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 TRO P I CA L S T R E AM S

Leaf-litter breakdown in urban streams

of Central Amazonia: direct and indirect eects
of physical, chemical, and biological factors
Renato T. Martins1,4, Adriano S. Melo2,5, Jos F. Gonalves Jr3,6, and Neusa Hamada1,7
1
Programa de Ps-Graduao em Entomologia, Coordenao de Biodiversidade, Instituto Nacional de Pesquisas da
Amaznia INPA, Avenida Andr Arajo, 2936, CP 478, CEP 69067-375, Manaus, Amazonas, Brazil
2
Departamento de Ecologia, ICB, Universidade Federal de Gois, CP 131, CEP 74001-970, Goinia, Gois, Brazil
3
Laboratrio de Limnologia, Departamento de Ecologia, IB, Universidade de Braslia, CEP 70910-900, Braslia, Distrito Federal,
Brazil

Abstract: Urbanization alters water physical and chemical variables and may aect leaf-litter breakdown in streams.
Higher temperature and nutrient inputs in urban streams can stimulate microbial biomass, which can increase leaf-
litter breakdown rates over rates in nonurban streams. On the other hand, urbanization can reduce leaf-litter break-
down rates by eliminating shredders. We evaluated physical, chemical, and biological factors that may directly and
indirectly aect leaf-litter breakdown of Coussapoa trinervia and Mabea speciosa in 42 urban streams in Central Ama-
zonia. We used structural equation modeling to assess whether: 1) shredder activity is more important than microbes
for leaf-litter breakdown of plant species with softer tissues, 2) microbes (as adenosine triphosphate [ATP] concentra-
tion) and fungi (as ergosterol concentration) positively inuence leaf-litter breakdown rate, 3) water velocity positively
aects leaf-litter breakdown rate, and 4) eects of shredders and microbes, including fungi, on leaf-litter breakdown
are mediated by the eects of urbanization. Leaf-litter breakdown of M. speciosa and C. trinervia was fastest in the least
urbanized streams. Fungi had a direct positive eect on leaf-litter breakdown of both species, but shredders were the
most important factor for leaf-litter breakdown in M. speciosa (softer leaf tissues). Water velocity had a slight in-
direct eect on leaf-litter breakdown of C. trinervia through its eect on fungi. Microbes were not important for
leaf-litter breakdown rates of either species. Urbanization indirectly aected leaf-litter breakdown via negative eects
on shredder and fungal biomass. Our study provides evidence for multiple direct and indirect pathways by which
urbanization can decrease leaf-litter breakdown rates in tropical streams, mainly through negative eects on the fungal
and shredder biomass.
Key words: aquatic invertebrates, fungi, leaf shredders, microorganisms, ecosystem functioning, leaf decom-
position

Decomposition of organic matter in streams may be aected
by 3 main groups of factors. First, leaf-litter breakdown may
depend on intrinsic properties of the organic matter. Previ-
ous studies have shown that shredders activity and, con-
sequently, leaf-litter breakdown are aected negatively by
toughness and the presence of secondary compounds, and
are aected positively by the nutrient concentration of tis-
sues (Graa 2001, Onoda et al. 2011, Ferreira et al. 2012).
Second, leaf-litter breakdown can be aected by environ-
mental conditions. For instance, high values of water tem-
perature, O2 concentration, velocity, and concentrations of
N and P usually have positive inuences on the loss of de-
trital mass (Pascoal and Cssio 2004, Ferreira and Chauvet
2011). Last, fallen organic matter is quickly colonized by
microorganisms (principally fungi and bacteria) that start
the decay process by producing enzymes that breakdown
leaf litter (Fenoglio et al. 2006). Microbial biomass, in turn,
favors colonization by invertebrate shredders that consume
the coarse particulate organic matter and increase the leaf-
litter breakdown rate (Graa and Cressa 2010, Pozo et al.
2011, Boyero et al. 2012).
Urban areas usually show an urban stream syndrome,
which is characterized by increased values of impervious
catchment cover, nutrient concentrations, water temper-
ature, stream width and depth in pool areas, and by reduced
invertebrate diversity, channel complexity, and stability (Paul

E-mail addresses: 4martinsrt@gmail.com; 5asm.adrimelo@gmail.com; 6jfjunior@unb.br; 7nhamada@inpa.gov.br

