Journal of Freshwater Ecology

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Microhabitat use of larval fish in a South Carolina

Piedmont stream

Luke M. Bower & Brandon K. Peoples

To cite this article: Luke M. Bower & Brandon K. Peoples (2022) Microhabitat use of larval
fish in a South Carolina Piedmont stream, Journal of Freshwater Ecology, 37:1, 583-596, DOI:
10.1080/02705060.2022.2144957

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JOURNAL OF FRESHWATER ECOLOGY
2022, VOL. 37, NO. 1, 583–596
https://doi.org/10.1080/02705060.2022.2144957

Microhabitat use of larval fish in a South Carolina

Piedmont stream
Luke M. Bowera and Brandon K. Peoplesb
a
U.S. Geological Survey, South Carolina Cooperative Fish and Wildlife Research Unit, Clemson
University, Clemson, SC, USA; bDepartment of Forestry and Environmental Conservation, Clemson
University, Clemson, SC, USA

ABSTRACT ARTICLE HISTORY

Understanding habitat use and nursery areas of larval fish is a key Received 18 August 2022
component to managing and conserving riverine fishes. Yet, Accepted 2 November 2022
freshwater researchers often focus only on adult fishes, resulting
KEYWORDS
in a limited understanding of the habitat requirements for the
Habitat use; nursery habitat;
early life stages of freshwater fishes. The goal of this study was to temporal changes; riffle-
quantify the larval fish microhabitat use of three fish families in pool sequence
Twelvemile Creek, a fifth-order tributary of Lake Hartwell
(Savannah River basin) in the Piedmont ecoregion of South
Carolina, USA. We used handheld dipnets to sample larval fishes
along 20 equidistant transects spaced 10 m apart weekly from
May to July 2021 along a 200 m stream reach. We also collected
microhabitat data at each larval fish capture location. Most cap-
tured individuals were in the metalarval stage and were identified
to the family level. A partial distance-based redundancy analysis
indicated that water velocity contributed to changes in larval fish
assemblage structure. Larval fishes occupied a subset of the avail-
able habitat that was characterized by low water velocity, non-
Podostemum substrate, and shallow habitats close to the shore or
bed rock structure. We also detected temporal patterns in larval
fish counts, with peak Percidae and Leuciscidae counts in late
July and the highest Catostomidae counts in late May–early June.
Our results suggest that larval fishes select habitats with low
water velocity and shallow habitats close to shore microhabitat
characteristics, and that riffle-pool sequences may serve as a
nursery habitat for Percidae, Catostomidae and Leuciscidae metal-
arvae.

CONTACT Luke M. Bower [email protected] U.S. Geological Survey, South Carolina Cooperative Fish
and Wildlife Research Unit, 261 Lehotsky Hall, Clemson University, Clemson, SC 29634, USA
Supplemental data for this article can be accessed online at https://doi.org/10.1080/02705060.2022.2144957
This work was authored as part of the Contributor’s official duties as an Employee of the United States Government and is therefore a work of
the United States Government. In accordance with 17 USC. 105, no copyright protection is available for such works under US Law.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/
4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
584 L. M. BOWER AND B. K. PEOPLES

GRAPHICAL ABSTRACT

Introduction
The management and conservation of riverine fishes have focused primarily on adult life
stages, generally excluding the early life stages of many fishes (Snyder 1990; Raborn et al.
2001). Yet, the population dynamics, assemblage structure and year-class strength of adult
fishes are strongly influenced by events in early life stages (Harvey 1987; Houde 1987;
Sogard 1997). Riverine larval fishes often have habitat requirements, diets and responses
to environmental stresses distinct from those of adult and juvenile fishes (Floyd et al.
1984; Snyder 1990; Scheidegger and Bain 1995). Studies of larval fishes have focused pre-
dominantly on marine, lake, or large riverine systems, with very little larval fish research
conducted in smaller streams. Yet, streams contain the highest fish diversity by volume
compared to other aquatic habitats (Helfman 2007). The lack of studies on larval stream
fishes likely stems from the absence of keys for most larval stream fish and the difficulty
of identifying individuals to species (Snyder 1990). Regardless of the cause, this lack of
studies has led to serious gaps in our understanding of the ecology and natural history of
critical life stages, preventing managers from adequately protecting fish populations and
assemblages.
Fish habitat use varies across life stages and among species (Schlosser 1991). For
example, larvae of more precocial species (sensu (Balon 1981) emerge from the egg well
developed and closely resembling adults and often remain near the spawning location or
use the same habitat types throughout their development cycle (Simon and Wallus 2003).
In contrast, larvae of more altricial species emerge poorly developed and can drift long
 JOURNAL OF FRESHWATER ECOLOGY 585

