The ITS region provides a reliable DNA barcode for identifying reishi/lingzhi (Ganoderma) from herbal supplements.

The dietary supplement industry is rapidly growing yet, a recent study revealed that up to 60% of supplements may have substituted ingredients, some of which can be harmful contaminants or additives. When ingredients cannot be verified morphologically or biochemically, DNA barcoding complemented with a molecular phylogenetic analysis can be a powerful method for species authentication. We employed a molecular phylogenetic analysis for species authentication of the commonly used fungal supplement, reishi (Ganoderma lingzhi), by amplifying and sequencing the nuclear ribosomal internal transcribed spacer regions (ITS) with genus-specific primers. PCR of six powdered samples and one dried sample all sold as G. lucidum representing independent suppliers produced single, strong amplification products in the expected size-range for Ganoderma. Both best-hit BLAST and molecular phylogenetic analyses clearly identified the presence of G. lingzhi DNA in all seven herbal supplements. We detected variation in the ITS sequences among our samples, but all herbal supplement samples fall within a large clade of G. lingzhi ITS sequences. ITS-based phylogenetic analysis is a successful and cost-effective method for DNA-based species authentication that could be used in the herbal supplement industry for this and other fungal and plant species that are otherwise difficult to identify.

Introduction

Molecular barcoding is an efficient tool for identifying macroscopic, microscopic and biochemically enigmatic samples [1]. It has been applied across the tree of life [2, 3] and is increasingly employed in identifying the provenance of unidentifiable food products in restaurants [4] and in retail [5-7]. Leveraging the DNA content of processed living organisms that is not otherwise identifiable holds great prospects for quality control—especially helpful for authenticating the ingredients and avoiding contaminants and additives that may cause allergic reactions for consumers [8], although DNA of a species can be present long after losing the biological activity of its compounds (i.e. after an aggressive processing). This is particularly relevant in the herbal supplement industry, where safety and effectiveness are loosely regulated by the FDA through the Dietary Supplement Health and Education Act of 1994, which requires the manufacturer to ensure the safety and effectiveness of a supplement [9]. The Federal Food, Drug, and Cosmetic Act requires that manufacturers and distributors who wish to market dietary supplements that contain "new dietary ingredients" (not marketed in a dietary supplements before October 15, 1994) notify the Food and Drug Administration about these ingredients (Section 413(d) of [10]). Under this Act, it is the responsibility of the manufacturer or distributor to assess whether a dietary supplement will be safe to use [10].

The herbal supplement industry is a growing enterprise, expected to amount to $104.78 billion dollars or more by 2025 [11, 12], yet a recent study revealed that up to 60% of herbal supplements have substituted ingredients not listed on their labels, some of which can be harmful contaminants or additives [8]. For both marketing advantage and ethical concerns, suppliers must ensure accurate identification of all ingredients in their products [9]. Moreover, dietary supplement regulations require a manufacturer to perform identity testing on 100% of incoming lots of dietary ingredients, except when it has petitioned the FDA for a special exemption [10, 13]. For some manufactures, accurate identification of species, complete listing of ingredients, and precise reporting of potency are paramount. Furthermore, retailers are expected to exercise due diligence regarding oversight of suppliers. This is especially important since a large portion of the population consuming herbal supplements are doing so because their health is already compromised [14].

Reishi (Ganoderma spp. P.Karst.) is one of the oldest herbal medicines in recorded history [15, 16] and estimated to represent 2% of the herbal supplement industry [14]. It is recommended as an anti-inflammatory and to enhance immunity [17, 18]. After being cultivated on rice, most reishi products are ground to a powder and sold in capsules as herbal supplements. Although the glossy, lignicolous, leathery, shelf-like polypore fruiting bodies of this group of laccate Ganoderma species are distinctive when fresh, once pulverized along with the rice medium (which often constitutes >50% of the dry weight), the powder is not easily differentiable macroscopically, microscopically, or biochemically [18, 19]. For example, using biochemistry, Wu et al. [19] could only verify 26% of 19 reishi supplements purchased in the United States as true reishi (they use “G. lucidum”, but we will use G. lingzhi Wu, Cao & Dai for true reishi heretofore).

Adding to the difficulty of identifying processed reishi is the taxonomic confusion surrounding the species within Ganoderma [20]. The genus consists of approximately 80 species that fall into five or six clades—one of which is centered around G. lingzhi (Clade A) and another clade includes the true G. lucidum P.Karst. (Clade B) [21-26]. Because of their wood-decaying capabilities, several Ganoderma species have been investigated for biopulping [14] and bioremediation [27], however, it is most prized as a “model medicinal mushroom” [28] because of the putative health benefits of the triterpenoids and polysaccharides [29, 30]. These clades include several well-supported phylogenetic lineages that received unstable taxonomic treatments in the past [22, 24, 25]. According to a thorough morphological and molecular investigation of the commonly cited G. lucidum and the actual medicinal mushroom, G. lingzhi “the most striking characteristics which differentiate G. lingzhi from G. lucidum are the presence of melanoid bands in the context, a yellow pore surface and thick dissepiments (80–120 μm) at maturity” [23]. Ganoderma lucidum (including G. tsugae Murrill) can be found in the wild from Europe to northeastern China (some have likely escaped from cultivation in California and Utah [22]), whereas G. lingzhi is restricted to Asia [18]. Fresh G. lingzhi has higher levels of triterpenoids than G. lucidum which may be responsible for the suggested physiological effects of G. lingzhi in humans. This biochemical result also supports the distinctiveness and the commercial importance of differentiating these often confused taxa [18, 19]. The history of taxonomic confusion surrounding G. lucidum and G. lingzhi [18, 26, 31] has been largely resolved by recent morphological comparisons [23], biochemical investigation [18], and molecular phylogenetic analyses [22–24, 32]. In particular, molecular phylogenetic analyses place G. lucidum and its closest relatives (G. oregonense, G. tsugae, and G. carnosum) in Clade B and the medicinally important G. lingzhi in Clade A with numerous other closely related species [22, 24].

The nuclear ribosomal internal transcribed spacer region (ITS) is an informative DNA region for barcoding plants and fungi [33-35]. It consists of two hypervariable spacers of approximately 200-250bp flanked by the 18S (small) subunit and 28S (large) subunit rDNA and separated by the 5.8S rDNA [36, 37]. Primers designed to bind to highly conserved portions of the 18S and 28S subunits have been widely used across plants and fungi [37]. However, lineage-specific primers have been developed for many groups of fungi to help diagnose the presence or absence of particular species [23]. Lineage-specific primers can also improve PCR specificity especially when working with compromised DNA templates that may be degraded, contain inhibitors, or be composed of a mixture of species. Ganoderma-specific primers developed by Cao et al. [23] have been shown to improve PCR specificity. These primers have been used for barcoding reishi herbal supplements in previous studies (see [38] with limited sampling of reishi samples (n = 4) and [14] with a broader retail sampling (n = 14), but unclear how many unique suppliers were represented in the latter).

Although considerable attention has been given to the identification of the best barcoding loci and the development of unique and creative applications [34], less explicit attention has been paid to the analysis of the data. The two main approaches for analysis of the DNA sequences arising from barcoding investigations are genetic distance-based measures (e.g., best-hit BLAST or nearest neighbor analysis) and phylogenetic methods (e.g., maximum likelihood or Bayesian tree-building algorithms). Some studies rely solely on best-hit BLAST [39] or otherwise crude phylogenetic approaches [40, 41] sometimes without assessment of the uncertainty [8, 42]. Genetic distance-based measures are known to fail in several common situations such as variable rates of molecular evolution [43, 44], gene duplication [44, 45] and changes in a gene’s composition [46]. Empirically, genetic distance-based approaches and phylogenetic methods for barcoding analysis are rarely compared explicitly even though they can produce conflicting identifications [47, 48].

Herein, we present an efficient method for unambiguous identification of the herbal supplement, reishi (G. lingzhi). We report successful DNA extraction, PCR amplification, and DNA sequencing of the ITS region from store-bought reishi samples. We compare the results from best-hit BLAST with two molecular phylogenetic approaches to determine if the species in the store-bought samples are correctly labeled or not.

Materials and methods

Sampling

Store-bought samples were collected from multiple nutritional supplement retailers representing seven distinct suppliers of cultivated fungal products (Table 1). Four samples were encapsulated powders and two were loose powders, all of which purport to contain reishi, or “Ganoderma lucidum”, based on the product’s labeling. Of the seven supplements sampled, four were labeled as containing only mushroom mycelial biomass, two samples claimed to contain both mycelia and fruiting body, and one sample did not specify. The powdered samples varied in color, texture, and smell. All powdered samples were macroscopically unidentifiable as a mushroom and for Powder #1, only mycelia were observed under compound microscope (40-100x) (Cresswell and McFerrin, unpublished data).

Table 1

Sampling information for powdered and fresh samples that were store-bought or wild-collected.

