Pseudowintera Colorata Research Articles

Antimicrobial Properties against Human Pathogens of Medicinal Plants from New Zealand

The emergence of resistant microorganisms towards standard antibiotics has stimulated an on-going exploration for new sources of antimicrobials. The microbial susceptibility of extracts produced from leaf, bark, or rhizome parts of nine different New Zealand bushes was investigated using liquid broth dilution and agar plating techniques. Minimum inhibitory (MIC) and lethal concentrations (MLC) were expressed in micrograms of dry extract per milliliters of solution. The lowest MIC of 62.5 μg/mL was determined for methanol extract of Kunzea ericoides against Bacillus cereus and Candida albicans, and ethyl acetate extract of Pseudowintera colorata against Staphylococcus aureus. Additionally, K. ericoides also presented the lowest MLC of 250 μg/mL against S. aureus and B. cereus (methanol extract), and against S. aureus (ethyl acetate extract). The methanol extract of Weinmannia racemosa was lethal to B. cereus (MLC = 250 µg/mL). Some of the extracts of Phormium tenax, Schefflera digitata, and Pomaderris kumeraho were antimicrobial against S. aureus and B. cereus (MIC = 500 µg/mL). The extracts of Geniostoma ligustrifolium and Melicytus ramiflorus plants did not exhibit antimicrobial activity.

Fungal Communities in the Native New Zealand Medicinal Plant Pseudowintera colorata (Horopito) Are Determined by Plant Organ Type and Host Maturity with Key Members Promoting Plant Growth.

The plant Pseudowintera colorata is well known for its antimicrobial and medicinal properties and is endemic to New Zealand. Using PCR-Denaturing gradient gel electrophoresis (DGGE), we investigated the factors influencing the composition of endophytic fungal communities in P. colorata from ten distinct sites across New Zealand. Our results showed that plant organs of P. colorata influenced the diversity and richness of endophytic fungi (PERMANOVA, p < 0.05). In addition, plant maturity and its interactions revealed that endophytic fungal communities formed discrete clusters in leaves, stems, and roots of mature and immature P. colorata plants (PERMANOVA; p = 0.002, p = 0.001 and p = 0.039, respectively). For identifying isolates with biocontrol potential, dual culture tests were set up against four different phytopathogenic fungi. Isolates with high activity (zone of inhibition > 10 mm) were sequenced and identified as Trichoderma harzianum, Pezicula neosporulosa, Fusarium tricinctum, Metarhizium sp., and Chaetomium sp. Applying selected endophytic fungi (n = 7) as soil drenchers significantly increased the growth of P. colorata seedlings and produced more internodes. Seedling shoots treated with Trichoderma sp. PRY2BA21 were 2.2 × longer (8.36 cm) than the untreated controls (3.72 cm). Our results elucidate the main plant factors influencing fungal community composition and demonstrate a role for endophytic fungi in P. colorata growth and further demonstrate that medicinal plants are a rich source of endophytes with potential as biocontrol agents.

Phytoremediation of microbial contamination in soil by New Zealand native plants

Novel research has demonstrated that the roots of some bioactive plants - called pathogen phytoremediation plants - enhance die-off of pathogenic organisms in the soil. Strategic establishment of pathogen phytoremediation plants may reduce the transport of human pathogens to water sources. Such plantings could be used in riparian margins, as buffer strips to protect drinking water supplies, or block planting in ‘critical source areas’ of microbial contamination, such as grazing paddocks, organic waste – including sewage sludge - amended land, animal feedlots and housing facilities, and manure storage areas. This work aimed to investigate the antimicrobial activity of a range of New Zealand native plants known for their antimicrobial potential from previous research or through indigenous knowledge, and to assess if any of them could potentially be used for pathogen phytoremediation. Two laboratory screening experiments demonstrated the antimicrobial activity of Leptospermum scoparium, including the local variety swamp mānuka, Kunzea ericoides, Pseudowintera colorata, and Metrosideros robusta against three human pathogens and two indicator organisms. A greenhouse experiment showed a 90% reduction of Escherichia coli numbers in dairy shed effluent irrigated pots after 14 days in soils under swamp L. scoparium and M. robusta, compared with 45 days in soil under Lolium perenne. The pH in the soil under swamp L. scoparium and M. robusta was significantly lower than under L. perenne, which could partially explain the faster E. coli reduction.

