suggests that a “complete” version of the lycodine-type biosynthetic route leading to huperzine-type alkaloids is likely an evolutionarily conserved trait within the Huperzia clade, which—besides Huperzia Bernh. —also includes the monotypic genus Phylloglossum Kunze [4, 5, 7–9].

From a biosynthetic perspective, Lycopodiaceae alkaloids are largely lysine-derived: L-lysine serves as an entry point into the alkaloid network, while cadaverine—the decarboxylation product of lysine—is regarded as an early, key “gateway” metabolite that channels flux toward multiple skeleton types in this group (Fig. 1) [5, 7]. In pathway reconstructions, cadaverine can be further transformed (e.g., via Δ¹-piperideine) to pelletierine, which is considered a central intermediate linking early lysine metabolism with the formation of complex alkaloid skeletons [5, 7]. This biogenesis connects nitrogen metabolism and polyamine-related physiology with the formation of specialized metabolites, making lysine and cadaverine rational candidates for experimental modulation of alkaloid biosynthesis under controlled conditions [5, 7].

Among Lycopodiaceae alkaloids, huperzine (selagine) derivatives are of particular practical importance, mainly huperzine A (HupA) and huperzine B (HupB), both potent, selective, and reversible acetylcholinesterase inhibitors with significant pharmacological potential [6, 10]. Despite the availability of total synthesis routes, HupA sourcing in practice still relies largely on plant biomass collected from natural populations, which—together with slow growth and limited material availability—creates raw-material pressure and standardization challenges [6, 8–11]. In this context, in vitro plant systems provide an attractive alternative both as research models and as potential production platforms.

Accordingly, in vitro plant platforms—including cell, organ, and gametophyte cultures—remain highly relevant because they provide controlled environments in which growth, regulatory cues, and the availability of biosynthetic substrates can be manipulated with precision [1, 3, 6, 12]. For club mosses, an additional advantage is the potential to radically shorten the developmental cycle and obtain biomass with reduced environmental variability: whereas under natural conditions the transition from spore germination to a mature sporophyte usually spans many years, axenic gametophytes can be obtained within several to a dozen months (approximately 7–18 months) and subsequently propagated in multi-month cycles, facilitating the identification of regulatory nodes and “temporal windows” of maximal metabolite accumulation [8, 12].

One of the simplest, least expensive, and economically justified approaches to intensifying specialized metabolism in in vitro cultures is precursor feeding, i.e., exogenous supplementation with metabolites that enter the target biosynthetic route upstream of key branch points [13, 14]. Despite major progress in systems-level analyses (genomics, transcriptomics, proteomics, and metabolomics) and metabolic engineering, this strategy remains attractive due to its operational simplicity, low cost, and transferability across culture systems [13, 14]. Classic examples—e.g., from Catharanthus roseus cultures—demonstrate that increasing the availability of selected precursors can markedly enhance alkaloid accumulation, while efficacy depends on dose and timing and may be greatest in systems where limiting regulatory nodes have been shifted or relieved [15–17].

Within this framework, Huperzia selago represents an especially compelling model for testing the impact of the lysine-derived “entry node” into the Lycopodiaceae alkaloid network. Although the species has been reported as a source of research-relevant alkaloids, its chemical profiles, biosynthetic regulation, and biological roles of these metabolites—particularly in ecological and phylogenetic contexts—remain insufficiently resolved in several aspects, supporting studies that integrate in vitro systems with targeted manipulation of precursor supply [5–8, 18, 19]. Moreover, the biosynthetic model used in this study—gametophytes as an autonomous haploid generation with distinct physiology and developmental priorities—remains comparatively underexplored with respect to alkaloid pathway regulation, which makes it a valuable subject for both mechanistic and applied research [6, 10, 18].

For these reasons, we systematically evaluated whether supplementation with L-lysine (Lys), cadaverine (Cad), and their combination (Lys + Cad) can enhance HupA and HupB biosynthesis in axenic in vitro gametophyte cultures of H. selago, and how this effect depends on culture age and plant growth regulator (PGR) regime. By testing three concentrations (5, 10, and 20 mg·L⁻¹) and quantifying time-dependent accumulation across consecutive sampling points, we formulated the hypotheses that (1) precursor supply increases HupA and HupB accumulation in a dose- and time-dependent manner, (2) lysine and cadaverine differ in the magnitude and kinetics of their effects (reflecting distinct metabolic entry points and potential physiological coupling), and (3) PGRs modulate these responses by shifting the growth–specialized metabolism balance and altering the temporal “window” of maximal accumulation.

Results

This section summarizes time-dependent changes in huperzine A (HupA) and huperzine B (HupB) content in Huperzia selago gametophytes in response to precursor supplementation (Lys, Cad, and Lys + Cad) and the plant growth regulator regime (–PGR vs. + PGR). Data are presented as mean ± SD (n = 3 biological replicates; each quantified in technical triplicate). Week 0 values were treated as baseline descriptive measurements and were not subjected to significance testing; inferential analyses were performed for weeks 4–24.

Two-way ANOVA revealed significant effects of culture time, medium variant, and the time × medium variant interaction for both HupA and HupB, indicating that the magnitude of supplementation effects depended on the culture stage. Temporal profiles of HupA and HupB are shown in Figs. 2 and 3, respectively, while detailed numerical values and statistical comparisons are provided in Supplementary Table S1.

Fig. 2.

Time-dependent changes in huperzine A (HupA) content in axenic Huperzia selago gametophyte cultures under lysine/cadaverine feeding and PGR regimes. A L-lysine (Lys), B cadaverine (Cad), and C combined supplementation (Lys + Cad). Precursors were supplied at 5, 10, and 20 mg L⁻¹ (for Lys + Cad: 5 + 5, 10 + 10, and 20 + 20 mg L⁻¹) in two medium variants: PGR-free (− PGR) and PGR-supplemented (+ PGR; 0.05 µM indole-3-butyric acid + 1.4 µM kinetin). HupA was quantified by HPLC–DAD and expressed as mg g⁻¹ DW. Bars represent mean ± SD (n = 3 independent biological replicates; technical triplicates were averaged prior to statistics). Asterisks indicate significant differences versus the unsupplemented control (Control, −PGR) within the same week (Dunnett’s test following two-way ANOVA): * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Week 0 values are shown as baseline descriptors and were not included in inferential statistics

Fig. 3.

Time-dependent changes in huperzine B (HupB) content in axenic gametophyte cultures of Huperzia selago under precursor feeding and PGR regimes. A L-lysine (Lys), B cadaverine (Cad), and C Lys + Cad supplied at 5, 10, or 20 mg·L⁻¹ (final concentrations), each cultured either without plant growth regulators (PGR-free) or with 0.05 µM IBA + 1.4 µM kinetin (IBA + KIN). HupB content is expressed as mg·g⁻¹ DW (mean ± SD, n = 3). Statistical significance (asterisks) indicates differences vs. the unsupplemented control under PGR-free conditions within the same sampling week (weeks 4–24; Dunnett’s post hoc test following two-way ANOVA); week 0 values are shown as baseline descriptors and were not included in inferential statistics. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Effect of supplementation on huperzine A (HupA) accumulation

In the control cultures (–PGR), HupA content increased steadily over time, reaching approximately 3.4 mg·g⁻¹ DW by week 24 (Fig. 2, Supplementary Table S1), which served as the reference for evaluating precursor feeding effects.

Already at week 4, the first significant differences relative to the control were observed. The highest HupA level was recorded after supplementation with L-lysine at 10 mg·L⁻¹ in the absence of PGRs (Lys-10, –PGR; approx. 3.4 mg·g⁻¹ DW), whereas other variants that differed significantly from the control generally ranged between approx. 1.9 and 2.4 mg·g⁻¹ DW (Supplementary Table S1). Under + PGR conditions at this time point, increases in HupA were less consistent and were evident only for selected combinations (Supplementary Table S1).

By week 8, differences among variants became more pronounced, and the highest HupA accumulation was still observed under lysine feeding (Lys-10, –PGR; approx. 4.5 mg·g⁻¹ DW), corresponding to an almost two-fold increase relative to the control at the same time point (Fig. 2, Supplementary Table S1). Among cadaverine-supplemented variants, the higher dose tended to show a clearer effect (Cad-20; Supplementary Table S1).

At week 12, a distinct “window” of peak HupA accumulation emerged for selected precursor combinations. The maximum value was recorded for Lys + Cad at 10 + 10 mg·L⁻¹ (–PGR; approx. 5.7 mg·g⁻¹ DW), significantly exceeding the control at the same time point (approx. 2.6 mg·g⁻¹ DW; Supplementary Table S1). Elevated HupA levels were also observed in selected cadaverine variants and in the Lys + Cad combination at the lower dose (Supplementary Table S1).

By week 24, the highest HupA levels in the entire experiment were obtained. The strongest accumulation was recorded under cadaverine supplementation (Cad-10 and Cad-20, –PGR; approx. 6.7–6.8 mg·g⁻¹ DW), i.e., about two-fold higher than in the control (Fig. 2, Supplementary Table S1). High values were also maintained in selected Lys + Cad combinations and in some + PGR variants (Supplementary Table S1), confirming that the ranking of the most effective medium variants shifted with culture duration.

Effect of supplementation on huperzine B (HupB) accumulation

In the control cultures (–PGR), HupB content remained low during the early culture stage and increased gradually over time, reaching 0.29 ± 0.03 mg·g⁻¹ DW at week 24 (Fig. 3, Supplementary Table S1). In contrast, variants supplemented with lysine and/or cadaverine showed a distinct accumulation dynamics, dependent on both culture time and medium configuration, consistent with a significant week × medium variant interaction (Fig. 3, Supplementary Table S1).