Sample	Species (as advertised)	Sample Information
Powder #1	Ganoderma lucidum	Store-bought: Oregon’s Wild Harvest Astragalus-Reishi
		Supplier: Oregon’s Wild Harvest
Powder #2	Ganoderma lucidum	Store-bought: Host Defense Reishi
		Supplier: Fungi Perfecti
Powder #3	Ganoderma lucidum	Store-bought: Solaray Reishi Mushroom
		Supplier: Nutraceutical Corp.
Powder #4	Ganoderma lucidum	Store-bought: The Vitamin Shoppe Reishi Mushroom
		Supplier: Gourmet Mushroom Inc.
Powder #5	Ganoderma lucidum	Store-bought: Eclectic Institute Fresh Freeze Dried Reishi Mushrooms
		Supplier: Eclectic Institute Inc.
Powder #6	Ganoderma lucidum	Store-bought: Now Rei-Shi Mushrooms
		Supplier: Now Foods
Fresh #1	Ganoderma brownii	Wild-collected: De Laveaga County Park, Redwood Loop Trail, approximately 50 m NE of crooked tree picnic area, common among dead Umbellularia californica, Santa Cruz, CA, USA (36.999720, -122.000360
Fresh #2	Fomitopsis pinicola	Wild-collected: Pogonip County park, Fern Trail, approximately 0.5 km south of junction with Spring Trail, on dead Quercus agrifolia, Santa Cruz, CA, USA (37.001568, -122.042023)
Fresh #3	Ganoderma lucidum	Store-bought: Staff of Life Organic Reishi Mushroom
		Supplier: Mycological Natural Products

A fresh mushroom sample advertised as “organic reishi mushroom” was collected from the bulk herb section of Staff of Life natural goods store, Santa Cruz, CA in July of 2018 and was also evaluated based on its morphological characteristics (Fresh #3 in Table 1). The sample had been cut into strips of approximately 6 x 1 cm from cross sections of the fruiting body. The sample appeared woody in texture with extensive pore-containing regions similar to morphologically identified samples of the complete fruiting body.

Two additional fresh samples were collected from the wild (Santa Cruz County, CA, USA; Table 1) and used as positive controls for DNA extraction, PCR and sequencing (Table 1, Fresh #1 and Fresh #2). Samples were morphologically identified as Ganoderma brownii (Murrill)Gilb. and Fomitopsis pinicola (Sw.)P.Karst. [49]. These two closely related genera can be distinguished by the presence (Ganoderma) or absence (Fomitopsis) of bruising on the white pores of the fruiting body’s underside [49]. All samples were stored at room temperature until the DNA could be extracted.

DNA extraction

For each nutritional supplement, two subsamples were taken from each sample and DNA was extracted from each of them. Encapsulated samples were opened and only the powder contained within was used. Field collected samples were dissected and cut into smaller pieces for further morphological evaluation and then prepared for DNA extraction. Fresh tissue was removed from the underside of the fruiting body and cut into 2mm x 5mm rectangles for homogenization. Approximately 30–100 mg of material was homogenized in QIAGEN’s DNEasy Plant Mini Kit extraction buffer using a BeadBeater with 4 x 3.2 mm steel beads in XXTuff 2mL O-ring screw cap tubes (Biospec, Bartlesville, OK, USA). Following homogenization, DNA extraction was performed using the Qiagen DNEasy Plant Mini Kit following the manufacturer’s protocol (QIAGEN, Valencia, CA, USA). Concentration and purity of extracted DNA was evaluated using a Nanodrop spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA).

PCR and sequencing

Several fungal ITS primer pairs were tested for initial success of amplification for both fresh and powdered samples (S1 Table) [50]. Among them, the Ganoderma-specific primers (G-ITS-F1 and G-ITS-R2) were selected based on consistently producing strong, single bands [23]. These primers were designed to prevent amplification from plant or other fungal DNA, which is a common problem with herbal supplements since they often include a plant-based growing medium or fungal contamination.

Extracted DNA was used as a template in 25 μL PCR reactions. Each reaction consisted of 2.5 μL of MgCl2 (25 mM), 2.5 μL of Taq Buffer B (Mg-free; 10X) (New England Biolabs, Ipswich, MA, USA), 2.5 μL of dNTPs (2.5 mM of each base), 2.5 μL of each of the aforementioned primers (10 μM), 0.25 μL of Taq polymerase (5U/μL) (New England Biolabs, Ipswich, MA, USA) and 1μL of extracted template DNA. A negative control (Milli-Q water in place of DNA template) was included in each PCR to ensure there was no contamination. Amplification took place under the following thermal cycling conditions: initial denaturation at 92°C for 2 min followed by 35 cycles of 94°C for 1 min, 55°C for 45 s, 72°C for 45 s and a final extension step at 72°C for 5 min. The PCR products were run on a 1% agarose gel stained with ethidium bromide alongside a 100 bp ladder (New England Biolabs, Ipswitch, MA, USA).

PCR reactions producing single, strong bands, were cleaned-up using shrimp alkaline phosphatase and directly sequenced in both directions using the Applied Biosystems 3730xl DNA Analyzer with the same primers used in PCR (Applied Biosystems, Waltham, Massachusetts, USA). Direct sequencing followed by BigDye Terminator or BigDye Primer methodologies per manufacturer recommendations (Sequetech, Mountain View, CA, USA). Forward and reverse chromatograms for each sample were trimmed to remove the opposing primer sequence and low-quality sequence at the beginning and end of each read and edited to correct any ambiguous base calls. Reads were then aligned to form a single contiguous sequence using the pairwise alignment tool in Geneious Prime (Geneious Prime 2019.0.4, Biomatters, Auckland, NZ).

Data analysis

BLAST

We used Basic Local Alignment Search Tool (BLAST) as the first method of identification for each sample. We used the megablast algorithm to search the nucleotide (nr/nt) collection to find the closest match to our sequences (BLASTDBv4) [51-53]. We compared the BLAST results from full-length sequence queries to the BLAST results of sequences trimmed to the portion of the alignment with maximum overlap with the reference database that were used in our phylogenetic approach (see below).

Multiple sequence alignment

We assembled an alignment of related sequences from Genbank (S2 Table). We started by including our top BLAST hits from the full sequence search query described above. If multiple Genbank accessions had equal coverage and identity as the top hit, we took at least one representative of each species which appeared. We also added all the unique Reishi samples (G. lingzhi and G. lucidum) of ITS using text searches in ENTREZ. Finally, we included representatives of as many Ganoderma species we could find using a filtered discontiguous megablast allowing us to limit ourselves to the most highly similar Ganoderma accessions, and increase our taxonomic coverage with a diverse and comprehensive reference set for nucleotide alignment and subsequent phylogenetic analysis. Several outgroup sequences were chosen which included other mushroom species belonging to the same order, Polyporales.

Sequences were aligned using the Geneious alignment tool (Biomatters, Auckland, New Zealand). All sequences were trimmed to approximately the same size producing an alignment of consistent length across the available ITS sequences of the Genbank reference set. Sequences with 100% nucleotide match to another sequence of the same species were removed so that only one representative sequence remained to simplify later phylogenetic analyses (S3 Table). If a sequence had a 100% match to a sequence belonging to a different species, both sequences were kept in the alignment to represent the additional taxonomic diversity. After all sequences were trimmed to approximately the same length we repeated the BLAST analysis of each sample to determine if sequence length affected the identity of the unknown samples.

Phylogenetic analyses

Maximum likelihood analysis was performed using the RAxML plug-in for Geneious (RAxML 8.2.11) [54]. We applied the GTR + CAT + I model of evolution and employed a rapid bootstrapping algorithm using 1,000 bootstrap replicates. Additionally, the MrBayes 3.2 plugin was used to build a Bayesian phylogenetic inference using Markov chain Monte Carlo (MCMC) algorithm (MrBayes 3.2.6) [55]. The GTR substitution model was used with a proportion invariable, remaining gamma rate variation model. Bayesian analysis ran for 2,000,000 generations. After removing the first 1,000,000 generations as burn-in, we sampled trees every 1000 generations creating a posterior distribution of 1000 trees. The intention of our study is not to disentangle the taxonomic uncertainty regarding G. lucidum sensu lato and G. lingzhi. Therefore, throughout the results and discussion we have chosen to report the scientific names as they are reported in Genbank although some of these have been suggested to be mislabeled (see the S2 Table in [56]).

Results

The average concentration of DNA in the nine samples was 34.1 ng/uL (range 3.9 to 175.2; S4 Table). The average purity of the DNA measured as the 260/280 ratio was 1.34 (range 0.66 to 1.91; S4 Table).

To assess successful amplification of the ITS region from newly extracted fungal DNA, PCR with three different primer pairs was performed and samples were visualized with gel electrophoresis. All primer pairs produced visible bands of expected size for the ITS region for both fresh and powdered samples. The PCR products using Ganoderma-specific primers were chosen for sequencing and all subsequent analyses based on their increased band intensity compared to other primers. After trimming these newly created sequences for seven samples, lengths ranged from 780 base pairs to 895 base pairs with an average of 854 base pairs. Quality scores (HQ%) for full contiguous sequences of the forward and reverse directions ranged from 75.4% to 96.5% and averaged 91.2%.

Our first approach for sample identification was to query Genbank for the top BLAST hit using the full length ITS sequence (Table 2). Of the seven store-bought samples, all yielded a top BLAST result that matched their labeled genus and species (“G. lucidum”, likely a mislabeled G. lingzhi, see Phylogenetic Analyses section below). Top BLAST hits changed to G. lingzhi for all fresh samples when using the trimmed sequences from the 618 bp alignment as described in more detail below (Table 2).

Table 2

BLAST results using full length ITS sequences compared to ITS sequences trimmed to the GenBank reference panel alignment (618 bp) used in phylogenetic analysis.

