plausible mechanism for the maintenance of
adult differentiated cell types. More broadly,
these results showcase the power of explorative
single-cell RNA-seq and point the way toward
future whole-brain and even whole-organism
cell type discovery and characterization. Such
data will deepen our understanding of the
regulatory basis of cellular identity, in development, neurodegenerative disease, and regenerative medicine.
REFERENCES AND NOTES
1. B. J. Molyneaux, P. Arlotta, J. R. Menezes, J. D. Macklis, Nat.
Rev. Neurosci. 8, 427–437 (2007).
2. T. Klausberger, P. Somogyi, Science 321, 53–57 (2008).
3. J. DeFelipe et al., Nat. Rev. Neurosci. 14, 202–216 (2013).
4. K. Sugino et al., Nat. Neurosci. 9, 99–107 (2006).
5. G. Fishell, B. Rudy, Annu. Rev. Neurosci. 34, 535–567 (2011).
6. E. S. Lein et al., Nature 445, 168–176 (2007).
7. A. Kepecs, G. Fishell, Nature 505, 318–326 (2014).
8. D. A. Jaitin et al., Science 343, 776–779 (2014).
9. B. Treutlein et al., Nature 509, 371–375 (2014).
10. A. A. Pollen et al., Nat. Biotechnol. 32, 1053–1058 (2014).
11. S. Islam et al., Nat. Methods 11, 163–166 (2014).
12. T. Kivioja et al., Nat. Methods 9, 72–74 (2011).
13. D. Tsafrir et al., Bioinformatics 21, 2301–2308 (2005).
14. C. Shi, E. G. Pamer, Nat. Rev. Immunol. 11, 762–774 (2011).
15. O. Kann, C. Huchzermeyer, R. Kovács, S. Wirtz, M. Schuelke,
Brain 134, 345–358 (2011).
16. H. W. Dong, L. W. Swanson, L. Chen, M. S. Fanselow,
A. W. Toga, Proc. Natl. Acad. Sci. U. S.A. 106, 11794–11799 (2009).
17. K. Mizuseki, K. Diba, E. Pastalkova, G. Buzsáki, Nat. Neurosci.
14, 1174–1181 (2011).
18. L. Tricoire et al., J. Neurosci. 30, 2165–2176 (2010).
19. M. Prinz, J. Priller, Nat. Rev. Neurosci. 15, 300–312 (2014).
20. I. Galea et al., Glia 49, 375–384 (2005).
21. Y. Zhang et al., Neuron 78, 785–798 (2013).
22. M. Kohyama et al., Nature 457, 318–321 (2009).
23. J. Thomas et al., Biol. Cell 102, 499–513 (2010).
24. E. R. Brooks, J. B. Wallingford, Curr. Biol. 24, R973–R982 (2014).
25. M. A. Zariwala et al., Am. J. Hum. Genet. 93, 336–345 (2013).
26. M. I. Chung et al., eLife 3, e01439 (2014).
The raw data have been deposited with the Gene Expression Omnibus
( www.ncbi.nlm.nih.gov/geo) under accession code GSE60361.
Annotated data are available at http://linnarssonlab.org/cortex. We
thank P. Ernfors, K. Harris, and R. Sandberg for useful comments on
the manuscript; F. Ginhoux for helpful discussions on microglia and
macrophages; A. Johnsson for laboratory management and support;
ALM/SciLife (H. G. Blom) for technical support; and Fluidigm Inc.
(R. C. Jones and M. Lynch) for generous technical and instrument
support. S.L. was supported by the European Research Council
(261063, BRAINCELL) and the Swedish Research Council (STARGET);
A.Z. was supported by the Human Frontier Science Program; A.B.M.-M.
was supported by the Karolinska Institutet (BRECT); C.R. was
supported by the Swedish Cancer Society (CAN2013/852); G.C.-B.
was supported by the Swedish Research Council, the European Union
(FP7/Marie Curie Integration Grant EPIOPC), the Åke Wiberg
Foundation, the Karolinska Institutet Research Foundations, Svenska
Läkaresällskapet, Clas Groschinskys Minnesfond, and Hjärnfonden;
J.H.-L. was supported by the Swedish Research Council, the European
Union [FP7/Marie Curie Actions (322304, Adolescent Development)],
StratNeuro, and the Jeanssons, Åke Wibergs, and Magnus Bergvalls
Foundations; C.B. was supported by the European Research Council
(294556, BBBARRIER), a Knut and Alice Wallenberg Scholar Grant,
the Swedish Cancer Society, and Swedish Research Council.
Supplementary materials contain additional data.