At week 4, the HupB response to supplementation was still selective: significant differences relative to the control were observed only in a subset of variants, with the clearest early effect associated with cadaverine-containing media at the higher dose (Fig. 3, Supplementary Table S1). In other combinations, changes were smaller and did not yet form a stable dose–response pattern.

By week 8, supplementation effects became more apparent and more frequently involved cadaverine-containing variants as well as selected Lys + Cad combinations. At this time point, the increase in HupB was still moderate (relative to later weeks), but it indicated a growing susceptibility of HupB accumulation to precursor availability over time (Fig. 3, Supplementary Table S1).

At week 12, HupB accumulation increased markedly in multiple supplementation variants. The highest values were observed in combined Lys + Cad variants, with the maximum recorded for Lys + Cad-5 + 5 in the presence of IBA + KIN (0.87 ± 0.06 mg·g⁻¹ DW), significantly above the control (0.24 ± 0.04 mg·g⁻¹ DW; p < 0.001) (Fig. 3, Supplementary Table S1). Increased HupB levels were also detected in selected cadaverine-only variants and in some lysine variants, indicating that during the mid-culture stage HupB levels depended on both cadaverine availability and the growth-regulator context in specific medium configurations (Fig. 3, Supplementary Table S1).

By week 24, HupB reached the highest values in the entire experiment and simultaneously showed the largest number of significant differences relative to the control (Fig. 3, Supplementary Table S1). The maximum was recorded for Lys + Cad-5 + 5 (0.97 ± 0.05 mg·g⁻¹ DW; p < 0.001), and comparably high levels were also observed in variants containing cadaverine at the higher dose (e.g., Cad-20) and in selected + PGR combinations. Overall, these results indicate that in the late culture stage HupB is particularly responsive to cadaverine and combined supplementation regimes, and that optimal configurations for HupB differ from those maximizing HupA (Figs. 2 and 3, Supplementary Table S1).

In summary, the HupB response to supplementation was delayed relative to HupA and became most evident from weeks 8–12, with the maximum at week 24. Cadaverine and Lys + Cad combinations (particularly at the lower dose) were most frequently associated with the highest HupB levels, while IBA + KIN acted as a response modulator and enhanced accumulation only in selected medium configurations and time points (Fig. 3, Supplementary Table S1).

Effect of IBA and kinetin as modifiers of the feeding response

Matched pairwise comparisons between precursor-supplemented variants cultured without plant growth regulators (–PGR) and their corresponding variants supplemented with 0.05 µM IBA + 1.4 µM kinetin (+ PGR) showed that PGRs modified huperzine accumulation in Huperzia selago gametophytes, with both the direction and magnitude of differences depending on culture time, supplementation regime, and dose (Figs. 2 and 3, Supplementary Table S1). Because an unsupplemented + PGR control (0 mg·L⁻¹ precursors) was not included, the effect of PGRs was evaluated only within supplemented conditions by direct –PGR vs. + PGR comparisons for matched pairs (identical precursor configuration and dose). Statistical significance of –PGR/+PGR differences was assessed separately within each week, and p-values were adjusted using the Holm method within the given time point.

For huperzine A (HupA), significant –PGR/+PGR differences were detected at weeks 4, 12, and 24, whereas no comparisons remained significant at week 8 after multiple-testing correction. At week 4, the presence of IBA + KIN was associated with lower HupA accumulation in the Cad-5, Lys-5, Lys-20, Cad-10, and Lys + Cad-5 + 5 variants, while in Cad-20 a significant increase in HupA was observed under + PGR relative to –PGR. At week 12, +PGR increased HupA in the Lys + Cad-20 + 20 variant, but decreased HupA in the Cad-10 and Lys + Cad-10 + 10 pairs. At week 24, +PGR was associated with higher HupA in Cad-5 and Lys-10, and with lower HupA in Lys-20, Lys + Cad-5 + 5, and in cadaverine-based variants (Cad-10 and Cad-20). In addition, directional –PGR/+PGR trends were apparent in selected pairs (e.g., Lys-10 at week 8), but these did not meet significance after correction.

For huperzine B (HupB), the influence of PGRs was more temporally restricted: significant –PGR/+PGR differences were detected only at weeks 4 and 8, while no comparisons remained significant at weeks 12 and 24 after correction. At week 4, IBA + KIN increased HupB in the Lys-5 variant, but decreased HupB in the Cad-20 and Lys + Cad-20 + 20 pairs. At week 8, a significant decrease in HupB persisted in Cad-20, accompanied by an increase in Lys-5. At later stages (weeks 12–24), although HupB values were high in some variants, matched –PGR/+PGR contrasts did not confirm significant differences after multiple-testing correction.

Overall, these results indicate that IBA + kinetin did not exert a uniform, unidirectional effect on huperzine accumulation across conditions, but instead modulated the precursor-feeding response in a manner dependent on medium configuration and culture stage (Figs. 2 and 3, Supplementary Table S1).

Qualitative alkaloid profiling by LC–MS/IT–TOF (ESI⁺)

Qualitative alkaloid profiling was performed by LC–MS/IT–TOF in positive-ion mode (ESI⁺) using representative extracts of Huperzia selago gametophytes obtained from cultures supplemented with L-lysine (Lys), cadaverine (Cad), and the Lys + Cad combination under plant growth regulator conditions (IBA + KIN), as well as from control cultures. The detected LC–MS/IT–TOF features are summarized in Table 1. In all cases, numerous signals attributable to alkaloids typical of the Lycopodiaceae family were detected.

Table 1.

LC–MS/IT–TOF features detected in representative Huperzia selago gametophyte extracts and annotated as Lycopodiaceae alkaloids based on accurate mass and MS/MS fragmentation where available

