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2006 SOUTHEASTERN NATURALIST 5(1):17–26
Relationship Between Physiochemical Factors and
Distribution of Stygobitic Crayfishes in
GENEVIEVE R. SPANJER1,2,* AND MARTIN L. CIPOLLINI1
Abstract - This study was conducted to determine the relationship between water
chemistry and presence of stygobitic (cave-obligate) crayfishes (Cambaridae) in
Tennessee, Alabama, and Georgia. We analyzed nine chemical factors in water
samples from twenty caves, twelve of which contained stygobitic crayfish and eight
in which none were found. A multiple analysis of variance using principal components
scores suggested that absence of crayfish was associated with lower dissolved
oxygen, higher ammonia, and higher water temperature. Caves with externally originating
streams supported no stygobitic crayfishes, and the chemical factors of the
water in these caves were more variable.
Crayfishes (freshwater members of the infraorder Astacidea) are found in
various lentic and lotic aquatic systems worldwide and are considered moderately
sensitive to pollution or alterations in water chemistry (Pennak 1989).
The southeastern United States is home to the US’s greatest crayfish diversity
(Taylor et al. 1996), including about 30 stygobitic species and subspecies
from three genera (Cambarus, Procambarus, and Orconectes) occurring in
Tennessee, Alabama, and Georgia (TAG) (Hobbs 2000, Hobbs et al. 2003).
Two stygobitic crayfish species are reported from the counties we surveyed.
Cambarus hamulatus Cope is reported from Jackson County, AL, and Marion
and Franklin Counties, TN, and Orconectes australis australis Rhoades is
reported from Jackson County, AL, and Franklin and Van Buren Counties,
TN. No stygobitic crayfish have been reported from Dade or Catoosa Counties,
GA (J. Buhay, Provo, UT, pers. comm.; Hobbs et al. 2003).
Unlike epigean (aboveground) environments, caves completely lack
light and may have relatively constant air and water temperatures (except
during flooding). Like some epigean environments, cave ecosystems depend
largely on allochthonous food sources. The scarcity of food in caves may
have been a selective pressure on populations in caves and resulted in
specialized adaptations to this environment (Hobbs 1992).
The distribution of stygobitic organisms may reflect island biogeography
(MacArthur and Wilson 1967) because caves function as isolated “islands.”
Extensive migration between such islands may be difficult (Hobbs 2000).
Therefore, species may evolve within a single cave or cave system.
However, Culver et al. (1973) found that aquatic species are less isolated
1Department of Biology, Berry College, PO Box 430, Mount Berry, GA. 30149.
2Current address - Department of Biology, University of Maryland, College Park,
MD 20742. *Corresponding author - firstname.lastname@example.org.
18 Southeastern Naturalist Vol. 5, No. 1
than terrestrial species because they may migrate between caves via hydrological
connections. Culver et al. (2000) reported that overall endemism was
high, as 61% of known cave-obligate (aquatic or terrestrial) species and
subspecies in the 48 continental states of the US and 44% of aquatic caveobligate
species and subspecies were endemic to a single county.
Stygobitic crayfishes feed on allochthonous organic debris (Hobbs 1974),
predate on cave isopods and amphipods, and exhibit adaptations as a consequence
of life in the dark. These adaptations include lack of pigment, reduced
or absent eyes and eye function, a slender body form, and attenuated appendages,
the last of which compensate for lack of visual senses (Hobbs 2000,
Hobbs et al. 1977). Loss of pigment and eyes that are functional in complete
darkness may be an evolutionary result of selection for efficiency (Hobbs et al.
1977). Stygobites tend to exhibit “K-selected” characteristics (late maturity,
small population size, low reproductive rates, extended lifespans; Hobbs et al.
1977), potentially making them more susceptible to disturbance than epigean
species. While the lifespan of epigean crayfish is about two to three years, some
stygobitic individuals may live for several decades (Taylor et al. 1996).
Most stygobitic organisms are highly sensitive to changes in water chemistry
and susceptible to contaminants in surface and ground water (Culver et
al. 2000). Mathews et al. (1977) found that mortality of the stygobitic
crayfish Orconectes australis australis increased with increasing chlorine
levels (with acclimated crayfish showing higher tolerance), but overall tolerance
of stygobites to various water chemical variables remains little known.
We designed this study to determine if water temperature, water chemistry,
or stream source relate to presence or absence of stygobitic crayfish while
further documenting their distribution in TAG. We predicted that caves with
an influent stream source (originating outside the cave) would be less likely
to support stygobitic crayfish than those with an effluent stream source
(originating inside the cave) because surface streams have a greater likelihood
of variability since their water chemistry and temperature are affected
by surface conditions (Hobbs 1992).
