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2012 SOUTHEASTERN NATURALIST 11(2):287–296
Southern Two-Lined Salamander Diets in Urban and
Forested Streams in Western Georgia
Kyle Barrett1,2,*, Stephen T. Samoray1, Brian S. Helms1, and Craig Guyer1
Abstract - Streams are heavily affected by watershed urbanization as increased stormwater
runoff changes their physical and chemical composition. Benthic macroinvertebrate
species richness has been consistently shown to decline with urbanization. Conversely,
biomass of macroinvertebrates can increase with urban development. We examined the
effect of such shifts in macroinvertebrate assemblages on the diet of larval Eurycea cirrigera
(Southern Two-lined Salamanders). Salamanders have been documented to decrease
in diversity in urban habitats; however, Southern Two-lined Salamander larvae which
persist in urban streams (at lower densities) tend to grow larger than larvae in forested
streams. Diet may play a role in these diversity and growth trends. We examined prey
consumed by larval salamanders during spring, summer, and winter seasons across urban
and forested watersheds. Prey diversity in salamander digestive tracts peaked during
summer. We found Chironomidae (Diptera) larvae to be the most common prey item, followed
by Ostracoda. Gastropoda were a common prey item during summer, which may
be indicative of nutrient requirements of premetamorphic larvae. Overall, we observed
minor differences in larval diet between urban and forested watersheds. A previous study
within these same watersheds found that larvae in urban watersheds grew larger than
those in forested watersheds, and the authors suggested prey availability may have contributed
to that finding. The diet data we present here do not support such a hypothesis.
Urbanization alters biomass, diversity, and species richness of biota occupying
formerly undeveloped habitats (Czech and Krausman 1997, Klein 1979,
Paul and Meyer 2001, Stratford and Robinson 2005). Streams are especially
influenced by watershed urbanization, as increased impervious surfaces (e.g.,
roads, roofs) cause increased overland flow, which can lead to extreme physical
alteration of instream habitats (Galster et al. 2008, Walsh et al. 2005). Ecologists
have repeatedly shown a decline in species richness of stream macroinvertebrates
following watershed urbanization (Klein 1979, Paul and Meyer 2001, Walsh et
al. 2005). Recently, Helms et al. (2009) documented a similar decline in species
richness of macroinvertebrates; however, they recorded an overall increase in
biomass of stream invertebrates with urbanization.
Like macroinvertebrates, species richness of stream-breeding salamanders declines
with urbanization (Barrett and Guyer 2008, Hamer and McDonnell 2008).
The altered hydrology that accompanies urban development has been linked to a
decline in density of Eurycea cirrigera Green (Southern Two-lined Salamander)
larvae, and also may contribute to a loss of other amphibian species (Barrett and
1Department of Biological Sciences, Auburn University, Auburn, AL 36849. 2Current address
- D.B. Warnell School of Forestry and Natural Resources, 180 East Green Street, The
University of Georgia, Athens, GA 30602. *Corresponding author - email@example.com.
288 Southeastern Naturalist Vol. 11, No. 2
Guyer 2008). A shift in trophic dynamics of urban communities is also likely
to be important in explaining species richness and abundance of top consumers
in urban habitats (Faeth et al. 2005). For example, Johnson and Wallace (2005)
demonstrated decreased biomass and density of larval Eurycea wilderae Dunn
(Blue Ridge Two-lined Salamander) as a result of diet shifts caused by experimental
Many benthic predators actively select particular prey based on nutritional
content (Schaefer et al. 2008). If, because of decreased invertebrate species richness,
preferred salamander prey disappears with urbanization, then the loss of an
important resource base may translate to negative effects on salamander larvae.
Conversely, stream-dwelling salamander larvae have been recorded to consume
a wide variety of prey items (Burton 1976, Caldwell and Houtcooper 1973,
Petranka 1984). If salamanders do not discriminate among available prey, then
an increase in invertebrate biomass associated with urbanization, which was observed
by Helms et al. (2009), could result in ample resources for the salamander
larvae that are able to persist in urban streams. Barrett et al. (2010) documented
higher growth rates in Two-lined Salamander larvae from urban watersheds
relative to forested ones, which is consistent with the hypothesis of prey as a
non-limiting resource for this species in urban streams.