DOI: 10.1086/681086. Received 6 June 2013; Accepted 6 October 2014; Published online XX Month 2015.
Freshwater Science. 2015. 34(2):000000. 2015 by The Society for Freshwater Science. 000
000 | Urbanization effects on leaf breakdown in Amazonian streams
and Meyer 2001, Walsh et al. 2005). The features of these
urban streams can inuence the leaf-litter breakdown rate
relative to in streams in preserved areas (Chadwick et al.
2006, Imberger et al. 2008, Kominoski and Rosemond 2012).
Moreover, high water temperature in urban streams increases
leaching of soluble compounds (Chergui and Pattee 1990) and
can stimulate fungal activity (Ferreira and Chauvet 2011),
thereby positively aecting the leaf-litter breakdown rate.
Increased concentrations of N and P caused by domestic
euents typically increase the breakdown rate by inducing
an increase in microorganism biomass (Suberkropp et al.
2010, Krauss et al. 2011). On the other hand, the increase in
water temperature and nutrients causes high rates of oxi-
dation of dissolved organic matter in urban streams and re-
duces the availability of O2, decreasing the activity of leaf-
associated microorganisms (Pascoal and Cssio 2004, Lecerf
and Chauvet 2008, Medeiros et al. 2009), thereby slowing
leaf-litter breakdown. Low O2 concentration also can reduce
or eliminate sensitive invertebrates, such as Trichoptera, which
is perhaps the main group of shredders in the tropics (Cou-
ceiro et al. 2007).
Urban areas present higher percentages of impervious
surfaces than nonurban areas, and during storms, the in-
put of urban pollutants into aquatic systems may be ampli-
ed by transport of organic compounds and heavy metals
into streams (Schueler 1994). Another eect of urbanization
is reduction or removal of riparian vegetation, causing a de-
crease in particulate organic matter input and an increase
in light input (Encalada et al. 2010, Pettit et al. 2012, Miller
2013). The low leaf input in urban streams can negatively
aect the food available for aquatic organisms in the stretch
where the eect occurs and for long distances downstream
(England and Rosemond 2004). The high input of light fa-
vors increases in temperature and algal biomass (Roy et al.
2005) and may alter the trophic structure of the ecosystem,
reducing its dependence on allochthonous sources (England
and Rosemond 2004).
We studied leaf-litter breakdown of 2 plant species that
are common in riparian zones of Central Amazonia streams.
The study was done in 42 streams that diered mainly in
the degree of human impact associated with urbanization.
We evaluated physical, chemical, and biological factors that
may potentially inuence leaf-litter breakdown in urban
streams. We built models for the 2 plant species with struc-
tural equations to disentangle direct and indirect eects of
the studied factors. We hypothesized that: 1) the contribu-
tion of shredder activity to leaf-litter breakdown should be
more important for plant species with soft than with tough
tissues; 2) microbes, including fungi, should positively inu-
ence leaf-litter breakdown rates directly and indirectly via
their positive eects on shredders; 3) water velocity should
have a direct positive inuence on leaf-litter breakdown rates
by enhancing physical fragmentation, and an indirect posi-
tive inuence by stimulating fungal colonization; and 4) the
R. T. Martins et al.
eects of shredders and microbes, including fungi, on leaf-
litter breakdown rates are inuenced by the eects of ur-
banization on stream sites.
M E T H O DS
Experimental design
We initially selected fty 1st- and 2nd-order terra rme
(upland) streams in the city of Manaus, northern Brazil
(Fig. 1). We excluded 8 streams from the study because of
loss of samples caused by vandalism or by spates.
We carried out the leaf-litter breakdown experiment be-
tween August and September 2010 (dry season) and used
leaves of Coussapoa trinervia Spruce ex Mildbr. (Cecropiaceae)
and Mabea speciosa Mller Argoviensis (Euphorbiaceae).
Coussapoa trinervia has hard leaves that required 348 66 g
to be penetrated, whereas M. speciosa is softer and required
only 154 45 g to be penetrated according to the method
suggested by Graa and Zimmer (2005).
We used senescent leaves of C. trinervia and fresh leaves
of M. speciosa because eld observations indicated that most
leaves of M. speciosa found in the studied streams were
green, whereas those of C. trinervia were always senescent.
Kochi and Yanai (2006) used green and senescent leaves of
Acer mono Maxim. and Alnus hirsuta Turcz. and did not
observe signicant dierences in shredder colonization and
leaf-litter breakdown between senescent and green leaves.
Such results cannot be easily extrapolated to our studied
species, but indicate that potential eects of leaf senescence
on leaf-litter breakdown in our study might be small.
For each plant species, we built 250 litter bags (10 20 cm)
with coarse mesh (10 10-mm openings) containing 3
0.05 g air-dried leaves. We incubated 5 litter bags of each
species in each stream and retrieved them after 30 d, a pe-
riod necessary to decompose 50% of the original mass of
M. speciosa (Landeiro et al. 2010).
Environmental variables and conservation
status of stream sites
We measured the pH (model PH90; WTW, Weilheim,
Germany), electrical conductivity (S/cm; model LF90; WTW),
dissolved O2 (mg/L; model 55; Yellow Springs Instruments,
Yellow Springs, Ohio), and water temperature (C) on the
day the experimental leaves were installed in the streams
and during their removal. We estimated total P (mg/L) and
total N (mg/L) concentrations using the method of Valder-
rama (1981). We measured water velocity with a ow meter
(model 2030R; General Oceanics, Miami, Florida).
We calculated the percentage of deforested and total im-
pervious area (TIA) around each stream from 2010 Landsat
satellite images classied into forested and deforested areas.
We delimited a circle with radius = 300 m around each col-
lection site and quantied the deforested area and the areas
with primary and secondary forests (Couceiro et al. 2007).
We opted to estimate the deforested areas within a circle
 Volume 34 June 2015 | 000