Materials and Methods
Cave selection and determination of presence/absence of crayfish
From 20 June to 30 September 2001, we sampled water one time each from
20 caves in six counties: Jackson County, AL; Franklin, Marion, and Van
Buren Counties, TN; and Catoosa and Dade Counties, GA (Fig. 1). Some caves
were pre-selected based on prior observations of presence or absence of
stygobitic crayfish. Crayfishes were not identified to the species level. Identification
in the field is very difficult and collection of specimens was not feasible
considering the protected status of some species. While identification of
crayfish species could be useful information, our purpose was to examine
factors potentially relating to distribution of stygobitic crayfish as a group,
rather than any particular species. In each cave, at least two individuals
searched thoroughly for stygobitic crayfish, looking in riffle and pool sections,
2006 G.R. Spanjer and M.L. Cipollini 19
under rocks and ledges, in stream sections with a variety of substrate types, and
in puddles separated from the flowing stream. In smaller caves, the entire
accessible length of the stream was searched. In larger caves, portions of the
stream approximately equal to the size of smaller caves were explored. Epigean
crayfish were also recorded when noticed.
Water collection and analysis of abiotic factors
In each cave, we collected one water sample from an undisturbed pool
section of the stream deep enough to submerge a 500-mL amber Nalgene
bottle. The bottle was capped while underwater to exclude all air. Samples
were taken from areas containing stygobitic crayfish, from sections resembling
crayfish habitat in other caves (pool section, non-bare rock bottom,
within dark zone), or, when possible, from sections containing epigean
crayfish or other aquatic organisms.
Water and air temperatures were taken onsite using a mercury thermometer.
Dissolved oxygen (DO) was tested onsite using a LaMotte Winkler Kit
(azide modification of Winkler Method; LaMotte 2004). Samples were
refrigerated until analysis for the remainder of the chemical parameters,
which occurred within the time frame appropriate for each factor (Hach
1989). We used a Hach EC10 pH meter to determine pH, and a Hach CO 150
conductivity meter to measure total dissolved solids (TDS) and conductivity.
We used Hach manual standard methods 8038, 8204, 8203, and 8171
Figure 1. Location of caves sampled for the presence of stygobitic crayfish. See
Table 1 for cave names and additional information.
20 Southeastern Naturalist Vol. 5, No. 1
(Hach 1989) and a Hach DREL/2000 spectrophotometer to measure ammonia
nitrogen, calcium hardness, alkalinity, and nitrate as nitrogen, respectively.
We measured DO saturation using a standard nomograph (Horne and
Goldman 1994) and calculated un-ionized ammonia from total nitrogen
ammonia using a standard table (Thurston et al. 1979).
We included the variables mentioned above for each cave in a principal
components analysis (PCA). This analysis allowed for the description of
overall relationships between water quality parameters and the presence or
absence of stygobitic crayfish. Principle components were extracted as linear
combinations of the raw data for the above parameters with maximum variance
rotation using the statistical package SPSS for Windows, version 12.0
(SPSS Inc., Chicago, IL). Factor loadings were calculated by correlating the
original variables with each factor extracted. Factor loadings indicate the
strength of association of each water quality parameter with each factor.
To test statistically for differences in water quality between caves
containing and lacking stygobitic crayfish, we used the PCA scores as
dependent variables in a multiple analysis of variance (MANOVA), using
presence of stygobitic crayfish as the independent variable (factor). We
used PCA components instead of raw data because many of the variables
tested were chemically related and thus correlated statistically. The use of
principle components avoids problems of collinearity among the numerous
independent variables. The MANOVA technique thus tested for overall
statistical differences between the two sets of caves.
We observed at least one stygobitic crayfish in 12 caves. In nine of these,
epigean crayfish were also observed. In three caves, only epigean (no
stygobitic) crayfish were observed (Table 1). No stygobitic crayfishes were
found in the four caves with an influent stream source.
Water chemistry overview
For every variable except nitrate (N-NO3), ranges of values were greater for
caves without stygobitic crayfish than for caves with these crayfish present
(Table 2). Dissolved oxygen was at or near saturation in all caves. Total and unionized
ammonia levels were greater for caves without stygobitic crayfish than
for those with stygobitic crayfish. A single cave without stygobitic crayfish
(Howard’s Waterfall Cave) had levels of several chemicals that differed greatly
from the remainder of sites. However, for each physiochemical parameter, the
mean value for caves without stygobites did not change significantly when this
cave was excluded (two-tailed t-tests; P ≥ 0.3 for each).
