First DNA barcode efficiency assessment for an important ingredient in the Amazonian ayahuasca tea: mariri/jagube, Banisteriopsis (Malpighiaceae)

Mariri or jagube (Banisteriopsis C. B. Rob.) is a vine of the Malpighiaceae family and is naturally found in the Brazilian Amazon. It is also an important ingredient of ayahuasca tea, which is used by some traditional, indigenous people and religious groups of Amazonian origin. Via DNA barcodes, this study aimed to evaluate the efficiency of six markers to access genetic diversity and use them in the identification of either ethnovarieties or species of Banisteriopsis used in the preparation of “ayahuasca” in the Amazon. The efficiency of three plastid genes (matK/trnK, ndhF, and rcbL) and three nuclear markers (MP3 and yage5 isolated in the present study and ITS—Internal Transcribed Spacer) were tested. For these six markers, a total of 4792 base pairs were sequenced. The plastid markers revealed that all the samples analyzed belong to the genus Banisteriopsis; however, they presented the lowest indices of genetic diversity and were not efficient for use in the identification of ethnovarieties. Nuclear DNA markers showed the highest levels of genetic diversity. The ITS showed the highest values of genetic distances between the identified lineages. This study demonstrates that the use of ITS for DNA barcoding was more efficient since the identified lineages showed correspondence with the ethnovarieties of Banisteriopsis spp. used in the preparation of ayahuasca. As such, we encourage new integrative studies like this one, which combines modern approaches and traditional knowledge, to contribute to the maintenance of traditional knowledge as well as the conservation of species.

T. Z. Luz · A. S. Cunha-Machado (*) · J. da Silva Batista Programa de Pós-Graduação em Genética, Conservação e Biologia Evolutiva (PPG-GCBEv), Laboratório Temático de Biologia Molecular (LTBM), Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus,  List 2019). Species of this genus have biologically active metabolites that present important pharmacological activities (Dos Santos and Hallak 2019). Among these species is the vine Banisteriopsis caapi (Spruce ex Griseb.) C. V. Morton, which is found in the Amazon Rainforest and which is one of the main ingredients of an entheogenic tea known as "ayahuasca". This tea has been used for centuries by indigenous groups of the Amazon in Brazil, Peru, Colombia, and Ecuador for ritual, religious and therapeutic purposes (Schultes et al. 1992;Ogalde et al. 2009). The popularly known formula of ayahuasca is the decoction of the macerated vine of B. caapi (mariri/jagube) with leaves from Psychotria viridis Ruiz & Pav (chacrona/ rainha). In its composition, the vine B. caapi contains alkaloids that belong to the group of β-carbolines, and the species P. viridis has in its composition N, N-dimethyltryptamine (DMT), which is considered to be the main entheogenic agent of the drink (Callaway et al. 2005).
Ayahuasca is also used by non-indigenous populations of these countries, such as syncretic religious organizations that originate in the northern region of Brazil, the main ones being Santo Daime and the Centro Espírita Beneficente União do Vegetal (CEBUDV), in which ayahuasca is called "Daime" and "Hoasca", respectively. The CEBUDV is present in all Brazilian states and ten countries: the United States, Canada, Peru, Portugal, Spain, the United Kingdom, Switzerland, Italy, the Netherlands, and Australia (CEBUDV 2022a, b). Although the CEBUDV is present in the main capitals of Brazil, many of its members in the Amazon region are descendants of traditional and indigenous peoples and were already familiar with ayahuasca before entering the religion, a fact that makes them holders of traditional knowledge.
Although Banisteriopsis caapi is the species traditionally that is attributed to the ayahuasca drink, there are reports that Banisteriopsis muricata (Cav.) Cuatrec. and other species of the genus are also used for the preparation of tea (Gates 1982;Schultes 1984;Schultes et al. 1992). Due to the scarcity of fertile collections and lack of detailed taxonomic studies, these species have not been properly discriminated and described. It is also important to consider that the floral morphology found in the genus Banisteriopsis has conserved characters and this hinders the clear identification and classification of the species of the genus (Gates 1982;Langdon 1986).