Figure 1. Stream locations (n = 42) in the city of Manaus used to study leaf-litter breakdown of Coussapoa trinervia and Mabea
speciosa. On the map, the triangle indicates a site where only litter bags of C. trinervia remained in place at the end of the study (n = 1),
the square indicates a site where only litter bags of M. speciosa remained in place at the end of the study (n = 1), circles indicate sites
where litter bags of both species were present at the end of the study (n = 40). The line indicates the limit of the urban area of Manaus.

because adult insects of some groups (e.g., Trichoptera)
have enough ight ability to colonize sampling areas from
downstream reaches (Petersen et al. 2004). We classied
deforested areas as urban, exposed soils, or agricultural land.
To reduce possible underestimation of TIA, we followed
the method of Chadwick et al. (2006) and pooled the exposed
soil fraction (usually small) and urban cover together as im-
pervious surface.
Sample processing
We removed samples from streams and placed them in
plastic bags for transport (3 h) to the laboratory in coolers
with ice. We stored the samples in a refrigerator (4C) until
processing. We selected 5 leaves from each litter bag and
cut 15 disks (16 mm diameter). We divided them into 3 sets
of 5 disks, and used the sets to measure ash-free dry mass
(AFDM), ergosterol concentration (to estimate fungal bio-
mass), and adenosine triphosphate (ATP) concentration (to
estimate microbial biomass). To obtain AFDM, leaf disks
were oven-dried at 60C for 72 h and subsequently com-
busted at 500C for 4 h.
Fungal and total microbial biomass
We estimated fungal biomass on decomposing leaves
by quantifying the ergosterol concentration in 1 set of disks.
The leaf disks were frozen at 20C until ergosterol extrac-
tion. We carried out the extraction at 80C for 30 min in
methanol and potassium hydroxide. We puried the ex-
tract by passing it through solid phase extraction (SPE) car-
tridges (model Sep-Pak Vac-RC-500 mg; Waters, Milford,
Massachusetts). The ergosterol retained on the column was
eluted with isopropanol and quantied by high-performance
liquid chromatography (HPLC) (model 625 LC System; Wa-
ters). The mobile phase was 100% methanol and the ow
rate was set to 1.4 mL/min. We calculated nal ergosterol
concentrations on the basis of g AFDM of the disks (Gessner
2005).
We quantied total microbial biomass in terms of ATP
concentration in the detritus. We stored the disks at 20C
until extraction. We ground and centrifuged the disks in a
solution of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES), sulfuric acid, and oxalic acid. We ltered the
samples (0.22-m pore size), neutralized them, and recorded
their volume. We measured ATP in a luminometer (model
TD-20/20; Turner Designs, Sunnyvale, California) via light
emission by luciferase. We obtained concentrations by mea-
suring relative changes in light peak values before and after
the addition of ATP solutions. We corrected nal ATP con-
centrations for extraction eciency determined by adding a
known amount of ATP to an extra disk set. Final ATP con-
centrations were calculated on the basis of g AFDM of the
disks (Abelho 2005).
000 | Urbanization effects on leaf breakdown in Amazonian streams
Invertebrates
We washed the material remaining in the litter bags in
running water and passed it through a 0.12-mm-mesh sieve
to retain detritus and invertebrates. We preserved inverte-
brates in 80% alcohol and sorted them under a stereomicro-
scope. We identied organisms to family or genus with the
aid of keys provided by Pes et al. (2005) and Hamada and
Ferreira-Keppler (2012).
We obtained shredder biomass as dry mass (60C for
48 h; balance accurate to 10 g). Because of the poor state
of knowledge of the functional feeding groups of inverte-
brates in the Neotropics, we followed the conservative deci-
sion of Landeiro et al. (2010) and considered only 2 caddisy
genera as shredders: Triplectides (Leptoceridae) and Phylloicus
(Calamoceratidae).
Leaf mass loss
After removing 15 disks from each litter bag, we oven-
dried the remaining leaves at 60C for 72 h and weighed
them to the nearest 0.001 g to measure dry mass remain-
ing. We estimated the dry mass of the 15 removed disks
based on the dry mass of the 5 disks used to obtain AFDM.
We obtained the nal dry mass by summing of the oven-
dried mass of the remaining leaves and the estimated dry
mass of the removed disks.
The leaves used in the experiment were air-dried, and
we lled bags based on air-dried mass (ADM). We estimated