To determine effects of urbanization on larval salamander diet, we quantified
dietary compositional shifts for Southern Two-lined Salamander larvae seasonally
and across land-cover categories for streams in forested, suburban, and urban watersheds.
Results from this analysis will contribute to our ability to examine shifts
in community interactions with urbanization. This area of urban ecology has received
little attention in stream systems; however, analyses from other community
types suggest it is a topic that warrants increased study (Faeth et al. 2005).
We examined the diet of larval Southern Two-lined Salamanders in nine second-
or third-order streams in western Georgia, all within the larger Chattahoochee
River Basin (Fig. 1). To evaluate larval diets in urban habitats, we selected three
streams within Columbus, GA (Bradley Creek [BR], Cooper Creek [BU2], and
Roaring Branch [RB]). These sites were heavily urbanized, with at least 25% of the
land cover in the watershed as impervious surface (mean = 32%, range = 25–40%).
For comparison, we also selected three streams (Blanton Creek [BLN], Cline’s
Branch [MO], and Turntime Branch [MU3]) within forested watersheds (Lockaby
et al. 2005) approximately 30 km north of Columbus (Meriwether County). We
refer to these streams as reference streams because they retain forested borders that
approximate the ancestral landscape. These sites had a minimum of 75% (mean =
79%, range: 76–81%) of the total watershed as forested area (evergreen + deciduous
forest), and no more than 1% of the total watershed land-cover as impervious
surface. Finally, to determine if watersheds subjected to small amounts of very
recent development contained larvae with altered diets, we examined larvae from
three streams within Harris County (developing streams), a rapidly developing
2012 K. Barrett, S.T. Samoray, B.S. Helms, and C. Guyer 289
suburban area adjacent to Columbus (Schley Creek [SB1], Standing Boy Creek
Tributary [SB2], and Standing Boy Creek [SB4]). The watersheds for these
streams all had relatively low impervious surface cover within the individual watersheds
(mean = 3%, range = 2–3%); however, this level of development relative
Figure 1. Location of study sites and associated waterways located within the
Chattahoochee River Basin of western Georgia. Sites were divided into three different
land-cover categories (see Study Area for descriptions). The white area in west central portion
of the inset map depicts the location of the two counties shown in the main map.
290 Southeastern Naturalist Vol. 11, No. 2
to forested watersheds appears to be sufficient enough that the biological character
of these streams has been altered (Barrett and Guyer 2008). The qualitative landcover
category delineations were supported by a principal components analysis
described in Barrett and Guyer (2008).
Southern Two-lined Salamander larvae were captured for gut content analysis
during four seasons. The captures from summer (July 2006 and 2007) and fall
(November 2006 and October 2007) were combined for this analysis, as this period
of warm temperatures likely represents the peak of salamander foraging, and
we refer to them as our summer sample. We also captured larvae during winter
(January 2007) and spring (April 2007). Upon capture, individuals were euthanized
in 0.04% unbuffered MS 222 solution and then preserved by freezing until
examined for gut contents in the laboratory.
To identify prey items consumed by salamanders, we made a sagittal incision
along the ventral midline of each individual and subsequently opened the
digestive tract so that contents could be removed by flushing with 70% ethanol
(Bardwell et al. 2007). We sorted prey items under a dissecting scope, counted
individuals, and identified them to the lowest possible taxonomic level (typically
We compared prey composition among land-cover categories and seasons
using a Fisher’s exact test (FET) in Program R (Version 2.13.0). This test is appropriate
for determining whether or not an association exists between categorical
variables, and it is particularly suited for situations where some of the expected
frequencies are very small (i.e., less than five; Crawley 2007). In short, the test
was used to determine if the number of prey observed in one category (e.g., land
cover) depended upon another category (i.e., taxa). A lack of independence between
categories implies a shift in prey composition as a function of either site
To evaluate prey composition shifts, we combined data from streams within
land-cover categories and used prey taxa categories that had at least five occurrences
across land-cover categories in a given season (Table 1). This procedure
resulted in the inclusion of the following prey groups: Coleoptera, Diptera, Gastropoda,
Ostracoda, and Other (a combination of taxa too infrequent to analyze
separately). Because of the complications involved in evaluating a three-way
interaction between taxon, season, and land-cover category, and because we a
priori expect variations in diet across seasons, we focused more detailed analyses
within seasons to evaluate shifts in taxon composition across land-cover categories.
