Amphibia-Reptilia (2018) DOI:10.

1163/15685381-17000209

You are what, where, and when you eat:

seasonal and ontogenetic changes in a tropical tadpole’s diet

Jéssica S. Kloh1 , Cleber C. Figueredo2 , Paula C. Eterovick1,∗

Abstract. Tadpole diet is likely to vary in response to environmental conditions and nutritional needs throughout growth and
development. We investigated seasonal variation in diet composition of Bokermannohyla saxicola tadpoles and compared
diets between two developmental stages with a significant difference in size. We found that the diet of B. saxicola tadpoles was
dominated by periphytic algae, in accordance with their benthic habits. Considering number of cells ingested, tadpole trophic
niches were broader in more advanced developmental stages. Tadpole trophic niches were narrower during the summer (wet
season) than during the winter (dry season), which may reflect increased consumption of more energetic food items during
the warm period when primary productivity is expected to be higher. Tadpole metabolism is likely to be higher in the summer
and increased energetic needs might be supplied in this manner. However, results differed when biovolume was considered
instead of number of cells ingested, with larger items assuming a greater importance and niches being usually larger in the
summer. In these cases, the increased ingestion of diatoms (likely to be more nutritive) in the summer may decrease the
relative importance of large algae (e.g., Mougeotia sp.) that form the bulk of the diet. Both food availability/accessibility and
tadpole feeding behaviour driven by nutritional needs may influence patterns of food acquisition. Given the importance of
biofilms to tadpole diet, studies on the mechanisms by which tadpole nutritional needs and environmental conditions interact
are likely to provide important insights into the dynamics of aquatic food webs.

Keywords: Bokermannohyla saxicola, niche overlap, periphyton, trophic niche.

Introduction and access to microhabitats with better forag-

The anuran larval phase is subject to strong se-
lective pressures imposed by predators, com-
petitors, and other factors (Heyer, 1979; Wells,
2007). Tadpole diets are an important com-
ponent of tadpole success because they can
vary in amounts of carbohydrates, proteins,
and lipids, which influence growth, develop-
ment, and metamorphosis (Kupferberg, 1997a;
Richter-Boix et al., 2007). For instance, a high
quality diet can decrease time to metamor-
phosis (Kupferberg, 1997a) and consequently
time exposed to aquatic predators (Heyer et al.,
1975). On the other hand, predator presence
may limit tadpole activity (Nomura et al., 2011)
1 - Programa de Pós Graduação em Biologia de Vertebra-
dos, Pontifícia Universidade Católica de Minas Gerais,
30535-610, Belo Horizonte, Brazil
2 - Departamento de Botânica, Universidade Federal de Mi-
nas Gerais, 31270-901, Belo Horizonte, Brazil
∗ Corresponding author;
e-mail: pceterovick@gmail.com
ing opportunities (high quality food; Kupfer-
berg, 1998).
Tadpoles can eat a variety of items that
include attached algae or biofilms essentially
composed of green algae, cyanobacteria, di-
atoms, and bacteria (Dickman, 1968), as well
as organic matter, protozoans, pollen (Diaz-
Paniagua, 1985), insects, and even other tad-
poles or anuran eggs (Alford, 1999; Schiesari
et al., 2009; Jefferson et al., 2014). Diatoms
and other algae, as well as cyanobacteria, are
rich in protein; green algae (Chlorophyta) also
store high quantities of carbohydrates (Bold
and Wynne, 1985) and represent important re-
sources for tadpoles, especially those with a
long larval stage (Kupferberg, 1997a). Diatoms
are also especially important for larval nutri-
tion due to their high lipid content (Gordon et
al., 2006). Increased algae ingestion is likely
to increase growth of consumers (Bowen et al.,
1995), which, on the other hand, influence the
dynamics of algae populations and aquatic food
webs (Dickman, 1968; Kupferberg, 1997b).

