Field Collection Guidelines

Gujarat Biodiversity Gene Bank Field Collection Guidelines For Biomaterial Submission [V.1.1.2014-15]

A GSBTM, DST, Government of Gujarat initiative

Field Collection Guidelines For Biomaterial Submission V.1.1.2014-15 BioGene Umbrella Programme

Table of Contents Foreword .................................................................................................4 1. Introduction to BioGene.........................................................................5 1.1 Aim and Objectives ..........................................................................5 1.2 Infrastructure ..................................................................................6 1.2.1. Key Equipments ...............................................................................6 1.3. Microbial Repository ........................................................................6 1.4 Plant Gene Bank ..............................................................................7 1.5 Animal gene bank ............................................................................7 1.6 BioGene Umbrella Program on Barcoding Biodiversity of Gujarat ............8 2. Scope and objectives .......................................................................... 10 3. General Guidelines .............................................................................. 11 4. Basic Requirements ............................................................................ 12 5. Plant specimen collection Guidelines ..................................................... 13 5.1 Pressing the plants ......................................................................... 15 5.2 Mounting voucher specimens ........................................................... 16 5.3 Graphical representation of herbarium preparation ............................. 16 6. Seed collection Guidelines.................................................................... 18 6.1 Containers for collecting samples ..................................................... 18 6.2 Processing of seeds in the field ........................................................ 19 6.3 Transporting the collected material to the laboratory .......................... 19 6.4 Minimum required information with plant / seed sample ..................... 19 7. Mushroom Collection Guidelines ........................................................... 21 7.1 Collection and drying of fungal specimens ......................................... 21 7.2 Data to be recorded in the field ........................................................ 23 7.2.1 Site information ....................................................................... 23

Gujarat Biodiversity Gene Bank | GSBTM | DST | GoG

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7.2.2 Specimen information (field) ...................................................... 23 7.2.3 Macroscopic description aid ........................................................ 25 8. Soil collection guidelines for fungus isolation .......................................... 27 9. Bryophyte, Lichen and Algae Collection Guidelines .................................. 28 9.1 Bryophyte specimens (mosses, liverworts) ........................................ 28 9.2 Lichen specimens ........................................................................... 28 9.3 Algae specimens ............................................................................ 28 9.4 Liquid preservation of mushroom, algae, lichen and bryophyte ............ 29 10. Invertebrate collection guidelines ........................................................ 30 10.1 Collecting Invertebrates for preservation and DNA barcoding ............. 30 10.2 Methods for Dry Mounting ............................................................. 32 10.3 Pining specimen on mount strips .................................................... 32 10.4 Gluing Specimens onto paper cards ................................................ 33 10.5 Preservation and transport of arthropods which are to be dry mounted ......................................................................................................... 33 11. Fish Collection Guidelines ................................................................... 35 11.1 Fixatives ..................................................................................... 36 11.2 Fixation Procedures ...................................................................... 36 12. Mammalian Tissue and Aves Feathers Collection Guidelines .................... 38 13. Amphibian and Reptile Samples Collection Guidelines ............................ 40 14. Key references ................................................................................. 41 Annexure I: Proforma for Deposition of specimen ....................................... 42 Annexure II: Essential information required for submission of specimen to Barcode of Life Database (BOLD).............................................................. 43 Annexure III: Optional information for submission of specimen to Barcode of Life Database (BOLD) ............................................................................. 44


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Annexure IV: Parameters for sampling, voucher preparation, transportation and preservation of biomaterial from various sources ........................................ 45


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Akshay Saxena, IFS Mission Director Gujarat State Biotechnology Mission Department of Science &Technology Government of Gujarat 11th Block, 9th Floor, Udyog Bhavan Gandhinagar - 382 011, Gujarat, INDIA Phone: +917923252197, Fax: +917923252195

Foreword Gujarat Biodiversity Gene Bank (BioGene) is an initiative of Gujarat State Biotechnology Mission, Department of Science & Technology and Government of Gujarat for research in ex-situ conservation of state floral, faunal and microbial biodiversity using tools of modern biotechnology. Biomaterial collection is a key task for various activities in biodiversity and conservation biotechnology research. This requires definite strategies and methods with detailed documentation. Any collection, without primary information, labeling and proper collection methodology, is of little importance and results in waste of time, effort and opportunity. As we set upon to explore, document and undertake research on this rich biodiversity of Gujarat, it is important to comply and follow the internationally acceptable norms and guidelines. This document is a compilation which captures the procedures and process required to be observed while undertaking survey and sample collection of various biological taxa. This document provides guidelines for research students, academicians and scientists working in the areas of conservation biotechnology of plants, microbes and animals. I am confident that “Field Collection Guidelines” document will serve to improve documentation and research in areas of bio-banking and DNA barcoding.

Akshay Kumar Saxena Mission Director


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1. Introduction to BioGene Gujarat Biodiversity Gene Bank [BioGene], is an initiative of Gujarat State Biotechnology Mission, Department of Science and Technology, Government of Gujarat. One of the prime mandates of BioGene is conservation of biodiversity of Gujarat in association with Department of Forest, Government of Gujarat. Gujarat holds a very unique position in biodiversity spread, having wide variations in eco-climatic and geographical conditions, which includes hot saline desert, humid hilly forests and 1600 km long coastline. An estimated 70 mammalian species and 2000 plant species are known to exist. However, Gujarat’s flora and fauna has been inadequately reported specially at the molecular level. BioGene was thus established with the vision of securing the biodiversity, by storing DNA, tissue of endangered species as well as socio-economically important species of Gujarat.

1.1 Aim and Objectives The aim of Gujarat Biodiversity Gene Bank [BioGene] is long term ex-situ conservation of plant, animal and microbial origin. BioGene actively involved in developing high end infrastructure facility, research, training and awareness program for use of molecular sciences for conservation biotechnology. For this purpose microbial repository, plant, seed and animal gene bank are established.


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1.2 Infrastructure BioGene is having a state of art research infrastructure with dedicated facilities for 1. DNA Banking, 2. Cryo preservation, 3. Culture facility for handling plant, animal and microbial biomaterial, 4. Molecular biology facilities and 5. Genomics facilities (capillary and next generation sequencing)

1.2.1. Key Equipments Ion Proton -40 Deg C Freezer(s)

Ion Torrent – 80 Deg C Freezer(s)

24 Capillary Sequencing Biosafety Cabinet Platform 4 Capillary Sequencing CO2 Incubator Platform PCRs

-20 Deg C Freezer(s)

TissueLyser II Cryo storage system

2D-IEF Anaerobic Chamber

Lyophilizer

Protein Purification System Automated Electrophoresis System RT-PCRs

High Speed Refrigerated Centrifuge(s) Geldoc System(s)

1.3. Microbial Repository Microbial Repository (BAB-BioGene) is microbial culture-collection of microorganisms in Gujarat. It is currently having 3107 bacteria, 942 fungi and 68 archaea. The repository is the third largest in India, comprising of more than 2000 NCBI accessions. Long term storage of microbes is done as lyophilized or glycerol stock. BioGene microbial repository is currently registered with World Data Center of Microbes (Reg. No. 1058) with the registered name “Bank A Bug”. It also maintains a web based catalogue for holding strains which is accessible from the following link: [biogene.in/Biogene/BAB_catalogue.pdf]. Present status of microbial repository Culture Type Bacteria Fungus Actinomycetes Algae Yeast Archaea Total

Total microbes 3107 942 39 1 3 68 4160

NCBI Submission 1585 613 24 0 2 57 2281


Accession number 1376 569 20 0 2 44 2011

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1.4 Plant Gene Bank Plant Gene Bank stores herbarium, silica dried plant tissue, seeds and DNA. The herbarium serves as reference for identifying plant and also fulfils the aim for dry plant tissue preservation and can also be utilized for DNA isolation. BioGene has more than 3000 herbarium specimens of 120 plant families. These herbariums are photographed to include them in BioGene e-herbarium collection. Plant tissues are stored in dry condition. The material is preserved in airtight container/pouch after freeze drying process. BioGene has tissue bank of more than 700 plant accessions. BioGene DNA bank is established to cater the need of long-term preservation of genetic material utilized for research purpose for the scientific community. At Present, DNA banking of 127 plant species is completed. BioGene activities also include Seed banking, varietal identification, Morphological, physiological and molecular characterization of seed accessions. BioGene seed repository has more than 750 accessions comprising of 640 samples covering 100 species of forest plants and 143 accessions of agriculturally important species which includes 74 cash crops accession covering 25 species. Seed Bank has 37 accessions covering 14 vegetable species and 3 horticultural species.