distances or actively migrate to more suitable habitats (Balon 1984; Snyder 1990;
Hoagstrom and Turner 2015). Larval fish habitat use can also be affected by the develop-
ment time required for larvae to become free-swimming and can differ among species.
For example, Hypentelium nigricans (Catostomidae) can require 8 days after hatching to
become free-swimming (Buynak and Mohr 1978), while Leuciscus leuciscus (Cyprinidae)
can start actively swimming only hours after hatching (Kupren et al. 2016). This develop-
ment time creates a time lag between spawning and movement to nursery habitats. Larval
fishes are often more sensitive to abiotic and biotic stresses than adults (Balon 1984;
Schlosser 1985; Sogard 1997). For example, some larval fish have lower larval survival
rates during high flows as a result of increased stress, abrasion, or inability to feed
(Raborn et al. 2001). Given that development time and spawning length and timing varies
among species (Floyd et al. 1984), understanding the temporal patterns of larval fish
abundance can provide insights on when fish populations may be most sensitive to
instream flow alterations and other environmental stressors (Marchetti and Moyle 2000;
Rodger et al. 2016).
Habitat requirements and specialization have been reported for some riverine larval
fishes (Dewey and Jennings 1992; Scheidegger and Bain 1995; Childs et al. 1998; Pusey
et al. 2002). For example, leuciscid larvae tend to occupy shallow microhabitats near to
shore and cover, indicating these habitats are a preferred nursery habitat for some larval
stream fish and important for species survival (Scheidegger and Bain 1995; Childs et al.
1998). Larval fish habitat requirements and specialization can also vary among taxa and
regions (Scheidegger and Bain 1995; Childs et al. 1998). Therefore, site-specific informa-
tion on larval fish habitat requirements and specialization is needed to better understand
how larval and adult fish assemblages will respond to environmental alteration.
Quantifying microhabitat use can also aid in identifying potential nursery habitats that
are needed for larval fish survival (Scheidegger and Bain 1995; Pusey et al. 2002).
However, information of the habitat requirements and specialization of riverine larval fish
is lacking, and quantitative analysis of microhabitat preference remains limited (Floyd
et al. 1984; Scheidegger and Bain 1995; Pusey et al. 2002).
In this study, we conducted single-season, spatially intensive larval fish surveys in a
rocky shoal complex of a mid-sized river in the Piedmont ecoregion of South Carolina,
USA. Our goal was to quantify microhabitat associations and temporal changes in metal-
arvae abundance of three freshwater fish families: Catostomidae, Leuciscidae and Percidae.
Specifically, our objectives were to: (1) identify the timing of peak larval fish counts; (2)
quantify the microhabitat preference of larval fish to identify potential nursery habitats;
and (3) quantify the influence of microhabitat characteristics on larval fish assemblage
structure. While this work is limited in spatiotemporal extent and taxonomic specificity, it
is intended as an initial step toward a better understanding of larval fish microhabitat
associations in streams.

Methods
Twelvemile Creek is a tributary to Lake Harwell located in the Piedmont ecoregion of
northwestern South Carolina (Figure 1a; Omernik 1987). This small river has a mean
annual discharge of 6.03 m3/sec and drains roughly 352 km2. Land cover in the
Twelvemile Creek watershed is made up largely of forest (57%) and agriculture (23%)
with some urban development (19%). Our sample site is located approximately 500 m
downstream of a small dam with a hydraulic height of 6 m. Adult fish assemblages at this
site have been sampled extensively for a previous study (Marion 2014), and ongoing
586 L. M. BOWER AND B. K. PEOPLES

Figure 1. A) A map showing the study site on Twelvemile Creek in northwestern South Carolina. Layers were col-
lected from ArcGIS. B) An image of the sampling site taken by Bower, LM.

