Applying the principles of isotope analysis in plant and animal ecology to forensic science in the Americas.

The heart of forensic science is application of the scientific method and analytical approaches to answer questions central to solving a crime: Who, What, When, Where, and How. Forensic practitioners use fundamentals of chemistry and physics to examine evidence and infer its origin. In this regard, ecological researchers have had a significant impact on forensic science through the development and application of a specialized measurement technique-isotope analysis-for examining evidence. Here, we review the utility of isotope analysis in forensic settings from an ecological perspective, concentrating on work from the Americas completed within the last three decades. Our primary focus is on combining plant and animal physiological models with isotope analyses for source inference. Examples of the forensic application of isotopes-including stable isotopes, radiogenic isotopes, and radioisotopes-span from cotton used in counterfeit bills to anthrax shipped through the U.S. Postal Service and from beer adulterated with cheap adjuncts to human remains discovered in shallow graves. Recent methodological developments and the generation of isotope landscapes, or isoscapes, for data interpretation promise that isotope analysis will be a useful tool in ecological and forensic studies for decades to come.

An automated system for stable isotope and concentration analyses of CO2 from small atmospheric samples.

We have developed an automated, continuous-flow isotope ratio mass spectrometry (CF-IRMS) system for the analysis of delta(13)C, delta(18)O, and CO(2) concentration (micromol mol(-1)) ([CO(2)]) from 2 mL of atmospheric air. Two replicate 1 mL aliquots of atmospheric air are sequentially sampled from fifteen 100 mL flasks. The atmospheric sample is inserted into a helium stream and sent through a gas chromatograph for separation of the gases and subsequent IRMS analysis. Two delta(13)C and delta(18)O standards and five [CO(2)] standards are run with each set of fifteen samples. We obtained a precision of 0.06 per thousand, 0.11 per thousand, and 0.48 micromol mol(-1) for delta(13)C, delta(18)O, and [CO(2)], respectively, by analyzing fifty 100 mL samples filled from five cylinders with a [CO(2)] range of 275 micromol mol(-1). Accuracy was determined by comparison with established methods (dual-inlet IRMS, and nondispersive infrared gas analysis) and found to have a mean offset of 0.00 per thousand, -0.09 per thousand, and -0.26 micromol mol(-1) for delta(13)C and delta(18)O, and [CO(2)], respectively.

The cost of trauma.

Trauma is a financial burden. For the 2634 trauma patients seen in 1999, the percentage of their hospital bill reimbursed and cost coverage (CC), whether that reimbursement covered their hospital costs, were analyzed. Student t tests to compare the mean percentage reimbursements (mPR) and logistic regression with CC (yes/no) as dependent variable with results as odds ratio (OR) were done. The overall mPR was 36 per cent. Among the 947 patients admitted (36%), there was no association between injury severity and mPR. For penetrating trauma, the mPR (25%) was lower than for blunt trauma (37%, P = 0.05). The assault mPR (21%) was lower than for motor vehicle crash (39%, P < 0.001). The mPR for patients transferred in (26%) was lower than for all others (37%, P < 0.001). Male sex (OR = 0.76), Hispanic ethnicity (OR = 0.46), admission (OR = 0.69), severe brain injury (OR = 0.58), abdominal injury (OR = 0.65), and extremity injury (OR = 0.69) were significant predictors for no CC. Reimbursement is better for blunt trauma. That transfers had a significantly lower mPR may represent "dumping" of patients. There is an association between anatomic regions injured and CC. No reimbursement was obtained for 26 per cent of the patients, and in 56 per cent the reimbursement did not cover costs. A change in financing for trauma is needed.

Rapid 18O analysis of small water and CO2 samples using a continuous-flow isotope ratio mass spectrometer.

High-frequency throughput is often needed in isotopic studies in biological and medical fields. Here we report that high-precision oxygen isotope ratio measurements of water (+/-0.13 per thousand) were rapidly and routinely made on small samples (40-100 microL) using an isotope ratio mass spectrometer operated in continuous-flow mode. Simple modifications to existing instrumentation allow for rapid manual analyses of dilute CO2 (10% CO2/90% N2), including the addition of a septum port and water trap prior to the gas chromatography (GC) column (elemental analyzer column in this study) and the extension of fused-silica capillary tubing between the mass spectrometer source and the effluent tubing from the GC column (located within the CONFLO unit on Finnigan mass spectrometers). We routinely analyzed 20 small-volume samples per hour using this technique, without sacrificing precision of the oxygen isotope ratio measurement.

Unusually low carbon isotope ratios in plants from hanging gardens in southern Utah.

