Applications of Stable Isotope Techniques to Ecosystem Research


Jeffrey S. Owen

Institute of Earth Sciences, Academia Sinica, China-Taipei



Stable isotopes studies could contribute much to our understanding of elemental cycles in Asian ecosystems.  As an example of an ongoing study in the Asia region that uses stable isotope techniques, results from an inventory of 15N natural abundance of soils and vegetation in a forested watershed in Taiwan are discussed.  Soil d15N values in the forest floor ranged from –1.8 to 1.8 ‰.  Mineral soils had higher d15N values (4.1 to 6.0 ‰).  Foliage d15N values for overstory trees ranged from –6.6 to –2.0 ‰, and understory foliage d15N values ranged from –5.0 to – 1.2.  Foliar d15N values of both groups were generally lower at the upper hillslope site.  Both among species and among site differences are likely related to differences in plant-mycorrhizal interactions.  A smaller difference between plant and soil d15N values at the lower elevation site is possibly related to increased N availability at this site, either through differences in mineralization/nitrification rates or differences in plant and fungal N cycling.  These results highlight the need for more information regarding the relationships between N stable isotopes and processes affecting rates of N cycling, especially as related to wider scale patterns in forest ecosystems within the East Asian region.





Biogeochemistry is by definition a multi-disciplinary field.  This adds to the complexity of studying challenging problems such as climate change, environmental pollution, and protecting the multiple functions of the earth’s biological diversity.  When we consider the fact that ecosystem processes, pollution, and natural catastrophic events ignore political boundaries, these problems become even greater challenges.  Asia, the world’s largest and most populated continent, offers such challenges at the highest level.  Despite the seemingly universal agreement regarding the notion that scientists and policy makers require more information about ecosystems in regions other than the temperate zone, relatively few examples of cross-site, comparative ecological studies can be found in the region.  Many gaps remain in our understanding of Asian subtropical and tropical ecosystem characteristics and function (Schlesinger, 1997, Howarth et al. 1996).  This report is intended to present a simple review of stable isotope techniques in ecosystem and biogeochemical research.  This approach is fairly new in ecosystem research and might be used to improve our understanding of patterns and function in a diverse range of ecosystems.  Some results from an ongoing project in Taiwan are given as examples.



Importance of Asian Region in Global Ecosystem Research


Human activity has changed global processing and patterns of many materials: nutrients, sediments, metals, pollutants, and organic matter (e.g. Smil, 1985, Galloway, et al., 1995, Berner and Berner, 1996).  Several trends indicate these changes will become more pronounced in Asia (and South America) in the future.  Global population estimates show that Asia has about 60 per cent of the world’s people (World Resources Institute, 1996).  The 1995 World Population Trends report indicate that most of the future population growth will take place in developing countries (United Nations Population Division, 1995).  This pattern has many implications for ecosystem nutrient cycling and global biogeochemistry.  Galloway et al. (1989) estimated that sulfur and nitrogen emissions from Asia could increase by 650 and 530 per cent, respectively, by 2020.  These trends, when taken together, reinforce the importance of understanding the present status and future projections for ecological systems in Asia.  One approach that can be incorporated into ongoing, as well as future, environmental monitoring networks and research programs is stable isotope ecology.



Brief Introduction to Stable Isotope Terminology


Isotopes are atoms of the same element that have a different number of neutrons.  Stable isotopes do not decay into radioactive daughter products, and this is advantageous since there are no concerns about radioactive contamination when preparing materials for stable isotope analysis.  Stable isotope compositions are measured using an isotope ratio mass spectrometer.  By convention, the stable isotopic composition is expressed as the per mil (‰) deviation relative to a standard:

d = (Rsample / Rstandard – 1) x 1000, where R refers to the ratio of heavy isotope to the light isotope.

