Ethnobotany genomics - discovery and innovation in a new era of exploratory research

The study site (longitude 6° 40' to 7° 10' E and latitude 10° 55' to 11° 10' N) is located within the Velliangiri holy hills, which forms a major range in the Western Ghats in the Nilgiri Biosphere Reserve. The research was conducted among seven hills with altitudes ranging from 520 m - 1840 m, which is bordered by the Palghat district of Kerala on the western boundary, the plains of Coimbatore district to the east, the Nilgiri mountains to the north, and the Siruvani hills on the southern boundary.

Floristic explorations were made within respective study areas within India [18, 29, 33, 38‐41]. Collections were made from April 2004-January 2009 and included all seasons in order to collect any ephemerals or specialized phenotypes. Six collections or "specimens" from each population were collected, labelled with locations and collection numbers for of 19 Biophytum species (Figure 1) and 12 Tripogon species (Figure 2). Corresponding field data included details of the specimens (habit, flower colour, phenology and presence or absence of latex) and environmental variables (habitat, latitude, longitude, altitude, soil type and plant associations). Multiple populations were sampled along transects separated by 2 km in order to insure that we were collecting distinct populations and not vegetative colonies. This also accounted for local morphological variants within the different ecosites. The survey used is that of earlier methodologies [12, 18, 33, 41] to identify local experts in traditional botanical knowledge. We interviewed over 120 informants from which we selected 80 informants. Vouchers were collected and labelled for all taxa identified (Figure 3). The data were gathered in a series of structured, semi-structured and unstructured interviews, and participatory approach regarding plant uses, identification, and nomenclature. To elucidate cultural domains and determine differences in knowledge or taxonomy among aboriginals, a cross check was made with other aboriginal respondents by using various research protocols such as free recall lists, pile sorts, and consensus analysis.

Plant samples were collected from the aboriginal community and preserved for both herbaria and DNA barcode analysis (Figure 4). Leaf, stem and flower parts collected in situ were fixed in silica gel, FAA (50% ethanol, 5% acetic acid, 10% formalin, 35% water) and stored in 70% ethanol for morphological study ex situ. Herbarium specimens were prepared as per Jain and Rao's [42] manual and deposited in the herbarium of Kongunadu Arts and Science College, Coimbatore. The isotypes of new taxa and other taxonomically significant plant species were deposited at Madras Herbarium (MH), Southern Circle, Botanical Survey of India, Coimbatore and Ontario Agricultural College (OAC) Herbarium, Biodiversity Institute of Ontario, University of Guelph, Canada.

Calculation of a Consensus Factor (Fic), and pile sorting relative frequency (RF) was used to test homogeneity of knowledge (SK & TK) in identifying specimens, revealing cryptic taxa or limitations of the classification without the use of molecular data. Voucher samples collected from five collection sites were systematically identified by the taxonomists and aboriginal informants. The relative frequency (RF) of each specimen from the interviews were calculated to determine a quantitative value for choosing a plant name (latin binomial or aboriginal ethno-taxon) from the pool of collected vouchers and placing it in a species concept [12]. RF is the simple calculation of the percentage of specimens associated with a taxon when taxonomists or aboriginal informants are presented with a pool of vouchers and asked to perform "pile sort". Trotter and Logan [43] provide the calculation of a Consensus Factor [Fic = Nur-Nt/(Nur-1)], which is adopted to evaluate the degree of partition into categories [44]. We have adopted this to include 'aboriginal utility' by the aboriginal informants [33, 18, 39, 41], where Nur is the number of use-reports of informants for particular category (TK plant use) factor, where a use-report is a single record for use of a plant mentioned by an individual, and Nt refers to the number of species used for that particular category for all informants [18].

Three DNA regions (rbcL, matK and trnL-F) were selected based on the previous plant barcoding studies [27, 30, 35, 36]. We isolated total genomic DNA from approximately 10 mg of dried leaf material from each sample using the kit, NucleoSpin^® 96 Plant II (MACHEREY-NAGEL). Extracted DNA was stored in sterile microcentifuge tubes at -20°C. The selected loci were amplified by PCR on a PTC-100 thermocycler (Bio-Rad). DNA was amplified in 20 μl reaction mixtures containing 1 U AmpliTaq Gold Polymerase with GeneAmp 106PCR Buffer II (100 mM Tris-HCl pH 8.3, 500 mM KCl) and 2.5 mM MgCl2 (Applied Biosystems, Foster City, CA), 0.2 mM dNTPs, 0.1 mM of each primer (0.5 mM for matK), and 20 ng template DNA. Amplified products were sequenced in both directions with the primers used for amplification, following the protocols of the University of Guelph Genomics facility. Products from each specimen were cleaned using Sephadex columns and run on an ABI 3730 sequencer (Applied Biosystems, Foster City, CA). Bidirectional sequence reads were obtained for all the PCR products. Sequences were assembled using Sequencher 4.5 (Gene Codes Corp, Ann Arbor, MI), and aligned manually using Bioedit version 7.0.9. The sequences were used in combination with the morphometric analysis to produce classification trees.