For these analyses, we first evaluated the FET for a table including all taxa
meeting our minimum requirement of at least 5 observations across land-cover
categories. If the test was significant (P < 0.05), then we dropped from the table
the taxon that appeared to contribute most to the lack of independence in counts
between land-cover category and taxa. This decision was made based on a qualitative
assessment of the data, and was done because there is no formal post hoc
2012 K. Barrett, S.T. Samoray, B.S. Helms, and C. Guyer 291
pairwise test available for categorical data with small expected frequencies. After
removing a taxon, we then performed the test again on the reduced taxa set, and
continued this process until the test was no longer significant. A lack of statistical
significance implied that counts of taxa did not depend on land-cover category
(i.e., there were no diet shifts observed across land-cover categories for those
groups included in the test). In addition to this analysis, we compared taxonomic
richness across seasons and land-cover categories with a goodness of fit (GOF)
test, and we calculated Shannon diversity index (H') for diets in each of the landcover
categories in each season. The Shannon diversity index is often used as a
measure of diet breadth (Levins 1968, Pianka 1986).
We captured a total of 145 Southern Two-lined Salamander larvae across all
seasons and land-cover categories (Table 1). Twelve individuals were found with
either no food in their guts, or contained no identifiable prey. Among all prey
taxa, Diptera larvae consistently made up the largest proportion of larval diets
(Table 2). The FET on a table of season x land-cover category x taxa revealed
that counts within a particular invertebrate taxon varied as a function of season
and land-cover category (P < 0.0001). As described in the Methods, we made
no attempt to investigate this table further, and focused instead on the within-
Table 1. Number of prey items (expressed as a sum per taxa) found in the diet of Southern Twolined
Salamander larvae in nine streams in western Gerogia. The number of digestive tracts
examined for each stream is represented below the stream name in parentheses.
Reference Developing Urban
BLN MO MU3 SB1 SB2 SB4 BU2 BR RB
Taxon (18) (10) (19) (14) (17) (17) (14) (21) (15)
Acari 1 - - 2 - 1 - 3 -
Amphipoda 2 - - - - - - - -
Cladocera 10 - 1 - - 1 - - -
Coleoptera 2 5 2 - 2 3 1 - 22
Collembola 1 - - - - 1 - - -
Copepoda 6 3 5 - - 9 - 3 -
Diptera 76 44 164 31 100 47 22 27 64
Ephemeroptera - - - - - 2 - 1 -
Gastropoda - 5 - - 6 1 7 10 5
Hemiptera - - - - - 1 - - -
Hymenoptera 1 - - 1 1 1 - 1 1
Lepidoptera 1 - 1 3 1 - - - -
Megaloptera 2 - 1 - - - - - -
Nematoda 1 - 1 - - - - 1 -
Odonata - 1 - 1 - - 1 - -
Ostracoda 7 1 1 10 1 56 9 2 13
Plecoptera - - - 1 - 1 - 1 -
Trichoptera - 1 - 1 10 - - - 2
Unidentified 2 4 7 4 7 - 1 9 3
Empty gut 1 - 3 - 3 - 2 3 -
292 Southeastern Naturalist Vol. 11, No. 2
season analyses. The analysis of spring prey items included Diptera, Ostracoda,
and Other. The FET on the 3 x 3 table (all spring taxa and the three land-cover
categories) was significant (P < 0.0001). We then eliminated Ostracoda from
the table (as this group was encountered in the diets of larvae from developing
streams in much higher proportion than in reference and urban streams; Table 2).