			Full Length				Trimmed to Alignment Length
Sample Name	Presumed Species1	Genbank Accession	Sequence Length (bp)	Top BLAST Hit2	GenBank Query Coverage	GenBank Percent Similarity	Top BLAST Hit2	GenBank Query Coverage	GenBank Percent Similarity
Powder #1	G. lucidum	MT994154	824	G. lucidum (MF476200.1)	100%	100%	G. lingzhi (MH160076.1)	100%	100%
Powder #2	G. lucidum	MT994155	739	G. lucidum (MF476201.1)	99%	99%	G. lingzhi (MH160076.1)	99%	99%
Powder #3	G. lucidum	MT994156	868	G. lucidum (MF476200.1)	100%	100%	G. lingzhi (MH160076.1)	100%	100%
Powder #4	G. lucidum	MT994157	868	G. lucidum (MF476200.1)	100%	100%	G. lingzhi (MH160076.1)	100%	100%
Powder #5	G. lucidum	MT994158	865	G. lucidum (MF476200.1)	100%	100%	G. lingzhi (MH160076.1)	100%	100%
Powder #6	G. lucidum	MT994159	844	G. lucidum (MF476200.1)	100%	99%	G. lingzhi (MH160076.1)	100%	100%
Fresh #1	G. brownii	MT994160	848	G. australe (MK968731.1)	100%	97%	G. brownii (MG279159.1)	100%	100%
Fresh #2	Fomitopsis pinicola	MT994161	780	F. pinicola (EF530947.1)	100%	99%	F. pinicola (EF530947.1)	100%	99%
Fresh #3	G. lucidum	MT994162	868	G. lucidum (MF476200.1)	100%	99%	G. lingzhi (MH160076.1)	100%	100%

1Presumed species is based on product label for store-bought samples and morphological identification [49] for wild-collected samples.

2All top BLAST hits had an E-value of 0.0.

To further assess the identity of our store-bought and field-collected samples, they were aligned with a reference panel (S2 Table). After trimming the alignment to the length of the shortest sequence in the reference panel and temporarily removing identical sequences (S3 Table), we created a final alignment of 93 sequences measuring 618 base pairs long with 52.2% identical sites (including outgroups). Among these unique sequences, the average pairwise percent identity is 87.6%. Within the G. lingzhi clade, there were 91 variable sites (mean pairwise identity = 99.3%). The average genetic identity between our store-bought samples and the most similar Genbank accession was 99.8% (range 99.5–100%).

The maximum likelihood analysis yielded a moderately resolved tree. Of the 91 distinct branches in the maximum likelihood tree, 31 branches (34%) had bootstrap values greater than 70%, a commonly used cut-off for 95% reliability (Fig 1) [57]. There is a moderately supported G. lingzhi clade containing nearly all of the samples labeled G. lingzhi, several G. lucidum samples, one likely misidentified G. sichuanense sample, and all seven of the store-bought herbal supplement samples (bootstrap = 88%) (Fig 1). We also reconstructed a strongly supported clade containing the real G. lucidum, G. tsugae, G. oregonense and G. carnosum (Clade B; 100% bootstrap) (Fig 1). We have applied clade names A and B from Loyd et al. [22] and Zhou et al. [24]. Clade A containing G. tuberosum and G. multipileum appears paraphyletic in Fig 1, however the deepest nodes are only weakly supported (<20% bootstrap) and therefore, not in conflict with previous studies [22, 24].

Fig 1

Maximum likelihood phylogenetic analysis.

RAxML phylogeny including store-bought samples, wild collected samples and the Genbank reference set. (A) Cladogram with branch support at critical nodes indicated along the branches as maximum likelihood bootstrap percentage/Bayesian posterior probability (asterisks indicate 100% bootstrap and 1.0 posterior probability). Clade names A and B are from Loyd et al. [22] and Zhou et al. [24]. The red rectangle identifies the true G. lucidum samples per Loyd et al. [14] and the green rectangle contains the samples referred to as the G. lingzhi clade (many G. lucidum sequences are misidentified G. lingzhi). (B) Maximum likelihood phylogram with unlabeled tips in the same order depicting branchlengths proportional to substitutions per site.

Although there is variation among the reishi samples, there is very little resolution within the G. lingzhi clade (a portion of Clade A; Fig 1). The mean pairwise genetic identity among our herbal supplement samples was 99.8%, yet there are only two branches with bootstrap values greater than 70%. The phylogenetic affinities of Powders #1–5 were completely unsupported (Fig 1). Only Fresh #3 had a weak to moderate affinity to “G. lucidum” (KX589244) with low bootstrap support (67%). Finally, Powder #6 appears sister to a clade of eight poorly resolved accessions named G. lingzhi and G. lucidum with very weak bootstrap support (54%; Fig 1).

As a control, we included two fresh samples of wild-collected polypores. Fresh #1 was morphologically identified as Ganoderma brownii and was 100% identical to two other G. brownii samples (MK883702 & MG279159). Fresh #2 was morphologically identified as Fomitopsis pinicola and only had one nucleotide difference (99.8% identical) when compared to the F. pinicola Genbank accession that it paired with (EF530947; Fig 1).

The maximum likelihood tree revealed two putatively incorrectly named Genbank accessions worth noting (Fig 1): (1) a G. lingzhi sample (AB811852) that is strongly supported as sister to G. multipileum (AB811849; 100% bootstrap), but both are very divergent from their Clade A conspecifics; (2) a G. sichuanense sample (KT693254) that is nested within the well supported G. lingzhi clade (88% bootstrap), yet deeply separated from another sample of the same species (JQ781878) (see [22] for discussion about this taxon).

The Bayesian phylogenetic analysis is largely consistent with the maximum likelihood tree, yet considerably less resolved. Twenty-four branches (26%) have posterior probabilities greater than 0.95 (S1 Fig). Within Clade A, there is a strongly supported subclade containing nearly all of the Genbank accessions named G. lingzhi, many erroneously named G. lucidum and all of the store-bought samples (posterior probability = 0.96; S1 Fig). Clade B is strongly supported as monophyletic (posterior probability = 1.0) containing a monophyletic lineage of correctly identified G. lucidum accessions (per [22]). The two putatively misidentified accessions described for the maximum likelihood analysis above had similarly unexpected phylogenetic affinities in the Bayesian analysis (S1 Fig).

In the Bayesian phylogenetic tree, six of the seven store-bought samples are part of a large unresolved polytomy of accessions named G. lucidum and G. lingzhi (the true G. lingzhi clade). The exceptional sample (Fresh #3) falls within a strongly supported subclade (posterior probability = 1.0) that is composed of an unresolved trichotomy with two other Genbank samples—one labeled G. lucidum and one labeled G. lingzhi (KX589244 and LC090753, respectively; S1 Fig).

For the Bayesian analysis, the two control samples allied with similar Genbank accessions as in the maximum likelihood analysis. Fresh #1 (morphologically identified as G. brownii) allies with the other two G. brownii samples with a posterior probability of 1.0 (S1 Fig). Fresh #2 (morphologically identified as Fomitopsis pinicola) is strongly supported as sister with a Genbank F. pinicola sample (EF530947; posterior probability = 1.0; S1 Fig).

Discussion

Our study demonstrates that the ITS region provides an efficient barcode for store-bought reishi herbal supplements thereby supporting the conclusions reached by earlier authors including Loyd et al. [14] and Raja et al. [38]. Amplifiable genomic DNA was successfully extracted from both powdered and fresh samples—all of which closely allied with established G. lingzhi samples within Clade A (even though many of those samples and the herbal supplement samples were sold as “G. lucidum”). Loyd et al. [14] found widespread label confusion in both “grow your own” kits (15/17) and manufactured herbal supplements (13/14) that were sold as “G. lucidum”. They used both ITS and tef1-alpha sequences to identify the manufactured supplements were all G. lingzhi, except one G. applanatum [14]. The label confusion surrounding G. lucidum and G. lingzhi was likely unintentional due to the taxonomic uncertainty, although there are clear biochemical differences (and therefore potential human physiological consequences) that differentiate the two taxa [18, 19]. In fact, Wu et al. [19] considered 26% of their 19 samples “verified” even though the labels read “G. lucidum” and not the correct species name, “G. lingzhi.” Rampant misidentification of true reishi is highlighted in the authoritative Herbs of Commerce [58] which indicates that the most important species commercially sold under the common name “reishi” are “G. japonicum, G. lucidum, and G. tsugae”–completely neglecting what is now considered true reishi, “G. lingzhi” [14, 22, 24, 32].

In general, herbal supplements are notoriously mislabeled—Newmaster et al.’s [8] study of plant herbal supplements found 59% (30 out of 44) had species substitutions and about 33% of these products had fillers or contaminants that were not listed on the product label—some of which could pose health risks to consumers. Herbal Commerce DNA barcoding will continue to be a valuable tool for manufacturers, retailers and consumer-watch groups, especially for herbal supplements like reishi where a lack of morphological and chemical distinctiveness once in powder form is compounded by underlying taxonomic confusion.

