MOLECULAR SYSTEMATIC STUDY OF TWO SOLANACEOUS GENERA DATURA L. AND BRUGMANSIA PERS. BASED ON ITS SEQUENCES OF NRDNA

Dhanya C. 1 , Shabir Rather 2 and Devipriya V 3 . 1. Research & PG Department of Botany, SN College, Kollam, Kerala, South India, 2. Department of Botany, Delhi University, New Delhi, India, 3. Department of Botany, SN College, Chempazhanthy, Kerala, South India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 13 August 2018 Final Accepted: 15 September 2018 Published: October 2018 Phylogenetic analysis was performed based on ITS 1 and 2 sequences to determine monophyly of Datura and Brugmansia and to understand their relationships. The results support the splitting of Datura and Brugmansia into two distinct genera (BS 100 & 91). D. ceratocaula earlier considered as the connecting link between the two genera, is nested within the Datura clade (BS 57) as a distinct species. Although the three conventional sections of Datura viz. Dutra, Ceratocaulis & Datura (Stramonium) stand well supported (BS 100, 100 & 83), D. kymatocarpa, D. leichhardtii and D. pruinosa appear shifted from Section Dutra to Section Datura, corroborating earlier observations. D. discolor remains nested within the Section Dutra, refuting earlier suggestions as intermediary or ancestral role. But the divergence of D. discolor from the remaining taxa within the section Dutra as a clearly supported subclade (BS 100) may be suggestive of the subdivision of Section Dutra into two subsections. This subdivision of section Dutra however remains to be appraised using cladistical studies incorporating both molecular and morphological data. The infrageneric grouping of Brugmansia into cold and warm groups viz. sections Sphaecarpium and Brugmansia exclusively on the basis of fruit shape and habitat lacks adequate support in the present analysis. But fruit shape appears not consistent as the warm climatic inhabitant B. aurea with trumpet shaped flowers sports egg-shaped fruits. Hence we propose subdivision of the genus into two sections on the basis of the more stable corolline form, and the proposition is well supported by molecular data viz. section Brugmansia (with trumpet and/or funnel shaped flowers, with fruits fusiform or egg-shaped) and section Sanguinea (with tubular flowers and consistently egg-shaped or short oval fruits).