The complete plastome sequences of Pseudowintera colorata and Tasmannia lanceolata (Winteraceae – Canellales)

In this study, we report the complete plastome sequences of two Winteraceae taxa, Pseudowintera colorata (MT555077) and Tasmannia lanceolata (MT555078). Both plastomes show typical quadripartite structure. The plastome size of P. colorata is 161,675 bp, which consists of 89,583 bp large single-copy (LSC), 18,606 bp small single-copy (SSC), and 26,743 bp inverted repeat (IR) regions. The plastome size of T. lanceolata is 160,424 bp, which consist of 88,589 bp LSC, 18,351 bp SSC, and 26,742 bp IR regions. Both plastomes contain 113 genes, including 79 protein-coding, 30 tRNA, and four rRNA genes. Sixteen genes contain one intron and two genes (clpP and ycf3) have two introns. Ninety-three and 89 simple sequence repeat (SSR) loci are scattered in the P. colorata and T. lanceolata plastomes, respectively. Our phylogenetic tree shows the relationship of (T. lanceolate (P. colorata, Drimys granadensis)) in the Winteraceae. The Canellales (incl. Winteraceae) are the sister group of Piperales.

Community Structure, Diversity and Potential of Endophytic Bacteria in the Primitive New Zealand Medicinal Plant Pseudowintera colorata.

Although the importance of the plant microbiome in commercial plant health has been well established, there are limited studies in native medicinal plants. Pseudowintera colorata (horopito) is a native New Zealand medicinal plant recognized for its antimicrobial properties. Denaturing gradient gel electrophoresis (DGGE) and Illumina MiSeq analysis of P. colorata plants from ten sites across New Zealand showed that tissue type strongly influenced the diversity and richness of endophytic bacteria (PERMANOVA, P < 0.05). In addition, two OTUs belonging to the genus Pseudomonas (Greengenes ID: 646549 and 138914) were found to be present in >75% of all P. colorata leaf, stem and root samples and were identified as the members of the P. colorata “core endomicrobiome”. Culture-independent analysis was complemented by the recovery of 405 endophytic bacteria from the tissues of P. colorata. Some of these cultured endophytic bacteria (n = 10) showed high antagonism against four different phytopathogenic fungi tested. The influence of endophytic bacteria on plant growth was assessed by inoculating P. colorata seedlings. The mean shoot height of seedlings treated with Bacillus sp. TP1LA1B were longer (1.83×), had higher shoot dry weight (1.8×) and produced more internodes (1.8×) compared to the control.

The potential use of pulsed electric field to assist in polygodial extraction from Horopito (Pseudowintera colorata) leaves

Horopito (Pseudowintera colorata) contains polygodial as an active compound that has many health beneficial properties. The potential of applying a continuous pulsed electric field (PEF) as a pretreatment step prior to solvent extraction of polygodial from Horopito leaves was studied. Horopito leaves suspended in water were subjected to PEF at electric field intensity ranging from 5 to 25 kV/cm and pulse frequencies from 200 to 800Hz. The interaction between electric field intensity and pulse frequency was found to have a significant role in extraction. Both electro-permeabilization and temperature increase from treatment caused some polygodial leaching from the leaves prior to solvent extraction. The study revealed that PEF at low electric field intensity and high frequency is the most effective way to achieve higher solvent extraction yield while minimizing the effect of leaching. The maximum improvement was obtained when PEF at 5 kV/cm and 800Hz for 348 μs were applied, giving a polygodial extraction yield of about 16.6% higher than that of non-PEF treated leaves.

Community Structure of Endophytic Actinobacteria in a New Zealand Native Medicinal Plant Pseudowintera colorata (Horopito) and Their Influence on Plant Growth.

