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Jammi L. Ladwig, M.A., Doctoral Candidate

March 13th, 2015

Introduction to Phytolith Analysis

◦ Definitions

◦ History of Phytolith Research ◦ Taxonomic Sensitivity and Morphotypes

◦ Laboratory Techniques and Analytical Considerations

“How it’s Done” … dependent on who is doing it, specific research questions …

Software Programs

• kounter (T2) •Tilia (1.7.16) • ImageJ (FIJI)

• LAS EZ (Leica Application Suite)

Current Methodological & Theoretical Issues / Topics

…Break (~10 or 15 mins)…

MICROSCOPY TIME!!!

Phytoliths (plant stones) are silicified plant cells. Monosilicic acid (H4SiO4) from the environment is taken up by plants (as groundwater) and is laid down in cell walls, cell interiors, and intercellular spaces as silicon dioxide (SiO2).

Some cells are active silica accumulators (short cells), while other portions of plants can be incidentally silicified (long cells, epidermal tissue, etc).

Different plant families, genera, and species produce different phytolith assemblages, and different parts of any given plant produce different phytolith morphotypes.

Many phytolith forms are highly redundant, with significant overlap in shape and size, though there are some forms which are diagnostic (or thought to be so).

Phyto = plant, Lython = stone

Hydrogenated Silicon Dioxide (SiO2 • H20)

Roots

Monosilicic Acid (H2SiO4)

Phytoliths - Plant stones made of opal (non-crystalline) silica

Cyperaceae (Sedge)

roots

Zizania palustris

(Wild rice)

Red pine (bark) White pine (bark)

Phragmites australis

(Giant Reed)

Credit: Chad Yost

Phalaris

arundinacea

(Reed canarygrass)

“I collected a little packet of this brown-coloured fine dust, which appeared to have been filtered from the wind by the gauze of the vane at the masthead. Professor Ehrenberg finds that this dust consists in great part of the infusoria with siliceous shields, and of the siliceous tissue of plants. In five little packets which I sent him, he has ascertained no less than sixty-seven organic forms.”

[Darwin 1909 as cited in Piperno 2006: 2-3].

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The Discovery and Exploratory Stage ◦ (1835 – 1895)

The Botanical Phase ◦ (1895 – 1936)

The Period of Ecological Research ◦ (1955 – 1975)

The Modern Period of Archaeological and

Paleoenvironmental Research ◦ (1975 – Present)

“Paleoethnobotanist: An archaeologist who analyzes and

interprets plant remains from archaeological sites in order

to understand past interactions between human

populations and plants.” (pg. 210)

Archaeobotany Refers only to the recovery and identification of plant

remains, unrelated to human activity, from archaeological

contexts by specialists.

Paleoethnobotany Introduced by Hans Helbaek in 1959, defined by

Richard Ford (1979: 286) as:

“The analysis and interpretation of the

direct interrelationships between humans

and plants for whatever purpose as

manifested in the archaeological record.”

Investigating plants from archaeological contexts

Macro-botanical (macroremains)

visible with the naked-eye or very low-powered microscopy

charred, desiccated, or waterlogged floral remains such as seeds, nut

shells, fruit pits, etc.

Micro-botanical (microremains, microfossil)

visible only with the assistance of microscopy

inorganic (generally) but also some organic compounds, such as

pollen, starch grains, phytoliths, diatoms, etc.

Pollen vs. Phytoliths Attribute Pollen Phytoliths

Anatomical origin Flowers All parts of the plant

Produced by Most vascular plants Only some groups of plants

Size range 10-100 μm 5-200 μm

Phenology Annual or biannual Continuously

Control Genetic Genetic and passive (E/T)

Morphotypes ~1:1 (order, family, genus, species) Highly variable

Dispersal Wind and insect Decay-in-place

2° transport Wind, water, herbivory Wind, water, herbivory, ash

Production/yr 104 (flax)-1010 (pine) 105-106 (grass)

Influx (cm2/yr) 101 (desert)-104 (pine forest) ~103 for lakes, soils likely higher

Spatial interpretation Local and regional Mostly local, limited regional

Best preservation Acidic, anoxic, abiotic, no T or ppt 𝚫 Acidic to neutral, alkaline if dry

Tree/Shrub resolution Good for most wind pollinated taxa Very limited (Pinus, Celtis, Arecaceae)

Grass resolution Family level (Poaceae) Subfamily, some genus and species IDs

Credit: Chad Yost

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Cucurbitaceae (squash/gourd; Cucurbita maxima)