Water chemistry PCA analysis
About 80% of the variation in abiotic factors among the caves was
accounted for by the first three principal components of the PCA (Table 3).
2006 G.R. Spanjer and M.L. Cipollini 21
The first component segregated caves predominantly along a gradient from
values representing high pH, ammonia, hardness, alkalinity, TDS, and conductivity
to low values for those parameters. The second component was
positively related to temperature and ammonia, and negatively related to
DO. The third component was positively related to nitrate nitrogen values
and negatively related to pH. None of the other extracted components were
deemed statistically significant, since their eigenvalues were < 1.0 (they
explained less of the overall variation than did the raw variables).
Table 1. Name, location, stream source, and presence or absence of stygobitic and epigean
crayfish in each cave during this study. # = number on map (Fig. 1)
crayfish crayfish Stream
# Cave County, state observed? observed? source
1 Buckets of Blood Franklin, TN No Yes Effluent
2 Walker Spring Franklin, TN Yes Yes Effluent
3 Wet Franklin, TN Yes * Effluent
4 Bible Spring Marion, TN Yes No Effluent
5 Catacomb Marion, TN No * Influent
6 Gourdneck Marion, TN Yes Yes Effluent
7 Owen Spring Marion, TN Yes * Effluent
8 Shakerag Marion, TN Yes Yes Effluent
9 Whiteside Marion, TN Yes Yes Effluent
10 Howard’s Waterfall Dade, GA No No Influent
11 Cueva Guapa del Norte Van Buren, TN Yes Yes Effluent
12 Jess Elliot Jackson, AL Yes Yes Effluent
13 Tate Jackson, AL Yes Yes Effluent
14 Bluff River Jackson, AL Yes Yes Effluent
15 Limrock Blowing Jackson, AL Yes Yes Effluent
16 Isbell Spring Jackson, AL No * Effluent
17 “Keener, Wright” Marion, TN No No Effluent
18 Sitton’s Dade, GA No Yes Effluent
19 Lost Creek Catoosa, GA No * Influent
20 Upper Tumbling Rock Jackson, AL No Yes Influent
*Caves not exhaustively searched for epigean crayfish.
Table 2. Descriptive statistics for physiochemical variables of water from caves with and
without stygobitic crayfish.
Stygobites present Stygobites absent
Parameter Mean ± SD Min.–Max. Mean ± SD Min.–Max.
Air temperature (oC) 15.7 ± 1.4 14.0–18.5 17.6 ± 2.6 14.5–23.0
Water temperature (oC) 14.0 ± 0.8 12.0–15.0 16.3 ± 3.1 14.0–21.0
DO (mg/L) 9.6 ± 0.4 9.0–10.2 9.0 ± 0.8 8.0–9.9
pH 7.5 ± 0.2 7.3–7.9 7.3 ± 0.3 6.8–7.8
NO3 (mg/L x 10-1 N) 3.4 ± 1.8 2.0–9.0 3.6 ± 2.1 2.0–8.0
NH3-N (mg/L x 10-2 N) (total) 1.4 ± 0.9 0.0–4.0 18 ± 28.0 1.3–81.0
NH3 (mg/L x 10-4 N) (un-ionized) 1.3 ± 1.0 0.0–4.0 18 ± 26.0 0.3–67.0
Hardness (mg/L x 102 CaCO3) 1.2 ± 0.4 0.6–1.6 1.0 ± 0.7 0.2–2.4
Alkalinity (mg/L x 102 CaCO3) 1.1 ± 0.4 0.5–1.5 0.8 ± 0.6 0.2–2.1
TDS (mg/L x 102) 1.1 ± 0.3 0.7–1.6 1.0 ± 0.6 0.2–2.0
Conductivity (μS x 102) 2.4 ± 0.6 1.5–3.2 2.0 ± 1.2 0.4–4.2
22 Southeastern Naturalist Vol. 5, No. 1
A 3-dimensional PCA graph (Fig. 2) depicting scores for each cave for
the first three components showed caves with stygobitic crayfish clustered
together, while those without them were more scattered. Moreover, segregation
between caves with and without stygobitic crayfish can be seen along
the second PCA axis (see MANOVA results below).
Multiple analysis of variance
The MANOVA analysis using components extracted from the PCA gave
a Wilks’ Lambda value of 0.561 (F3,16 = 4.175; P = 0.023) for the overall
effect (differences between caves with and without stygobitic crayfish).
