2007 SOUTHEASTERN NATURALIST 6(4):693–704
Observations on the Reproductive Biology of Two
Notropis Shiner Species
Bruce Stallsmith1,*, Kevin Butler1,2, Amy Woodall1,2, and Bob Muller3
Abstract - Important details of the reproductive ecology of many freshwater fishes
of the species-rich southeastern USA are still poorly known. Two such species are
Notropis asperifrons (Burrhead Shiner), and Notropis stilbius (Silverstripe Shiner).
Both are endemic to the Mobile Basin, AL. To determine timing and patterns of
reproductive effort, collections were made of as many as 20 individuals of each
species at roughly four-week intervals from March through September. Female
gonadal somatic index (GSI) data averages for each collection indicate that both
species peak in reproductive activity in April. Both species still had reproductively
competent female GSI values as late as July 31. Examination of ovarian tissue indicates
that Burrhead Shiner oocytes are usually larger at equivalent developmental
stages. Burrhead Shiners exhibit sexual size dimorphism with larger females, while
Silverstripe Shiners do not.
Important details of the reproductive ecology of many freshwater fishes
of the species-rich southeastern USA are still poorly known. Many of these
species are under growing pressure from anthropogenic activities such as
urban expansion, agricultural uses, and waterway modification (Butler 2002,
Powell 2003, Scott 2006). Proper management and protection of existing
aquatic ecosystems requires the fullest possible knowledge of the life history
of resident species.
Two such species are the cyprinids Notropis asperifrons Suttkus and
Raney (Burrhead Shiner) and N. stilbius Jordan (Silverstripe Shiner). Both
are endemic to the Mobile Basin of Alabama, Mississippi, and Georgia, and
are typically found in the riffles and pools of upland streams. Both species
are often locally abundant, but little is known of the details of their reproduction.
Boschung and Mayden (2004) state that the Silverstripe Shiner spawns
from mid-April to mid-June. Burrhead Shiner, based on observations of
tuberculate males, spawns from April to June. No information exists on
whether these two generally similar species, often found in the same creeks,
have different reproductive strategies such as producing fewer, larger eggs,
or temporal spawning differences. A species’ schedule of reproduction
(sensu Heins and Rabito 1986) is a key aspect of its life history and ecological
relationships. Females in the genus Notropis are known to be multiple
1Department of Biological Sciences, University of Alabama in Huntsville, Huntsville,
AL 35899. 2Current address - Open Biosystems, Huntsville, AL 35806. 3Royal
Oak Nature Society, Royal Oak, MI 48067. *Corresponding author - fundulus@
694 Southeastern Naturalist Vol. 6, No. 4
spawners, spawning more eggs in a season than are found in the ovary at any
one time (Dahle 2001, Heins and Clemmer 1976).
The objective of this study is to assess seasonal variation in the reproductive
competence of gonadal tissues for sympatric populations of Burrhead
Shiners and Silverstripe Shiners.
Materials and Methods
Collection site and field collection
Burrhead Shiners and Silverstripe Shiners were collected monthly from
March 2004 to October 2004, and March 2005. Two collections were made
in July, early and late, because the planned collection in June was cancelled
due to heavy rains. A collection was made in March 2005 because few fish
were collected in March 2004 due to high water. The collection site was at
the Borden Creek Trailhead (34º18.569'N, 87°23.670'W; elevation 200 m)
inside the Sipsey Wilderness Unit of the Bankhead National Forest in Lawrence
County, AL. Borden Creek is a second-order tributary to Sipsey Fork
of the Warrior River.
Twenty individuals of each species were taken in each collection using
seine nets (3.0-m L x 1.3-m D; 3.0-mm mesh). No effort was made to collect
individuals of specific sex or size. Collected fishes were euthanized
in a solution of MS-222 (tricaine methanesulfonate) and later transferred
to 5% phosphate buffered formalin for transport and storage prior to histological
Water temperature (ºC), total dissolved solids (TDS, parts per million),
and pH were recorded on each visit to Borden Creek. Temperature was measured
by placing an alcohol thermometer on the streambed in a shady area
of steady flow. TDS was measured using a Hanna Instruments TDS 1 meter.
Water pH was measured using a Hanna Instruments pHep 2 meter.
Length and mass data collection
Standard length (0.01 mm) was recorded for each fish with digital
calipers. Individuals were then weighed (0.01 g) after the removal of
excess surface fluid by wrapping in a paper towel. Gonads (if present)
and the digestive tracts were removed, and gonad mass was recorded to
the nearest 0.01 g. The gonadal somatic index (GSI) was calculated as:
(gonad mass/somatic mass) x 100. A Mettler H18 balance was used for
the collection of all mass data.