Table 1. The mean, weighted average, standard error (SE), and range of water depth, velocity, and distance
weighted by abundance from shore for each fish family in Twelvemile Creek, South Carolina.
Taxa Microhabitat Mean Average SE Range
Catostomidae Depth (cm) 20.953 28.092 1.884 5–76
Distance (cm) 92.392 106.082 11.707 7–470
Velocity (cm/sec) 3.12 4.652 0.562 0–19
Leuciscidae Depth (cm) 23.623 21.720 2.500 6.5–76.0
Distance (cm) 84.821 87.622 10.415 13–320
Velocity (cm/sec) 2.360 1.927 0.537 0–11
Percidae Depth (cm) 37.091 35.563 6.784 8–87
Distance (cm) 117.909 129.650 25.993 32–320
Velocity (cm/sec) 6.091 3.690 2.029 0–19

routine sampling provides of a list common species present at this sampling site
(Supplemental Table 1). The sampling site included two shoal sequences made up of rif-
fles, runs and pools divided by large bedrock outcroppings (Figure 1b; henceforth riffle-
pool sequence), creating a complex habitat network with varying water velocities. The
primary substrates in the riffle-pool sequences were bedrock and sand.
We actively collected larval fishes along a 200 m stream reach of Twelvemile Creek
below the U.S. Geological Survey gage 02186000. We created transects every 10 m along
the 200 m stream reach, sampling for fish in all microhabitats along these transects with
velocities less than 0.5 m/sec. We limited our sampling from the highest velocities because
larval fish generally cannot persist for long periods in habitats with water velocities more
than 3 to 7 times their body lengths per second (Webb 1975). Microhabitats were desig-
nated as areas of relatively homogeneous depth, current velocity, and substrate compos-
ition and were generally less than 2
2 m. We sampled the stream reach weekly from
late May to mid-July 2020. However, we excluded the mid-July sample from analyses
 JOURNAL OF FRESHWATER ECOLOGY 587

because too few larval fish specimens were collected. Owing to the complex nature of the
riffle-pool sequence habitat, traditional collection methods such as drift nets or ichthyo-
plankton pulls would be ineffective since many of the larval fish were located in small,
shallow rocky pools. All active sampling was done during daylight hours with either a 1-

1-m seine (500-mm mesh) or a 250-mm-mesh dip net. We used the seine in larger,
deeper habitats, whereas the dip nets were used in smaller, shallower habitats. Dipnets
and seining are effective methods for collecting larval fish in clear shallow streams (Pusey
et al. 2002; Hanks and Hartman 2019). A collection of a microhabitat was considered
complete after three samples produced no new fish individuals. All individuals collected
from a given microhabitat were placed in a labeled Whirlpak to be later identified and
counted in the lab. Once fishes were captured from a given microhabitat, we marked this
point with labeled flagging tape attached to 5-ounce pyramid fishing weights to allow for
microhabitat data to be collected at the point of fish capture. All individuals were imme-
diately preserved in 10% buffered formalin. All specimens were identified to the family
level (Hogue et al. 1976; Auer 1982; Holland-Bartels et al. 1990). Analysis at the family
level can reduce the possibility of inaccurate conclusions from misidentifications. No keys
exist for many species of stream fishes with most keys focusing on marine, lake, or large
river species. Identification of fish larvae to species often requires costly molecular tools
such as DNA barcoding (Peoples et al. 2017).
To estimate the microhabitat occupied by larval fishes (hereafter, occupied microhabi-
tat data), we recorded the following microhabitat data at each sample point containing
larval fish: distance from shore (cm), water velocity (cm/sec), depth (cm), and percentage
of substrate in the sample area. All microhabitat variables were measured at each flagged
point after sampling for fish to minimize disturbing the sampling points. Current velocity
was measured at 0.6 depth to the nearest 0.01 m/s with an electromagnetic flow meter
using a Hach FH950. Distance from shore was measured from the point of capture to the
closest large bedrock outcroppings or shoreline. We estimated the percentage of substrate
composition within a 1 m2 quadrant centered at each sampling point. Substrate types
were categorized based on a coarsened Wentworth scale: mud (<1 mm), sand (2–6 mm),
fine gravel (7–32 mm), gravel (33–64 mm), cobble (65–128 mm), rock (128–512 mm), bed-
rock (>512 mm), large woody debris (diameter > 20 cm), small woody debris (diameter
< 20 cm) and small aquatic vegetation (Podostemum sp.). Microhabitat data were