Fabaceae (bean; Phaseolus vulgaris)

Poaceae (grass; Leersia oryzoides – rice cutgrass)

Poaceae (grass; Triticum sp. – wheat)

Poaceae (grass; Zea mays – corn)

(Figure developed from Novello et al. 2012, Review of Palaeobotany and

Palynology, Phytolith signal of aquatic plants and soils in Chad, Central

Africa, V. 178, 15 June 2012, pp. 43-58)

Morphotypes: (phytolith “shapes”)

Ro: rondels

Bi: bilobate

Cr: cross

Poly: polylobate

S: saddles

Tra: trapeziform

Phytolith Shape Description by Plant Anatomical Location

(Developed from Madella et al. 2005, ICPN Working Group,

"International Code for Phytolith Nomenclature 1.0")

Phytolith Morphotypes

(Developed from Madella et al. 2005, ICPN Working Group, "International Code

for Phytolith Nomenclature 1.0")

• 3 species of wild rice native to North America:

• Zizania aquatica, Z. palustris, Z. texana (endemic to Texas) – 1 species, Z. latifolia, is found in Japan (native), Asia, India, Russia, Korea, and is grown as an exotic

species in Hawaii

• Grows annually (Z. aquatica + Z. palustris) in lakes and rivers; • [Z. latifolia and Z. texana perennial]

• Seed shatters (is ripe and drops) in late summer/early

autumn

• Taxonomic position:

Kingdom Plantae – Plants Subkingdom Tracheobionta – Vascular plants Superdivision Spermatophyta – Seed plants Division Magnoliophyta – Flowering plants Class Liliopsida – Monocotyledons Subclass Commelinidae Order Cyperales Family Poaceae – Grasses Genus Zizania L. – wild rice

(http://www.fsl.orst.edu/forages/projects/regrowt

h/print-section.cfm?title=Grass%20Structures)

(http://www.fsl.orst.edu/forages/projects/regrowth/seed.jpg)

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Credit: Chad Yost

Inflorescence Type-1 (rondel) Phytoliths on Wild Rice Hull (pistillate spikelet lemma)

Credit: Chad Yost

Inflorescence Type-1 (rondel) Phytoliths on Wild Rice Hull (pistillate spikelet lemma)

Credit: Chad Yost

Leaf Type 1 Leaf Type 1 Leaf Type 1 Leaf Type 2 & 1

Leaf Type 2 Leaf Type 2 Leaf Type 2 Leaf Type 2

Leaf Type 3 Leaf Type 3 Leaf Type 4 Leaf Type 5

Leaf Type 5 Leaf Type 6

A B C

C

D

E F G H

I J K L

M N O P

Zizania palustris (wild rice) Leaf Phytoliths

Leaf Type 1 Leaf Type 1 Leaf Type 1 Leaf Type 2 & 1

Leaf Type 2 Leaf Type 2 Leaf Type 2 Leaf Type 2

Leaf Type 3 Leaf Type 3 Leaf Type 4 Leaf Type 5

Leaf Type 5 Leaf Type 6

A B C

C

D

E F G H

I J K L

M N O P

Zizania Morphotype ICPN* Nomenclature % Morphotypea

Leaf Type 1 Cross 15.1

Leaf Type 2 Bilobate 22.3

Leaf Type 3 Trapeziform 19.6

Leaf Type 4 Interstomatal Ground Cell 12.1

Leaf Type 5 Blocky and cuneiform bulliform 22.7

Leaf Type 6 Elongate tuberculate long cell 8.2 a n=515; *ICPN=International Code for Phytolith Nomenclature

Leaf Type 1

Leaf Type 1 Leaf Type 1 Leaf Type 1 Leaf Type 2 & 1

Leaf Type 2 Leaf Type 2 Leaf Type 2 Leaf Type 2

Leaf Type 3 Leaf Type 3 Leaf Type 4 Leaf Type 5

Leaf Type 5 Leaf Type 6

A B C

C

D

E F G H

I J K L

M N O P

Leaf Type 1 Leaf Type 1 Leaf Type 1 Leaf Type 2 & 1

Leaf Type 2 Leaf Type 2 Leaf Type 2 Leaf Type 2

Leaf Type 3 Leaf Type 3 Leaf Type 4 Leaf Type 5

Leaf Type 5 Leaf Type 6

A B C

C

D

E F G H

I J K L

M N O P

Leaf Type 1 Leaf Type 1 Leaf Type 2 & 1 Leaf Type 2 Leaf Type 2 Leaf Type 2 Leaf Type 2

