1

CHAPTER 1

INTRODUCTION

India is well known for its rich heritage of natural diversity, as it intersects

four biodiversity hotspots such as Eastern Himalaya, Western Ghats, Indo-Burma,

and Andaman & Nicobar Islands. It is one among the 17 mega diverse countries

which ranking tenth in the world and fourth in Asia [1]. India harbors nearly 11% of

the world’s floral diversity which documented over 17,500 flowering plants, 6,200

endemic species, 7,500 medicinal plants and 246 globally threatened species in only

2.4% of world’s land area [2]. This is due to varied eco-climatic conditions together

with unique geography and cultural features have contributed to an astounding

diversity of habitats. India’s biogeography is diverse with ten different bio-

geographic zones [3], of which 23.4% of the land area is under forest and tree cover

(Table 1.1).

The Tropical Dry Evergreen Forest (TDEF) is part of the costal bio-

geographic zone in India that is narrowly confined to the East coast province. The

forests are regarded as national treasure that is responsible for India’s rich

biodiversity. “Forest is a unit of vegetation which possesses characteristics in

physiognomy and structure sufficiently pronounced to permit of its differentiation

from other units”. India’s forests were first classified by Sir H.G. Champion in 1936

and later revised by Champion and Seth in 1968 [4]. According to this classification,

there are 6 major types, 16 minor types and about 221 sub-types of forest reported in

India. Tropical Dry Evergreen Forest (TDEF) is one of the minor forest types

classified within the major forest type, Tropical Dry Forest (TDF). The TDEF is

relatively under-studied on aspects of structural and functional ecology, as compared

to the other forests [5].

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Table 1.1 Biogeographic zones of India

Biogeographic

ZonesBiogeographic Provinces

% of

geographical

area of India

Trans Himalaya

1A: Himalaya - Ladakh Mountains

1B: Himalaya -Tibetan Plateau

1C: Trans - Himalaya Sikkim

3.3

2.2

<0.1

The Himalaya

2A: Himalaya - North West Himalaya

2B: Himalaya - West Himalaya

2C: Himalaya - Central Himalaya

2D: Himalaya - East Himalaya

2.1

1.6

0.2

2.5

The Indian

Desert

3A: Desert – Thar

3B: Desert – Katchchh

5.4

1.1

The Semi Arid4A: Semi - Arid - Punjab Plains

4B: Semi - Arid - Gujarat Rajputana

3.7

12.9

The Western

Ghats

5A: Western Ghats - Malabar Plains

5B: Western Ghats -Western Ghats Mountains

2.0

2.0

The Deccan

Peninsula

6A: Deccan Peninsular - Central Highlands

6B: Deccan Peninsular - Chotta Nagpur

6C: Deccan Peninsular - Eastern Highlands

6D: Deccan Peninsular - Central Plateau

6E: Deccan Peninsular - Deccan South

7.3

5.4

6.3

12.5

10.4

The Gangetic

Plains

7A: Gangetic Plain - Upper Gangetic Plains

7B: Gangetic Plain - Lower Gangetic Plains

6.3

4.5

The Coasts

8A: Coasts - West Coast

8B: Coasts - East Coast

8C: Coasts – Lakshdweep

0.6

1.9

<0.1

Northeast India9A: North - East - Brahamputra Valley

9B: North - East – North East Hills

2.0

3.2

Islands10A: Islands – Andamans

10B: Islands – Nicobars

0.2

0.1

3

1.1 Tropical Dry Evergreen Forest (TDEF)

The TDEF was described as a thin belt of degraded forest occurring on the

south-eastern coastal region of peninsular India [6]. It was first recognized by Sir A.

Wimbush in 1935 the chief conservator of forest of Madras Presidency [7]. He stated

met which is semi- evergreen in character. Many of these species are quite different

from there to the west and although many individual trees are leafless during part of

hot weather much of underground and shrubs retain their leaves with the result that

we never get completely leafless appearance so characteristic of true deciduous

. This vegetation was predominantly composed of trees and

shrubs which have thick dark green foliage throughout the year.

The term TDEF was first officially applied by Sir H.G. Champion in his

classical book on Forest Types of India published in 1936 [4]. The most explicit

analysis of the forest types of India was provided by Champion and Seth in 1968 on

the basis of structure, physiognomy and floristic diversity. These authors were

basically foresters and they had broadly treated all Indian forests under six

categories, viz. Moist tropical forests, Dry tropical forests, Montane sub-tropical

forests, Montane temperate forests, Sub-alpine forests and Alpine scrub [4,8].

Among these the dry tropical forests have three distinct subtypes: tropical dry

deciduous forests, tropical thorn forests and tropical dry evergreen forests (Table

1.1.1).