Tests of between-subjects effects indicated that the second PCA component
was significantly related to presence/absence of stygobitic crayfish
(Table 4). Based upon the results of the PCA factor loadings for this component
(Table 3), lower values for temperature, higher values for DO, and
lower values for total and un-ionized ammonia were associated with cave
streams containing stygobitic crayfish.
Stygobitic crayfish survival may be limited to a relatively narrow range
of acceptable values with regard to water chemistry and temperature. In
our study, we sampled water from each cave only once, and the range of
physiochemical values found in water from the group of caves not supporting
stygobitic crayfish was greater than the range of values recorded from
caves known to be inhabited by stygobitic crayfish (Table 2, Fig. 2).
Additionally, we did not find stygobitic crayfish in any cave with an
influent stream (Table 1), and water chemistry in this stream type is known
to be more variable than in effluent streams because water chemistry is
directly affected by surface conditions (Hobbs 1992).
Table 3. Component matrix for PCA with presence of stygobites as the independent variable.
Parameter 1 2 3
Water temperature -0.20 0.81 0.37
Air temperature 0.05 0.89 -0.12
DO 0.18 -0.87 -0.08
pH 0.50 -0.23 -0.61
NO3 0.24 -0.04 0.77
NH3-N (total) 0.68 0.50 -0.11
NH3 (un-ionized) 0.64 0.54 -0.20
Hardness 0.93 -0.12 0.12
Alkalinity 0.93 -0.10 0.14
TDS 0.93 -0.10 -0.06
Conductivity 0.94 -0.11 -0.05
Eigenvalue 4.74 2.85 1.19
% variance 43.1 25.9 10.8
Cumulative % variance 43.1 70.0 79.8
Note: Factors with values ≥ 0.50 or ≤ -0.50 are in boldprint.
2006 G.R. Spanjer and M.L. Cipollini 23
We found stygobitic crayfish only in caves with water ≤ 15 °C. We believe
that temperature, which is important for growth and survival of many crayfish
species, is one of the most important factors contributing to this distribution
pattern (Table 3, Table 4). Taylor (1984) found that three species of epigean
crayfish preferred temperatures ranging from about 20 to 26 °C in a lab setting,
while Hobbs (1974) reported that a species found both inside and outside caves
lived only in water ≤ 20 °C. Optimum growth ranges and lethal limits regarding
temperature vary by species, but 10 °C is the lowest temperature at which many
epigean species can grow (Biggs 1980, Brewis and Bowler 1983, Pratten
1980). While an increase in temperature has been positively correlated with
crayfish growth (Aiken and Waddy 1992), crayfishes also have an upper
temperature limit, and stygobitic crayfishes may be adapted to groundwater
(lower) temperatures because they have evolved in subterranean environments.
H. Hobbs III (pers. comm., Springfield, OH) reports that he kept stygobitic
crayfish at “room temperature” for years without apparent harm. These
individuals did not reproduce, but other factors may have been involved,
Table 4. MANOVA tests of between-subjects effects, indicating the relationship of each factor
to the overall statistical distinction between caves with and without stygobitic crayfish. Factors
with P ≤ 0.05 were significantly related to this difference.
Source Factor Type III SS df MS F P
Intercept PCA1 0.01 1 0.01 0.01 0.944
PCA2 0.32 1 0.32 0.53 0.478
PCA3 0.01 1 0.01 0.01 0.933
Stygobite PCA1 0.13 1 0.13 0.13 0.728
(presence/ PCA2 8.02 1 8.02 13.15 0.002
absence) PCA3 0.19 1 0.19 0.18 0.675
Error PCA1 18.87 18 1.05
PCA2 10.98 18 0.61
PCA3 18.81 18 1.05
Figure 2. Plot of PCA
scores for caves with
and without stygobitic
crayfish. PCA scores
were based on the
Table 2. For interpretation
of PCA factor
loadings, refer to
24 Southeastern Naturalist Vol. 5, No. 1
making the true link between temperature and stygobite distribution difficult
to discern. In our study, caves with an external stream source had a higher
mean water temperature and a wider range in water temperature than did caves
with an internal stream source. It is possible that the amount of variability in
water temperature may be more important than the mean water temperature
when it comes to predicting the presence of stygobitic crayfish.
