Secondary Growth Rings & Leaf Trait Analysis

Lab 3: Secondary Growth Rings & Leaf Trait Analysis

Part A: Secondary Growth Analysis - Tree Rings

Background: What Are Growth Rings?

Simple explanation: Each year, a tree adds a new layer of wood under its bark. This creates visible rings you can see when a tree is cut. By studying these rings, we can:

• Determine the tree's age

• See which years had good/bad growing conditions

• Detect droughts, fires, and other environmental events

• Understand the tree's life history

Ring Anatomy

Each annual ring has TWO bands:

1. Earlywood (Springwood)

• What it is: Light-colored band formed in spring/early summer

• Why it's light: Cells are large with thin walls (less dense)

• Function: Rapid water transport during growing season

• In Arizona: Forms during spring moisture period

2. Latewood (Summerwood)

• What it is: Dark-colored band formed in late summer/fall

• Why it's dark: Cells are smaller with thick walls (more dense)

• Function: Structural support

• In Arizona: Forms as growth slows during hot, dry summer

One complete ring = 1 year of growth

Materials for Part A

Field/Collection:

• Camera or smartphone

• Metric ruler (with millimeters)

• String or flexible measuring tape

• Field notebook and pencil

• Hand lens or magnifying glass (10× magnification)

• Pencil for marking rings

What you need to find:

• 3 tree stumps or thick branches (>5 cm diameter)

• Mix of species if possible

• Showing clear annual rings

Finding Tree Stumps in Mohave County

Best sources:

1. Tree removal services (BEST option)

   • Companies in Kingman, Bullhead City, Lake Havasu City

   • Call ahead and ask to collect fresh-cut sections

   • They often have ponderosa pine, juniper, cottonwood from local work

2. Parks maintenance departments

   • City parks sometimes remove trees

   • Ask permission first

   • May have sections available

3. Landscape/firewood suppliers

   • Often cut local trees

   • May provide cross-sections

4. Private property (with permission)

   • Dead/fallen trees

   • Old fence posts (juniper, pine)

   • Recently cut firewood

Arizona-appropriate species to look for:

Conifers (excellent ring clarity):

• Ponderosa pine (Pinus ponderosa) - BEST for this lab, clear rings

• Pinyon pine (Pinus edulis) - common, clear rings

• Utah juniper (Juniperus osteosperma) - very common, sometimes unclear rings

• Arizona cypress (Cupressus arizonica) - good ring clarity

Hardwoods (more challenging but workable):

• Velvet mesquite (Prosopis velutina) - common but rings can be hard to see

• Fremont cottonwood (Populus fremontii) - near water, clear rings

• Ash (Fraxinus) - landscape trees, clear rings

• Arizona sycamore (Platanus wrightii) - near streams, good rings

Avoid:

• Creosote bush (doesn't form clear rings)

• Most desert shrubs (too small, unclear rings)

• Weathered wood (rings deteriorated)

• Spalted or rotted wood (rings obscured)

Procedure: Part A

Step 1: Stump Assessment

For each stump or branch section you collect:

a. Initial documentation:

• Take photo with ruler for scale

• Show entire cross-section

• Include close-up of ring detail

b. Identification:

• Species (if known): _____

• If unknown, photograph bark texture and any attached needles/leaves

• Identification tip: Conifers have resin canals (small holes), hardwoods have vessels (pores)

c. Measurements:

• Diameter (if circular): _____ cm

• If oval/irregular, measure:

  • Maximum diameter: _____ cm

  • Minimum diameter: _____ cm

• Circumference: _____ cm

d. Condition assessment: / Fresh cut (within weeks) / Weathered (months old) / Partially decayed / Good condition

e. Visual inspection - note obvious features: / Fire scars (charred areas, distorted rings) / Insect damage (galleries, holes) / Asymmetric growth (wider rings on one side) / Rot or decay areas / Reaction wood (compressed/stretched rings) / Missing bark on sections

f. Initial ring count estimate:

• Count visible rings by eye: approximately _____ rings

• Note clarity: / Very clear / Moderately clear / Difficult to see

![Photo example: Well-prepared stump surface with visible rings]

Step 2: Surface Preparation (if needed)

For weathered stumps:

• Lightly sand surface with fine-grit sandpaper

• Sand in direction of rings (not across them)

• Brush away sawdust

• Slightly dampen surface with water to enhance contrast

• Photograph before and after

Note: Fresh cuts need no preparation!