Most of the world's tropical and subtropical broadleaf forest trees have

tendency to lose their leaves during the dry season to conserve moisture. However,

the TDEF retain their leaves year round hence, named as TDEF. The vegetation of

TDEF occupies different forms of elements includes trees, shrubs, lianas, epiphytes,

herbs, and tuberous species. Blasco and Legris [9] in 1972 reported that, there were

approximately 500 dicotyledonous species, including aquatic, mangrove and

terrestrial species in the whole region. Meher-Homji [10] in 1974 claimed that there

was a total of only 266 species in the TDEF region and contained over 160 woody

species. At present, it is estimated that the TDEF contains about 1,500 species and

4

over half of these species have a medicinal use, timber use and other cultural or

religious uses. Champion and Seth [4] was described TDEF as low forest of trees

with 9–12 m high that forms a complete canopy and they usually grow in lateritic

and sand dune soils. This forest comprised of several evergreen, semi-evergreen and

deciduous species. According to this scheme of classification, TDEF are under type 7

and they include subtypes of ‘typical dry evergreen forest’ (7/C1) and ‘tropical dry

evergreen scrub’ (7/DS1).

Table 1.1.1 Types of forests in India

Major Types Minor TypesArea

(m ha)

% of

forest

area

Moist Tropical Forest

Tropical Wet Evergreen Forest

Tropical Semi-Evergreen Forest

Tropical Moist Deciduous Forest

Littoral and Swamp Forest

4.5

1.9

23.3

0.7

5.8

2.5

30.3

0.9

Dry Tropical Forest

Tropical Dry Deciduous Forest

Tropical Thorn Forest

Tropical Dry Evergreen Forest

29.4

5.2

0.1

38.2

6.7

0.1

Montane Subtropical

Forest

Subtropical Broadleaved Hill Forest

Subtropical Pine Forest

Subtropical Dry Evergreen Forest

0.3

3.7

0.2

0.4

5.0

0.2

Montane Temperate

Forest

Montane Wet Temperate Forest

Himalayan Moist Temperate Forest

Himalayan Dry Temperate Forest

1.6

2.6

0.2

2.0

3.4

0.2

Sub-Alpine Forest Sub-alpine Forest - -

Alpine ScrubMoist Alpine Scrub

Dry Alpine Scrub

3.3

-

4.3

-

5

The ‘typical dry evergreen forest’ was treated under sub-type 7/C1 which are

dominated by trees like Manilkara hexandra, Memecylon umbellatum, Diospyros

ferrea, Chloroxylon sweitenia, Albizzia amara and other evergreen species. Further,

the sub-type 7/DS1 were described as ‘tropical dry evergreen scrub’ which are

dominated by Memecylon edule, Ziziphus xylopyrus, Dichrostachys cinerea, Psydrax

dicoccos, Carissa spinarum, Albizzia amara, Buchanania axillaris, Dodonea viscosa

and other species.

The phyto-sociological classifications of the TDEF vegetation were also

described by several authors as, the Manilkara hexandra series [11], the Manilkara

hexandra – Drypetes sepiaria – Chloroxylon swietenia – Memecylon umbellatum

series [12], the Manilkara hexandra – Memecylon umbellatum – Drypetes sepiaria –

Pterospermum suberifolium – Carmona microphylla facies of the Albizia amara

community [13], the Manilkara hexandra – Chloroxylon swietenia vegetation type

within the Albizia amara zone [14] and the Memecylon edule – Atalantia

monophylla series [15].

However, to distinguish this special forest type of the Coromandel Coast

region from the other forest types, the physiognomic term “dry evergreen” was

retained and officially classified as “Tropical Dry Evergreen Forest” by Champion

and Seth. After a span of 47 years, even today this classification is still followed by

Indian Council of Forestry Research Education (ICFRE) for conservation and

management purposes [8].

1.2 Distribution of TDEF

The TDEF type is considered as rare and unique and as no unified features;

but they are chosen based on local climatic, biotic and edaphic factors [5]. These

factors largely influence the forests physiognomy, stand structure, species

composition, and dynamics. The species of this region were highly variable in terms

of height depending on site location, soil type and the level of human impacts. The

TDEF has restricted global distribution which occurs in some parts of Tropical

America, Africa and Asia.

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The systematic review made through wide survey of literatures showed the

occurrence of TDEF, either as a vegetation formation or as a forest type in the world.

The distribution of dry evergreen forest is given in Table 1.2.1.

Table 1.2.1 Distribution of TDEF in the world

TROPICS LOCATION REFERENCES

America

Antigua

Bahamas

British Guiana

Jamaica

Trinidad

Tobago

[16]

[17]

[18]

[19]

[20]

[21]

AfricaEthiopia

Tanzania

Zambia

[22]

[23]

[24]

AsiaThailand

Sri Lanka

INDIA

[25]

[26,27]

[4,28,29]

In India, TDEF occurs as a thin patch along the Coromandel Coast of

southern India. Historically, the forest extended from Visakhapatnam in Andhra

Pradesh to Ramanathapuram in Tamil Nadu as a belt of vegetation about a length of

1,800 km and a width of about ca. 60 km and it covers a total area of ca. 1,08,000

km2 [30,31]. It is found in the rain shadow of the Western Ghats and Eastern Ghats.