The test of a table with only Diptera and Other was not significant (FET: P =
0.70). Within all land-cover categories during summer, we observed a notable
increase in the proportion of Gastropoda (snails, primarily Physidae and Planorbidae)
within larval diets (Table 2). Analysis of summer data included Diptera,
Ostracoda, Coleoptera, Gastropoda, and Other. No combination of taxa resulted
in a non-significant FET; therefore, we concluded that counts for all taxa groups
showed a lack of independence with the land-cover category variable (FET: P less than
0.02 for all tests; Table 2). In summer, we observed significantly fewer Diptera
in larvae captured in the urban land-cover category relative to the other two
land-cover groups. Developing sites had larvae with higher counts of Other invertebrates
relative to larvae in reference and urban streams, while reference sites
had significantly lower counts for Ostracoda relative to the other two land-cover
categories. Coleoptera constituted 14% of the summer diet for larvae found in
urban streams, but made up less than 5% of larval diets in the other land-cover categories.
Finally, the winter analysis included Diptera, Coleoptera, and Other. The FET
was significant with all three groups included in the analysis (P = 0.05); however,
when the combined taxonomic group of Other was removed, there was no evidence
that counts of Diptera or Coleoptera varied across land-cover categories.
Table 2. Proportion of prey items found in the diet of Two-Lined Salamanders across seasons in
nine west Georgia streams. Values were calculated by combining data from streams within land
cover categories (Ref = Reference, Dev = Developing, Urb = Urban).
Spring Summer Winter
Taxon Ref Dev Urb Ref Dev Urb Ref Dev Urb
Acari - - - 0.01 0.02 0.02 - - -
Amphipoda - - - 0.01 - - 0.01 - -
Cladocera 0.01 - - - 0.01 - 0.13 - -
Coleoptera 0.02 0.02 0.10 0.02 0.01 0.14 0.03 0.04 0.12
Collembola - - - 0.01 - - - 0.01 -
Copepoda 0.05 0.03 0.05 0.01 0.01 - 0.06 0.06 0.06
Diptera 0.78 0.29 0.52 0.74 0.62 0.47 0.71 0.72 0.67
Ephemeroptera - - - - 0.01 0.01 - 0.01 -
Gastropoda - - - 0.02 0.04 0.13 - - -
Hemiptera - - - - 0.01 - - - -
Hymenoptera - - - 0.01 0.01 0.01 - 0.01 -
Lepidoptera 0.01 - - - 0.03 - 0.01 - -
Megaloptera - - - 0.01 - - - - -
Nematoda - - 0.05 0.01 - - - - -
Odonata 0.01 - - - 0.01 0.01 - - -
Ostracoda 0.07 0.62 0.05 0.03 0.13 0.13 0.04 0.06 0.03
Plecoptera - - - - 0.01 0.01 - - -
Trichoptera - - - 0.01 0.07 0.01 - - -
Uidentified 0.04 0.01 0.19 0.01 0.03 0.05 0.01 0.07 0.06
2012 K. Barrett, S.T. Samoray, B.S. Helms, and C. Guyer 293
Figure 2. Shannon diversity (a) and taxa richness (b) for prey items found in the digestive
tracts of Southern Two-lined Salamanders in reference (n = 47 larvae), developing (n =
48 larvae), and urban (n = 50 larvae) streams during three seasons in western Georgia.
Larval sample sizes are the same as in Figure 1.
294 Southeastern Naturalist Vol. 11, No. 2
Counts for Other were far higher in larvae from reference streams than those
from either developing or urban streams (Table 2).
Diet breadth, as measured by the Shannon index, was highest in urban and
lowest in reference streams during spring and summer seasons; however, during
winter, niche breadth was lowest in urban streams and highest in reference
streams (Fig. 2a). Taxonomic richness (primarily assessed at the order level)
showed high variability among land-cover categories, but was significantly
higher in all categories during summer (GOF test: df = 2, P = 0.007; Fig. 2b).
