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MATERIALS AND

METHODS

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MATERIAL AND METHODS

All the lichen specimens belonging to the Kumaun Himalaya, preserved in the

lichen herbarium of National Botanical Research Institute, Lucknow (LWG), were

segregated. The localities within different district earlier not explored for their lichen

wealth were listed and more than six field trips were organized for collection of lichens.

The different localities of lichen specimens collected during various field trips are listed

in tables (1-5). The unidentified specimens preserved in the herbarium, collected in past

from the study area were identified up to their species level with the help of recent

literature (Awasthi, 1988; 1991; 2000, Divakar and Upreti , 2005; S. Nayaka, 2004 and

Y. Joshi, 2008).

Table 1. Localities of Almora district, surveyed for collection of Lichens (Map 2).

S. No. Localities Altitude (m) Common vegetation

1. Binsar forest 1500–1800

Rhododendron arboreum, Qurecus gloca, Quercus leucotricophora, Pyrus pashia, Madhuca longifolia.

2. Jageshwar 1600–1800 Rhododendron arboretum, Pinus roxburghii, Betula alnoides, Quercus floribunda, Quercus leucotricophora, Alnus nepalensis.

3. Kasar Devi 1500–1700 Pinus roxburghii, Dalbergia sissoo, Engelhardtia spiicata and Quercus trees.

4. Karbla Pine forest 1400–1600 Pinus roxburghii and Rhododendron arboretum.

5. Ranikhet-Chaubattia

1800–2300

Rhododendron, Cedrus deodara, Alnus nepalensis, Betula, Quercus leucotriochophora, Quercus floribunda, Pyrus pasia.

6. Shikhar 1400–1600 Pinus roxburghii, Quercus leucotrichophora

7. Sitoli forest 1600–1800 Quercus leucotricophora, Madhuca longifolia, Dalbergia sissoo.

8. Takula forest 1500–1700 Pinus roxburghii and Quercus leucotricophora.

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PLATE 1

Different altitudinal zones of Kumaun Himalaya

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Localities in Almora district explored for lichens (A) Near Dhaulchhina (B) Around Jageshwar Temple

A

B

PLATE 2

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Localities of Bagaeshwar district (A) Pindari Glacier (B) Phurkia forest in alpine region

A

B

PLATE 3

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Table 2. Localities of Bageshwar district, surveyed for collection of Lichens (Map 3). S.

No. Localities Altitude

(m) Common vegetation

1. Dhakuri – Jatoli 2000–2800 Quercus semecarpifolia, Abies pindrow, Betula utilis and shrubs.

2. Loharkhet – Dhakuri 1700–2600

Rhododendron arboreum, Quercus leucotrichofora, Q. Semecarpifolia, Chirita pumila, Thalictrum javanicum, Stellaria semivestita and Fagopyrum cymosum.

3. Dhakuri – Khati 2100–2600 Alnus nepalensis, Rhododendron, Lyonia ovalifolia and Betula utilis.

4. Dwali – Kafni 2200–3000 Pinus roxburghii, Quercus semecarpifolia, Abies pindrow and Salix.

5. Dwali – Khati 2200–2700

Rhododendron arboreum, Pinus roxburghii, Alnus nepalensis, Cedrus deodara, Acer caudatum, Juglans regia and Quercus floribunda.

6. Dwali – Phurkia 2700–3300

Abies pindrow, Rhododendron, Taxus baccata, Betula utilis, Polygonum polystachyum, Lonicera alpigena and shrubs.

7. Bogudiyar – Naher Devi 2400–2700 Quercus semecarpifolia, Quercus leucotrichofora and Rhododendron arboretum.

8. Phurkia – Zero Point 3200–3700

Rhododendron and Juniperus, with the increase in altitude the plant shape become smaller and cushion like.

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Forest localities in Champawat district A. Kimtoli forest (Cedrus deodara dominant tree), B. Devidhura Pinus roxburghii forest

A

B

PLATE 4

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Localities of Champawat district A. Purnagiri Parwat B. Ghat Bridge

PLATE 5

A

B

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Localities of Champawat district (A) In and around Champawat High Court, (B) Gurauli Hill

PLATE 6

A

B

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Table 3. Localities of Champawat district, surveyed for collection of Lichens (Map 4). S.