The role of plant endophytic Actinobacteria remains poorly understood with no reports of these communities in New Zealand native plants. This first investigation of endophytic Actinobacteria in New Zealand targeted the culturally significant medicinal shrub Pseudowintera colorata (horopito) as a model plant. Community analysis in plant tissues collected from ten geographically distinct sites showed that tissue type had the strongest influence on diversity and richness of endophytic Actinobacteria. More denaturing gradient gel electrophoresis (DGGE) bands were obtained from stems (n = 18) compared to roots (n = 13). Sequencing analysis of the major bands (n = 20) identified them as uncultured bacteria, Streptomyces sp. and Angustibacter peucedani. Using two Actinobacteria-specific media, nine isolates were recovered from surface-sterilised P. colorata tissues. This was approximately 12% of the total taxa and correlated well with culturable numbers in international studies. In vitro analysis of the functionality of these strains showed that Streptomyces sp. PRY2RB2 inhibited all the tested phytopathogenic fungi (n = 4), Streptomyces sp. UKCW/B and Nocardia sp. TP1BA1B solubilised phosphate and produced siderophores. The functionality of the phosphate solubilising strains (n = 2) in vivo was investigated by inoculation of P. colorata seedlings. After 4months, the mean shoot height of seedlings treated with Nocardia sp. TP1BA1B was 1.65× longer, had higher shoot dry weight (1.6×) and number of internodes (1.67×) compared to control. This study identified for the first time a key group of endophytic Actinobacteria that are likely to be important in the ecology of New Zealand flora.

Approach for Polygodial Extraction from Pseudowintera colorata (Horopito) Leaves Using Deep Eutectic Solvents

Polygodial is a bioactive compound that is present in an ancient native plant known as “horopito” (Pseudowintera colorata) in New Zealand, and it possesses antifungal, antibacterial, antifeedant, insecticidal, and antithelmintic properties. In this work, an approach for polygodial extraction using deep eutectic solvents (DESs) is presented. Two newly prepared DESs based on 1-dodecanol and polyethylene glycol were found to be comparable with ethanol in their polygodial extractability. However, they exposed a superior ability to protect polygodial from degradation and also a better solvent reusability for extraction of polygodial as compared to ethanol. The Box-Behnken design (BBD) in combination with response surface methodology (RSM) was used to design the experiment and optimization of polygodial extraction using the dodecanol-based DES. The optimal condition was acquired at 47.13 °C, for 1.03 h, and 5.01% w/v biomass, where 12.35 ± 0.05 mg of polygodial/g of dried horopito leaf was extracted.

The significance of shared leaf shape in Alseuosmia pusilla and Pseudowintera colorata

Leaf shape, size, and colour are used by herbivores to identify sources of palatable foliage for food. It is possible, therefore, that an undefended plant might gain protection from herbivores by matching leaf characteristics of a chemically defended species. We demonstrate the use of a geometric morphometric approach to quantify spatial variation in leaf shape and size across populations of Pseudowintera colorata (Raoul) Dandy, and a putative Batesian mimic, Alseuosmia pusilla (Colenso) A. Cunningham. These are unrelated, sympatric species that, to the human eye, bear strikingly similar foliage. Using the Cartesian coordinates of leaf margins as descriptors of leaf shape, we found that in the chemically defended P. colorata, leaves were morphologically distinct from all of the neighbouring species except for the undefended A. pusilla. Alseuosmia pusilla individuals were more similar to neighbouring than to distant P. colorata, and 90% of leaf shape variation in the two species varied similarly across an altitudinal gradient. The data are consistent with Batesian mimicry, wherein the conspicuous characteristic of a defended model is replicated by an undefended mimic across its entire growing range. Our study provides the first detailed and powerful quantitative leaf shape evidence of leaf shape being matched between an undefended plant species to a chemically defended unrelated species across a shared growing range, and highlights the importance of using a spatially explicit morphometric method when investigating leaf shape, especially in relation to plant mimicry.

Leaf colour polymorphisms: a balance between plant defence and photosynthesis

Summary Whether plants use leaf colour to deter herbivores remains controversial. The warning signal hypothesis predicts that red pigmentation is adaptive by reducing herbivory; plants with predominantly red foliage should have higher fitness than those with green leaves. Despite many discussions, this prediction has rarely been tested, and alternative, non‐exclusive hypotheses cannot be ruled out. We have exploited leaf colour polymorphism in Pseudowintera colorata to test the warning signal hypothesis and to address possible alternative explanations. Consistent with warning signals, redder foliage contained higher concentrations of polygodial, a sesquiterpene dialdehyde with strong antifeedant properties, and incurred less herbivory than green leaves. Redder plants hosted 22% fewer lepidopteran leafroller larvae than neighbouring green plants. However, contrary to the predictions of the hypothesis, there were no differences in fitness parameters between red and green plants. Overall leaf canopy colour was not a significant predictor of the number of seeds per fruit or of mean seed weight. This may be explained by differences in photosynthesis: green P. colorata leaves had 47% higher maximum CO2 assimilation rates than matched red leaves from neighbouring plants. These results indicate that the benefits of deterring insect herbivores by signalling may be balanced by the higher photosynthetic rate of non‐signalling plants. A balance between signalling and photosynthesis is a novel mechanism for the maintenance of leaf colour polymorphisms in nature. Synthesis. Anthocyanin pigments may simultaneously serve multiple functions within leaves, and individuals of the same plant species may use different strategies to cope with insect herbivores. Therefore, investigations into the role of these pigments in plant–insect interactions need to consider plant physiology and the diversity of plant defence mechanisms.