Present Status of Plant Gene Bank Number of vouchers/ herbarium Number of Taxa barcoded Number of Forest seeds accessions Number of Agriculture seeds accessions

Family Genus Species

3100 108 381 572 750 143

1.5 Animal gene bank Animal gene bank is established with a purpose to store DNA and its amplified products which may be used for the characterization of genotypes, assessment of genetic diversity, estimation of genetic relationships within, identification of duplicates, establishment of co-relation as well as monitoring genetic stability and integrity. DNA banks also help in obtaining knowledge to improve the efficiency of some conservation activities or to scientifically inform decisions related to the conservation of species. BioGene stores animal tissue samples in the form of blood, swab and other forms such as feathers. Currently 444 tissue samples are preserved in animal gene bank.


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Animal Gene Bank Status

Mammals and Reptiles

Blood

Tissue

266

90

50

8

Aves Insect

Feather

Whole specimen

33

73

Fish

43

Mollusc

43

1.6 BioGene Umbrella Program on Barcoding Biodiversity of Gujarat The shortage of trained taxonomists and access to the essential information resources (especially museum and herbarium collections, taxonomic publications, databases on the Web) are most acute in developing countries having rich biodiversity. DNA barcoding is process for determining sequence variation within short and standardized regions of genome. It is a tool for species identification providing additional information along with identification based on traditional taxonomy. In view of above BioGene launched an umbrella program from August 2013 in collaboration with various academic professionals of state on barcoding biodiversity of Gujarat. It provides opportunity to post graduates students to pursue higher studies through M. Phil and Ph. D. In the short span of one year the number of barcodes submitted to BOLD reached 1130 and are publicly accessible in the GENG project on BOLD. A summary table is given below: Barcodes submitted to Barcode of Life Database [BOLD]

BOLD Project Marker rbcL matK trnH-psbA ITS COI cytB atp6

Fungi

Plant

Animal

Total

MGEN

GENG

ANGEN

GENG

673 15 13 7

673 15 13 319 326 124 124 1 1 1 1 Total submissions 1153* *BioGene is leading in the country with highest number of DNA barcode submissions


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Bio materials deposited in Gujarat Biodiversity Gene Bank (BioGene)

Eranthemum roseum

Lantana rugosa

Senna alata

Ganoderma multipileum

Flavodon flavus

Phellorinia herculeana

Trimerotropis sp.

Aplysia fasciata

Eulalia viridis

Moringa olifera

Oroxylum indicum

Peltophorum ferrugineum


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2. Scope and objectives The “Field collection guidelines” intend to provide students/ researchers/ academicians specific guidelines for plant/ fungal and animal collection. We encourage adoption of these guidelines to improve the quality, efficacy and accuracy of biomaterial collection for voucher preparation and for the purpose of DNA barcoding. The main objective of these guidelines is to provide the user appropriate methods for collection and documentation. It offers general protocols for the collection of plants, fungus, insects, fishes, amphibian, reptile and mammalian tissue. It also provides explanation for sample collection for voucher preparation and highlights the difference in sample collection for DNA barcoding, DNA banking and voucher preparation. The manual also is designed to provide a detailed description of data entry rules and data entry procedures in order to standardize data entry. This standardized form is a prime requirement for submission to Barcode of Life Database (BOLD). This is an ongoing document, which will be updated and modified periodically. The document is structured in a way that any user can easily get a step by step guide to collection strategies for preparing both vouchers and DNA barcoding. A summary table is also appended to give a snapshot of the entire guidelines. Moreover tables are also provided for documentation and description of the data before submitting the specimen.


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3. General Guidelines 1. 2.

Always carry the collection kit. Wherever required, collector(s) should take prior written permission from the authorized agency for collection. 3. Collections must be done in two sets: a. First for Voucher preparation in triplicates (find details in the respective guidelines). b. Second for DNA barcoding (NO FIXATIVE BE ADDED). 4. A review of the collection site including its habitat, area and diversity will help giving an insight into its usefulness to the collector. 5. Collection should not harm the environment. 6. When submitting the specimen to BioGene it is mandatory to submit the proforma attached in this guideline as Annexure I and Annexure II. 7. Any submission without these proforma will not be considered. 8. Avoid duplication of the samples. Check BioGene specimen list. 9. While collecting the samples, avoid cross-contamination. 10. Surface sterilize the specimen, if required and avoid diseased/ pathogenic specimens. 11. Collected specimens should be transported immediately to the lab avoiding direct sunlight. Refer to details mentioned in the respective guidelines for plants, animals and microbes. 12. Take high resolution photographs (preferably 300 dpi) with appropriate ISO and background settings.


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4. Basic Requirements Zip seal bags/ sterile containers

Label Sticker

Cellotape

Pen

Field record book

GPS with spare batteries

Camera

Rucksack Bag

Blotting / Newspapers/ Butter Paper

First aid box

Scale

Forceps

Gloves

Field shoes


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5. Plant specimen collection Guidelines 

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Before setting out to collect plants, be sure to familiarize yourself with potentially injurious plants. Both native/naturalized species and cultivated plants of known provenance can be collected. Determine whether or not the plant(s) you wish to collect are planted or spontaneous. Always collect from an area where there are numerous examples or sufficient plant material of the species or cultivated variety you wish to collect. Always collect enough plant material for preparation of at least 3 voucher specimens (herbarium). Along with the material collected for voucher specimen 3-5 young leaves (minimum 1 g, preferably in between 10-100g, if available) must be collected in silica gel filled in a zip seal bag for DNA barcoding. If plant material is collected in rainy season then ensure that plant material is free of moisture (except in the case of aquatic species) before you collect and proper care should be taken to avoid fungal infection. Herbarium specimens should have the plant features or characteristics required for positive identification. For most plants, this means flowers or fruit/seeds should be present. Make sure your specimen is representative of the plant’s stem and leaf patterns. Therefore, the timing of plant collection should be an important consideration. In addition to flowers and fruit, other identifying features of the plant should be sampled at the time of collection. If collecting herbaceous plants (i.e. wild flowers, graminoids or ferns) roots or underground plant parts (rhizomes, bulbs or tubers) should be collected where possible, along with the above ground plant parts (leaves, stems, flowers, fruit, thorns etc). Cyperaceae, Liliaceae, Zingiberaceae, Costaceae and plants with underground roots should be preferably collected with roots, rhizome, corm or underground part. If you are collecting trees, shrubs and other woody plant material, take a cutting of a branch with several representative leaves and flowers and/or fruit. It is often useful to include a small sample of a tree’s bark. Do so by carefully removing a piece of bark, not larger than 2 x 2 cm, with a knife. Sterilize secateurs or other cutting tools with a little rubbing alcohol before taking a sample from a different plant to avoid the spread of pathogens and disease. Herbarium specimens should be pressed between newspaper or blotting paper using herbarium press. Large or long-trailing herbaceous plants can be cut into sections and pressed and mounted separately (see pressing the plants). For each plant specimen collected, brief information for taxonomic identification should be noted in the field at the time of sampling as given below: 1. Plant name - Scientific name (if not known, some kind of identifier should be used). 2. Collection number, optional. 3. Date of collection. 4. Habit (Herb, Shrub, Tree, Climber, Epiphyte, Parasite, Saprophyte, Insectivore, Symbiont)


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5. 6. 7. 8.

