LC-HRMS/MS-Based Metabolomics Approaches Applied to the Detection of Antifungal Compounds and a Metabolic Dynamic Assessment of Orchidaceae

The liquid chromatography–mass spectrometry (LC-MS)-based metabolomics approach is a powerful technology for discovering novel biologically active molecules. In this study, we investigated the metabolic profiling of Orchidaceae species using LC-HRMS/MS data combined with chemometric methods and dereplication tools to discover antifungal compounds. We analyze twenty ethanolic plant extracts from Vanda and Cattleya (Orchidaceae) genera. Molecular networking and chemometric methods were used to discriminate ions that differentiate healthy and fungal-infected plant samples. Fifty-three metabolites were rapidly annotated through spectral library matching and in silico fragmentation tools. The metabolomic profiling showed a large production of polyphenols, including flavonoids, phenolic acids, chromones, stilbenoids, and tannins, which varied in relative abundance across species. Considering the presence and abundance of metabolites in both groups of samples, we can infer that these constituents are associated with biochemical responses to microbial attacks. In addition, we evaluated the metabolic dynamic through the synthesis of stilbenoids in fungal-infected plants. The tricin derivative flavonoid- and the loliolide terpenoidfound only in healthy plant samples, are promising antifungal metabolites. LC-HRMS/MS, combined with state-of-the-art tools, proved to be a rapid and reliable technique for fingerprinting medicinal plants and discovering new hits and leads.


Introduction
Orchidaceae is one of the largest and most diverse families in the plant kingdom, with more than 28,000 species and 763 genera [1,2]. Orchidaceae species, popularly known as orchids, are sold commercially for presenting beautiful and diverse flowers and the economically important spice known as vanilla [3]. Many species have been studied for their chemical composition and pharmacological activities, including anticancer, antiinflammatory, antioxidant, neuroprotective, antivirus, and antimicrobial [3]. Several orchid species are known to produce stilbenoids, phytoalexins responsible for protection against predation and antimicrobial activities [4][5][6]. Orchinol and hircinol are two stilbenoid compounds isolated from the Orchis and Loroglossum genera, respectively, which were reported with antifungal activity and play a role in the defense of orchid tubes [7].
Considering the antifungal activity of the secondary metabolites present in orchids and the low rate of new drug discovery, there is an urgent need to develop fast and efficient methodologies for screening new antimicrobial agents. Since the classic process of screening bioactive natural products is a time-consuming and labor-intensive step, analytical techniques applied to separating and characterizing phytocompounds stand out for their high ability to dereplicate complex samples and detect constituents even at low concentrations that can be useful in planning new drugs [8,9].

Results
In this work, we used ultrahigh-resolution mass spectrometry coupled with liquid chromatography to investigate the metabolic dynamic of healthy and fungal-infected plants of the same botanical family to identify substances with antifungal properties. Metabolome-based antifungal screening approach was evaluated in twelve species of the Orchidaceae family by evaluating the spectral similarity of samples and applying chemometric methods. Initially, the ions were discriminated using data mining and organization tools from the GNPS-Global Natural Products Social Molecular Networking (https://ccms-ucsd.github.io/GNPSDocumentation/ (accessed on 16 August 2022) platform, and the structural annotation of the metabolites was based on accurate mass (m/z), MS/MS fragmentation pattern, chromatographic retention time, and chemotaxonomy data from Orchidaceae family. We used metadata and data mining tools according to the GNPS platform documentation, and the structural information and metabolic coverage data were inspected in detail. All library hits classified as gold (thoroughly characterized as struc-tures), silver (compound crude extract), and bronze (partial annotation) [20] were evaluated, and chemophenetics data from the Orchidaceae family confirmed the metabolic annotation.
The structural annotation of the metabolites was confirmed through the analysis of candidates and their analogs suggested by the natural products databases, reference mass spectral libraries, and spectral similarity networks resulting in level 2 identification according to the Metabolomic Standard Initiative-MSI [30].
Lyophilized extracts from two groups of samples (healthy and fungal-infected plants) of species of the genus Vanda (five samples) and Cattleya (five samples) were submitted to the evaluation of the presence of metabolites through metabolite annotations were based on searching the experimental spectra against the GNPS spectral library using the tools Classical Molecular Networking-MN [20], Dereplicator+ [21], Network Annotation Propagation-NAP [22], Moldiscovery [23], MS2LDA [31], MolNetEnhancer [32], and analysis of chromatographic data such as retention time and UV spectra unit of absorbance. The ion abundance assessment was performed using the Feature-Based Molecular Networking-FBMN [33] tool obtained from MS/MS data in positive ionization mode (ESI(+)). Annotations with high spectral similarity were prioritized, resulting in the structural annotation of 50 compounds.

