Palynology: History, branches, basic principles and application, collection of polleniferous materials from modern samples (i.e. living flowers, spider webs, tree barks and honey samples) and sediments

1. Palynology: History, branches, basic
principles and application, collection of
polleniferous materials from modern samples (i.e.
living flowers, spider webs, tree barks and honey
samples) and sediments
Birbal Sahni Institute of Palaeosciences, Lucknow
53 University Road, Lucknow-226007, U. P., India

2.  Organisms of the whole living world:
Plantae (entire Plant Kingdom) and Animalia
(entire Animal Kingdom).
 Linnaeus (1753), based on the absence or
presence of flower: Cryptogams- Plants
without flowers and Phanerogams- Plants
bearing flowers.
 Cryptogams (flowerless and hence
seedless): Thallophyta (plant body is an
undifferentiated thallus), Bryophyta (plant
body thalloid or a leafy axis lacking roots)
and Pteridophyta (plant body differentiated
into roots, stem and leaves).

3.  Phanerogams (plants bearing flowers and
hence also known as seed bearing plants:
Spermatophyta: Gymnosperms (plants bearing
naked seeds) and Angiosperms (plants bearing
closed seed).
 Tippo (1942): Plant Kingdom- Thallophyta (no
embryo: Bacteria, Lichen, Algae-Phycophyta and
Fungi-Mycophyta) and Embryophyta (with embryo).
 Embryophyta: Bryophyta (no vascular tissue and
Trachaeophyta (with vascular tissue).
 Trachaeophyta: Pteridophyta (no seed) and
Spermatophyta (with seed).
 Spermatophyta: Gymnosperms (naked seed) and
Angiosperm (covered seed).

4. Copeland’s (1956) four Kingdom Classification
 Monera (prokaryotes: Bacteria and
Cyanobacteria)
 Protista (Eukaryotes: all other plants
and animals)
 Metaphyta (multicellular plants:
bryophytes, pteridophytes, gymnosperms
(archegoniates) and angiosperms
 Metazoa (entire animal kingdom excluding
protozoa).

5. Whittaker’s (1969) Five Kingdom Classification
 Monera (prokaryotes)
 Protista (unicellular Eukaryotic
organisms, unicellular and colonial algae,
dinoflagellates, diatoms, slime moulds, and
protozoa)
 Plantae (multicellular plants)
 Fungi
 Animalia (multicellular animals)

6.  Organisms: Protobiota (acellular organisms-
Viruses) and Cytobiota (all cellular organisms)
 Cytobiota (based on absence or presence
of well organised (true) nucleus)- nuclear
membrane and nucleolus: Prokaryotes and
Eukaryotes

7. Plant Kingdom: a Historical account
 Otto Brunfels (1464-1534) was the first
grouped the plants into Cryptogams
(imperfecti) and Phanerogams (perfecti).
 Theophrastus (370-285 BC) – Father of
Botany ; first classified the plants into
herbs, undershrubs, shrubs and tress.
 Albert Magnus (1193-1280) for the first
time distinguished between monocot and dicot
plants.
 John Ray (1628-1705) for the first time
established the presence of one or two
cotyledons in angiosperm, which were grouped
into dicots and monocots.

8.  Carolus Linnaeus (1753) : Binomial system
of Nomenclature (Species Plantarum-1753). ;
Sexual System of Classification; Father of
Systematic Botany.
 Robert Brown (1773-1858) : Gymnosperms were a
separate group with naked ovules.
 Artificial System (based on only one or a
few characters; Theophrastus, Magnus,
Brunfels, Ray, Linnaeus)
 Natural System (all important characters
are taken into account; Adanson, A. P. de
Candole, Bentham and Hooker (Genera
Plantarum- 1862, Brown)

9.  Phylogenetic System (classification
is based on the genetic relationship of
the organisms and evolutionary sequences
(primitive vs. advanced characters;
Sachs, Eichler, Engler & Prantl-Die
naturlischen Pflazenfamilies, Wettstein,
Bessey, Hutchinson-Families of Flowering
Plants, Cronquist, Takhtajan, Dalgrehn)

10. PALYNOLOGY
 Study of pollen grains and spores
 Greek verb: palunō, which means I strew
or sprinkle; palunein: to strew or
sprinkle;
 Greek noun: pale- dust, fine meal
 Latin word: pollen- fine flour, dust
 and Greak noun: logos- to study, word,
speech
 The term palynology was coined by Hyde and
Williams (1944).