oven-dried initial mass of leaves based on a correction factor
(CF) obtained from the oven-dried mass (ODM) (60C, 72 h)
of leaves in 15 litter bags that were not used in the exper-
iment. We calculated the CF as mean ODM/mean ADM.
The CF values were 0.918 and 0.442 for C. trinervia and
M. speciosa, respectively. We calculated the leaf-litter break-
down coecient (k) according to the negative exponential
model:
k lnfinal mass=initial mass=time: (Eq. 1)
Mass was expressed in grams and time in days (Petersen
and Cummins 1974).
Statistical analyses
Principal Components Analysis (PCA). We used PCA of
environmental variables to synthesize stream conditions
along the urbanization gradient. The environmental vari-
ables were water velocity, dissolved O2 concentration (DO),
temperature, pH, electrical conductivity, total dissolved N
(TDN), total dissolved P (TDP), % deforested area, and TIA.
Prior to the analysis, we standardized all variables to unit
variance ([observed mean]/standard deviation).
Structural Equation Modeling (SEM). We used SEM to
model the relationship between the leaf-litter breakdown
rates of both species and the explanatory (exogenous) vari-
R. T. Martins et al.
ables. This analysis enables estimation of the direct and in-
direct eects of exogenous variables on a response variable.
The indirect eect is the contribution of an exogenous vari-
able to the response variable through the inuence of a me-
diator variable (Grace 2006). SEM also allows inclusion of
latent variables, which are variables that are dicult or im-
possible to measure directly but can be dened from several
measurable variables. In addition, SEM allows inference of
causal factors (Shipley 2004, Grace 2006).
We constructed a model that represented the 4 hypothe-
ses raised in our study. First, a latent variable was created
to represent the urbanization factor. We used the following
variables to dene urbanization: water temperature, pH,
DO, electrical conductivity, TDN, TDP, % deforested area,
and TIA. The regression part of the model included the
direct eects of microbial, fungal, and shredder biomass
and water velocity on leaf-litter breakdown. Indirect eects
of water velocity on the leaf-litter breakdown rate were in-
cluded through mediated factors: fungal and shredder bio-
mass. We also included indirect eects of fungal and micro-
bial biomass through their eects on shredder biomass. Last,
we included the eects of the latent variable (urbanization)
on microbial, fungal, and shredder biomass.
We ran the SEM analysis using the lavaan package in R
(version 0.4; R Project for Statistical Computing, Vienna,
Austria; Rosseel 2012). In cases of poor t of the model to
the data, we removed variables to obtain a model that rep-
resented the data. This procedure was done carefully (Grace
2006), by rst removing dening variables from the latent
urban variable. Model ts were evaluated by the 2 test and
its associated p-value. The 2 represents the dierence be-
tween the observed data and the hypothesized model. Thus,
an adequate model should have a low 2 statistic and a non-
signicant p-value. We also evaluated the t of the mod-
els with the comparative t index (CFI). This test compares
each proposed model with an alternative baseline model. A
common rule-of-thumb is that good models should have
values >0.95 (maximum = 1) (Shipley 2004). The response
and explanatory variables for each stream site are available
in Appendix S1.
R E S U LT S
Environmental variables and the conservation
status of stream sites
The 1st axis of the PCA based on environmental variables
explained 70.76% of the total variation and separated streams
along an urbanization gradient with the most-impacted
streams scoring at the right of the ordination (Fig. 2A, B). Axis
1 was positively associated with high values of pH (r = 0.897),
TDN (r = 0.874), TDP (r = 0.792), water temperature (r =
0.898), electrical conductivity (r = 0.946), % deforested area
(r = 0.916), and TIA (r = 0.908). On the other hand, the least-
impacted sites scored to the left of the ordination (Fig. 2A,
B) and were associated with high DO (r = 0.930). Water
velocity was associated with axis 2 (r = 0.960).
 Volume 34 June 2015 | 000

Figure 2. Principal Components Analysis (PCA) of environmental data in 42 streams in Manaus, Central Amazonia. Axis 1 reects the
eects of urbanization in Amazonian streams. The sizes of circles are proportional to leaf-litter breakdown rates of Coussapoa trinervia
(A) and Mabea speciosa (B). Cond = electrical conductivity, def = % deforested area, Temp = water temperature, TIA = total impervious area.