Comprehensive Structural Annotation of Orchidaceae Species Using Molecular Networking
To obtain hits with higher structural similarity and the biosynthetic origin within molecular families from molecular networking, the threshold for the cosine score similarity was set to 0.7.
The library matches using the classical molecular network (MN) from Cattleya and Vanda genera assessment in both physiological conditions (healthy and fungal-infected) yielded a total of 1.220 hits with 315 unique library compounds. We found 60 hits with a high confidence level, a high number of hits with gold classification, represented by spectral similarity greater than 90% (cosine score > 0.9), mz error (ppm) less than 5, and a high amount of shared peaks in the MS/MS spectrum. The analysis of the candidates suggested by the spectral library after inspection of the profile of MS/MS fragmentation patterns, evaluation of high-resolution calculation of empirical formula, and chromatographic analysis data, yielded the compounds described in Table 1. All the annotated metabolites have been previously described in the Orchidaceae family [34,35].
From the MS/MS fragmentation pattern analysis, it was observed that the O-glycosylated phenolic compounds were more abundant than the C-glycosylated compounds. O-glycosylated flavonoids exhibited a neutral loss of a sugar moiety, which corresponds to a loss of 162 ) were identified by the consecutive losses of sugar moieties, and the flavonoids aglycone Acacetin, 4 -Methoxy-6-methylflavone, and Liquiritigenin were identified by fragments originating from retro Diels-Alder reactions, and data from chromatographic analysis such as UV spectra and retention times. In addition, it was possible to perform structure-based propagation and guided detection of phenolic compounds not annotated by the spectral library. The stilbenoids Rhapontin, Erianin, 3-O-Methylgigantol, Gigantol, Dendrosinene B, Tristin, 3 -O-Methylbatatasin III, 3-[2-(3-hydroxyphenyl)ethyl]-5-methoxyphenol, and Thunalbene, metabolites found in both Vanda and Cattleya genera, were putatively annotated through accurate mass precursor and characteristic product ions such as consecutive losses of C 2 H 2 O and methoxyl groups. To narrow the focus to identify metabolites with antifungal potential using the strategy of investigating the metabolome of healthy plants compared with the metabolome of fungal-infected plants from the Cattleya and Vanda genera, we eliminate metabolites common to all species. The two set plants shared about 75% of their metabolome. Healthy plant species exhibited a richness of secondary metabolites (approximately 60% of hits) compared to fungal-infected plants (approximately 40% of hits), evidencing the role of these metabolites in defense against microbial attack. The attack of pathogens on plants generates a biochemical response. Therefore, the production of these chemical constituents in infected plants belonging to the same genus or species may be suppressed by environmental factors [36][37][38]. In addition, the quantitative alterations of these metabolites may also be a consequence of environmental stimuli, explaining the low abundance of some compounds in plants of the same species affected by microbiological attacks. Moreover, other factors, such as nutrient and water availability in the soil, can influence metabolic pathways and considerably affect the synthesis of secondary metabolites with antimicrobial action [39,40]. The chromatographic profile and molecular networking show subtle differences between the sample groups for both species analyzed under the same chromatographic and spectrometric conditions ( Figure 2). The library matches using the classical molecular network (MN) from Cattleya Vanda genera assessment in both physiological conditions (healthy and fungal-infec yielded a total of 1.220 hits with 315 unique library compounds. We found 60 hits wi high confidence level, a high number of hits with gold classification, represented spectral similarity greater than 90% (cosine score > 0.9), mz error (ppm) less than 5, a high amount of shared peaks in the MS/MS spectrum. The analysis of the candid suggested by the spectral library after inspection of the profile of MS/MS fragmenta patterns, evaluation of high-resolution calculation of empirical formula, chromatographic analysis data, yielded the compounds described in Table 1. All annotated metabolites have been previously described in the Orchidaceae family [34,  To narrow the focus to identify metabolites with antifungal potential using the strategy of investigating the metabolome of healthy plants compared with the metabolome of fungal-infected plants from the Cattleya and Vanda genera, we eliminate metabolites common to all species. The two set plants shared about 75% of their metabolome. Healthy plant species exhibited a richness of secondary metabolites (approximately 60% of hits) compared to fungal-infected plants (approximately 40% of hits), evidencing the role of these metabolites in defense against microbial attack. The attack of pathogens on plants generates a biochemical response. Therefore, the production of these chemical constituents in infected plants belonging to the same genus or species may be suppressed by environmental factors [36][37][38]. In addition, the quantitative alterations of these metabolites may also be a consequence of environmental stimuli, explaining the low abundance of some compounds in plants of the same species affected by microbiological attacks. Moreover, other factors, such as nutrient and water availability in the soil, can influence metabolic pathways and considerably affect the synthesis of secondary metabolites with antimicrobial action [39,40]. The chromatographic profile and molecular networking show subtle differences between the sample groups for both species analyzed under the same chromatographic and spectrometric conditions ( Figure  2). The metabolites cinnamic alcohol (phenylpropanoid), tricin 5-glucoside (flavonoid), loliolide (terpenoid), and allobetulinol (terpenoid) were detected only in samples of healthy plants. In contrast, the metabolites 3-(4-hydroxy-3-methoxyphenyl)propyl 3-(4hydroxyphenyl)propanoate (phenol), salidroside (phenol), isovitexin (flavonoid), rutin (flavonoid), homoorientin (flavonoid), hyperoside (flavonoid), 2,3,5,7-tetramethoxy-9,10- The metabolites cinnamic alcohol (phenylpropanoid), tricin 5-glucoside (flavonoid), loliolide (terpenoid), and allobetulinol (terpenoid) were detected only in samples of healthy plants. In contrast, the metabolites 3-(4-hydroxy-3-methoxyphenyl)propyl 3-(4hydroxyphenyl)propanoate (phenol), salidroside (phenol), isovitexin (flavonoid), rutin (flavonoid), homoorientin (flavonoid), hyperoside (flavonoid), 2,3,5,7-tetramethoxy-9,10dihydrophenanthrene (stilbenoid/phenanthrenoid), 4 -methoxy-6-methylflavone (flavonoid), isoshaftoside (flavonoid), sinapyl alcohol (phenylpropanoid), liquiritigenin (flavonoid), gigantol (stilbenoid), 3-O-Methylgigantol (stilbenoid), 4-methylcinnamic acid (phenylpropanoid), and isorhamnetin 3-galactoside (flavonoid) were detected only in samples of fungal-infected plants. Although the sample sets share a wide metabolic diversity, the metabolites were found at different levels of abundance since the activation of the plant defense mechanism can result in the greater activation of a biosynthesis pathway and suppress the synthesis of other metabolites. The flavonoids afrormosin, acacetin, and stilbenoids were primarily found in fungal-infected plants, while nitrogen compounds were found predominantly in samples from healthy plants. Since the infection by microorganisms induces biochemical alterations in the host organism in an attempt to defend itself from the aggressive agent [41][42][43][44], a wide diversity of metabolites was found in fungalinfected plants (Figure 3). The tricin derivative flavonoid, and the loliolide terpenoid, found only in healthy plant samples, are reported in the literature with promising antifungal activity [9,45,46].