11.  Palynology is the study of microscopic
organic structures which are resistant to
treatments with HF (hydrofluoric acid).
 All entities found in palynological
preparations.
 Microscopic structures: Palynomorphs
(Tschudy, 1961), which include, other than
pollen grains and spores, acritarchs,
dinoflagellate cysts, scolecodonts.
 Palynofacies (Combaz, 1964): assemblage
of phytoclasts (plant-derived, more or less
resistant-walled particles occurring in a
sediment). e.g. palynomorphs, wood
fragments, cuticle

12.  Palydebris (Manum, 1976): Palynofacies
minus palynomorphs; all palynomorph-sized
particle in a sediment excluding those that
actually are palynomorphs but include wood
fragments, cuticle and some animal remains

13. POLLEN
Pollen grain is the first cell of male
gametophyte which is unicellular, haploid
and performs the function of pollination.
Pollen is formed in the anther of
flowers by the reduction division of pollen
mother cell (PMC). Initially, they are
formed in tetrad. Most pollen are liberated
as a single grain (monad), but in some
families such as Ericaceae, Orchidiaceae,
Typhaceae, etc. the grains remain united in
tetrads.

14. .
Stamen
T.S of anther
Pollen development

15.  Nature’s Fingerprints of
Plants
 found from Late Ordovician
(445-458 Ma to the Holocene
(11.7 ka)
Plants produce either pollen
or spores
Pollen - carries the male sex
cells of flowering plants
(angiosperms) and cone
producing plants (gymnosperms).
Spores – asexual reproductive
bodies of cryptogams (ferns,
mosses, algae, fungi, etc).
Pollen grains: Golden Dust, Wonder Dust of
Nature

16. Dinoflagellate Alga Fungal spores

17. Dicotyledonous tetrad
Monocotyledonous tetrad
POLLEN TETRAD
Polar axis: An imaginary line connecting the
proximal and distal poles.
Equatorial axis: The line perpendicular to
polar axis, dividing the pollen into two
equal halves.
Proximal face: The part of pollen which faces
proximal pole.
Distal face: The part of pollen away from the
centre of tetrad i.e. between the equator
and distal pole.
Proximal pole
Distal pole
Distal face
Proximal face
Monad

18. POLLEN MORPHOLOGY
 Each plant produces pollen or spores that are
distinctive from those of other plants; the
uniqueness can sometimes only be seen at the SEM
or TEM level.
 Pollen and spores can usually be identified to
the plant family, genus, and sometimes to the
species level with LM.
 Characters considered to identify pollen:
 Shape
 Size
 Aperture
 Wall structure (layers)
 Sculpture (surface ornamentation)

19. Oblate 50 -75
Sub-spheroidal 75-133
Prolate 133-200
Oblate spheroidal 88-100
Prolate spheroidal 100-114
Perprolate >200
Suboblate 75-88
SHAPE : P/EX100(G. Erdtman,1954)
Pollen grains are of various shapes i.e. spherical, oval, elliptical,
etc. In equatorial view by taking the measurements of polar and equatorial
axes, the grain shapes are categorized by applying the formula P/Ex100.
Peroblate <50
Subprolate 114-133
E
P

20. Pollen size: 5-250 µm
Minute grains < 10 µm (smallest grain- Myosotis sp., 5 µm)
Small grains 10-24 µm Medium-sized grains 25-49 µm
Large grains 50-99 µm Very large 100-199 µm
Gigantic grains >200 µm
(Walker & Doyle, 1975)

21. APERTURE
Morphologically it is opening or thinning of exine, where the
intine is usually thick, physiologically it is a germination zone or a
harmomegathus or both.
APERTURE STRUCTURE
Aperture may be simple or composite.
Simple aperture: ‘Simple’ opening or thinning of exine overlying a thick
intine. The simple denotes the lack of differentiation into outer and inner
units.
Composite aperture: Composite opening or thinning of exine overlying a thick
intine. ‘Composite’ signifies the differentiation into outer unit i.e.
ectoaperture (aperture of the outer exine) and an inner unit i.e.
endoaperture (aperture of inner exine) which are not congruent.
APERTURE TYPES
Based on their forms ( elliptic/  circular) and their position in the
pollen (polar, distal/proximal, global, meridional, equatorial) with
reference to the centre of tetrad, they can be distinguished as colpus,
sulcus, ulcus, porus and spiral)

