Molecular Approach to the Classification of Medicinally Important Actinorhizal Genus Myrica

Indian Journal of Biotechnology Vol 12, January 2013, pp 133-136

Molecular approach to the classification Many nitrogen fixing plants are woody perennials or of medicinally important actinorhizal trees (NFTs), most of these being found in the tropics. genus Myrica Frankia, the actinomycete involved in symbiotic BNF, nodulates the roots of dicotyledonous plants Mhathung Yanthan and Arvind K Misra* belonging to 8 plant families and 25 genera, and they are called actinorhizal genera. The plant families Department of Botany, North-Eastern Hill University, Shillong 793 022, India belonging to such genera are Betulaceae, Casuarinaceae, Coriariaceae, Datiscaceae, Elaeagnaceae, Myricaceae, Actinorhizal genus Myrica, found in Meghalaya, India, has Rhamnaceae and Rosaceae. Although taxonomically some dispute with respect to its classification. The present paper diverse, the actinorhizal plants have some common reviews the status of this genus. Fruits of Myrica sp. are features. All are perennial dicots and all except commercially available in some parts of the country. It is a useful medicinal plant rich in vitamin C. Being actinorhizal, it is also Datisca, which has herbaceous shoots, are woody 2 useful in regeneration of nitrogen depleted soils. shrubs or trees . Actinorhizal plants are widely distributed and are found in every continent, except Keywords: Actinorhizal, molecular phylogeny Myrica, Antartica. They are mostly found in the temperate zone, while some species of Casuarinaceae and Nitrogen is required in large amounts as an important Myricaceae are considered tropical. In the Indian component of proteins, nucleic acids and other subcontinent, they are confined to higher altitudes in cellular constituents, and is frequently found limiting Himachal Pradesh, Jammu and Kashmir, Arunachal to the growth of the green plants. This is because of Pradesh, Sikkim, West Bengal hills, Meghalaya and the continual loss of nitrogen from soil reserves by to some extent in the coastal regions and plains. Some processes like microbial denitrification, soil erosion, of the genera commonly found in India are Alnus, leaching, chemical volatilization and removal of Casuarina, Coriaria, Elaeagnus, Hippophae and nitrogen-containing crop residues from the land. The Myrica. Actinorhizal plants are pioneers on poor replacement of soil nitrogen is brought about by either nitrogen soils including sandy and gravelly sites, adding nitrogen containing chemical fertilizers shores of streams and lakes, wetlands and exposed directly in the soil or by the activity of biological raw mineral soils. Actinorhizal plants find economical nitrogen fixation (BNF) systems. The conversion of use as timber, fuel wood, in land reclamation, biomass atmospheric nitrogen into nitrate, a form of nitrogen production and in forestry3. In western Europe, that can be metabolized into amino acids and proteins actinorhizal plants are used primarily for reclamation by terrestrial plants, is called nitrogen fixation. of industrial wastelands and for land stabilization. In the living world, major conversion of N into 2 Myrica ammonia is brought about by certain prokaryotes by a About 97 species of Linn. (Family: process called as BNF. The BNF involves two Myricaceae) are reported globally and this genus types of microorganisms: Non-symbiotic or free is widely distributed in both the temperate and living microorganisms like Clostridium, Klebsiella, sub-tropical regions with maximum species diversity Azotobacter, etc., and symbiotic microorganisms like in Africa and Boreal America (Index Kewensis, 1977- Frankia and Rhizobium. In symbiotic fixation, the 1985), and only one representative has been reported nitrogen fixed by the microorganisms is made in Australia (Myrica australiasica F. Muell). Its available to the host plant, whereas the microsymbionts, geographical distribution in India can be seen in sub- in turn, use organic compounds supplied by the plant tropical Indian Himalaya from Ravi eastwards to 1 Assam and in Khasi, Jaintia, Naga and Lushai Hills at as an energy source . Nitrogen fixing plants are key 4 constituents in many natural ecosystems in the world. altitudes between 900-1200 m above mean sea level . However, they have also been seen in higher reaches ———————— of Himachal Pradesh. Trees of Myrica grow well in *Author for correspondence: E-mail: [email protected]; [email protected] nitrogen depleted soils and are common associates of Part of Professor K B Dutta memorial lecture delivered by A K Misra pine (Pinus sp.) and oak (Quercus leucotrichophora). 