Antimicrobial potential of bacterial endophytes that inhabit New Zealands medicinal plant &lt;i&gt;Pseudowintera colorata&lt;/i&gt;

Medicinal plants are valued for the naturally bioactive compounds they produce International research has demonstrated that these plants contain endophytic microorganisms that through close interaction with the plants physiology contribute to the production of host metabolites either directly or via involvement in biochemical pathways Some endophytes can produce bioactive compounds that are either the same or similar to those originating from their host plants Pseudowintera colorata (horopito or NZ pepper tree) an endemic New Zealand plant used in traditional Maori medicine (rongo257;) is recognised for its antimicrobial properties The biologically active chemical constituent polygodial is used for treating candidiasis A total of 340 endophytic bacteria were recovered from surface sterilised horopito leaves stems and roots from nine sites across New Zealand; strains were assessed for their ability to inhibit the phytopathogens Neofusicoccum parvum N luteum Ilyonectria liriodendri Nectria galligena Pectobacterium atrosepticum and P brasiliensis using a dual culture assay Inhibition zones 1048726;3 mm were considered a positive result Out of the strains tested seven showed activity against all the phytopathogenic fungi tested and ten strains were active against at least three phytopathogens Future work will determine if any of the endophytic bacteria produce polygodial

Distribution of Drimane Sesquiterpenoids and Tocopherols in Liverworts, Ferns and Higher Plants: Polygonaceae, Canellaceae and Winteraceae Species

The liverwort, Porella vernicosa complex produces a very hot tasting polygodial, a drimane-type sesquiterpene dialdehyde. The same compound has been isolated from two ferns, Thelypteris hispidula and Blechnum fluviatile, as well as from the higher plants Polygonum hydropiper, P. hydropiper f. purpurascens (Polygonaceae), Cinnamosma, Caspicodendron, Canella and Warburgia species (Canellaceae), and Pseudowintera colorata, Tasmannia lanceolata, Drimys and Zygogynum species (Winteraceae). In addition, the liverworts and higher plants which elaborate polygodial and its related pungent drimane dials contain a small amount of alpha-tocopherol, gamma-tocopherol or delta-tocotrienol. The present paper gives the results of a comparative study on the drimane-type sesquiterpenoids in some liverworts, ferns and higher plants, and the role of tocopherols in these plant groups.

Red leaf margins indicate increased polygodial content and function as visual signals to reduce herbivory in Pseudowintera colorata

Red-pigmented leaf margins are common, but their functional significance is unknown. We hypothesized that red leaf margins reduce leaf herbivory by signalling to herbivorous insects the presence of increased chemical defences. Leaves were collected from a natural population of Pseudowintera colorata. Margin size, herbivory damage, anthocyanin content and concentrations of polygodial, a sesquiterpene dialdehyde with antifeedant properties, were quantified. Feeding trials involving larvae of Ctenopseustis obliquana, a generalist herbivore, were conducted on red- and green-margined P. colorata leaves in darkness, or under white, green or red light. Leaves with wider red margins contained higher concentrations of polygodial and anthocyanins, and incurred less natural herbivory. In trials under white light, C. obliquana consumed disproportionately more green- than red-margined leaf laminae. Larvae exhibited no feeding preference when light was manipulated such that leaf colour discrimination was impaired. Red leaf margins provide a reliable and effective visual signal of chemical defence in P. colorata. Ctenopseustis obliquana larvae perceive and respond to the colour of the leaf margins, rather than to olfactory signals. Our study provides direct experimental evidence for aposematic coloration in red leaves.