2.2.Structural Annotation Strategy Using In Silico Fragmentation Tools to the Fingerprinting of Healthy and Fungal-Infected Plants from Orchidaceae
From the evaluation of the secondary metabolism of the genera Vanda and Cattleya, 145 and 202 unique metabolites were detected in samples of fungal-infected plants and healthy species from the genus Cattleya, respectively. For the genus Vanda, 146 and 166 unique metabolites were detected in fungal-infected plants and healthy species samples, respectively. The molecular diversity found in healthy plant samples evidences a greater capacity of plants to synthesize secondary metabolites with biological action. In order to obtain greater metabolic coverage, more sophisticated state-of-the-art tools were applied, such as Moldiscovery, which allows obtained structural candidates with greater accuracy and reliability. The Moldiscovery tool yielded a total of 2044 unique metabolites and 1348 unique metabolites at a cutoff score of 15 from samples of fungal-infected plants. While for samples of healthy plants, 2706 unique metabolites and 1768 unique metabolites were detected at a cutoff score of 15.
Furthermore, a greater number of unique metabolites were found in healthy plant samples (351) compared to fungal-infected plants (377) using the in silico dereplication tool termed Dereplicator+. To obtain a more comprehensive chemical overview of both sets of samples and chemical structural information, the MolNetEnhancer tool was applied to detect the chemical classes present in the samples and to assist in annotating metabolites that did not show a matching MS/MS spectrum. The analysis showed mostly a higher number of nodes for all classes at the "superclass" level for healthy plant samples, except for the classes of organic acids derivatives and phenylpropanoids and polyketides, which were more abundant in fungal-infected plants ( Figure 5).