The indigenous peoples and traditional populations of the Amazon recognize diverse ethnovarieties of the vines of Banisteriopsis spp., which are used for the preparation of ayahuasca. Among the Siona indigenous peoples, at least eighteen ethnovarieties have been identified; however, it was only possible to collect reproductive branches from five varieties and they were identified as B. caapi (Langdon 1986). Among the Colombian Tukanos of the Vaupés River, six ethnovarieties of the ayahuasca vine are recognized; though botanical identification has not yet been possible (Schultes et al. 1992).
The CEBUDV recognizes two ethnovarieties of Banisteriopsis spp.: mariri tucunacá and mariri caupuri, which are recognized primarily by the shape of the stem. On the other hand, some of the people at CEBUDV identify more ethnovarieties (Cattanio et al. 2011), for example, pajézinho, caupuri without nodes, tucunacá with nodes, mariri nativo, and amarelinho, among others. Santo Daime recognizes 11 varieties (Monteles 2020), though this can reach up to 20 varieties (personal information from R. Monteles), and at least 30 ethnovarieties are recognized by the natives throughout the western Amazon (Schultes 1986). Schultes (1986) described a botanical enigma, which concerns the "mysterious" ability of the Amazonian natives to distinguish between varieties of plants, which he could not distinguish at the botanical level, especially those used to make ayahuasca. The author reports that the members of different tribes, who live significant distances from each other, identify the varieties of the vine of ayahuasca with incredible consistency. Schultes (1986) noted that it was "botanically impossible" to discern morphological differences to separate these varieties from each other and lamented the fact that so little research has been dedicated to "this fascinating aspect of ethnobotany". This is a situation that remains to the present day.
Historically, ethnobotanists have documented, described, and explained the complex relationships between crops and the usefulness of plants (Newmaster and Ragupathy 2010). As such, ethnobotany identifies plants that may have agricultural, industrial or pharmacological value (Schultes 1979). In other situations, ethnobotanical knowledge can be used as a base to aggregate indigenous and traditional knowledge within their own scientific and cultural structures. This approach is based on the concept of an assembly of knowledge on biodiversity, which includes a union of different ways of identifying and valuing species variation in a new approach that seeks to add value to traditional and scientific knowledge (Newmaster and Ragupathy 2010;Hibert et al. 2011).
In this sense, the DNA barcode approach was used to evaluate the efficiency of three plastid markers (matK/trnK, rcbL, and ndhF) and three nuclear markers (two of these isolated in the present study and the ITS), and assess the genetic diversity of the vines used in the preparation of ayahuasca in the Amazon. The correspondence of the identified genetic lineages with the ethnovarieties known in the traditional context of the CEBUDV and with the DNA sequences (matK/trnK, ndhF, and rcbL) of the species of Banisteriopsis spp., which have been identified at the morphological level by Davis and Anderson (2010), was also evaluated. We primarily take into account ethnovarieties that are recognized by differences in shape of the stem. The final objective of the study was to present the most efficient markers for future genetic studies for the conservation of the ethnovarieties known by members of the CEBUDV in the Amazon and contribute to the maintenance of traditional knowledge.

DNA sampling and extraction
A total of 20 samples of mariri vines (Banisteriopsis spp.) were obtained. The collections were carried out between the years of 2017 and 2018 in Manaus, Novo Airão, and Presidente Figueiredo, which are three municipalities of the state of Amazonas, Brazil ( Fig. 1). In Manaus, 15 samples were collected in three plantations in three nuclei (Núcleo Mestre Vicente Marques, Núcleo Mestre Angílio and Núcleo Flor do Norte) of the CEBUDV (Table S1). For the collection of these samples, different morphological variations of the vine stem were prioritized (Figs. 2d and S1). In Presidente Figueiredo (4 samples) and Novo Airão (1 sample) (Table S1), the collections were carried out in a natural environment and had the support of CEBUDV leaders, who are holders of the traditional knowledge of the vines that are used for the preparation of ayahuasca tea.This work was For the molecular analysis, the cambium tissue or leaves of vines (Banisteriopsis spp.) were stored in containers containing silica gel and subsequently stored in the freezer. Genomic DNA was extracted from 120 mg of plant material following the CTAB plant tissue extraction protocol (Oliveira et al. 2017).