© Koninklijke Brill NV, Leiden, 2018. DOI:10.1163/15685381-17000209

2 J.S. Kloh, C.C. Figueredo, P.C. Eterovick
Many aspects of the trophic ecology of tad-
poles remain understudied, such as food se-
lection, nutritional demands throughout on-
togeny and even their actual trophic level in
aquatic communities (Dutra and Callisto, 2005;
Schiesari et al., 2009). Tadpoles have varied
morphologies that enable them to forage in dif-
ferent microhabitats and likely consume differ-
ent diets (Altig and Johnston, 1989; Altig and
McDiarmid, 1999). Tadpoles are known to se-
lect microhabitats (Eterovick and Barata, 2006)
and may alter their microhabitat use/selection
throughout the year. However, assessed preda-
tion risk or microhabitat availability did not ex-
plain changes in microhabitat use, the causes
of which remain to be understood (Eterovick et
al., 2010). A recent study showed the availabil-
ity of items consumed by tadpoles (unicellular
and filamentous Zygnematophyceae and Bacil-
lariophyceae, Cyanophyceae, pollen and tes-
tate amoebae) to vary among different types of
microhabitats in streams (including the stream
where the present study was conducted) and
influence the diets of species using these mi-
crohabitats (JSK, CCF, and PCE, unpublished
data). Diet is also likely to change with tad-
pole developmental stage (Souza-Filho et al.,
2007). On the other hand, food items differ in
digestibility, as well as in susceptibility to for-
aging consumers (Peterson and Jones, 2003).
Thus, comprehensive studies on tadpole diet
need to account for spatial and temporal vari-
ation in food consumption and take digestibility
into account in order to understand the trophic
status and ecological roles of tadpoles.
We chose the tadpole of Bokermannohyla
saxicola (Bokermann, 1964) as a model to study
diet composition and variation throughout the
year of two different developmental stages. At
four periods throughout the year, we selected
five tadpoles in each of developmental stages 25
and 30 (sensu Gosner, 1960) to represent an ini-
tial stage and a more advanced stage (with de-
veloping hindlimbs), respectively. We assumed
our stage 25 tadpoles were demanding a lot
of energy for growth, while stage 30 tadpoles
were investing more in limb development. We
aimed to test the hypotheses that (1) tadpole
diets will vary between the two developmental
stages, which will likely differ in size and nu-
tritional needs, and (2) tadpole diets will vary
throughout the year due to expected changes in
food availability (Ferragut et al., 2010) and pat-
terns of microhabitat use by tadpoles (Eterovick
et al., 2010). We expected tadpoles to prioritize
consumption of items of higher nutritional value
in the rainy, warm season, when productiv-
ity/food availability is likely to increase, which
would result in narrower niches and greater
niche overlap. On the other hand, we expected
tadpole consumption to be less specific in the
dry, cold season, when productivity/food avail-
ability decreases, or at larger sizes, due to con-
straints to attend the greater demands of a large
body. We also expected niche overlaps to be
greater within developmental stages than be-
tween them.
Materials and methods
Study site
The study was conducted at a third-order section (sensu
Strahler, 1957) of Água Escura stream (19°16 1.2 S;
43°32 48.5 W, 1200 m alt., datum WGS 84). The stream
is located inside the Parque Nacional da Serra do Cipó
(PNSC), Brazil, and belongs to the Doce river basin. The
stream has dissolved oxygen saturation from 77 to 137%,
conductivity between 5.5 and 6.4 μS/cm, total alkalinity
of 0.03 mEq/l and low nutrient concentrations, being char-
acterized as oligotrophic (Mendes, 2003). Total phospho-
rus concentration is 15μg/l, and pH is between 4.23 and
6.88, corresponding to Type 4 in the classification of acidic
streams proposed by Coring (1996). The stream bottom is
mainly rocky with sand deposits, pebbles, or debris and
some patches of aquatic vegetation. The climate presents
cyclic seasonality, with a dry, cold season from April to
September and a wet, warm season from October to March.
Mean air temperature is 21.1°C and mean annual rainfall
is 1622 mm (Eterovick and Sazima, 2004). The historical
mean minimum and maximum temperatures in the sampling
months, October, February, May and June (see next section),
are 17 and 27°C, 18 and 27°C, 13 and 24°C, 11 and 24°C,
respectively. Most of the rainfall is concentrated between
October and February, with usually much less than 50 mm
or no rainfall in May and June together (ICMBio, 2009).
Changes in tadpole diet
Tadpole sampling
Tadpoles of Bokermannohyla saxicola (Anura, Hylidae)
are present year-round in permanent streams, which makes
them suitable for this study. The tadpoles take at least five
months to develop (Eterovick and Brandão, 2001). They
are benthic, mostly nocturnal and usually rest on rocks
(Eterovick and Sazima, 2004). These tadpoles remain on
the stream bottom (Eterovick and Sazima, 2004) and have
eight (most commonly) to 11 denticle rows (Eterovick and
Brandão, 2001) that enable them to scrape food items from
surfaces of rocks, pebbles, and other substrates (see Altig
and Johnston, 1989). The great number of denticle rows may
enable them to eat even small diatoms of difficult access that
remain closely attached to the substrate.
We collected tadpoles twice during the rainy season (Oc-
tober 2012 and February 2013) and twice during the dry sea-
son (May and June 2013). We expected the sampling months
to represent the onset (October and May) and ongoing (Feb-
ruary and June) periods of the rainy and dry seasons, respec-
tively. From now on we refer to these periods as OW (onset
wet: October), MW (middle wet: February), OD (onset dry:
May), and MD (middle dry: June).
Bokermannohyla saxicola tadpoles hatch in stage 25
which has a relatively long duration (Eterovick and
Brandão, 2001), and is when most growth takes place. We
thus collected tadpoles within a size range that would prob-
ably be reached about one month after hatching (PCE, pers.
obs.), thus avoiding recently hatched tadpoles that could
have not filled their guts yet. We collected tadpoles with a
dipnet, immediately euthanized them with 10% lidocaine,
and preserved them in 5% formalin.
Data analyses
We measured tadpole total length, tail length, tail width,
body length, and mouth width according to Altig and Mc-
Diarmid (1999). We performed t-tests to check for size dif-
ferences in these measurements between the developmen-
tal stages considered when normality was observed in the
dataset. For data violating assumptions of parametric tests,
we performed Mann-Whitney tests. In order to make sure
our sample was standardized throughout the year or ac-
count for increased growth at any specific sampling period,
we conducted Kruskal-Wallis tests separately for stages 25
and 30 to compare tadpole dimensions among the four sam-
pling periods. These analyses were performed in the soft-
ware Systat (SYSTAT 12 for Windows. SYSTAT Software
Inc., USA; https://systatsoftware.com).
We analyzed separately three segments of equal size (an-
terior, median, and posterior) of the digestive tract in order
to account for digestion level throughout the gut. The ante-
rior segment corresponded to the manicotto and most of the
anterior small intestine; the median segment corresponded
to the final portion of the anterior small intestine, inflection
and the initial portion of the posterior small intestine; and
the posterior segment corresponded to the rest of the pos-
terior small intestine, colon and rectum (sensu Pryor and
Bjorndal, 2005).
3
We macerated each segment, diluted its contents in 1 ml
of distilled water and counted food items in a Sedgewick-
Rafter counting chamber under a microscope with 10×
magnification. We previously identified food items in a mi-
croscope with 100× magnification in order to be able to
identify them correctly at the smaller magnification used
with the counting chamber. We used diatoms to compare
breakage/digestion level in different segments of tadpole
guts because they were the most abundant food items and
their frustules are resistant enough to be found in all por-
tions of the gut, whereas other food items may suffer fast
breakage/digestion and leave no traces. We quantified only
intact diatoms (we considered empty frustules as digested
diatoms). According to Altig et al. (2007), it is also pos-
sible that tadpoles may accidentally ingest empty diatom
frustules while foraging on a substrate where these are abun-
dant. We identified ingested items according to Bicudo and
Menezes (2005) and Moresco et al. (2011). We used data
from the anterior segment of the gut in subsequent analyses.
We represented tadpole diets based on the quantification
of (1) number of cells and (2) biovolume of each food type
ingested by each tadpole in two non-metric multidimen-
sional scalings (NMDSs) with the package Vegan (Oksanen
et al., 2016) in R (R Core Team, 2016). We used the same
data to test for significance of developmental stage (25 or
30) and sampling period (OW, MW, OD, and MD) as well
as their interactions to explain variation in diet composition
with a PERMANOVA in the Vegan package. We calculated
biovolume in μm3 based on Hillebrand et al. (1999), and
estimated ingested biovolume as μm3 /ml.
We tested our predictions on tadpole dietary niche
breadth variation based on the number of cells and biovol-
ume. We calculated niche breadth for each sampling period
and each tadpole developmental stage using the Probability
of Interspecific Encounter (PIE) diversity index in EcoSim
7.0 (Gotelli and Entsminger, 2001). The value of the PIE
index ranges from 0 to 1 and can be interpreted as the prob-
ability that two randomly picked items (cells or μm3 , re-
spectively) from the sample represent two different items.
We applied the index to food item diversity in the guts. We
considered as significantly different niche breadths that did
not overlap within the 95% range of estimated values (based
on 1000 simulations).
To test our predictions on niche overlap, we calcu-
lated niche overlap (Ojk ) between developmental stages and
among the four sampling periods using Pianka’s (1973) in-
dex in EcoSim 7.0 (Gotelli and Entsminger, 2001). Esti-
mates were obtained based on 1000 simulations and data
from different stages/periods were compared through rar-
efaction considering the smallest number of food items as
reference to standardize sample size. Values of p were ad-
justed through Bonferroni correction to minimize chances
of type I error (Rice, 1989).
Results
Based on counts of cells, tadpole gut contents
were dominated by diatoms (Bacillariophyceae)
4 J.S. Kloh, C.C. Figueredo, P.C. Eterovick