      In ecological research, we are usually concerned with stable isotopes of carbon (13C/12C), nitrogen (14N/15N), and sulfur (32 S/ 34S).  Several recent reviews on stable isotopes in environmental and ecological studies have been published (Peterson and Fry, 1987, Lajtha and Michener, 1994, Griffiths, 1998).  One research area in biogeochemistry that has exploited stable isotope approaches especially well is in ecosystem nitrogen cycling studies.  Attention has been focused on the possibility of “nitrogen saturation” in forested ecosystems, especially in North America (Aber et al. 1989; Stoddard, 1994) and Europe (Skeffington, 1990; Gundersen et al., 1999).  Recent work has indicated that differences in hydrology and patterns of N cycling from those in watersheds in North America and Europe were responsible for the differences in temporal patterns in stream chemistry found in several watersheds in Japan (Mitchell et al. 1997).  Much more information about rates of N cycling from areas other than North America and Europe is needed to increase our understanding of the variation in forest ecosystem nitrogen cycling processes among different forest types and regions.  In this example, I analyze patterns of d15N natural abundance for soils and vegetation in a forested watershed in Taiwan.  The objective is to investigate 15N natural abundance of soils and vegetation collected along a hillslope gradient in a forested watershed.

Since there have been few stable isotope studies conducted in forests within the East-Asia Pacific region, data from tropical and temperate forests in other regions are used to compare with results from this subtropical forest.  Overall, there is much evidence that there are differences in rates and patterns in N cycling between tropical and temperate forests.  Because many N cycling processes result in isotopic fractionation, some predictable patterns in d15N among components of forested ecosystem occur (Nadelhoffer and Fry, 1994).  Recently, Martinelli et al. (1999) used this approach to test the hypothesis that tropical forests, in general, contain N that is more enriched in 15N.  This should occur because tropical forests are often relatively rich in N and the mechanisms for N losses in forests leave the N remaining in the forest (soil and vegetation N pools) enriched in 15N.



Study Site


The Fu-shan Experimental Forest is located about 40 km southeast of Taipei, Taiwan (24°34’ N, 121°34’ E).  Fu-shan is a moist, subtropical, evergreen forest with a rich flora of over 500 species (Mabry et al., 1998).  Soil and vegetation samples for this study were collected from experimental watershed 1 (37 ha).  The watershed has a SE facing aspect and elevation ranges from about 670 to 1100 m.  Dominant tree species in the study area include Castanopsis carlesii (Helmsl.) Hayata, Litsea acuminata (Blume) Kurata, and Diospyros morrisiana Hance.  Common understory plants are Lasianthus microstachys, Helicia formosana, Alsophila podophylla Hook, and Blatus cochinchinensis Lour.  Mean annual precipitation is about 4200 mm and mean annual temperature is 20.4 °C.

Three hillslope locations were selected and soil pits were excavated by hand  to a depth approaching 100 cm.  Mineral horizons were sampled at 10 cm intervals, sieved, and oven-dried prior to analysis.  Foliage samples were collected from the middle third of the tree canopy in dominant tree species in the immediate area adjacent to each soil pit.  Understory foliage was sampled in a similar manner from two to four randomly clipped branches or bracts and composited.  Foliage samples were oven-dried prior to analysis.  Foliage was pulverized to a fine mesh using a dental amalgamator device.  Elemental and stable isotopic composition were determined using a Finnigan Delta-Plus isotope ratio mass spectrometer interfaced to an elemental analyzer.  Typical precision for duplicate isotopic analyses is 0.2 ‰.



Results and Discussion


Soil d15N profiles showed a sharp increase below the litter horizon at each location (Figure 1).  Soil d15N values in the litter horizon ranged from –1.8 to 1.8 ‰.  For mineral soils, higher d15N values were found (4.2 to 6.0 ‰).  Below the litter horizon, d15N values changed very little with depth at all sampling locations.  Soil N concentration in the forest floor was lowest at the middle hillslope location and ranged from 0.80 to 1.86 % N (Figure 1).  In mineral soil horizons, soil N concentrations did not change notably with depth or among the three soil profiles and ranged from 0.11 to 0.83 % N.