No. Localities Altitude

(m) Dominant vegetation

1. Purnagiri

300–550

Plains are vegetated with plenty of exotic and traditional variety of plants such as Eucalyptus, Babul, Teak, Sagon, Bans, and Madar.

2. Chalthi 500–700 Mangifera indica, Cedrus deodara, Madhuca longifolia and Syzygium Cumini.

3. Shukhidak 1100–1400 Pinus roxburghii, Alnus nepalensis and Rhododendron.

4. Kimtoli 1100–1400 Quercus leucotricophora, Pinus roxburghii, Cedrus deodara and Quercus floribunda.

5. Marodkham 1300–1500 Rhododendron arboreum, Alnus nepalensis, Quercus floribunda and Pinus roxburghii.

6. Banlekh 1400–1600 Rhododendron, Taxus, Alnus nepalensis, Quercus floribunda and Pinus roxburghii, Quercus leucotricophora and shrubs.

7. Champawat Proper 1450-1650 Quercus leucotrichofora, Pinus roxburghii, Betula alnoides, Alnus nepalensis and shrubs.

8. Devidhura 1500–1700 Rhododendron, Pinus roxburghii and Quercus leucotricophora.

9. Lohaghat 1500–1700 Alnus nepalensis, Pinus roxburghii and Quercus leucotricophora.

10. Dunaghat 1500–1700 Pinus roxburghii, Quercus leucotricophora, Quercus floribunda and shrubs.

11. Pati 1600–1750

Quercus leucotricophora, Quercus floribunda, Pinus wallichiana, Pinus roxburghii and shrubs.

12. Abbot Mont 1800–2700

Rhododendron arboreum, Alnus nepalensis, Quercus floribunda, Pinus roxburghii, Quercus semecarpifolia and lower plants.

Table 4. Localities of Nainital district, surveyed for collection of Lichens (Map 5).

S. No. Localities

S. No. Sub localities

Altitude (m) Dominant Tree

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1. Tiffin Top No 2292 Quercus semecarpifolia

2. Ayarpatta (2278 m)

1 High Court 1938 Q. leucotrichophora 2 Kumaun University 1940 Q. leucotrichophora 3 Modi Field 2100 Q. leucotrichophora 4 Modi Guest House 2100 Q. leucotrichophora

3. Naina Peak No 2611 Q. semecarpifolia

4. Kilbury (2500 m)

1 Nand Kutter 2250 Q. semecarpifolia 2 Plan`s View 2264 Q. semecarpifolia 3 Amla Cottage 2270 Q. semecarpifolia 4 Tankey Band 2300 Q. semecarpifolia

5. Snow View (2270 m)

1 Birla Field 2250 Q. semecarpifolia 2 Backnbery Hall 2263 Q. semecarpifolia

6. 36 Sheeree (2258 m)

1 Tower Compound 2255 Q. semecarpifolia 2 Duffan Lodge 2230 Q. semecarpifolia 3 Kumaun Lodge 2220 Q. semecarpifolia 4 Devi Lodge 2200 Q. semecarpifolia 5 Budda Temple 2200 Q. semecarpifolia 6 Draw Paul 2210 Q. semecarpifolia

7. Mangawali (2280 m)

1 Ramji Hospital 2240 Q. semecarpifolia 2 Ratan Cottage 2150 Q. leucotrichophora

8. D.S.B. Campous (2010 m)

1 Kane Field Hospital 2075 Q. leucotrichophora

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Nainital District collection sites (A) Birla Top (B) Rajbhawan

A

C

PLATE 7

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Nainital district localities (A) Snow View (B) Tiffin Top

PLATE 8

A

B

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Table 5. Localities of Pithoragarh district, surveyed for collection of Lichens (Map 6). S.

No. Localities S.