Morphological data variables, were recorded for all specimen collections. A matrix of specimens and morphological characters were used in a multivariate phenetic analysis. Canonical ordination was used to detect groups of specimens and to estimate the contribution of each variable to the analysis. A cluster analysis was used to classify the specimens because it is better at representing distances among similar specimens [45]. Cluster analysis was carried out using NTSYS [46]. A distance matrix was generated from the specimens and characters using an arithmetic average (UPGMA) clustering algorithm and standardized data based on average taxonomic distance subjected to the unweighted pair-group method. The resulting distance matrix from the cluster analysis used in combination with the sequence data above to produce classification trees.

The genus Biophytum DC. (ca. 80 species, Oxalidaceae) is predominantly pantropical to subtropical in distribution [47]. Biophytum is one of only eight genera in three families of flowering plants (Lythraceae, Oxalidaceae, and Potederiaceae) that are tristylous [48]. The genus is poorly studied with limited floristic treatment in Knuth's [49] monograph of Oxalidaceae, which was later revised by Veldkamp [50]. The genus has been confused with that of Oxalis. Linnaeus described Oxalis sensitiva [51], from a neotype later classified as Biophytum sensitivum (L.) DC. Veldkamp [52] noted that the genus Biophytum appears to be first described in a treatise by Acosta [53], which later appeared with a plate in Clusius' treatment (1605) of Herba viva. A brief narrative of the historical nomenclature on Biophytum of the old world is provided by Veldkamp [52]. Veldkamp [52] states that there is no comprehensive treatment of the genus, which contains many undescribed species.

The contribution of the local aboriginal knowledge concerning variation in Biophytum within India is considerable. India has a high diversity of Biophytum; the Biophytum flora of India is currently represented by 17 species and two varieties of which four species are endemic, representing taxa that are in need of conservation status and protection [54]. Recent floristic surveys are reporting considerable diversity within protected religious areas in India, some of which preserve a significant portion of the Biophytum flora [55, 18, 33]. All 19 species and varieties of Biophytum in this study are found in the Western Ghats, which is part of Nilgiri Biosphere Reserve (NBR) in Tamil nadu. The Velliangiri hills of India are also known for their rich anthropogenic diversity. The aboriginals living in the Velliangiri hills are the "Malasars, Mudhuvars and Irulas" [11, 12, 18, 33, 41]. They have accumulated extensive ethnobotanical knowledge by their long association with their diverse, local flora [38]. In our floristic study within the Velliangiri hills we recorded 177 plants, which are used by the local people for various purposes [12, 18]. These aboriginals recognize plants of the genus Biophytum ("thottal sinungi", trnsl. 'touch me not') naming and identifying many ethnotaxa including an ecological knowledge of them [11, 12, 38]. It is this TK that provided clues to the identity of several new species [29, 55] while working with the aboriginals in the Velliangiri hills. The respective classifications of the genus Biophytum using both SK and TK are not homogeneous. Taxonomists identified taxa with 84% (RF) accuracy, while the Aboriginal informants identified the same specimens with 97% (RF) accuracy [38]. Consensus factors were high (Fic = .94-.99) and not partitioned among the Aboriginal informants. The TK classification recognizes considerable fine scale variation among Biophytum samples (Figure 1). The TK classification of Biophytum is hierarchical, employing several TK classification characters; morphology, ecology, experience, gestalt and utility including 4 secondary classification mechanisms (e.g., nutritional, medicinal, technical or ritual). Interestingly these new species corresponded to unique aboriginal taxa with respective nomenclature and medicinal use [29, 30, 12, 33, 18, 41].