Development of the genomic library
The DNA genomic library was constructed for Banisteriopsis spp. using a sample (MT1, tucunacá without nodes) cultivated in the Mestre Angílio nucleus (Manaus, AM). A genomic library was constructed according to the methodology described by Billotte et al. (1999). DNA extracts (50 ng) were digested with RsaI (Invitrogen) and ligated to the RSA adapters. DNA fragments containing putative markers were selected by hybridization with (CT)8 and (GT)8 biotin-linked probes and were recovered with streptavidin-linked particles (Streptavidin Magne-Sphere Paramagnetic Particles, Promega). Selected fragments were linked to a pGEM-T easy vector (Promega), and transformed into Escherichia coli XL1blue competent cells, which were subsequently inoculated in plates with X-Gal/IPTG Luria-Bertani (LB) agar and ampicillin (100 mg/ml) for overnight growth at 37 °C. Single white colonies were transferred onto microplates with a HM/FM medium with ampicillin (100 mg/ml) to grow overnight. Plasmid DNA was recovered (Green and Sambrook, 2012) from 96 insert-containing colonies and then bi-directionally sequenced in a DNA Analyzer (ABI 3130xl Applied Biosystems) using T7 and SP6 primers and the Big Dye® Terminator V3.1 Kit (Applied Biosystems).
The sequences were edited using The SEQSCAPE v2.7 (Applied Biosystems) program and analyzed in the basic local alignment search tool (BLAST N) (Altschul et al. 1990) in the sequence database of the National Center for Biotechnology Information (NCBI) for verification and confirmation of anonymous DNA regions and protein-coding regions. The design of the primers was carried out with the help of the program PRIMER3 web version 4.0.0 (Koressaar and Remm 2007;Untergasser et al. 2012).
Amplification and sequencing of nuclear and plastid DNA markers Three markers were amplified for nuclear DNA, which comprised two nuclear markers resulting from the genomic library that was built in the present study and the internal transcribed spacer (ITS) ( Table 1). Three plastid markers were also amplified: maturase K and trnK intron (matK/trnK), ribulose-1,5-bisphosphate (rbcL), and dehydrogenase F (ndhF) ( Table 1). Reagent conditions used for the PCR were as follows: 1X PCR buffer, 0.18 mM dNTPs, 3.0 mM MgCl 2 , 0.3 μM of each primer (forward and reverse), 0.25 μg/μL of BSA, 0.5U Taq DNA polymerase (Invitrogen) and 0.6 ng/μL g DNA. ddH 2 O was added to achieve the final volume (17 μL). For the rbcL, the primers rbcL-01F and rbcL-1367r were used and for the matK/trnK the primers trnK-2R and matK-400F were used ( Table 1). The amplification was performed in a thermal cycler (Veriti®, ThermoFisher) and followed the cycles given in Table 1. PCR products were stained with nucleic acid dye (Diamond™, Promega) and their size and quality were checked in 1% agarose gel. The PCR products were purified using the GFX purification kit (GE Healthcare).

Analysis of DNA sequence data
The SEQSCAPE v2.7 (Applied Biosystems) program was used to edit the sequences of nuclear and plastid markers. For the alignment of the sequences, the program MAFFT v7.307 (Katoh and Standley 2013). SeqPHASE (Flot 2010) and PHASE v2.1.1 (Stephens et al. 2001) was used to determine nuclear markers alleles, with a 90% posterior probability per heterozygous site, using homozygous sequences as prior information.
Genetic diversity indices were estimated for all markers using the DNASP 6.0 program (Rozas et al. 2017). Phylogenetic trees were constructed for all markers using the neighbor-joining (NJ) method using the K2P model in MEGA X (Kumar et al. 2018) and also by the maximum likelihood (ML) method in RAXML v8.2.10 (Stamatakis 2014). The reliability of each node of the NJ and ML analyses was estimated by the bootstrap method with 1000 replicas (Felsenstein 1985). The genetic distances for each marker were estimated and considered the clades and/or genetic lineages identified in the respective phylogenies. Genetic distances were calculated using the Kimura-2-parameters model (Kimura 1980) with bootstrap support with 1000 replications in the MEGA X program (Kumar et al. 2018).