Leaf Type 3 Leaf Type 3 Leaf Type 4 Leaf Type 5 Leaf Type 5 Leaf Type 6

Credit: Chad Yost

Zizania palustris (wild rice) Sheath Phytoliths

Sheath Type 1 Sheath Type 1 Sheath Type 1 Sheath Type 1

Sheath Type 2 Sheath Type 2 Sheath Type 2 Sheath Type 3

Sheath Type 4 Sheath Type 4 Sheath Type 4 Sheath Type 4

Sheath Type 4 Sheath Type 4 Sheath Type 5 Sheath Type 5

A B C

C

D

E F G H

I J K L

M N O P

Sheath Type 1 Sheath Type 1 Sheath Type 1 Sheath Type 1

Sheath Type 2 Sheath Type 2 Sheath Type 2 Sheath Type 3

Sheath Type 4 Sheath Type 4 Sheath Type 4 Sheath Type 4

Sheath Type 4 Sheath Type 4 Sheath Type 5 Sheath Type 5

A B C

C

D

E F G H

I J K L

M N O P

Sheath Type 1 Sheath Type 1 Sheath Type 1 Sheath Type 1 Sheath Type 4 Sheath Type 4 Sheath Type 4 Sheath Type 4 Sheath Type 1 Sheath Type 1 Sheath Type 1 Sheath Type 1

Sheath Type 2 Sheath Type 2 Sheath Type 2 Sheath Type 3

Sheath Type 4 Sheath Type 4 Sheath Type 4 Sheath Type 4

Sheath Type 4 Sheath Type 4 Sheath Type 5 Sheath Type 5

A B C

C

D

E F G H

I J K L

M N O P

Sheath Type 1 Sheath Type 1 Sheath Type 1 Sheath Type 1

Sheath Type 2 Sheath Type 2 Sheath Type 2 Sheath Type 3

Sheath Type 4 Sheath Type 4 Sheath Type 4 Sheath Type 4

Sheath Type 4 Sheath Type 4 Sheath Type 5 Sheath Type 5

A B C

C

D

E F G H

I J K L

M N O P

Sheath Type 2 Sheath Type 2 Sheath Type 2 Sheath Type 3 Sheath Type 4 Sheath Type 4 Sheath Type 5 Sheath Type 5

Zizania Morphotype ICPN* Nomenclature % Morphotypea

Sheath Type 1 Bilobate with ampliate basal lobes 28.5

Sheath Type 2 Reniform rondel with basal sinus 5.3

Sheath Type 3 Polylobate with ampliate basal lobes 3.4

Sheath Type 4 Cross with 4-6 ampliate basal lobes 7.2

Sheath Type 5 Trapeziform with bilobate base 55.6 a n=207; *ICPN=International Code for Phytolith Nomenclature

Credit: Chad Yost

Zizania palustris (wild rice) Culm (stem) Phytoliths

Culm Type 1 Culm Type 1 Culm Type 2 Culm Type 2

Culm Type 3 Culm Type 4 Culm Type 5 Culm Type 6

Culm Type 7

A B C D

E F G H

I J K L

Culm Type 1 Culm Type 1 Culm Type 2 Culm Type 2

Culm Type 3 Culm Type 4 Culm Type 5 Culm Type 6

Culm Type 7

A B C D

E F G H

I J K L

Culm Type 1 Culm Type 1 Culm Type 2 Culm Type 2 Culm Type 3 Culm Type 4

Culm Type 1 Culm Type 1 Culm Type 2 Culm Type 2

Culm Type 3 Culm Type 4 Culm Type 5 Culm Type 6

Culm Type 7

A B C D

E F G H

I J K L

Culm Type 1 Culm Type 1 Culm Type 2 Culm Type 2

Culm Type 3 Culm Type 4 Culm Type 5 Culm Type 6

Culm Type 7

A B C D

E F G H

I J K L

Culm Type 5 Culm Type 6 Culm Type 7

Zizania Morphotype ICPN* Nomenclature % Morphotypea

Culm Type 1 Cross with ampliate basal lobes 36.3

Culm Type 2 Cross without ampliate basal lobes 12.6

Culm Type 3 Polylobates & bilobates 11.6

Culm Type 4 Reniform rondel with basal sinus 6.9

Culm Type 5 Trapeziform with 1-cleft base 26.1

Culm Type 6 Bilobate with cleft lobes 5.1

Culm Type 7 Polylobate with 6 ampliate basal lobes 1.4 a n=215; *ICPN=International Code for Phytolith Nomenclature