Figure 2. Bar graph of larval fish abundance by family in Twelvemile Creek, South Carolina for each sampling event
scaled between 0 and 1. Line shows daily mean discharge scaled between 0 and 1.
588 L. M. BOWER AND B. K. PEOPLES

collected during the first three samples. In addition, we collected the same microhabitat
variables every 1 m along each transect after the collection of larval fish specimens in a
grid design, providing a representative sample of available microhabitat space within the
sampling site (hereafter, available microhabitat data). Points falling on dry bed rock were
not included in the analysis of the available microhabitat data.
We used a principal component analysis (PCA) to determine if larval fish occupy a
subset of the microhabitat available to the larval fish. The PCA was run using a combined
dataset of available microhabitat data from the grid design and occupied microhabitat
data. To evaluate associations between larval fish assemblage structural and microhabitat
environmental variables, we used a partial distance-based redundancy analysis (pRDA)
based on Bray–Curtis dissimilarities on log(x)þ1 transformed larval fish count within
each microhabitat using the occupied microhabitat data. A partial RDA explores the linear
effects of the microhabitat variables on the assemblage composition while accounting for
the effects of a covariable. We used the number of days since the first sampling event as
the covariable to account for temporal autocorrelation. The pRDA was performed using
the function ‘rda’ in the VEGAN package (Oksanen et al. 2019) in R version 4.0.0 (R
Core Team 2020). We tested the statistical significance of the environmental variables on
assemblage structure using a permutated analysis of variance of 999 permutations
(Legendre et al. 2011).

Results
We collected 893 Catostomidae, 784 Leuciscidae and 393 Percidae individuals across all
seven sampling events for a total of 2,077 individuals. The highest number of Leuciscidae
and Percidae larvae collected occurred in late June, whereas the highest counts of
Catostomidae were found in late May to early June (Figure 2). The number of Percidae
and Catostomidae larvae peaked and decreased rapidly, while Leuciscidae counts gradually

Figure 3. Principal component analysis of all microhabitat variables for larval fish in Twelvemile Creek, South Carolina
colored by used habitat and available habitat with 95% confidence ellipses. Microhabitat variables: distance from
shore (Dis), water depth (Depth), % bedrock substrate (Bed), % Podostemum (Moss), % sand-silt substrate (Sand-silt),
and water velocity (Velocity).
 JOURNAL OF FRESHWATER ECOLOGY 589

Table 2. The loading scores, eigenvalues, and variance explained for the first two axes of the distance-based redun-
dancy analysis (RDA) of larval fish assemblage structural and microhabitat environmental variables in Twelvemile
Creek, South Carolina. Variables are given as: distance from shore (Dis), water depth (Depth), % Podostemum (Moss),
% sand-silt substrate (Sand-silt), water velocity (Velocity), proportion of variance explained (Proportion), and cumula-
tive variance explained.
RDA1 RDA2
Dis 0.384 0.442
Depth 0.627 0.429
Velocity 0.865 0.092
Sand-silt 0.229 0.357
Moss 0.187 0.190
Leuciscidae 0.544 1.243
Percidae 0.240 0.444
Catostomidae 1.474 0.252
Eigenvalue 0.922 0.352
Proportion 0.633 0.242
Cumulative 0.633 0.875

Figure 4. Distance-based redundancy analysis (RDA) for larval fish abundance in Twelvemile Creek, South Carolina
(log þ1) using Bray–Curtis dissimilarity and constrained by microhabitat variables: distance from shore (Dis), water
depth (Depth), % bedrock substrate (Bed), % Podostemum (Moss), % sand-silt substrate (Sand-silt), and water vel-
ocity (Velocity).

increased and decreased (Figure 2). Catostomidae larval occupied microhabitats with
higher water velocities than Leuciscidae (Table 1). Percidae larvae tended to be collected
in greater water depth, whereas Leuciscidae larvae tended to be found in microhabitats
with the lowest depth, velocity, and greatest distance from shore (Table 1).