The species diversity of Datura is centred on Mexico and southwestern United States in North America (Hammer et al 1983;Mace et al 1999;Jiao et al 2002). The three species of Datura (D. inoxia, D. metel and D. ferox) that occur in South America are believed to have been introduced there in pre-Columbian times (Hunziker 1979;Nee 1991). Its occurrence in Australia and India are recent and human-mediated (Symon and Haegi, 1991). However, the Old World origin of Datura metel from the Indian Subcontinent has been advocated by Indian researchers on the basis of Indian lexical evidence, Arabic and Indic texts, and Southern Indian iconographic representations (Siklos, 1993;Geetha and Gharaibeh, 2007). The genus Brugmansia includes woody species, several of which are considered to be hybrids, unknown in the wild state.
The plants belonging to Brugmansia and Datura contain tropane alkaloids, mainly atropine and scopolamine. Several members are hallucinogenic used by indigenous people in the new world (Schultes, 1979;Dabur et al. 2004). Some are sources for the commercial production of hyoscine, an alkaloid of medicinal interest as a spasmolytic, anaesthetic and anti-motion sickness drug. Datura has been used to treat skin ulcers, arthritis, rheumatism, hemorrhoids, asthma, lung infections, etc. Brugmansia is widely cultivated as ornamentals, because of their attractive large tubular flowers. Bristol (1966Bristol ( , 1969 reported that species of Brugmansia are used for medicinal and psychotropic preparation by the Sibundoy Indians. Brugmansia is closely related to the genus Datura in both its morphology and chemistry, and was earlier considered to be synonymous with Datura. Persoon (1805) collated all arboreal perennial varieties into a separate genus Brugmansia. Bernhardi (1833) opined that the differences recorded by Persoon are not sufficient for separating out the taxon into a separate genus, and included Brugmansia as a section within the genus Datura. Lagerheim (1895) elevated Brugmansia to the status of an independent genus. This uncertainty remained for a long time until Lockwood (1973) transferred all woody members of Datura in a separate genus, Brugmansia.
Majority of the morphological characters are conserved in both, and differences between the two are mainly confined to the habit and fruit characters (Fig. 1). Datura ceratocaula which is restricted to Mexico and Central America, is a semi-aquatic hollow-stemmed prostrate creeping plant. It is considered as the connecting link between herbaceous Datura and shrubby Brugmansia due to its unique combination of characters (Bernhardi, 1833; Safford, 1921;Griffin and Lin, 2000). The plant is a herbaceous annual with erect flowers, circumscissile calyx, tetralocular ovary and carunculate seeds like Datura, but has smooth, pendulous, fleshy fruits characteristic of Brugmansia.
Although the chloroplast gene rbcL has been utilized for solving plant phylogenetic controversies at higher taxonomic levels (Chase et al. 1993;Qui et al. 1998), the nrDNA ITS region is being increasingly depended upon to resolve inter-generic and inter-specific phylogenetic concerns (Baldwin, 1992;Kron and King, 1996). This region is fast-evolving and serves as a method of direct DNA sequence analysis for detecting reticulate phenomena which would be appropriate for subgeneric or subspecific cladistic studies (Gaut et al. 2000). 1125 Bye and Sosa (2013) studied the molecular phylogeny of the genus Datura using matK, ndhF, trnH-psbA, trnL-F and morphological markers, and supported the previous recognition of Brugmansia as sister of Datura in the subtribe Datureae by Olmstead et al, 2008. The analyses also supported the recognition of Datura as monophyletic with the monospecific section Ceratocaulis as the sister to the remaining species of Datura. Mace et al. (1999) performed cluster analysis of AFLP data for seven species of Datura and six species of Brugmansia and considered the genera as sister groups. However both these studies have focused largely on the distinct generic status of Brugmansia and the subgeneric classification within the genus Datura. The present study while addressing the systematic and phylogenetic relationships between the members of the two genera Datura and Brugmansia using ITS markers, focuses more on the infrageneric interrelationships between the members of Brugmansia.

Materials and Methods:-
Taxon sampling:-Five taxa of Brugmansia and four taxa of Datura from Kerala and Tamil Nadu, India, are included in the present study. Author citations are given in Tables 1. The voucher specimens of the taxa collected have been deposited in SNCH herbarium.
DNA extraction, amplification, and sequencing:-Genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, Amsterdam, The Netherlands). DNA amplification and sequencing of the ITS region were performed using the primers ITS1 and ITS2 (White et al. 1990).
The polymerase chain reaction (PCR) was performed with standard methods using AccuPower HF PCR premix (Bioneer, Daejeon, Korea) in 20 µl volumes containing 2 µl of 10X buffer, 300 µM dNTPs and 1 unit of HF DNA polymerase. To this 1 µl of a 10 pM solution of each primer, 1 µl of genomic DNA and distilled water added to make a total volume of 20 µl. PCR amplification was performed with 40 cycles (Denaturation for 1 min at 94ºC, annealing for 1 min at 49º C, and 1 min of extension at 72ºC followed by a last cycle of final extension for 5 min at 72ºC). PCR products were checked for the presence of appropriate bands on a 0.8 % agarose gel, purified, and sequenced at Sci-Genome, Kochi. Sequences comprised of ITS1, 5.8S, and ITS2 regions. Forward and reverse sequences were edited and assembled using the computer program DNA Baser (vers. 3) (2011). All sequences have been deposited in GenBank (Table 2).