Sexual size dimorphism was evaluated by comparing mean standard
length between all collected males and females for each species. Equality of
variances was evaluated with a F-test and mean difference between samples
was assessed with a 2-tailed t-test (Sokal and Rohlf 1973).
Ovarian tissue from up to four females from each monthly collection,
March through late July, were prepared histologically. Samples for
2007 B. Stallsmith, K. Butler, A. Woodall, and B. Muller 695
histological examination were fixed in Bouin’s fixative for 12 hours, then
transferred through a series of ethanol dehydrations. Following dehydration,
the samples were cleared in toluene and xylene. Samples were then
embedded in paraffin by soaking in a paraffin bath overnight and allowed
to cool. The embedded samples were faced off and then soaked in a solution
of Downy® fabric softener and water (≈200 ml deionized H2O : ≈2
drops of Downy®) for 15 minutes before sectioning. The samples were
sectioned at 4 μm, mounted on glass microscope slides, and stained with
Gill-Modified Hematoxylin and Eosin Y.
Examination and measurement of oocytes and ova
To visually determine the status of oocyte development, stained slides
of ovarian tissue were examined with a Wolfe Digivu TM CVM digital
compound microscope using the software package Motic Images 2000 version
1.2. Four random digital images at 40X magnification were captured
for each fish from among slides of an individual’s fixed tissue (Maddock
and Burton 1999). Each developing ooctye fully visible in each of these
images was characterized to stage following the methods of Rinchard and
Kestemont (1996) and Maddock and Burton (1999). These four stages are:
perinucleolar (visible nucleus but no vacuoles in the cytoplasm), cortical
alveolar (appearance of yolk vesicles forming rings in the cytoplasm periphery),
early exogenous vitellogenesis (oocyte full of yolk vesicles and
the differentiation of cellular and follicular layers), and the nearly mature
stage of late exogenous vitellogenesis (accumulation of yolk globules at
the periphery of the cytoplasm). Mature oocytes were characterized by the
appearance of the micropyle and the migration of the germinal vesicle to
The area (A) of each fully visible oocyte in a captured image was calculated
in square micrometers by tracing the outer edge of the on-screen
image using the computer mouse. Diameter of each oocyte was then calculated
as: Diameter = 2(√A/π).
Fully mature ova released from the ovarian lumen by the visceral dissection
of randomly selected females of both species from April, May, and July
2004 were also examined with the above microscope and software combination
and measured for diameter.
Aquarium spawning of Burrhead Shiner
A group of four Burrhead Shiners, sex ratio unknown, was kept in an allglass
37-L aquarium. To simulate natural conditions, the aquarium was kept
at about 17 ºC starting in January 2004, and allowed to rise to about 22 ºC by
May 2004. The aquarium was kept on an elongated rack to allow the bottom
of the aquarium to be viewed from underneath. The bottom of the aquarium
was covered by a piece of plastic egg-crate light diffuser from a fluorescent
light fixture and covered with netting. Stones (15–30 mm) were placed on
696 Southeastern Naturalist Vol. 6, No. 4
top of the diffuser to serve as spawning medium. This allowed the tank to
be examined from the underside to see if any eggs had been deposited in the
gravel and fallen through the egg crate diffuser. The bottom of the aquarium
was examined for the presence of eggs on a near daily basis. A pipette was
used to remove any eggs from the aquarium. Six eggs had their diameter
measured microscopically, and all were removed to another aquarium for
observation to determine incubation time.
Water quality of Borden Creek
Water temperature varied from a low of 12 ºC in March to a high of 23 ºC
in late July. Total dissolved solids ranged from 76 ppm in March during a period
of high water to 133 ppm in September during low flow. The pH varied
from 8.2 to 8.5.
Oocyte development and size
Measurements of the different stages of oocyte development for
both species are summarized in Table 1. Mature oocytes at the stage
of late exogenous vitellogenesis were found in females of both species
from April through late July, as well as oocytes at all three of the earlier
stages of development. In March, females of both species carried
developing oocytes, but none were beyond the stage of early exogenous
The monthly proportions of oocyte developmental stages for Burrhead
Shiner and the Silverstripe Shiner are shown in Fig. 1. In both species,
over 80% of March oocytes were in early stages of development, more
than 50% of May oocytes were in the advanced maturation stages of
vitellogenesis, and roughly 20% of the late July oocytes were in vitellogenesis.
The major difference between the species was that Burrhead
Shiner ooctyes were more mature in April with over 50% in vitellogenesis
compared to roughly 25% for Silverstripe Shiner. Gonadal tissues from
females collected in August was sufficient to weigh, but too small for
successful histological preparation.