Microhabitat patterns
The PCA visualizing microhabitats occupied by larval fishes and overall available micro-
habitat indicated that larval fishes tended to occupy a subset of the available habitat space
(Figure 3). The first two PC axes accounted for 37% and 28% of the variance in the
microhabitat data, respectively. Water velocity and Podostemum had high positive
590 L. M. BOWER AND B. K. PEOPLES

loadings on PC1; PC2 was negatively associated with bedrock substrate and positively
related to sandy substrate, depth, and distance from shore (Figure 3). We found used
habitat space to be characterized by low water velocity, non-Podostemum, shallower habi-
tats, and shorter distance to shore (Figure 3).
The first two axes of the distance-based RDA explained 87% of the variation in larval
fish assemblage structure. Water velocity, depth, sand-silt and distance were positively
correlated with RDA1. Depth, sand-silt, and Podostemum had positive loadings on RDA2
(Table 2; Figure 4). We found negative loading scores for Leuciscidae on RDA1, indicat-
ing counts of this family were negatively related to depth, distance to shore, and velocity
(Table 2; Figure 4). The RDA2 axis was negatively associated with Catostomidae, suggest-
ing Catostomidae counts were positively related to distance to shore. The microhabitat
variables explained a significant proportion of the larvae count variation based on the full
RDA model at the microhabitat scale (Adjusted R2 ¼ 0.12, p ¼ 0.05). Tests of the individ-
ual microhabitat variables showed a significant effect of water velocity on larval fish
assemblage structure (F ¼ 2.760, df¼ 1, p ¼ 0.033).

Discussion
Our study suggests distinct habitat use by metalarvae of three freshwater fish families, and
provides insights on microhabitat use in a riffle-pool sequence of a southeastern USA
Piedmont stream. Larval fishes in our study tended to occupy a subset of the available
habitat characterized. These microhabitats were characterized by lower water velocity,
absence of Podostemum sp. vegetation and, to a lesser extent, shallower habitats closer to
shore. Depth and water velocity played a role in determining larval fish assemblage struc-
ture at the microhabitat scale, indicating these variables helped determine the habitats lar-
val fish can occupy. We also observed temporal changes in the number of larvae at a
weekly time step for all three families, with peaks of larvae counts in late May for
Catostomidae and in late June for Leuciscidae and Percidae.
Temporal changes in larval fish counts varied among families in our study. For
example, counts of Leuciscidae and Percidae larvae counts peaked in late June, whereas
the number of Catostomidae larvae was highest in late May and early June. However, the
abundance peak of Catostomidae may have occurred earlier before we started sampling,
as the catostomid species in this assemblage spawn earlier than the percids and leuciscids.
Larvae abundance changed at a weekly time step, suggesting sampling larval fish at a
monthly time step may miss substantial variation in abundance patterns. The abundance
of leuciscid larvae increased and decreased gradually across our study period, whereas the
larval abundance of Percidae peaked and decreased rapidly. This rapid peak of percid
abundance suggests a single large spawning event in line with an equilibrium life history
strategy rather than the serial spawning of an opportunistic species (Winemiller and Rose
1992). Turner et al. (1994) also noted an abrupt reduction in percid abundance after
abundance peak. Abundance peaks observed in this study generally correspond with
spawning times for species found in Twelvemile Creek after accounting for hatching time.
Percina nigrofasciata and Etheostoma inscriptum, the dominate darter species in
Twelvemile Creek, spawning is reported from March to June and May to June, respect-
ively (Mathur 1973; Rohde et al. 2009). For two species in the genus Etheostoma (E. bel-
lum and E. camurum), incubation time was reported to be 7–9 days. Our results generally
match the larvae count peak seen in Twelvemile Creek after accounting for hatching time.
Spawning of the dominant leuciscid species, Hybopsis rubrifrons, Nocomis leptocephalus,
Notropis hudsonius and Notropis lutipinnis (Marion 2014), occurs from April to June
 JOURNAL OF FRESHWATER ECOLOGY 591

