4.1. Patterns of Variation of Physico-Chemical Factors
The overall physico-chemical characteristics of epikarst drip water are that of water in contact with carbonate rock, with resulting high levels of conductivity, a predominance of Ca2+
cations, slightly basic pH, and temperatures close to the mean annual air temperature (Table 2
The differences among samples were caused by a combination of location (Figure 2
), seasonality (Table 4
), and water retention time. We did not measure residence time of water in this study, but previous studies in Postojnska jama [32
], found that residence time of water varied from 2.5 months to over a year. Similarly Kluge et al. [33
] found retention times of one to three years in three German caves. Three caves—Dimnice, Postojnska jama, and Škocjanske jame—have relatively thick ceilings [20
], so that, all things being equal, residence time of water may be greater in these caves. If residence time is correlated with presence (and abundance) of copepods, then differences in residence time will result in differences among the caves. The underlying geology of the caves is nearly identical, with all caves formed in Jurassic limestones, although there may be some dolomite in Županova jama [34
There are factors that vary on the scale of the projected area of a cave onto the surface that influence the physico-chemical variables that we measured. For example, sites in Pivka jama are high in NO3−
]. We suspect, but cannot demonstrate, that this is the result of the presence of a campground and associated structures [35
]. Other potentially anthropogenically causes include high Cl−
in Dimnice [20
], which is also correlated with NO3−
), and may indicate water from wells with high concentrations of both [36
]. There is no commercial activity and minimal agricultural activity in the area, but elevated levels of Cl−
nonetheless suggest anthropogenic inputs because of the relative rarity of naturally occurring Cl−
. High Cl−
concentrations may be from animal waste, human wastewater, or other sources such as salt licks.
Given variation in residence time of water underground, it is not surprising that there is almost no seasonality, but high temporal variability with respect to physico-chemical variables. One manifestation of the variability of residence time is the response to the flow rate of drips to rainfall events, according to Kogovšek [32
]. After a dry period or drought, the flow rate of drips does not increase after a rainfall event, but once the epikarst layer is saturated with water, the increase of flow can be in a matter of hours. Of course, the water exiting the drip is not the precipitation water itself, but some of the water previously stored. That is, rainfall has a piston effect on the water in epikarst [37
Temperature showed a rather different pattern than the chemical variables (Figure 3
). The differences in temperature among caves are likely the result of details of the differences in sampling times (Table 1
), climate differences (especially with respect to Škocjanske jame, which is in region of more Mediterranean climate than the others), and vertical distance from the surface. Except for Postojnska jama, for which there were no winter samples (Table 1
), all caves showed a seasonal pattern of reduced temperatures from January to April (Figure 3
, upper panel). In some cases, this difference was small. In Županova jama, the monthly least squares means (see Figure 3
), varied less than 1 °C, but the coldest months were between January and March. While these differences were not statistically significant, the seasonal pattern in Črna jama, Dimnice, Pivka jama, and Škocjanske jame was statistically significant, and the temperature differences were greater. Kogovšek [32
] also found a seasonal pattern in temperature for two drips in Postojnska jama, although the range was less than 1 °C. The relationship between temperature and residence time is a complex one (see [39
]) for a more detailed discussion, but the temperature of resident water eventually approaches the mean annual temperature [39
]. Temperature also reflects season, as seen in this study. The lower temperatures in winter are indicative of other events, such as changes in evapo-transpiration and precipitation.
4.2. The Epikarst Copepod Physico-Chemical Niche
A useful beginning point of the analysis is to see which individual physico-chemical factors can account for presence or absence of epikarst copepods in a sample. If the effect of cave is included as a covariate, only temperature and conductivity were significant predictors of the presence or absence of copepods in a sample. However, when a non-parametric multivariate approach, using variables shown to be important in a random forest analysis (Figure 4
) in a classification tree (Figure 5
, Table 6
), was employed, the most important correlates of copepod presence were:
Conductivity itself was unimportant in the classification tree, presumably because some its major components, especially Ca2+
, were exposed by the random forest analysis, which teased apart correlated variables (see Table 3
). Correlation analysis indicated that conductivity was not pairwise correlated with any of the cations (Table 3
). The results of the classification tree argue that the relationship of conductivity with the other variables is more complicated than simple pairwise relationships. In fact, a regression tree (not shown) using conductivity as the response variable and the other five variables as predictors indicated that conductivity is explained in order by Ca2+
, and Mg2+
which is the same order that these variables enter the tree for predicting presence of copepods.