134 INDIAN J BIOTECHNOL, JANUARY 2013

They are also found in mixed forests and in agricultural Himalayan tract, Khasi hills, Sylhet and Southwards and marginal lands5. up to Singapore and in the Malayan islands at an Myrica has been used variedly. Fruits of this tree altitude of 1600-2000 m above mean sea level. are used in making jams, syrups, and juices and M. esculenta is a small to moderate sized, can also be consumed raw. Bark is used in making evergreen, dioecious tree with height ranging from paper and rope. Mention of M. esculenta in the 3-15 m. Leaves are crowded and aromatic and are medicinal system of ayurveda is noteworthy. The lanceolate to oblanceolate or obovate, nearly entire or extract from the root and bark is known to be sharply serrate, obtuse or acute, almost glabrous with astringent, carminative and antiseptic, and especially resinous dots beneath. New leaves usually start a decoction of the bark is considered useful in asthma, sprouting in February-March. Flowers are minute, toothache, diarrhoea, fever, lung infection, cough, unisexual, male and female flowers are borne on chronic bronchitis and dysentery. Bark is chewed to different trees. Male flowers occur in catkin and the relieve toothache and headache6, and is also used as female flowers in axillary erect spikes. Fruits are fish poison7. Tannin extracted from the bark is used as edible and ellipsoid or ovoid drupes of the size a tanning and dyeing agent8. The oil obtained from of cherry or bigger, tubercled, reddish or cheese the flowers of Myrica is also reported to have coloured when ripe with rugose nut. They are covered medicinal effect, especially in ear-ache, inflammation with a crust of white waxy material, permeated and paralysis6. Myriconol isolated from stem bark is with brown and black spots. They ripen during reported to have lesser toxicity than related rotenone9. summer and possess a pleasant sourish sweet taste. Smith10 gave the following classification for genus Fruiting starts at the beginning of April and lasts till Myrica: Division- Magnoliophyta; Class- Magnoliopsida; the month of June. Subclass- Hamamelidae; Family- Myricaceae; Order- Interestingly, morphological diversity within Myricales. The subclass Hamamelidae comprises of M. esculenta exists with regard to fruit size, fruit Myricales, Fagales, Juglansadales, Hamamelidales, colour, leaf serration, leaf size, tree height, etc. This Urticales, Leitneriales and Casuarinales. This is a has led to confusion among various workers. Some phylogenetic grouping of orders, which is characterized workers claimed that different morphotypes of the by strongly reduced, often unisexual flowers, which plant belong to different species. One of the either lacks or produces a poorly developed perianth11. morphotypes was referred to as M. nagi14 and the Phylogenetically, the Myricales are thought to be other one as M. esculenta15. Others also claimed that most closely related to the Juglandales and the M. nagi is synonym of the species, M. esculenta16,17. Fagales11,12. The Myricaceae is considered to be an These claims were based on morphological ancient family by taxonomists, dating to the Tertiary descriptors. Therefore, in an effort to resolve Epoch of the Cretaceous Period with the living this dispute, a need was felt to study molecular members representing relics of once extensive tracts phylogeny of this genus using nucleotide sequence of subtropical forest that spread across the