No. Sub localities Altitude

(m) Dominant Tree

1. Pithoragarh (1500-1800 m)

1 Kujauli Village 1500 Pyrus, Prunus, Celtis

2 Bisar 1500 Cedrus, Quercus leucotrichophora

3 Kuchigar Quercus forest

1500 Pyrus, Prunus, Q. leucotrichophora

4 Pagama forest 1500 Pyrus, Prunus, Q. leucotrichophora

5 Pandegaon 1600 Q. leucotrichophora 6 Barabey 1600 Q. leucotrichophora 7 Narain Nagar 1800 Q.leucotrichophora

8 Nainipatal forest 1800 Cedrus, Q. leucotrichophora

2. Gori-Ganga (1600-2300 m)

1 Kauli 1600 Q. leucotrichophora 2 East Ghandhura 1800 Q. leucotrichophora 3 Thakala forest 1800 Q. leucotrichophora 4 Dhaphiyadhura 2200 Quercus semecarpifolia 5 Thali 2300 Q. semecarpifolia 6 Salyari 2300 Q. semecarpifolia 7 Mwani–Dwani 2300 Q. semecarpifolia 8 Majthan 2300 Q. semecarpifolia

3. Askote–Sandev (1550-2300 m)

1 Churani 1500 Q. leucotrichophora 2 Jamtri 1500 Q. leucotrichophora 3 Singali 1500 Q. leucotrichophora

4 Chaurani 1650 Q. leucotrichophora, Alnus

5 Deochula 1650 Q. leucotrichophora, Alnus

6 Lamagharh 1650 Q. leucotrichophora, Alnus

7 Shantikunj 1650 Q. leucotrichophora, Alnus

8 Dhanlekh range 1950 Q. leucotrichophora, Alnus

9 Adhichura 2300 Q. semecarpifolia, Alnus

4. Chandak No 1500-1600

Q. leucotrichophora, Cedrus, Pinus

5. Dhwaj No 1800-2100

Q. leucotrichophora, Rhododendron

6. Haat Kali No 1500-1700

Cedrus deodara, Q. leucotrichophora

7. Hokra sacred grove No 1500- Cinnamomum tamala,

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1700 Zanthoxylum

8. Malay Nath No 1800-2000

Rhododendron, Q. leucotricophora

9.

Nakuleshwar

No

1300-1550

Albizia, Cedrus, Cinnamomum

10. Narayan Swami Ashram

No 2200-3000

Cedrus, Rhododendron, Q. semecarpifolia

11. Patal Bhuvaneshwar

No 1400-1650

Cedrus, Berberis, Pyracantha

12. Munsiyari (1800-3000 m)

1 Virthi Fall 1800 Q. leucotrichophora 2 Nain Singh Top 2700 Q. semecarpifolia

3 Kalamuni 3000 Q. semecarpifolia, Cedrus deodara

4 Khaliya Top 3000 Q. semecarpifolia, Cedrus deodara

13. Milam Glacier (3000-3500 m)

1 Naher Devi to Bugdiyar

3000 Alpine Grasland

2 Naher Devi to Mapamg

3100 Alpine Grasland

3 Rilkot to Milam 3250 Alpine Grasland 4 Moritoli 3390 Alpine Grasland 5 Berfu 3450 Alpine Grasland 6 Bejiu 3450 Alpine Grasland 7 Milam Village 3450 Alpine Grasland

14. Dharchula Sobhla No 1800-2000

Cedrus deodara, Q. leucotrichophora

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Localities of Pithoragarh district (A) Alpine region of Munsiyari, (B) Temperate region forest of Munsiyari.

PLATE 9

A

B

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Localities on way to Munsiyari in Pithoragarh district (A) Sela Ghat, (B) Birthi fall

PLATE 10

A

B

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Localities of Pithoragarh district (A) Narayan Swami Ashram (B) Dam on way to Narayan Swami Ashram

PLATE 11

A

B

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Localities of Pithoragrh district (A) Rilkot (B) Beiju village near Munsiyari

A

B

PLATE 12

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Localities of Pithoragarh district (A) Khuliya Top forest, (B) Narayan Swami Ashram hill

PLATE 13

A

B

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Localities Udham Singh Nagar district A. Tanda Forest B. Khatima Forest

PLATE 14

A

B

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The dried samples were packed on hard card sheets inside a lichen herbarium

packet (17cm X 13 cm) with details of the locality, date of collection, substratum and are

preserved at the lichen herbarium of National Botanical Research Institute, Lucknow

(LWG). The study is based on the lichen material collected and the material collected

earlier and preserved in the lichen herbarium of National Botanical Research Institute,

Lucknow (LWG), lichen herbarium of Lucknow University, Lucknow (LWU) and

personal herbarium of Dr. D.D. Awasthi (AWAS).