Chemotaxonomy of Pseudowintera: Sesquiterpene dialdehyde variants are species markers

Two sesquiterpene dialdehydes, the 1β- E-coumaroyl-5α-hydroxypolygodial plus the known 1β- E-cinnamoyl-5α-hydroxypolygodial, were isolated from the recently described species Pseudowintera insperata. This discovery is a further example of the rare sesquiterpene dialdehyde coumarate/cinnamate combination being found exclusively in the family Winteraceae. Another sesquiterpene dialdehyde, isopaxidal, with the rare rearranged drimane skeleton, was isolated from Pseudowintera axillaris. The sesquiterpene dialdehyde contents of leaves of 25 individual plants of the four Pseudowintera species, all endemic to New Zealand, were measured by HPLC. P. insperata individuals all had high levels (3.0–6.9% of leaf dry wt.) of the coumarate, P. axillaris had high levels (2.2–6.9%) of paxidal, and Pseudowintera colorata from different areas of New Zealand contained varying levels of polygodial (1.4–2.9%) and 9-deoxymuzigadial (0–2.9%). Therefore the sesquiterpene dialdehydes are good species markers.

Contrasting chemistry of fruits and leaves of two Pseudowintera species: Sesquiterpene dialdehyde cinnamates and prenylated flavonoids

Two new sesquiterpene dialdehydes, cinnamate 7 and coumarate 8, were isolated from the fruits of Pseudowintera colorata. The known sesquiterpene dialdehydes polygodial 1 and 9-deoxymuzigadial 2 were also found in these fruits, at 5% w/w compared to 0.5% in the leaves. Fruits of Pseudowintera axillaris contained no sesquiterpene dialdehyde cinnamates, even though these are present in the leaves, but did contain prenylated flavanones 11– 13. Compounds 7 and 8 are further examples of the rare sesquiterpene dialdehyde cinnamate combination, found exclusively in the family Winteraceae. This is the first report of the uncommon prenylated flavanones in Winteraceae.

An evaluation of new treatment options for candidiasis using natural products

The aim of this paper is to evaluate new treatment options for candidiasis using natural products. It discussed clinical trials, which found that combination of two phytochemicals isolated from common food spices (anethole and polygodial) was able to suppress the growth of candida more rapidly and effectively than the conventional anti-fungal drugs, without any side effect. Candidiasis is the name given to common yeasts that live within human intestines and certain mucous membranes. We as humans are born with candida. They usually live in harmony with other intestinal fauna in our body when kept in check by acidophilus and other beneficial micro flora. In the absence of these checks, candida would grow rapidly and its overgrowth has been suggested as the origin of a complex medical syndrome called chronic Candidiasis also known as thrush. Family doctors either remain sceptical about intestinal Candidiasis or prescribe anti-fungal drugs with unpleasant side effects such as blurred vision, dry mouth and nausea. Scientists have found that these natural products could treat the condition without the side effects. In conclusion, the paper recommended that the natural treatment should be followed by drinking lots of water, eating diets low in carbohydrate but high in fibre and supplementing with acidophilus to stop recurrence of candidiasis. Keywords: candidiasis, natural products, Pimpella anisum, polygodial, Pseudowintera colorata Nigerian Journal of Natural Products and Medicine Vol. 9 2005: 11-13

Dynamics of upland conifer/broadleaved forest at Waihaha, central North Island, New Zealand

Regeneration in upland Podocarpus hallii‐Prumnopitysferruginea/Quintinia serrata‐Weinmannia racemosa forest, central North Island, New Zealand, was sampled in the three phases of the forest growth cycle. Mean age, determined from the largest stem present, differed significantly between gaps (13 yr), building‐phase (58 yr), and mature forest (221 yr), which comprised 8, 28, and 65%, respectively, of the forest area. Current diameter growth rates among species (Griselinia littoralis > W. racemosa, Q. serrata > P. hallii, P. ferruginea) did not differ between phases. Canopy P. hallii sampled were up to 500 yr old and P. ferruginea were 550 yr old. Broadleaved species were all younger on average (<250 yr). Three replacement strategies among major species involve establishment in different stages of the forest growth cycle, reflecting specific shade tolerances. Relatively intolerant W. racemosa and G. littoralis establish solely in gaps (gap‐phase replacement), Dacrydium cupressinum mostly in gaps. More tolerant P. ferruginea and Q. serrata establish in gaps and building‐phase forest. Shade‐tolerant P. hallii, Nestegis cunninghamii, Elaeocarpus hookerianus, Pseudowintera colorata, and Neomyrtus pedunculata establish throughout the forest growth cycle, Myrsine salicina only in later stages. A conifer “regeneration gap” is locally evident in relatively shade‐intolerant and fertility‐demanding Prumnopitys taxifolia. Basal area recovery after gap formation is slow in relation to overall basal area here and in other forests in the region, and may result from a relatively long history of browsing by introduced mammals.