The examination of fully mature but unhydrated ova found during dissection
of Silverstripe Shiners from May and early July yielded a mean diameter
of 1.4 mm, with a range of 1.2–1.6 mm. Fully mature ova of Burrhead Shiner
from the same time periods had a mean diameter of 1.8 mm, with a range of
Female GSI and length
Reproductively active female Burrhead Shiners were present in Borden
Creek from April through late July, as indicated by GSI values and the presence
of oocytes in the advanced developmental stage of late exogenous
vitellogenesis (Fig. 2, Table 1). Peak reproductive condition was in April
2007 B. Stallsmith, K. Butler, A. Woodall, and B. Muller 697
with a mean GSI of 16.3. At least some females showed evidence of being
reproductively active in late July, with a mean GSI value of 5.6 and the
continued presence of oocytes in the developmental stage of late exogenous
Table 1. Oocyte size at different developmental stages during the spawning season. Mean diameter
in μm is reported monthly for each developmental stage for each species.
diameter Standard Range (fish/
Oocyte stage Month Species (μm) error (μm) oocytes)
March Burrhead Shiner 69.3 0.81 39–99 1/289
Silverstripe Shiner 68.3 1.25 37–99 1/121
April Burrhead Shiner 66.2 1.26 33–105 3/161
Silverstripe Shiner 70.1 1.04 30–97 2/173
May Burrhead Shiner 65.1 1.11 34–129 3/185
Silverstripe Shiner 75.2 1.57 42–131 3/119
Early July Burrhead Shiner 64.3 1.59 34–94 1/77
Silverstripe Shiner 68.0 0.99 33–100 2/231
Late July Burrhead Shiner 61.7 1.26 32–91 1/133
Silverstripe Shiner 62.6 0.72 32–90 2/388
March Burrhead Shiner 144.7 2.04 90–237 1/247
Silverstripe Shiner 123.3 1.67 90–183 1/142
April Burrhead Shiner 142.5 2.80 90–204 3/85
Silverstripe Shiner 153.9 2.80 88–240 2/149
May Burrhead Shiner 138.0 4.21 86–213 3/58
Silverstripe Shiner 121.2 1.96 92–219 3/83
Early July Burrhead Shiner 167.5 8.02 121–221 1/17
Silverstripe Shiner 147.8 3.45 89–227 2/102
Late July Burrhead Shiner 120.9 4.02 90–211 1/43
Silverstripe Shiner 119.5 1.71 82–201 2/201
Early exogenous vitellogenesis
March Burrhead Shiner 256.3 5.66 159–341 1/44
Silverstripe Shiner 224.5 5.28 137–317 1/62
April Burrhead Shiner 265.8 4.24 153–410 3/180
Silverstripe Shiner 285.8 5.08 206–382 2/59
May Burrhead Shiner 275.5 4.47 133–439 3/188
Silverstripe Shiner 252.9 3.70 154–355 3/142
Early July Burrhead Shiner 322.2 14.74 216–450 1/24
Silverstripe Shiner 284.2 7.76 198–401 2/51
Late July Burrhead Shiner 270.0 11.94 181–390 1/24
Silverstripe Shiner 251.9 5.54 124–348 2/78
Late exogenous vitellogenesis
March Burrhead Shiner none n/a n/a n/a
Silverstripe Shiner none n/a n/a n/a
April Burrhead Shiner 454.4 7.88 298–779 3/116
Silverstripe Shiner 466.5 8.84 340–636 2/60
May Burrhead Shiner 537.9 10.54 340–762 3/109
Silverstripe Shiner 423.3 7.98 305–564 3/83
Early July Burrhead Shiner 566.1 16.60 316–813 1/46
Silverstripe Shiner 459.8 6.41 283–596 2/97
Late July Burrhead Shiner 522.7 17.41 366–677 1/24
Silverstripe Shiner 436.9 7.50 249–565 2/67
698 Southeastern Naturalist Vol. 6, No. 4
vitellogenesis. By August, GSI values were lower than in March. Sexually
mature females varied in size between 37.8–59.3 mm.
Female Silverstripe Shiners show evidence of reproductive activity from
April to late July, with a May peak mean GSI of 12.2 and the presence of
oocytes in the developmental stage of late exogenous vitellogenesis (Fig.2,
Table 1). Some individuals seemed to be reproductively active into late July
with a mean GSI of 4.8. As with Burrhead Shiners, the mean August GSI
value was lower than that for March. Sexually mature females varied in size
from 41.7 to 70.1 mm. Individuals of both species collected in September
had such reduced gonads that we were unable to determine gonadal mass to
report GSI values for them.
