Nitrogen Isotopes in Animals: Systematics Timothy Lambert (adapted from 2007 presenter) Earth 229, Winter

Roadmap Why are animals enriched in 15 N? 1.Physiology 2.Model What causes variability in this discrimination? 1.Dietary protein 2.Environmental controls 3.Growth vs. catabolism

N Cycle (human) amino acid pool throughout body significant mixing protein turnover Some proteins turnover faster than others Enzymes break dietary protein into amino acids.

N Cycle (human) Fate #2: Metabolized -N excreted as ammonia/urea -C skeleton converted to fat/glucose Fate #1: Body protein Amino acid pool

Nitrogen excretion Ammonia NH 3 Simplest form, but toxic Bony fish, amphibian larvae Urea (NH 2 ) 2 CO More complex but still toxic Mammals, some herps (frogs), cartilagenous fish Uric Acid C 5 H 4 N 4 O 3 Least toxic Birds, insects Water efficiency

Moving N in the body: Transamination α-keto acidsAmino acids Transfer of amine group

First transfer amine group to carrier Ketoglutarate → Glutamate Deamination Then deaminate Glutamate to produce ammonia in liver or kidney Deamination fractionates N! Ammonia product is depleted in 15 N.

The Urea Cycle Requires CO 2, NH 3, and aspartate Glutamate = source of NH 3 and aspartate Glutamate fractionates N ( 14 N is preferentially reacted)

N in the Body Kinetic Fractionation, Open System Urea (depleted in 15 N) Hair, milk, feces… Diet -6 per mil Body (enriched in 15 N) Animals are enriched in 15 N relative to diet because urea is depleted in 15 N relative to body.

Model for  15 N in the Body

Diet = Excretion Products No significant depletion of waste products relative to diet Claimed it contradicted theory of enrichment due to depleted 15 N in urea Explanations? Urea is not urine (contains creatinine, etc.) High protein diet In equilibrium, inputs = summer outputs (always!) Body tissue is elevated relative to diet, urea is depleted relative to body. Pretty llama pictures Llama Study (Sponheimer et al. 2003)

~3 per mil for every trophic level Trophic Ecology

Amino Acids in Trophic Ecology Bimodal  15 N distribution Source amino acids (essential) Trophic amino acids (nonessential) Martinez del Rio et al. 2009

Trophic Ecology But lots of variation. Why?

What causes variability in N isotope fractionation? Low vs. high protein Herbivore vs. carnivore ↑ quality decreases fractionation ↑ quantity increases fractionation 1. Protein in the Diet Koch 2007

Effects of elemental composition on the incorporation of dietary nitrogen and carbon isotopic signatures in an omnivorous songbird. (Pearson et al., 2003) Yellow-rumped warbler High vs. low protein diets Food: Bananas and insects in varying proportions Sampling of mass, blood, feathers

Diets: %Insect, Isotopes, & Concentrations Attempted to create diets along a linear continuum of increasing a) isotopic signature ( didn’t quite work for  15 N ) b) elemental concentration by increasing the % insect protein in diet

Only 0.12‰ difference in δ 15 N values among diets. Diet containing most insects did not have highest δ 15 N value (diet with lowest proportion of insects did not have the lowest δ 15 N value) Diets: %Insect, Isotopes, & Concentrations

Turnover Rates: Half-life Plasma & Blood Half-life estimates plasma: δ 13 C daysδ 15 N: days Half-life whole blood: δ 13 C ~4-6 days (diet 1=33 days!) δ 15 N days Whole blood is variable!

Discrimination: Plasma, Feather, and Blood  15 N values plasma & whole blood enriched 1.7 to 3.0‰ “Apparent” fractionation factor for feathers  15 N enriched ( ‰) Fractionation factors increased linearly with elemental concentration in diet for N

↑ %N ↑ uric acid w/ ↑ 14 N in out ↑ tissue δ 15 N

High Protein = Large Fractionation Due to larger loss of 15 N- depleted urea %N in diet Results 1.Diet: Linear relationship between elemental concentration and fractionation factor. 2. Tissue: Discrimination and turnover rates vary. Solution: Concentration dependent, multi-compartment mixing models

What causes variability in N isotope fractionation? 1.High vs. low protein diets 2.Water availability? Correlation between bone collagen  15 N and aridity

Why does ↓ Water availability ↑ δ 15 N in Animal Tissue? 1. Diet/plant δ 15 N increases in arid habitats –↑ aridity = larger relative 14 N-rich gas loss (soil denitrification) 2. Metabolic enrichment theories –↑ urine excreted is isotopically heavy (rich in δ 15 N) (Ambrose & DeNiro 1986, Sealy 1987) –↓ protein diets in arid regions promote urea recycling for N

Kangaroo metabolism does not cause the relationship between bone collagen δ 15 N and water availability (Murphy & Bowman, 2006) Motivating question: Can ↑ δ 15 N be explained by herbivore diet alone?

Methods Big study! 779 road killed roos –  15 N,  13 C of bone collagen –Macropus spp, grazers, small ranges 173 grass collections –3-4 primary spp at each site,  15 N Water Availability Index

data = +

Results 4.74‰ to 4.79 ‰ enrichment

What about C3 vs C4 grasses? Both C3 and C4 plants show decreased δ15N with increased water availability. δ 13 C of bone collagen as proxy Negative and weak relationship Lower δ 15 N in C4 plants (1.1‰) C4 C3 C4 C3 A : No! Can’t explain isotope trend by differences in C3:C4. Q: Can dietary C3:C4 explain the δ 15 N vs. water availability trend?

Strong negative relationship of herbivore δ 15 N bone collagen and water availability. Near identical negative pattern of δ 15 N in grass and kangaroo bone collagen with water availability Plant δ 15 N is main cause, with no change in metabolism Huge support for historic trophic ecology and past climate change data that rely on direct relationship between herbivores and plants which not confounded by animal metabolism Summary

What causes variability in N isotope fractionation? 1.High vs. low protein diets 2.No aridity effects (but understand environmental effects on  15 N of the food chain’s base!) 3.Starvation! Growth vs. catabolism

Nitrogen Balance: Starvation Body Mass Lost Urea Body 6‰6‰ Generalization: Starvation increases  15 N of tissue. Inconsistent results (Martinez del Rio et al.) Assumes well-mixed pool Reality: tissues vary in growth Some continue protein synthesis (e.g. splanchnic organs, liver), others shut off (e.g. muscle) Solution: Multiple compartments Kinetic Fractionation, Closed System Urea (depleted in 15 N) Hair, milk, feces… Diet -6 per mil Body (enriched in 15 N)

Nitrogen Balance: Starvation Body Mass Lost Urea Body 6‰6‰ Generalization: Starvation increases  15 N of tissue. Inconsistent results (Martinez del Rio et al.) Assumes well-mixed pool Reality: tissues vary in growth Amino acid pool becomes enriched; Some tissues continue protein synthesis (e.g. liver), others shut off (e.g. muscle) Solution: Multiple compartments Kinetic Fractionation, Closed System

Summary 1.Animals retain 15 N, excreting 14 N preferentially (~6‰) 1.Useful in trophic ecology 2.Differences between source and trophic amino acids 2.Discrimination affected by: 1.Protein quality and quantity 2.Aridity affects food chain, not physiology 3.Starvation increases δ 15 N