(Rohde et al. 2009; Kim and Kanno 2020). Hatching of leuciscid eggs, such as Nocomis
micropogon, is reported to occur 5–6 days after fertilization and measured 5.7–6.1 mm
after hatching (Cooper 1980). We found leuciscid individuals within this size range in all
our samples from early June to early July, indicating multiple spawning events of a single
species or multiple spawning events of multiple species during this time period. The tem-
poral patterns observed for leuciscid and percid larvae match Spawning of the most
prevalent sucker species in Twelvemile Creek (Marion 2014), H. nigricans (Catostomidae),
occurs primarily in April and May (Jenkins and Burkhead 1994). Eggs take about 10 days
to hatch and H. nigricans larvae require about 8 days to become free-swimming (Buynak
and Mohr 1978). Other catostomid species found in Twelvemile Creek, such as
Minytrema melanops, Moxostoma collapsum, and Moxostoma rupiscartes (Marion 2014),
are reported to spawn from March and May (Jenkins and Burkhead 1994). Given the
delay in hatching and growth, the peak larvae counts found in our study appear to match
reported spawning times of H. nigricans and most of the catostomid species.
Based on the PCA of the combined microhabitat datasets, larval fish families occupied
a subset of the available microhabitat space in our study area. The larvae selected shal-
lower habitats with lower water velocity and nearer to stream margins than was available.
Similar to our results, Pusey et al. (2002) reported a strong preference for microhabitats
associated with low water velocity, shallow to moderate depth, and cover. However, we
observed no large macrophytes or woody debris within our study site. Water velocity is
thought to be one of the main environmental filters for larval fishes (Webb 1975; Turner
et al. 1994; Scheidegger and Bain 1995), restricting individuals from occupying areas with
high velocities. A majority of our specimens were collected along the margins of stream
or near a bedrock outcropping, generally less than 2 m from any given stream bank or
outcrop. Other studies also found greater larval fish densities closer to the margins of
streams, where larvae abundance was up to seven times greater near the margins than in
the main channel (Raborn et al. 2001). This may be a result of larval fish avoiding high
water velocities that occurred at high frequency away from stream margins in our riffle-
pool sequence. In our study, greater distances from shore were generally associated with
runs or riffles with high water velocities where larval fish could not persist (Webb 1975).
Larval fish tended to occupy shallower microhabitats than were available in our study. In
streams and rivers, larval fish are often associated with moderate to shallow depths
(Scheidegger and Bain 1995; Pusey et al. 2002). Interestingly, we did occasionally collect
high numbers of larval fish in deep (>1 m), non-flowing pools as well as deep areas
between two runs or riffles where the water swirled or formed pools. The riffle-pool
sequence site in our study was made up of a series of small pools, riffles, runs divided by
large bedrock outcroppings. These habitats provide high densities of shallow, low velocity
microhabitats with greater marginal habitat area, suggesting riffle-pool sequence habitats
may be well suited as nurseries for Percidae, Catostomidae and Leuciscidae metalarvae.
However, further study is needed to determine the use of riffle-pool sequence as a nursery
habitat by larval fish compared to other sections of a river. These riffle-pool sequences
may also shelter larval fish from predators. Predation is known to influence larval fish
microhabitat use (Schlosser 1987; Harvey 1991a; Harvey 1991b). Our study did not
account for the presence of predators that could potentially alter larval fish habitat use.
The distance-based pRDA indicated that microhabitat variables influenced larval fish
assemblage structure in our study site. Water velocity was the most important microhabi-
tat variable affecting the assemblage structure of larval fish families in our study. Water
velocity can strongly influence larval fishes’ ability to persist in a given habitat. For
example, Percidae larvae were reported as being vulnerable to transport downstream at
592 L. M. BOWER AND B. K. PEOPLES