Different phorophytes for good growth of Lichens in Kumaun Himalaya. (A) Rhododendron (B) Quercus (C) Mellotous Philipensis (D) Shorea robusta

A B

C D

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Identification:-

The external morphology has invariably been studied under dissecting binocular

microscope. The anatomy of the thallus and apothecia were studied under compound

microscope.

The external morphology was examined generally in dry condition but dark

brown to bluish specimens of Leptogium and Collema were studied in wet condition. The

anatomical structures were studied after cutting the sections of dry material by microtome

or with the help of safety razor blade. The thin dry sections of the thallus or apothecia

were immersed in 90% ethyl alcohol to drive off the intercellular or inter-hyphal air

bubbles and the sections were mounted in water or in cotton blue in lactophenol. The

colour of medulla, epithecium, hypothecium and ascus were recorded. The asci and

ascospores were taken from the sections when sections were mounted in water and

shapes, sizes were recorded. The measurements of the thallus, medulla, epithecium,

hymenium were generally taken in the sections mounted in cotton blue. The thallus sizes

were measured in centimetre, lobe size and apothecia in millimetre and thallus medulla,

epithecium, hymenium thickness, asci and ascospores size in millimicron.

Chemistry of the specimens includes colour tests and thin layer chromatography.

(I) Colour tests

Chemical reagents have performed colour test by applying it on thallus and

medulla resulting change in colour. A positive change is denoted by a positive symbol

(+), followed by the colour produced and no change in colour is denoted by a negative

symbol (-). The chemical reagents used are as follows:

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K test: 10-25% aqueous solution of potassium hydroxide (10 g KOH pellets + 100 ml

distilled water), applied to cortex, medulla and part of apothecia. Also used as a clearing

agent for sections of fruiting bodies and thalli, as it often dissolves the crystalline lichen

substances and removes some mucilage that may obscure detail in sections.

Pd test: Solution of paraphenylene diamine is prepared in ethanol or alcohol in a small

quantity for the use of a day. It is unstable and cannot be used for the next day. A more

stable solution called Steiner's Pd is prepared by dissolving 1.0 gm of paraphenylene

diamine and 10 gm of sodium sulphite in 100ml of distilled water with 1.0ml of a liquid

detergent. This reagent keeps well for about a month.

C test: A freshly prepared aqueous solution of calcium hypochlorite or bleaching powder

or modern commercial bleaching fluid containing active chlorine. It is prepared by

dissolving calcium hypochlorite in the distilled water in 2% ratio.

KC test: At a particular spot of thallus, K is applied first and immediately followed by C.

I test: 2-5 gm of iodine is dissolved in water with 0.5 gm of potassium iodide. It does not

react with lichen secondary metabolites but rather with starch like polysaccharides in the

thallus or fruiting body. The reagent keeps well for several days and is to be renewed

when colour fades.

(II) Micro-crystallography

Microcrystallography was introduced by Asahina (1936; 1938). The method does not

need elaborate equipment. A small fragment of lichen to be investigated is placed on the

middle part of a microscopic glass slide and one-two drops of acetone or any other

organic solvent are dripped on to the fragment by means of dropper pipette. Lichen

substances if present gets dissolved in the solvent and extracted on the slide as residue in

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a ring from around the fragment as soon as the solvent evaporates. The thallus fragment

is blown off. A micro-cover glass is placed over the residue and a drop of one of the

crystallizing fluids (detailed below) is placed at the edge of the cover glass. The fluid

gradually seeps in. The slide is then heated gently over a sprit lamp. The residue

dissolves in the fluid and lichen substances gradually crystallize into their characteristic

of shapes on cooling. These crystals are observed under low power of microscope and

identified by comparison with the photographs or line diagram published by Asahina

(1950; 1952), Hale (1967), Thomson (1967), Krog (1951) and others. Identification of

depsides, depsidones and dibenzo-furans is usually confirmed by this method. The

crystallizing fluids used are as:

a) G.E. Glycerol: acetic acid, 1:3

b) G.A.W- Glycerol: ethanol: water, 1:1:1

c) G.A.Ot- Glycerol: ethanol: ortho-toluidine, 2:2:1

d) G.A.An- Glycerol: ethanol: aniline, 2:2:1

e) G.A.Q- Glycerol: ethanol: quinoline, 2:2:1

(III) Chromatography:

It was mainly performed in solvent system A (Toluene: 1, 4-dioxane: acetic acid::

180: 60: 8 ml) but sometimes in solvent system B (Hexane: Diethyl ether: Formic acid ::

130: 100: 20 ml). The chemical substances were extracted in acetone and loaded on silica

gel pre-coated aluminium plates. After running in solvent system A, the TLC plates were

sprayed with distilled water for checking the presence of fatty acids. Later the plates were

sprayed with 10% H2SO4 solution and heated in hot air oven at 110°-120°C till the colour

spots were developed due to charring. Parmelinella wallichiana (Taylor) Elix & Hale,

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having Salazinic acid (Rf class 2), Norstictic acid (Rf class 4) and Atranorin (Rf class 7),

was used as reference materials. The TLC was observed under UV radiations at 350 nm

wavelength before and after charring. The guidelines of Walker and James (1980) were

followed for colour spot test and TLC.

(IV) Other colour tests:

A dilute aqueous solution of nitric acid and an aqueous solution of ferric chloride

are sometime used for identification of Melanelia and Buellia species. The spot tests can

be done on any part of the thallus but younger parts give better results. Colour test is done

to a small fragment of the desired lichen thallus part or thallus or ascocarp. A definite

colour comes showing the presence of any lichenic acid.

UV test: A number of secondary metabolites in lichens exhibit a characteristic

fluorescence under UV light. The response of (+ or -) these metabolites plays a vital role

in the lichen identification.

Ecological methods:-

The relative importance of organisms in a community is not determined by its

taxonomic position but rather by the number, size and other relationships. The degree of

importance of a species is usually expressed by an index of dominance. Community

analysis may be carried out in any given location on the basis of distinct zones or

gradients that may exist in that area. Generally, the steeper the environmental gradient,

the more distinct are the communities since sharp boundaries are formed by the abrupt

changes in the physical nature of the environment.

The ratio between the numbers of individuals in a community is termed as species

diversity. This is related to the stability of the environment and it varies with different

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communities. Species diversity is of great importance in assessing the extent of damage

done to natural systems by human interference.

Species diversity may be taken to denote the number of species in a given area or

as the number of species among the total number of individuals of all species present.

This relationship may be expressed as diversity index. The number of species in a

community is important ecologically since the species diversity seems to increase as the

community becomes more stable. Several disturbances cause a marked decline in the

diversity. A great diversity also indicates the availability of a large number of niches.

Frequency:

This term refers to the degree of dispersion of individual species in an area and is

usually expressed in terms of percentage occurrence. It can be defined as the chance or

probability of an individual of a given species to be present in a randomly placed

quadrate. This can be studied by sampling the study area at several places at random, or

in a desired pattern to cover the site adequately, and recording the names of species that

occur in each sampling.

The formula to analyze frequency is: -

Total number of quadrates in which species occurs × 100 Frequency (F) =

Total number of quadrates sampled A species most abundantly spread all over the area will have chance of occurring

in all the samplings and therefore, its frequency will be hundred percent. A poorly spread

even with large number of species in one corner will have a chance of occurrence in only

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few quadrates and its frequency value will be low. Thus higher frequency value shows

greater uniformity of its spread or dispersion.

Density:

Density is also an expression of the numerical strength of a species where the total

number of individuals of each species is divided.

The formula to calculate density is:-

Total number of individuals in all the quadrates Density (D) =

Total number of quadrates sampled

Abundance:

Abundance is an appreciation of the relative number of individuals of each

species entering into constitution of the plant population of the territory under study, but

thus obtained in quantitative terms gives little idea of the distribution of the species.

Forumula to calculate density is: -

Total number of the individuals of the species Abundance (A) =

Total number of quadrate in which the species occurred

The 10 × 10 m quadrates were laid in different sites of Kumaun Region for the

ecological studies of lichens. In each locality equal numbers of three quadrates were laid

and within each quadrate total number of lichen species were counted weather on tree

(T), rock (R) or soil (S).

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