It is considered as one of the ecoregions in India that is home to number of cities,

including metropolis of Chennai, Pondicherry, Thanjavur, Kanchipuram and Nellore.

The TDEF on the Coromandel Coast of India, which occur as patches, are short-

statured, largely three-layered, tree-dominated evergreen forests with a sparse and

patchy ground flora [32].

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The TDEF is composed of several indigenous species which are scatteredly

distributed along the Coromandel Coast in three different habitats such as (1) Sacred

groves, (2) Reserve forests, and (3) Isolated hillocks.

1.2.1 Sacred groves

Sacred groves are forest fragments of varying sizes, which are conserved by

local communities based on religious belief, taboos and social sanctions that have

cultural and ecological implications [33,34]. Sacred groves are the home to native

keystone species of plants and animals which represent mini-biosphere reserve,

making them an essential part of the conservation process. There are about 448

scared groves reported in Tamil Nadu and their sizes ranging from less than an acre

to little more than ten acre [35]. Most of the TDEF patches are considered as scared

groves and are still preserved as a result of the religious belief of the local people

[36] (Figure 1.2.1.1). It is believed that one of the prime utilities of sacred grove is

the protection and supply of medicinal plants. It is usually occurs in red lateritic and

clayey soil when intact acts as an effective sponge for the monsoon rains that are

characteristic of the area.

The natural vegetation is dominated by large number of evergreen, and semi-

evergreen tree species such as, Atalantia monophylla, Aegle marmelos, Alangium

salviifolium, Garcinia spicata, Diospyros chloroxylon, Phoenix pusilla, Calophyllum

inophyllum, Strychnos potatorum, Suregaeda angustifolia, Pterospermum canescens,

Pamburus missionis, Mimusops elengi, Lepisanthes tetraphylla, Syzygium cumini,

Diospyros ferrea, Wrightia tinctoria. Considerable areas of TDEF have long been

significantly degraded and fragmented [37] and nearly 80% of the remnants are

conserved as sacred grove [38].

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9

1.2.2 Reserve forests

Reserve forests are protected on the basis of judicial and various legal

constitution of India. The term reserved forest was used to designate protected forest

areas in India, under the Indian Forest Act, 1927. The vegetation of reserve forests

are usually undisturbed and occur in the clayey, red lateritic and alluvial soil.

Plantation of native tree species and other deciduous species were done by forest

department to enrich the vegetation (Figure 1.2.2.1). It supports evergreen, deciduous

and thorny trees includes Acacia planiformis, Acacia auriculiformis, Acacia

chundra, Butea monosperma, Hardwickia binata, Helicteres isora, Holoptelea

integrifolia, Terminalia bellirica, Pterocarpus santalinus, Dichrostachys cinerea,

Catunaregam spinosarum, Euphorbia antiquarum, Flaucortia indica, Ziziphus

xylopyrus.

1.2.3 Isolated hillocks

A hillock or knoll is a small hill usually similar in their distribution and size

to small mesas or buttes. Several patches of TDEF vegetation found as isolated

hillocks and are scatteredly found along the Coromandel Coast (Figure 1.2.2.1).

Many places it is considered as the megalithic burial sites that indicates the ancient

human colonization and are protected by Archaeological survey of India [31].

Isolated hillocks are usually consists of gravelly and red lateritic soil and the run-off

from the hillocks during the rainy season enriches the soil as well as surrounding

place. The vegetation comprises of dense bushes and scattered stunted trees. Species

such as Buchanania axillaris, Manilkara hexandra, Premna corymbosa, Bauhinia

racemosa, Ziziphus mauritiana, Psydrax dicoccos, Premna mollissima, Catunaregam

spinosa, etc are commonly found in the hillocks. It also supports large number of

animals include mammals and reptiles.

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1.3. Characteristic features of TDEF

The TDEF is floristically distinguished by a fair representation of

characteristic and preferential species, exclusively or mostly confined to this

vegetation type. The preferential tree species such as Atalantia monophylla, Lannea

coromandelica, Lepisanthes tetraphylla, Manilkara hexandra, Memecylon

umbellatum, Psydrax dicoccos, Pamburus missionis, Sapindus emarginatus,

Putranjiva roxburghii, Dolichandrone falcata, Buchanania axillaris, etc signifies

this TDEF vegetation [5,32,38]. Species of climbing habits and liana such as

Combretum albidum, Ventilago maderaspatana, Grewia orientalis, Hugonia mystax,

are also commonly found in this vegetation. Several such species are dominated in

sacred groves that are conserved by local people based on the religious belief.