Structural Annotation Strategy Using In Silico Fragmentation Tools to the Fingerprinting of Healthy and Fungal-Infected Plants from Orchidaceae
From the evaluation of the secondary metabolism of the genera Vanda and Cattleya, 145 and 202 unique metabolites were detected in samples of fungal-infected plants and healthy species from the genus Cattleya, respectively. For the genus Vanda, 146 and 166 unique metabolites were detected in fungal-infected plants and healthy species samples, respectively. The molecular diversity found in healthy plant samples evidences a greater capacity of plants to synthesize secondary metabolites with biological action. In order to obtain greater metabolic coverage, more sophisticated state-of-the-art tools were applied, such as Moldiscovery, which allows obtained structural candidates with greater accuracy and reliability. The Moldiscovery tool yielded a total of 2044 unique metabolites and 1348 unique metabolites at a cutoff score of 15 from samples of fungal-infected plants. While for samples of healthy plants, 2706 unique metabolites and 1768 unique metabolites were detected at a cutoff score of 15.
Furthermore, a greater number of unique metabolites were found in healthy plant samples (351) compared to fungal-infected plants (377) using the in silico dereplication tool termed Dereplicator+. To obtain a more comprehensive chemical overview of both sets of samples and chemical structural information, the MolNetEnhancer tool was applied to detect the chemical classes present in the samples and to assist in annotating metabolites that did not show a matching MS/MS spectrum. The analysis showed mostly a higher number of nodes for all classes at the "superclass" level for healthy plant samples, except for the classes of organic acids derivatives and phenylpropanoids and polyketides, which were more abundant in fungal-infected plants ( Figure 5).
In a more comprehensive investigation at the "class" level, the coumarin derivatives were found only in healthy plant samples, while the aurone class was found only in fungal-infected plant samples. Other chemical classes were found in both sets of samples. However, they were detected in different proportions, such as stilbenes and cinnamic acid derivatives that were detected mainly in samples of fungal-infected plants, and the flavonoids, phenols, and steroids that were detected in a more significant proportion in healthy plant samples. Through molecular networking, we observe that the fungal-infected plants synthesized twice as many metabolites from the shikimate natural products pathway than healthy plants. This is because the variation of environmental conditions can silence or activate biosynthetic pathways, which impact the synthesis or reduction of the production of secondary metabolites. In a more comprehensive investigation at the "class" level, the coumarin derivatives were found only in healthy plant samples, while the aurone class was found only in fungal-infected plant samples. Other chemical classes were found in both sets of samples. However, they were detected in different proportions, such as stilbenes and cinnamic acid derivatives that were detected mainly in samples of fungal-infected plants, and the flavonoids, phenols, and steroids that were detected in a more significant proportion in healthy plant samples. Through molecular networking, we observe that the fungal-infected plants synthesized twice as many metabolites from the shikimate natural products pathway than healthy plants. This is because the variation of environmental conditions can silence or activate biosynthetic pathways, which impact the synthesis or reduction of the production of secondary metabolites.
The propagation of structural annotations was also performed from unknown fragmentation mass spectrum analysis using the in silico tool Network Annotation Propagation (NAP), which yielded 62 metabolites putatively annotated from NAP-Fusion in silico prediction for fungal-infected plants and 45 for healthy plants samples. A Venn diagram represents the unique metabolites number and overlapped unique metabolites putatively annotated by classical Molecular Networking (MN), Dereplicator+, Moldiscovery, and NAP-fusion annotated metabolites obtained from fungal-infected plants (F) and healthy (H) plants ( Figure 6). A significant number of flavonoids and phenols were found in high abundance in healthy plant samples, which may be responsible for antimicrobial potential since they are metabolites that play a central role in plant defense against pathogen attacks [49]. The A significant number of flavonoids and phenols were found in high abundance in healthy plant samples, which may be responsible for antimicrobial potential since they are metabolites that play a central role in plant defense against pathogen attacks [49]. The dereplication and data mining tools employed in this study provided chemical refinement of metabolomics results for exploration and guided the selection of candidates with antimicrobial potential.