DNA barcoding validated three new cryptic species to science that were previously recognized by TK classifications of the Irulas and Malasars. These species include 1) 'Vishamuruchi' (translation - detoxification of the poison; Biophytum coimbatorense sp. nov.), which is used as an antidote for poisonous scorpion bites, 2) 'Thear chedi' (translation - Chariot umbrella; Biophytum tamilnadense sp. nov) is used as a bait plant for fish and crab and 3) 'Idduki poondu' (translation - between the rock; Biophytum velliangirianum sp. nov.) is used for curing ear aches. A Classification tree from DNA barcoding sequence data (rbcL, matK and trnH-psbA + 41 quantitative variables) resolved 19 Biophytum species and varieties including the three new species (Figure 1). DNA barcoding discriminated the cryptic ethnotaxa Biophytum coimbatorense sp. nov. ('Vishamuruchi') from the morphologically similar species of B. longipedunculatum Govind. ('Thotal sinungi'). Amplifications were highly specific with a clear background in the agarose gel. Although there were no differences in the rbcL or atpF sequences for these two cryptic species, the matK and more variable non-coding spacer regions such as trnH-psbA sequences were consistently different. Several segregating sites in the matK sequences are found consistently among the five distant populations. Several other studies [30, 36] have also found that closely related species are not distinguished by several plastid regions like rbcL or atpF.

The genus Tripogon Roem. & Schult. consists of nearly 40 species in tropical and subtropical regions [56‐58]. The diversity of this genus of grass has been described thoroughly within the catalogue of world grasses by Peterson et al. [59], a revision of African species of Tripogon [60, 61], the description of new species of Tripogon from Africa [62], a summary of grass genera worldwide [56], an online world grass flora by [58], and nomenclature changes by Veldkamp [63]. Rúgolo de Agrasar & Vega [64] reported that Indo-Asia constitutes the centre of diversity for this genus, with 23 species of which 16 species are native to China and 21 species including eight endemics are native to India [29]. Most of what has been published within the Indian flora and includes three new species of Tripogon[65‐67, 29].

We recently discovered a new species of Tripogon (T. cope Newm.) during an ethnobotany genomics study in the Nilgiri Biosphere Reserve, Western Ghats, India [29]. We worked with aboriginal informants who are members of the local hill tribes (Irulas and Malasars). The informants revealed ethno taxa that we later confirmed to be a new species. The ability of our field taxonomists and the Hill Tribe informants to identify species in the genus Tripogon was high, but the respective classifications of SK and TK are not homogeneous. Our taxonomists identified seven taxa from the 40 specimens with 96% (RF) accuracy among individuals. Aboriginal informants identified eight taxa from the same 40 specimens with 98% RF among the informants. A closer investigation of the voucher samples revealed that what we called T. wightii the informants split into two distinct ethnotaxa; 'Sunai pul' and 'Kattai pul'. The TK classification of Tripogon is hierarchical, employing several TK classification characters; ecology, experience, gestalt and utility including 4 secondary classification mechanisms (e.g., nutritional, medicinal, technical or ritual). An additional TK character used to distinguish 'Sunai pull' was that it is a 'hot' plant (see discussion below).

Further research validated that the cryptic ethnotaxa 'Sunai pul' was indeed a new species. Morphometric [29] and genetic studies [33] confirmed that the cryptic ethnotaxa 'Sunai pul' (Tripogon cope Newm.) was distinct from the morphologically similar species of Tripogon wightii ('Kattai pul'). We looked at herbarium vouchers and found that the close resemblance of T. cope to T. wightii has resulted in misidentifications by taxonomists during previous botanical surveys. Although the hill tribes can easily identify these species, these cryptic species are only differentiated by minor floral characters; slight variation (1 mm) in the rachilla internodes and the number (1-3) of awns at the lemma apex. The local aboriginal classification systems species are clearly discriminated by different life cycles. We grew the plants in the greenhouse and found that 'Sunai pul' (T. cope) is an annual and 'Kattai pul' (T. wightii) is a perennial. We also used DNA barcoding to discriminate the new species (Fig 2). Our classification tree from DNA barcoding sequence data (rbcL, matK and trnH-psbA) clearly distinguished the 12 Tripogon known species from T. cope (Fig. 2). Intraspecific variation within the classification tree are recognized by the hill tribes as ethnotaxa of which 'Sunai pul' (T. cope) and 'Kattai pul' (T. wightii) are clearly differentiated. The DNA amplifications were highly specific with a clear background in the agarose gel. The matK and trnH-psbA sequences had several segregating sites in sequences that were found consistently among the distant populations. There is a gross interspecific variation (p-distance 0.00234) and no intraspecific variation among T. cope and T. wightii. Interspecific variation among all eight species ranged from (p-distance 0.002-0.003). Intraspecific p-distance was 0.00 for all regions within all eight species.