The key characteristics of TDEF are,

Mostly TDEF occurs in patches and as sacred groves

They extend from the coast to at least 60 km inland

The vegetation is approximately varies between 9-12 m and forming a

complete canopy

Canopy forming trees are coriaceous-leaved, evergreen and posses short

trunks and spreading crown

Undergrowth of the canopy also possess similar features

They occur in climatically dry areas and dry season may extend from three to

six months

It receives an annual rainfall of 900 mm to 1500 mm per year

Little monotypic dominance

Deciduous species, thorny shrubs and climbers are common

Numerous species with climbing habit

Liana is common

Bamboos sparsely represented

Presence of infrequent grass

12

Physiognomically, evergreen species dominates the forest that forms strong

association between the qualitative reproductive traits, pollination and dispersal

spectrum exists among the TDEF species. Ecological niche of the emergent canopy

tree is more suitably filled by deciduous species which can tolerate the drying

atmosphere of the upper canopy better than the evergreen species. It is also contains

mixture of brevi-deciduous and semi-evergreen tree species.

The climate pattern of the TDEF area is significant in determining the

vegetation forms and it is influenced by rainfall of winter season, dry months and

dew that falls between September and April [39,40]. The climate is distinguished by

its inconstancy rainfall pattern that varies in intensity, amount, and distribution both

within and between years. A dissymmetric rainfall regime of rainy season is from

October to January and over 50% of the annual rainfall could fall in these months.

The number of rainy days during October and November vary between 2 and 21

days. The intensity of the rain may also reach 100 mm in a day. Such inconsistency

in the rainfall pattern will inevitably have effects on the vegetation of this region.

The average rainfall varies greatly, but an approximate range could be described as

900- 1500 mm per year [14,40]. It is 2.5 times more that of June to September. The

dry season may extend from January to March or from December to May and is

limited to six months based on the geography.

1.4 Ecological Importance of TDEF

The TDEF are ecologically important because of their unique biotic

communities [25]. The TDEF ecosystems are home to local flora and fauna that

represent a mini-biosphere reserve. It is considered as one of the ecoregions in India

and has evolved as an important reservoir of biological diversity. The TDEF provides

several ecosystem services such as soil conservation, water conservation, seed

preservation, and carbon sequestration [35]. It is an important refuge for rare,

endangered, endemic and threatened medicinal plants (Figure 1.4.1).

The forest with its dense and evergreen characteristic is an excellent

conservator of soil, and when intact acts as an effective sponge for the monsoon rains

13

that are characteristic of the area. In watershed management the forest is very

effective, particularly due to its evergreen nature, maintaining a constant ground

cover that breaks up the rain’s impact. The soil seed banks in TDEF play an

important role in the natural environment of ecosystems. The increase of species

richness in a plant community due to a species-rich and abundant soil seed bank is

known as the storage effect.

The components of TDEF form a complex diverse habitat that is home to a

myriad of animal species such as birds, insects, reptiles and mammals that help to

control the pest population in the agro-ecosystem and promote regeneration of tree

species by dispersing seeds [6,39]. It also facilitate cross pollination of many tree

species that play a significant role in balancing the natural ecosystem. Apparently,

69% of the trees in the coastal forests are dispersed by jackals, civets, bats and

rodents.

Figure 1.4.1 Ecosystem services of TDEF

14

1.5 Economic importance of TDEF

The TDEF biodiversity provides a variety of goods and services to the user

for generating use values. The values are further divided into Timber Forest Product

(TFP) & Non-Timber Forest Product (NTFP) value [41].

1.5.1 Timber Forest Product (TFP)

Several tree species occurring in TDEF area provides wood as source of

timber that are utilized for commercial commodities like building purposes,

agricultural implements, ply wood, packing cases, furniture, musical instruments,

railway sleepers etc. Some of the highly valued timber trees [42] found in the TDEF

is listed in Table 1.5.1.1.

Table 1.5.1.1 High value timbers from TDEF

Botanical Name Family English Name Tamil Name

Albizia lebbeck Fabaceae Lebbek Tree Vagai

Dalbergia sissoo Fabaceae Indian rosewood Sissoo

Eucalyptus tereticornis Myrtaceae Eucalyptus Thailamaram

Gmelina arborea Verbenaceae Gamhar Marakumizh

Holoptelea integrifolia Ulmaceae Indian Elm Aaya

Pterocarpus santalinus Fabaceae Red Sandal Wood Senchandanam

Santalum album Santalaceae Sandalwood Sandhanam

Swietenia macrophylla Meliaceae Mahogany Magogani

Syzygium cumini Myrtaceae Jamon Tree Naaval

Tamarindus indica Fabaceae Tamirind Tree Puliya maram

Tectona grandis Lamiaceae Teak Thekku

Thespesia populnea Malvaceae Portia Tree Poovarasu

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1.5.2 Non-Timber Forest Product (NTFP)

The NTFP are obtained from the TDEF that does not require harvesting of

trees. These commodities include medicinal plants, foods, fibres, fuel wood, fodder

etc. The TDEF contains several medicinal that are highly traded as raw drugs for

their medicinal value. According to the National Medicinal Plants Board (NMPB) of

India, about 1,289 raw drugs derived from 960 plant species are actively traded in

India to serve domestic as well as international markets [43]. These medicinal plants

raw drugs obtained from plant parts (leaves, stem bark, seeds, roots etc) are widely

used in the preparation natural health products. It is also reported that over 80% of

the medicinal plant raw drugs are collected from the wild with the help of local

farmers or collectors [43]. Some of the highly traded medicinal trees are listed in

Table 1.5.2.1.