All of the samples we examined had BLAST and phylogenetic results suggesting they were clearly members of Clade A sensu Zhou et al. [24] and Loyd et al. [14]. None of our nine distinct distributors sampled contained material belonging to Clade B (G. lucidum). Technically all of our samples are misidentified since they are being sold as “G. lucidum”, yet are molecularly allied with the true reishi samples in Clade A (“G. lingzhi”). A similar case of mistaken identity is reported by Loyd et al. [14]. We assume the mislabeling was unintentional and arose from the history of taxonomic confusion surrounding G. lucidum vs. G. lingzhi (yet recently and lucidly clarified by [32]). This level of mistaken identity (100%) is relatively rare among herbal supplement barcoding studies in general [8]. Although we only included seven store-bought samples, these represent seven distinct suppliers thereby broadening the implications of our study to all the retailers using those suppliers as well, something previous Ganoderma retail barcoding studies have not reported (using different retail samples from the same supplier could be considered pseudoreplication; see [14, 38]). Misidentifications can arise at any of the multitude of links that connect the growers with the retailers. Our targeted sampling at the supplier stage clearly indicates that the misidentifications are likely applied early in the process and inherited by the retailers.

Our study does not attempt to resolve the taxonomic ambiguity among closely related species within the genus Ganoderma which permeates the available sequence data in Genbank [23, 32, 56]. However, we do not wish to contribute to the confusion so will attempt to reconcile some of the Genbank names with the recent literature in regard to reishi here. Clade A includes a strongly supported lineage of the medicinally important reishi (also known by the common name “lingzhi”) which is properly named G. lingzhi and restricted to Asia (see [32] for nomenclatural justification; also see Correction in [56]). These samples should all be identified as G. lingzhi according to Zhou et al. [24], Patterson & Lima [32], and Loyd et al. [22]. Alternatively, we have recovered a clade of four genetically distinct G. lucidum sequences (bootstrap = 60%) which ally with three other taxa to comprise the very strongly supported Clade B (100%). The real G. lucidum is native to Europe, closely related to North American G. oregonense and G. tsugae, and most likely introduced to Utah and California, USA according to Loyd et al. [22]. In comparison to Cao et al. [23] who examined four nuclear genes including ITS (yet only four samples of G. lingzhi and G. lucidum and a total of 13 species), our analysis has 10 G. lingzhi and 11 G. lucidum samples) and more species overall (n = 29), yet limited to the single barcoding locus, the ITS region.

More broadly, our phylogenetic results (Fig 1) are generally congruent with previous studies employing the ITS region [22–24, 56]. They all report similar clades that we have identified in our results, yet they often report higher confidence likely due to the inclusion of more loci. Although we have chosen to report the Genbank organism fields as they are in the database, we highlight the taxonomic confusion around these lineages and anticipate their realignment in Genbank in the near future.

ITS variation within the G. lingzhi clade allowed us to further partition our store-bought samples. Most samples were part of a large unresolved polytomy, but in two cases, there were distinct phylogenetic affinities suggesting separate sources. The intraspecific variation in ITS could prove valuable for tracing the intraspecific provenance of some reishi herbal supplements, but will likely need to be complemented with additional rapidly evolving loci (e.g., tef1-alpha, see [14, 22, 24]).

Methodologically, BLAST and phylogenetic analyses agreed on the provenance of all of the store-bought samples. When the rates of molecular evolution are relatively constant among the samples, in the absence of gene duplication, and when gene structure is conserved (such as for the ITS region), BLAST and phylogenetic methods are predicted to converge on similar identifications [43, 44, 46]. However, when any of those characteristics are violated, genetic distance-based approaches, such as BLAST, that rely on a local alignment algorithm (some modification of [59]) can be misleading. Alternatively, phylogenetic analysis relies on a global alignment algorithm [60] spanning the entire length of the locus being compared and is more likely to identify the evolutionary history of the samples for that locus [44], yet is most rigorously employed with a model-based approach (e.g. maximum likelihood and Bayesian methodologies) compared to a distance-based approach that is commonly found in the barcoding literature [61]. UNITE is a noteworthy database and search tool for identifying fungal ITS sequences to species using some objective sequence-based cutoffs that should be considered in future barcoding studies [62]. Our results were generally robust to whether we used the entire ITS region or the trimmed region of overlap used in the multiple sequence alignment and subsequent phylogenetic analysis (all results point to G. lingzhi in Clade A). However, because of the nomenclatural issue associated with many Genbank samples, it appears that our results change from G. lucidum to G. lingzhi (Table 1). This points to the importance of rectifying the Genbank taxonomy to avoid future, honest misidentifications.

Bayesian phylogenetic analysis.

(A) Bayesian cladogram with Genbank accession numbers preceding species names for the reference panel. Samples are identified with reference to Table 1. Posterior probabilities greater than 0.50 are indicated along the branches. Branches with less than 0.50 posterior probability have been collapsed. Clade names A and B are from Loyd et al. [22] and Zhou et al. [24]. The red rectangle identifies the true G. lucidum samples per Loyd et al. [14] and the green rectangle contains the samples referred to as the G. lingzhi clade (many G. lucidum sequences are misidentified G. lingzhi). (B) Bayesian phylogram with unlabeled tips in the same order depicting branchlengths proportional to substitutions per site.

(TIFF)

Click here for additional data file.

Three ITS primer pairs tested for amplification from reishi herbal supplements.

(DOCX)

Click here for additional data file.

Genbank reference panel sampling.

(DOCX)

Click here for additional data file.

Duplicate sequences removed from reference panel.

Identical sequences from the same species were removed to compress the alignment and facilitate phylogenetic analysis.

(DOCX)

Click here for additional data file.

DNA concentration and purity for herbal supplement powder samples and fresh samples.

(DOCX)

Click here for additional data file.

3 Sep 2020

PONE-D-20-21583

The ITS region provides a reliable DNA barcode for identifying reishi/lingzhi (Ganoderma) from herbal supplements

PLOS ONE

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2. In addition, please ensure that you have deposited the sequencing data and provide the Genbank Accession numbers in Table 1 which are currently listed as XXXXX

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"TG received funding from Oregon's Wild Harvest, an herbal supplement company included in our sampling. MC and JM are employees of Oregon's Wild Harvest. The senior author, JW received no funds from Oregon's Wild Harvest, nor any other herbal supplement company."

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Reviewer #1: The manuscript PONE-D-20-21583 ‘The ITS region provides a reliable DNA barcode for identifying reishi/lingzhi (Ganoderma) from herbal supplements’ presents the results of sequencing ITS rDNA from commercial samples of powdered Ganoderma and several fresh control specimens, and compares two different downstream analyses to identify the sequences obtained: distance-based BLAST searches in public databases, and likelihood-based phylogenetic analyses. Unfortunately, I think that the manuscript does not provide enough scientific or technical novelties to be published in PLOS One. ITS rDNA is already known to discriminate most species of Ganoderma, and it has been already tested to identify powdered commercial samples. The most relevant result obtained is that the commercial samples analyzed are all mislabeled, suggesting that many marketed products need to be checked. This is important from a commercial point of view, but does not constitute a relevant scientific or technical novelty.

Therefore, I recommend the editors to reject the manuscript. I suggest the authors to look for a different journal to publish this work, or else explore other issues that could provide more interesting results in order to submit it again to PLOS One:

1) It would be great if the authors could quantify the different DNAs present in the samples by means of NGS, in order to estimate the amount of target fungus, additives and contaminants.

2) Another interesting issue is the great amount of misidentifications in public databases (what about UNITE?), and the need for reference sequences (preferably obtained from type collections) to stabilize the taxonomy of these species and provide more accurate database-guided identifications.

3) Authors could focus also on the comparison between distance-based and likelihood-based phylogenetic methods, and discuss cases where both methods produce different results (maybe comparing Ganoderma with other marketed species, i.e. Morchella?).

Below, I provide some more comments about the text:

Keywords = I would shorten the list of keywords. Please remove the words already included in the title. Please replace ‘nuclear ribosomal internal transcribed spacer region’ for ‘ITS nrDNA’. Correct ‘reishi’ (or remove it, as it is present in the title)

Abstract = The first 10 lines could be moved to the Introduction, or substantially reduced to one or two sentences. Please use cursive for species names.

‘clearly identified the predominant fungal DNA was G. lingzhi’ = the PCR product is supposed to belong to the predominant DNA present in the sample, but this could not be the case, as genus-specific primers were employed. Other species could be present and not amplified because of the primer specificity. You can change the sentence to ‘clearly identified the presence of G. lingzhi DNA’. Did you obtain a mixed signal after sequencing the PCRs done with universal primers?

‘ITS is a successful and cost-effective method for DNA-based species authentication’ = ITS is not a method, but a region of the rDNA. ‘ITS-based phylogenetic analysis is a successful and cost-effective method for DNA-based species authentication’ would be more correct.

Introduction

‘Barcoding is an efficient molecular tool’ --> better ‘Molecular barcoding is an efficient tool’

‘identifying morphologically, anatomically and biochemically enigmatic samples’ = Anatomy is a specific type of morphological study. Maybe you mean ‘macroscopically, microscopically and biochemically enigmatic samples’? Please correct other similar sentences.

‘especially helpful for maintaining the validity of active ingredients’ = DNA barcoding can detect the presence/absence of some target taxa, but it does not provide any info about the activity of ingredients. DNA of a species can be present after losing the biological activity of its compounds (i.e. after an aggresive processing).

‘avoiding contaminants that may cause allergic reactions for consumers’ = other species can be present also as additives. DNA testing can be employed also to check for their presence.

‘Reishi’ = please add a latin binomial with authors when this species is mentioned for the first time, or at least Ganoderma spp. if the vulgar name is applied to more than one species. Please add the authors of all scientific names when they are first mentioned.