Leaf-litter breakdown 13.93, p = 0.001). Microbial biomass ranged from 13 to

The leaf-litter breakdown rates in the studied streams
ranged from 0.0086 to 0.0243/d in C. trinervia (tough
leaves) and from 0.0071/d to 0.0386/d in M. speciosa (soft
leaves) (Appendix S1). Leaf-litter breakdown of the 2 plant
species tended to decrease with urbanization, although at
dierent rates (analysis of covariance [ANCOVA]; leaf spe-
cies urbanization interaction: F1,78 = 21.30, p < 0.001).
The leaf-litter breakdown rates for C. trinervia (tough leaves)
were lower than those for M. speciosa (soft leaves) in the
least urbanized streams, but this pattern tended to reverse
in the most urbanized streams (Fig. 3).
115 nmol/g AFDM in C. trinervia and was negatively
associated with urbanization (R2 = 0.12, F1,40 = 6.41, p =
0.016). Microbial biomass ranged from 16 to 118 nmol/g
AFDM in M. speciosa and was not associated with the
urbanization gradient (R2 = 0.02, F1,39 = 0.09, p = 0.769).
Structural equation modeling
The original SEM was unable to properly describe the
hypothesized causal relationships for both plant species
(2 test, p < 0.05). We simplied the model by dropping

Invertebrate colonization
We collected 15,683 invertebrates in all litter bags of
both plant species (33.4% were in C. trinervia bags). The
number of invertebrate families ranged from 0 to 15 in
C. trinervia and from 0 to 24 in M. speciosa bags. The re-
lationships between urbanization and the number of in-
vertebrate families in C. trinervia (R2 = 0.52, F1,40 = 44.83,
p < 0.001) and M. speciosa (R2 = 0.65, F1,3= 73.74, p <
0.001) were negative. Shredder biomass was negatively as-
sociated with the urbanization gradient in C. trinervia
(R2 = 0.07, F1,40 = 4.04, p = 0.051) and M. speciosa (R2 =
0.36, F1,39 = 23.30, p < 0.001). We did not record shred-
ders (Triplectides and Phylloicus) in the most urbanized
streams. Instead, the invertebrate fauna in these streams
were dominated by oligochaetes and dipterans (collector-
gatherers).

Microbial colonization
Fungal biomass ranged from 83 to 392 g/g AFDM in
C. trinervia, and from 22 to 374 g/g AFDM in M.
speciosa. Fungal biomass was negatively associated with
the urbanization gradient in C. trinervia (R2 = 0.42, F1,40 =
30.01, p < 0.001) and M. speciosa (R2 = 0.24, F1,39 =
Figure 3. Eects of urbanization on leaf-litter breakdown
rate of Coussapoa trinervia and Mabea speciosa in 42 streams
in Manaus, Central Amazonia. Urbanization was quantied as
scores of the 1st axis of a Principal Components Analysis (PCA)
on environmental variables.
000 | Urbanization effects on leaf breakdown in Amazonian streams
single environmental variables dening the latent urban
variable. For C. trinervia, we obtained an adequate model
t (2 = 43.39, df = 38, p = 0.252, CFI = 0.98; Fig. 4) by
dropping electrical conductivity and % deforested area. In
the case of M. speciosa, a good model t was obtained only
after dropping electrical conductivity and % deforested area
from the latent urban variable and removing microbial bio-
mass (2 = 39.83, df = 31, p = 0.133, CFI = 0.97; Fig. 5).
The SEM for C. trinervia indicated that fungal biomass
was the exogenous variable with the greatest direct eect on
leaf-litter breakdown rate (path coecient = 0.65, p < 0.001;
Fig. 4, Table 1). Water velocity aected leaf-litter breakdown
rate negatively by its indirect eect on fungal biomass (in-
direct eect = 0.19, p = 0.030). Shredder and microbial
biomass did not aect leaf-litter breakdown rate of C. triner-
via. The latent urban variable had a strong eect on fungal
biomass (path coecient = 0.62, p < 0.001), indirectly af-
fecting leaf-litter breakdown rate (sum of indirect eects =
0.49, p = 0.001; Table 1).
The SEM that described the causal relationships for
M. speciosa indicated that shredders (path coecient =
0.47, p < 0.001) and fungal biomass (path coecient = 0.33,
p = 0.008) were the only direct eects on leaf-litter break-
down rate (Fig. 5, Table 1). Water velocity did not have a
direct (path coecient = 0.02; p = 0.884) or indirect (path
coecient = 0.09, p = 0.220) signicant eect on leaf-
litter breakdown rate of M. speciosa. The urban latent vari-
Figure 4. Structural model showing the relationships of water
velocity, fungi, shredders, microbial biomass and the latent urbani-
zation variable to leaf-litter breakdown of Coussapoa trinervia in
42 streams in Manaus, Central Amazonia. Solid lines indicate sta-
tistically signicant relationships ( p < 0.05). Dotted lines indicate
statistically nonsignicant relationships ( p > 0.05). The width of
each line is proportional to the strength of the relationship, and
values next to arrows indicate path coecients. Temp = water
temperature, TIA = total impervious area.
R. T. Martins et al.
Figure 5. Structural model showing the relationships of wa-
ter velocity, fungi, shredders, and the latent urbanization vari-
able to leaf-litter breakdown of Mabea speciosa in 42 streams
in Manaus, Central Amazonia. Solid lines indicate statistically
signicant relationships ( p < 0.05). Dotted lines indicate statis-
tically nonsignicant relationships (p > 0.05). The width of
each line is proportional to the strength of the relationship, and
values next to arrows indicate path coecients. Temp = water
temperature, TIA = total impervious area.
able negatively aected shredders (path coecient = 0.58,
p < 0.001) and fungal biomass (path coecient = 0.52,
p < 0.001), resulting in a net negative eect on leaf-litter
breakdown rate (sum of indirect eects = 0.52, p < 0.001;
Table 1).
DISCUSSION
Urbanization and leaf-litter breakdown
We observed lower rates of leaf-litter breakdown of
M. speciosa and C. trinervia in highly urbanized streams.
In these streams, litter bags did not harbor shredders,
whereas Phylloicus and Triplectides occurred in less urban-
ized streams. Thus, our results indicate that these caddis-
ies are sensitive to urban impacts (high concentrations of
P and N, low availability of O2, high water temperature,
high TIA, and removal of riparian vegetation) (Schueler
1994, Couceiro et al. 2007). Increases in P and N result-
ing from environmental degradation usually are accom-
panied by a reduction in O2 and increases in other vari-
ables (e.g., conductivity, pesticides; Woodward et al. 2012).
These changes can reduce shredder abundance and con-
comitantly slow leaf-litter breakdown (Woodward et al.
2012). In our most urbanized streams, invertebrate com-
munities were composed mostly of chironomids (prepon-
derantly Chironomus spp.) and oligochaetes, which usually
are present in high densities in the detritus. However, these
organisms do not directly aect leaf-litter breakdown be-
 Volume 34 June 2015 | 000