Step 3: Ring Counting and Marking

Why we use multiple radii: Trees don't always grow evenly. Counting three different radii helps us catch missing or false rings.

a. Locate the pith (center):

• Look for the darkest spot in the middle

• May be slightly off-center

• If missing or decayed, estimate where it would be

![Diagram: Cross-section with pith marked in center]

b. Mark three measurement radii:

• Draw three lines from pith to bark

• Space them about 120° apart (roughly equal spacing around circle)

• Label them: Radius A, Radius B, Radius C

• Choose paths that avoid:

  • Knots (dark circular features)

  • Obvious damage or rot

  • Branch attachments

  • Fire scars

![Diagram: Stump with three radii marked 120° apart]

c. Count rings along each radius:

Counting method:

1. Start at pith (center)

2. Count outward toward bark

3. Use hand lens to see fine rings

4. Mark every 10th ring with a pencil dot (makes recounting easier!)

5. Count carefully: One ring = one light band + one dark band together

Watch out for:

• Missing rings: Very narrow or absent rings (compare radii)

• False rings: Incomplete bands that don't go all the way around

• Wedging rings: Extremely narrow rings (easy to miss)

• Double rings: Two bands in one year due to stress

e. Age estimate:

• Average ring count: _____ rings

• Add 3-5 years if stump cut above ground level

• Estimated tree age: _____ years

f. Verify your count:

• Recount at least once

• If counts differ by >3 rings, count again

• If one radius has fewer rings, it likely has missing rings

Confidence level:  High (counts agree within 2 rings) / Medium (counts differ by 3-5 rings) / Low (counts differ by >5 rings)

Step 4: Ring Width Measurements

Why measure ring width? Wide rings = good growing year (wet). Narrow rings = stress year (drought, fire, competition).

a. Select your best radius:

• Choose the clearest, most complete radius

• Should avoid damage, knots, irregular areas

• This is your "primary measurement radius"

• Mark it clearly: "PRIMARY"

b. Measure each ring width:

Measurement technique:

1. Start at pith (center), work outward

2. Measure from beginning of one earlywood to beginning of next earlywood

3. Measure to nearest 0.5 mm (or 0.1 mm with calipers)

4. Be consistent with your starting/stopping points

![Diagram: Showing how to measure ring width consistently]

c. Create data table:

d. Photograph your measurements:

• Show ruler alongside radius

• Mark where you're measuring

• Include close-ups of interesting patterns

Step 5: Earlywood/Latewood Analysis

Purpose: Understanding the ratio of earlywood to latewood tells us about growing season length and conditions.

a. Select 5 representative rings:

Choose rings from different life stages:

• Ring(s) near pith: _____ (juvenile growth, often narrow)

• Ring(s) from widest section: _____ (prime growth years)

• Ring(s) near bark: _____ (recent growth)

b. Detailed measurements for selected rings:

For each selected ring:

Calculate Earlywood %: (Earlywood width ÷ Total width) × 100

c. Observe qualitative features:

• Color contrast:  Strong / Moderate / Weak

• Earlywood vessels/cells:  Large / Medium / Small

• Ring boundary sharpness:  Sharp / Gradual / Diffuse

• Rays visible (radial lines):  Yes, conspicuous / Faint / Not visible

d. Take detailed photos:

• Close-ups showing earlywood/latewood contrast

• Use hand lens if available

• Include scale bar

Step 6: Growth History Reconstruction

Now interpret what the rings tell you about the tree's life!

a. Calculate growth statistics:

1. Mean ring width:

   • Add all ring widths: _____ mm

   • Divide by number of rings: _____ mm

   • Average ring width: _____ mm

2. Find extremes:

   • Maximum ring width: _____ mm (which ring? #_____)

   • Minimum ring width: _____ mm (which ring? #_____)

   • Range: _____ mm (max - min)

3. Calculate standard deviation: (measure of variability)

   • Use calculator or Excel: _____ mm

4. Coefficient of variation (CV):

   • Formula: (Standard deviation ÷ Mean) × 100 = _____%

Interpretation:

4. • CV > 30%: "Sensitive" tree - responds strongly to environment (common in dry areas!)