22. SIMPLE APERTURE
COLPUS (pl. colpi, adj. colpate)
Meridional simple furrow like aperture with length/breadth ratio >2.
Polar view
Equatorial view
Depending upon the numbers of such
apertures the grains are known as
monocolpate, bicolpate, tricolpate and
stephanocolpate.
Colpate pollen are found in Brassicaceae,
Lamiaceae, etc.
polar view
Polar view
Sesamum indicum
Tricolpate Brassica pollen
Equatorial view
Polar view
Equatorial view
Stephanocolpate pollen-
Lamiaceae
Apocolpium
Mesocolpium

23. SULCUS (pl. sulci, adj. sulcate)
Simple furrow like distally located aperture with
length/breadth ratio >2.
This type of pollen are found Annonaceae, Magnoliaceae, etc.
ULCUS (pl. ulci, adj. ulcerate)
A spheroidal aperture at the distal face of pollen with
length/breadth ratio<2. This type of aperture is found in
Sparganium and Typha of Typhaceae and Poaceae.
Typha angustifolia Sparganium Poaceae
Crinum americanum
(Amaryllidaceae)

24. Triporate pollen
Biporate-Morus alba
PORE (pl. pori, adj. porate)
Equatorial simple circular or elliptic aperture with
length/breadth ratio <2. Depending upon the numbers of such apertures,
the grains are known as monoporate, biporate, triporate and
stephanoporate/polyporate.
Polar view Equatorial view
Equatorial view
Caryophyllaceae Chenopodium
Betula utilis
-Polar view
Cannabis sativa
-polar view
Malvaceae Holoptelea
Polyporate or stephanoporate

25. Thunbergia fragrans
(Acanthaceae)
SPIRAL APERTURE
Pollen grains with more or less spiral aperture are
found in Thunbergia fragrans (Acanthaceae) and Eriocaulon
quinquangulare (Eriocaulaceae)
Eriocaulon quinquangulare
(Eriocaulaceae)

26. COMPOSITE APERTURES
COLPORUS (pl. Colpori, adj. Colporate):
Compound aperture consisting of an ectocolpus with more or less pore like
endoaperture known as ‘Os’ (pl. ‘ora’). Grains bearing such apertures are
known as colporate grains.
Colpus
Pore or os
Madhuca indica
Adina cordifolia
Equatorial view
Polar view
Os- circular Os-lalongate Os-lolongate
Polygala chinensis
Synorate pollen
Tricolporate pollen
Stephanocolporate pollen

27. PORORUS (pl. porori, adj. pororate)
Pollen grains with compound aperture in which both the
ectoaperture and endoaperture are pores and the two are not
congruent. This type of apertures are found in the Himalayan
plant, Myrica esculanta and Casuarina equisetifolia.
Pororate
Casuarina equisetifolia

28. P
E
Zonocolpate
Zonoporate
Betula
Pantoaperturate
A prefix for global
distribution
Synonym of peri-.
Pantoaperturate (adj.): Pollen
grain with apertures spread
over the surface sometimes
forming a regular pattern.
Such grain may be, for example
pantocolpate, pantocolporate or
pantoporate
Evolvulus
alsinoides
Zonocolporate
Zonoaperturate
A prefix for equatorial
arrangement of apertures.
Zonoaperturate (adj.): Pollen
grain with apertures situated
only at equator. Such grain may
be, for example zonocolpate,
zonocolporate or zonoporate
Pantocolporate Pantoporate
Pantocolpate
Chenopodium
Caryophyllaceae
Holoptelea
p
E
Zonocolpate
Zonoporate

29. AMB (L. Ambitus)
Outline of pollen when it is viewed from one of the poles
Loranthus Bombax ceiba
Angulaperturate
Subtringular
Spheroidal Tringular
Sinu-aperturate
Planaperturate
Angulaperturate: Apertures are situated at the angles of the amb.
Planaperturate: Apertures are situated at the mid-points of the
sides.
Sinuaperturate- Apertures are situated in middle of concave sides.

30. Tectum or Tegillum (Adj. Tectate or Tegillate)
The layer of sexine which forms a roof over the columella.
Tectum imperforatum/Eutectum: With a continuous tectum, without
perforations.
Tectum perforatum/ Punctum: A tectum with perforations < 1µm in diameter.
Semitectum: A partially discontinuous tectum with perforations >1µm
diameter.
Atectate: Grain without tectum i.e. exine without any internal structure.
Tectatum perforatum Semitectum
Tectatum imperforatum
Exine without tectum
With suprategillar structure Without suprategillar structure