Table 1. Correlation coecient (r), and standardized path coecients for direct (d), indirect (i) and total (e) eects
for the structural models of Coussapoa trinervia and Mabea speciosa leaf-litter breakdown rate (k) in 42 streams in
Manaus, Central Amazonia. * indicates signicant p-value ( p < 0.05). In M. speciosa, only the correlation coecient for
microbial biomass is shown because this variable was not included in the structural model.
Coecient

Dependent variable Explanatory variable r d i e=d+i

C. trinervia k Fungal biomass 0.58 0.65* <0.01 0.67*

Shredder biomass 0.07 0.05 0.05
Microbial biomass 0.11 0.07 <0.01 0.07
Water velocity 0.13 0.22 0.19* 0.04
Urbanization 0.49* 0.49*
M. speciosa k Fungal biomass 0.50 0.33* 0.03 0.36*
Shredder biomass 0.59 0.47* 0.47*
Microbial biomass 0.11
Water velocity 0.15 0.02 0.09 0.11
Urbanization 0.52* 0.52*

cause they feed mainly on ne particulate organic matter
(Walsh et al. 2005). Landeiro et al. (2008) observed that free-
living chironomids were not associated with leaf break-
down of M. speciosa. However, leaf-mining chironomids
were positively associated with leaf breakdown (Landeiro
et al. 2008). We did not nd chironomids inside the leaf
matrix in the most urbanized streams, reinforcing the low
importance of these organisms in leaf-litter breakdown in
urban streams.
Despite a general negative eect of urbanization on leaf-
litter breakdown, our results indicate that the mechanisms
were not identical for the 2 plant species studied. For C.
trinervia (tough leaves), the negative eects of urbaniza-
tion on leaf-litter breakdown were mediated by decreased
fungal biomass. This mechanism also was important for
M. speciosa. However, for M. speciosa, urbanization also
aected leaf-litter breakdown by reducing shredder bio-
mass. Thus, in the least urbanized streams, leaf-litter break-
down of M. speciosa was mediated by both fungi and
shredders, resulting in faster leaf-litter breakdown than
observed for C. trinervia. However, in the most urbanized
streams, the lack of shredders caused the 2 litter species
to have more similar breakdown rates.
We also observed a negative relationship between fun-
gal and microbial biomass with urbanization. Several authors
have observed increased microbial biomass and high break-
down rates in environments enriched with inorganic nutri-
ents, especially N and P (Pascoal et al. 2001, 2003, 2005, Gulis
and Suberkropp 2003). However, we found the lowest leaf-
litter breakdown rates in the most urbanized streams, where
nutrient concentrations were high. This nding indicates
that urbanization can impair the functioning of ecosystems
by altering microbial and invertebrate community struc-
tures. Our results should be interpreted by taking into ac-
count variables associated with general stream condition.
For instance, the high values of nutrients (P and N) in the
most urbanized streams were associated with low DO val-
ues (r = 0.78). Low O2 availability inhibits the positive
stimulus of N on microbial activity and biomass and can
exclude some species of aquatic hyphomycetes (Pascoal and
Cssio 2004, Medeiros et al. 2009, Krauss et al. 2011). More-
over, a potential additional negative inuence of urban wa-
ters on leaf-litter breakdown rates is the deleterious eects
of NH4+ on invertebrate taxa (Lecerf et al. 2006, Woodward
et al. 2012).
Shredder effects on leaf-litter breakdown
Shredders were not important for leaf-litter breakdown
of C. trinervia. This species has hard leaves that may have
prevented colonization by shredders. Previous investigators
of temperate and tropical streams have found weak eects
of shredders on tough leaves (Rincn and Martnez 2006,
Li and Dudgeon 2008, Graa and Cressa 2010). Tough leaves
usually require long periods of conditioning by microorgan-
isms before they become attractive to shredders (Gonalves
et al. 2006). In contrast, shredders exerted a strong direct
eect on M. speciosa leaf-litter breakdown despite the low
abundance and biomass of shredders. In previous studies of
leaf-litter breakdown of M. speciosa in the same region, this
species was colonized rapidly by shredders and had rapid
leaf-litter breakdown (Landeiro et al. 2008, 2010). We ob-
served that leaf toughness inuenced the eects of shred-
ders on leaf-litter breakdown of the 2 species. On the other
hand, Treplin and Zimmer (2012) studied leaf-litter break-
down in aquatic and terrestrial environments and found
that detritus quality (according to breakdown rates and nu-
tritional value) was not important for the breakdown pro-
cess in the presence of decomposers (shredders and grazers).
Moreover, in the absence of decomposers, leaf quality tended
to be less important in aquatic than in terrestrial environ-
000 | Urbanization effects on leaf breakdown in Amazonian streams
ments because of leaching of soluble components. We found
that shredders were rare on the tough leaves and that the
breakdown rates of such leaves were low. A possible ex-
planation is that the duration of the experiment (30 d) was
not sucient to condition C. trinervia. In a study by Ligeiro
et al. (2010), the duration of exposure was the principal factor
explaining invertebrate colonization during the leaf-breakdown
process.
Fungal and microbial effects on leaf-litter breakdown
We observed that fungal biomass tended to increase the
breakdown rate of the soft leaves of M. speciosa, but the
magnitude of the eect was lower than that exerted by