territory data of different morphotypes. The first molecular that is now central and southern Europe11,12. Plants of phylogenetic study on the family Myricaceae was the family Myricaceae are considered to be promiscuous done by Huguet and others18 where the molecular hosts because several species are effectively phylogeny of 13 species of the family Myricaceae nodulated by most isolated strains of Frankia. The was established based on rbcL gene and the 18S-26S base chromosome number throughout the family is ITS. Their results showed that some species of the eight, with various levels of ploidy present13. genus Myrica, such as, M. gale and M. hartwegii, and Myrica esculenta Buch.-Ham. ex D. Don (Syn. genus Comptonia belong to a distinct phylogenetic M. farquhariana Wall., M. sapida Wall., M. nagi cluster distinct from some other Myrica species. They Thunb., M. integrifolia Roxb.) belongs to the family transferred the latter Myrica species to a new genus, Myricaceae and is commonly known as ‘Soh-Phi’ in Morella. However, the taxonomy within this family Khasi, ‘Nagatenga’ in Assamese and ‘Kaiphal’ in is highly controversial because of morphological Hindi. Common name in English is Box myrtle. variation exhibited by many species. This is the only species of the genus Myrica reported Approach to both fundamental and applied to be found in India16-18. This tree is distributed in biological problems has been transformed by the the Chinese-Japanese region including the Sub- emergence of many new techniques. Amplification of SHORT COMMUNICATIONS 135

specific regions of DNA to a million fold has been M. esculenta. They considered morphotype 3 to be made possible by the polymerase chain reaction intermediate between these two species. They found (PCR) technique. This method was first invented supporting evidence in the form of projected by Kary Mullis in 198319. PCR has revolutionized secondary folding of the 5.8S rRNA, which was approaches to molecular study in many fields. similar for morphotypes 2 and 3 and different for Restriction site analysis of amplified DNA is a morphotype 1. valuable method for detecting genetic variation in some cases20. References 1 Raven P H, Evert R F & Eichhorn S E, Biology of plants, The ribosomal RNA genes family comprises of th very conserved regions (18S and 26S gene) that can 4 edn (Worth Publishers, Inc., New York, USA) 1986, pp 775. be used to infer phylogeny at higher taxonomic levels, 2 Tjepkema J D, Schwintzer C R & Benson D R, Physiology as well as more rapid evolving segments (ITS, IGS) of actinorhizal nodules, Annu Rev Plant Physiol, 37 (1986) that may be useful at the generic, specific and even 209-232. (in case of IGS) at the population level. Ribosomal 3 Chauhan V S, Generation of molecular signatures for Alnus DNA cistrons typically are located in the nucleolar nepalensis genotypes with high nitrogenase activity in symbiotic association with Frankia. Ph D Thesis, North organizing region (NOR) and may be present on Eastern Hill University, Shillong, India, 2000. several different chromosomes. The internal 4 Osmaston A E, Forest flora of Kumaon (Bishen Singh transcribed spacer (ITS) region of the 18S-5.8S-26S Mahendra Pal Singh, Dehradun, India) 1987. nuclear ribosomal cistron is one of the most popular 5 Bhatt I D, Rawal R S & Dhar U, Improvement in seed sequences for phylogenetic inference at the generic germination of Myrica esculenta Buch.-Ham. ex D. Don—A 20 high value tree species of Kumaun Himalaya, India, Seed Sci and infrageneric levels in plants . The ITS-1 and Technol, 28 (2000) 597-605. ITS-2 regions are part of the nuclear rDNA transcript, 6 Kirtikar K R & Basu B D, Indian medicinal plants, vol III but are not incorporated into ribosomes, and appear to (Bishen Singh Mahendra Pal Singh, Dehradun, India) 1984. play a role in the maturation of nuclear rRNAs, 7 Anonymous, The wealth of India—A dictionary of Indian bringing the large and small subunits into close raw materials and industrial products, Raw materials, vol 20,28 VI, L-M (CSIR Publications, New Delhi) 1962, 