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Habitat (Grassland, wasteland, weed of cultivated field, stream, water bodies) Root (Tap root, Adventitious roots) Stem (Aerial, climbing, underground, specialized stem) Leaf (Bearing of leaves) a. Phyllotaxy (Arrangement of leaf on stem) i. a. Alternate b. opposite c. whorled b. Type of leaves (simple, compound) Inflorescence (Arrangement of flower on floral axis) 1. Racemose (an inflorescence where the main axis does not terminate in a flower) 2. Cymose (an inflorescence where the main axis terminate in a flower) 3. Special type (Cyathium, Thyrsus, Verticillaster, Hypanthodium) Flower (prefer to not floral formula in field) 1. Colour 2. No. of petals 3. No. of stamen 4. No. of carpels Fruit a. Simple fruit 1. Dry fruit (Capsule, legume, siliqua) 2. Fleshy fruit (Drupe, Pome, Berry, Pepo, Hesperidium) b. Multiple or composite fruit (syconus, sorosis) 1. color 2. shape 3. seeds Special notes a. Aroma, b. Association with other plant sp., c. Morphological features i.e. height, d. vernacular name, e. number/quantity of plants growing in the area; f. health of the plants; g. record photo number(s) h. Parts of economic importance and for cultivated plants, provide information on provenance, breeders, garden location, accession number, etc. i. Digital photos of the plants in their habitat or growing medium may be taken. Take close-up photograph of plant with details of reproductive organs. Digital photos are complimentary to voucher specimens and can be stored in a database and/or printed and linked to the Herbarium specimens for additional reference.


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14. A preformatted collection data sheet to register all the information that is important to annotate during sampling may be useful.

5.1 Pressing the plants 

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Plants should be pressed as soon as possible after collection. Plant material can be stored in large polyurethane bags while in the field, and some plants will survive overnight in bags in a refrigerator if they cannot be pressed immediately. Press plants using a standard plant press. If no plant press is available, smaller plants can be pressed between several sheets of newspaper and placed under a stack of heavy books or blocks. Label each sheet of newspaper according to the species name and date or collection number that corresponds to your notes. This is important so that there is no mix up when it comes to mounting and labelling the plants at a later date. When pressing plants, blot any moisture away. Lay your specimens out on one side of an opened piece of newspaper.  Plant parts such as leaves and petals should be laid out flat.  The flowers should be placed so that the flower parts are distinguishable.  Flowers and fruit may be cross-sectioned prior to drying.  It is helpful to turn some leaves over so that examples of the top and underside surfaces are visible.  Close the newspaper and sandwich it between two pieces of blotting paper and cardboard.  If the specimen is particularly thick then a piece of foam can be folded inside the newspaper. Alternatively, thick or bulky plant parts such as roots, seeds, bark could be pressed in separate sheets.  Larger plants can be cut into sections and components can be pressed individually (i.e. base, middle & top of plant). If plants are sectioned into two or more, label newspapers accordingly (i.e. for a plant cut into three parts, specimen 1 of 3, 2 of 3, and 3 of 3 should be used for the top, middle and lower parts of the plant).  If the specimens are succulent or wet (e.g. aquatic submerge or floating plants) then they can be pressed between pieces of parchment paper. Layer all of your specimens into a plant press and tighten the press firmly. Store press in a dry place with adequate air circulation. After a day you can check on your plants while they are still not completely dry and rearrange them if any leaves were folded over while pressing. Change newspapers and blotting paper as necessary to prevent molding until plants are dry. The length of time it takes for a species to dry depends on the plant’s water content and on the drying conditions. The quicker a specimen is dried, the better its colour will be preserved. After several days plants will be dry and ready to mount.


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5.2 Mounting voucher specimens     

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Specimens should be mounted on acid-free standard Herbarium mounting paper. Poison the plant material with 0.01% HgCl2 in 60% Ethanol and 40% distilled water solution or in spirit. Stamp the herbarium with label POISON For epiphytic orchids possibly dry the plant, inflorescence and flowers immediately in Microwave oven to avoid abscission. Possibly stitch the plants to the herbarium. Place your dried plants onto a piece of 42 cm x 28 cm standard handmade paper. Arrange them in such a way that the main features used to identify the plant are evident. Ensure there is room for a label in the lower right hand corner. Use thick paper strips to place plant specimen on sheet. Arrange plant specimen and flower / fruit parts on handmade paper sheet. Apply glue on both the ends of strip and stick the plant. Use large strips for bigger plant parts such as twig and stem. While fixing the plant on sheet additional flowers could opened or dissected to display flower parts. Alternatively low glue tape can be used to reinforce areas that do not stick as well (ex. nodes, root balls, flower heads) Cover the specimen with waxed paper, place it under a heavy block or between hard boards and leave it to dry overnight. Store specimens in a cool, dry place and minimize exposure to light.

5.3 Graphical representation of herbarium preparation 1. Collecting: Select a typical plant and if possible two or three extra flowers to supplement the specimen and for dissection. Ensure the plant is healthy and collect average-sized leaves and flowers typical of the plant, not the biggest. Photograph the plant habit and a close-up. Attach a label with the name and date and location. Avoid collecting material in wet weather. 2. Describing: When collecting, record the following: name of plant, date of collection, collector, site of collection, original source of plant. Note other details that may be lost by pressing: overall size, habit and form, leaf or flower scent. Record color of the fresh plant using standard color chart.


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3. Pressing: Remove soil from around the material. Use a press made with a pair of boards of hardboard or plywood cut to the same size as the drying paper. Dip the specimen in mercury chloride preservative solution. Place some corrugated card on one board of press, and then place two sheets of blotting paper on top of this. Arrange plant material on blotting paper retaining the character of the plant. Put flowers and dissected parts in flimsy paper. Remove leaves and flowers of congested specimens to reduce the bulk without losing the character of the plant. 4. Pressing: Cover the sample with two further sheets of blotting paper and corrugated card. With both bulky and fleshy specimens, add a sheet of foam between the blotting paper and corrugated card. Any absorbent fabric may be useful in drawing out moisture; place it on top of the plant material, with a thin sheet of paper between the plant material and the fabric to prevent sticking.

5. Pressing: Once all samples have been included, cover with top board and place bricks or heavy object, applying pressure evenly throughout or use straps to keep the press tight. Place in a warm place, such as a drying cabinet, airing cupboard.

6. Pressing: Inspect the material 24 hours later, replacing the corrugated card and top layer of blotting paper with dry card. This is your last chance to re-arrange any material while the plant material is still moist and pliable. Inspect regularly - at least once a week. Depending on the plants being pressed and the drying conditions a dry specimen will be ready in anything from two to three days to two to three weeks.


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7. Mounting: The BioGene Herbarium uses handmade acid-free paper, measuring 419 x 276mm. Good quality A4 paper, preferably acid-free, is sufficient for the needs of a domestic herbarium in absence of handmade paper sheet.

8. Mounting: Attach the specimen to the paper using a combination of neutral pH PVA adhesive and gummed linen hanging tape. The label should the bottom right-hand corner. Alternatively paper strips can be used to simply hold the specimen on herbarium sheet. Further stitching and/or non-stick low friction transparent tape can be used to place specimen on paper sheet. 9. Storage and conservation: Place the prepared specimen in a sealed plastic bag and freeze for 72 hours. Ideally the temperature should be -32 ºC, although most domestic freezers have a minimum of -18 ºC. Freezing is the only method open to combat pests. The most common pest of the herbarium specimen is the biscuit beetle, Stegobium paniceum. Regular freezing (every six months) is recommended, as is regular inspection to check for infestation and damage.

6. Seed collection Guidelines Seeds should be collected at optimum maturity when seed vigor, desiccation tolerance and longevity are expected to be highest.

6.1 Containers for collecting samples  Use paper bags for collecting seeds.  Use cloth bags that allow circulation of air (such as muslin bags) for collecting panicles or dry fruits.  Use open containers, such as baskets made of wire or bamboo or tubs to collect fleshy fruits.  Ensure that fruits are not squashed.  During transport, do not let fruits become too hot and ferment.  Use nylon-net bags for collecting samples as they allow air to circulate freely. Besides their use for collecting seeds, pods and fruits, they can be used for seed extraction and for drying extracted seeds. They are available in a range of mesh sizes.