Phylogenetic analyses:-
A total of 45 nucleotide sequences (including all outgroups) were aligned using Clustal X vers. 2.0.11 (Thompson et al. 1997) followed by manual adjustments in Clustal W (Thompson et al.1994). All positions containing gaps and missing data were eliminated. Phylogenetic analyses were done using neighbour-joining (NJ) methods and RAxML (maximum likelihood, ML). ML analyses were done using RAxML-HPC BlackBox 8.0.9 (Stamatakis 2006;Stamatakis et al. 2008). The best-fit model was determined using jModel Test vers. 0.1.1 ascuel 2003) by the Akaike Information Criterion (AIC) and the maximum likelihood method; the lowest AIC score and highest log-likelihood score. Relative support for the clades recovered was assessed via bootstrap analyses using 1,000 replicates in both analyses. The following criteria were used to assess bootstrap support percentages (BP): 50-70 %, low; 71-84 %, moderate; 85-100%, strong. The results of MP and NJ analyses are congruent with those from Bayesian analyses.

Results:-
Sequence analyses: -45 accessions of 21 species were used for the analysis and out of 21 species 9 were generated new, 9 sequences consists of five Brugmansia and four Datura species. All the sequences have been deposited in GenBank (Tables 2).

Alignment:-
The ITS alignment comprises of total of 45 accessions of 21 species and has an aligned length of 636 base pairs. The ITS sequences used for analysis were having 362 (56.9%) identical sites and remaining variable and informative sites -Major variations were observed in ITS1 and ITS2, while the 5.8s region remained conserved. Mean pairwise percentage is 89.6%. Of the total sites, 3.1% consists of gap.

Maximum Likelihood analyses:-
Monophyly of both the genera (Datura and Brugmansia) is proved and is well supported (BS 100 and 91). Datura ceratocaula once considered to be a hybrid or connecting link between Datura and Brugmansia is very well placed in 1126 Datura clade (BS 57) and is grouped along with other species. The genus Brugmansia appears to be split into two with B. sanguinea and B. vulcanicola diverging out as a distinct cluster (BS 91).

Discussion:-
The results obtained from the present study support the separation of Brugmansia from Datura by earlier taxonomists, and the tree topology obtained using ITS data is also congruent to their study (Text Fig. 2). In the light of the present observations, the systematic inter-relationships between and within the two genera are discussed below.

Elevation of Brugmansia as a new genus:-
Earlier taxonomic studies based on morphological as well as phytochemical data consider Brugmansia to be synonymous with Datura (Bernhardi, 1833;Bristol 1966), the grouping being challenged by several others who advocated its elevation as an independent genus (Persoon, 1805; Lagerheim 1895). The erection of Brugmansia as a separate genus by Lockwood (1973) was generally accepted, yet doubted by some taxonomists despite it being recognized under the International Code of Botanical Nomenclature (Nee, 1991). The protein chemotaxonomy study based on amino acid sequences of ferredoxin supported the conventional classification that included Brugmansia within the genus Datura (Mino 1994(Mino , 1995. Phylogenetic study using ITS1 sequences, isozymes and morphology on 12 species of Datura  The taxonomic status of Datura ceratocaula:-Datura ceratocaula has been considered to be a hybrid of Datura and Brugmansia, owing to its unique combination of traits being a herbaceous annual with erect flowers, circumscissile calyx, tetralocular ovary and carunculate seeds like Datura and bearing smooth, pendulous, fleshy berries similar to Brugmansia. The fruits however dehisce irregularly like some species of Datura. This reticulation of characters from the two major genera in D. ceratocaula was raised as a major argument in support of the inclusion of Brugmansia as a section under the genus Datura. Consequently, D. ceratocaula was considered as the connecting link between the two genera (Bernhardi, 1833; Safford, 1921). However, later molecular studies revealed that the species is well nested within the genus Datura The three conventional sections of Datura stand well supported in the phylogenetic tree from the current study also (BS 100, 100 and 83). D. kymatocarpa, D. leichhardtii and D. pruinosa from Section Dutra appear shifted to Section Datura, corroborating the observations by Bye and Sosa (2013). The only difference is seen with regard to D. discolour, which remained stationed within the Section Dutra and yet diverged from the remaining members of the section, forming a clearly defined subclade (BS 100). The nodding, septifragal capsules of D. discolor led Barclay (1959) to consider the species as an intermediate between the sections Dutra and Stramonium (Datura). This was supported by Mace et al. (1999 and2000) from isozyme and ITS-1 analyses, who considered that D. discolor might be a recombinant from taxa of both sections or it may represent the ancestral form of the two groups. However in the present study, D. discolor was well-nested within the Section Dutra, and the molecular data do not support its intermediary or ancestral ranking. The divergence of the species from the remaining taxa within the section Dutra observed in the phylogenetic tree (Fig.2) is not sufficient enough for the erection of a new section as was proposed earlier (Mace et al. 1999). All the same it forms a clearly supported subclade (BS 100) which may be suggestive of the subdivision of Section Dutra into two subsections and this remains to be appraised using cladistical studies incorporating both molecular and morphological data.

