1. Animal Nitrogen Overview of N cycling farm animals a few unfortunate songbirds road-kill down under Nitrogen Isotopes in Mammalian Herbivores: Hair  15 N Values from a Controlled Feeding Study (Sponheimer et al., 2003) Effects of elemental composition on the incorporation of dietary nitrogen and carbon isotopic signatures in an omnivorous songbird (Pearson et al., 2003) Kangaroo metabolism does not cause the relationship between bone collagen δ 15 N and water availability (Murphy et al., 2006)

2. N Cycle (human) amino acid pool throughout body significant mixing protein turnover Some proteins turnover faster than others some tagged (oxidized or other means)

3. High conversion to fat/glucose Ammonia/urea excretion N Cycle (human) Dietary protein Low deficiency of essential aa’s

4. First transfer amine group to carrier Ketoglutarate → Glutamate Deamination Then deaminate Glutamate to produce ammonia in liver or kidney

5. First transfer amine group to carrier Ketoglutarate → Glutamate Synthesis Then to amino acid in liver or kidney

6. *Direction of these reactions controlled by [ ] of Glutamate Ketoglutarate Ammonia ratio of oxidized to reduced enzymes Synthesis Deamination

7. Urea cycle Urea cycle controlled by acetyl CoA and glutamate increase in [ ] after protein rich meal kidney liver

8. Nitrogen excretion animals Ammonia NH 3 Simplest form, but toxic fully aquatic animals Urea (NH 2 ) 2 CO Still toxic more complex than ammonia mammals some herps (frogs), cartilaginous fish Uric Acid C 5 H 4 N 4 O 3 Least toxic egg layers (bird, reptiles, insects) precipitates from egg

9. A few things 1.animals assimilate dietary components with varying efficiencies 2.animal tissues fractionate the isotopes in their diet 3.animals allocate nutrients in their diet differentially to specific tissues ‘isotopic routing’ 4.animals retain δ 15 N, excreting δ 14 N (~6‰) 5.protein balance is a key to fractionation low dietary protein = “protein sparing” reserve dietary protein for tissue maintenance rather than catabolizing it for energy (Castellini and Rea 1992). high dietary protein = use diet protein for tissue synthesis and catabolize excess

10. Nitrogen Isotopes in Mammalian Herbivores: Hair  15N Values from a Controlled Feeding Study (Sponheimer et al., 2003) Goals: Determine the importance of 1.hindgut vs foregut fermentors 2.dietary protein levels on herbivore δ 15 N values.

11. Nitrogen uptake herbivores Hindgut Horses, rabbits, birds, iguanas, green turtle Limited cycling of urea nitrogen fermentation, N cycling, protein balance Foregut Ruminants (can synthesize proteins from inorganic nitrogen compounds) multi compartmental stomachs cows, llamas Ruminant-like kangaroos, wallabies, hoatzin cycle/mix N from diet and self deamination and de novo protein syntheses

12. Diet-Hair Fractionation Same diet, fair bit of variation rabbits and alpaca vary 3.6‰, > 1 trophic level! Foregut fermenters are enriched vs hindgut fermenting rabbits But not to horses…

13. ↑ dietary protein (9-19%) causes enrichment δ 15 N (1.5- 2.8‰) Not what they expected! This refutes N cycling hypo (states that low protein group ↑δ 15 N) feces explanation poor feces is δ 15 N enriched (0.5- 3.0‰), low protein = less urine loss and greater relative (not absolute) %N loss via feces, ↑δ 15 N loss High Protein vs Low Protein Herbivores

14. Effects of elemental composition on the incorporation of dietary nitrogen and carbon isotopic signatures in an omnivorous songbird. (Pearson et al., 2003) Goals Determine turnover rates of δ 15 N and δ 13 C in whole blood and plasma. δ 15 N and δ 13 C diet-tissue fractionation factors for plasma, whole blood, and feathers. Influence of high protein (%N) and low protein (%C) concentrations on fractionation factors. yellow-rumped warbler

15. 32 captive wild-caught migratory birds ‘controlled’ for age & sex Acclimation diet 32% insect Experimental diet 20%,49%,73%, 97% insects Sampled 21 days, mass, blood (plasma, wb), feathers (entire) Determined C&N δ values of different diets turnover rates Discrimination Isotopic signatures of diet on different tissues Materials and methods

16. Diets: %Insect, Isotopes, & Concentrations Attempted to created 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

17. 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) Banana Effect ( δ 15 N 0.5 - 5.3‰) Diets: %Insect, Isotopes, & Concentrations

18. Turnover Rates Isotope incorporation kinetics model (O’Brien et. Al 2000) Δ dt = discrimination factor r = fractional turnover rate Half-life =

19. Turnover Rates: Half-life Plasma & Blood Half-life estimates plasma: δ 13 C 0.4-0.7 daysδ 15 N: 0.5-1.7 days Half-life whole blood: δ 13 C ~4-6 days (diet 1=33 days!) δ 15 N 7.45-27.7 days Whole blood is variable!

20. 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 (3.2-3.6‰) Fractionation factors increased linearly with elemental concentration in diet for N

21. ↑ %N ↑ urine w/ ↑ 14 N in out ↑ tissue δ 15 N

22. Importance of Elemental Concentrations Both isotopic signature of diet and fractionation factors influence the ultimate isotopic signature of tissues (at least plasma). Supports the importance of using concentration-dependent mixing models when reconstructing diet.