Table 1.5.2.1 High trade value medicinal tress from TDEF

Botanical Name Family English Name Tamil Name

Alstonia scholaris Apocynaceae Indian Devil tree Ezhilai palai

Cassia fistula Fabaceae Golden Shower Manja konnai

Ficus religiosa Moraceae Cluster Fig Arasmaram

Helicteres isora Malvaceae Indian screw tree Valampuri

Mimusops elengi Sapotaceae Indian Medaller Mahizhamaram

Pongamia pinnata Fabaceae Indian beech Pungai Maram

Strychnos nux-vomica Loganiaceae Strychnine tree Etti

Terminalia bellirica Combretaceae Belliric Myrobalan Thanrikkai

Wrightia tinctoria Apocynaceae Dyers's Oleander Vetpalai

Zizyphus xylopyrus Rhamnaceae Woody fruite jujube Kottaiilanthai

Butea monosperma Fabaceae Flame of the Forest Kattu Thee

Strychnos potatorum Loganiaceae Clearing Nut Tree Thetthan Kottai

16

Besides, several exotic species are represented in TDEF that are planted

artificially for various purposes such as for horticulture value (Anacardium

occidentale, Manilkara zapota and Psidium guajava), commercial value (fiber/cotton

-Ceiba pentandra), green manure (Delonix elata and Leucaena latisiliqua),

beautification (Delonix regia, Sterculia foetida, Kigelia africana, Nerium oleander,

Tecoma stans), medicinal value (Bixa orellana), or traditional reasons (Cocos

nucifera). These trees are planted either by forest department or by some social

agencies for the benefit of forest to increase the green cover and to enrich the

vegetation. Several introduced species are naturalized and regenerated through seed

dispersal mainly by birds and bats within the TDEF.

1.6 Major threats to TDEF

It is reported that illegal logging is particularly high in tropical countries like

Cameroon 50%, Brazil 80%, Indonesia >90% and Cambodia >90% [44]. The TDEF

is vulnerable in India because of their very narrow geographic boundary, which is

under considerable development pressures. The tree species with timber and

medicinal value are quickly disappearing in TDEF due to overexploitation of forest

resources. Earlier study has reported that the impending threat to the rich native

biodiversity in the TDEF is partly due to its inherent abundance of natural resources

[5,30]. Human activities such as dead wood collection, biomass gathering, lopping of

tender branches and green leaves for cattle’s, creation of footpaths, grazing, mining

of sand and clay, brick-making and collection of wild fruits and vegetables and

indiscriminate collection of plants for medicinal use are greatly affecting the

biodiversity of the TDEF. Increased economic activities along coastal regions have

led to severe fragmentation of these forests and threaten the gene pool of a unique

forest ecosystem.

Moreover, tree species of timber and medicinal value are more concentrated

in Tropical Dry Forest (TDF) and TDEF is one of the minor forest types of TDF

which is greatly affected by commercial harvesting of timber. Illegal logging of

17

timber is become a routine occurrence and considered to be a major problem in the

conservation of TDEF biodiversity. Illegal trade of timber is not only reducing the

economy of the country but also affects the ecological balance of the forest. Besides,

invasion of exotic species has become serious problem in the TDEF. The domination

of the exotic species often threatens and depletes the native tree species.

1.7 Need for conservation strategies in TDEF

Tropical forests are being degraded at a fast pace and it is estimated that over

11,000 tree species may facing a direct risk of extinction [45,46]. Thus, large-scale

biodiversity inventories are critically needed in order to develop informed

conservation strategies for these diverse ecosystems. The TDEF is an endangered

forest type in India, therefore conservation strategies are greatly demanded in order

to conserve this valuable ecosystem [29,47]. Notable scientists have reported that the

impending threat to the rich native biodiversity in TDEF of India is partly due to its

inherent abundance of natural resources [29,32]. The TDEF needs to be given high

priority for natural resource planning strategies that conserve biodiversity as

envisioned in National Environment Policy. Quick and reliable documentation of tree

species is essential in order to conserve the natural resources within TDEF. Plant

taxonomists with botanical field experience are very much limited for the reliable

identification of the tree species in TDEF. Moreover, the tree species are not easy to

identify when the specimen is incomplete, damaged or derived from plant parts such

as leaves, roots, bark, wood and seeds. Currently, it is very much difficult to monitor

the illegal trade of rare, endemic, threatened and medicinal tree species. Species

identification by conventional taxonomy is often challenging in many cases due to

the lack of experienced experts.