Peak ID	RT (min)	Ion*	m/z (exp.)	m/z (calcd.)	Mass error (ppm, abs.)	Formula	Putative annotation (primary)	Alternative candidates	Alkaloid class/skeleton	MSI level	Evidence**	MS/MS fragments (m/z)	Notes
1	2.03	[M + H]⁺	249.2318	249.2325	2.90	C16H28N2	Dihydrodeoxycernuine	—	miscellaneous Lycopodiaceae alkaloid	3	MS1	Low intensity; reliable MS/MS spectrum not acquired	Accurate mass/formula match; previously reported among Lycopodiaceae alkaloids [7,20].
2	3.20	[M + H]⁺	264.1947	264.1958	4.16	C16H25NO2	Fawcettimine	—	fawcettimine-type	3	MS1; MS/MS	264, 246, 228, 213, 188, 172, 150, 136, 122, 105, 91	Accurate-mass/formula match; MS/MS fragmentation supports a fawcettimine-type alkaloid, but assignment remains putative without an authentic standard/reference MS/MS spectrum [7,12].
3	3.20	[M + H]⁺	266.2104	266.2115	4.13	C16H27NO2	Deacetylfawcettine	—	fawcettimine-type	3	MS1; MS/MS	266, 248, 230, 202, 176	Accurate-mass/formula match; MS/MS acquired and consistent with a fawcettimine-type feature. Assignment remains putative without an authentic standard/reference spectrum [12].
4	5.22	[M + H]⁺	282.2057	282.2064	2.48	C16H27NO3	Deacetyllycofawcine	—	fawcettimine-type	3	MS1; MS/MS	282, 264, 128, 91	Accurate-mass/formula match; MS/MS acquired. Reported as a Lycopodiaceae alkaloid in the literature; definitive confirmation would require a reference spectrum or standard [21].
5	5.58	[M + H]⁺	280.1900	280.1907	2.50	C16H25NO3	Lycoposerramine G	Serratezomine C; Lycoposerramine P	lycopodine-type	3	MS1; MS/MS	280, 262, 244, 216, 188, 174, 150, 91	Accurate-mass/formula match; MS/MS acquired. Several isomeric Lycopodiaceae alkaloids are plausible; annotation remains putative [22,23].
6	5.79	[M + H]⁺	278.1755	278.1751	1.44	C16H23NO3	Hydroxylated serratidine isomer (4α- or 6α-hydroxyserratidine)	Lycoposerramine E; Huperzine O	huperzine/lycodine-type	3	MS1; MS/MS	278, 260, 146, 113	Accurate-mass/formula match; MS/MS acquired, but positional/isomeric candidates cannot be resolved without authentic standards or reference MS/MS spectra [24].
7	9.70	[M + H]⁺	264.1953	264.1958	1.89	C16H25NO2	Lycoposerramine L / Lycoposerramine M (isomeric assignment)	Lucidioline; Lycodoline	lycopodine-type	3	MS1; MS/MS	264, 246, 188, 150, 122	Reported in H. selago as ‘lycoposerramine-L or -M’; MS/MS was acquired, but isomer-level discrimination requires standards/reference spectra [25].
8	21.20	[M + H]⁺	257.1636	257.1648	4.67	C16H20N2O	N-desmethylhuperzinine-related isomer (tentative)	N-desmethylhuperzinine isomeric assignment	huperzine/lycodine-type	3	MS1	Low intensity; reliable MS/MS spectrum not acquired	Earlier-eluting m/z 257.1636 feature, distinct from the HupB peak confirmed at RT 25.96 min. Assignment remains tentative based on accurate mass/formula and retention behavior.
9	21.20	[M + H]⁺	262.1790	262.1802	4.58	C16H23NO2	Lycoposerramine C–type / lycopodine-type isomer (tentative)	Huperzine I isomeric candidate	huperzine/lycodine-type	3	MS1; MS/MS	262, 244, 189, 160, 105	Accurate-mass/formula match; MS/MS acquired. Annotation remains putative due to isomeric alternatives reported for Lycopodiaceae/Huperzia [26].
10	25.96	[M + H]⁺	257.1638	257.1648	3.89	C16H20N2O	Huperzine B	—	huperzine/lycodine-type	1	MS1; MS/MS; HPLC-DAD; Std	257, 240, 212, 198, 184, 158, 154, 130, 115, 91	Confirmed as HupB based on diagnostic MS/MS fragmentation and agreement with an authentic standard in HPLC-DAD. This resolves the isomeric ambiguity of m/z 257.16 features in the dataset.
11	25.96	[M + H]⁺	305.2226	305.2224	0.66	C18H28N2O2	Serratinidine	—	lycopodine-type	3	MS1; MS/MS	305, 264, 246, 228	Accurate-mass/formula match; MS/MS acquired. Previously reported in H. selago gametophytes; annotation remains putative without a standard/reference spectrum [12].
12	26.30	[M + H]⁺	264.1960	264.1958	3.03	C16H25NO2	C16H25NO2 isomer (e.g., lycodoline / lucidioline / lycoposerramine J/M)	—	lycopodine-type	3	MS1; MS/MS	264, 246, 218, 202, 176, 91	Multiple structural isomers share this elemental composition. MS/MS was acquired, but definitive isomer-level assignment requires authentic standards or reference spectra.
13	27.23	[M + H]⁺	243.1485	243.1492	2.88	C15H18N2O	Huperzine A	—	huperzine/lycodine-type	1	MS1; MS/MS; HPLC-DAD; Std	243, 227, 210, 196, 192, 178, 165, 154, 115, 91	Confirmed as HupA by agreement with an authentic standard in HPLC-DAD and MS/MS fragmentation consistent with HupA.
14	27.62	[M + H]⁺	259.1805	259.1805	2.31	C16H22N2O	11-hydroxylycodine	—	lycodine-type	3	MS1; MS/MS	259, 227, 210, 190, 180, 167, 165, 152, 130, 91	Accurate-mass/formula match; MS/MS acquired and compatible with a lycodine-type alkaloid. Matches literature HRMS for 11-hydroxylycodine; assignment remains putative without standard/reference spectrum.
15	27.62	[M + H]⁺	287.2114	287.2118	1.39	C18H26N2O	Des-N-methylfastigiatine (N-demethylfastigiatine)	—	fastigiatine-type	3	MS1	Low intensity; reliable MS/MS spectrum not acquired	Accurate-mass/formula match; previously reported in H. selago gametophytes by HRMS [12].
16	27.74	[M + H]⁺	261.1955	261.1961	2.30	C16H24N2O	N-desmethyl-α-obscurine / lycodine-type isomer (putative)	—	huperzine/lycodine-type	3	MS1	Low intensity; reliable MS/MS spectrum not acquired	Accurate-mass/formula match; annotation remains tentative because multiple isomers share this formula.
17	27.74	[M + H]⁺	303.2067	303.2067	1.65	C18H26N2O2	Oxygenated fastigiatine-type alkaloid (putative des-N-methylfastigiatine N-oxide / hydroxy-des-N-methylfastigiatine)	—	fastigiatine-type	3	MS1; MS/MS	303, 228, 183, 172, 146, 91	Mass shift + 15.995 Da relative to des-N-methylfastigiatine suggests a mono-oxygenated analogue. MS/MS was acquired, but assignment remains putative without reference spectra/standard.
18	27.74	[M + H]⁺	319.2017	319.2016	0.31	C18H26N2O3	Di-oxygenated fastigiatine-type alkaloid	possible N-oxide / hydroxylated analogue; isomeric	fastigiatine-type	3	MS1	Low intensity; reliable MS/MS spectrum not acquired	Accurate-mass/formula match; mass shift suggests a di-oxygenated analogue. Assignment remains tentative.
19	28.37	[M + H]⁺	248.1998	248.2009	4.43	C16H25NO	Lycopodine / Selagoline	isomeric assignment; possible co-elution	lycopodine-type	2	MS1; MS/MS	248, 230, 200, 188, 174, 145, 131, 105, 91	MS/MS supports a lycopodine/selagoline-type assignment, but the two isomers share the same elemental composition, and co-elution cannot be excluded under the applied LC conditions.
20	28.74	[M + H]⁺	333.2165	333.2173	2.40	C19H28N2O3	Unidentified C19 Lycopodiaceae alkaloid	—	lycodine-type	4	MS1; MS/MS	333, 289, 247, 230, 176, 145, 91	MS/MS was acquired, but available evidence remains insufficient for a confident structural annotation.
21	29.43	[M + H]⁺	246.1852	246.1852	3.66	C16H23NO	Fawcettidine	—	fawcettimine-type	3	MS1; MS/MS	246, 228, 204, 186, 172, 148, 131, 105, 91	Accurate-mass/formula match; MS/MS acquired and compatible with a fawcettimine-type alkaloid. Assignment remains tentative without a standard/reference spectrum [27].
22	29.43	[M + H]⁺	276.1588	276.1594	2.17	C16H21NO3	C16H21NO3 Lycopodiaceae alkaloid	huperzine F / annotine / annotinine isomers	huperzine/lycodine-type	3	MS1; MS/MS	276, 234, 188, 160, 145, 134, 105, 91	Accurate-mass match; MS/MS acquired, but multiple isomers are reported in Lycopodiaceae. Definitive assignment requires authentic standards or reference spectra.
23	29.43	[M + H]⁺	287.2111	287.2118	2.44	C18H26N2O	Des-N-methylfastigiatine isomer (putative)	isomeric feature possible	fastigiatine-type	3	MS1	Low intensity; reliable MS/MS spectrum not acquired	Accurate-mass/formula match; putative isomeric feature.
24	29.84	[M + H]⁺	264.1961	264.1958	1.14	C16H25NO2	C16H25NO2 isomer (e.g., lycodoline / lucidioline / lycoposerramine J/L/M)	—	lycopodine-type	3	MS1; MS/MS	264, 246, 218, 203, 176	Same elemental composition occurs in multiple structural isomers. MS/MS was acquired, but definitive assignment requires standards or reference spectra.
25	30.32	[M + H]⁺	289.2273	289.2274	0.35	C18H28N2O	Flabelline	—	putative Lycopodiaceae feature	3	MS1; MS/MS	289, 230, 146, 105	Accurate-mass/formula match; MS/MS acquired. Compound reported as a Lycopodiaceae alkaloid; assignment remains putative without standard/reference spectrum [7,28].
26	31.75	[M + H]⁺	276.1964	276.1958	2.15	C17H25NO2	Lycoflexine	magellanine / lobscurinol / epilobscurinol isomers	fawcettimine-type	3	MS1	Low intensity; reliable MS/MS spectrum not acquired	Accurate-mass match supports annotation as a C₁₇H₂₅NO₂ Lycopodiaceae alkaloid, with lycoflexine as a plausible candidate reported from Huperzia. Related fawcettimine-type isomers cannot be excluded, and because no reliable MS/MS spectrum or authentic standard was available, the assignment remains tentative at the isomer-specific level.
27	31.98	[M + H]⁺	292.1898	292.1907	3.08	C17H25NO3	Annopodine	lyconnotine isomer	lycopodine-type	3	MS1; MS/MS	292, 274, 218, 186, 174, 162, 158, 147	Accurate-mass match; MS/MS acquired. Annopodine was previously reported in H. selago gametophytes, but isomeric ambiguity with lyconnotine cannot be fully excluded without authentic standard/reference spectrum.

* m/z (calcd.) refers to the monoisotopic [M + H]⁺ ion; mass error is reported as absolute Δppm

** Evidence codes: MS1, accurate-mass measurement/formula assignment; MS/MS, tandem mass spectrum acquired; HPLC–DAD, photodiode-array detection; Std, authentic standard used for target-compound confirmation

MSI confidence: Level 1, confirmed by authentic standard (RT agreement in HPLC–DAD and MS/MS where available); Level 2, putatively identified compound or isomer set supported by diagnostic MS/MS; Level 3, putative annotation/class based on accurate mass/formula with or without non-diagnostic MS/MS, isomers possible; Level 4, unassigned or insufficient structural evidence

Abbreviations: RT retention time, ESI electrospray ionization, ppm parts per million, MSI Metabolomics Standards Initiative

Comparison of total ion chromatograms (TIC) and LC–MS signal lists indicated that qualitative profiles were broadly consistent across the analyzed media: no alkaloid peaks were observed that were exclusive to a single medium variant (Lys vs. Cad vs. Lys + Cad+PGR). Differences among variants primarily reflected changes in signal intensity and relative contributions of individual compounds rather than the presence or absence of unique constituents (Table 1).

In total, 27 LC–MS signals (RT–m/z pairs) corresponding to alkaloid pseudomolecular ions [M + H]⁺ were recorded within the retention time range RT 2.03–31.98 min. Annotations were based on accurate mass matching, calculated elemental compositions of neutral molecules, comparison with literature data, and MS/MS fragmentation where reliable spectra could be acquired [7, 12, 19–28]. Additional MS/MS data were obtained for 20 of the 27 detected features, substantially improving the evidence supporting the qualitative profile. Confidence levels were assigned according to MSI guidelines and are summarized together with evidence codes and diagnostic MS/MS fragments in Table 1. MS/MS spectra were used primarily to support alkaloid-class assignment and, where possible, to strengthen compound-level annotation. Diagnostic or characteristic fragment ions were especially important for confirming the target compounds HupA and HupB and for supporting selected lycopodine-, lycodine- and fawcettimine-type assignments. For example, HupB showed a fragmentation pattern including ions at m/z 240, 212, 198, 184, 158, 154, 130, 115 and 91, whereas HupA produced fragments including m/z 227, 210, 196, 192, 178, 165, 154, 115 and 91. In several non-target features, MS/MS fragmentation supported the proposed alkaloid class but did not allow unambiguous discrimination between closely related structural isomers; such annotations were therefore retained as putative.