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6.2 Processing of seeds in the field  Seeds collected with surface moisture should be dried in shade or a well-ventilated room. Use newspaper or blotting paper for drying. After drying transferring them to cloth or paper bags.  Seeds from dry dehiscent fruits (such as okra, sesamum) can be extracted by spreading the fruits on a tarpaulin under shade.  Mature fruits split open and release their seeds as they dry. Sometimes, additional impact such as raking or shaking is needed.  Remove empty fruits and debris, and transfer the seeds into cotton, nylon-net or paper bags.  Pulpy fruits (such as tomato and cucumber), extract the seeds carefully by hand, wash them under running water to remove pulp and mucilage, spread them in a thin layer to maximize aeration and allow them to dry in the shade.  Always maintain seeds in moisture-permeable containers such as cotton or paper bags, and ensure that air circulates freely between and through them.  Under hot and humid condition and during long collection mission dry seeds using desiccants such as silica gel (3:1).

 Keep alternate layers of packed silica gel and packed seeds in a large airtight container to reduce seed moisture.  Hold seeds inside fruits when seed material is small and inside fleshy fruits.  Avoid seed extraction if fruits require after-ripening or if seeds are delicate or recalcitrant.  Do not collect seeds naturally fallen on ground.

6.3 Transporting the collected material to the laboratory   

Maintain proper temperature and safe moisture content during transport. Use cushioned seed container to avoid damage during transportation. Take extra care while acquiring unique / rare seeds.

6.4 Minimum required information with plant / seed sample  

Samples should be accompanied by adequate information, especially plant/cultivar name, details of variety etc. with collection code. The minimum required information for plant collection is as below;  Common name and/or genus and species.  Collecting number (in prescribed format).  Location of collection site (Habitat description with GPS coordinates and elevation).  Collecting date (dd-mm-yyyy).  Morphological features of plant with habit.  Field photographs (preferably with habitat and close-up of reproductive organs).  Numbers of vouchers prepared (Minimum 3 with reproductive organs).


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Approximate quantity of seed supplied. Name and contact details of specimen collector ((Address, phone no. and e-mail). Name and contact details of specimen identifier (Address, phone no. and e-mail).


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7. Mushroom Collection Guidelines 7.1 Collection and drying of fungal specimens   

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The common proforma given in annexure-1 should be filled in for all specimens to be submitted. Obtain all necessary permits before setting off for any collection. Basic requirements for collecting macrofungi:  Suitable collecting boxes of varying size. Small collecting boxes may be required for smaller fungi, such as the Ascomycetes.  Roll of aluminium foil (preferred), greaseproof paper or greaseproof bags can be very useful for wrapping larger macrofungi. Do not use plastic bags, fungi deteriorate rapidly when wrapped in plastic.  Pocket knife, or trowel, to remove entire specimens from their substrate without damaging tissues.  GPS for recording accurate latitude, longitude and altitude readings. Alternatively, mark the position on a topographic map and include the map date field notebook. This can be a pocket-sized notebook, or a book of pre-printed specimen labels or the QMS field recording sheet (See Table-1).  Camera for photographing the form, colour and natural habitat of the fungi. Photographs should be linked to each specimen by a unique reference number.  Set of gloves, for protection when collecting in litter.  Tags for labelling the collection. Choose specimens which are in good condition and representative of the population’s variability. Ideally, the fruiting bodies should be produced by an individual mycelium (some of these can cover an extensive area) and collection from an isolated patch is therefore a preferred practice. A good specimen includes all parts of the fungus cap, hymenium (spore-bearing tissues) and stipe; and ring and volva where present. For some species the colour of the attached mycelium is an important diagnostic character and should be noted. The fungal material should be fertile i.e. it should have mature spores, as these are vital for identification. Note carefully the substrate (what the specimen was growing on/in) and the associated organisms (the species or genera of plants it was growing with). High Resolution Photographs should include the specimen growing in its natural habitat, a display of the specimen which shows the gills or pores, any unusual, distinctive or interesting features, a tag to provide scale and its unique reference number.


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Specimens should be wrapped in waxed paper or foil (Never use plastic) and placed in a suitable container for transport back to the laboratory. Good collection of medium–large fungi normally consists of at least five fruiting bodies in which representatives of each stage of development are present. For medium sized fruiting bodies, at least 10 would be required, for small-medium, at least 20, and for very small about 30 or so. In the case of extremely large fungi, a single sporocarp may suffice. Ensure that sufficient photographs are taken, however, to capture any variations that may occur in the taxon. Use a pocket-knife or a trowel to extract specimens from the substrate, taking care to collect the whole specimen including the base. Don’t collect by cutting the stalk of the fungus. Never press the specimen as done for plants. The transport to the laboratory should be done as quickly as possible in not more than two days so that mushrooms are as fresh as possible. If it is not possible to transfer them quickly to the lab morphological characters including size, color, and spore print should be taken and then the mushroom should be dried in a cool oven with a temperature not more than 40-42°C. Two to three mushrooms should be stored as museum specimen in glass jars containing 70:20:10 (ethanol: water: formaldehyde) as described in the liquid preservation section. Never freeze fresh samples. Clean all tools and implements that you have used on your foray/survey with 70% methylated spirits (or ethanol, if you have access). This is necessary in order to prevent the possibility of any transfer of contaminants, e.g. pathogens, between sites. For taking spore print follow the below given steps:  Spore prints are generally made on acid free white paper. In case the spore color is very pale or pink black paper can be used.

Steps to create a spore print:

Fold a square piece of white paper.

Cut in the middle.


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Place the stipe through the slot in the paper and suspend on the mouth of a plastic beaker/cup.

Once a spore print has been obtained, label it and place this on the dryer with the collection and other labels.

7.2 Data to be recorded in the field Fungi collections unaccompanied by field notes are of very little use.

7.2.1 Site information 1. Site name: If the name is not known, then describe how to get to the site from the nearest known locality. 2. GPS location: This can be recorded as lat/long. Most GPS devices will also record and store an altitude reading. 3. Habitat data: Should include landforms, slope, dominant plant species, and vegetation structure, for example ‘open forest’, ‘open woodland’ or ‘grassland’. 4. Record any evident management system or any disturbance, for example grazed paddock, recently burnt, or timber extraction.

7.2.2 Specimen information (field) The following information about fungal specimens should be recorded in the field: 1. Reference/voucher number: to uniquely denote each collection. 2. Genus and/or species name: if the name is not known, then a description of the type of fungus, e.g. agaric. 3. Substrate: what the fungus was growing on. e.g. leaf, insect, soil, log, twig, sawdust pile. 4. Associated organism: which can be either the species name for the vegetative material on which the fungus was growing, or in the case of mycorrhizal fungi, the name of the nearest likely mycorrhizal associate. 5. Color of the fruiting body, hymenium, and smell should be noted down.


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6. 7. 8. 9. 10.

Collector: the name and initials of the collector. Determiner: Name and initials of the person who identified the fungus in the field. Photographer: Name and initials of the photographer, if different from the collector. Morphological characters: Record the morphological characters as given in Table 1. Do not forget to fill in the Annexure II before submitting the specimen.

Table 1: Morphological characters required of identification of mushrooms (refer section 7.2.3) Locality: Associated species: Substrate: Cap: Shape: Diameter: Texture: Surface: Color: Cap margin: Flesh: Smell: Color: Gills (Gills/ Pores/ Teeth) Color: Attachment: Stipe Color: Size: Height ____mm Texture: Shape: Ring or Volva Spore Color: Size: Height ____mm

Width ____mm

Width ____mm


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7.2.3 Macroscopic description aid


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8. Soil collection guidelines for fungus isolation 

   



     

Ten to 30 g of soil should be collected using soil coring tube or similar device sterilized in 70% ethanol and dried just prior to collection. Before sampling the overlying litter or humus should be removed. The sample should be placed in sterile container or bag, labeled and stored in an ice box or cooler. The soil sample must be plated out within 4 days or frozen if plating cannot be done. The soil and sediment sample may be collected from terrestrial or shallow water environment using sterile containers. Presence of filamentous fungi is common in these environments. Standard plating techniques using serial dilution, four flame streaking or pour plate method on standard fungal media generally results in isolated colonies. Two to three sub-cultures generally result in pure colonies of the fungus. Once a pure culture is obtained the spore morphology must be recorded. A high resolution photograph must be taking showing the spores as well as mycelia. For DNA isolation freshly grown mycelia work best. For long-term preservation the fungal spores must be cryopreserved in glycerol or lyophilized. Permanent slide of spore should also be prepared.