Male GSI and length
Male mean GSI values for both species started relatively high in March
at 0.8 (Fig. 3). Males of both species had variably high mean GSI values
from April through early July, with a sharp drop in late July and very low
Figure 1. Mean proportion of maturing oocyte stages observed in Notropis asperifrons
(Burrhead Shiner) and N. stilbius (Silverstripe Shiner) over a breeding season.
Different oocyte developmental stages are represented in the graphs as follows: PN
= perinucleolar, CA = cortical alveolar, EEV = early exogenous vitellogenesis, LEV=
late exogenous vitellogenesis.
2007 B. Stallsmith, K. Butler, A. Woodall, and B. Muller 699
mean GSI values in August. Reproductively active male Burrhead Shiners
ranged in size between 28.1–49.7 mm, and male Silverstripe Shiners ranged
between 38.2–64.5 mm. As with females, males collected in September had
such reduced gonadal tissue that it was not possible to determine gonadal
mass for the reporting of GSI values.
Sexual size dimorphism
For each species, average standard length of all collected males was
compared to average standard length of all collected females. In both species,
females were longer than males. For Burrhead Shiners, mean standard
length of males was 40.8 mm (n = 29, standard error = 0.96) and mean
standard length of females was 46.2 mm (n = 42, standard error = 0.81).
For Silverstripe Shiners, mean standard length of males was 55.2 mm (n
= 70, standard error = 0.76) and mean standard length of females was 56.7
mm (n = 63, standard error = 0.82). A two-tailed t-test comparing standard
length of males to females for each species showed a significant difference
in Burrhead Shiners (p < 0.001) but not in Silverstripe Shiners (p = 0.19).
Figure 2. Mean monthly GSI values for female Notropis asperifrons (Burrhead
Shiner) and N. stilbius (Silverstripe Shiner) from Borden Creek, Bankhead National
Forest, Lawrence County, AL. Error bar indicates one standard error. Sample size is
indicated by a number above each bar.
700 Southeastern Naturalist Vol. 6, No. 4
Aquarium observations of spawning by Burrhead Shiners
All four adults in the spawning group were observed to spend much of
their time buried in the gravel on the bottom of the aquarium. No color differences
were discernible between the individuals, so the sex ratio was not
determined. Individuals were not observed in the act of spawning. Freshly
spawned eggs were clear, 2 mm in diameter, and non-adhesive. Eggs removed
to another aquarium hatched in 82 hours at 22 ºC. Fry were observed
to remain in the bottom 50 mm of the aquarium. At 60 days, the fry were
about 15 mm long.
Both Burrhead Shiners and Silverstripe Shiners show evidence of being
reproductively active from April into July based on GSI values for both
sexes and microscopic examination of ovarian tissue. Reproductive competence
into July is later than what has been reported for these two species.
Other Notropis species from both warmer and colder climates have been
observed to have a spawning season of three or more months. Notropis
Figure 3. Mean monthly GSI values for male Notropis asperifrons (Burrhead Shiner)
and N. stilbius (Silverstripe Shiner) from Borden Creek, Bankhead National Forest,
Lawrence County, AL. Error bar indicates one standard error. Sample size is indicated
by a number above each bar.
2007 B. Stallsmith, K. Butler, A. Woodall, and B. Muller 701
longirostris Hay (Longnose Shiner) from warmer south Mississippi were
in breeding condition from late March until October as indicated by GSI
of females and males, and male tuberculation and coloration (Heins and
Clemmer 1976). In colder Minnesota, Notropis topeka Gilbert (Topeka
Shiner) individuals were observed to be in reproductive condition from
mid-May to early August based on gonadal development, GSI, and field
observations (Dahle 2001). These differences in breeding-season length
seem to be correlated with variation in local climates, especially the onset
of milder weather in the spring.
The fact that at least some individuals of both species have elevated
GSI and well-developed gonadal tissues including maturing oocytes
of all stages in July is also consistent with multiple spawnings over a
reproductive season. This asynchronous ovarian development is a trait
observed in many other cyprinids (Heins and Rabito 1986, Roberts and
Grossman 2001). Many other North American cyprinids show evidence
of producing 6 or more clutches in a continuous spawning season, including
Longnose Shiner (Heins and Clemmer 1976), Cyprinella leedsi
Fowler (Bannerfin Shiner; Heins and Rabito 1986), Rhinichthys cataractae
Valenciennes (Longnose Dace; Roberts and Grossman 2001) and
Topeka Shiner (Dahle 2001).