NJ and ML phylogenetic analyses were also performed for the three markers (rbcL, matK/trnK, and ndhF) with concatenated sequences, including seven species of Banisteriopsis that have sequences for the same markers, which were also identified morphologically by Davis and Anderson (2010). The sequences of plastid markers (matK/trnK, rbcL, and ndhF) generated in the present study were compared with the sequences of 19 species of Banisteriopsis (Davis and Anderson 2010). For these comparisons, BLAST N analyses were used in the NCBI sequence database. The ITS nuclear marker sequences were also subjected to the same analysis.

Genomic library
From the 96 clones in the DNA genomic library, good quality sequences were obtained for 53 clones that ranged from 570 to 1004 base pairs (bp) and, of these, 52 were nucleotide sequences of non-redundant clones. In all, 10 pairs of primers were designed, seven amplified for all samples and two (MP3, yage5) presented good quality sequences and were used in the genetic analysis. The clone sequence used for the design of primers for the yage5 marker of nuclear DNA (accession ON168504) did not show significant similarity with DNA sequences of the NCBI database after analysis in BLAST N. However, the clone sequence used for the design of primers for the MP3 marker of nuclear DNA (accession ON168519) presented up to 82.72% identity percentage (45% query cover) with the protein kinase (accession LN713255).

Nuclear markers
The alignments of the three nuclear markers MP3, yage5, and ITS resulted in a length of 425 bp, 566 bp, and 523 bp, respectively, and resulted in a total of 1514 bp. For these markers, the number of polymorphic sites ranged from 9 (MP3) to 170 (ITS), haplotypic diversity ranged from 0.727 (MP3) to 0.970 (yage5), nucleotide diversity ranged from 0.00326 (MP3) to 0.11724 (ITS), with indels (inserts or deletions) ranging from 2 (MP3) to 6 (ITS). The highest numbers of phased nuclear haplotypes were observed for yage5 and ITS, which presented 22 and 15 phased nuclear haplotypes, respectively (Table 2). Three clades were identified (A, B, and C) with bootstrap support values > 99 and 7 genetic lineages (I to VII; Fig. 2) based on the topology of the NJ and ML phylogenetic tree using the ITS marker. Clade A is made up of lineage I with a sample of the tucunacá ethnovariety that was collected in a cultivation environment (MA8), and lineage II with 3 samples of the native tucunacá ethnovariety, which was collected in a natural environment (Presidente Figueiredo). These two lineages (I and II) had a genetic distance of 3.86% (Table 3). Clade B is represented by lineages III and IV (Fig. 2). Genetic lineage III has half of the sequences analyzed in the present study and includes only samples of the ethnovariety caupuri with and without nodes, which were collected at the CEBUDV nuclei. Lineage IV is also part of the ethnovariety Fig. 2 The phylogenetic trees were performed with 16 mariris samples (Banisteriopsis spp.) used for the preparation of ayahuasca tea. Trees were obtained by the a maximum likelihood (ML) and b neighbor-joining (NJ) method for the internal transcribed spacer (ITS) molecular marker with phased sequences.
c The genetic lineages identified are represented by different colors and Roman numerals, followed by the names of the ethnovarieties. d Photos of different morphological variations of the mariri stem were used for analysis caupuri with nodes (sample MOAC3) and is a sister lineage to lineage III, which presented a 4.09% genetic distance (Fig. 2 and Table 3). Clade C is represented by the ethnovarieties that are more related to pajezinho, and include three lineages (V, VI, and VII), two of which are native (Presidente Figueiredo and Novo Airão) and one that is cultivated.
The greatest genetic distance was 28.99%, which was observed between the ethnovarieties caupuri with nodes and caupuri without nodes (lineage III) and native mariri (lineage VI). The shortest genetic distance (2.17%) was observed between lineage V (sample PF5) and lineage VI (sample SA3) (Table 3).