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9. Bryophyte, Lichen and Algae Collection Guidelines 9.1 Bryophyte specimens (mosses, liverworts)       

Collect fertile material with capsules, if possible. If the bryophyte is growing on a tree collect some bark. Collect at least a 4 × 4 cm patch. Place collection in a paper envelope (never plastic) and air dry as rapidly as possible in a wellventilated area. Do not press the specimens. Bryophytes can often be found growing intertwined. It is important to check carefully that capsules belong to the plant that they seem to be growing from. Bryophytes should be kept cool and moist or air dried immediately. Liquid preservation is done in commercial formalin as described in liquid preservation section.

9.2 Lichen specimens  

Simply place the lichen specimen in a paper bag or envelope. Do not place in plastic bags. Dry in a well-ventilated area, as rapidly as possible. Collect some of the substrate, such as rock or wood, but trim excess substrate.

9.3 Algae specimens    

 



Aquatic algae can be temporarily stored in water from the collecting site for a couple days but must be transported on ice at the earliest and no later than two days to the lab Marine algae must be covered by waxed paper or muslin before pressing between newspaper (which otherwise adheres to the specimen). Preparation of vouchers must be done in field itself. For preparing the voucher specimen use the floating out technique followed by pressing. With the specimen in a shallow dish of water, place a piece of stiff white paper under the specimens and then carefully lift out the paper with the specimen on top. Press between newspapers. If possible one should go with a field microscope to do a general microscopy Specimens collected should be in three sets (i) Specimens for microscopic analyses should be fixed in 4%formaldehyde solution, (ii) unfixed subsamples dried on herbarium paper as voucher and (iii) another in a vial with silica gel for DNA extraction and barcoding. Liquid preservation is done in commercial formalin as described in Liquid preservation section.


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9.4 Liquid preservation of mushroom, algae, lichen and bryophyte Algae/Mushroom/Bryophyte can be stored initially in a bucket, jar, bottle or plastic bag, with some water from the collecting site. The container should be left open or only half filled with liquid and wide shallow containers are better than narrow deep jars. Note that glass is reportedly not satisfactory for some due to its inherent alkalinity damaging cells. However, glass vessels are commonly used to collect specimens. Samples can also be refrigerated or kept on ice soon after collection from field and can be kept alive for short periods (a day or two). Place collected sample at cool place with reduced light. For long-term storage, specimens can be preserved in liquid (see below), dried, or made into a permanent microscope mount (preferably all three). Even with ideal preservation, examination of fresh material is sometimes essential for an accurate determination. For liquid preservation the following preservatives/ fixative can be used.

1.

2. 3. 4. 5.

Specimen can be directly preserved in formalin (40% formaldehyde) at 1/10 to use as fixative. (Note: Formaldehyde is thought to be carcinogenic hence avoid contact with skin, eyes and air passages). Specimen can be preserved in FAA (by volume, 40% formaldehyde 1: glacial acetic acid 1: 95% alcohol 8: water 10) or 6-3-1 (by volume, water 6: 90% alcohol 3: 40% formaldehyde 1) solutions Instead of FAA solution, standard alcohol and water mix (e.g. 70% ethyl alcohol or industrial methylated spirit) can also be used to preserve specimen. Algae can be kept in diluted formalin for a number of years, but the solution is usually replaced by 70% ethyl alcohol with 5% glycerin (the latter to prevent accidental drying out). Lugol's solution is commonly used for short-term (e.g. a few months, but possibly a year or more) storage. Dissolve one gram of iodine crystals and two grams of potassium iodide in 300 ml of water. Use three drops of this solution in a 100 ml sample.


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10. Invertebrate collection guidelines 10.1 Collecting Invertebrates for preservation and DNA barcoding 

 

All collections must be processed in duplicate sampling: (i) for voucher preparation and (ii) for DNA barcoding. o For voucher preparation: At least three specimens must be collected, fixed and stored as per the table given in Annexure-IV. o For DNA barcoding: The sample size depends on the size of the specimen collected. For small insects 3-5 specimens are required corresponding to a minimum of 100 mg material for DNA extraction. The sample which is to be used for DNA extraction should not be stored in formalin. The invertebrates should be collected using proper techniques to prevent damage. Damaged or incomplete specimens should be avoided. The collections can be made using passive and active technique. Passive technique uses Pitfall trapping, Leaf Litter extraction, Yellow pan trap, Flight intercept traps, glue trap, pheromone trap. Yellow pan trap The collection fluid attracts insects in search of water but more specifically many wasps and certain flies are attracted to the color yellow, which is essential for the method.

Leaf litter extraction

Flight Intercept trap A variety of intercept traps have been designed to catch flying insects but all work on the same principle, that is an insects flight is impeded and in the process the insect is channeled into a container with preservative.


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

Active technique involves hand picking, chemical knockdown, beat sampling, light sampling, sweep sampling.



Beating tray, sweeping net, butterfly net and pond net may also be used

Beating Tray





Butterfly Net

Pond Net

Several kind of traps may also be set such as pitfall trap, Aspirator, Malaise trap, Mercury Vapor Lamp, Tullgren funnel

Aspirator

 

Sweeping Net

Pitfall Trap

Mercury Vapor Lamp

Tullgren funnel

After collection the invertebrate must be immobilized in a relaxed state. Insects must never be transported in zip seal bags. Always use sterile containers of suitable size for the transfer of insets. Invertebrates destined for DNA isolation should ideally be collected in 70-95% ethanol while still alive. Container must be leak-proof. Container should be kept in ice-box with icepack immediately after collection and while transport.


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   



Invertebrates must be labeled with collection place, unique collection number and collection date. High resolution photograph of the insect in its habitat, dorsal and ventral photographs should accompany the invertebrate submission. For voucher preparation and long term storage of invertebrates group specific fixatives and preservatives are given in Annexure IV: The preservation procedure generally follows three steps: Step I: Formalin - 1 week (fix soft tissue) Step II: One day (leach out the formalin) Step III: Alcohol - long term storage All specimen stored in liquid medium must be transport in cool and dark condition.

10.2 Methods for Dry Mounting Dry-mounting and pinning is recommended or even necessary for the Lepidoptera, Coleoptera, Hymenoptera, Diptera, Heteroptera, Orthoptera, Odonata and Neuropterida. All other taxonomic insect groups as well as insect larvae, Arachnida, Myriapoda, and Crustacea are best killed and preserved in 70 -80% ethanol. Two methods for dry mounting are well known; best suited alternative according to the insect type should be used.

10.3 Pining specimen on mount strips 



Direct pinning of specimens should be done with an insect pin that fits to the specimen’s size. The standard size for insect pins is 1 or 2 which fits for most Lepidoptera, large Hymenoptera and many Coleoptera. Larger specimens should be pinned with size 3, 4 or 5, while for smaller specimens, pins with size 0, 00, or even 000 are available. However, it should be noted that pins with size 0 or smaller are difficult to handle. Pinning through the labels or through the paper layer of insect boxes should be done with great care as the thin pins are easily twisted. Direct pinning of insects should be done in a way that the pin is in a right angle to the body. The insect specimens should rest about 1/3 of the pin length away from the top. This gives enough space to handle the specimens, i.e. to grip the top of the needle by the thumb and the index finger without damaging the specimens with the fingertips or fingernails the specimens should not rest further away from the top of the needle as the bottom space is needed for collection and determination labels. The pin is usually inserted Inserted needle is in a through the mesothorax but the exact insertion point depends on right angle to the body the insect group. Bugs (Heteroptera) are pinned submedially of the insect through the scutellum. In Hymenoptera and Diptera the insertion point is slightly removed laterally from the median axis. This allows median sculpture or bristle


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patterns to remain intact and visible medially and also on one side. Beetles (Coleoptera) are pinned through the right elytron. In butterflies and moths (Lepidoptera) the insertion point is in the middle of the mesothorax.