Credit: Chad Yost

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Zizania palustris (wild rice) Inflorescence Phytoliths

Rachis Type 1 Rachis Type 1

Rachis Type 1 Rachis Type 2

Zizania Inflorescence Morphotype ICPN* Nomenclature % Morphotype

Rachis Type 1 = Inflorescence Type 1a Reniform rondel with basal sinus 95.0

Rachis Type 2 = Inflorescence Type 3 Rondel w/opposing basal sinuses 5.0 (n=500)

Spikelet Type 1 = Inflorescence Type 1a Reniform rondel with basal sinus 60.0

Spikelet Type 2 = Inflorescence Type 2 Rondel with 2-3 basal clefts 30.0 (n=500)

*ICPN=International Code for Phytolith Nomenclature a Rachis Type 1 and Spikelet Type 1 are equivalent and reclassified here as Inflorescence Type 1

Spikelet Type 1 Spikelet Type 1 Spikelet Type 1

Spikelet Type 1 Spikelet Type 1 Spikelet Type 1

Spikelet Type 2

Dendriform

Credit: Chad Yost

Sheath Type 2Sheath Type 3

Sheath Type 4

Sheath Type 5

Culm Type 1

Culm Type 5

Culm Type 6

Culm Type 7

Inflorescence Type 1

Leaf Type 6Leaf Type 5

Leaf Type 4

Sheath Type 1

Leaf Type 2Leaf Type 3 Other (FL Type 5)

Inflorescence Type 3

Inflorescence

Type 2

Culm Type 3Culm Type 4

Culm Type 2

Leaf Type 1

Wild Rice Phytolith Morphotype Assemblage (Entire Plant)

29%

Floating Leaf Phytoliths: 0.8%

Culm Phytoliths: 25%

Inflorescence Phytoliths: 37%

Leaf Phytoliths: 9%

Sheath Phytoliths: 28%

Not locally diagnostic

Credit: Chad Yost

Spring

Summer

Late summer

Late fall/Winter

Wild Rice Phenology and Taphonomy

current current

core

core

core

%

Floating Leaf Types

Leaf, Sheath,

CulmTypes

Sheath Type 1 Sheath Type 1 Sheath Type 1 Sheath Type 1

Sheath Type 2 Sheath Type 2 Sheath Type 2 Sheath Type 3

Sheath Type 4 Sheath Type 4 Sheath Type 4 Sheath Type 4

Sheath Type 4 Sheath Type 4 Sheath Type 5 Sheath Type 5

A B C

C

D

E F G H

I J K L

M N O P

% % Inflorescence types

Credit: Chad Yost

Sedge (Cyperaceae):

1) Achene (seed/fruit)

Cyperaceae:

2) Rhizome (root)

Making sense of mixed assemblages

and interpreting the phytolith

record.

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Control for contamination from the field to the lab.

Usually only a small amount of sediment required

(1 cubic centimeter or ~1 gram) but this is variable!

Questions to consider:

What analysis (or analyses) are most appropriate given the

nature of the site and sampling contexts? Can multiple analyses be conducted on samples?

Can quantitative data be gathered through the addition of an exotic spike?

Were there adequate samples taken from contexts of interest? Are samples taken appropriate to answer research questions?

Preservational biases? Acidic/alkaline soils? Taphonomic considerations?

Diagnostic plant functional type phytoliths

C3 grass phytolith Xeric C4 grasses

Mesic C4 grasses

C3 grasses (cool, shade,

wet)

C3 grasses (cool, shade,

wet)

Credit: Chad Yost

Lake Ogechie:wild rice phytoliths first appear ~3500 BP

• Grass pollen diameter as a Zizania indicator? Possible but equivocal.