from five genera: Cymbella, Eunotia, Navicula,
Nitzschia, and Sellaphora. Together, diatoms
represented 86% and 80.9% of all cells ingested
by stage 25 and stage 30 tadpoles, respectively
(fig. 1). Other algae were also present, includ-
ing Zygnemataceae (Mougeotia), Desmidiaceae
(Actinotaenium, Bambusina, Cosmarium, Euas-
trum, Xanthidium), Mesotaeniaceae (Cylindro-
cystis), and Cyanobacteria (Phormidium) (fig.
1A, 1C, 1E, 1G). Each genus was represented
by a single species, except for Euastrum, which
was represented by five different species, and
Xanthidium, which was represented by two. We
also recorded pollen and testate amoebae (Ar-
cella sp.), vegetal remains, sand grains, eggs,
and limbs and antennae of aquatic invertebrates
and microcrustaceans (whose digestion level
impaired identification). When we evaluated
biomass instead of number of cells, Bambusina
and Mougeotia (Zygnematophyceae) accounted
for most of the volume in gut contents due
to their large cells (60.9% of volume in stage
25 and 65.1% in stage 30 tadpoles); this was
not the case for some small Zygnematophyceae,
such as Euastrum, Cosmarium, Cylindrocys-
tis, Actinotaenium and Xanthidium. Large non-
photosyntesizing items such as testate amoebae
(Rhizopoda) and pollen represented together