10.4 Gluing Specimens onto paper cards Very small insects (body length below 3 mm) should never be directly pinned as specimens will always be damaged or lost over time. Card mounting is the method of choice for small beetles, bugs and Micro-Hymenoptera.  Beetles and bugs should be glued on rectangular cards.  Small Hymenoptera can either be glued on rectangular cards or on the tip of card points, which are small triangles of stiff paper.  The paper cards with the mounted insects should be pinned with common insect pins of larger size (sizes 3 to 5). Pins of that size can easily be inserted through the paper cards. The glue should be water-soluble or ethanol soluble so that specimens can easily be removed from the card in case they need to be re-mounted without being damaged. Seccotine (fish glue) is a water soluble glue and also recommended for rectangular cards and shellac (a resin produced by lac bugs, Coccoidea) is recommended point cards.

10.5 Preservation and transport of arthropods which are to be dry mounted 



Use cardboard tubes of different diameter for transport. Both sides of a tube are to be closed with a cotton plug. The freshly killed sample should be placed directly inside the tube. It can be transferred into a soften chamber afterwards for preparation of setting and mounting. This method is feasible for strongly sclerotized specimens (e.g., beetles) which need to be stored during fieldwork before they can be dry mounted in the laboratory. However, scaled, pilose, and coated specimens could be rubbed off during transport. Cardboard tubes are preferred over glass or plastic ones as they are lightweight, fracture-proof and absorb moisture. The tubes are to be stored inside of feasible sealed transport boxes containing crumbs of thymol which prevents moulding. Use butterfly envelopes of different size, made of vellum. This method should be used to transport or even store dry unset Macro-Lepidoptera and winged insect orders like Odonata and Neuroptera. It is important to “close” the specimens inside of the envelope with the wings folded upwards. This protects the more important upper sides of the wings (as identification characters) against rubbing and facilitates later setting and spreading. If this is not possible in case of rigor mortis, the specimens have to be injected by syringe with ammonium chloride to soften rigor. Placing more than one specimen into one envelope should be avoided as they may damage each other during transport.


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Card Mounting. A label can be sticked to the other card and inserted in parallel to the specimen card.

Card points are small triangles of Cardboard tubes are ideal for stiff paper that allow specimens to hard bodied insects, such as be observed from all sites if beetles. specimens are glued laterally to the tip of the triangle.


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11. Fish Collection Guidelines    

An important part of any field program is to obtain and carry all relevant permits for sampling activities in the study area. It is advised to take help of a local person as a guide. Always carry a field identification key/ guide. It is important to acquaint with the general knowledge of the different body parts of the fish.

Typical fish labeled with the body parts is given below;



As soon as the fish is captured the length of the fish should be measured. Fork length, total length and standard length should be measured as shown below


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 

If possible the weight of the fish should also be taken after draining the excess liquid. Where-ever possible all collections of fish should be made in two sets, one for (i) Voucher preparation and the other for (ii) DNA barcoding.

For voucher preparation the fish must be anaesthetized to kill so that it remains in relaxed condition. Thereafter it must be fixed and stored in isopropyl alcohol.

11.1 Fixatives 





Formalin is commonly used to preserve collected specimens and it is available in liquid or powder forms (Full strength liquid formalin is actually 37% formaldehyde dissolved in water). It is recommended that a solution of 10% formalin be used for the preservation of fish specimens. To make a 10% solution of buffered formalin, combine 1 part full strength formalin with 9 parts distilled water and add approximately 3 ml of borax (buffering agent) per liter of solution. Formalin is slightly acidic and will de-calcify and soften bony structures. The addition of a buffering agent helps to slow down this process. Alcohols, such as ethanol and iso-propanol, are also commonly used to fix and preserve fish specimens, especially if skeletal structures such as otoliths are to be examined. Alcohol is a poor fixative and is not recommended for fixation. An alternative for preserving specimens is to quickly freeze them in dry ice or liquid nitrogen. This is one of the best methods to preserve the colors and tissues of the specimen. Samples must remain frozen until they arrive at the laboratory and can be permanently preserved.

11.2 Fixation Procedures 







Specimens should be fixed soon after collection to limit deterioration of the tissues. All specimen must be killed prior to fixation. To fix the specimen, place it in a wide-mouthed glass, and fill the jar with the fixative solution. Specimens should be inserted into jars head first to make them easier to remove from the jars in the laboratory. Different species captured in the same set can be fixed and stored together. The fish must be preserved in as natural a state as possible. Where possible, the specimen should float freely in the jar to avoid curling or bending. Before immersing large specimens, fixative should be injected directly into the body cavity to facilitate penetration and preservation of the internal organs. The stomach should also be incised for internal fixation in order to prevent rotting due to digestive juices. Two labels are needed for the specimen: a waterproof specimen attached to the jaw or inserted into the mouth or opercular area of each specimen and a waterproof data label on the outside of each jar. All labels must be written in pencil. The specimen label contains.  the fish identification number  species name of the fish


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  

collection method code collection date A brief description of the habitat and catch may also be included.

For DNA barcoding Tissue samples for DNA extraction should be frozen or preserved in fresh 95% EtOH and stored in a cool place, preferably in a freezer.  





Large pieces of tissue should be cut into small pieces (5-7 mm) to permit adequate fluid penetration. Two archival quality tissue samples will be immediately collected from each specimen;  one frozen to preserve the broadest array of molecular characters possible,  one placed into a preservation fluid, such as EtOH, to serve as a back-up in case of a meltdown or loss of the frozen specimen. Several tissues are suitable for DNA extraction from fishes. These include the following:  Musculature: remove one or more cubes (5 – 7 mm) of lateral muscle from the right side of the specimen.  Gill tissue: remove one or more gill arches with attached filaments from the right side.  Eye: remove the right eye from extremely small specimens such as larvae. For species with small body size, entire specimens can be placed in preservative in lieu of subsampling. This should be avoided unless a series of conspecifics are available for fixation in formalin for standard morphological analysis.


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12. Mammalian Tissue and Aves Feathers Collection Guidelines 

  



Because of their important economical, conservation, and biomedical status, animals are among the most highly regulated living organisms when it comes to collecting, deposition, and international transfer. It is imperative that any prospective contributor to the animal barcoding campaign is well aware of the national, international, and institutional regulations pertaining to the collection, storage and distribution of specimens and any derivative biomaterials and conducts his/her operations in a compliant manner. All necessary permits must be obtained prior to any collection. Any infection to the animal should be notified while submitting the tissue. Vital field for DNA barcoding involve detailed locality with coordinates and elevation, collection date, name(s) of collector(s), sex and age of the vertebrate. When collecting tissue for DNA barcoding.  Avoid tissue from kidney or liver as they are enzymatically rich which adversely effects DNA. If kidney or liver cannot be avoided then they should be submitted within 24h to the lab for processing.  Preferred source of tissue for barcoding studies is muscle or gonads stored in 95100% ethanol. Ethanol preserved tissue should be stored at -20 ˚C or lower.  The tissue can also be stored in tissue protectant which comprise of 70% PBS (NaCl 8g, KCl 0.2 g, Na2HPO4 1.44g, KH2PO4 0.24 g in 1L) and 30% glycerol.  The tissue must be minced thoroughly before fixation and the fixative should be changed after the initial first week.  The volume of tissue to fixative should be less than 1:10.  If the tissue is dry i.e. obtained from a carcass, museum can be stored as it is and fixative should not be added. When collecting feather (in case of birds)  Make sure that the superior umbilicus of the feather is present.  A minimum of 3-4 feathers should be collected.  In case of large feathers (rectrices) only the section of the feather shaft containing the superior umbilicus is used as sample for DNA isolation and barcoding.  If possible freshly plucked feathers (from breast or retrices) should be preferred.  Feather characteristics include macroscopic attributes like plumage pattern, texture and size should be noted followed by microscopic attributes of the plumulaceous barbs.  High resolution photograph of the animal in its habitat should accompany the tissue/ feather submission.