Chemometrics Methods Applied to Orchidaceae Plants Spectral Analysis
Principal component 1 (PC1) and PC2 explained 24.31% and 15.34% of the variation, respectively, as shown in Figure 7. Both Figure 7A,B are biplot scores of the same PCA, with (A) showing the genus (Vanda and Cattleya) differentiation and (B) the healthy and fungal-infected samples separation. In fact, only the healthy and fungal-infected separation was successful, mainly in the PC1. In this way, PLS-DA and OPS were applied to classify samples as healthy or fungal-infected to find the variables that contribute more to this separation. A significant number of flavonoids and phenols were found in high abundance in healthy plant samples, which may be responsible for antimicrobial potential since they are metabolites that play a central role in plant defense against pathogen attacks [49]. The dereplication and data mining tools employed in this study provided chemical refinement of metabolomics results for exploration and guided the selection of candidates with antimicrobial potential.

Chemometrics Methods Applied to Orchidaceae Plants Spectral Analysis
Principal component 1 (PC1) and PC2 explained 24.31% and 15.34% of the variation, respectively, as shown in Figure 7. Both Figure 7A,B are biplot scores of the same PCA, with (A) showing the genus (Vanda and Cattleya) differentiation and (B) the healthy and fungal-infected samples separation. In fact, only the healthy and fungal-infected separation was successful, mainly in the PC1. In this way, PLS-DA and OPS were applied to classify samples as healthy or fungal-infected to find the variables that contribute more to this separation.  Figure 8A shows the PLS-DA scores with two latent variables (LV), explaining 58.61% of the variance with the selected variables by OPS. In this case, with the selection of only 80 variables for both classes (healthy and fungal-infected), the separation was  Figure 8A shows the PLS-DA scores with two latent variables (LV), explaining 58.61% of the variance with the selected variables by OPS. In this case, with the selection of only 80 variables for both classes (healthy and fungal-infected), the separation was accentuated in LV1, with 27.62% of explained variance. These 80 variables can be divided into three groups: 62 common variables between healthy and fungal-infected samples, and 10 healthy and 8 fungal-infected variables, as shown in Figure 8B. For each group (healthy or fungal-infected samples), the most important variables were the 10 and 8 different variables highlighted in the loadings plot ( Figure 8B). These 18 variables were previously described in Table 1.  Figure 8B. For each group (healthy or fungal-infected samples), the most important variables were the 10 and 8 different variables highlighted in the loadings plot ( Figure 8B). These 18 variables were previously described in Table 1.  In summary, our findings in chemometric data analysis were supported by the comprehensive structural annotation of the metabolites, being an essential tool for discovering antifungal compounds from the Orchidaceae family.

Plant Material
Healthy and fungal-infected fresh leaves from five Vanda sp. and Cattleya sp. plants were collected from a private greenhouse in Lençois Paulista, São Paulo, Brazil (22 • 36 46.2 S 48 • 50 02.1 W). The plants were maintained at −80 • C until freeze-drying and sample preparation.

Sample Preparation
The fresh leaves were frozen at −80 • C and then freeze-drying for 72 h. The freezedrying material was extracted with methanol. The solvent was removed using a Speedvac concentrator (Thermo Scientific Savant SPD131DDA). Subsequently, the extracts were submitted to a cleanup step (using SPE C18 cartridges) to eliminate chlorophyll and other interferences. The final extracted material (2 mL of methanol) was concentrated in a Speedvac concentrator. One milligram of dried extracts was made-up to 1 mL of methanol. This solution was diluted to 1:5 (v/v) in methanol, filtered (0.45 µm), and used in LC-MS analysis. Before the injections, the samples were spiked with a mixed standard solution (caffeine-13 C 3 and progesterone-d 9 , at 2.5 µg mL −1 ).