In the early retention time region, signals corresponding to relatively more polar Lycopodiaceae alkaloids were detected. The peak at RT 2.03 min (m/z 249.2318, C₁₆H₂₈N₂) was annotated as dihydrodeoxycernuine (MSI level 3), although the signal intensity was too low to acquire a reliable MS/MS spectrum. Within RT 3.20–5.22 min, three signals assigned to fawcettimine-type alkaloids were recorded: fawcettimine (RT 3.20; m/z 264.1947; C₁₆H₂₅NO₂), deacetylfawcettine (RT 3.20; m/z 266.2104; C₁₆H₂₇NO₂), and deacetyllycofawcine (RT 5.22; m/z 282.2057; C₁₆H₂₇NO₃). For these features, MS/MS fragmentation was acquired and supported their assignment to Lycopodiaceae alkaloids; however, in the absence of authentic standards and because of possible structural isomerism, the annotations were retained as putative (MSI level 3; Table 1).

Between RT 5.58 and 9.70 min, signals corresponding to lycopodine/lycodine-type alkaloids and oxygenated derivatives were recorded. At RT 5.58 min, an ion at m/z 280.1900 (C₁₆H₂₅NO₃) was annotated as lycoposerramine G, with serratezomine C and lycoposerramine P considered as possible isomeric candidates. At RT 5.79 min, m/z 278.1755 (C₁₆H₂₃NO₃) was annotated as a hydroxylated serratidine isomer, with alternative candidates including lycoposerramine E and huperzine O. At RT 9.70 min, a signal at m/z 264.1953 (C₁₆H₂₅NO₂) was annotated as a lycoposerramine L/M-type isomer, with lucidioline or lycodoline as possible alternative assignments. MS/MS spectra were obtained for these features, but the observed fragmentation did not allow unambiguous discrimination between closely related structural isomers. Therefore, these assignments remain putative at the compound/isomer level (MSI level 3; Table 1).

In the later part of the chromatogram (RT ≥ 21.20 min), multiple signals corresponding to huperzine/lycodine-type alkaloids and more hydrophobic compounds were detected. Two signals at m/z 257.164 (C₁₆H₂₀N₂O) were recorded at RT 21.20 min (m/z 257.1636) and RT 25.96 min (m/z 257.1638), consistent with isomeric huperzine/lycodine-type alkaloids. The later-eluting signal at RT 25.96 min was identified as huperzine B (HupB), based on accurate mass, diagnostic MS/MS fragmentation, and agreement with an authentic standard in HPLC–DAD analysis (MSI level 1). The earlier signal at RT 21.20 min represents a distinct isomeric feature with the same elemental composition; because its intensity was too low to acquire a reliable MS/MS spectrum, it was tentatively assigned as an N-desmethylhuperzinine-related isomer based on accurate mass, formula and retention behavior (MSI level 3; Table 1).

In the same RT region, an ion at m/z 262.1790 (RT 21.20; C₁₆H₂₃NO₂) was annotated as a putative lycoposerramine C / huperzinine / huperzine I-type isomer. MS/MS fragmentation was acquired for this feature, but the assignment remains putative because structurally related Lycopodiaceae alkaloids may share similar elemental compositions and overlapping fragmentation behavior. A signal at m/z 305.2226 (RT 25.96; C₁₈H₂₈N₂O₂) was assigned as serratinidine and supported by MS/MS fragmentation, although it also remains a putative annotation in the absence of an authentic standard (MSI level 3; Table 1).

The signal corresponding to huperzine A (HupA) was detected at RT 27.23 min (m/z 243.1485, C₁₅H₁₈N₂O) and confirmed by agreement with an authentic standard in HPLC–DAD analysis and by matching MS/MS fragmentation (MSI level 1). In the adjacent RT region, signals corresponding to 11-hydroxylycodine (RT 27.62; m/z 259.1805; C₁₆H₂₂N₂O), des-N-methylfastigiatine (RT 27.62; m/z 287.2114; C₁₈H₂₆N₂O), and a signal at m/z 261.1955 (RT 27.74; C₁₆H₂₄N₂O) annotated as a putative lycodine-type isomer were recorded. MS/MS fragmentation supported the 11-hydroxylycodine assignment, whereas the des-N-methylfastigiatine-related and C₁₆H₂₄N₂O features remained MS1-based because their intensities were too low to acquire reliable MS/MS spectra. In addition, two signals interpreted as oxygenated analogues of the compound at m/z 287.2118 were detected: m/z 303.2067 (RT 27.74; C₁₈H₂₆N₂O₂; putative mono-oxygenated analogue/N-oxide) and m/z 319.2017 (RT 27.74; C₁₈H₂₆N₂O₃; putative di-oxygenated analogue). MS/MS fragmentation was obtained for the mono-oxygenated analogue, whereas the di-oxygenated feature remained MS1-based due to low signal intensity (Table 1).

Within RT 28.37–31.98 min, further signals corresponding to more hydrophobic Lycopodiaceae alkaloids were detected. The signal at m/z 248.1998 (RT 28.37; C₁₆H₂₅NO) was annotated as lycopodine/selagoline and supported by MS/MS fragmentation (MSI level 2); however, these isomeric candidates share the same elemental composition, and possible co-elution cannot be excluded under the applied LC conditions. An ion at m/z 333.2165 (RT 28.74; C₁₉H₂₈N₂O₃) was retained as an unidentified C₁₉ Lycopodiaceae alkaloid (MSI level 4), despite the acquisition of MS/MS fragments, because the available evidence was insufficient for a more specific structural assignment. Additional compounds included fawcettidine (RT 29.43; m/z 246.1852; C₁₆H₂₃NO), a C₁₆H₂₁NO₃ signal (RT 29.43; m/z 276.1588; possible huperzine F / annotine / annotinine isomer), a signal at m/z 287.2111 (RT 29.43; C₁₈H₂₆N₂O; putative des-N-methylfastigiatine isomer), flabelline (RT 30.32; m/z 289.2273; C₁₈H₂₈N₂O), lycoflexine (RT 31.75; m/z 276.1964; C₁₇H₂₅NO₂; with possible isomers), and annopodine (RT 31.98; m/z 292.1898; C₁₇H₂₅NO₃; possible lyconnotine isomer). MS/MS fragmentation was acquired for most of these late-eluting features, including fawcettidine, the C₁₆H₂₁NO₃ signal, flabelline, and annopodine; nevertheless, definitive isomer-level assignments would require authentic standards or reference MS/MS spectra.

Overall, the expanded MS/MS analysis substantially increased the confidence of the qualitative alkaloid profiling compared with MS1-only annotation. HupA and HupB were confirmed as target compounds using authentic standards and MS/MS evidence, and several additional Lycopodiaceae alkaloid features were supported by MS/MS fragmentation. However, because structural isomerism is frequent among Lycopodiaceae alkaloids and authentic standards were not available for most detected compounds, many annotations remain putative at the isomer-specific level. Thus, the LC–MS/IT–TOF data should be interpreted as a qualitative alkaloid profile supporting the complexity of the biosynthetic background rather than as a complete definitive structural characterization of all detected alkaloids.

Morphological and histological characterization of gametophytes

Across most experimental conditions, Huperzia selago gametophytes progressively produced gametophyte-derived callus tissue, which developed either as friable/loose callus or as more compact, nodular callus. Callus typically formed on the prothallus surface and/or at the base of gametophyte aggregates (Fig. 4). Callus formation was consistently more pronounced under the IBA + KIN regime than under the PGR-free regime and became increasingly evident with prolonged culture duration, being clearly visible by week 24 (Fig. 4).

Fig. 4.

Macroscopic morphology of Huperzia selago gametophyte cultures under precursor feeding and PGR regimes (week 24). Representative plate images of gametophyte-derived biomass maintained on W/S medium supplemented with (A) L-lysine (Lys), (B) cadaverine (Cad), or (C) Lys + Cad at 5, 10, and 20 mg L⁻¹. For each precursor regime, cultures were maintained either PGR-free (upper row) or in the presence of IBA + KIN (lower row; 0.05 µM indole-3-butyric acid + 1.4 µM kinetin). Cultures show varying proportions of organized prothalli and gametophyte-derived callus (friable to compact/nodular), with cadaverine-supplemented variants frequently retaining typical organized gametophyte morphology. Scale bar = 26 mm (applies to all subpanels). For improved visualization of morphological details, the figure should be viewed at higher magnification in the online version

Importantly, cadaverine-supplemented cultures frequently retained the typical dorsiventral, strap-shaped prothallus morphology, and the proportion of morphologically typical gametophytes visibly increased with cadaverine dose, particularly at 10–20 mg L⁻¹ (Fig. 4B). In contrast, in several non-cadaverine conditions, the relative contribution of callus tissue to the overall biomass increased. No callus morphology unique to a single feeding regime was observed; variation among treatments was mainly associated with the extent of callus proliferation (friable vs. compact) and the relative abundance of callus versus organized gametophyte tissue within plates (Fig. 4). Stereomicroscopic observations further illustrated the coexistence of organized prothalli (pro), rhizoids (rh), and callus sectors (cal) within the same cultures (Fig. 5). Juvenile, compact thallus bodies with abundant rhizoids (rh) were frequently observed (Fig. 5A). In addition, more differentiated, strap-shaped thallus fragments (pro) were recorded, sometimes accompanied by adjacent callus proliferations (cal) (Fig. 5B). Overall, typical gametophytes reached several millimetres in length (approximately 4–10 mm) and showed morphological variability ranging from dorsiventrally flattened/discoid forms to more cylindrical or irregular outlines, consistent with the plasticity reported for in vitro-grown Lycopodiaceae gametophytes (Figs. 5 and 6). Histological sections confirmed a dorsiventral organization and the presence of an apical meristematic region associated with a meristematic groove/notch (Fig. 6A–B; label mg). The outer surface displayed filamentous structures consistent with paraphyses on the dorsal side (Fig. 6A–B; label a) and rhizoids associated with the ventral side (Fig. 6A; label rh; Fig. 6B; label b). In cross-section, the thallus commonly appeared discoid to “hourglass-like”, with a smaller ventral region in some specimens (Fig. 6). Tissue differentiation included a peripheral region with palisade-like cells (p), a more internal parenchymatous/medullary region (m), and a surrounding cortical tissue zone (co) (Fig. 6A–B). Cells in the internal region were generally larger and more elongated along the thallus axis, whereas cells closer to the ventral surface were smaller and more isodiametric (Fig. 6).