31. Based on the staining of pollen with fuchsin-B, the exine is divided in two
distinct layers i.e. the outer one ectexine, which takes the dark pink colour and
the inner layer endexine, which takes light pink colour, if not over stained.
Pollen wall
The wall of angiosperm pollen is constituted of three layers:
 Living cell membrane: It is made up of lipo-protein and gets lost after
death.
 The middle wall or intine: This is primarily composed of pecto-cellulose.
It is not usually preserved in the fossilized pollen grains.
 The outer wall or exine: It is made up of sporopollenin (Zetsche, 1932),
which is the most resistant organic compound. Due to presence of this compound,
the pollen remains preserved with all its surface features in the sediments for
longer period of time. Sporopollenin is a complex polymer of carotenoid and
carotenoid esters (Brooks and Shaw, 1968, Brooks, 1971, Shaw, 1971).
Its chemical formula is (C90 H142 O36)n.
Cell membrane
E
X
I
n
e
(Faegri & Iversen, 1975)
(Erdtman, 1952)

32. Lumen
Striate
Reticulate
Murus
Brochus
EXINE STRATIFICATION AND PATTERN
Striato-reticulate
Retipilate
Granulate Granulate
Lannea coromandelica
Striate
Lumen
Brassica

34. GYMNOSPERMOUS POLLEN
Abies
Cedrus Pinus
Picea
Ephedra
Juniperus
Pinus
Saccate pollen of conifers Saccus
Corpus
Marginal crest
Re-entrant angle

35. HISTORY
 Simple Microscope was invented by J.
Janssen and Z. Janssen in 1590.
 Compound Microscope was developed by
Hooke(1665).
 N. Grew (1682) , first recognised stamens
as the male organs of flower, also noted
constancy of pollen characters within the
same species (The Anatomy of Plants).
 Marcello Malpighi (1901) was the first to
describe pollen grains as having germination
furrow (Anatomia Plantarum).

36.  Camerarius (1665) first recognised
carpels as the female organs of flower, also
described several pollination experiments
(about the plant sexuality).
 Carl von Linnaeus first used the term
“Pollen” and stated that male ‘seed dust’ is
necessary for seed development.
 Kolreuter (1761-1766) and Sprengle
(1793), founder of floral biology, perceived
the importance of insects in flower
pollination and discovered that pollen plays
an important role in determining the
characters of offsprings. Kolreuter (1806,
1811) first classified pollen based on their
morphology.

37.  Amici (1824): pollen tube
 J E Purkinje (1830) worked on
palynological terminology and classified
pollen based on their morphology.
 Wodehouse (1935: ‘Harmomegathy’; 1945:
“Pollen Grains” (Book)
 Robert Brown (1828, 1833) first described
the origin and role of pollen tube.
 Göppert (1837, 1848) and Ehrenberg (1838)
first described the fossil pollen grains and
spores.
 Hugo von Mohl (1834) “On the Structure
and diversity of pollen grains”.

38.  Fritzsche (1837) established the terms:
exine and intine.
 Zetzsche (1931) first coined the term
“sporopollenin” for the resistant chemical
substance present in the outer wall (exine)
of both pollen grains and spores.
 Sporopollenin is a complex polymer of
carotenoid and carotenoid esters (Brooks and
Shaw, 1968, Brooks, 1971, Shaw, 1971).
Its chemical formula is (C90 H142 O36)n.

39.  Palaeopalynology established at the end
of the nineteenth century when P. Reinsch
(1884) published the first photomicrographs
of fossil pollen and spores of Russian
coals.
 Lennart von Post (1916) published the
first pollen diagram using exclusively the
arboreal pollen from Sweden.
G. Erdtman (1921) used pollen as a tool to
study the Quaternary vegetation succession
and climate change.

41. Prof. Dr. Birbal Sahni
 Drs. Bhardwaj, Lele, Navale,
Venkatachala, Vishnu-Mittre, A.K. Kar,
P. K. K. Nair, A. B. Singh, S. T. Tilak,
Sunirmal chanda, Sudhendu Mandal, M. S.
Chauhan

42.  G.Erdtman (1952): Pollen morphology and
plant taxonomy/Angiosperms
 K. Faegri and H. Iversen (1950, 1964.,
1975). Textbook of Pollen Analysis

43. Branches of Palynology
Basic Palynology or Actuopalynology- It deals
with the study of pollen grains and spores of extant plants.
It is also known as Neopalynology. This study is used in
taxonomy to understand the affinities of the modern plant
groups/families as well as in ascertaining the precise
status of plant species in various taxonomic groups.
Applied Palynology- It deals with the use of
pollen-based information in understanding the other aspects
of science, particularly relevant to human society. It is
further divided into following categories i.e.:
Aeropalynology: Study of pollen/spore content of the air in
relation to allergy.
Melissopalynology: Study of pollen of honey, which provides
information regarding the botanical provenance of honey, its
purity and seasonality.