Long-term influences of introduced deer on the composition and structure of New Zealand Nothofagus forests

New Zealand’s Nothofagus (southern beech) forests evolved in the absence of mammalian herbivores. The deliberate introduction of deer in the 19th century caused a rapid decline in the abundance of palatable understorey herbs and shrubs, but the longer-term consequences of deer herbivory remain poorly understood. This study uses data from permanent plots established in two separate surveys in northern and southern Kaimanawa Forest Park, central North Island, New Zealand to relate the impacts of two post-irruptive populations of deer to two decades of change in forest composition and canopy species regeneration. Plots were established on randomly located transects in Nothofagus forest between 1979 and 1982. Some were re-measured in 1987/1988, and all were re-measured between 1998 and 2000. Our objective was to gain a better understanding of the long-term effects of deer on Nothofagus forests. In the northern forests dominated by Nothofagus fusca, N. menziesii and Weinmannia racemosa, there was an expansion in the stem densities (stems ≥20 mm diameter at breast height) of highly unpalatable species of small trees. Numbers of unpalatable Pseudowintera colorata stems increased on north-facing slopes. The unpalatable Neomyrtus pedunculatus and Leucopogon fasciculatus increased in number at many sites. Analysis of seedling densities indicated that two palatable species ( W. racemosa and Griselinia littoralis) were failing to recruit into the >75 cm height class. In the southern Kaimanawa forests of N. solandri var. cliffortioides, understorey composition shifted towards browse-tolerant herbs. This is consistent with deer influencing the competitive interactions of herb and shrub communities, but storm-induced disturbances dominated tree demographics in the southern Kaimanawa, making the effects of deer difficult to ascertain. It is possible, but less likely, that factors other than deer, such as other herbivores and normal successional processes, are responsible for the changes in forest structure and composition that were observed in this study. There is some evidence that deer were affecting gap-phase regeneration of Nothofagus, but not strongly enough to prevent recruitment on a large scale. We believe that there is sufficient evidence of deer browsing shifting the composition of Kaimanawa Forest Park Nothofagus forests for managers to justify increased deer control.

Do anthocyanins function as antioxidants in leaves? Imaging of H2O2 in red and green leaves after mechanical injury

AbstractPurified anthocyanin extracts show strong antioxidant properties in vitro, but it is not known whether they can scavenge reactive oxygen in living cells. The oxidative responses in red and green portions of Pseudowintera colorata leaf laminae were compared by the real‐time imaging of H2O2 in cells after mechanical injury. An oxidative burst was elicited almost immediately from chloroplasts in the palisade mesophyll, as evidenced using the fluorochromes dichlorofluorescein and scopoletin. H2O2 accumulated in green lamina regions for 10 min, and then decreased slowly. By contrast, red regions recovered rapidly, and maintained consistently low levels of H2O2. Infusion of cells with N‐acetyl‐l‐cysteine accelerated the depletion of H2O2 from green regions. Wounded leaves ultimately developed a localized necrotic lesion and an intense anthocyanic band. The red regions were enriched in anthocyanins, flavonols, dihydroflavonols, and hydroxycinnamic acids. Only the anthocyanins were suitably located to account for the enhanced rates of H2O2 scavenging. The data support the hypothesis that red cells have elevated antioxidant capabilities in vivo.

Infraspecific variation of insecticidal sesquiterpene dialdehydes in Pseudowintera colorata

HPLC and NMR methods are described for determining the levels of the sesquiterpene dialdehydes polygodial and 9-deoxymuzigadial in the foliage of Pseudowintera colorata. Analyses of 25 individual plants, from four populations on the South Island of New Zealand, showed two chemotypes: a mixed chemotype with similar levels of polygodial and 9-deoxymuzigadial, and a polygodial chemotype with very low levels of 9-deoxymuzigadial. Only the polygodial chemotype was found in northern and southwestern populations, both chemotypes were found in a central eastern population, and only the mixed chemotype was found in a southeastern population.