Materials and Methods
Figs. S1 to S11
Tables S1 and S2
30 October 2014; accepted 30 January 2015
Published online 19 February 2015;
Experimental nutrient additions
accelerate terrestrial carbon loss
from stream ecosystems
Amy D. Rosemond,1 Jonathan P. Benstead,2 Phillip M. Bumpers,1 Vladislav Gulis,3
John S. Kominoski,1† David W. P. Manning,1 Keller Suberkropp,2 J. Bruce Wallace1
Nutrient pollution of freshwater ecosystems results in predictable increases in carbon (C)
sequestration by algae. Tests of nutrient enrichment on the fates of terrestrial organic C, which
supports riverine food webs and is a source of CO2, are lacking. Using whole-stream nitrogen
(N) and phosphorus (P) additions spanning the equivalent of 27 years, we found that average
terrestrial organic C residence time was reduced by ~50% as compared to reference
conditions as a result of nutrient pollution. Annual inputs of terrestrial organic C were rapidly
depleted via release of detrital food webs from N and P co-limitation. This magnitude of
terrestrial C loss can potentially exceed predicted algal C gains with nutrient enrichment
across large parts of river networks, diminishing associated ecosystem services.
Nutrient pollution of freshwater ecosystems is pervasive and strongly affects carbon (C) cycling. Excess nutrients stimulate the pro- duction of C-rich algal biomass but can also stimulate C loss through increased
organic C mineralization that releases CO2
instead of supporting production of higher trophic
levels and other ecosystem functions (1, 2). Production of aquatic life in freshwater ecosystems
is based on algae and organic C of terrestrial
origin. Currently, consideration of nutrient effects on C cycling in inland waters has focused
on enhancement of algal C sinks in lakes and
less on fates of terrestrial C that may experience
accelerated loss in river networks (3–5).
The processes that lead to nutrient stimulation
of algal C production and terrestrial C mineralization are fundamentally different. Algal production increases relatively predictably with the
availability of growth-limiting nutrients (1, 6). In
contrast, mineralization of particulate organic C
(POC) is the more complex result of activity by
multiple trophic levels consisting of microbial
decomposers and detritivorous animals (hereafter
detritivores) (7). Inputs of leaves and wood are
the main sources of POC in many rivers, supporting production of animals and uptake of inorganic pollutants (8–10). Nutrients stimulate
microbial processing of POC, which results in increased losses of CO2 to the atmosphere (2, 11).
Consumption of microbially colonized POC by
detritivores further contributes to its breakdown
and conversion to smaller particles, which affect
its subsequent transport and processing downstream (7).
To determine how moderate nutrient pollu-
tion affects terrestrially derived POC at stream-
reach scales, we tested how long-term (2- to 5-year),
continuous, flow-proportional nitrogen (N) and
phosphorus (P) additions affected its loss rates
and fates in headwater forest streams (12). We
measured the response of terrestrial C loss rates
in whole 70- to 150-m stream reaches (tables S1
and S2). Carbon loss rates at this spatial scale
are a function of biologically driven breakdown
and hydrological export and have not been pre-
viously assessed in response to human-influenced
stressors (13). We conducted two manipulative
experiments at large spatial and temporal scales
and focused our measurements on forest-derived
leaf litter, because it is the most biologically ac-
tive pool of terrestrial C in forest streams and is
renewed annually (7). After a pretreatment year,
we enriched one stream with N and P at a set
ratio for 5 years in a paired watershed design (N+P
experiment; a second stream acted as a control)
and used expanded N and P gradients in a second
experiment in five other streams for 2 years after
a pretreatment year (N×P experiment) (table S1).
Reach-scale terrestrial C loss rates increased
with N and P enrichment across all the concentrations we tested (Fig. 1). Discharge, N, P, temperature, and associated random effects (stream
and year) explained 83% of the variation in C loss
rates across 27 annual measurements (table S3).
Standardized regression coefficients indicated that
our moderate additions of N and P contributed
roughly three-fourths of the effect on litter loss
rates as annual cumulative discharge, which varied 87-fold across streams and years (table S3).
Nitrogen and P (r = 0.79) and discharge and
temperature (r = –0.76) were correlated, so their
effects and relative significance cannot be teased
apart fully. However, roughly similar-sized effects of N and P on loss rates are strong evidence
of co-limitation (Fig. 2 and table S3). Comparisons of loss rates from corresponding enriched
and reference streams indicate that median C
loss rates increased 1.65 times with nutrient enrichment (table S4); the range in these values
(1.02 to 4.49 times) reflects variation due to N
1Odum School of Ecology, University of Georgia, Athens, GA
30602, USA. 2Department of Biological Sciences, University
of Alabama, Tuscaloosa, AL 35487, USA. 3Department of
Biology, Coastal Carolina University, Conway, SC 29528, USA.
*Corresponding author. E-mail: email@example.com †Present
address: Department of Biological Sciences, Florida International
University, Miami, FL 33199, USA.