Fig. 5.

Stereomicroscopic views illustrating the coexistence of organized prothalli, rhizoids, and callus sectors in axenic Huperzia selago cultures. A Cluster of juvenile gametophytes with compact thallus bodies bearing abundant rhizoids (rh) and visible organized prothallus tissue (pro). Scale bar = 300 μm. B More differentiated, strap-shaped prothallus fragment (pro) accompanied by adjacent callus proliferation (cal). Scale bar = 500 μm

Fig. 6.

Histological organization of H. selago prothalli showing dorsiventral structure and the apical meristematic region. A Transverse section of a juvenile prothallus with the meristematic groove (mg) and dorsiventral polarity (dorsal/ventral). B Section of a more mature prothallus fragment showing differentiated tissue zones. Labels: a, dorsal paraphyses (surface filaments); rh, rhizoids on the ventral side; p, palisade-like zone; co, cortex; m, central parenchymatous/medullary region; b, ventral region at the rhizoid-bearing surface. Scale bars: 700 μm (A) and 500 μm (B)

Discussion

Precursor supplementation of culture media (“feeding”) remains one of the simplest and most cost-effective approaches to modulate specialized (secondary) metabolism in plant in vitro systems, despite major advances in molecular biology and large-scale omics analyses [1–4, 29, 30]. Review papers consistently emphasize that the effectiveness of this strategy depends, among other factors, on (1) the tissue type and its developmental status, (2) the entry point of the supplied precursor into the pathway, and (3) downstream constraints that may become apparent only after the substrate pool has been expanded [3–5, 14, 31, 32]. The outcomes of the present study follow this general framework: exogenous L-lysine and cadaverine modulated huperzine A and huperzine B biosynthesis in axenic gametophyte cultures of Huperzia selago, and the magnitude and direction of the response were clearly dependent on culture age, dose, and the plant growth regulator context.

The rationale for applying feeding in H. selago—and potentially in other Lycopodiaceae—stems from the lysine-derived nature of club moss alkaloids [6, 7]. From a biosynthetic perspective, lysine serves as a substrate for early steps leading to piperidine-type intermediates, whereas cadaverine, the decarboxylation product of lysine, is frequently regarded as a key intermediate that enables further channeling of metabolic flux toward multiple alkaloid skeleton types [5, 7]. In the model examined here, increasing the availability of this “lysine node” indeed elevated the levels of the target metabolites (HupA and HupB), yet the response was non-linear and changed over time. This is consistent with observations from other alkaloid-producing models (e.g., Catharanthus roseus), in which feeding efficacy depends on dose and timing and on whether the primary limitation is substrate availability or the capacity of downstream enzymatic steps during culture progression [5, 12, 16, 17].

A key finding of this work is the temporal separation of HupA and HupB responses to supplementation: HupA responded earlier and more strongly during the early and intermediate phases, whereas HupB reached its highest levels predominantly at later sampling points, toward the end of culture maintenance (weeks 12–24). This pattern suggests that, despite belonging to the same lycodine-type alkaloid group, their accumulation may be governed by different time-dependent constraints, for example through distinct coupling to the metabolic maturation of cultures or through competition with parallel branches of the pathway [5, 12, 33].

For HupA, the most pronounced early-phase effect was observed under lysine supplementation at 10 mg·L⁻¹, pointing to an optimum substrate supply in younger cultures. At week 8, the Lys-10 variant reached an approximately two-fold higher HupA level compared with the control. Notably, the highest dose (20 mg·L⁻¹) did not confer a consistent advantage at the same time point, suggesting that excessive precursor supply may trigger mechanisms that limit efficient redirection of biosynthetic flux toward specialized metabolites, such as metabolic costs, transport limitations, or a shift of bottlenecks to downstream steps [3, 13, 14].

In contrast to lysine, cadaverine showed a more progressive, time-amplified effect on HupA accumulation, with the highest values observed at week 24 (best-performing variants Cad-10 and Cad-20). This indicates that in later culture stages cadaverine may support flux toward huperzine A more effectively than lysine. One plausible explanation is the “dual” nature of cadaverine: it acts both as an intermediate in lysine-derived alkaloid biosynthesis and as a compound linked to polyamine-related metabolism, potentially influencing nitrogen balance and broader physiological status of cultures [13, 14, 17]. Irrespective of the mechanistic basis, the practical conclusion is that the “best” precursor for HupA depends on culture stage: lysine promotes a rapid rise in HupA during the early phase, whereas cadaverine emerges as the dominant factor supporting high long-term accumulation, in this study from week 12 onwards.

Combined supplementation with lysine and cadaverine revealed additive tendencies but also pronounced non-linearity dependent on time and dose. Importantly, the maximum HupA level within the “intermediate window” was obtained for Lys + Cad-10 + 10 at week 12, exceeding the effects observed for the corresponding single-precursor variants (Lys-10 and Cad-10) at the same time point. By week 24, however, the combined variants were not consistently superior to the best cadaverine-based regime. This distribution of outcomes suggests that parallel supplementation may accelerate the attainment of high HupA levels, whereas in very long cultures the final outcome may be shaped by factors other than the availability of early precursors, including limitations at late steps, cofactor supply, or branch competition within the alkaloid network [3, 13, 14].

For HupB biosynthesis, the most favorable outcomes were associated primarily with combined lysine–cadaverine regimes at lower doses, with maxima occurring at late time points (weeks 12–24). This indicates that conditions supporting HupB accumulation may require both an increased input at the lysine entry node and a sufficiently stable throughput across intermediate/late steps, while excessive precursor supply may favor competing branches or reduce the efficiency of conversions leading to HupB [5, 12, 34, 35].

The interpretation of these feeding responses should be considered in the context of the current state of knowledge on Lycopodiaceae alkaloid biosynthesis. Classical biogenetic and isotope-labelling studies support the view that Lycopodium alkaloids are largely lysine-derived and that cadaverine, formed by lysine decarboxylation, represents an early intermediate in the pathway leading to piperidine-type precursors such as pelletierine and subsequently to more complex alkaloid skeletons [5, 7, 13]. At the same time, the biosynthetic routes to individual Lycopodiaceae alkaloids, including huperzine-type compounds, remain incompletely resolved, and most direct precursor-incorporation evidence has been obtained from isotope-feeding studies performed on sporophytic shoots rather than on axenic gametophyte cultures. Therefore, the present results should not be interpreted as direct proof of incorporation of exogenously supplied lysine or cadaverine into HupA and HupB molecules. Rather, they show that supplementation with these compounds modulates huperzine accumulation in the gametophyte culture system. The observed increases may reflect direct utilization of the supplied precursors, indirect effects on nitrogen metabolism, polyamine-related physiology and the developmental state of the cultures, or a combination of these mechanisms. This is particularly relevant for cadaverine, which can be viewed both as a biosynthetic intermediate of lysine-derived alkaloids and as a polyamine-related compound involved in the regulation of plant growth and metabolism. Future experiments using stable isotope-labelled lysine and cadaverine would be required to distinguish direct metabolic incorporation from broader physiological regulation and to trace carbon and nitrogen fluxes within the Lycopodiaceae alkaloid network.

The differential temporal responses observed after Lys and Cad supplementation may also reflect differences in uptake, compartmentation and intracellular availability of the two compounds. Lysine, as a proteinogenic amino acid, is expected to enter plant cells mainly through amino acid transport systems, including broad-specificity and cationic amino acid transporters [36]. Cadaverine, by contrast, is a diamine related to polyamine metabolism and may additionally depend on transport and compartmentation processes associated with polyamine homeostasis [5, 37]. Since cellular polyamine levels in plants are shaped by biosynthesis, transmembrane transport and apoplastic catabolism, differences in uptake, intracellular handling and metabolic turnover may influence the apparent efficiency of Cad as a feeding agent [5]. Transporter identity and uptake kinetics have not been characterized in H. selago and other Lycopodiaceae gametophytes; therefore, this interpretation remains hypothetical. Nevertheless, different transport routes and metabolic coupling may partly explain why Lys produced a stronger early response, whereas Cad showed a more delayed and time-amplified effect on HupA accumulation.