Figure 1. Material recorded in the anterior portion of Bokermannohyla saxicola tadpole guts at developmental stages 25
(black bars) and 30 (grey bars) throughout the year considering number of cells (A, C, E, G) and biovolume (B, D, F, H).
A and B: Onset wet, C and D: middle wet, E and F: onset dry, G and H: middle dry (see text for details).
Changes in tadpole diet
17.9% and 12.6% of ingested volume in stage
25 and 30 tadpoles, respectively (fig. 1B, 1D,
1F, 1H). On the other hand, diatoms repre-
sented only 12.8% and 10.7% of the volume
ingested by stage 25 and stage 30 tadpoles, re-
spectively.
The amount and variety of food items was
markedly greater in the anterior segment of tad-
pole guts compared to median and posterior seg-
ments, so we conducted all the subsequent anal-
yses based only on the anterior segment. The
only remaining identifiable items in median and
posterior gut segments were diatoms, while all
the other items seemed to be digested before
they reached these segments. Tadpoles in de-
velopmental stage 25 were significantly smaller
than tadpoles in stage 30 (table 1). However,
tadpole dimensions did not differ significantly
among sampling periods for stages 25 or 30 (re-
sults not shown).
Tadpoles differed in their diets between de-
velopmental stages 25 and 30 considering num-
ber of cells (MS = 0.325, F = 3.950, df = 1,
p = 0.002) but not biovolume (MS = 0.254,
F = 1.175, df = 1, p = 0.284) of food types. On
the other hand, tadpoles differed in their diets
among sampling periods considering both num-
ber of cells (MS = 0.536, F = 6.505, df = 3,
p = 0.001) and biovolume (MS = 0.461, F =
2.135, df = 3, p = 0.022) of food types. The
interactions between developmental stage and
sampling periods were significant considering
both number of cells (MS = 0.374, F = 4.544,
df = 3, p = 0.001) and biovolume (MS = 0.482,
F = 2.233, df = 3, p = 0.009) of food types. The
Table 1. Measurements of Bokermannohyla saxicola tadpoles in developmental stages 25 (n = 20)
and 30 (n = 20) (sensu Gosner, 1960). Data is presented as mean ± SD. Stages were compared
with Mann-Whitney or t-tests for non-normal and normal data, respectively (significance level
considered as p < 0.05).
Developmental stage 25
Total length 44.8 ± 2.2
Body length 15.3 ± 1.0
Tail length 29.3 ± 1.2
Tail heigth 9.5 ± 0.7
Mouth width 4.4 ± 0.5
Gut length 261.8 ± 13.1
5
NMDSs provided a reasonable spatial represen-
tation of the data considering that it included
20 dimensions, each one represented by a food
type (number of cells: stress = 0.233, rmse =
0.035, max. resid. = 0.125; fig. 2A; biovolume:
stress = 0.234, rmse = 0.001, max. resid. =
0.005; fig. 2B).
Tadpole dietary niches based on number of
cells ingested were broader during the dry sea-
son (OD and MD) than during the wet season,
and differed among all sampling periods, except
for stage 30 tadpoles between OD and MD (ta-
ble 2). Niches were always broader for stage 30
compared to stage 25 tadpoles within the same
period (table 2). Niche overlap was higher than
expected by chance among sampling periods for
both stage 25 (Ojk = 0.895, p < 0.0001) and
stage 30 tadpoles (Ojk = 0.766, p < 0.0001).
Niche overlap was also higher than expected
by chance between the tadpole developmental
stages considered in the middle of both seasons
(MW: Ojk = 0.974, p < 0.0001; MD: Ojk =
0.972, p < 0.0001). In the onset of both sea-
sons niche overlap between tadpole stages did
not differ from random (OW: Ojk = 0.501, p =
0.052; non-significant after Bonferroni correc-
tion; OD: Ojk = 0.799, p = 0.258).
Tadpole dietary niches showed a different
pattern when biovolume was considered instead
of number of cells. Niches were broader dur-
ing the wet season, except for stage 25 tad-
poles in MW, whose niches were the narrow-
est. Niches were broader for stage 25 than stage
30 tadpoles, except for MW, when stage 30 tad-
poles had broader niches than stage 25 tadpoles.
30 Statistical comparison between stages
73.1 ± 2.4 U = 0.0; p < 0.005; df = 1
22.4 ± 1.8 U = 0.0; p < 0.005; df = 1
50.5 ± 1.4 U = 0.0; p < 0.005; df = 1
14.0 ± 0.7 U = 0.0; p < 0.005; df = 1
6.6 ± 0.6 U = 3.0; p < 0.005; df = 1
299.1 ± 15.4 t = −8.23; p < 0.005; df = 38
6 J.S. Kloh, C.C. Figueredo, P.C. Eterovick