‘The G. lucidum clade consists of several species that are in taxonomic flux’ = maybe better ‘The G. lucidum clade includes several phylogenetic lineages that received an unstable taxonomic treatment in the past’.

‘Ganoderma lucidum sensu lato (including G. ‘tsugae’)’ = Ganoderma tsugae is a valid name, so it does not need quotation marks. You can correct it as ‘(including G. tsugae Murrill)’.

‘can be found in the wild from Europe to northeastern China (likely escaped from cultivation in California and Utah, see [22])’ = there are three lineages within the G. lucidum clade (G. lucidum s. str., G. tsugae and G. oregonensis). Do you mean that G. tsugae and G. oregonensis could have escaped from cultivation? Or maybe you refer to Ganoderma luciudm s. str. instead?

‘According to several recent molecular phylogenetic studies, the taxonomy of G. lucidum and G. lingzhi remains uncertain [18, 26, 31].’ = I disagree, both species can be easily discriminated genetically. The whole paragraph needs to be corrected.

‘The nuclear ribosomal ITS region is a powerful tool for barcoding’ = better ‘the nuclear ribosomal internal transcribed spacer region (ITS) is an informative DNA region for barcoding...’

‘lineage-specific primers have been developed for many groups of fungi in order to improve PCR success’ = in many cases (especially for rDNA) the lineage-specific primers were not designed to improve PCR success, but to provide diagnostic primers to check the presence/absence of some species, or else to avoid the amplification of contaminant organisms.

‘Lineage-specific primers improve PCR success especially when working with compromised DNA templates that may be degraded, contain inhibitors, or be composed of a mixture of species’ = If DNA is degraded, specific primers by themselves will not improve PCR success. Maybe this could happen as a consequence of a different PCR approach, a smaller amplicon, or other factors unrelated with primer specificity. However, lineage-specific primers can improve PCR success when designed for non-conserved annealing regions if the ‘universal’ primers available do not work. In case that inhibitors are present, specific primers will not provide any improvement to PCR success. If a mixture of species is present, then specific primers can bypass the contaminants, improving PCR specificity (but not PCR success, which was also successful with the universal primers).

‘The two main approaches for analysis of the DNA sequences arising from barcoding investigations are similarity-based measures (e.g., best-hit BLAST or nearest neighbor analysis) and phylogenetic methods (e.g., maximum likelihood or Bayesian tree-building algorithms).’ = ‘distance’ is usually employed instead of ‘similarity’. You probably refer to genetic distance, but other types of distances can be measured, so please specify ‘genetic distance’. In addition, distance-based methods are employed also to build phylogenies, so they are phylogenetic methods. You could maybe call the second kind ‘likelihood-based methods’, since ML and bayesian approaches make use of the likelihood function.

‘Herein, we present an efficient barcoding method for unambiguous identification of the herbal supplement, reishi (G. lingzhi).’ = barcoding refers to the sequencing itself, but the identification needs also some kind of analysis, so maybe better ‘‘Herein, we present an efficient method for unambiguous identification of the herbal supplement, reishi (G. lingzhi).’

‘All powdered samples are morphologically unidentifiable as a mushroom’ = did you check for the presence of spores?

Table 1

I think it would be better to hide the name of stores and suppliers, unless you have the explicit consent of these companies to publish the results of your study. The species name is the one provided by the seller, or the results obtained from your analyses? Please clarify.

DNA extraction

‘Each nutritional supplement was extracted twice’ = you mean that two subsamples were taken from each sample and DNA was extracted from each of them, right? Please clarify.

‘Ganoderma-specific primers (G-ITSF1 and G-ITS-R2) were selected based on consistently producing strong single bands’ = you mean that the other primers produced multiple bands or weak bands?

‘These primers were designed to prevent amplification from plant or other fungal DNA, which is a common problem with herbal supplements since they often include a plant-based growing medium.’ = what about the fungal-specific primer ITS1F and the basidiomycete-specific primers ITS4B? Is contamination with other fungi a real issue in commercial samples of powdered Ganoderma?

‘ethidium bromide’ = I strongly recommend you to replace ethidium bromide with GelRed or other less toxic and contaminating DNA stain.

‘Forward and reverse chromatograms for each sample were trimmed to remove primer sequence and low quality sequence’ = chromatograms are trimmed to remove low quality reads. Primer sequences are rarely reached by the chromatogram, and they are almost always poorly resolved. However, they can be recovered in some cases. Also, you should correct ambiguous reads due to noise, dye blobs, and heteromorphic sites. So the most correct would be to say ‘Forward and reverse chromatograms for each sample were trimmed to remove low quality reads at the extremes, and edited to correct ambiguous reads and heteromorphic sites between them.’

BLAST = from which platform did you launch BLAST algorithm? Please cite Cochrane et al. (2011) if accessed from INSDC.

‘We also added all the unique Reishi samples (G. lingzhi and G. lucidum) of ITS using ENTREZ’ = what do you mean by ‘all the unique Reishi samples’? I dont understand, please clarify.

‘Finally, we included representatives of as many Ganoderma species we could find using a filtered discontiguous megablast’ = Maybe this is not the case, but there could be sequences related to your samples that are not listed in the BLAST results, especially if some species are overrepresented in GenBank. Maybe you could have just included those species more closely related to your samples by checking the phylogenetic studies available.

‘Several outgroup sequences were chosen’ = the outgroup should be selected from the clade most closely related to the sequences to be analyzed. In your case, this could be another species of Ganoderma, or the type of a sister genus.

‘Bayesian analysis used 1,500,000 Markov chains’ = usually 4-6 chains are employed. You probably mean 1.5 M generations.

‘and after a burn-in length of 750,000 samples.’ = 1.5 M generations sampled each 750 generations make a total 2000 sampled trees. So, you cannot remove 750.000 samples. You probably mean that you removed the samples taken during the first 750.000 generations (1000 samples, a 50% burn-in).

‘All primer pairs produced visible bands of expected size for the ITS region for both fresh and powdered samples. The Ganoderma-specific primers were chosen for all other analyses.’ = you should explain why these primers are chosen instead of the universal ones. Did you find problems in the sequences produced by the universal primers? Were they chosen to avoid putative contaminants? Please remove cursive from ITS.

‘Nucleotide sequences were recovered from the ITS region from all of the samples.’ = this sentence is superfluous. You should remove it and reorganize the paragraph.

‘After trimming the sequences, lengths ranged from 780 base pairs to 895 base pairs with an average of 854 base pairs.’ = you probably refer to the sequences obtained from GenBank, but it seems like you speak about the sequences produced de novo. Please clarify.

‘31 branches (36%) were greater than 70%’ = 70% is the bootstrap support, please specify it.

Fig. 1 = it would be better to show a phylogram instead of a cladogram. Also, you could add bayesian PP support to the nodes.

‘Fresh #3 had a sister relationship (to “G. lucidum” KX589244)’ = it is better so say ‘a significant relationship’. Change also in the following sentence. These relationships are based on very few bases from a single marker, so they should be interpreted cautiously.

‘Ganoderma brownii and falls clearly outside the G. lingzhi clade in a poorly resolved cluster of Ganoderma accessions in Clade A’ = why none of the two G. brownii ITS sequences in GenBank appear in the tree? Probably due to the sampling procedure. You could have ordered BLAST results by % similarity (removing those <50% coverage).

‘phylogenetically aberrant Genbank accessions’ = maybe better ‘putatively incorrectly named GenBank accessions’

‘a G. lucidum sample (MG654066) falls within a small, yet moderately supported clade of mostly North American samples’ = but in Fig. 1 you report that ‘The red rectangle [MG654066] identifies the only true G. lucidum sample per Loyd et al. [14]’ So, this is not ‘a phylogenetically aberrant Genbank accession’

‘Our study demonstrates that the ITS region provides an efficient barcode for store-bought

reishi herbal supplements as previously described by Loyd et al. [14] and Raja et al. [38].’ = maybe better to say that your study supports the conclusions reached by earlier authors.

‘Clade A sensu Zhou et al. [24] and Loyd et al. [14] which only includes “G. lucidum” as defined in the broadest sense’ = this is very confusing. Clade A sensu Loyd et al. includes multiple species, but not G. lucidum. You could say that clade A includes species morphologically similar to G. lucidum. A sensu lato always includes the sensu stricto plus other clades.

‘Technically all of our samples are misidentified since they are being sold as “G. lucidum”, yet are molecularly allied with the G. lingzhi samples in Clade A.’ = So, MG654066 is not an aberrant accesion, but the correct concept of G. lucidum.

‘Genbank sample MG654066 named G. lucidum (in Clade B) is the only sample that represents G. lucidum sensu stricto’ = this seems to mean that MG654066 is the only known sequence of G. lucidum s. str. However, there are others (at least 8 more in Loyd et al.). It is the only one in your tree, probably because of the sampling process employed.

‘there were distinct phylogenetic affinities clearly indicating separate sources’ = this could be true, but not necessarily.

‘Our results were generally robust to whether we used the entire ITS region or the trimmed region’ = not for G. brownii. This should be enough to say that the sampling method employed to obtain closely related sequences from GenBank was not the most suitable one. I think you should have ordered GenBank results by their similarity with the query, not the BLAST score.