In the NCBI database, there is only one ITS accession for B. caapi (accession number LC496605), and this showed an identity percentage ranging from 85.33 to 95.43% after comparison with sequences presented (Table S2). Trees were constructed using the NJ and ML methods in which this accession of the ITS for B. caapi from the NCBI was included and this grouped with the samples of clade A (Fig. S2). The ITS marker presented 168 informative sites for parsimony and an average genetic distance of 14.26% (Table 4). The phylogeny of the anonymous marker yage5 presented two clades (A and B) (Fig. S3). Clade A represents most of the samples that were collected in the plantations at the CEBUDV. Only the VM5 sample, which was collected in a cultivation environment, was grouped with the samples collected in a natural environment, in clade B. The genetic distance between clades A and B for the yage5 marker was 7.35% and the mean genetic distance was 3.61% (Table 4).
Based on the NJ and ML phylogenies, it was not possible to observe clades that were divergent with the nuclear marker MP3 (Fig. S4). The mean genetic distance was 0.34% (Table 4), which is the shortest distance when compared with the other nuclear markers analyzed in the present study.
The mean genetic distances estimated for each of the three markers (rbcL, matK/trnK, and ndhF) were less than 0.15% (Table 4). The NJ and ML trees for these three markers (rbcL, matK/trnK, and ndhF) showed no more than two bootstrap values greater than 90 (Fig.S5). BLAST N analysis for these markers identified all samples as being of the genus Banisteriopsis (Tables S3, S4, and S5).
The phylogenetic trees (NJ and ML) with the three concatenated plastid markers (rbcL, matK/trnK, and ndhF) included nucleotide sequences of 19 samples of Banisteriopsis spp. that are used in the preparation of ayahuasca and the seven species of Banisteriopsis that have sequences for the three markers available in GenBank (Davis and Anderson 2010). Although the phylogenetic analyses (NJ and ML) supported a clade with a bootstrap of 99, it included all of the 19 samples analyzed in the present study and B. caapi and B. schwannioides (Griseb.) B. Gates, which are species identified at the morphological and genetic level by Davis and Anderson (2010) (Fig. S6). The mean genetic distance for the concatenated plastid markers was only 0.09% (Table 4).

Discussion
This is the first report of the efficiency of six molecular markers as potential DNA barcodes for the evaluation of their genetic diversity and the identification of ethnovarieties of Banisteriopisis spp. used for the preparation of ayahuasca in the Amazon in the religious context of CEBUDV. Our combined results of the genetic diversity, the ability to differentiate groups of ethnovarieties in the phylogenetic trees, and patterns of genetic divergence, support the designation of ITS as the main DNA barcode marker for the identification of ethnovarieties of Banisteriopisis spp. In addition, the inclusion of at least one of the three analyzed plastid markers (matK/trnK, rbcL or ndhF) as an additional marker is recommended, since the combination of nuclear and plastid information gives more reliability to the dataset. Plastid markers did enable the confirmation of the genus Banisteriopsis, although they do not increase the taxonomic power of identification of species and ethnovarieties.

Genetic diversity and BLAST N
The nuclear markers presented the highest rates of genetic diversity when compared to the plastid markers, which is a fact that has been observed in over 54 families of Amazonian angiosperms such as Sapotaceae Juss. and Rubiaceae Juss. (Gonzalez et al. 2009;Mezzasalma et al. 2017). In forty-nine genera of Amazonian angiosperms, the highest rates of genetic diversity were observed with the ITS (Gonzalez et al. 2009). In eighteen samples of piri-piri (Cyperus L. spp.), which are also used in traditional Amazonian medicine, eight haplotypes have been observed for the ITS (Mezzasalma et al. 2017). High genetic diversity was observed with 14 haplotypes for the 15 and 16 samples of Banisteriopsis spp. that were sequenced in the present study for yage5 and ITS, respectively, although 80% (16 samples) sequencing efficiency for ITS and 75% (15 samples) for yage5 was achieved. The difficulty in sequencing using ITS has already been observed in other studies (Gonzalez et al. 2009).