Figure 2. NMDS showing classification of tadpole diets based on number of cells (A) and biovolume (B) of food items for
eight groups given by the combination of two developmental stages (25 and 30 sensu Gosner, 1960) and four sampling periods
(OW = onset wet, MW = middle wet, OD = onset dry, MD = middle dry; see text for additional details). Species included
in the polygon representing each group are those consumed more by that group than by other groups. Each dot represents one
tadpole, and colors correspond to groups (stage × sampling period). Sampling periods within the wet season are marked in
green for stage 25 tadpoles and in orange for stage 30 tadpoles. Sampling periods within the dry season are marked in blue
for stage 25 tadpoles and in yellow for stage 30 tadpoles.

Niche overlap was still higher than expected
by chance among sampling periods for both
stage 25 (Ojk = 0.604, p < 0.0001) and stage
30 tadpoles (Ojk = 0.966, p < 0.0001). Niche
overlap was higher than expected by chance
between tadpole developmental stages consid-
ered in the middle of the wet season (MW:
Ojk = 0.977, p < 0.0001) and at the onset of
both seasons (OW: Ojk = 0.808, p < 0.0001;
OD: Ojk = 0.934, p = 0.006), but not in the
middle of the dry season (MD: Ojk = 0.375,
p = 0.113).
Changes in tadpole diet 7

Table 2. Estimatives of niche breadth based on (1) number of cells and (2) biovolume of food types for Bokermannohyla
saxicola in developmental stages 25 and 30 (sensu Gosner, 1960) throughout the year: OW = onset wet, MW = middle wet,
OD = onset dry, MD = middle dry (see text for further details). Estimatives were obtained based on 1000 simulations and
data from different stages/periods were compared through rarefaction considering the smallest number of items/biovolume
ingested (n = 18 440 and n = 1 213 210.2 μm3 /ml, respectively) as reference. Different letters (last column) indicate
significantly different results (p < 0.05) within the analyses (number of cells or biovolume).