Because of caves’ low biological and chemical oxygen demand and cool
water temperatures, DO is usually not a limiting factor for crayfish survival
(Hobbs 1992), and DO levels in all caves tested were well above the threshold
of 1.0–1.5 mg/L reported by Biggs (1980). Un-ionized ammonia (NH3) may
harm crayfishes. Levels of 0.09 mg/L NH3 can reduce growth, and 5.71 mg/L
can kill some crustacean species (Chin and Chen 1987, Colt and Armstrong
1979). The lowest NH3 level considered hazardous to most aquatic organisms is
0.02 mg/L (Department of Agricultural and Biosystems Engineering 1996),
and the range of the values in this study was substantially lower (0–0.0067
mg/L). Total ammonia can also be harmful, and Lee et al. (1985) reported that
concentrations of 0.5 to 1.0 mg/L total ammonia nitrogen (not just NH3) can
cause histological damage in Procambarus clarkii Girard. Our highest recorded
total ammonia value (in Howard’s Waterfall Cave, no stygobites
present) was 0.81 mg/L, but high ammonia levels usually are not problematic
when DO levels are sufficiently high (H. Hobbs III, Springfield, OH, pers.
comm.), as they were in all study sites. Because stygobitic crayfishes are
relatively understudied, their tolerance to various chemicals is not well known.
It is possible that ammonia affects distribution among stygobitic crayfishes, but
most likely only if levels exceed the values we recorded in this study.
Calcium, which is related to both alkalinity and hardness, is an important
parameter for crayfishes, particularly during molting, because it is a major
component of the exoskeleton. Our results show a correlation between
higher alkalinity and hardness values and stygobitic crayfish presence
(Table 3). In limestone caves, the calcium ion is very common and usually
sufficient for crayfishes (McGregor et al. 1997), and we have no data
indicating calcium was harmfully low in any of the sampled caves.
Pollution seemed a likely factor for crayfish survival at Howard’s Waterfall
Cave, and a potential factor in Upper Tumbling Rock Cave (neither site
contained stygobitic crayfish). In the former, which is frequently visited by
humans and near a highway, garbage was found around the cave and directly in
the stream inside the cave. Additionally, even the undisturbed stream water was
cloudy in appearance (unlike other caves sampled), and we saw no organisms in
this stream despite its near-saturation DO and low temperature (14.5 °C). This
stream had a total ammonia level more than three times greater than the next
highest value and > 25 times higher than most values. Its alkalinity, hardness,
conductivity, and TDS levels also deviated greatly from that of other caves
sampled. In Upper Tumbling Rock Cave, much garbage was dumped uphill of
the cave and its stream, though less was found within the cave. However, the
presence of epigean organisms here, in combination with the occurrence of an
external stream source, suggest that the latter, rather than pollution, was
responsible for the absence of stygobitic crayfish.
2006 G.R. Spanjer and M.L. Cipollini 25
Based upon frequency of precipitation, water level and chemical properties
fluctuate seasonally (McGregor et al. 1997). Therefore, multiple water samples
from different times of year should have been collected. Further, while this
study examined factors potentially related to presence of stygobitic crayfish as
a group, identification to the species level is recommended in future studies, as
tolerance to various chemical parameters may vary by species.
For more insight into the ideal environment for stygobitic crayfish, testing
of both biotic and additional abiotic parameters, such as turbidity and concentrations
of heavy metals, should be conducted. Excess sediment can clog
crayfish gills (Holdich 2003), and high levels of certain heavy metals, which
may be present as a result of human influence, are harmful to crayfish
(McGregor et al. 1997). Two additional factors potentially affecting stygobite
distribution include predation from epigean fish or crayfish, particularly in
caves with influent streams, and competition from epigean relatives. Also, a
study including only caves with an effluent stream source would eliminate the
factor of stream source evidently influencing water chemistry. Finally, lab or
field experiments could address whether some caves with seemingly ideal
conditions for stygobitic crayfish contain none because of chance alone.
We thank W.T. Davin for use of his equipment and general information and
assistance regarding water chemistry and aquatic biology. R.F. Spanjer provided
cave locations/information, photographed some of the specimens, and assisted with
fieldwork; M.J. Wright assisted with fieldwork, provided resources necessary to
create the map, provided cave locations/information, and initially suggested this type
of project. J. Buhay, K. Crandall, H.H. Hobbs III, and D. Withers provided guidance
and help obtaining pertinent literature for the project. D.B. Conn, M.B. Fenton, R.
Fiorillo, H.H. Hobbs III, P.L. Klerks, T. Luszcz, M. Papazian, H.M. ter Hofstede, and
four anonymous reviewers provided helpful comments on earlier versions of the
manuscript. J. Graham and J. Orprecio provided assistance with statistical analysis,
and A.B. Spanjer assisted with fieldwork.
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