water velocities greater than 3 cm/sec (Houde 1969). Larvae mortality may result from
increased stress, abrasion, predation on disoriented larvae, and inability to feed in higher
flow habitats (Raborn et al. 2001). We also observed Leuciscidae larvae associated with
lower water velocities. Scheidegger and Bain (1995) reported similar velocity preferences
for leuciscid and catostomid larvae (mean ¼ 2 to 2.6 cm/sec and 1.6 to 3.2, respectively).
Catostomidae larvae tended to occupy microhabitats with higher water velocity than pre-
viously reported by Scheidegger and Bain (1995). However, a previous study showed an
average prolonged swimming speed of 10.6 cm/sec for 16 mm catostomid larvae
(Moxostoma robustum), which is much greater than the mean velocity found for
Catostomidae in our study (Ruetz and Jennings 2000).
Many species of larval fishes are reported to select areas of moderate to shallow depths
(Scheidegger and Bain 1995; Raborn et al. 2001; Pusey et al. 2002). We found that
Leuciscidae and Catostomidae tended to prefer shallower microhabitats, similar to the
findings of Scheidegger and Bain (1995). However, these trends were not significant.
Scheidegger and Bain (1995) observed leuciscid larvae occupying deeper microhabitats
than found in our study. This difference in depth preference may be due to our study
occurring within riffle-pool sequences where only a few deep, low water velocity habitats
occur. We observed Percidae larvae occupying deeper microhabitats relative to
Leuciscidae or Catostomidae. Additionally, Percidae larvae tended to be found in deeper
areas compared to the other two families. Similar results were found for Percidae larvae
in the Tallapoosa and Cahaba Rivers, where larvae tended to select deeper microhabitats
than other families (Scheidegger and Bain 1995).
It is important to acknowledge several limitations of this study. We collected larval
fishes only within riffle-pool sequences at a single stream reach, preventing conclusions
about habitat use and temporal patterns of larval count across different streams or habitat
types within a stream. Habitat preferences of larval fish can vary across sites (Scheidegger
and Bain 1995; Pusey et al. 2002), yet little is known about how much habitat use varies
among streams for different taxa or regions, particularly for the species in our study.
Additional studies are needed to explore the relative role of intraspecific variation in habi-
tat preference across and within sites. Ontogenetic shifts in microhabitat use are also
common among freshwater fishes (Pusey et al. 2002), adding to the need for additional
studies on microhabitat use. Additionally, our samples were only collected during the day,
potentially missing important diel patterns. The diversity and number of larvae captured
have been shown to increase at night as drift occurs more often after dark (Reeves and
Galat 2010). Our study highlights the need for future studies to explore intraspecific vari-
ation in habitat use across streams, habitat types within a stream, and regions to deter-
mine the intraspecific variability in habitat use. Our study started in late Spring, missing
the full spawning season of many species in Twelvemile Creek. For example, our results
suggest that peak spawning of Catostomidae likely occurred earlier in the season.
Additionally, sampling for a single season may have missed potential temporal variation
in spawning times and habitat use. To fully capture the temporal patterns of larval fish
abundance and habitat use, a study would need to be conducted across the entire spawn-
ing season of all fishes in the creek and across multiple years.
The use of higher taxonomic levels restricts our ability to draw specific conclusions
about larval fish ecology, preventing collections of more precise data needed for robust
conservation and management efforts. This coarse taxonomic resolution likely masks pat-
terns of interspecific variation in spawning phenology and larval habitat use. The lack of
comprehensive keys for larval stream fish prevents the identification of specimens to the
species level and restricts research on early life stages of stream fishes. The management
 JOURNAL OF FRESHWATER ECOLOGY 593