The main differences in prey composition of Southern Two-lined Salamanders
among land-cover categories were not from the presence or absence of a given
prey item, but rather the proportions in which they were consumed. For example,
Ostracoda appeared to be especially important in developing streams, particularly
during spring. Gastropods and coleopteran larvae were consumed more in
urban streams than in any other category. Other aspects of larval diet composition
showed some consistency. Diptera (primarily in the family Chironomidae) was
the main prey taxon of larvae in nearly all streams and seasons. This finding is
consistent with several other foraging studies on larval Eurycea (Burton 1976,
Caldwell and Houtcooper 1973, Johnson and Wallace 2005, Muenz et al. 2008,
Petranka 1984). Ostracoda was the next most abundant prey taxon (and the most
abundant during spring at developing sites). In previous studies, this taxon was
either not observed in the guts of other larval Eurycea (Burton 1976, Johnson
and Wallace 2005), or was observed with few occurrences (Caldwell and Houtcooper
1973, Muenz et al. 2008, Petranka 1984). Plecoptera larvae, which were
important in the diet of Southern Two-lined Salamanders studied by Caldwell and
Houtcooper (1973), were not predominant prey in the organisms we examined
or in those examined in pasture and forested habitats by Muenz et al. (2008).
Taxa richness of prey consumed and dietary niche breadth of salamander larvae
were both found to increase in summer across all land-cover categories. This
result does not correspond to the period of greatest macroinvertebrate diversity,
which was found to be during spring (Helms 2008) for samples taken from these
same study streams during 2004. The greater diversity of prey items consumed
by salamanders during summer may represent a general increase in biomass
consumption during warmer months when metabolic rates are likely increased
and growth rates are high (Barrett et al. 2010).
Gastropoda were one prey group occurring only during summer that contributed
to the high species richness, and they were found in all land-cover categories.
The only other study with a seasonal component during which gastropods were
observed as prey for Southern Two-lined Salamanders also recorded the presence
of snails in the diet during summer (Caldwell and Houtcooper 1973). Many of the
larvae we captured during the summer were pre-metamorphic. In tadpoles, calcium
deposits increase dramatically during the pre-metamorphic stage (McDonald et al.
1984). Presumably the increase occurs because of calcification of the skeleton as
larvae prepare for increased skeletal demands associated with terrestrial life. It is
2012 K. Barrett, S.T. Samoray, B.S. Helms, and C. Guyer 295
possible that larval Southern Two-lined Salamanders consume snails, which have
extensive calcium deposits in the shell, for similar reasons.
Previously, Barrett et al. (2010) documented increased growth rates of Twolined
Salamander larvae from urban streams relative to reference streams within
the same western Georgia system we describe in this study. Several potential
explanations for the observed growth differential were explored as part of that
study, and one of them was a positive correlation between growth rate and
invertebrate abundance (Helms 2008) within a stream. Little support for that
relationship was found by Barrett et al. (2010). The relatively minor differences
we observed in diet composition and overall invertebrate counts within larvae as
part of this study further suggests that diet composition is not a suitable explanation
for why Two-lined salamander larvae in these urban streams exhibit higher
Our description of salamander diets provides the information necessary to begin
constructing and comparing stream food webs in urban and forested habitats.
Studies demonstrating a change in species richness or abundance of taxa with
urbanization have accumulated rapidly, and sufficient information now exists to
begin examining changes in multi-trophic interactions that result from urbanization
(Faeth et al. 2005, Helms 2008). Such an approach will increase our ability
to understand how management strategies for one trophic level will cascade (up
or down) to other trophic levels.
We would like to thank Shannon Hoss, John Peterson, Christina Romagosa, John
Peterson, and Matt Williams for assistance in the field. We are grateful to an anonymous
reviewer and Glen Mittelhauser for offering insightful comments on previous drafts of the
manuscript that greatly improved the final version. Support was provided by the Center for
Forest Sustainability and the Department of Biological Sciences at Auburn University.
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