• Pollen and phytolith

records are complimentary

Credit: Chad Yost

A B C D

E F G H

Phragmites australis (Giant Reed)

I J K L

M N O P

Phalaris arundinacea (Reed Canary Grass)

Credit: Chad Yost

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A

A

B C D

E F G H

Calamagrostis sp. (Canada Bluejoint)

I J K L

M N O P

Glyceria sp. (Manna Grass)

Credit: Chad Yost

A B C D

E F G H

I J K L

M N O P

Muhlenbergia glomerata (Spike Muhly)

Credit: Chad Yost

A B C D

E F G H

I J K L

M N O P

Poa palustris (Waterfowl Meadow Grass)

Dulichium, Carex & Equisetum

Credit: Chad Yost

Inflorescence Type 1 Inflorescence Type 1 Inflorescence Type 2 Inflorescence Type 2

Inflorescence Type 3 Inflorescence Type 3 Inflorescence Type 4 Inflorescence Type 4

Culm Type 1 Culm Type 1 Culm Type 2 Culm Type 2

Culm Type 3 Culm Type 4 Culm Type 5 Culm Type 6

A B C

C D

E F G H

I J K L

M N O P

Leersia oryzoides (Rice cut-grass)

Infl

ore

scen

ce

Culm

(st

em)

Credit: Chad Yost

Leersia oryzoides (Rice cut-grass)

Leaf Type 1 Leaf Type 1 Leaf Type 2 Leaf Type 2

Leaf Type 3 Leaf Type 4 Leaf Type 5 Leaf Type 6

Leaf Type 7 Leaf Type 7 Leaf Type 8 Leaf Type 8

Leaf Type 8 Leaf Type 8 cuneiform bulliform tuberculate long cell

A B C

C

D

E F G H

I J K L

M N O P

Leaf Phytoliths

Credit: Chad Yost

Series of keys designated by analyst

Ctrl keys are OUTSIDE of count, as are TRACERS

Old computer??? Put it to “good” use!

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Zea

Tower 1:1 Rondel

Tower 2:1

Tower 3:1

Blocky Artemesia-

type Trichome Globular w indents

Quercus branched

Dicot w/parallel

ridges

CYPERACEAE 0

Cereus CYPERACEAE ACHENE

PHYTO

Chloridoid

Rondel Δ Agropyron-

type

Rondel pyramidal Rondel

Rondel keeled

Rondel Phalaris-

type

Trapeziform sinuate

Oblong w/oblong

platform

Irreg. w/irreg.

platform

CYPERACEAE ROOT PHYTO

Chloridoid thin saddle

Phragmites-type saddle

Dicot knobby

Bilobate fat w/bilobate

platform

Cross Bilobate Polylobate

Elongate smooth

TR

Elongate castillate

Lowercase –KOUNTER

Elongate spiny

Straight Hair

Dicot angular

Bilobate fat

Square w/square

platform

Elongate Ridged

Bulliform Bulliform rectangular

Segmented Hair

Elongate sinuate

Epidermal wavy

Epidermal spiny

Epidermal polyhedral

Epidermal anticlinal

large

Oblong w/circular

platform

Dicot thin w/sharp

ridges

“add .5 to which” 0

CYPERACEAE

Chlor 1 2 sides

Rondel elongate

Rondel horned

Circular w/circ.

platform

Circular w/oblong

platform

Thin w/ridges

Crenate platform

Oblong long w/ob.

platform

Interstomatal cell PALMAE

Stipa-type bilobate

Circular w/saddle

platform

Parallelipiped BLOCKY LARGE

Cross w/angular X

platform

Commelina dianthifolia

Globular

Wavy top

Elongate castillate

Uppercase (shift) –KOUNTER

Elongate dendritic

Confier needle

stomata

Dicot w/wings Opuntia

Dicot long w/branched

end

Commelina erecta

Bulliform triangular

Conifer globular

Dicot wedge Elongate sinuate

Epidermal anticlinal

medium

Epidermal anticlinal

small

Charred Asteraceae

Tissue Frag.

“^E”

Wood phytolith

Helianthus

“no”

Spheraste Dendritic Dicot bulliform

Sponge spicule

Diatom round

Fiber Long diatom

Elongate castillate

Control key (ctrl)–KOUNTER

Volcanic

Parallelipid Opuntia Dicot long

w/branched

end

Centric Volcanic Volcanic Elongate sinuate

Meant for palynology, but hey, we’ll make it work …

A mess at first, but with enough modification…

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Freeware!

Using Plugins to automate gathering data, but only 2-D…

ASIG

Dr. Gilliane Monnier

Dr. Katherine Hayes

LacCore: National Lacustrine Core Facility

Chad Yost (Univ. of Arizona)

Dr. Mikhail Blinnikov (SCSU)

Evolutionary Anthropology Labs

◦ (Matt Edling and Keith Manthie)

Robert Thompson and Rob Lusteck

Anthropology Graduate Students