23. Results Discrimination factors depend on diet and tissue Fractionation factors to reconstruct diet requires an estimate of elemental concentrations in the diet. Turnover rates Plasma 1 day (short)Whole blood 1 wk (longer) Carbon and nitrogen fractionation factors increase linearly with elemental concentration in the diet. Relationship between the isotopic signature of the diet and the sum of a given tissue’s (at least plasma) isotopic signature + fractionation factor was also positive & linear. USE CONCENTRATION-DEPENDENT MIXING MODELS WHEN ATTEMPTING TO ESTIMATE THE RELATIVE CONTRIBUTION OF DIFFERENT FOOD SOURCES TO AN ANIMAL’S DIET!!!

24. Kangaroo metabolism does not cause the relationship between bone collagen δ 15 N and water availability (Murphy & Bowman, 2006) Goals Evaluate importance of water availability and dietary δ 15 N in determining δ 15 N values in herbivore bone collagen Indirectly determine if ↑ δ 15 N linked to animal metabolism Assessed if δ 15 N in grass and Kangaroo bone collagen are constant with respect to a Water Availability Index Examine other factors influencing δ 15 N in herbivore bone collagen

25. Does ↓ Water availability ↑ δ 15 N in Animal Tissue? Plants enriched in arid environs ‘openness’ N cycle theory (Austin & Vitousek 1998) –↑ water in system = ↓ in ratio of N loss to intrasystem N turnover Cryptobiotic crusts Why ↑ animal δ 15 N when in water limited systems? Metabolic enrichment ‘theories’ –↑ Urea osmolarity, urine excreted is more nitrogen (δ 15 N) concentrated (Ambrose & Deniro 1986, Sealy 1987) excrete more δ 15 N deplete urea when arid (Sponheimer 2003) not experimentally shown for rats (Ambrose 2000) –not tested rigorously… BUT… can ↑ δ 15 N be explained by herbivore diet alone?

26. Methods 173 grass collections (3-4 primary spp/collection) 779 road killed roos –macropus sp, grazers… Water Availability Index estimated from mean annual actual and potential evapotranspiration Akaike’s Information Criterion (AIC) Big study!

27. → +

28. data = +

29. Results Found relationship of δ 15 N and WAI similar between grass and kangaroo bone collagen 4.74‰ to 4.79 ‰ enrichment ~0.05‰ variation over entire range of data

30. When plotted against annual rainfall Murphy & Bowman’s δ 15 N relationship fits with Previous Kangaroo work Eutherian herbivores North America & Africa matches Sealey et al 1987 follows similar pattern

31. What about C3 vs C4 grasses? Model gave little support for other variables: slope chenopod δ 13 C of bone collagen as proxy negative and weak relationship Found lower δ15N in C4 plants (1.1‰) C4 diet (high δ 13 C, low protein) = lower consumer δ 15 N C4 C3 C4 C3

32. 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 (near constant offset in slopes) Suggest dietary δ 15 N is main cause of negative relationship between δ 15 N of kangaroo bone collagen, with water availability and metabolic factors having little discernible effect. Summary

33. Importance… Ties water availability directly to plant δ 15 N to animal δ 15 N values, with little ‘animal’ affect 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

34. Marine food webs are enriched in δ 15 N (Kelly 2000)

35. Trophic Systems (Hobson & Welch 1992) Marine systems 3-4‰/ trophic level Herbivores ~3.2‰ Carnivores 5‰

36. Marine food chains tend to have longer food webs Diet affects, as ascend trophic chain, ↑ %N in diet expect more catabolism = discrimination @ high trophic level Trophic enrichment commonly produces 3:1 slope for δ 15 N and δ 13 C ratios Trophic Systems

40. N15 to get at rainfall abundance

41. Diet-Hair d15N Equilibration Dietary  15 N values changed from 2.5‰ to 7.8‰. Dietary equilibration took ~8-10 weeks Hair

42. Kelly

43. δ 15 N in top consumers in C3, C4 and Marine food chains (Kelly 2000)

44. Discrimination Tissue effects Feathers more enriched than plasma or wb

45. Diet Tissue Relationship C & N signatures linearly related with tissue signatures + discrimination factors

46. Correlated linearly with metabolic rate of tissue Different species have different turnover rates for same tissues Likely related to size, mass specific metabolic rates, life history factors half-life for wb C in bear > crow > quail > warbler Turnover Rates

47. Plasma (1-5 days) Whole Blood (5-35 days) Feces ( Feathers, Hair, Nails, Hoof (time when grown, maybe a lag here) Bone Teeth Turnover Rates

48. Importance of Elemental Concentrations Phillips & Koch 2002

49. Implications CO2WUEN demand NPP δ 15 N in plants ‘openness’ of N cycle δ 15 N in herbivores

50. Pearson Funk/questions -variability in initial mass and mass change following dietary switch among treatment groups (shows they like carbs -Diets did not have ↑ δ 15 N values w/ ↑ % insects -Fractionation vs. discrimination

51. CO2 effects on δ 15 N (Coltrain et. Al. 2004)