   • CV 20-30%: Moderate sensitivity

   • CV < 20%: "Complacent" tree - stable conditions (rare in Arizona)

Your tree is:  Sensitive / Moderate / Complacent

b. Identify growth periods:

Divide the tree's life into phases based on ring widths:

1. Juvenile phase (first years of life)

• Years: 1 to _____

• Average ring width: _____ mm

• Characteristics: _____

2. Prime growth phase (widest rings)

• Years: _____ to _____

• Average ring width: _____ mm

• Characteristics: _____

3. Mature phase (recent years)

• Years: _____ to _____ (present)

• Average ring width: _____ mm

• Characteristics: _____

4. Suppression events (very narrow rings, growth nearly stopped)

• Year(s): _____, _____, _____

• Possible causes:  Drought / Competition / Insect attack / Fire

5. Release events (sudden increase in growth)

• Year(s): _____, _____, _____

• Possible causes:  Increased water / Neighbor removed / Fire thinning / Other

c. Diameter growth over time:

Calculate when tree reached specific sizes:

Method: Add ring widths cumulatively from center outward

Example calculation:

• Ring 1: 2 mm → radius = 2 mm → diameter = 4 mm = 0.4 cm

• Ring 2: 3 mm → radius = 5 mm → diameter = 10 mm = 1.0 cm

• Ring 3: 4 mm → radius = 9 mm → diameter = 18 mm = 1.8 cm

• (Continue...)

Record milestones:

• Diameter at age 5: _____ cm

• Diameter at age 10: _____ cm

• Diameter at age 15: _____ cm

• Diameter at age 20: _____ cm

• Age when reached 5 cm diameter: _____ years

• Age when reached 10 cm diameter: _____ years

• Age when reached 15 cm diameter: _____ years

d. Create growth curve:

On graph paper, plot:

• X-axis: Age (years from pith to bark)

• Y-axis: Cumulative diameter (cm)

Curve shape reveals growth strategy:

• Steep early slope: Fast juvenile growth (common in open areas)

• Gradual leveling: Growth slowing with age (normal)

• Stepped pattern: Periods of growth and suppression

• Sudden increases: Release after competitor removal

e. Cross-check between radii:

Compare your three radii:

• Do they show the same pattern of wide/narrow rings?

• Distinctive patterns (signature years) should appear in all three

• If a pattern appears in only one radius → may be false ring or local damage

• If one radius has fewer rings → likely has missing rings

Final refined age estimate: _____ years

Confidence:  High / Medium / Low

Step 7: Environmental Interpretation (Arizona-specific)

Connect your ring patterns to Mohave County climate history:

Known drought years in Arizona:

• 2002, 2011-2013, 2020-2021 (recent severe droughts)

• 1950s "Dust Bowl" drought

• Look for clusters of narrow rings during these periods

Wet years (El Niño events):

• 1982-1983, 1997-1998, 2015-2016 (strong El Niño winters)

• Look for wider rings following these years

Fire history:

• Fire scars appear as charred areas or distorted rings

• Mohave County has frequent wildfire history

• Post-fire rings often show release (wider growth)

Questions to answer:

1. Can you identify any major drought years in your tree's rings? Years: _____

2. Are there any wet years showing unusually wide rings? Years: _____

3. Does your tree show evidence of fire?  Yes - describe: _____ / No

4. What was the best growth year for this tree? Year (ring #): _____ Ring width: _____ mm

5. What was the worst growth year? Year (ring #): _____ Ring width: _____ mm

Part B: Leaf Functional Traits

Background: Why Study Leaves?

Simple explanation: Leaves are where photosynthesis happens, but they also lose water. Different environments require different leaf strategies. By measuring leaf traits, we can understand how plants adapt to their habitats.

