The LINX study (Lotic Intersite Nitrogen eXperiment) is a collaborative effort aimed at understanding the dynamics of nitrogen in stream ecosystems. Begun as a workshop at Coweeta in 1995, it currently involves parallel investigations in 10 streams ranging from the Arctic to the tropics. For additional information on the project, please see the LINX webpages at http://coweeta.ecology.uga.edu/webdocs/linx.

Mack Creek was the last of the 10 streams investigated, with sampling completed in October 1998. We measured several key hydrodynamic, chemical and metabolic parameters using the same techniques employed at the other nine LINX sites. These measurements, in addition to the stable isotope work described below, have been used in the LINX group integration efforts, which will combine data from all sites to try to identify broad patterns in nitrogen dynamics in stream ecosystems. These cross-site syntheses also include an examination of factors controlling stream metabolism (Mulholland et al 2001), microbial biomass variability (Findlay et al 2002) as well as methods development (Mullholland et al 2001). The role of biophysical factors in controlling the uptake and export of nitrogen across biomes was quantified and a model developed based on the LINX cross-site analyses (Peterson et al 2001).

After quantifying background parameters, each LINX site added the stable isotope of nitrogen, ¹⁵N in the form of ¹⁵N labeled ammonium for approximately 6 weeks. The amounts added were sufficient to track uptake and assimilation, but not enough to achieve a fertilization effect. At Mack Creek, we sampled various components of the food web weekly along a 225 m downstream gradient. Each of the LINX sites tried to sample approximately similar portions of the food web. For example, epilithon (organic matter and organisms such as algae and bacteria growing on rocks) and grazing invertebrates were sampled at all sites; wherever possible, we all tried to sample similar taxa. At Mack Creek, we also attempted to sample as broad a representation of the food web as possible. We collected riparian plants (leaves, roots, stems), small mammals that forage in the riparian zone (in cooperation with the Oregon Cooperative Fisheries & Wildlife Unit of the NBS/USGS), and aquatic birds (in cooperation with USFWS). In addition, Diane Sanzone, a doctoral student at the University of Georgia, collected spiders and emerging aquatic insects.

Water samples taken along the downstream transect over the course of the ¹⁵N addition reveal a rapid uptake of the dissolved enriched ammonium in the first 100 m of the release (Figure 1). This was used to calculate an uptake length of ¹⁵N-ammonium uptake length, which was much less than that predicted by the initial enrichment release study. Dissolved nitrate (Figure 2) increased downstream from the drip site, a pattern that increased over the duration of the release. Both declined within 12 hours of turning off the dripper (the day 43 values).

We analyzed ¹⁵N uptake by the in-stream components according to their presumed trophic status. By the final day of ¹⁵N addition (day 42), the herbivores and their presumed food sources showed similar spatial patterns to that of the ¹⁵N-ammonium (Figure 3). We sampled three primary producers, liverworts, mosses, and epilithon. Interestingly, the liverworts and mosses became much more enriched than the faster-growing epilithon. The grazers, including two mayflies and tailed-frog tadpoles, also showed different spatial patterns, with the mayflies incorporating more ¹⁵N than the larger, slower growing tadpoles. The mayflies showed a higher degree of enrichment than their presumed food source, the epilithon, a pattern that has been seen at other LINX sites (Mulholland et al 2000).

We observed somewhat different patterns with the organisms that feed on decaying leaves or woody materials. Of the detrital components we sampled, only the epixylon (biofilm on wood) became significantly enriched (Figure 4). Epixylon probably consists primarily of fungi and bacteria, but may include some algal cells as well. In contrast, fine benthic organic material (FBOM), decaying conifer needles, and wood pieces showed relatively little enrichment. The spatial pattern for these compartments, as well as their consumers (a stonefly and a caddisfly) was similar to that seen for the grazing food web. The enrichment levels and uptake rates seen for the wood biofilm suggest that this component of stream ecosystems may play an important role in nitrogen uptake and retention.