Copepods tend to be in samples with lower temperatures (Table 6
), and so the connection between temperature and copepod presence is likely a seasonal one. It is very likely that the relationship between occurrence probability and temperature is driven by copepods getting washed out of epikarst in greater numbers in winter because flow rates are greater. We did not measure flow rates but Kogovšek [32
] continuously monitored discharge from two epikarst drips in Postojnska jama for a period of more than two years, and demonstrated that discharge rates were highest in the winter. Rouch [42
] observed a similar pattern of copepod drift from a karst spring.
Of the other variables shown to be important in the classification tree, Ca2+
has a strong connection with the biology of copepods. It is critical in the molting process, and some subterranean crustacean species, like the amphipod Gammarus minus,
are limited to carbonate springs [43
]. Additionally, of interest is that copepods tend not to be found in water with temperature greater than 8.2 °C and Ca2+
concentrations greater than 57.5 mg/L. Water in epikarst can be supersaturated with respect to Ca2+
(part of the mechanism of deposition of CaCO3
in caves (e.g., stalactites)) and this may cause physiological problems for animals in this water. While carbonate geochemists have long focused on the Ca2+
system, we suggest it deserves more attention from biologists working in the same systems. Mg2+
is also a critical nutrient [44
], and it is possible that it is limiting in some contexts.
concentration may or may not be biologically significant, it seems likely that the correlation of epikarst copepod abundance and Cl−
is due to some other unmeasured variable, one that varies at the scale of cave. Cl−
concentrations in Dimnice are twice as high (5.54 ± 1.12 mg/L) as in any other cave [26
]. We suspect that it is not Cl−
that is important but some other unmeasured factor.
The correlation with NO3−
is perhaps also the result of some other unmeasured variable, but nitrate is also biologically important. It is a frequent contaminant of karst aquifers, resulting from agricultural runoff, septic tanks, and perhaps atmospheric deposition [45
]. There are few studies of the nitrogen cycle in caves or epikarst, but available evidence suggests that it is not a limiting nutrient [15
]. However, there are still a number of puzzling aspects of the nitrogen cycle (see, [45
]), such as whether nitrogen fixation occurs in caves. If not, Barton [47
] points out it is likely to be limiting in some circumstances.
If we take classification trees as the most general approach to the understanding of the connections of copepod occurrence to physico-chemical parameters, then we have the following factors, listed in order of importance:
Temperature, which is likely a reflection of flow velocities rather than community structure.
Calcium and perhaps magnesium ions, which are important, both as essential nutrients and in molting.
Anthropogenically augmented ions—Cl− and perhaps NO3−—may indicate contamination from upgradient well water, or they may be surrogates for particular epikarst sites, where some unmeasured variable is important.
The multi-faceted statistical approach, combined with an emphasis on the overall community rather than individual species, has made some sense of the complex patterns of variation of physico-chemical variables.
4.3. The Relationship between Community Physico-Chemical Niche and Individual Physico-Chemical Niches
] and Pipan et al. [27
] analyzed the same data from a different perspective, one that emphasized niche separation among the epikarst copepod species. Using the same variables with the addition of ceiling thickness, they found that the following parameters were significant factors in distinguishing individual species in a Canonical Correspondence Analysis (CCA): ceiling thickness; NO3−
; and Na+
. Of these, only NO3−
was important on a community-wide basis. Thus, the parameters by which the species are separated are, for the most part, distinct from the parameters that predict the presence or absence of one or more species (see Figure 5
An example of individual niche analysis for NO3−
is shown in Figure 6
. This is reflected in the presence of some species, especially Brycocamptus dacicus, Bryocamptus
n.sp., Moraria varica,
and Maraenobiotus brucei
, only in high NO3−
concentrations and only in Pivka jama (Figure 6
). Whatever the source, it points to nitrate as an important factor in organizing communities (see [44
]). What is also apparent in Figure 6
is the difficulty in separating or even characterizing the physico-chemical niches of the different species. Of the 27 species found in drips, there were data on more than 100 individuals for only three species, and only an additional four species had more than 10 individuals for which nitrate data were available.
The data we used were not originally collected for the purpose of elucidating physico-chemical niches, and this is the case for many ecological and bioinventory studies. In many of these studies data collected on basic water chemistry remains unconnected and often unanalyzed with respect to the organisms being studied. The results of our study suggest that a careful field study exploring the impact of variation in the physical and chemical characteristics of water on the likelihood of copepods being present may yield additional insights into the forces that control aquatic community structure and dynamics. These impacts could be implicit such as when variables act as surrogates for other factors (such as temperature) or explicit, i.e., with direct effects (such as is likely the case with Ca2+
). There are other constraints on the epikarst copepod community, especially the lack of light and low levels of organic carbon, that are a formidable barrier for not just copepods, but any species to survive in epikarst. Thus, the physico-chemical constraints suggested by the classification tree (Figure 5
), are not absolute, but constraints in the context of no light and little organic carbon, among the extreme conditions that characterize epikarst in general.