and conservation of stream fishes would greatly benefit from the creation of comprehen-
sive larval fish keys. Until then, species-level information on larval stream fishes can only
be attained through molecular approaches such as DNA metabarcoding (Buckwalter et al.
2019); these tools have proven to be highly effective, but can be costly, especially for
small-scale natural history studies. Furthermore, we did not separate specimens into
development stage due to limited numbers of protolarvae for some families, likely missing
potential habitat use differences by among larval development stages. For example, proto-
larvae of many species may occupy interstitial spaces of coarse substrate before developing
to swim to favorable habitats, while other species drift as ichthyoplankton for days or
weeks before gaining the ability to select for favorable habitats (Snyder 1990; Hoagstrom
and Turner 2015). Accordingly, the results presented in this study cannot be applied
across all larval fish stages.
Our results provide insights on the microhabitat preference for three families of larval
fish and the temporal changes in their counts within a riffle-pool sequence of a southeast-
ern USA Piedmont stream. Evidence from this and other studies show that larval fish
occupy microhabitats with specific environmental characteristics (Scheidegger and Bain
1995; Childs et al. 1998; Falke et al. 2010), such as shallow habitats with low water vel-
ocity close to stream margins, and microhabitat use of larval fish can differ among taxa
(Scheidegger and Bain 1995; Childs et al. 1998). The high proportion of marginal micro-
habitats with low water velocity and shallow depths in riffle-pool sequences suggests that
these habitats may function as nursery areas for Percidae, Catostomidae, and Leuciscidae
larvae. However, additional studies are needed to determine the suitability of riffle-pool
sequences as nursery habitats relative to other habitats within a river. Anthropogenic
alterations of the flow regime, such as dams, can increase discharge and hydropeaking,
resulting in reductions of suitable nursery habitat and reduced larval fish density
(Scheidegger and Bain 1995; Erickson et al. 2021). These impacts are likely greater when
fish larvae are small and their swimming ability is poor (Ruetz and Jennings 2000). For
example, flooding events were found to be fatal to larval fish under 10 mm (Harvey 1987).
However, future studies are needed to determine the impact of hydrologic alteration on
larval fish habitat and assemblage structure. The statistical association between microhabi-
tat variables and larval fish counts in ours and previous studies indicate active orientation
of the larvae to suitable microhabitats rather than pure random drift of larvae
(Scheidegger and Bain 1995; Falke et al. 2010), particularly riffle-current species such as
those in Percidae and Leuciscidae (Floyd et al. 1984). Our study suggests conservation
and management efforts can benefit from focusing on low water velocity habitat close to
stream margins.

Acknowledgements
The authors thank Andrew Peel and Colby Denison for field work assistance, Ty’Celia Young, Daniel St.
Amand, Anna Pereda, Charles Jackson, and Brianna Taylor for help identification and measurement of
specimens. This work represents technical contribution number 7100 of the Clemson experiment station.
Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorse-
ment by the U.S. Government. This study was performed under the auspices of Clemson University
protocol # 2020-006.

Disclosure statement
No potential conflict of interest was reported by the authors.
594 L. M. BOWER AND B. K. PEOPLES

Funding
This work was supported by the South Carolina Water Resources Center, the Creative Inquiry
Undergraduate Research Program at Clemson University, and by the NIFA/USDA, under project number
SC-1700599.

Notes on contributors
Luke M. Bower is a freshwater ecologist broadly interested in community assembly and the drivers of bio-
diversity spatial patterns. His research focuses on understanding how changes in environmental gradients
influence assemblage and functional trait structure of aquatic systems across multiple scales, and applying
community, evolutionary, and functional ecology concepts to inform the management and conservation
of freshwater fishes. Another research focus is to understand the roles of stream flow in driving the ecol-
ogy and evolution of fishes across landscape gradients to better inform state flow standards.

Brandon K. Peoples’ research is broadly focused on cross-scale approaches to freshwater ecology and con-
servation. His research ranges from applied issues in fisheries ecology and conservation to using fishes as
a model system for examining general models of ecology. He uses observational field studies, experiments,
and modeling of large datasets to investigate key questions regarding reproductive ecology, invasive spe-
cies, population characteristics, and movement.

Data availability statement

The data that support the findings of this study will be openly available as a supplemental document once
paper is accepted.

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