Leaf carbon isotope ratios (δ13C) and photosynthetic gas exchange were measured on plants growing in hanging garden communities in southern Utah, USA. Hanging gardens are unusual, mesic cliff communities occurring where water seeps from the sandstone bedrock in an otherwise extremely arid region; there is very limited overlap in species distributions inside and outside these gardens. Solar exposure in hanging gardens varied with orientation and one of the gardens (Ribbon Garden) was shaded throughout the day. The leaf δ13C values of plants in hanging gardens were significantly more negative than for plants from either nearby ephemeral wash or riparian communities. In Ribbon Garden, the observed δ13C values were as low as -34.8‰, placing them among the most negative values reported for any terrestrial plant species growing in a natural environment. Hanging garden plants were exposed to normal atmospheric CO2 with an average δ13C value of -7.9‰ and so the low leaf δ13C values could not be attributed to exposure to a CO2 source with low 13C content. There was a seasonal change toward more negative leaf δ13C values at the end of the growing season. The observed leaf δ13C values were consistent with photosynthetic gas exchange measurements that indicated unusually high leaf intercellular CO2 concentrations associated with the relatively low light levels in hanging gardens. Thus, extremely negative leaf δ13C values would be expected if significant amounts of the seasonal carbon gain occur at light levels low enough to be near the light compensation point. Maximum observed photosynthetic rates varied with light levels at each of the gardens, with maximum rates averaging 20.3, 14.6, and 3.1 µmol m-2 s-1 at Double Garden, Lost Garden, and Ribbon Garden, respectively. Leaf nitrogen contents averaged 18.5 mg g-1 in species from the more shaded hanging gardens (Lost and Ribbon). When expressed on a leaf area basis, nitrogen contents averaged 117 mmol N m-2 at Lost Garden and 65 mmol N m-2 at Ribbon Garden (shadiest of the two gardens). Leaf nitrogen isotope ratios averaged -2.3‰ (range of -0.7 to -6.1‰), suggesting that most of the nitrogen was derived from a biological fixation source which is most likely the Nostoc growing on the sandstone walls at the seep. These values contrast with leaf nitrogen isotope ratios of 5-9‰ which have been previously reported for arid zone plants in nearby ecosystems.

Seasonal carbon isotope discrimination in a grassland community.

Grassland communities of arid western North America are often characterized by a seasonal increase in ambient temperature and evaporative demand and a corresponding decline in soil moisture availability. As the environment changes, particular species could respond differently, which should be reflected in a number of physiological processes. Carbon isotope discrimination varies during photosynthetic activity as a function of both stomatal aperture and the biochemistry of the fixation process, and provides an integrated measure of plant response to seasonal changes in the environment. We measured the seasonal course of carbon isotope discrimination in 42 grassland species to evaluate changes in gas exchange processes in response to these varying environmental factors. The seasonal courses were then used to identify community-wide patterns associated with life form, with phenology and with differences between grasses and forbs. Significant differences were detected in the following comparisons: (1) Carbon isotope discrimination decreased throughout the growing season; (2) perennial species discriminated less than annual species; (3) grasses discriminated less than forbs; and (4) early flowering species discriminated more than the later flowering ones. These comparisons suggested that (1) species active only during the initial, less stressful months of the growing season used water less efficiently, and (2) that physiological responses increasing the ratio of carbon fixed to water lost were common in these grassland species, and were correlated with the increase in evaporative demand and the decrease in soil moisture.

Characteristics of Encelia species differing in leaf reflectance and transpiration rate under common garden conditions.

The performance of coastal and desert species of Encelia (Asteraceae) were evaluated through common garden growth observations. The obectives of the study were to evaluate the roles of leaf features, thought to be of adaptive value (increased leaf reflectance and/or transpirational cooling), on plant growth in the hot, arid, desert garden versus their impact on growth under cooler, relatively more moist coastal garden conditions. E. californica native to the coast of southern California and E. farinosa, and E. frutescens, interior desert species, were grown in common gardens at coastal (Irvine, California) and interior (Phoenix, Arizona) sites under both irrigated and natural conditions. Although all species survived in both gardens during the two and a half year study period, there were large differences in their sizes. In the desert garden, leaf conductance and leaf water potential were both lower than at the coastal site. E. californica shrubs were leafless much of the time under natural conditions in the desert garden and had the smallest size there as well. Under natural conditions, E. farinosa, with its reflective leaf surface, was able to maintain lower leaf temperatures and attained a large size than the other two species in the desert garden. The green-leaved species (E. californica and E. frutescens) were not able to maintain leaves into the drought periods in the desert garden, with the exception of the irrigated E. frutescens which did maintain its leaf area if provided with supplemental watering to maintain transpirational leaf cooling. In the coastal garden, all species survived and there were few clear differences in the physiological characteristics among the three species. E. californica, the coastal native, attained a larger size in the coastal garden when compared with either of the two desert species.

Comparative water use and nitrogen relationships in a mistletoe and its host.

The impact of the xylem-tapping mistletoe Phoradendron juniperinum on the nitrogen and water relations of its host Juniperus osteosperma was investigated under natural field conditions. Leaf conductance, leaf water potential, and leaf Kjeldahl nitrogen contents were followed through the growing season on mistletoes, infected junipers (separating infected from uninfected stems) and uninfected junipers. Infected trees experienced lower leaf water potentials than uninfected trees and also had lower leaf conductances and lower leaf nitrogen contents. Infected juniper stems had higher conductances than uninfected stems. Mistletoes had higher leaf nitrogen contents than their hosts and much of this nitrogen appeared as arginine, a potential nitrogen storage compound. Photosynthetic rates (per unit leaf area) were significantly higher in junipers than in the mistletoe, and higher in the uninfected than infected junipers. Water use efficiencies as estimated by carbon isotope ratios were significantly lower in mistletoes than in their hosts. Increased mistletoe infestation appeared to increase absolute water use efficiency of both host and mistletoe.