LC-HRMS Analysis
LC-HRMS/MS analyses were performed on an HPLC-UV 1220 Infinity II (Agilent Technologies) coupled with a Q-Exactive hybrid Quadrupole-Orbitrap high-resolution mass spectrometer (Thermo Scientific) as well as an electrospray ionization source. The column used in this study was an Infinity Lab Poroshell 120 EC-C18 column (4.6 × 100 mm × 2.7 µm Agilent). All samples were analyzed using a gradient elution program. The binary mobile phase comprised A (water with 0.1% formic acid) and B (methanol). The gradient elution started at 5% (B) and linearly increased to 100% (B) in 40 min and kept constant for 10 min at 100% (B). The eluent was restored to the initial conditions in 10 min. The flow rate was set at 0.3 mL min −1 . The injection volume was 30 µL, and the column temperature was set at 35 • C. The electrospray ionization was operating with the following parameters: spray voltage 3.5 kV; capillary temperature: 269 • C; S-lens RF level 50 V; sheath gas flow rate at 53 L min −1 ; aux gas flow rate at 14 L min −1 ; sweep gas flow rate 3 L min −1 . The high-resolution mass spectrometry (HRMS) was obtained under full MS/dd-MS 2 mode. The mass range in the full MS scanning experiments was m/z 80-1200. The top 5 (TopN, 5, loop count 5) most abundant precursors were sequentially transferred for collision for fragmentation acquisition. The collision energy for target analytes was 20, 30, and 35 eV. Resolving power was set at 140,000 and 70,000 for full MS and dd-MS 2 acquisitions, respectively.

Compound Characterization
The files acquired in the Q-Exactive hybrid Quadrupole-Orbitrap mass spectrometer for the methanolic extracts were converted from raw into (.mzML) format using MSConvert software (ProteoWizard, Palo Alto, CA, US) before being processed using MZmine software, version 2.53. We used metadata to organize compound information according to the Global Natural Products Social Molecular Networking (GNPS) online workflow (https: //ccms-ucsd.github.io/GNPSDocumentation/ (accessed on 16 August 2022).

Chemometric Data Analysis
Twenty samples were used in data analysis: ten Vanda and ten Cattleya samples, using five healthy and five fungal-infected of each genus. The obtained .raw files were converted into .cdf format and imported to Matlab R2020a (Math Works, Natick, MA, USA), where all chemometric tools were applied.
An exploratory data analysis was carried out using principal component analysis (PCA) [50] to visualize the Vanda and Cattleya genus and healthy and fungal-infected samples in multidimensional space. PCA was carried out on the normalized and autoscale dataset with twenty samples and 1,021,894 variables (m/z 80 to 1200).
Additionally, partial least squares for discriminant analysis (PLS-DA) [51], a classification method based on PLS regression, was applied in healthy and fungal-infected samples. The ordered predictors selection (OPS) [52], a variable selection method adapted for classification, was applied to find the more essential and interpretative variables for healthy and fungal-infected classification.

Conclusions
In this study, we presented the application of the integrated metabolomics approach and the state-of-the-art computational tools to provide acute insights regarding the characterization of phytochemicals of healthy and fungal-infected leaves from Vanda and Cattleya species. The data analysis pipelines of untargeted metabolomics analysis enabled the characterization of a considerable variation in compounds belonging primarily to the class of flavonoids, phenolic acids, chromones, stilbenoids, tannins, terpenoids, and steroids. Flavonoids were identified as the major compounds in both species independent of the physiological condition (healthy or fungal-infected). The flavonoids afrormosin, acacetin, and stilbenoids were found chiefly in fungal-infected plants, while nitrogen compounds were found predominantly in samples from healthy plants. The tricin derivative flavonoid, and the loliolide terpenoid were found only in healthy plants. From the dereplication and in silico fragmentation tools, 202 and 145 unique metabolites were detected in healthy, and fungal-infected Cattleya leaves. For the genus Vanda, 166 and 146 unique metabolites were detected in healthy and fungal-infected leaves. The integrated metabolomics approach and the combination of structural annotation, data mining, and chemometric tools could be applied as a reference dataset for MS/MS-based untargeted metabolomic analysis of species from the Orchidaceae. In addition, based on the interesting results shown in the present study, the model data on the molecular annotation adopted here may be further explored with high efficiency through other species from the Orchidaceae family. Data Availability Statement: All support data used in this study are available from the authors.