The overall pattern of the timing of reproduction as indicated by mean
GSI values is similar between the two species, but there appears to be
differences of timing and investment in the quality and quantity of eggs
between the species within the shared spawning season. Silverstripe Shiner
is the larger of the two species, but produces smaller mature ova on average
(1.4 mm) than Burrhead Shiner (mean of 1.8 mm). Our data on mean
oocyte size at different development stages reveal two tendencies. First,
Silverstripe Shiners produce on average slightly larger perinucleolar
oocytes, the first stage in maturation. Secondly, Burrhead Shiner produces
larger oocytes at later developmental stages, except in the peak GSI month
of April. The mean size of Burrhead Shiner late-exogenous vitellogenesis
oocytes increases by ≈100 μm for the three months after April, while those
of Silverstripe Shiner slightly decrease in size. Figure 1 show that Burrhead
Shiner ovaries contain a higher fraction of oocytes in advanced stages
of development than those of Silverstripe Shiner. Our interpretation is that
Burrhead Shiner females produce larger eggs more evenly over the reproductive
season than Silverstripe Shiner females, which show evidence of
peak egg production in May.
Previous studies have found no inverse relationship between clutch
size and egg size in North American cypriniformes (Winemiller and
Rose 1992). These authors describe Notropis species as showing an
opportunistic reproductive strategy as small fish with seasonal spawning,
moderately large clutches, small eggs, and relatively few spawning bouts
per year. This description fits both Burrhead Shiner and Silverstripe Shin702
Southeastern Naturalist Vol. 6, No. 4
er. Data presented in this paper illustrate potential differences between
the two species’ reproductive biology that may reflect trade-offs with
other life-history parameters. During seining to collect these fish in Borden
Creek, we collected many more Silverstripe Shiners than Burrhead
Shiners, often by a ratio of ten to one. The Silverstripe Shiner was by far
the most abundant species at the research site. One possible explanation
for this difference in abundance may be that Silverstripe Shiner females
produce a larger number of smaller eggs than Burrhead Shiner females,
and this larger number of larvae can better use available microhabitat(s).
In addition, we observed habitat partitioning between adult Burrhead
Shiners and Silverstripe Shiners in the creek. While snorkeling in
Borden Creek, we saw Burrhead Shiners in small groups near the substrate,
hiding under rocks or burrowing in sand, while large schools of
Silverstripe Shiners occupied the upper water column. In our aquarium,
we provided few options for spawning habitat and did not observe any
spawning activity of Burrhad Shiner. However, spawning in Burrhead
Shiner is likely more closely associated with the substrate rather than the
water’s surface given that we noted a greater propensity for adults to be
buried in the aquarium substrate, and that spawned eggs were retrieved
from the bottom of the aquarium below the substrate. There are no published
spawning observations of Silverstripe Shiners, but based on the
lack of sexual size dimorphism, it is likely that the reproductive mode is
egg scattering during group spawning as has been observed with another
member of the subgenus Notropis, N. atherinoides Rafinesque (Emerald
Shiner) (summarized in Boschung and Mayden 2004). From our GSI data,
neither Burrhead Shiners nor Silverstripe Shiners have testes that are unusually
enlarged, an expectation if a species’ reproductive mode involves
sperm competition (Pyron 2000). This is consistent with observations of
other North American minnows (Pyron 2000). Differences between reproductive
mode in the observed two species seem to be differences between
female reproductive effort. Other cyprinid minnows present in Borden
Creek have more elaborate spawning strategies, such as crevice spawning
by Cyprinella species (Heins 1990, Rabito and Heins 1985) and the
construction and guarding of stone nests by dominant male Campostoma
oligolepis Hubbs and Greene (Largescale Stoneroller).
Neither the Burrhead Shiner nor Silverstripe Shiner is in immediate danger
of extinction. The key to management for species such as these two with
an opportunistic reproductive strategy is to protect their habitat from largescale
or chronic disturbances that would eliminate all refugia (Winemiller
and Rose 1992). A better understanding of what aspects of these species’
life-history strategies have made them tolerant to human disturbance to date
can help our efforts to understand why other, often closely related, species
are in decline.
2007 B. Stallsmith, K. Butler, A. Woodall, and B. Muller 703
We would like to thank Ruth Fledermaus, Bill Garstka, Dewey Mason, Amy
Bishop, and members of NANFA for help with the field collection and laboratory
preparation necessary to this project. Jim Daniels and two anonymous reviewers
offered suggestions that much improved this manuscript. Fishes were collected
under Special Use Permit BAN700114 from the US Department of Agriculture,
US Forest Service.
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