Key Leaf Traits

1. Specific Leaf Area (SLA)

• What it is: Leaf area per unit dry mass (m²/kg)

• What it means:

  • High SLA: Thin, large leaves per unit weight → "cheap" leaves, fast growth

  • Low SLA: Thick, small leaves per unit weight → "expensive" leaves, long-lived

2. Leaf Size

• Why it matters: Larger leaves catch more light but heat up more

• Desert adaptation: Small leaves reduce water loss and overheating

3. Leaf Thickness

• Thick leaves: Store water, resist drying (succulents, desert plants)

• Thin leaves: Efficient gas exchange but dry quickly

4. Leaf Margin

• Entire (smooth): Less evaporative surface

• Toothed/serrated: More surface area, found in moist climates

![Diagram showing leaf margin types: entire, serrate, dentate, lobed]

Materials for Part B

Field collection:

• Pruning shears or scissors

• Plastic bags (ziplock, labeled)

• Permanent marker

• Field notebook

• Camera

• Cooler with ice (optional but helpful)

• Scale

• Metric ruler

• ImageJ software (free download)

• Plant press

Leaf Collection Strategy

Target: 30 leaves from at least 10 different species

You need variety in:

1. Habitats: Sun vs. shade, wet vs. dry

2. Growth forms: Trees, shrubs, herbs, vines

3. Leaf types: Monocots and eudicots, simple and compound

4. Leaf sizes: Small, medium, large

Leaf Collection Guide

Category 1: Full Sun, Dry Habitat (Xeric) - Collect 6-9 leaves

Trees/Shrubs (woody plants):

• Creosote bush (Larrea tridentata) - small, waxy, resinous

• Brittlebush (Encelia farinosa) - silvery, hairy

• Catclaw acacia (Senegalia greggii) - compound leaves, small leaflets

• Desert willow (Chilopsis linearis) - narrow, linear leaves

• Fourwing saltbush (Atriplex canescens) - gray, scaly

Herbs:

• Desert marigold (Baileya multiradiata) - hairy, dissected

• Desert chicory (Rafinesquia neomexicana) - spring annual

Expected traits: Small size, thick, hairy, waxy coatings, low SLA

Category 2: Shade, Moist Habitat (Mesic) - Collect 6-9 leaves

Trees/Shrubs:

• Fremont cottonwood (Populus fremontii) - near streams, large, thin

• Arizona sycamore (Platanus wrightii) - large, lobed, thin

• Goodding's willow (Salix gooddingii) - riparian, narrow leaves

• Arizona ash (Fraxinus velutina) - compound leaves

Understory/Shade plants:

• Western poison ivy (Toxicodendron rydbergii) - BE CAREFUL! Compound leaves

• Wild grape (Vitis arizonica) - large, lobed

• Shade-grown landscape plants in irrigated areas

Expected traits: Larger size, thinner, less hairy, higher SLA

Category 3: Mixed Habitats - Collect remaining leaves

Grasses (monocots):

• Big galleta (Pleuraphis rigida)

• Indian ricegrass (Achnatherum hymenoides)

• Bermuda grass (Cynodon dactylon) - in lawns

Succulents (modified leaves):

• Yucca (Yucca spp.) - thick, fibrous

• Agave (if available) - very thick, fleshy

Landscape/Garden plants (with permission):

• Mulberry - large, sometimes lobed

• Olive - small, waxy

• Various ornamentals

Agricultural weeds:

• Pigweed (Amaranthus spp.)

• Puncture vine (Tribulus terrestris)

• Russian thistle (Salsola tragus)

Procedure: Part B

Step 1: Leaf Collection and Initial Assessment

Best collection practices:

a. Timing:

• Collect in early morning (leaves fully hydrated)

• Avoid midday heat (leaves may be wilted)

• Spring or fall are best seasons

b. Selection criteria:

• Choose mature, healthy leaves

• Avoid damaged, diseased, or insect-eaten leaves

• Select typical size for the species

• One leaf per plant (spread out collection)

c. Handling:

• Cut with clean shears

• Place immediately in labeled plastic bag

• Keep cool and moist until processing

• Label bag with: species, location, habitat, date

For each of your 30 leaves:

1. Assign unique ID number: Leaf #_____

2. Species identification:

• Scientific name: _____

• Common name: _____

• If unsure, take photos and use field guide later

3. Habitat information:

• Collection location: _____

• Light environment:  Full sun / Partial shade / Deep shade

• Moisture regime:  Xeric (dry) / Mesic (moderate) / Hydric (wet)