shredders. However, for the tough leaves of C. trinervia,
shredders had no eect, and fungi carried out most of the
leaf-litter breakdown. Previous investigators have shown that
fungi play a dominant role in the breakdown of detritus
and can represent up to 96% of total microbial biomass
during this process (Baldy et al. 1995, Findlay et al. 2002,
Das et al. 2007). In fact, most investigators who quan-
tied fungal biomass recorded positive relationships with
leaf-litter breakdown rates (Pascoal and Cssio 2004, Imber-
ger et al. 2008, Lecerf and Chauvet 2008). In tropical streams,
some data indicate that shredders are poorly diversied
and not abundant and that leaf-litter breakdown in these
streams is carried out preponderantly by fungi, organisms
capable of degrading complex polysaccharides (Irons et al.
1994, Dobson et al. 2002). Our study was restricted to 2
species, but our results for C. trinervia support the view
that fungi can compensate for a low abundance or absence
of shredders during leaf-litter breakdown.
Microbial biomass was not important in the leaf-litter
breakdown of either plant species. ATP values can repre-
sent the biomass of various microorganisms, including fungi.
Several investigators have used the relationship between
ATP and ergosterol to obtain fungal participation in mi-
crobial biomass (Suberkropp et al. 1993, Ruzicka et al. 2000,
Abelho 2009). We recorded a low association between ATP
and ergosterol concentrations (r < 0.1) for both plant spe-
cies. Accordingly, fungi were not the principal group of mi-
croorganism in the total microbial biomass (measured as
ATP) in our study. After 46 d of leaf submersion, Abelho
(2009) found a weak relationship (r = 0.04) between er-
gosterol and ATP and attributed this result to greater
importance of bacteria and other nonfungal microorgan-
isms in the microbial biomass.
In a nutrient-enriched river with low values of DO and
high sedimentation, Pascoal et al. (2005) observed low fun-
gal activity and increased bacterial production. We did not
observe a positive relationship between microbial biomass
and stream urbanization. However, in the most urbanized
streams, substitution of fungi by bacteria in the microbial
communities may have occurred, as indicated by our low
relationship between ergosterol and ATP. These results are
similar to those of Quinto et al. (2013) who found low
R. T. Martins et al.
fungal biomass and an increase in facultative anaerobic bac-
teria biomass in eutrophic waters. Thus, increased impor-
tance of bacteria in urbanized streams (Paul and Meyer 2001)
may indicate changes in ecological processes in these eco-
systems. Moreover, we think that the relationship between
ergosterol and ATP can be an important tool in assessing
health of tropical stream ecosystems.
In contrast to our hypothesis, fungal and microbial bio-
mass did not aect leaf-litter breakdown indirectly through
positive eects on shredders. Fungi and bacteria can degrade
leaf detritus and immobilize dissolved N and P from the
water column, making leaf litter more palatable to shredders
(Gessner et al. 1999). Plausible explanations for the absence
of these relationships may be the nature of the leaf species
studied. Coussapoa trinervia is a plant species with tough
leaves that few shredders were able to colonize. Thus, the
potential increase in palatability apparently was not enough
to enable high-level colonization of the species by shred-
ders. On the other hand, the higher densities of shredders
colonizing the soft leaves of M. speciosa (3 more than
C. trinervia) may have occurred independently of fungal
colonization. This speculation is based on the nding of
Landeiro et al. (2008) that this species was colonized by
shredders 1 d after submersion. In addition, these authors
observed that leaves of this species are used intensively by
Phylloicus to construct their leaf cases.
Water velocity effects on leaf-litter breakdown
Water velocity, in general, has a positive eect on leaf-
litter breakdown by increasing physical fragmentation (Paul
et al. 2006, Dewson et al. 2007). However, in our study,
its direct eect was weak for C. trinervia and absent for
M. speciosa. Our results should be interpreted cautiously
because detritus in the most urbanized streams was buried
by ne sediment and, thus, not subjected to water ow over
the substrate surface (Sponseller and Beneld 2001, Navel
et al. 2010). Moreover, the detritus burial may in part have
been a result of the method used because under natural
conditions, a portion of the detritus would be carried down-
stream and deposited at the surface of the substrate.
Water velocity also can aect leaf-litter breakdown by
its indirect eect on organisms. For instance, increased
water velocity can increase oxygenation and turbulence,
which positively aect the production of conidia, fungal
biomass, and the number of fungal species, and in turn,
may increase leaf-litter breakdown (Ferreira and Graa
2006, Schlief and Mutz 2009). However, we found a negative
eect of water velocity on fungal biomass for C. trinervia
and unimportant eects for M. speciosa. Again, these un-
expected eects probably are a result of burying of detri-
tus as a consequence of urbanization.
A 2nd indirect eect of water velocity is on the biomass
of shredders. We did detect weak negative eects of wa-
ter velocity on shredders. These negative eects probably
are related to behavior of 2 shredder genera from the study