Limitations of conventional taxonomy includes,

Species identification often requires reproductive characters (Flowers &

Fruits) that are not easy to obtain from tree species due to seasonal flowering

patterns and many of them are large trees

18

Accurate species level identification requires considerable amount of time

Tree species can be incorrectly identified due to variability in the

morphological characters used for species identification

Morphological keys could be used only by the experts and an inexperienced

person may not correctly identify a species

Seedlings and young plants are mostly difficult to identify

Medicinal plant raw drugs is in incomplete or damaged form and derived

from dried plant parts such as leaves, roots, bark, and wood are also difficult

to identify

Other methods include advanced microscopy, and chemotaxonomy was

showed only limited success in the species identification. Limitations of these

methods are owing to the involvement of complex chemistries, lack of unique

compounds, influence of environmental factors, plant’s age and geographical

variations. Therefore, it is necessary to utilize an alternate method for species

identification.

DNA barcoding is emerging as a valuable tool for quick assessments of

biodiversity that provides high quality data for developing conservation strategies

[48,49]. Large scale DNA barcoding studies are very much limited in bio-diverse

countries like India. In order to speed up the species inventories, DNA barcoding

should be utilized to promote conventional method of species identification.

1.8 DNA barcoding a promising tool for plant species identification

The emerging molecular technology known as DNA barcoding was proposed

by group of scientists lead by Paul Hebert at University of Guelph in Canada in

2003. The team discovered the use of DNA fragment from mitochondrial gene as a

universal ‘identification’ marker for animal species [50,51]. They targeted CO1 gene

(Cytochrome oxidase gene) as a DNA barcode system to identify animal life from

658 base pair fragment of the mitochondrial gene. This region has successfully been

19

implemented in DNA barcoding studies discriminating between species in 95% of

the cases [51,52].

Though the mitochondrial genome has a rapidly changing gene structure,

plant mitochondria has very little variation in most genera [53,54]. It transfer genes

between the nuclear, plastid and mitochondrial genomes in the angiosperms and

estimated over 1,000 previous horizontal transfer events of the CO1 gene. For these

reasons the mitochondria CO1 gene is unsuitable as a source for DNA barcoding in

plants. Therefore, the search for DNA barcodes for plants starts with chloroplast and

nuclear genome.

1.8.1 Characteristics of DNA barcodes

The selection of plant DNA barcoding region involves choosing one or a few

standard loci that can be sequenced routinely and reliably in very large and diverse

sample sets, resulting in easily comparable data which enable species to be

distinguished from one another. It is necessary for DNA barcode should possess

certain characteristics for using them as universal marker [55,56].

The ideal characteristics of DNA barcoding region is listed below,

DNA barcode should be a conserved region

DNA barcode should provide low intra-specific and ample inter-specific

variation

DNA barcodes should be short, universally and easily amplifiable across all

taxa

DNA barcode sequences should align readily and contain a limited number of

INDELS

20

1.9 Plant DNA barcoding

The focus for choosing a universal plant DNA barcode has began on

chloroplast because of high copy number and easy recoverability. Several coding and

non-coding regions were screened for the species identification of plants. The

historical overview of the search for a plant barcode is shown in Figure 1.9.1

(retrieved from Hollingsworth et al. [57]). The colours in figure represent an

informal measure of enthusiasm among DNA barcoding researchers in the

systematics community for CBOL and iBOL adoption of different markers [57]. The

dashed lines indicate the year of three international barcoding conferences in London

(2005), Taipei (2007) and Mexico City (2009). Based on the various studies different

markers were considered as barcodes for plant barcoding [58-66].

The use of DNA barcodes for species identification has led to the

establishment of an international initiative, the Consortium for the Barcode of Life

(CBOL) to develop and promote DNA barcoding. The CBOL established the Plant

Working Group (PWG) which joins researchers from the 50 countries and 6

continents. The main objectives of this group were to establish a suitable gene region

for DNA barcoding based on the multi-national studies, and to address the problems

of DNA barcoding. In 2009, CBOL plant working group officially announced rbcL

and matK marker as core DNA barcode for plant barcoding [54].

21

Figure 1.9.1 Screening of DNA barcodes from chloroplast and nuclear

genome. The symbol represents CBOL recommended marker. Retrieved

from Hollingsworth et al. [57]

22

1.9.1 rbcL gene

The rbcL (ribulose-bisphosphate carboxylase) is part of a large subunit of the

RuBisCO protein in land plants. This protein consists of eight small subunits and

eight large subunits which are encoded by a single gene in the chloroplast [67].