Some regions of the plastid genome have relatively slow rates of nucleotide replacement (Clegg et al. 1994), which may have favored the results that showed lower indices of genetic diversity in the analyzed samples (Table 2). Despite the plastid markers matK/trnK and rbcL being recommended as DNA barcodes for plants (Hollingsworth et al. 2009;Mishra et al. 2016), they were not efficient for Banisteriopsis spp vines, because were highly conserved in the 20 samples analyzed in the present study. The three plastid markers analyzed (matK/trnK, rbcL, and ndhF) presented only three polymorphic sites and four haplotypes (Table 2) and, in this sense, did not vary enough to discriminate the ethnovarieties. In addition, the comparison of plastid markers in the NCBI sequence database with the 19 species of Banisteriopsis identified at the morphological level by Davis and Anderson (2010) presented a percentage of identity ranging from 99 to 100% for all 19 species (Tables S3, S4, and S5). Therefore, for Banisteriopsis spp., the plastid markers on their own do not vary enough to be able to separate and identify species.
On the other hand, for the nuclear marker ITS, the BLAST N analysis resulted in a percentage of identity ranging from 85.33 to 95.43% after comparison with the only accession of the ITS for B. caapi (Table S2). These molecular identification applications depend on the existence of complete reference libraries of DNA sequences with which new sequences can be compared to perform taxonomic identification (De Lima et al. 2018). If a reference library has poor coverage in terms of species with sequences already available, the implementation of DNA-based identification techniques will remain inefficient. Thus, this work presents an important contribution by providing new accessions of nuclear and plastid sequences of Banisteriopsis spp. for the NCBI database, especially when we consider Banisteriopsis spp., which is used in the preparation of ayahuasca tea.
Testing the identification of ethnovarieties based on phylogenetic trees and genetic distance methods The three clades (A, B and C) that were differentiated by the molecular marker ITS are represented by lineages of the three main ethnovarieties of Banisteriopsis spp.; tucunacás, caupuris, and pajezinho, respectively (Fig. 2). These ethnovarieties are recognized by traditional knowledge in the context of CEBUDV, but they can also be recognized by other names when we consider the indigenous context and other religions.
In clade A, it was possible to identify two lineages of Banisteriopsis spp. of the tucunacá ethnovariety; one is a lineage (I) cultivated in the Mestre Angílio nucleus in Manaus and the other a native lineage (II) of Presidente Figueiredo (Fig. 2). The only accession of B. caapi available in the NCBI database for the ITS presented the highest percentage values of identity related to specimens of lineage II, from the native tucunacá ethnovariety (ranging between 95.24 and 95.43%; Table S2).
The Banisteriopsis spp. varieties that were identified in lineage III and IV (clade B) are ethnovarieties of caupuri with nodes and without nodes. These presented the highest phenotypic plasticity of the stem (Figs. 2d and S1) and all were collected in cultivations in Manaus. Of the eight samples (lineage III), four presented the phenotype with nodes and the other four without nodes, corroborating the traditional knowledge that recognizes the caupuri ethnovariety by this characteristic (with nodes and without nodes). The caupuri with nodes was recognized and introduced to the CEBUDV in the 1960s. At this time, marked by the beginning of the CEBUDV in the city of Manaus, Mestre Florêncio Carvalho (in memoriam), who was a rubber tapper in the Amazon Rainforest for many years, carried out some searches for Banisteriopsis spp. in the forests of the banks of the Negro River, where the caupuris (with nodes) were encountered.
Clade C presented the greatest number of lineages (V, VI, and VII) and these are represented by the ethnovariety identified as pajezinho, which comprises two native (V and VI) and one cultivated lineage (VII). Pajezinho is recognized for having one of the strongest effects in ayahuasca after its preparation (Fig. 2). As such, the pajezinho ethnovariety can be confused with the tucunacá ethnovariety because neither possess nodes. However, the lineages of clade C were genetically differentiated from the others, including within the same clade (Table 3), in which it is possible to observe that the cultivated lineage (VII) resulted in a genetic distance of 3.19 and 2.79% for the native lineages V and VI, respectively.