Subgeneric classification of Brugmansia:-Hay et al (2012) divided the genus Brugmansia into two sections -Section
Brugmansia -the warm group with long, spindle shaped fruits and Section Sphaerocarpiumthe cold group with short, egg-shaped fruits. They observed that hybridization is not possible across the sectional limits. The former includes B. aurea, B. x candida, B. insignis, B. suaveolens and B. versicolor, while the latter includes B. arborea, B. x rubella, B. sanguinea and B. vulcanicola. This classification based mainly on habitat and fruit shape, ignores coralline features. But the short, egg shaped fruit of B. arborea makes it a misfit in Section Brugmansia characterized by long spindle shaped fruits. Hence fruit shape alone cannot be depended upon as a distinguishing character for subdividing the genus into sections. Haegi (1976) observed that in Brugmansia large trumpet or funnel shaped corollas are characterized by flared throats and limbs, while they were scarcely flared out in tubular corollas. Morphological observations reveal a close association between tubular corollas and short egg shaped fruits, while large flared out corollas are not linked to any particular fruit form. So when the two traits are considered together, the members of the genus diversify into two clear morphologically distinct groups -(1) a larger group including taxa having large trumpet or funnel shaped and flared out corollas along with long spindle shaped or short egg shaped fruits and (2) a second group with narrow tubular corollas and short egg shaped fruits.
The phylogenetic tree observed in our study shows a clear subdivision of Brugmansia Clade into two sublcades -a larger subclade with majority of taxa conforming to the larger morphological group based on corolline and fruit characters and a second subclade including the taxa with narrow tubular corollas and short egg shaped fruits. The two subclades stand well supported (BS 100 and 91). Hence it is proposed that the genus Brugmansia may be subdivided into two sections based on corolline and fruit characters as Section Brugmansia and Section Sanguinea, as explained above.

Conclusions:-
The present molecular analysis based on ITS 1 and ITS 2 sequences supports the splitting of Datura and Brugmansia and also establishes the distinctiveness of the two genera (BS 100 and 91). D. ceratocaula earlier considered as the connecting link between the two genera, is nested within Datura clade (BS 57) as a distinct species. The three conventional sections of Datura viz. Dutra, Ceratocaulis and Datura (Stramonium) stand well supported in the phylogenetic tree (BS 100, 100 and 83). D. kymatocarpa, D. leichhardtii and D. pruinosa from Section Dutra appear shifted to Section Datura, corroborating earlier observations. D. discolor was nested within the Section Dutra, and the molecular data do not support its intermediary or ancestral ranking, contrary to earlier molecular reports. The divergence of D. discolor from the remaining taxa within the section Dutra is not sufficient enough for the erection of a new section as was proposed earlier. The egg shaped fruit of B. arborea makes it a misfit in Section Brugmansia 1128 characterized by long spindle shaped fruits. Hence fruit shape alone cannot be depended upon as a distinguishing character for subdividing the genus Brugmansia. The phylogenetic tree observed in the present study shows a clear subdivision of Brugmansia Clade into two well supported sublcades (BS 100 and 91). Hence we propose the subdivision of the genus Brugmansia into two sections based on corolline and fruit characters as Section Brugmansia comprising majority of taxa under the genus characterized by large trumpet or funnel shaped and flared out corollas along with long spindle shaped or short egg shaped fruits and Section Sanguinea with narrow tubular corollas and short egg shaped fruits including B. sanguinea and B. vulcanicola.