RuBisCO is involved in photosynthesis and interacts with its substrates CO2, O2 and

ribulose 1,5 bisphosphate (RuBP) [67].

Figure 1.9.1.1 Schematic representation of rbcL gene (not drawn to scale) [68]Boxes denotes the coding regions, and the connecting lines represent intronregions

The coding region rbcL is situated between the atpB and trnR regions of

chloroplast genome [68] (Figure 1.9.1.1). The rbcL barcode consists of a 600 bp

region at the 59 end of the gene, located at bp 1–600 in the complete Arabidopsis

thaliana plastid genome sequence (gi 7525012:54958–56397). The rbcL sequences

have been used in various systematic studies and phylogenetic tree analysis [67,69].

1.9.2 matK gene

The chloroplast maturase K gene (matK) is situated within an intron of the

trnK gene (Figure 1.9.2.1) [70]. The gene is approximately 1,535 bp long and

encoded for group II intron maturase [71]. The matK barcode region consists of a ca.

841 bp region at the center of the gene, located between bp 205–1046 in the

complete A. thaliana plastid genome sequence (gi 7525012:2056–3570). Only a 600-

800 bp region of the matK gene are utilized for DNA barcoding [72].

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The matK gene evolves three times faster than rbcL [73] and several studies

suggest it can effectively discriminate between species in the angiosperms. It is also

widely used in many systematic studies.

Figure 1.9.2.1 Schematic representation of matK gene (not drawn to scale) [70,71,73] Boxes denotes the coding regions and the connecting lines representintron regions and intergenic spacer

The choice of rbcL and matK as a core DNA barcodes was based on the

straight forward recovery of the rbcL region and the discriminatory power of the

matK region. However, species level identification with rbcL and efficient PCR

success of matK are limited in many plant groups [57].

1.9.3 Multigene tiered approach

There is no single DNA barcode that can perform well for barcoding plants. It

is generally agreed that the multigene tiered approach would perform better than the

single maker in discriminating plant groups [54,64,74,75]. Newmaster et al. [75] and

Purushothaman et al. [76] described this as the multigene tiered approach wherein

barcodes are constructed from two ‘tiered’ gene regions; an easily amplified and

aligned region is used for the first tier (rbcL) that acts as a scaffold on which data

from a more variable second-tier region are interpreted for species identification.

The chloroplast rbcL was proposed as the first tier marker because of its

universality and demonstrated success for differentiating congeneric plant species

[58,63]. The second tier variable marker may be chloroplast trnH-psbA (non-coding)

and matK (coding) or nuclear ITS2. The rbcL+matK combination were reported to

give better discrimination than other marker, and the combination were gave

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appreciably greater species resolution than the other combination [57]. These coding

regions have easy alignable nature of the data that also facilitates character based

analyses and comparative analyses of DNA barcode diversity among taxonomic

groups and geographical regions. Other studies also supported the importance of

using combined analyses which increases the species identification success

[61,64,77].

1.10 DNA barcoding studies on tropical, subtropical and temperate forests

DNA barcoding has been used in many botanical studies ranging from

detailed study on single genus to ecosystem level surveys in tropical, subtropical and

temperate forests. DNA barcoding of 1,073 trees from tropical forest of French

Guiana suggested that it could increase the quality and the speed of biodiversity

surveys [77]. DNA barcoding was found to be useful for detecting errors in

morphological identifications and increased the identification rate of juveniles from

72% to 96%. DNA barcoding of 200 accessions from tropical forest plots in

Northeast Queensland also showed that it could rapidly estimate species richness in

forest communities [78]. Tripathi et al. [79] have studied 300 specimens from

tropical trees of North India, and suggested that DNA barcoding will be useful in

large-scale biodiversity inventories.

Vegetation surveys in four equally sized temperate forest plots in the Italian

pre-alpine region of Lombardy, Valcuvia by morphological identification and DNA

barcoding revealed that the later could save time and resources [80]. Parmentier et al.

[81] have assessed the accuracy of DNA barcoding in assigning a specimen to a

species or genus by studying 920 trees from five lowland evergreen forest plots in

Korup and Gabon, Africa. DNA Barcoding was found to be useful in assigning

unidentified trees to a genus, but assignment to a species was less reliable, especially

in species-rich clades. In a large study that included 2,644 individuals representing

490 vascular plant species, mostly from the Canadian Arctic zone, again showed that

DNA barcoding differentiated the taxa more at the genus level than at the species

level [82].

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In another interesting study of tropical forest, DNA barcoding was applied on

1,035 samples representing all the 296 species of a Forest Dynamics Plot on Barro

Colorado Island in Panama [83]. DNA barcode data from rbcL, matK and trnH-psbA

were found to be sufficient to reconstruct evolutionary relationships among the plant

taxa that were congruent with the broadly accepted phylogeny of flowering plants.