The biological role of HupA and HupB in Lycopodiaceae should also be considered in a broader ecological and signalling context. As nitrogen-containing specialized metabolites, Lycopodiaceae alkaloids may contribute to chemical defence against herbivores, microorganisms and pathogens; however, their function is probably not limited to defence [2, 5]. HupA and HupB belong to the lycodine-type alkaloids and are among the strongest acetylcholinesterase inhibitors reported from club mosses [5–7, 10, 11]. The structural similarity of HupA and related lycodine-type compounds to acetylcholine and to the active site of acetylcholinesterase suggests a possible connection with plant cholinergic signalling [5, 7]. Earlier experimental observations in plant models showed that acetylcholinesterase inhibitors may increase tissue sensitivity to red light and acetylcholine, which points to a possible link between acetylcholine metabolism, phytochrome-controlled responses and morphogenetic regulation [5]. In Lycopodiaceae, changes in alkaloid content and qualitative profile may therefore be associated not only with defence, but also with light-dependent developmental responses, stress signalling and transitions between developmental states [5]. This aspect remains fragmentarily understood, particularly in the gametophyte generation [5, 6]. Consequently, future studies should combine field observations with controlled in vitro experiments testing selected biotic and abiotic cues, including wounding, microbial or fungal signals, oxidative stress, light quality and defence-related hormonal signalling, to determine whether these factors can modulate HupA and HupB accumulation in axenic gametophyte cultures [3, 16, 17].

The addition of plant growth regulators (IBA + kinetin) in gametophyte cultures did not produce a uniform, consistent direction of change, indicating that their role is more likely to reshape the balance between growth, differentiation, and specialized metabolism than to simply “stimulate” alkaloid biosynthesis (HupA and HupB). The direction of the response depended on both culture age and the feeding configuration: in some variants alkaloid accumulation increased in the presence of IBA and kinetin, whereas in others it decreased. Such divergence is consistent with the concept that the auxin–cytokinin balance can indirectly reallocate resources toward specialized metabolites by altering the architecture and developmental dynamics of in vitro cultures [1, 3, 35].

Importantly, the absence of an internal unsupplemented + PGR control in the present experiment should be interpreted in the context of our earlier work on the same axenic H. selago gametophyte culture system [12]. In that study, W/S medium without growth regulators supported the highest huperzine accumulation among the tested culture variants, with HupA reaching 4.73 mg·g⁻¹ DW at week 8 and remaining above 4 mg·g⁻¹ DW between weeks 8 and 24, whereas the mean HupA content over the 24-week culture period was 3.84 ± 0.27 mg·g⁻¹ DW. HupB showed a broadly similar pattern, reaching up to 0.52 mg·g⁻¹ DW, with a mean value of 0.36 ± 0.05 mg·g⁻¹ DW. Under the corresponding W/S medium supplemented with IBA + kinetin, huperzine levels were lower than in the PGR-free W/S variant, indicating that PGRs can modify alkaloid accumulation patterns in axenic H. selago gametophytes even in the absence of exogenous alkaloid precursors [12]. These earlier data indicate that PGRs can influence the basal metabolic state of H. selago gametophytes in the absence of exogenous precursor feeding. Thus, the previous study provides an external reference for the basal PGR response in this culture model. However, because that dataset originated from a separate experiment, it was not used as an internal statistical control for the present study. Accordingly, PGR-related effects in the present work are interpreted conservatively and restricted to matched − PGR/+PGR comparisons within identical precursor supplementation regimes and doses.

An important context for the time-dependent and PGR-dependent variability of feeding effects is the morphological plasticity of H. selago gametophytes under in vitro conditions. Under most regimes, cultures exhibited callus formation and proliferation of gametophyte-derived callus—ranging from friable/loose tissue to more compact, nodular structures—typically developing on the prothallus surface and/or at the base of prothallus aggregates. Callus proliferation was more pronounced under the IBA + KIN regime than under PGR-free conditions and increased with culture duration, becoming particularly evident by week 24. Notably, cadaverine-supplemented cultures more frequently retained the typical dorsiventral, strap-shaped prothallus morphology, and the proportion of morphologically typical gametophytes increased with cadaverine dose (especially 10–20 mg·L⁻¹). Stereomicroscopic observations further confirmed the coexistence, within the same cultures, of organized prothallus fragments (pro), rhizoids (rh), and callus sectors (cal), indicating a variable contribution of tissues differing in the degree of organization (Figs. 4 and 5).

Histological sections corroborated the maintenance of dorsiventral organization and the presence of a meristematic region associated with a meristematic groove/notch, along with characteristic structural features (paraphysis-like filaments on the dorsal side, rhizoids on the ventral side, and differentiation of tissue zones into palisade-like, cortical, and inner parenchymatous/medullary regions). Collectively, these observations suggest that, over time and depending on the PGR regime, the relative contribution of biomass with distinct organization (organized prothalli vs. callus) shifted, which may in turn contribute to variability in metabolite profiles and responsiveness to modifying factors. An important limitation of the present study is related to the heterogeneous morphology of the analyzed biomass. The phytochemical analyses were performed on total harvested material and therefore do not allow us to determine whether the observed increases in HupA and HupB occurred preferentially in morphologically organized prothalli, in callus tissue, or in both fractions simultaneously. At the same time, both fractions belonged to the gametophyte generation: the cultures contained organized prothallial tissues as well as callus derived from gametophytic biomass. Thus, the limitation concerns the relative contribution of different gametophyte-derived morphological fractions rather than a distinction between sporophytic and gametophytic sources of alkaloids. This is particularly relevant from a biotechnological perspective, because organized prothalli and callus may differ in developmental status, growth rate, and metabolic activity. Our morphological and histological observations confirmed the coexistence of organized prothalli, rhizoids, meristematic regions, and callus sectors within the same culture system. Moreover, previous studies on H. selago prothallus cultures demonstrated that gametophyte cultures are capable of producing both HupA and HupB, supporting the use of this generation as an alkaloid-producing model. However, tissue-specific allocation of huperzine biosynthesis remains unresolved. These cultures similarly contained both morphologically organized gametophytic tissues and gametophyte-derived proliferative tissues, suggesting that alkaloid biosynthesis is not restricted to a single morphological component of the gametophyte generation [12]. Future work should therefore consider separating morphological fractions (organized tissue vs. callus) and comparing their alkaloid profiles to better resolve the contribution of this variability to the quantitative differences observed here [12, 35].

Although the manuscript focuses on quantitative changes in HupA and HupB, the LC–MS/IT–TOF analysis confirmed that these changes occurred against a complex alkaloid background, with 27 signals corresponding to multiple Lycopodiaceae alkaloid skeleton types. The expanded MS/MS analysis, obtained for 20 of the 27 detected features, strengthened the qualitative profile and indicated that lysine/cadaverine supplementation may re-tune a broader alkaloid biosynthetic network rather than affect only the two quantified marker compounds. At the same time, many non-target assignments remain putative at the isomer-specific level because structurally related Lycopodiaceae alkaloids may share elemental compositions and overlapping fragmentation patterns; definitive confirmation of these minor features would require authentic standards, reference MS/MS spectra or isolation-based structural elucidation [5–7, 38].

From a bioprocess perspective, the present results indicate that optimization of HupA and HupB production in H. selago gametophyte cultures requires simultaneous consideration of the production target (HupA vs. HupB), culture duration, and the type and dose of supplementation, and—where relevant—the plant growth regulator regime [1, 6, 12, 29]. The time-course profiles allow several practical scenarios to be delineated. When the priority is rapid HupA intensification and shortening of the production cycle, the most favorable outcomes were achieved with an intermediate lysine regime (Lys-10) applied in the early window, typically around week 8. When the goal is to reach a mid-phase HupA maximum, peak levels were observed at week 12 under the intermediate combined regime (Lys + Cad-10 + 10), suggesting that a concurrent increase in both the upstream substrate pool and the key intermediate particularly favors accumulation at this stage. By contrast, in long-term cultures aiming at a late HupA maximum, the most robust and reproducible effects were obtained with cadaverine-based regimes (Cad-10/Cad-20) evaluated at week 24.

Distinct trends were observed for HupB. The highest levels of this alkaloid were recorded primarily in combined Lys + Cad variants at the lower dose (Lys + Cad-5 + 5), within the window spanning weeks 12 to 24. Thus, the production optimum for HupB is shifted relative to that for HupA and does not require the highest supplementation doses, further supporting the need for separate optimization strategies for the two compounds.

Overall, our data clearly show that a regime maximizing HupA is not necessarily optimal for HupB; therefore, production strategies may require either a compromise or a staged approach in which culture conditions are selected according to the planned harvest time and the target product. Moreover, translating these findings into typical bioprocess metrics—such as total yield or volumetric productivity—requires explicit consideration of biomass accumulation [1, 6, 29]. A high alkaloid content per unit dry weight does not necessarily correspond to the highest total production, highlighting the importance of integrating metabolite data with growth parameters when designing optimized production protocols.

Taken together, the present study defines a physiological and metabolic response framework for precursor feeding in axenic H. selago gametophyte cultures and provides a basis for further work on the regulation of lysine-derived alkaloid biosynthesis. Within the design applied here, PGR-related effects were interpreted through matched − PGR/+PGR comparisons within identical supplementation regimes, whereas the qualitative LC–MS analysis was focused on representative samples capturing the major response patterns. Future studies may extend this framework by incorporating isotopic labeling approaches, integrating metabolite data with growth and differentiation parameters, including the relative contribution of callus biomass versus organized prothalli, and testing staged feeding–elicitation strategies and scaling in semi-continuous or bioreactor systems [3, 13, 16, 17, 39].