• Position on plant:  Upper canopy / Mid / Lower / Understory

• Soil type:  Sandy / Rocky / Clay / Cultivated

4. Photograph leaf:

• Place on white background

• Include metric ruler for scale

• Flatten leaf gently (don't damage)

• Photo of upper surface (adaxial - top side)

• Photo of lower surface (abaxial - bottom side)

• Ensure entire leaf including stem (petiole) is visible

5. Qualitative trait observations:

Special features (check all that apply): / Glands (dots or bumps) / Stipules (small leaf-like structures at base) / Aromatic (strong smell when crushed) / Sticky/resinous / Color: / Green / Blue-green / Gray-green / Silvery / Other: _____

6. Linear dimension measurements:

Use metric ruler, measure to nearest mm:

For simple leaves:

• Leaf blade length (base to tip along midvein): _____ mm

• Leaf blade width (widest point, perpendicular to midvein): _____ mm

• Petiole length (base of blade to stem attachment): _____ mm

• Total leaf length (blade + petiole): _____ mm

For compound leaves:

• Number of leaflets: _____

• Whole leaf length (base to tip including all leaflets): _____ mm

• Whole leaf width (widest spread): _____ mm

• Largest leaflet length: _____ mm

• Largest leaflet width: _____ mm

Note: For analysis, you can use either whole leaf or individual leaflet measurements depending on the question

7. Thickness estimate:

Gently feel leaf between thumb and forefinger:

Thickness rating: / 1 - Very thin (tissue paper-like) / 2 - Thin (typical broadleaf) / 3 - Moderate / 4 - Thick (leathery) / 5 - Very thick (succulent)

Arizona note: Desert plants often rate 4-5; riparian plants often rate 1-2

Step 2: Fresh Mass Measurement

Why measure fresh mass? Allows calculation of water content, which relates to habitat moisture.

Procedure:

1. Gently blot leaf with paper towel to remove surface moisture

2. Weigh immediately on digital balance

3. Fresh mass: _____ g

4. Record to 0.01 g precision

Handle leaf carefully - oils from fingers can affect mass!

Step 3: Begin Drying Process

Purpose: Dry mass is needed for calculating SLA (most important leaf trait).

Drying procedure:

Method 1: Drying oven (fastest, most reliable)

1. Place leaf in labeled paper envelope

2. Write leaf ID on envelope in pencil (pen may run)

3. Place in drying oven at 60-70°C

4. Dry for 48-72 hours

5. Weigh daily until mass is constant (change <0.01 g between days)

Method 2: Air drying (no oven available)

1. Place leaf in labeled paper envelope

2. Place in warm, dry, well-ventilated location

3. Good spots: on top of refrigerator, near (not on) heater, sunny windowsill

4. Will take 5-7 days

5. Check daily until completely crispy-dry

While leaves are drying, continue to Steps 4 & 5...

Step 4: Leaf Area Determination

Why is area important? SLA = Area ÷ Mass. It's the key trait linking leaf structure to function.

You'll need ImageJ software (free download): https://imagej.net/ij/download.html

Scanning/photographing leaves:

Method 1: Scanner (best accuracy)

1. Place leaves flat on scanner bed

2. Include metric ruler in scan

3. Scan at 300 dpi minimum (600 dpi better for small leaves)

4. Can scan multiple leaves per image if they don't overlap

5. Save as JPEG

Method 2: Camera (if no scanner)

1. Place leaves flat on white/contrasting background

2. Include ruler

3. Camera directly overhead (perpendicular to leaves)

4. Good even lighting, no shadows

5. High resolution setting

Processing images with ImageJ:

Video tutorial: https://www.youtube.com/watch?v=kXFShlKSXbM

Step-by-step:

1. Open image in ImageJ

   • File → Open → select your image

2. Set scale (tells ImageJ what size things are)

   • Analyze → Set Scale

   • Use "Straight Line" tool to draw along ruler in image

   • Enter known distance (e.g., 10 cm)

   • Select unit (cm)

   • Click "Global" if all images same scale

   • Click OK

3. Trace leaf outline

   • Zoom in for precision (use + tool)

   • Select "Polygon Selection" tool (for straight edges) OR

   • Select "Freehand Selection" tool (for curved edges)

   • Carefully trace around entire leaf perimeter

   • Close the selection (double-click or return to start)

4. Measure area

   • Analyze → Measure (or press 'M')

   • Area appears in Results window in cm²

   • Record area: _____ cm²

5. Repeat for all 30 leaves

Step 5: Leaf Perimeter Measurement

Why measure perimeter? Quantifies leaf complexity and dissection. More complex leaves have higher perimeter:area ratios.