Reviewer #2: Comments

The authors employed a molecular phylogenetic analysis for species authentication of the commonly used fungal supplement, reishi (Ganoderma lingzhi), by amplifying and sequencing the nuclear ribosomal internal transcribed spacer regions (ITS) with genus-specific primers.Their investigation indicated that ITS region could be used in the herbal supplement industry for fungal and plant species that are difficult to identify. This research is of general interest. There are some major comments for authors’ revision.

1. Could the authors please simplify the introduction? Eg. [reviewed in 15, 16], (yet see [18, 19] for biochemical profiles of reishi and close relatives) could be shorted as citations (just keep the reference number).

2. Could the authors please show the features of all the samples? It will be more intuitive for the readers to know the difference of the samples.

3. Please fill in the GenBank Accession numbers in Table 3 (the third column).

4. Could the authors please show the inter/intra-specific distance among these samples?

5. In view of the primers used in this study was Ganoderma-specific ones, how could the authors determine if there is any adulteration derived from other genera in the commercial samples?

6. The discussion section could be separated into several parts according to a clear logic.

**********

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: Review.docx

Click here for additional data file.

19 Oct 2020

Response to Reviewer’s Comments

Gunnel’s et al. Ganoderma barcoding

PONE-D-20-21583

Dear Dr. Tzen-Yuh Chiang, Academic Editor, PLOS ONE

We were glad to see your email indicating that you think our paper “has merit”. We are thankful for the two reviews, especially Reviewer #1’s thorough comments and valiant efforts in helping us improve our manuscript. We know this revised version is much better for their careful attention and thoughtful suggestions. A question for you:

1. Reviewer #1 wrote in regard to Table 1, “I think it would be better to hide the name of stores and suppliers, unless you have the explicit consent of these companies to publish the results of your study.” Please advise.

We have addressed all of the “Additional Journal Requirements” included in your email including amended Statements regarding Funding and Competing Interests that you can change in the online submission on my behalf.

#1 – checked formatting and reformatted as necessary

#2 – Genbank Accession numbers now in Table 2

#3.1 Amended Funding Statement: “There is a commercial affiliation of some authors (MC and JM) and the herbal supplement industry. Oregon’s Wild Harvest provided funding for supplies, sequencing and hourly wages to an undergraduate researcher (TG). MC and JM contributed to the study design and preparation of the manuscript, but in a completely unbiased way. They just wanted to know if their product was correctly identified or not.”

#3.2 Amended Competing Interests Statement: "MC and JM are employees of Oregon's Wild Harvest, an herbal supplement company included in our sampling. As an undergraduate researcher, TG was partially funded by Oregon’s Wild Harvest. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.”

Below we have summarized both reviewers’ comments in italics followed by our indented responses quoting from the revised manuscript whenever we felt it would be helpful in interpreting our revision.

Reviewer #1

General Comment: Unfortunately, I think that the manuscript does not provide enough scientific or technical novelties to be published in PLOS One.

Our understanding of the seven criteria for publication at PLOS ONE does not include anything about “scientific or technical novelties”.

1) It would be great if the authors could quantify the different DNAs present in the samples by means of NGS, in order to estimate the amount of target fungus, additives and contaminants.

We are currently pursuing this as a follow-up study, but the COVID19 response has prevented us from entering the lab since February, 2020. Therefore, we chose to report our Sanger-based results first, then if/when the pandemic-response allows, we will get back to trying NGS approaches on these samples. We feel our results are robust and a worthy contribution to the scientific literature.

2) Another interesting issue is the great amount of misidentifications in public databases (what about UNITE?), and the need for reference sequences (preferably obtained from type collections) to stabilize the taxonomy of these species and provide more accurate database-guided identifications.

Thank you for pointing out this very interesting database - UNITE. We found numerous records for G. lucidum and were initially hopeful to gain some valuable insights from its integration with “species hypotheses” based on 0.5-3% ITS sequene divergence. Unfortunately, the search tool retrieved zero records for G. lingzhi. Furthermore, I defer to Ganoderma taxonomic experts in circumscribing species boundaries that should include geographic, morphological and biochemical data when available to make inferences about reproductive isolation and species status (three forms of data that UNITE does not include in its results). Therefore, I don’t think using the database will contribute anything to our study since our study focuses on trying to differentiate these two taxa, but one is not in the database. We have added a reference to this tool in our Discussion of barcoding with the ITS region so readers will be more likely to attempt to utilize it (hopefully with more success than we had) (Nilsson et al. Nucleic Acids Research 2018).

We agree that reference sequences from type collections would be helpful, but we don’t have access to these and so their sampling is beyond the scope of our current project.

3) Authors could focus also on the comparison between distance-based and likelihood-based phylogenetic methods, and discuss cases where both methods produce different results (maybe comparing Ganoderma with other marketed species, i.e. Morchella?).

Although we feel that the comparison of using genetic distance-based approaches to barcoding identifications vs. phylogenetic ones are important discussions, since our results showed very little differences (qualitatively at least), we don’t think it’s appropriate to dive deep into this topic in this manuscript. We also cite some foundational papers that identified the circumstances in which the two will disagree in our penultimate paragraph of the Introduction (see refs 43-46).

Regarding the suggestion to pursue a comparison with morels, thank you for pointing us to this very interesting example. We have integrated Du et al.’s 2012 study “How well do ITS rDNA sequences differentiate species of true morels (Morchella)?” into our Discussion on using ITS for fungal species identifications. Interestingly, their webtool produces distance-based trees (UPGMA and Neighbor Joining), yet they report parsimony trees in their manuscript. We have left the distance vs. likelihood debate to be resolved in the phylogenetics literature as we are not qualified to address that here.

More comments about the text from Reviewer #1:

Keywords = I would shorten the list of keywords. Please remove the words already included in the title. Please replace ‘nuclear ribosomal internal transcribed spacer region’ for ‘ITS nrDNA’. Correct ‘reishi’ (or remove it, as it is present in the title)

PLOS ONE General Information for Authors states, “Add keywords to help expedite processing of your manuscript (optional). You will not have an opportunity to make changes, so make sure to add concise, accurate keywords now.” There is no additional guidance on the number or redundancy with the title in this regard. We have removed any terms that are redundant with the title. We kept nuclear ribosomal internal transcribed spacer region since it was abbreviated in the title.

Abstract

The first 10 lines could be moved to the Introduction, or substantially reduced to one or two sentences.

We removed several sentences at the beginning of the abstract in response to this comment.

Please use cursive for species names.

We have confirmed that all Latin names are italicized. I think some formatting is lost in the abstract during the submission process, but it is correct in the downloadable WORD version of the manuscript.

‘clearly identified the predominant fungal DNA was G. lingzhi’ = the PCR product is supposed to belong to the predominant DNA present in the sample, but this could not be the case, as genus-specific primers were employed. Other species could be present and not amplified because of the primer specificity. You can change the sentence to ‘clearly identified the presence of G. lingzhi DNA’. Did you obtain a mixed signal after sequencing the PCRs done with universal primers?

Good point. We changed the text as suggested. We only sequenced PCR products from the Ganoderma-specific primers, but all primer pairs tested (including universal primers) produced single strong bands.

‘ITS is a successful and cost-effective method for DNA-based species authentication’ = ITS is not a method, but a region of the rDNA. ‘ITS-based phylogenetic analysis is a successful and cost-effective method for DNA-based species authentication’ would be more correct.

Fixed.

Introduction

‘Barcoding is an efficient molecular tool’ --> better ‘Molecular barcoding is an efficient tool’

Done.

‘identifying morphologically, anatomically and biochemically enigmatic samples’ = Anatomy is a specific type of morphological study. Maybe you mean ‘macroscopically, microscopically and biochemically enigmatic samples’? Please correct other similar sentences.

Done.

‘especially helpful for maintaining the validity of active ingredients’ = DNA barcoding can detect the presence/absence of some target taxa, but it does not provide any info about the activity of ingredients. DNA of a species can be present after losing the biological activity of its compounds (i.e. after an aggressive processing). ‘avoiding contaminants that may cause allergic reactions for consumers’ = other species can be present also as additives. DNA testing can be employed also to check for their presence.

Done. Now reads, “…especially helpful for authenticating the ingredients and avoiding contaminants and additives that may cause allergic reactions for consumers [8], although DNA of a species can be present long after losing the biological activity of its compounds (i.e. after an aggressive processing).”

‘Reishi’ = please add a latin binomial with authors when this species is mentioned for the first time, or at least Ganoderma spp. if the vulgar name is applied to more than one species. Please add the authors of all scientific names when they are first mentioned.

Done.

‘The G. lucidum clade consists of several species that are in taxonomic flux’ = maybe better ‘The G. lucidum clade includes several phylogenetic lineages that received an unstable taxonomic treatment in the past’.

Done

‘Ganoderma lucidum sensu lato (including G. ‘tsugae’)’ = Ganoderma tsugae is a valid name, so it does not need quotation marks. You can correct it as ‘(including G. tsugae Murrill)’.

Done

‘can be found in the wild from Europe to northeastern China (likely escaped from cultivation in California and Utah, see [22])’ = there are three lineages within the G. lucidum clade (G. lucidum s. str., G. tsugae and G. oregonensis). Do you mean that G. tsugae and G. oregonensis could have escaped from cultivation? Or maybe you refer to Ganoderma luciudm s. str. instead?

Unsure, so changed to “…some have likely escaped…” to be conservative.