Tadpole stage and Number of cells Mean niche breadth Mean niche breadth 95% confidence interval
sampling period ingested (n) (PIE) ± SD (PIE) ± SD (n = 18 440)
(n = 18 440)

25 OW 35 320 0.599 ± 0.000 0.599 ± 0.000 0.595-0.604 a

25 MW 36 780 0.616 ± 0.000 0.616 ± 0.000 0.611-0.621 b
25 OD 27 120 0.804 ± 0.000 0.804 ± 0.000 0.802-0.806 c
25 MD 27 720 0.797 ± 0.000 0.797 ± 0.000 0.794-0.799 d
30 OW 34 120 0.760 ± 0.000 0.760 ± 0.000 0.756-0.764 e
30 MW 74 131 0.747 ± 0.000 0.747 ± 0.000 0.742-0.751 f
30 OD 18 440 0.812 ± 0.000 – 0.812-0.812 g
30 MD 34 980 0.811 ± 0.000 0.809 ± 0.000 0.809-0.814 g

Tadpole stage and Biovolume in- Mean niche breadth Mean niche breadth 95% confidence interval
sampling period gested (μm3 /ml) (PIE) ± SD (PIE) ± SD (n = 1 213 210.2
(n = 1 213 210.2 μm3 /ml)
μm3 /ml)

25 OW 1 213 210.2 0.821 ± 0.000 – 0.821-0.821 a

25 MW 2 842 556.4 0.459 ± 0.000 0.459 ± 0.000 0.458-0.459 b
25 OD 3 148 417.6 0.625 ± 0.000 0.625 ± 0.000 0.624-0.625 c
25 MD 2 263 377.6 0.713 ± 0.000 0.713 ± 0.000 0.712-0.713 d
30 OW 4 311 106.6 0.640 ± 0.000 0.640 ± 0.000 0.639-0.641 e
30 MW 3 953 119.6 0.668 ± 0.000 0.668 ± 0.000 0.667-0.669 f
30 OD 3 616 998.0 0.442 ± 0.000 0.442 ± 0.000 0.441-0.443 g
30 MD 2 617 273.6 0.623 ± 0.000 0.623 ± 0.000 0.622-0.623 h