In addition to the phenotype of the vine, which aids in the identification of ethnovarieties, the quality of the visions under the influence of ayahuasca tea is often described as an important characteristic for identification (Schultes 1986). Therefore, we must emphasize that, although the phenotype of the vine is very important for the identification of ethnovarieties, these characteristics should be used with caution. When we specifically consider the phenotype of the vine, the ethnovarieties of native lineages (lineages II, V, VI) have phenotypes that are similar to each other (Fig. S1), but are not recognized as being the same ethnovariety by members of the CEBUDV (Table S1), and are genetically different from each other.
Thus, the results presented partially unravel the enigma described by Schultes (1986), who reported the difficulty in recognizing morphological differences as a way of separating ethnovarieties from each other. In our genetic analyses, all known ethnovarieties in the traditional context of the CEBUDV were separated by genetic analyses based on the nuclear molecular marker ITS. However, the comparison of our results with analyses of ethnovarieties known in other religions and traditional peoples is important. Since the revision of the genus Banisteriopsis, there are reports of difficulties involved in differentiating species morphologically, due to the morphological floral characters being conserved (Gates 1982). Therefore, part of the botanical riddle described by Schultes (1986) remains.
All the vines analyzed are used in the preparation of ayahuasca tea; however, we still do not have morphological evidence that all the samples analyzed in fact belong to the botanical species B. caapi. Nonetheless, the phenotype of the with nodes stem of two samples (FN2 and MA1) of lineage III (clade B) is similar to the phenotype of the ethnobotanical specimen of B. caapi (formerly Banisteria caapi Spruce ex Griseb.) held in the Economic Botany Collection, at the Royal Botanic Gardens, Kew (EBC 67,428), which was collected in the Amazon by Richard Spruce in 1852 (Nesbitt 2014). These observations lead us to believe that representatives of clade B do in fact belong to the botanical species B. caapi.
The genetic distance thresholds, resulting from the ITS marker, between the lineages and ethnovarieties identified for the Banisteriopsis spp. vines were greater than we could expect within a single species and presented values of up to 28.99% (Table 3) of genetic distance (mean 14.26%). The assumption for the effectiveness of DNA barcode is that the genetic distances within the species are always smaller than the genetic distances between species, which is called the barcode gap. It is critical to determine these ranges of genetic distances between species of different taxonomic groups (Hebert et al. 2003). However, the genetic distances found in the present study for the vines used in the preparation of ayahuasca tea ( Table 3) may indicate that more than one species of Banisteriopsis is used, and not only B. caapi, as most studies mention (Gates 1982;Schultes 1984;Schultes et al. 1992). It is evident that integrative taxonomy studies are necessary to test this hypothesis.
For the piri-piri, another Amazonian species that was previously thought to be only a Cyperus articulatus Benth species used in the traditional context, four species were identified, with genetic distance values ranging from 2.7 to 4.7% (Mezzasalma et al. 2017). The genetic distance with the ITS gene for tropical trees of 49 families of angiosperms was 6.23%, which is a value that is lower than the maximum found in the present study (28.99%) for the genus of this Amazonian liana (Banisteriopsis).
Although, in the results, we did not present combinations of two markers (nuclear and plastid) in a single barcode to discriminate the ethnovarieties, it was possible to observe that this did not improve the overall performance when compared to a single marker (ITS), which has also been observed for other Amazonian angiosperms (Gonzalez et al. 2009).

Conclusion
In regards to the identified genetic lineages, the ITS presented the highest indices of genetic diversity among the markers evaluated and showed a greater correspondence between the ethnovarieties of Banisteriopisis spp. used in ayahuasca tea and recognized by the traditional knowledge of the CEBUDV. These results may be relevant for the establishment of conservation measures for the ethnovarities of Banisteriopisis spp. that have traditional and religious use. We also demonstrate that traditional knowledge is remarkable and can be tested by scientific methods. New integrative studies like this one, which combines modern approaches and traditional knowledge, should be encouraged since they contribute to the maintenance of traditional knowledge as well as the conservation of species.