Video tutorial: https://www.youtube.com/watch?v=CFpNrOSFU60

In ImageJ (after tracing outline for area):

• The Results window automatically shows "Perimeter" in addition to Area

• Record perimeter: _____ cm

For complex/lobed leaves:

• Measure entire outer perimeter (not individual lobes separately)

• Don't trace into deep sinuses

Step 6: Dry Mass Measurement

After 48-72 hours of drying (or 5-7 days air drying):

Check if fully dry:

• Leaf should be crispy, not flexible

• Weigh, wait 24 hours, weigh again

• If mass unchanged (within 0.01 g) → fully dry

• If still losing mass → continue drying

Final dry mass: _____ g

Step 7: Calculate Derived Traits

These calculated values are often more meaningful than raw measurements!

For each of your 30 leaves, calculate:

a. Specific Leaf Area (SLA) - THE MOST IMPORTANT LEAF TRAIT

Formula: SLA = Leaf area (cm²) ÷ Dry mass (g)

Example calculation:

• Leaf area = 25 cm²

• Dry mass = 0.18 g

• SLA = 25 ÷ 0.18 = 138.9 cm²/g

Convert to standard units (m²/kg):

• Multiply cm²/g by 10

• 138.9 × 10 = 1389 m²/kg

Your calculation:

• SLA = _____ cm²/g = _____ m²/kg

What SLA means:

• High SLA (>20 m²/kg, or >2000 cm²/g):

  • Thin, "cheap" leaves

  • Fast growth strategy

  • Shade-adapted or wet-habitat plants

  • Short leaf lifespan

• Low SLA (<10 m²/kg, or <1000 cm²/g):

  • Thick, "expensive" leaves

  • Slow growth, conservative strategy

  • Sun-adapted or dry-habitat plants

  • Long leaf lifespan (months to years)

• Medium SLA (10-20 m²/kg):

  • Intermediate strategy

Expected in Mohave County:

• Desert plants: LOW SLA (thick, long-lived leaves)

• Riparian plants: HIGH SLA (thin, fast-growing leaves)

b. Water Content

Formula: Water content = [(Fresh mass - Dry mass) ÷ Fresh mass] × 100

Example:

• Fresh mass = 0.45 g

• Dry mass = 0.18 g

• Water content = [(0.45 - 0.18) ÷ 0.45] × 100 = 60%

Your calculation:

• Water content = _____%

Interpretation:

• High water content (>70%): Mesophytes (moderate moisture plants)

• Medium water content (60-70%): Typical

• Low water content (<60%): Xerophytes (drought-adapted) or sclerophylls (thick, tough leaves)

c. Leaf Shape Index (Length:Width Ratio)

Formula: Length ÷ Width

Example:

• Length = 80 mm

• Width = 35 mm

• Ratio = 80 ÷ 35 = 2.3

Your calculation:

• Shape index = _____

Interpretation:

• Ratio close to 1: Round or ovate leaves

• Ratio 2-3: Elliptical leaves

• Ratio >3: Narrow, linear leaves (common in dry habitats)

d. Petiole:Blade Ratio

Formula: Petiole length ÷ Blade length

Your calculation:

• Ratio = _____

Why it matters:

• Long petioles allow leaves to adjust position for light

• Short/no petioles: more conservative water strategy

e. Leaf Dissection Index (Perimeter:Area Ratio)

Formula: Perimeter (cm) ÷ √Area (cm²)

Example:

• Perimeter = 45 cm

• Area = 25 cm²

• √25 = 5

• Dissection index = 45 ÷ 5 = 9.0

Your calculation:

• Dissection index = _____

Interpretation:

• Low values (4-6): Simple, entire margins

• High values (>8): Complex, lobed, or deeply toothed

• Higher dissection = more boundary layer disruption = better cooling

Step 8: Data Compilation

Create a master data table with all 30 leaves: