Invertebrate biodiversity in cold groundwater fissures in Iceland

Abstract Iceland has an abundance of fissures that are parallel to the Mid‐Atlantic Ridge where bedrock cracks as a result of continental rifting. Some fissures penetrate the aquifer and expose the groundwater within the bedrock, becoming springs. As such, groundwater fissures have uniform and constant physical and chemical environment but they can differ greatly in morphology. In addition, there is often great variation in depth within fissures and substrate types contrast between vertical rock wall and more heterogenous horizontal bottom. The variation in morphological environment may create dissimilar habitats with unique characteristics and/or influence distribution of resources. Our objective was to study macrozoobenthos communities in cold groundwater fissures in Iceland in relation to physical habitat by comparing invertebrate diversity and density both between fissures with different morphological characteristics as well as between substrate types and depths within fissures. Samples were collected in two fissures in SW Iceland, Silfra and Flosagjá. Assemblages were similar between fissures except for higher densities of cladocerans in Flosagjá fissure. Within fissures, there was significant difference in Shannon diversity between substrate types in Flosagjá, and ostracods were found in significantly higher densities on the bottom. The distribution of all other taxa groups was homogenous in both fissures regardless of depth gradient and substrate. Invertebrates were found to be living within and around a biofilm that covered the entire substrate. These biofilm mats are made from Cyanobacteria and benthic diatoms, which are successful under low light conditions and may minimize any effect of the heterogeneous habitat creating a uniform and suitable microhabitat for invertebrates regardless of depth and substrate type.

water fissures have uniform and constant physical and chemical environment but they can differ greatly in morphology. In addition, there is often great variation in depth within fissures and substrate types contrast between vertical rock wall and more heterogenous horizontal bottom. The variation in morphological environment may create dissimilar habitats with unique characteristics and/or influence distribution of resources. Our objective was to study macrozoobenthos communities in cold groundwater fissures in Iceland in relation to physical habitat by comparing invertebrate diversity and density both between fissures with different morphological characteristics as well as between substrate types and depths within fissures. Samples were collected in two fissures in SW Iceland, Silfra and Flosagjá. Assemblages were similar between fissures except for higher densities of cladocerans in Flosagjá fissure. Within fissures, there was significant difference in Shannon diversity between substrate types in Flosagjá, and ostracods were found in significantly higher densities on the bottom. The distribution of all other taxa groups was homogenous in both fissures regardless of depth gradient and substrate. Invertebrates were found to be living within and around a biofilm that covered the entire substrate. These biofilm mats are made from Cyanobacteria and benthic diatoms, which are successful under low light conditions and may minimize any effect of the heterogeneous habitat creating a uniform and suitable microhabitat for invertebrates regardless of depth and substrate type.