‘According to several recent molecular phylogenetic studies, the taxonomy of G. lucidum and G. lingzhi remains uncertain [18, 26, 31].’ = I disagree, both species can be easily discriminated genetically. The whole paragraph needs to be corrected.

Thank you for clarifying. We’ve restructured the last two sentences of the paragraph that now read, “The history of taxonomic confusion surrounding G. lucidum and G. lingzhi [18, 26, 31] has been largely resolved by recent work that clearly identifies two distinct lineages based on morphology [23], biochemistry [18], and molecular phylogenetics [22, 23, 24, 32].” The first part about Ganoderma in general and introducing the two species at hand is not invalidated by the reviewer’s comment.

‘The nuclear ribosomal ITS region is a powerful tool for barcoding’ = better ‘the nuclear ribosomal internal transcribed spacer region (ITS) is an informative DNA region for barcoding...’

Done.

‘lineage-specific primers have been developed for many groups of fungi in order to improve PCR success’ = in many cases (especially for rDNA) the lineage-specific primers were not designed to improve PCR success, but to provide diagnostic primers to check the presence/absence of some species, or else to avoid the amplification of contaminant organisms. ‘Lineage-specific primers improve PCR success especially when working with compromised DNA templates that may be degraded, contain inhibitors, or be composed of a mixture of species’ = If DNA is degraded, specific primers by themselves will not improve PCR success. Maybe this could happen as a consequence of a different PCR approach, a smaller amplicon, or other factors unrelated with primer specificity. However, lineage-specific primers can improve PCR success when designed for non-conserved annealing regions if the ‘universal’ primers available do not work. In case that inhibitors are present, specific primers will not provide any improvement to PCR success. If a mixture of species is present, then specific primers can bypass the contaminants, improving PCR specificity (but not PCR success, which was also successful with the universal primers).

Corrected. These sentences now read, “However, lineage-specific primers have been developed for many groups of fungi to help diagnose the presence or absence of particular species [23]. Lineage-specific primers can also improve PCR specificity especially when working with compromised DNA templates…”

‘The two main approaches for analysis of the DNA sequences arising from barcoding investigations are similarity-based measures (e.g., best-hit BLAST or nearest neighbor analysis) and phylogenetic methods (e.g., maximum likelihood or Bayesian tree-building algorithms).’ = ‘distance’ is usually employed instead of ‘similarity’. You probably refer to genetic distance, but other types of distances can be measured, so please specify ‘genetic distance’. In addition, distance-based methods are employed also to build phylogenies, so they are phylogenetic methods. You could maybe call the second kind ‘likelihood-based methods’, since ML and bayesian approaches make use of the likelihood function.

Changed “similarity-based” to “genetic distance-based” here and throughout.

‘Herein, we present an efficient barcoding method for unambiguous identification of the herbal supplement, reishi (G. lingzhi).’ = barcoding refers to the sequencing itself, but the identification needs also some kind of analysis, so maybe better ‘‘Herein, we present an efficient method for unambiguous identification of the herbal supplement, reishi (G. lingzhi).’

Fixed.

‘All powdered samples are morphologically unidentifiable as a mushroom’ = did you check for the presence of spores?

We were able to check one of the powdered samples and only saw mycelium. No fruiting bodies = no spores. We changed this section of the Methods to: “All powdered samples were macroscopically unidentifiable as a mushroom and for Powder #1, only mycelium was identifiable under compound microscope (40-100x) (Creswell and McFerrin, unpublished data).”

Table 1

I think it would be better to hide the name of stores and suppliers, unless you have the explicit consent of these companies to publish the results of your study. The species name is the one provided by the seller, or the results obtained from your analyses? Please clarify.

We emailed the PLOS ONE handling editor (Dr. Chiang) on Sept. 5, 2020 regarding the naming of retailers and suppliers in our manuscript. We are still waiting to hear from him so have not removed it yet. In the meantime, we have clarified that the Species names listed in the table are “as advertised”.

DNA extraction

‘Each nutritional supplement was extracted twice’ = you mean that two subsamples were taken from each sample and DNA was extracted from each of them, right? Please clarify.

Yes, clarified.

‘Ganoderma-specific primers (G-ITSF1 and G-ITS-R2) were selected based on consistently producing strong single bands’ = you mean that the other primers produced multiple bands or weak bands?

We mean both. Therefore, our explanation about “consistently producing strong, single bands” shouldn’t be misinterpreted. We added a comma to clarify these two characteristics.

‘These primers were designed to prevent amplification from plant or other fungal DNA, which is a common problem with herbal supplements since they often include a plant-based growing medium.’ = what about the fungal-specific primer ITS1F and the basidiomycete-specific primers ITS4B? Is contamination with other fungi a real issue in commercial samples of powdered Ganoderma?

Our statement refers specifically to the Ganoderma-specific primers (G-ITS-F1 and G-ITS-R2) as stated in the prior sentence. We list the fungal specific primers mentioned in the Reviewer’s comment in Table 1 as having tried them, but chose the Ganoderma-specific primers for reasons mentioned above.

Regarding contamination with other fungi in powdered Ganoderma, Loyd found only close relatives (e.g. G. applanatum). As thoughtfully mentioned, our choice of Ganoderma-specific primers would reduce the likelihood of detecting non-Ganoderma contamination. However, (1) previous studies have shown if there is any adulteration it is within the polypores and (2) we prove that these primers can amplify other distantly related Ganoderma species (Fresh #1 = G. brownii) and even species from another polypore genus (Fresh #2 = Fomitopsis pinicola). Therefore, I think we are safe using these primers to test for accidentally or intentionally substituted ingredients in reishi herbal supplements.

‘ethidium bromide’ = I strongly recommend you to replace ethidium bromide with GelRed or other less toxic and contaminating DNA stain.

Thanks for the suggestion. I didn’t know ethidium bromide was susceptible to contamination. We are planning to switch as soon as our stocks of EtBr run out.

‘Forward and reverse chromatograms for each sample were trimmed to remove primer sequence and low-quality sequence’ = chromatograms are trimmed to remove low quality reads. Primer sequences are rarely reached by the chromatogram, and they are almost always poorly resolved. However, they can be recovered in some cases. Also, you should correct ambiguous reads due to noise, dye blobs, and heteromorphic sites. So the most correct would be to say ‘Forward and reverse chromatograms for each sample were trimmed to remove low quality reads at the extremes, and edited to correct ambiguous reads and heteromorphic sites between them.’

Thank you for trying to clarify this section. However, the ends are not “reads”, but portions of a single contiguous sequence. We changed this to “…trimmed to remove the opposing primer sequence and low-quality sequence at the beginning and end of each read and edited to correct any ambiguous base calls.” We did not have any heteromorphic sites (and this term has multiple meanings so we want to avoid confusion). Furthermore, we definitely sequenced the opposing primer sequence and had to remove it so we clarified that this is the primer that lands on the opposing side of the direction of sequencing.

BLAST = from which platform did you launch BLAST algorithm? Please cite Cochrane et al. (2011) if accessed from INSDC.

We used BLAST directly through NCBI’s portal (no secondary references required). Therefore, the original references (51-53 listed below) seem most relevant:

51. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403-10.

52. Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol. 2000 Feb 1;7(1-2):203-14.

53. Morgulis A, Coulouris G, Raytselis Y, Madden TL, Agarwala R, Schäffer AA. Database indexing for production MegaBLAST searches. Bioinformatics. 2008 Aug 15;24(16):1757-64.

‘We also added all the unique Reishi samples (G. lingzhi and G. lucidum) of ITS using ENTREZ’ = what do you mean by ‘all the unique Reishi samples’? I dont understand, please clarify.

ENTREZ is a text search portal to NCBI’s Genbank. It’s a good way to get started finding sequences, but BLAST is an essential follow-up to ensure you have all similar sequences even if the text doesn’t match (e.g. misidentified sequences). We clarified the use of ENTREZ by writing, “…using text searches in ENTREZ.”

‘Finally, we included representatives of as many Ganoderma species we could find using a filtered discontiguous megablast’ = Maybe this is not the case, but there could be sequences related to your samples that are not listed in the BLAST results, especially if some species are overrepresented in GenBank. Maybe you could have just included those species more closely related to your samples by checking the phylogenetic studies available.

If the sequences were “related” to our sample, BLAST would have found them. I don’t know of any errors in the search algorithm regarding “overrepresentation”. As long as one expands the number of reported sequences so it doesn’t max out, they will get everything that is similar given the search parameters. Furthermore, we checked some key phylogenetic studies to ensure we had all the relevant sequences in our reference panel.

‘Several outgroup sequences were chosen’ = the outgroup should be selected from the clade most closely related to the sequences to be analyzed. In your case, this could be another species of Ganoderma, or the type of a sister genus.

Since we surveyed Ganoderma species broadly, we chose a more distant outgroup. Since we recovered the main three clades of Ganoderma that are previously described, it doesn’t appear to make a difference. I would be more concerned about accidentally choosing something too close and then the outgroup rooting would have severe phylogenetic repercussions. No changes made in this regard.

‘Bayesian analysis used 1,500,000 Markov chains’ = usually 4-6 chains are employed. You probably mean 1.5 M generations.

‘and after a burn-in length of 750,000 samples.’ = 1.5 M generations sampled each 750 generations make a total 2000 sampled trees. So, you cannot remove 750.000 samples. You probably mean that you removed the samples taken during the first 750.000 generations (1000 samples, a 50% burn-in).