Mack Creek is home to many predators, both invertebrate and vertebrate. Because of relatively low numbers, especially of the vertebrate consumers, only two species were sampled on a weekly or biweekly basis; all others were collected only on the final day of the addition. The two species that showed the greatest ¹⁵N enrichment were a predaceous stonefly, Calineuria, and young-of-the-year cutthroat trout fry (Onchorhynchus clarki). These two species showed almost identical patterns of uptake (Figure 5. These fish hatched only a few weeks before beginning of the enrichment period. In contrast, the older fish and Pacific giant salamanders (Dicamptodon tenebrosus) showed slight enrichment, but only at sites closest to the dripper.

We examined links from the aquatic ecosystem to the adjacent terrestrial system by sampling riparian vegetation on the last day of ¹⁵N inputs. Preliminary results of roots, stems and leaves of four different perennial species reveal different patterns of ammonium uptake (Figure 6). The ladyfern, Athyrium felix-femina (ATFI) showed strong enrichment of the roots, but not in leaves or stems. Wood-sorrel (Oxalis oregona, OXOR) was equally labeled in roots, stems and leaves, while piggy-back-plant (Tolmiea menziesii, TOME) showed little enrichment in the roots compared to stems and leaves. Roots of the hedge-nettle, Stachys cooleyae (STCO) were not sampled at this particular site, but the leaves and stems showed some enrichment, though not as much as the Oxalis or Tolmiea.

The results from this study are currently in draft form, and focus on the dynamics of nitrogen in streams of old growth forests. This work builds on earlier studies on stream nitrogen budgets at the H.J. Andrews Forest, with an additional emphasis on the structure of stream food webs. The most striking results from the Mack Creek study were the significance of in-stream bryophytes and the previously-overlooked role of biofilms on large wood in uptake and retention of nitrogen, and the transfer of dissolved, in-stream nitrogen into components of the terrestrial ecosystem.

The second phase of the LINX study (LINXII), involving the same group of stream researchers, will be focusing on the impacts of land use type (urban/suburban, agriculture and forest/reference) on nitrate dynamics in small streams. Recent research has suggested (Alexander et al) that small streams may be key players in retention and processing of nitrogen. This study will operate at a variety of scales across 8 different biomes, and will allow us to assess the effects of human disturbance on nitrogen dynamics in stream networks.

Literature Cited:

Findlay, S., J. Tank, S. Dye, H.M. Valett, P. Mulholland, W.H. McDowell, S. Johnson, S.K. Hamilton, J. Edmonds, W.K. Dodds, and W.B. Bowden. Bacterial and fungal biomass in detritus-based microhabitats of headwater streams. In Press. Microbial Ecology. 2002.

Mulholland, P.J., J.L. Tank, D.M. Sanzone, W. Wollheim, B.J. Peterson, J.R. Webster, and J.L. Meyer. 2000. Food resources of stream macroinvertebrates determined by natural-abundance stable C and N isotopes and a 15N addition. Journal of the North American Benthological Society 19:145-157.

Mulholland, P.J., J.L. Tank, D.M. Sanzone, B.J. Peterson, W. Wollheim, J.R. Webster, and J.L. Meyer. 2001. Ammonium uptake length in a small forested stream determined by 15N tracer and ammonium enrichment experiments. Verh. Internat. Verein. Limnol. 27:1320-1325.

Mulholland, P.J., C.S. Fellows, J.L. Tank, N.B. Grimm, J.R. Webster, S.K. Hamilton, E. Marti, L. Ashkenas, W.B. Bowden, W.K. Dodds, W.H. McDowell, J.L. Meyer, and B.J. Peterson. 2001. Controls on stream metabolism examined in an inter-biome comparison. Freshwater Biology 46:1503-1517.

Peterson, B.J., W. Wollheim, P.J. Mulholland, J.R. Webster, J.L. Meyer, J.L. Tank, Marti, E., W.B. Bowden, H.M. Vallet, A.E. Hershey, W.H. McDowell, W.K. Dodds, S.K. Hamilton, S.V. Gregory, and D.J. D'Angelo. Control of nitrogen exports by small headwater streams. Science 292:86-90.