K E Y W O R D S
biofilm, macrozoobenthos, species assemblages, spring, substrate

| INTRODUC TI ON
In freshwater systems, invertebrates merit special consideration as secondary producers are important for many ecological mechanisms (Richardson & Jackson, 2002). Too often however, invertebrates are overlooked and their role in ecosystem functioning is not considered until sudden ecological changes have occurred (Covich, Palmer, & Crowl, 1999). In freshwater systems in Iceland, invertebrate diversity tends to be greatest in spring-fed lakes and rivers compared to other freshwater systems (Gíslason, Ólafsson, & Aðalsteinsson, 1998;Malmquist et al., 2003). Biodiversity and density are generally high in springs due to environmental stability and complex habitat structure, which offers abundant and variable microhabitats for invertebrates (Cantonati, Gerecke, & Bertuzzi, 2006;Glazier, 1991). As a result, springs often contain a larger number of rare, endemic, and endangered species than other aquatic habitats (Cantonati, Füreder, Gerecke, Jüttner, & Cox, 2012).
Despite this, the study of invertebrates in these systems is insufficient and springs remain the least studied of aquatic ecosystems (Ferrington, 1998). Springs are also typically the least protected which leaves them more vulnerable to anthropogenic impact (Cantonati et al., 2012).
Most of the species found in springs are macrozoobenthos, and in cold groundwater systems in Iceland, chironomid larvae are the dominant taxon (Govoni, Kristjánsson, & Ólafsson, 2018). Springs are most common within the neovolcanic rock formations, originating from a rifting tectonic plate boundary and mantle plume, and these geological processes have caused the formation of numerous fissures (Jóhannesson & Saemundsson, 2009;Marshak, 2008).
Some of Iceland's rift fissures penetrate the aquifer and provide an opening into groundwater and thus act as spring habitats. Most of the fissures have cold and clear water (Ólafsson, 1992) and resemble freshwater systems in the High Arctic (Rautio, Bayly, Gibson, & Nyman, 2008). Since the fissures are often narrow and can reach up to 60 m in depth (Chowdury, 1998), most of the substrate is vertical rock wall while horizontal substrate is a smaller strip at the bottom ( Figure 1). The bottom of fissures is often covered with rubble that has fallen from the walls. In some areas, larger pieces of rock form caverns with dim light conditions and/or caves where light does not penetrate. The fissures have been thoroughly studied in a geological and geophysical aspect (Guðmundsson, 1986), but the ecosystems of water-filled fissures in Iceland are poorly known.
Our objective was to assess the biodiversity in cold groundwater fissures in Iceland in relation to their morphological habitat. Fissures in the same location typically have similar physical and chemical properties, and many environmental factors are uniform and constant as is characteristic of spring systems (Ólafsson, 1992;Van der Kamp, 1995). However fissures can differ greatly in morphology such as degree of cavern formation, uniformity of the bottom, and connectivity to other systems (some fissures are landlocked while others connect to water bodies). This variation may shape the diversity and density of invertebrates found within them.
Habitat heterogeneity has been shown to influence invertebrate community structure in springs (Ferrington, 1998). Within fissures, there is often great variation in depth and substrate types contrast between vertical rock wall and more heterogenous horizontal bottom. Light is absorbed and scattered in water which influences the distance it can travel (Jonasz & Fournier, 2007). Therefore, depth could influence light availability to varying degrees which in turn would affect primary production and available resources for benthic heterotrophs. Also, substrate types within fissures may create distinct niches and influence the distribution of organisms. Many studies on freshwater ecosystems have found that substrate is a principal environmental variable shaping macroinvertebrate assemblages (Alexander & Allan, 1984;Arunachalam, Nair, Vijverberg, Kortmulder, & Suriyanaraynan, 1991;Cobb, Galloway, & Flannagan, 1992 F I G U R E 1 A map of Lake Þingvallavatn in SV-Iceland and the two groundwater fissures studied, Flosagjá (a) and Silfra (b). The approximate location of the two fissures is defined by a black rectangle. The location and number of transects within fissures are specified with black lines 1980) although few of these studies have focused on continuous rock substrate. The different types of substrate found in fissure may influence the distribution of organisms and the availability of resources. For example, detritus is likely to accumulate readily on the bottom while little detritus would settle on rock walls. Since detritus is an important food source for most collectors (Hall & Meyer, 1998;Petersen, Gíslason, & Vought, 1995;Vannote, Minshall, Cummins, Sedell, & Cushing, 1998) its distribution can create distinct niches and may reflect the microhabitat of collectors (Covich et al., 1999).
As habitat diversity is known to influence biodiversity (Covich et al., 1999), we investigated whether the morphological habitat in fissures shapes assemblages of macroinvertebrates. We compared the density and diversity of invertebrate assemblages between fissures with different morphological characteristics. Furthermore, we investigated the distribution of invertebrates within fissures by comparing density and diversity between substrate types (rock wall and bottom) and at variable depths. Here we provide a detailed first look at macroinvertebrate communities within cold groundwater fissures in Iceland. In the laboratory, invertebrates were counted and identified to various taxonomic groups. The four most common invertebrate groups (chironomids, cladocerans, copepods, and ostracods) were identified to the lowest taxonomic level possible. Only a subsample of copepods was identified to species level, and therefore, the subclass "Copepod" was treated as a taxonomic group in statistical analysis. Detailed species identification followed taxonomic keys by Cranston (1982) and Schmid (1993) for Chironomidae, Alonso () for Cladocera, Alekseev and Defaye (2011) for Copeoda, Meisch (2000) for Ostracoda, Gíslason (1979) for Trichoptera, and Hynes (1955) for The 2 L samples were filtered through a 47 mm glass microfiber filter (GE Healthcare Life Sciences) and stored in 5 ml ethanol in a cooler for 24 hr. Subsequently, the samples were put in a centrifuge (Hettich Rotanta type 3500) at 1,660 g for 5 min and put into a 10 × 10 mm cuvette, and the absorbance was measured in a spectrophotometer (Hach DR 5000) at 665 and 700 nm. This was repeated after acidification with 0.1 N HCL which was used to convert Chla to phaeophytin.  (Oksanen et al., 2013) and Canoco Version 4.5.