Thank you for helping clarify our methods. In our reanalysis, we ran more generations. It now reads, “Bayesian analysis ran for 2,000,000 generations. After removing the first 1,000,000 generations as burn-in, we sampled trees every 1000 generations creating a posterior distribution of 1000 trees.”

Results

‘All primer pairs produced visible bands of expected size for the ITS region for both fresh and powdered samples. The Ganoderma-specific primers were chosen for all other analyses.’ = you should explain why these primers are chosen instead of the universal ones. Did you find problems in the sequences produced by the universal primers? Were they chosen to avoid putative contaminants? Please remove cursive from ITS.

We have removed the italics for ITS and clarified our primer selection in the following revised sentence, “The PCR products using Ganoderma-specific primers were chosen for sequencing and all subsequent analyses based on their increased band intensity compared to other primers.”

‘Nucleotide sequences were recovered from the ITS region from all of the samples.’ = this sentence is superfluous. You should remove it and reorganize the paragraph.

Done.

‘After trimming the sequences, lengths ranged from 780 base pairs to 895 base pairs with an average of 854 base pairs.’ = you probably refer to the sequences obtained from GenBank, but it seems like you speak about the sequences produced de novo. Please clarify.

No, this statement refers to the seven new samples from herbal supplements. We have clarified in the following revised sentence, “After trimming these newly created sequences for seven samples…”

‘31 branches (36%) were greater than 70%’ = 70% is the bootstrap support, please specify it.

Fixed.

Fig. 1 = it would be better to show a phylogram instead of a cladogram. Also, you could add bayesian PP support to the nodes.

Fig 1 has been completely redone since we included two G. brownii sequences and three additional G. lucidum sequences. We have included a phylogram as an inset (B) without labels (to save space) to depict the relative branchlengths as suggested. We also superimposed the Bayesian posterior probabilities onto the maximum likelihood tree at critical nodes.

‘Fresh #3 had a sister relationship (to “G. lucidum” KX589244)’ = it is better so say ‘a significant relationship’. Change also in the following sentence. These relationships are based on very few bases from a single marker, so they should be interpreted cautiously.

Completely rewritten.

‘Ganoderma brownii and falls clearly outside the G. lingzhi clade in a poorly resolved cluster of Ganoderma accessions in Clade A’ = why none of the two G. brownii ITS sequences in GenBank appear in the tree? Probably due to the sampling procedure. You could have ordered BLAST results by % similarity (removing those <50% coverage).

Thank you for noticing that oversight on our part. We have redone the phylogenetic analyses with these G. brownii sequences and the missing G. lucidum sequences mentioned below in regard to MG654066. They all fall out in the tree as expected, but the study is much more robust having updated this portion.

‘phylogenetically aberrant Genbank accessions’ = maybe better ‘putatively incorrectly named GenBank accessions’

Done (also at the end of the next paragraph describing the consistencies in the Bayesian analysis).

‘a G. lucidum sample (MG654066) falls within a small, yet moderately supported clade of mostly North American samples’ = but in Fig. 1 you report that ‘The red rectangle [MG654066] identifies the only true G. lucidum sample per Loyd et al. [14]’ So, this is not ‘a phylogenetically aberrant Genbank accession’

Correct. We have now added the additional G. lucidum sequences and removed any references to this single sample being “aberrant”.

Discussion

‘Our study demonstrates that the ITS region provides an efficient barcode for store-bought reishi herbal supplements as previously described by Loyd et al. [14] and Raja et al. [38].’ = maybe better to say that your study supports the conclusions reached by earlier authors.

Done.

‘Clade A sensu Zhou et al. [24] and Loyd et al. [14] which only includes “G. lucidum” as defined in the broadest sense’ = this is very confusing. Clade A sensu Loyd et al. includes multiple species, but not G. lucidum. You could say that clade A includes species morphologically similar to G. lucidum. A sensu lato always includes the sensu stricto plus other clades.

We agree that this portion of the Discussion was poorly written. We have reduced it to the essential details in the following revised sentence, “All of the samples we examined had BLAST and phylogenetic results suggesting they were clearly members of Clade A sensu Zhou et al. [24] and Loyd et al. [14].”

‘Technically all of our samples are misidentified since they are being sold as “G. lucidum”, yet are molecularly allied with the G. lingzhi samples in Clade A.’ = So, MG654066 is not an aberrant accession, but the correct concept of G. lucidum.

Correct. We have now added the additional G. lucidum sequences and removed any references to this single sample being “aberrant”.

‘Genbank sample MG654066 named G. lucidum (in Clade B) is the only sample that represents G. lucidum sensu stricto’ = this seems to mean that MG654066 is the only known sequence of G. lucidum s. str. However, there are others (at least 8 more in Loyd et al.). It is the only one in your tree, probably because of the sampling process employed.

Correct. We have now added the additional G. lucidum sequences and removed any references to this single sample being “aberrant”. Since several sequences were identical, we added a row to S3 Table indicating which G. lucidum samples were removed as identical (see below for detailed explanation).

We searched Genbank for “Ganoderma lucidum[organism] AND Loyd[author]” and got 10 hits (including our original MG654066). A quick alignment showed that of these sequences there were only four unique sequences as you can see from the distance matrix appended below. Therefore, we added three additional sequences and indicated in Supplemental Table 3 that there are seven identical sequences of which we chose MH160071 as our representative. Our new tree has four G. lucidum sequences instead of the single placeholder in our original submission.

Table S3 now has the following row for the G. lucidum sequences included in the phylogenetic analysis (MH160071) and the five other sequences identical to it:

MH160071 MG654067, MG654070, MG654071, MG654072, MG654073

Our sampling table for the reference panel (S2 Table) has also been updated with the new G. lucidum sequences as well.

‘there were distinct phylogenetic affinities clearly indicating separate sources’ = this could be true, but not necessarily.

OK, we changed “clearly indicating” to “suggesting” leaving room for alternative interpretations yet emphasizing the most likely conclusion.

‘Our results were generally robust to whether we used the entire ITS region or the trimmed region’ = not for G. brownii. This should be enough to say that the sampling method employed to obtain closely related sequences from GenBank was not the most suitable one. I think you should have ordered GenBank results by their similarity with the query, not the BLAST score.

Yes, as Reviewer #1 pointed out above, G. brownii (and some G. lucidum sequences) were accidentally removed from the alignment. I don’t think this was an inherent flaw in the sorting method (Genbank reports BLAST results based on MaxScore which is a composite of the E-value, % identity and % coverage and is widely used to identify most likely matches in Genbank).

Instead of a methodological error, I believe the missing sequences are simply an oversight on our part. This happened when we removed identical sequences from the reference panel because they were stalling the phylogenetic analyses (and by definition provide no additional phylogenetic information). When these identical sequences were removed, they were to be placed in Supplemental Table for reference, but thankfully Reviewer #1 noticed that some were lost in the process. We have reincluded the missing G. brownii and G. lucidum sequences, triple checked the results for any other missing sequences (none found) and updated the alignment & phylogenetic analyses, produced new trees for Figure 1 and the Supplemental Figure (Bayesian tree), and rewritten the relevant sections of the Results and Discussion. Qualitatively, we didn’t change our major findings, but the reference panel is now complete and the results even more robust than before.

Reviewer #2

1. Could the authors please simplify the introduction? Eg. [reviewed in 15, 16], (yet see [18, 19] for biochemical profiles of reishi and close relatives) could be shorted as citations (just keep the reference number).

Done.

2. Could the authors please show the features of all the samples? It will be more intuitive for the readers to know the difference of the samples.

We are awaiting feedback from the Associate Editor in regard to what to show and what to keep confidential. If the Associate Editor suggests we show the products, then we can try to assemble a multi-panel plate as suggested.

3. Please fill in the GenBank Accession numbers in Table 3 (the third column).

Genbank Accession numbers have been added to the table.

4. Could the authors please show the inter/intra-specific distance among these samples?

We have integrated this into the Results section when discussing the genetic variation detected among reishi samples and the field collected controls.

5. In view of the primers used in this study was Ganoderma-specific ones, how could the authors determine if there is any adulteration derived from other genera in the commercial samples?

Great point. We assumed that any adulteration intentional or accidental would have been by substitution of G. lingzhi with another polypore (mostly likely in the same genus, but possibly in an allied genus like Fomitopsis) based on the findings of Loyd. Fortunately, the primers are optimized for polypores and we have confirmed this empirically by successful amplification and sequencing of the control samples that represent a distant relative within Ganoderma and another sample that amplified with these primers and was identified Fomitopsis. We have clarified this point in the Discussion.

6. The discussion section could be separated into several parts according to a clear logic.

According to PLOS ONE Guide to Authors, subheadings in the Discussion are optional. Since there are only six paragraphs, most subheadings would only apply to a single paragraph and be less of an organizational tool. They would break-up the flow of the Discussion that we were shooting for. No change made.

Submitted filename: Response to Reviewers Comments.docx

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28 Oct 2020

The ITS region provides a reliable DNA barcode for identifying reishi/lingzhi (Ganoderma) from herbal supplements

PONE-D-20-21583R1

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4 Nov 2020

PONE-D-20-21583R1

The ITS region provides a reliable DNA barcode for identifying reishi/lingzhi (Ganoderma) from herbal supplements

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