Limitations and outlook

Two design-related aspects should be emphasized when interpreting the present dataset. First, an unsupplemented + PGR control (0 mg·L⁻¹ precursors under IBA + KIN) was not included; therefore, PGR-related effects should be interpreted primarily from matched − PGR/+PGR contrasts within identical precursor regimes and doses, rather than as a standalone “PGR effect” relative to the control. Second, the qualitative LC–MS/IT–TOF assessment was performed on representative samples and should therefore be interpreted as qualitative profiling rather than exhaustive metabolomic coverage of all treatment/time-point combinations. Although additional MS/MS spectra were acquired for most detected features, several non-target annotations remain putative because structurally related Lycopodiaceae alkaloids may show isomerism and overlapping fragmentation behavior, and authentic standards or reference spectra were not available for most minor compounds. Thus, inferred shifts in the broader alkaloid network should be treated as provisional at the level of individual non-target alkaloids. Finally, the current study provides a phenotypic response framework; incorporation of isotope tracing and fraction-level analyses would help distinguish direct precursor incorporation from indirect physiological effects and clarify the contribution of morphological heterogeneity to the observed metabolite dynamics.

Conclusion

Exogenous supplementation with L-lysine and cadaverine effectively modulated huperzine A and huperzine B accumulation in axenic Huperzia selago gametophyte cultures, with responses depending on culture duration and on the dose and configuration of the applied precursors. The distinct time-course patterns observed for HupA and HupB indicate that these alkaloids differ in their responsiveness to precursor supply and support the view that the lysine-derived entry point represents an important level of control within the Lycopodiaceae alkaloid network.

Plant growth regulators (IBA + kinetin) influenced this response in a context-dependent manner, varying with precursor regime and culture stage rather than acting as a uniform stimulant of huperzine accumulation. In parallel, the morphological plasticity of the cultures and the histologically confirmed organization of the prothalli suggest that tissue differentiation status may contribute to the metabolic heterogeneity of the biomass. LC–MS/IT–TOF profiling further showed that changes in HupA and HupB occurred within a broader Lycopodiaceae alkaloid background, indicating that precursor feeding may re-shape specialized metabolism beyond the two quantified target compounds.

Taken together, the present study establishes H. selago gametophytes as an experimentally tractable and underused model for probing precursor-dependent regulation of lysine-derived alkaloid biosynthesis in an early-diverging vascular plant lineage, and it provides a practical baseline for designing controllable culture strategies aimed at reproducible production of pharmaceutically relevant Lycopodiaceae alkaloids.

Methods

Chemicals

L-Lysine (L-lysine monohydrochloride) and cadaverine (cadaverine dihydrochloride) were obtained from Sigma-Aldrich and were of analytical grade or higher. Indole-3-butyric acid (IBA) and kinetin were purchased from Grand Island Biological Company (USA). Reference standards of huperzine A (HupA) and huperzine B (HupB) with confirmed purity (> 98%) were obtained from ChromaDex Inc. (Laguna Hills, CA, USA) and were used for calibration and quantitative method validation. Solvents for chromatographic analyses, including methanol (Chromasolv^®) and acetonitrile, were of HPLC or LC–MS grade (Merck). Sodium hexafluorophosphate (NaPF₆) as well as volatile salts used for mobile-phase preparation (ammonium acetate and/or ammonium formate; LC–MS grade) were purchased from Merck. HPLC-grade water was purchased from Merck KGaA (Darmstadt, Germany), whereas LC–MS-grade ultrapure water (18.2 MΩ·cm) was produced using a water purification system (Millipore Merck, Darmstadt, Germany).

For culture preparation and ancillary procedures (where applicable), formaldehyde solution (37%) and glacial acetic acid were purchased from POCH (Gliwice, Poland), Bacto™ agar from Becton, Dickinson & Company (Franklin Lakes, NJ, USA), and Plant Preservative Mixture (PPM) from Plant Cell Technology (Washington, DC, USA). Toluidine Blue was purchased from Acros Organics (Geel, Belgium), while periodic acid and Schiff’s reagent were obtained from Merck KGaA.

Stock and working solutions of plant growth regulators and amino supplements were prepared in sterile conditions, filter-sterilized through 0.22-µm membrane filters, and aseptically added to autoclaved media after cooling, where appropriate.

Plant material (source of spores) and culture conditions

Axenic Huperzia selago gametophyte cultures used in this study were originally established from spores obtained from sporangia-bearing sporophytes collected from wild populations in Babia Góra National Park (Beskid Mountains, southern Poland; 49.574175 N, 19.531518 E). Sampling was conducted on public land within Babia Góra National Park under the relevant permissions issued by the competent authorities (details and permit/decision numbers are provided in the ‘Ethics approval and consent to participate’ section). The taxonomic identity of the plant material was verified by Wojciech J. Szypuła (Medical University of Warsaw) based on diagnostic morphological characters of H. selago. Voucher specimens (sporophytes with sporangia) were deposited in the Herbarium of the Faculty of Biology, University of Warsaw (WA; voucher no. 0000074900). The established axenic gametophyte line was subsequently maintained in vitro and used as the starting material for the present precursor-feeding experiment [12]. Gametophytes were cultivated on Whittier–Storchová (W/S) [18] medium supplemented with glucose (5 g L⁻¹) and solidified with agar (7–8 g L⁻¹). The pH of the medium was adjusted to 5.6 before autoclaving. Cultures were incubated in a phytotron in continuous darkness under a programmed 24-h temperature regime of 18 ± 1 °C and 16 ± 1 °C. Gametophytes were subcultured onto fresh medium of identical composition every 8 weeks. Details of precursor supplementation with L-lysine (Lys), cadaverine (Cad), and their combination, dose levels, sampling scheme, and PGR regimes are provided in "Experimental design and precursor feeding" section.

Experimental design and precursor feeding

The experiment was designed as a factorial study including: (1) precursor supplementation variant (Control, Lys, Cad, Lys + Cad), (2) precursor dose (5, 10, or 20 mg L⁻¹; control without supplementation), and (3) plant growth regulator (PGR) regime (PGR-free vs. IBA + KIN). Cultures were sampled at predefined time points (weeks 0, 4, 8, 12, and 24) and, at each time point, gametophyte biomass was harvested for phytochemical analyses. Basal culture conditions were selected based on our previous medium screening, in which W/S variants (PGR-free and IBA + KIN-supplemented) supported high viability and among the highest biomass growth performance; therefore, the present study focused on secondary-metabolism readouts (HupA/HupB content expressed per DW and alkaloid profiling) rather than repeating comprehensive biomass-growth analyses for each treatment [12].

To avoid ambiguity and to document the intrinsic variability of the starting material, baseline (week 0) samples were collected separately for each experimental line (i.e., for each treatment × dose × PGR regime) from the gametophyte inoculum allocated to that line at the moment of transfer onto fresh medium, before precursor addition. Consequently, week 0 alkaloid levels could differ among lines, reflecting heterogeneity of the initial culture material rather than treatment effects. For subsequent time points (weeks 4, 8, 12, and 24), unsupplemented controls (0 mg L⁻¹ precursors) were maintained under the PGR-free regime and harvested at each sampling time alongside the supplemented variants. A PGR-supplemented control (0 mg L⁻¹ precursors under IBA + KIN) was not included, as the PGR-only condition had been evaluated previously for axenic H. selago gametophyte cultures [12]. Therefore, the effect of IBA + KIN was assessed only within supplemented treatments by pairwise comparisons of matched precursor conditions (identical supplementation variant and dose) in the absence vs. presence of PGRs (− PGR vs. +PGR).

Stock solutions of L-lysine and cadaverine were prepared under sterile conditions and filter-sterilized using 0.22 μm membrane filters. Aliquots were aseptically added to autoclaved W/S medium after cooling to achieve final concentrations of 5, 10, or 20 mg L⁻¹ (Table 2). For the PGR treatment, indole-3-butyric acid (IBA; 0.05 µM) and kinetin (KIN; 1.4 µM) were added to the medium (IBA + KIN variant). The PGR-free variant contained no exogenous IBA or kinetin. For each treatment × dose × PGR regime × time point, three independent biological replicates were maintained (n = 3). For each biological replicate, alkaloid quantification was performed in three technical replicates (analytical repeats) per sample, unless stated otherwise in the analytical sections.

Table 2.

Experimental variants (precursor supplementation and PGR regime). Abbreviations: Lys, L-lysine; Cad, cadaverine; PGR-free, no exogenous IBA/KIN; IBA + KIN, 0.05 µM IBA + 1.4 µM kinetin. All precursor doses are final concentrations in the culture medium (mg L^-1)

Experimental condition	L-lysine (mg L⁻¹)	Cadaverine (mg L⁻¹)	PGR regime
Control	0	0	PGR-free
Lys-5 (PGR-free)	5	0	PGR-free
Lys-5 (IBA + KIN)	5	0	IBA + KIN
Lys-10 (PGR-free)	10	0	PGR-free
Lys-10 (IBA + KIN)	10	0	IBA + KIN
Lys-20 (PGR-free)	20	0	PGR-free
Lys-20 (IBA + KIN)	20	0	IBA + KIN
Cad-5 (PGR-free)	0	5	PGR-free
Cad-5 (IBA + KIN)	0	5	IBA + KIN
Cad-10 (PGR-free)	0	10	PGR-free
Cad-10 (IBA + KIN)	0	10	IBA + KIN
Cad-20 (PGR-free)	0	20	PGR-free
Cad-20 (IBA + KIN)	0	20	IBA + KIN
Lys + Cad-5 + 5 (PGR-free)	5	5	PGR-free
Lys + Cad-5 + 5 (IBA + KIN)	5	5	IBA + KIN
Lys + Cad-10 + 10 (PGR-free)	10	10	PGR-free
Lys + Cad-10 + 10 (IBA + KIN)	10	10	IBA + KIN
Lys + Cad-20 + 20 (PGR-free)	20	20	PGR-free
Lys + Cad-20 + 20 (IBA + KIN)	20	20	IBA + KIN

Morphological and histological characterization of gametophytes

Gametophyte development and morphology were monitored at weeks 4, 8, 12, and 24 using stereomicroscopy and light microscopy. Macromorphological traits of intact gametophytes were assessed with a Zeiss Discovery V12 stereomicroscope (Zeiss, Germany) equipped with Apochromat S 0.63× and PlanApo S 1.5× objectives; images were recorded with a digital camera. Representative micrographs were acquired using a Zeiss Axio Lab A1 light microscope (Zeiss, Germany) equipped with a digital camera. Developmental stage (juvenile vs. mature gametophytes/prothalli) was assigned based on published morphological criteria [18], including overall thallus morphology and, where present, the appearance of reproductive structures. Photographs presented in the Results section represent typical examples of the observed structures.