region. Generally, species of Phylloicus and Triplectides are
found in both ries and pools, but they occur at high den-
sities only in pools (Prather 2003, Landeiro et al. 2008,
2010).
Conclusions
We conclude that, in urban streams, the positive eect
of shredders on leaf-litter breakdown may depend on the
plant species. On the other hand, fungal biomass showed
positive eects on the leaf-litter breakdown of both plant
species. However, the eects of shredders and fungi were
negatively modulated by the eects of urbanization, which
slowed down leaf-litter breakdown. In our model, micro-
bial biomass did not aect leaf-litter breakdown. Water
velocity only weakly aected the breakdown of both plant
species, and this result may have been an indirect eect
of urbanization, which reduced physical abrasion by bury-
ing detritus in the impacted streams. Last, urbanization
exerted strong negative eects on leaf-litter breakdown by
reducing the biomass of shredders and fungi in the Ama-
zonian streams.
AC K N OWL E D G E M E N T S
We thank Cludio S. Monteiro, Jr, Jefrson O. Silva, Gizelle
Amora, and Valdeana S. Linard for eld help, Bianca M. P. Ottoni
for help in analyses of ATP and ergosterol, Vivian C. Oliveira for
preparation of the map, Luciano F. Sgarbi for help with R scripts
for PCA gures, Philip M. Fearnside for the English review, and
Marcelo Moreira and the Fundao Vitria Amaznica for the
Landsat images and deforestation analyses. The Laboratrio de
Recursos Hdricos of the Instituto Nacional de Pesquisas da Ama-
znia provided analyses of N and P. We are also thankful to
Associate Editor Eric Chauvet and 2 anonymous referees who
provided comments and suggestions that improved the quality
of the manuscript. RTM received a doctoral scholarship (proc.
143624/2009-1) from Conselho Nacional de Desenvolvimento Cien-
tco e Tecnolgico (CNPq) and a fellowship from the Programa
de Apoio Fixao de Doutores no Amazonas FIXAM/AM
(FAPEAM). ASM and NH received research grants (procs. 558187/
2009-9 and 504223/2010-0, respectively) and research fellow-
ships (procs. 307479/2011-0 and 306328/2010-0) from the CNPq.
The CT-Amaznia/CNPq (Proc. 575875/2008-9), Pronex/CNPq/
Fapeam Aquatic insects, CT-Hidro/Climatic Changes/Water
Resources/CNPq (Proc. 403949/2013-0) and INCT/ADAPTA
Amazon projects supported the eld experiment and laboratory
analyses.
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