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Figure: Feather specimen with umbilicus


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13. Amphibian and Reptile Samples Collection Guidelines    





 







All necessary permits must be obtained prior to any collection. Any infection to the animal should be notified while submitting the tissue. For amphibians and reptiles, a broad spectrum of tissue samples can be used for the DNA extraction, amplification, and sequencing of the DNA barcoding process. Wherever possible the DNA barcoding study on amphibians and reptiles should be supplemented with adequate collection of representative voucher specimens for subsequent taxonomic study. In some cases, however, the killing of the animals and their deposition in public collections is undesirable or impossible; for instance, in critically endangered species blood samples (most reptiles with middle to large body size), tail tips (e.g., lizards and snakes), toe clips (frogs and salamanders), fin clips (aquatic salamanders and tadpoles), and scale clips (snakes) can be taken. In moderate- to large-sized specimens, buccal saliva swabs give good results. Swabs can be air-dried and then stored, but in humid environment, storage in pure ethanol is preferable. A further alternative source of DNA are the exuviae (shed skin, especially of snakes) but if encountered in the wild, the identity of the specimen will not always be ascertainable. For dead animals including museum specimens that have not been fixed using formalin, liver, heart, or muscle tissues represent optimal sources of genomic, and especially mitochondrial genomic DNA. In general, ethanol-fixed and ethanol-preserved museum specimen are ideal for DNA barcoding. All tissues must be stored in 95% ethanol or tissue protectant 70% PBS (For preparing 1 L of PBS: NaCl 8 g, KCl 0.2 g, Na2HPO4 1.44 g, KH2PO4 0.24 g) and 30% glycerol. Whenever samples are collected, documentation of color in life should be a crucial component of each study. Digital high resolution photos should be made both of dorsal and ventral sides. If voucher specimens are collected, the photos will later serve to know their life color since many amphibians and lizards (like colorful geckos or chameleons) will lose their color within hours in preservative (and sometimes in life while being handled). If voucher specimens are not collected, photographic documentation becomes even more important and should include lateral, dorsal, and ventral photos as well as close-ups (e.g., of head and ventral sides of hands and feet), to make sure diagnostic characters will be recognizable and allow to subsequently verify species identification of barcoded specimens. If possible in case of frogs vocalizations should be noted i.e. observation of the movement of the vocal sac must be noted and recorded as “call voucher”. If this can be achieved, this “call voucher” specimen should be collected separately, documented photographically, and its call recording and tissue sample marked unambiguously. Preferably capture amphibians by hand but wash hand frequently and in between two specimens. It is recommended that gloves be used and changed between two specimens.


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14. Key references 

CCAC species-specific recommendations on: amphibians and reptiles (Canadian Council of Animal Care), www.ccac.ca/Documents/Standards/Guidelines/Add_PDFs/Wildlife_Amphibians_Reptiles.p df



Fish sampling techniques, http://nptel.ac.in/courses/120108002/module5/lecture11.pdf



How To Preserve Fish Specimens for Long-Term Storage or Shipment, http://research.amnh.org/vz/ichthyology/congo/other05.html



http://www.ento.csiro.au/education/preserving.html#spiders



http://www.lib.ncsu.edu/agnic/sys_entomology/preserve.html



Iliffe R, 2006, British Mycological Society Recording Network: Guidance Notes In: Collecting and recording fungi, British Mycological Society.



Invertebrate Collection Manual: A guide to traditional invertebrate collection methods, http://australianmuseum.net.au/Uploads/Documents/9382/The%20Invertebrate%20Collec tion%20Manual.pdf



Iwanycki N, 2009, Guidelines for Collecting Herbarium Specimens of Vascular Plants, Royal Botanical Garden, https://www.rbg.ca/Document.Doc?id=125



Lijtmaer DA, Kerr KCR, Stoeckle MY, Tubaro PL, 2012, DNA Barcoding Birds: From Field Collection to Data Analysis, Methods in Molecular Biology, Vol. 858, pp 127-152.



Patrick Leonard (2010) A Guide to Collecting and Preserving Fungal Specimens for the Queensland Herbarium (Version 3.2).



Rudnick JA, Katzner TE, Bragin EA, Woody JA, 2007, Species identification of birds through genetic analysis of naturally shed feathers, Molecular Ecology Notes, 7(5), 757-762.



Steinke D, Hanner R, 2011, The FISH-BOL collaborators’ protocol. Mitochondrial DNA, 22(S1): 1014.



Vences M, Nagy ZT, Sonet G, Verheyen E, 2012, DNA barcoding amphibians and reptiles, Methods in Molecular Biology, Vol. 858, pp 79-107.



Wilson R, 2005, Marine invertebrate sample processing procedures, http://researchdata.museum.vic.gov.au/amit/Marine_sample_processing_8Sep2005.pdf


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Annexure I: Proforma for Deposition of specimen 1. Name of the depositor: 2. Designation: 3. Address of the organization: 4. Phone (with STD Code): 5. Mobile: 6. E-mail address: 7. Number of samples to be submitted: 8. Collection site with GPS details: Terms and conditions for submission: 1. The material/ specimen should be collected as per the guidelines. 2. All the details of the specimen should be provided in prescribed format (Annexure II). 3. The sole responsibility of sample deposition will be of the depositor. It is understood that samples/ specimens submitted to Gujarat Biodiversity Gene Bank (BioGene) are collected in compliance of national/ international regulatory requirements. 4. All specimen(s) once deposited to BioGene will be sole belongings of BioGene, GSBTM, DST,GoG and will not be returned back. 5. I/we own the copyright and have the consent and permission of each author to transfer copyright of the said contribution. I hereby assign to the Gujarat Biodiversity Gene Bank (BioGene) full copyright and all rights under it, including, but not limited to all rights for publication in paper, electronic, and facsimile, formats, and for electronic capture, reproduction, and licensing in all formats, in whole or in part, now and in perpetuity, in the original and all derivative works. 6. BioGene will make best efforts for the preservation and maintenance of the specimen; but it will not take any responsibility of loss of specimen because of any accidental hazards. 7. The detailed information asked in proforma reflects the academic authenticity. This will help to develop the information base which is required and desirable for match making, or culture exchange and culture transfer for potential organisms. Hence the depositor is requested to provide the detailed scientific information of the organism. I/ We have read the above terms and conditions and hereby agree to above terms and conditions for depositing specimen. Signature with official stamp Date: Place:


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Annexure II: Essential information required for submission of specimen to Barcode of Life Database (BOLD) Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Collection data Habitat including details of sample collection like place, ecological description, season Sample ID Field ID Collection code Collection Date Collector Phylum Class Order Family Genus Species Identifier Identifier Email Identifier Institution Identification Method Voucher Status Country State Region Sector Exact Site Latitude (in degree decimal format) Longitude (in degree decimal format) Elevation (meter) Photographer Digital photos of specimen in habitat (preferably in 300 dpi) Close-up photograph of specimen with taxonomic / identification features (preferably with scale in 300 dpi)

Note: Refer BOLD Handbook for submission parameter (http://www.boldsystems.org/libhtml_v3/static/BOLD_Handbook_Oct2013.pdf)


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Annexure III: Optional information for submission of specimen to Barcode of Life Database (BOLD) Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Collection data Vernacular name Identification method Taxonomy notes Voucher status Tissue descriptor Associated taxa Associated specimen Sex Reproduction Life stage Specimen collection depth / elevation (meter) Habit Sampling protocol Measurement GPS source Coordinate Accuracy Event Time Collection Date accuracy Collection notes Site Code

Note: Refer BOLD Handbook for submission parameter (http://www.boldsystems.org/libhtml_v3/static/BOLD_Handbook_Oct2013.pdf)


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Field Collection Guidelines BioGene Umbrella Programme

Annexure IV: Parameters for sampling, voucher preparation, transportation and preservation of biomaterial from various sources Required Sample and Information Sr. No.