For histological characterization, representative specimens were selected based on (1) the typical morphology observed for a given time point and culture regime and (2) the presence of reproductive structures (when present), such as archegonia and/or antheridia. Samples from both culture regimes (PGR-free and IBA + KIN) were processed to visualize internal anatomy and developmental features. For each histological assessment, at least 10 gametophytes were analyzed. Specimens were vacuum-infiltrated and fixed in FAE solution (formaldehyde 37% : glacial acetic acid : ethanol 50% = 6.5 : 3.5 : 100, v/v/v) for 72 h at room temperature, dehydrated through a graded ethanol series (40, 50, 70, 90, and 100% v/v; 30 min per step), embedded in Paraplast Plus (McCormick Scientific), and sectioned at 5–10 μm. Sections were stained with periodic acid–Schiff (PAS) or double-stained with PAS and Naphthol Blue Black (NBB) and examined under transmitted light. Where applicable, additional observations were made using tissue autofluorescence (e.g., blue autofluorescence of mature archegonia and red chlorophyll autofluorescence) under standard fluorescence filter settings.

Plant material for phytochemical analyses

Plant material intended for phytochemical analyses was harvested over the 24-week culture period at predefined sampling points (weeks 0, 4, 8, 12, and 24) from each experimental variant (feeding treatments and corresponding controls). Immediately after collection, gametophytes were stored at −80 °C until further processing.

Preparation and purification of extracts

Alkaloids were extracted using a protocol adapted from previously described methods [12, 19]. Briefly, frozen biomass was lyophilized, finely powdered, and an accurately weighed portion of dry material (typically ~ 0.25 g DW) was transferred to glass tubes. Samples were extracted three times with HPLC-grade methanol using decreasing solvent volumes (5 mL, 4 mL, and 3 mL). Each extraction step was assisted by sonication in a Sonorex ultrasonic bath at 40 °C for 30 min. After each step, the methanolic supernatant was collected and pooled. Combined extracts were evaporated to dryness under reduced pressure. The residue was reconstituted in HPLC-grade methanol to obtain a final extract concentration of approximately 1 mg·mL⁻¹. Before chromatographic analyses, samples were clarified by centrifugation and filtered through 0.22-µm syringe filters to remove particulate matter.

HPLC–DAD qualitative and quantitative determination of HupA and HupB

Qualitative profiling and quantitative determination of huperzine A (HupA) and huperzine B (HupB) were performed by HPLC–DAD using a Shimadzu HPLC system equipped with a diode-array detector and LabSolutions software. Chromatographic separation was carried out on a Hypersil GOLD C18 column (250 × 4.6 mm) with a compatible guard column (10 × 4 mm, 5 μm). The mobile phase consisted of (A) water containing 30 mM sodium hexafluorophosphate (NaPF₆) and (B) acetonitrile. The gradient program was: 0–5 min, 0→25% B; 5–20 min, 25→45% B; 20–30 min, 45→80% B [19]. The column temperature was maintained at 24 ± 1 °C, the flow rate was 1.0 mL·min⁻¹, and the total run time was 30 min. DAD chromatograms were recorded at 210, 230, 260, and 308 nm.

Quantification was performed using external calibration with certified HupA and HupB reference standards, and alkaloid contents were expressed as mg·g⁻¹ DW. For reference, the mean HupA content in the gametophyte biomass used to initiate subcultures was previously determined as 2.20 ± 0.11 mg·g⁻¹ DW [10]. For each biological replicate, three technical (analytical) replicates were prepared and analyzed by HPLC–DAD; technical replicates were averaged for each biological sample. Final results are reported as mean ± SD across biological replicates, unless stated otherwise.

LC–MS/IT–TOF analysis (alkaloid profiling)

For MS-based alkaloid profiling and confirmation of HupA/HupB signals, analyses were performed using a Shimadzu Prominence HPLC system coupled to an LC–MS/IT–TOF mass spectrometer equipped with an ion trap (IT), a time-of-flight (TOF) detector, and an electrospray ionization (ESI) source (Shimadzu). Mass spectra were acquired in positive-ion mode using LC-MS solution software (Shimadzu). Chromatographic separation was conducted on a Kinetex C18 column (2.6 μm, 2.1 × 100 mm, Phenomenex). The injection volume was 5 or 10 µL, the column oven temperature was set to 40 °C, the flow rate was 0.15 mL·min⁻¹, and the analysis duration was 70 min. PDA data were recorded over 200–800 nm. The mobile phase consisted of (A) water containing 5 mM ammonium formate (HCOONH₄; pH 4.0) and (B) acetonitrile. The gradient was: 0–1 min, 0→3% B; 3–40 min, 3→30% B; 40–50 min, 30→95% B; 50–55 min, 95% B; 55–57 min, 95→3% B, followed by 13 min re-equilibration at 3% B.

MS acquisition parameters were as follows: polarity positive, mass range m/z 100–800, accumulation time 10 ms, interface temperature 220 °C, heat block temperature 220 °C, nebulizing gas flow 1.5 L·min⁻¹, drying gas pressure 100 kPa, and ion spray voltage (IS) + 4.5 kV. The LC–MS/IT–TOF system was calibrated using the manufacturer’s calibration mixture prepared in ACN : 5 mM ammonium formate (pH 4.0) at 1:1 (v/v). Calibration samples were vortex-mixed and centrifuged; the supernatants were transferred to LC vials.

LC–MS/IT–TOF profiling was performed on selected representative extracts to confirm the presence of HupA/HupB signals and to compare qualitative alkaloid profiles between conditions. Representative samples were chosen to reflect typical HPLC–DAD chromatographic patterns within each condition; where applicable, samples representing distinct quantitative responses were also included.

Statistical analysis

All quantitative data are reported as mean ± SD. For each treatment × time point, three independent biological replicates were analyzed (n = 3). For each biological replicate, HPLC–DAD quantification was performed in technical triplicate; technical replicates were averaged prior to statistical testing, and all inferential statistics were conducted on biological replicates. Graphing and statistical analyses were performed in GraphPad Prism (GraphPad Software), with independent verification in Python (pandas, NumPy, SciPy, statsmodels).

To document intrinsic variability of the starting material, week 0 measurements were treated as baseline descriptive values and were not subjected to significance testing. Therefore, global hypothesis testing and post hoc comparisons were performed for weeks 4, 8, 12, and 24 only.

For each analyte (HupA and HupB), global effects were assessed using a two-way ANOVA with fixed factors time (weeks 4, 8, 12, 24) and medium variant (a single categorical factor representing each feeding × dose × PGR condition, plus the PGR-free unsupplemented control), including the time × medium variant interaction. Model assumptions were evaluated by inspection of residuals (normality and homoscedasticity); where necessary, data were transformed prior to analysis.

Planned comparisons versus control were performed within each week using Dunnett’s multiple comparisons test against the PGR-free unsupplemented control, controlling the family-wise error rate within that week. Because an unsupplemented PGR-supplemented control (0 mg L⁻¹ precursors under IBA + KIN) was not included, comparisons of + PGR variants to the unsupplemented control should be interpreted as the combined effect of precursor supplementation under IBA + KIN relative to the PGR-free control at the same time point. To evaluate whether IBA + KIN modified the feeding response, matched planned contrasts were performed within each precursor condition (same supplementation variant and dose) by comparing –PGR vs. + PGR at each time point; p-values were adjusted for multiple testing using the Holm method (within each week). Statistical significance was set at α = 0.05 (two-sided).

Authors’ contributions

WS conceived and designed the study, performed the experimental work, and wrote the manuscript. MM conducted the HPLC–DAD analyses and contributed to the interpretation of phytochemical data. BW carried out the HPLC–MS analyses and participated in the interpretation of phytochemical results. MM and BW critically reviewed the manuscript. All authors read and approved the final manuscript.

Funding

The initial stages of this research were supported by a grant from the Ministry of Science and Higher Education of Poland (grant no. N N405 362237). The funding body had no role in study design, data analysis, or manuscript preparation.

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files. Additional raw data and analytical outputs are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Sampling of Huperzia selago spores was performed on public land within Babia Góra National Park (Poland) with permissions from the competent authorities (Minister of the Environment, decision No. DOPpn-4102-307/16938/11/RS; Regional Directorate for Environmental Protection in Kraków, decision No. OP-I.6400.30.2011.PWi; Babia Góra National Park, permit/reference No. ZDB-0-060-02/04/10/12). All field sampling and handling of plant material were conducted in accordance with the applicable Polish regulations and the rules of Babia Góra National Park. This study did not involve human participants or vertebrate animals; therefore, ethics approval and consent to participate were not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.