471-472. proximity within a processing domain . The need 8 Dhyani P P & Dhar U, Myrica esculenta, Box Myrtle, for sequence data from nuclear genome at lower Kaiphal: A promising underexploited tree crop of the taxonomic levels makes the ITS region a popular site Himalaya, Himavikas, 3 (1994) 3-8. for this study. Since the ITS region is G+C rich and 9 Rastogi R P & Mehrotra B N, Compendium of Indian prone to secondary structure, sequencing can be medicinal plants, vol I (1960-1969) (Central Drug Research Institute, Lucknow, India) 1991. difficult21. Different protocols have been used to 22-24 10 Smith J P, Vascular plant families, (Mad River Press, CA, amplify and sequence the ITS regions . Since ITS USA) 1977. sequences are present in-between highly conserved 11 Cronquist A, The evolution and classification of flowering regions of the ribosomal RNA genes, it is possible to plants (Allen Press, Inc., Lawrence, Kansas, USA) 1978. design primers that are complementary to the 12 Takhtajan A, Flowering plants: Origin and dispersal (Oliver and Boyd, Edinburgh, UK) 1969, pp 310. conserved sequence for amplification purpose. 13 Sporne K R, The morphology of Angiosperms (St. Martin's Three different morphotypes of Myrica sp. are Press, New York, USA) 1975. found in Meghalaya. Morphotype 1 trees are 14 Murray J A, Systema Vegetabilium: Secundum classes considered as M. nagi by some workers. Morphotype ordines genera species cum characteribus et differentiis, 2 trees are considered as M. esculenta. Morphotype 3 Editio decima quarta, edn 14 (Typis et impensis Jo. Christ, trees have not been described separately and may Dieterich, Gottingae) 1784, 884. 15 Buchanan-Hamilton ex D. Don, Prodr. Fl. Nepal. 56. 1825. have been considered as variants of morphotype 2 16 MacDonald A D, The morphology and relationships of the trees. As stated above, some workers consider all Myricaceae, in Evolution, systematics and fossil history of these three morphotypes as members of the species M. the Hamamelidae, vol 2: Higher Hamamelidae, edited by esculenta. Recently, Yanthan and others25 used P R Crane & S Blackmore, Syst Assoc Spl Vol No. 40B ribosomal operon to develop a system for ascertaining (Clarendon Press, Oxford, UK) 1989, 147-165. 17 Haridasan K & Rao R R, Forest flora of Meghalaya, vol II. the boundaries of species and genera. Based on a Caprifoliaceae to Salicaceae (Bishen Singh Mahendra Pal scale developed by them, they have proposed that Singh, Dehradun, India) 1987, 851-852. morphotype 1 trees indeed should be classified as M. 18 Huguet V, Gouy M, Normand P, Zimpfer J F & Fernandez M nagi, while morphotype 2 trees be classified as P, Molecular phylogeny of Myricaceae: A reexamination of 136 INDIAN J BIOTECHNOL, JANUARY 2013

host-symbiont specificity, Mol Phylogenet Evol, 34 (2005) 22 Baldwin B G, Phylogenetic utility of the internal transcribed 557-568. spacers of nuclear ribosomal DNA in plants: An example 19 Mullis K B & Faloona F, Specific synthesis of DNA in vitro from the compositae, Mol Phylogenet Evol, 1 (1992) 3-16. via a polymerase-catalyzed chain reaction, Methods Enzymol, 23 Wen J & Zimmer E A, Phylogeny and biogeography of 155 (1987) 335-350. Panax L. (the Ginseng Genus, Araliaceae): References from 20 Varghese R, Chauhan V S & Misra A K, Hypervariable ITS sequences of nuclear ribosomal DNA, Mol Phylogenet spacer regions are good sites for developing specific PCR- Evol, 6 (1996) 167-177. RFLP markers and PCR primers for screening actinorhizal 24 Soltis D E & Kuzoff R K, Discordance between molecular symbionts, J Biosci, 28 (2003) 437-442. and chloroplast phylogenies in the Heuchera group 21 Baldwin B G, Sanderson M J, Porter J M, Wojciechowski (Saxifragaceae), Evolution, 49 (1995) 727-742. M F, Campbell C S et al, The ITS region of nuclear ribosomal 25 Yanthan M, Biate D & Misra A K, Taxonomic resolution of DNA: A valuable source of evidence on angiosperm actinorhizal Myrica sp. from Meghalaya (India) through phylogeny, Ann Mo Bot Gard, 82 (1995) 247-277. nuclear rDNA analyses, Funct Plant Biol, 38 (2011) 738-746.