44. Forensic palynology: Study of pollen in
detecting crimes and nabbing criminals.
Archaeopalynology: Study of pollen of
settlement sites, which furnish the data related
to inception of agriculture, anthropogenic
impact on natural resource and cultural shifts
owing to climate change.
Palaeopalynology: It deals with the study of
fossil pollen grains and spores, which help in
the reconstruction of past vegetation and
climate, phylogeny of various plant groups, etc.
in terms of geological time scale.

45. Basic principles
 The widespread transport and mixing of
pollen grains by wind and water is
generally believed to form a pollen
assemblage, which represent the
characteristics of vegetation, climatic
conditions or sedimentary environment at a
specific time or area (Erdtman, 1952).
 The number of pollen grains deposited
per unit time at a given point is directly
related to the abundance of the associated
species in the surrounding vegetation
(Davis, 1963)

46. Application
 in Biostratigraphy
 in exploration of fossil fuels, such as
oil and coal
 in understanding the sea-level
fluctuations
 in reconstruction of past vegetation and
climate (i. e. Pollen Analysis)

47. Collection of polleniferous materials
from modern samples (i.e. living flowers,
spider webs, tree barks and honey
samples) and sediments
 Forceps and/or needle (living
flowers/Herbarium specimens)
 Spatula/spade, mattock and other related
apparatus
 All the samples are put in zip-lock
polythene bags, labeled in the field and
registered in the museum of the Birbal Sahni
Institute of Palaeosciences (BSIP), Lucknow,
India.

48. Thank you!
Further readings…
 Palynology: A Treatise
M. R. Saxena
Oxford & IBH Publishing Co. Pvt Ltd.,
Calcutta
 Palynology and its Applications
S. A. Agashe
Oxford & IBH Publishing Co. Pvt Ltd., New
Delhi

49. Extraction of pollen grains from modern
polleniferous materials and fossil
sediments, reference pollen slide
preparation

50. PREPARATION OF POLLEN SAMPLES (Erdtman, 1952)
Centrifugation and decantation
Boil the measured amount of sample with 10% aqueous KOH solution to
dissolve the humus and to deflocculate pollen/spores from the sediments
Washing & decantation
Sieve the sample with 150 mesh to remove coarse debris
Washing & decantation
Treat the sample with 40% HF to remove silica
Washing & decantation
Treat the sample with glacial acetic acid to dehydrate
Acetolysis (9:1, acetic anhydride & conc. sulphuric acid)
Centrifugation and decantation
Treat the sample again with glacial acetic acid
washing & decantation
Prepare the sample in 50% glycerin solution and add few drops of phenol
as preservative
Sieve the sample with 600 mesh to remove fine debris
Treat the sample with 10% HCL to remove carbonates, if present
Washing & decantation

51. Modern polleniferous materials
 Preserve the material (dried floral
part) in glacial acetic acid
 After crushing gently in a plastic
centrifuge tube by means of a glass rod,
sieve the material 150 mesh sieve.
 treatment of the material with glacial
acetic acid
 Acetolysis is carried out.

52.  For honey samples, 20 g sample was taken
out from the bulk honey sample and dissolved
in 20 ml distilled water in a beaker and
centrifuged.
 treatment with glacial acetic acid to
dehydrate them
 Acetolysis is carried out.

53.  For spider web samples, HCl treatment is
given before conducting acetolysis; No HF
treatment.
 For tree barks, treat small pieces (10x5
cm; sample diameter) of the modern tree bark
samples with 10% KOH in a beaker with
distilled water. The beaker was heated
thereafter on a laboratory heating plate for
10 minutes and/or at boiling temperature in
order to deflocculate the pollen/spores from
the barks. Washing (with distilled water),
centrifugation and decantation were carried
out after sieving. Further, acetolysis was
carried out.

54. Reference pollen slide preparation
 In Quaternary palaeoclimatic studies,
temporary slides are prepared as we have
identify the palynomorphs, which sometimes
show distortion, so we change the shape
(actual) of palynomorphs through creating
some disturbance in the slide.
The residues of all the processed samples
from various substrates are put on a slide.
After that, Polyvinyl alcohol is mixed for
spreading. And mounting is done with the use
of Canada Balsam.