Discussion planktonic or periphytic autotrophic unicellu-

The prevalence of pennate diatoms, Zygnemat-
aceae and Desmidiaceae in tadpole guts sug-
gests reliance of Bokermannohyla saxicola tad-
poles on biofilms for feeding. Thus, the diet of
Bokermannohyla saxicola tadpoles seems to be
in accordance with their ecomorphological type
(benthic tadpoles, sensu Altig and Johnston,
1989). Biofilm algae are important primary pro-
ducers in several aquatic habitats, including
rivers (Stevenson, 1996; Vadeboncoeur et al.,
2008; Pellegrini and Ferragut, 2012), and are
affected by light, nutrient availability (Steven-
son, 1996; Boëchat et al., 2011; Pellegrini and
Ferragut, 2012) and pH (Müller, 1980). The
low pH of the studied stream was likely favor-
ing a high abundance of diatoms (JSK, CCF
and PCE, unpublished data). Diatoms are small
lar organisms that possess a cell wall (frustule)
composed of two silicate shells. Under exper-
imental conditions, they dominated under low
pH values (Müller, 1980). They are very abun-
dant in aquatic systems and important in the di-
ets of other trophic levels, such as zooplankton
(Liu and Wu, 2016) and tadpoles (Souza-Filho
et al., 2007; Santos et al., 2016).
Tadpoles can also be opportunistic and ex-
ploit nutritious food items that become avail-
able to them at different periods or habitats
(Petranka and Kennedy, 1999; Schiesari et al.,
2009). For instance, benthic tadpoles are also
likely to exploit other microhabitats for food,
such as the water surface for pollen (Wag-
ner, 1986), known to be a nutritive food for
several groups of organisms (Filipiak, 2016).
Bokermannohyla saxicola ingested pollen (from
8 J.S. Kloh, C.C. Figueredo, P.C. Eterovick
plants of the family Asteraceae) in both sea-
sons and in both developmental stages consid-
ered in the present study. Alternatively, pollen
could have sank and become available for them
on the bottom. Sand grains were also present in
the guts and could have been ingested acciden-
tally or intentionally, to take advantage of the
film of bacteria covering them or to physically
improve the breakage of food in the gut (Sousa-
Filho et al., 2007).
Some previous studies have analyzed the en-
tire digestive tract of tadpoles (e.g., Sousa-Filho
et al., 2007), which may overestimate items that
are difficult to digest. Rossa-Feres et al. (2004)
used the initial portion of tadpole intestines to
analyze diet and stated that they observed no
differences in food quantity and composition
throughout the entire intestine, although they
provided no data or detailed information on how
this comparison was done. Our comparison of
level of digestion (i.e. food breakage) through-
out the gut had to be based on diatoms due to
their predominance in the diet of B. saxicola,
but showed high digestibility of these algae. Al-
though our results were based only on diatoms,
we recommend that tadpole diet studies use the
anterior portion of the gut in order to mini-
mize the problem of overlooking small easily
digested items (see Hoff et al., 1999). This prob-
lem is corroborated by the fact that we only ob-
served other food items (likely to be more eas-
ily broken/digested) in the initial portion of the
gut.
Bokermannohyla saxicola tadpoles in both
developmental stages exhibited a narrower
trophic niche (considering number of cells) dur-
ing the rainy season than during the dry season,
as we expected. That is, increased algae pro-
ductivity and diversity (e.g. Souza et al., 2015)
may favor increased consumption of more en-
ergetic items, as we predicted. However, con-
sidering biovolume, stage 25 tadpoles had the
narrowest niches in MW, otherwise niches were
broader in the rainy season. Mougeotia is one of
the diet components with the largest total vol-
ume, thus the increased ingestion of more nutri-
tive items (like diatoms) during the rainy, warm
season may reduce the dominance of Mougeo-
tia and other large items in biovolume, explain-
ing the increase in niche breadth. Higher tad-
pole metabolism during the rainy, warm sea-
son would explain an increased consumption
of energetic food items, like diatoms (see fig.
1A, 1C). Diatoms are an important source of
organic nutrients, mainly lipidic compounds
such as PUFA (polyunsaturated fatty acids)
and EPA (eicopentaenoic acid; Napiórkowska-
Krzebietke, 2016). Diets enriched with diatoms
can boost development and allow tadpoles to
metamorphose into larger froglets (Kupferberg,
1997a).
Changes in tadpole diet are likely favored by
the dynamics of benthic communities and avail-
ability of food items, which show temporal vari-
ations (Sandefur et al., 2011; Souza et al., 2015).
Tadpoles can ingest high amounts of carbohy-
drates and lipids to capitalize growth (Richter-
Boix et al., 2007) and development. Food types
vary in nutrient content and can contribute to
complement nutritional needs of tadpoles (Gor-
don et al., 2006). Knowledge on the dynamics
of benthic communities on natural substrates is
rare in general, but seasonal changes have been
shown to occur (Ferragut et al., 2010; Pellegrini
and Ferragut, 2012). For instance, periphyton
algae biovolume, biomass and species richness
are likely to be higher in the summer (Souza et
al., 2015). The initial development of periphy-
ton includes colonization of exposed substrates
and development of a diatom biofilm. Filamen-
tous algae will then colonize mature commu-
nities that may lose fragments due to stream
current and allow recolonization (Sigee, 2005).
Filamentous green algae, like Mougeotia and
Bambusina, usually grow in clouds in micro-
habitats with slow flowing water because they
usually lack structures to attach themselves to
the bottom (CCF, pers. obs.). On the other hand,
periphyton consumers are expected to change
the composition of this benthic community due
Changes in tadpole diet
to direct consumption or nutrient enrichment via
excretion (Furey et al., 2012). Periphyton graz-
ers may also promote stability by avoiding ex-
cessive growth and thus reducing susceptibil-
ity of the thin biofilm to removal by currents
(Pringle and Hamazaki, 1997).
We consider that the consumption of diatoms
by tadpoles of B. saxicola in this study seems
indeed to be greater at the onset of the rainy sea-