Sample Type

Required Biomaterial

1

Soil

Pure Fungus cultured on Petri Plate (preferably in mycellial form)

2

Mushroo m

3

Lichen

Complete Fruiting body with fungus cap, hymenium (spore-bearing tissues) and stipe; and ring and volva where present Thallus with some amount of substratum

Voucher

DNA Barcoding (5 replicates)

Specimen (Preferably 3-5) 3 lyophilized copy and 3 live copy on solid media; 3 permanent slide of spores

Photograph (Pref. 300 dpi) Plate photograph having mycelia and spores; microscopic photograph of spore

3 dried mushrooms and 3 preserved in FAA or Lugol solution (see detailed guidelines); Spore print

Dorsal and ventral photographs showing color/ shape of hymenium

20-100 mg of tissue from the internal part of fruiting body collected under sterile conditions and proper sterilization

100-200 mg tissue in paper bag

Photographs in habitat, close-up of thallus

100-200 mg of tissue


20-100 mg mycellia for each replicate

Specimen Storage/ Preservation Medium

Specimen Transportation

Glycerol stock for cryopreservati on; Generally PDA (may be specific for some species) Mycellial form on PDA, Dried/ FAA preserved / lyophilized fruiting body

Plates containing full mycellial growth under ice conditions; glycerol stock in dry ice/ liquid nitrogen

Dried in paper bag preserving the structure

In a paper bag under dry conditions

Remarks

Fresh mushroom wrapped in butter paper in cold dark condition; dried / lyophilized mushroom at RT

Page | 45


4

Algae

Thallus

5

Bryophyt e

Fertile material with capsule

6

Higher Plants

Plant specimen collected during reproductive stage (with flower and/or fruiting organ)

Specimens for microscopic analyses should be fixed in 4%formaldehyde solution, and unfixed subsamples dried on herbarium paper as voucher Specimens for microscopic analyses should be fixed in 4%formaldehyde solution, and unfixed subsamples, air dried as voucher Plant vouchers on herbarium sheet


Photographs in habitat, close-up of thallus


Voucher specimen in dark/ dry condition; sample in silica containing zip seal bags for DNA isolation

Dried material in dry dark condition/ if material fresh then ship in ice box

Photographs in habitat, close-up of the sporophytic stage


Voucher specimen in dark/ dry condition; sample in silica containing zip seal bags for DNA isolation

Dried material in dry dark condition/ if material fresh then ship in ice box

Plant photograph in habitat/ closeup photograph covering details of reproductive organs

5-7 young leaves in zip seal bag containing dried silica (minimum 20 mg for each replicate)

Herbarium under dark/ dehumidified conditions; Silica gel sample under dark condition; Lyophilized sample at RT

Voucher sample from field with herbarium press and DNA sample with silica gel or in icebox with ice pack

Page | 46


7

Porifera

Whole animal (colony) as tissue and voucher source

70% ethanol

Photo in habitat

100-200 mg of tissue in ethanol

70% ethanol

8

Cnidaria( Octocora llia)

Whole animal

70% ethanol

Photo in habitat


70% ethanol

9

Cnidaria (Scyphoz oa)

Whole animal

Fixed in 4% formalin and preserved in 70% ethanol

Photo in habitat


70% ethanol

10

Cnidaria (others)

Whole animal


Photo in habitat


70% ethanol


Preferably snap chilled samples in liquid nitrogen; Alternatively it can be preserved and shipped on ice or with cool packs Preferably snap chilled samples in liquid nitrogen; Alternatively it can be preserved and shipped on ice or with cool packs Preferably snap chilled samples in liquid nitrogen; Alternatively it can be preserved and shipped on ice or with cool packs Preferably snap chilled samples in liquid nitrogen; Alternatively it can be preserved and shipped on ice or with cool packs

Formalin will render most sponges unidentifiabl e

Formalin will dissolve spicules and render many octocorals unidentifiabl e.

Page | 47


11

Platyhel minthes

Mature stage of animal

12

Annelida

13

Arthropo da (Crustace a)


Dorsal and ventral side of animal. Prefer to take photo in habitat


70% ethanol

Preferably snap chilled samples in liquid nitrogen; Alternatively it can be preserved and shipped on ice or with cool packs

Whole animal as Specimen fixed in tissue source 4% formalin followed by 70% ethanol (see detail guideline)

Dorsal and ventral side of animal. Prefer to take photo in habitat.


70% ethanol


Whole animal / muscle tissue



70% ethanol


Specimen fixed in 4% formalin followed by 70% ethanol (see detail guideline)


Fix living specimens on frozen 4% formalin or narcotise (freezing or propylene phenoxytol or MgCl2). Otherwise probably unidentifiabl e. Leeches and some polychaete families are easier to identify if anaesthetize d

Page | 48


14

Arthropo da (Insects)

Leg with muscle / whole animal

Insect must be stored in dry condition using mounting and pinning Fixed in 4% formalin, 70% ethanol or in 1% Chloral hydrate, Store at -20˚C

Dorsal and ventral side of animal. Prefer to take photo in habitat. Photo in habitat


Dry mounted specimen

Room Temperature

See detail guidelines

15

Mollusca (Opistho branchia)

Whole animal / muscle tissue (Use knife or sharp blade to remove animal from shell)


70% ethanol


Narcotise (freezing or propylene phenoxytol or MgCl2) if at all possible; recording colour in life are also very useful

16

Mollusca (Other)

Whole animal / muscle tissue (If required use hand digging tools)

Fixed in 4% formalin, 70% ethanol or in 1% Chloral hydrate, Store at -20˚C

Photo in habitat


70% ethanol, Outer shell

Soft tissue

70% ethanol

Dorsal and ventral side of animal. Prefere to take photo in habitat.


75-85% ethanol

Preferably snap chilled samples in liquid nitrogen; Alternatively it can be preserved and shipped on ice or with cool packs Preferably snap chilled samples in liquid nitrogen; Alternatively it can be preserved and shipped on ice or with cool packs

17

Echinode rmata


Formalin will render many echinoderms unidentifiabl e, especially holothurians

Page | 49


18

Tunicata

Soft tissue




19

Fish

Entire fish if smaller than 1215 inch

Lateral photographs with fin details; dorsal and ventral photographs for flat fishes

20

Amphibia and Reptiles

Preferably mature animal, if collecting developmental stages prefer to collect it with a mature organism for identification

3-5 fishes for smaller specimens (12 inch) take field photograph Fixed and preserved in FAA

Tissue of Storage in FAA Musculature (5 – 7 mm) from right lateral muscle; Gill tissue; Eye tissue preserved in Ethanol Prefer 1-2 ml Storage in FAA blood samples with EDTA; For large animals take 3-5 buccal saliva swabs;Minimum 500 mg tissue sample from tail tips (e.g., lizards and snakes), toe clips (frogs and salamanders), fin clips (aquatic salamanders and tadpoles), and



70% ethanol

Preferably snap chilled samples in liquid nitrogen; Alternatively it can be preserved and shipped on ice or with cool packs Preferably snap chilled samples in liquid nitrogen; Alternatively it can be preserved and shipped on ice or with cool packs Preferably snap chilled samples in liquid nitrogen; Alternatively it can be preserved and shipped on ice or with cool packs

Page | 50

Aves and Mammal s

Blood, tissue, feather, hair or nail with Follicle tissue

Stuffed bird, Feather having superior umbilicus, Muscle tissue


21

Front and lateral photograph. Prefer to take photo in habitat.

scale clips (snakes) Prefer 1-2 ml blood samples with EDTA; Minimum 500 mg muscle tissue; 3-5 feathers / hairs / nail with follicle tissue Stuffed Bird, FAA preserved material


Page | 51

BioGene Umbrella Programme

Field Collection Guidelines

Gujarat Biodiversity Gene Bank Gujarat State Biotechnology Mission Department of Science & Technology, Government of Gujarat 9/11, Udyog Bhavan, Gandhinagar – 382011 Ph. 91-79-23252197, Fax: 91-79-23252195, url: btm.Gujarat.gov.in; biogene.in

Field Collection Guidelines

Field Collection Guidelines

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