There was a significant relationship between soil N concentration and soil d15N value at each of the hillslope positions.  The best fit regression lines for the three soil profiles were d15Nsoil = -4.275 [%Nsoil] + 6.668 (p<0.001); d15Nsoil = -5.858 [%Nsoil] + 6.445 (p<0.001); and d15Nsoil = -4.345 [%Nsoil] + 6.559 (p<0.001) for the upper, middle, and lower hillslope positions, respectively.  Forest soil profiles often show a general trend of increasing d15N with depth; this pattern has been observed in many forest soils throughout the world (Hogberg et al., 1996).  This pattern occurs because of isotopic fractionation during nitrogen cycling leading to plant uptake of N that is depleted in 15N, and because of internal N cycling through mycorrhizal fungi that also results in the transfer of N that is depleted in 15N to host plants (Hobbie et al, 2000).  Both of these mechanisms result in the transfer N that is depleted in 15N  to the soil surface via litterfall.  Thus, for nitrogen, plants are nearly always isotopically lighter than the soil supporting their growth (Nadelhoffer and Fry, 1994).  Fractionation during decomposition and foodweb processes may also contribute to this pattern.

Foliage d15N values for overstory trees ranged from –6.6 to –2.0 ‰, and understory foliage d15N values ranged from –5.0 to – 1.2 (Figure 2).  Foliar d15N values of both groups were generally lower at the upper hillslope site.   The smaller difference between foliage and soil re was a trend for a smaller difference between foliage and soil d15N values at the lower hillslope position may be related either to differences in the d15N of available N or differences in mycorrhizal-plant N cycling.

Rates of microbial N transformations should affect the isotopic composition of soils (Garten, 1993; Hogberg et al., 1996).  Topographic position may affect the rates of many microbial processes in soils and thereby affect soil and foliar d15N values.  For example, nitrification rates in soils are influenced by variables such as temperature, moisture, and nitrate concentration.   During nitrification, the remaining ammonium should become enriched in 15N and the nitrate produced depleted in 15N (Hogberg et al., 1996). At our study site, preliminary evidence suggests that net nitrification rates are higher at the lower elevation site (Owen, unpublished data).  Differences in net nitrification and mineralization rates are likely related to the pattern we found in foliar d15N values.  At the lower site, if net nitrification rates are higher, then the isotopic composition of available NH4+ would be expected to be enriched in 15N.  This mechanism may help explain the trend of higher foliage d15N values found at the lower hillslope site.  In general, rates of net nitrification and mineralization are often higher in tropical forests compared to forest soils in temperate regions (Martinelli et al., 1999).  Information about stable isotopic composition of specific forms of N are needed to better understand patterns in N cycling in forested ecosystems.  The few data that are available (e.g. Koba et al., 1998) suggest that measurements of both soil extractable and solution NO3- and NH4+ could contribute to understanding regional differences in rates and patterns in N cycling.  Investigating the relationships between rates of microbial N transformations and soil isotopic composition is one focus of our ongoing research at this site.

Differences in foliar d15N values among the three hillslope sites can be plausibly explained by isotopic fractionation during mycorrhizal transfer and differences in mineralization and nitrification rates, with either greater effective mycorrhizal fractionation at the upper hillslope site with lower N availability, or smaller losses of inorganic N that is depleted in 15N  relative to that in the lower hillslope site.

In summary, these results for soil and vegetation stable N isotopic composition from a subtropical forest in Taiwan are consistent with similar data from other forest ecosystems.  Isotopic patterns in this sub-tropical forest seem to conform more closely to patterns in temperate forest than to patterns in tropical systems.  d15N values of bulk N for soil profiles among the three hillslope positions did not vary greatly.  d15N values of understory and overstory vegetation generally decreased from the lower site to the upper hillslope site.  The difference between foliage and soil d15N values was smaller at the lower hillslope site and this difference is probably related to a complex suite of variables with differences in inorganic N availability, mycorrhizal-plant N cycling, and rates of microbial N transformations among the more important factors. Intensive studies on N cycling in subtropical forests in the East Asia region, including stable isotope approaches can help improve our understanding of patterns in global N cycling.





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