son, when diatoms would be expected to colo-
nize substrates scoured by heavy rains (Sigee,
2005). The consumption of Mougeotia subse-
quently increased, probably with its availabil-
ity, as periphyton succession progressed. We no-
ticed the filamentous green algae Mougeotia to
be most consumed by tadpoles of both stages
in the middle of the wet season and at the on-
set of the dry season. Mougeotia was present in
large amounts in the diet of B. saxicola tadpoles
(present study) and has been detected in the di-
ets of tadpoles of other species as well (Dick-
man, 1968; Huckembeck et al., 2016), despite
having low nutritional value (Kupferberg et al.,
1994).
Bokermannohyla saxicola tadpoles in stage
30 were larger and had broader niches than tad-
poles in stage 25, considering number of food
items, as we expected. Increased size and de-
velopmental stage also resulted in a more di-
versified diet in tadpoles of Scinax angren-
sis (Sousa-Filho et al., 2007). Schiesari et al.
(2009) detected ontogenetic shifts in the diets
of Lithobates species as well. Mouth size alone
may not explain the variation in tadpole diet
during different developmental stages because
tadpoles are usually efficient in consuming a
broad array of food items of different sizes (Al-
ford, 1999). However, increased size and expe-
rience may enable tadpoles to move more and
explore microhabitats with different food avail-
ability. The gut of B. saxicola tadpoles in de-
velopmental stage 30 had a 14.6% increase in
length compared to stage 25, which could also
influence the amount of ingested items stored
9
in the intestine, contributing to the character-
ization of a broader dietary niche. The qual-
itative compositions of diets of stage 25 and
stage 30 tadpoles were similar (see fig. 1), but
the relative amounts of each food item ingested
changed between stages and among sampling
periods. We did not notice an increase in con-
sumption by stage 30 tadpoles of food items that
are hard to digest and would require a longer
gut to be efficiently broken and digested. Actu-
ally, developmental stages did not differ consid-
ering volume of food items ingested. Gut size is
likely to present allometric variation with tad-
pole size, and the associated changes in diet
composition/digestion deserve further study.
Niche overlaps (based on number of cells)
between tadpole developmental stages were
higher than expected in the middle of both sea-
sons but did not differ from random at the on-
set of either season. The onset of seasons may
represent a transition period, before the patterns
of food availability and consumption of the sea-
son are well established. Considering biovol-
ume, however, the pattern changed, with niches
overlapping more than expected by chance be-
tween tadpole developmental stages in all sam-
pling periods but MD. The similarities or dif-
ferences in the consumption of a few large food
items (e.g. Mougeotia sp., Bambusina sp., tes-
tate amoebae) are likely responsible for the ob-
served pattern. We recommend that biovolume
is also considered when studying tadpole di-
ets, since food items vary greatly in size. The
niche analyses based on biovolume seemed to
be highly influenced by the consumption of
Mougeotia sp. (see fig. 1B, 1D, 1F, 1H), which
is by far the largest food item (5457.9 μm3 ). The
remaining food items varied from 5.1 μm3 (Eu-
notia) to 982 μm3 (Actinotaenium).
Tadpoles of the same stage showed higher
than expected dietary niche overlap across all
sampling periods, considering both number of
cells and biovolume, despite any eventual sea-
sonal changes in food availability (not mea-
sured). Besides, stage 30 tadpoles did not dif-
fer in size throughout the year, showing ability
10
to grow equally in likely different temporal con-
texts of food availability. These results indicate
an ability to exploit available resources based
on the nutritional needs of each developmen-
tal stage (Richter-Boix et al., 2007). Although
we found the same result for size of stage 25
tadpoles, this should be a methodological arti-
fact because we made an effort to standardize
tadpole size when sampling this developmental
stage (that encompasses a great size range).
In conclusion, biofilm communities are im-
portant for B. saxicola tadpole development,
since they constitute the main source of food
for these tadpoles. We have shown that propor-
tions of ingested food items are likely to change
throughout the year and interact with develop-
mental stages. That is, where and when tadpoles
eat determine what they eat. The causes could
be food availability/accessibility through time
and space, behavioral adjustments to chang-
ing nutritional needs, or a combination of both,
which remains to be investigated. Knowledge
of tadpole diet is important for understanding
aquatic food webs (Kupferberg, 1997b; Dutra
and Callisto, 2005; Schiesari et al., 2009) and
understanding the ecological consequences of
the extinction of these grazers in ecosystems
(Ranvestel et al., 2004; Altig et al., 2007). We
believe there is a need for more studies on the
diet of tropical tadpoles, as well as internal (e.g.
food preference, nutritional needs) and exter-
nal (e.g. food availability, microhabitat use vs.
predator avoidance) factors that control its com-
position. Then we will make progress in under-
standing how impacts on aquatic habitats and
periphyton communities affect tadpole diet and
development.
Acknowledgements. We are grateful to G. Rodrigues and
I. Moreira for help during field work; to Sarah Kupferberg,
Dale Jefferson and two anonymous reviewers for comments
on a previous version of the manuscript; to Erik R. Wild for
revision of the English; to the Parque Nacional da Serra do
Cipó staff for logistic support; to Fundação de Amparo à
Pesquisa do Estado de Minas Gerais (Fapemig) and Pontifí-
cia Universidade Católica de Minas Gerais for finantial sup-
port; and to Conselho Nacional de Desenvolvimento Cien-
tífico e Tecnológico (CNPq) for a Research Productivity
J.S. Kloh, C.C. Figueredo, P.C. Eterovick
grant (304422/2014-2) provided to P. C. Eterovick. Collec-
tion permits were provided by Sisbio/ICMBio (31398-1).
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