The same research group studied another Forest Dynamics Plot in the

Luquillo Mountains of Northeast Puerto Rico that encompassed a mix of old growth

and secondary forest that has been largely free from human disturbance since the

1940s. This study again reinforced the congruence of the barcode phylogeny with the

phylogeny of flowering plants as per APG III classification [84]. DNA barcoding

was also used to construct community phylogeny in order to understand the patterns

of species occurrence in forest habitats [85].

Community phylogeny which was constructed for the Dinghushan Forest

Dynamics Plot in China by sequencing rbcL, matK, and trnH-psbA loci from 183

species showed that closely related species tend to prefer similar habitats. The

patterns of co-occurrence within habitats are typically non-random with respect to

phylogeny. While phylogenetic clustering was observed in valley and low-slope,

phylogenetic over-dispersion was characteristic of high-slope, ridge-top and high-

gully habitats. However, there is no such large scale DNA barcode study has been

carried out in rich biodiversity country of India.

1.10.1 Tree Barcode of Life (Tree-BOL)

The importance of DNA barcoding of tree species was highlighted by 4th

International DNA barcode Conference in 2012. A global initiative “Tree-BOL,”

a Tree Barcode of Life as created to DNA barcode all tree species in the world. The

primary objective of the Tree-BOL is to DNA barcode 100,000 species of trees. The

main purpose of DNA barcoding is to monitor CITES-listed trees and medicinal

plants from the illegal trade of Africa.

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1.11 Need for reference DNA barcode library

Identification of plant species is of critical importance in conservation and

utilisation of biodiversity, but this may be hindered by a lack of taxonomic expertise

[86]. Therefore, establishment of an appropriate reference DNA barcode library is

essential for reliable species identification. DNA barcode library is primarily depends

on the authentic reference sequence from well identified voucher sample. Global

search engines like GenBank and Barcode of Life Database (BOLD) are commonly

used for the purpose of species identification. However, most of the DNA barcode

sequences in the GenBank database are not linked to voucher specimen for

verification and database curation is largely left in the hands of individual users,

making it difficult to detect and remove mis-identified specimens or contaminated

sequences [57].

In contrast, BOLD database addresses this issue they contains link between

vouchers, sequences, trace files and other metadata but it is poor in species richness,

especially for plants. The database needs to expand with DNA sequences for

successful species identification. Therefore, developing regional reference DNA

barcode library with authentic DNA sequence will be helpful to resolve these issues

and give accurate species identification. At the same time, reference DNA barcode

library will be highly useful in monitoring the rare and threatened species of

particular region. Developing regional reference DNA barcode library could be

effectively used for the floristic assessments that are essential for enforcing

conservation measures.

1.12 Benefits of DNA barcoding

DNA barcoding plays a crucial role in the plant systematics which will

accelerate the discovery of many new species in near future [9]. Specimen

identification involves assigning taxonomic names to unknown specimens using a

DNA reference library of morphologically pre-identified vouchers.

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DNA barcoding is well established method, and has been shown to bring

important benefits to applications such as monitoring illegally sourced wood

products from the forest [87] and medicinal plants identification [59,75,76,88-92].

One of the major problems in wood products identification is it does not possess the

diagnostic features required for plant identification and hence reliable identification

is extremely challenging. In these cases, DNA barcoding will help lawmakers to set

enforcement and to seize the illegal wood products particularly form CITES

appendix plants.

Instead of identifying the whole plants, many cases DNA barcoding is useful

in the identification of medicinal plant parts such as dried leaves, roots, seeds,

rhizomes, fruits, and powders [89-92] although it is difficult or impossible using

traditional morphological taxonomy. DNA barcoding will be helpful to promote the

conventional taxonomy by establishing species identification from any form of the

sample. It is highly desirable for rapid species identification, since it does not rely on

morphology of the plants, not affected by the external factors and identification can

be done from live or dead tissues.

Besides, DNA barcoding can also helps to develop the biological knowledge,

increases the research interest on conservation biology and understanding the

concepts of plant evolution. It helps many professions involve making or using plant

identifications such as taxonomists, ecologists, conservationists, foresters,

agriculturalists, forensic scientists, customs and quarantine officers [93]. DNA

barcoding strategies have been employed for the verification of plant products

ranging from medicinal plants [94] to kitchen spices [95], berries [96], and tea

products [97]. Ecological applications include the identification of invasive species

[98,99], and reconstruction of past vegetation and climate from plant remains in the

soil [100]. DNA barcoding have been used to create phylogenetic trees for use in

phylogenetic community ecology [83,84].

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1.13 Objectives of the study

Major objectives of this study includes,

To assemble a reference DNA barcode library for trees and medicinal plants

occurring in TDEF

To utilize the TDEF reference barcode library for species identification of

wood samples

To utilize the TDEF reference barcode library for species identification of

medicinal plant raw drugs