| RE SULTS
A total of 32 invertebrate taxa were found during the study (Table 1).
Taxa richness was 24 and 25 in Flosagjá and Silfra, respectively. Of these taxa, 18 were shared between both fissures. The most common species was the chironomid Diamesa zernyi (Figure 4). Small benthic Arctic charr was also observed in all three fissures.
Shannon diversity was 2.1 in Flosagjá fissure and 1.7 in Silfra, and there was no significant difference in Shannon diversity between fissures ( Figure 5). Macroinvertebrate assemblages were similar except for cladocerans, which were found in greater densities in Flosagjá compared to Silfra (W = 87, p = 0.005, Wilcoxon signed-rank test). This difference is mostly attributed to Chydorus sp. which was the most common cladoceran, while rare in Silfra. Alona werestschagini, which was recently documented in Iceland for the first time (Novichkova, Chertoprud, & Gíslason, 2014), was also entirely absent from Silfra while being the second most common cladoceran in Flosagjá. Copepods were treated as one taxonomic group in analysis as only a subsample was identified to the species level. Copepods were found in similar densities in the two fissures (W = 58.5, p = 0.54, Wilcoxon signed-rank test) but species assemblages varied and fewer copepod species were identified in Flosagjá (Table 1). Chironomid and Ostracod assemblages were similar between the two fissures and found in similar densities (Chironomidae: W = 57, p = 0.63; Ostracoda: W = 66, p = 0.24, Wilcoxon signed-rank test). Although Tricoptera and Plecoptera were more common in Silfra, there was no significant difference in densities between the two fissures (Trichoptera: W = 13.5, p = 0.12; Plecoptera: W = 10, p = 0.45). A PCA ordination revealed that taxa composition between the fissures TA B L E 1 List of species of Chironomidae, Cladocera, Copepoda, Ostracoda, Plecoptera, Trichoptera, and other taxa groups collected and identified in two cold groundwater fissures in Iceland (Flosagjá and Silfra) is distinct although assemblages overlap to some degree ( Figure 6).
Crustacean taxa were more common and found in higher densities in Flosagjá fissure while aquatic insect larvae were more abundant in  (Table 2) making both fissures oligotrophic (Carlson & Simpson, 1996). No zooplankton was observed in water samples collected for chla measurements. The conductivity, pH, and total dissolved solids in the fissures were not significantly different. Detailed identification of flora in fissures was not conducted but a subsample of biofilm collected along invertebrate samples from both fissures was examined. The biofilm was mostly composed of Cyanobacteria and benthic diatoms. The green algae Tetraspora cylindrica and Klebsormidium sp. were also observed in Silfra fissure. fissures with different morphological characteristics. A second main aim of this study was to determine whether different substrate types and variable depth in fissures were influencing species diversity and distribution of organisms. Shannon diversity was higher in Flosagjá but no significant difference was found in Shannon diversity between fissures. Assemblages did, however, vary to some degree, and cladocerans were found in higher densities in Flosagjá. The fissure Silfra opens into Lake Þingvallavatn (Malmquist, 2012) and

| D ISCUSS I ON
has a more rapid water current compared to the landlocked Flosagjá, where a slower water current is likely to create a more suitable habitat for smaller crustaceans such as cladocerans. In a study investigating how environmental variables shape invertebrate communities in Icelandic springs, Govoni et al. (2018) found a similar pattern where crustaceans and in particular cladocerans were most common in limnocrene springs. Meanwhile, Diamesa spp. were most abundant in rheocrene springs. Although water current is more pronounced in Silfra compared to Flosagjá, Silfra cannot be considered a true rheocrene spring (Springer & Stevens, 2008). Chironomids including D. zernyi were more common in Silfra but there was no significant difference in chironomid density between the two fissures and all taxa groups besides crustaceans were found in similar densities. The taxa composition in the fissures is comparable to other freshwater habitats in Iceland, with chironomids being the dominant invertebrate group (Gíslason et al., 1998).
One might expect the distribution of taxa within a fissure to be influenced by variation in depth and the different types of substrate (rock wall and bottom), which could create dissimilar habitats with unique characteristics and influence the distribution of resources.
There was a difference in Shannon diversity between substrate types in Flosagjá fissure, and this was attributed primarily to higher densities of ostracods found at the bottom. The distribution of all other taxa groups was relatively even within fissures. The biofilm mats that cover the entire substrate in fissures may help to explain this homogeneity and obscure the effects of the morphological differences.
While water samples had very low concentrations of chla and contained no zooplankton, these wall/benthic mats seem to create favorable habitat for invertebrates and are akin to those commonly found in high latitude freshwater systems. In systems where the water temperature is below 5°C, the primary production of phytoplankton has been shown to be nutrient limited, while benthic cyanobacterial mats on the contrary act as a type of microenvironment that is nutrient sufficient, rich in biomass, and is home to many species (Bonilla, 2005).
Detailed identification of flora in fissures is still lacking but based on subsamples that were analyzed during the project the biofilm mats appear to be mostly consist of Cyanobacteria and benthic diatoms.
Mat-forming diatoms and Cyanobacteria have been documented in cold freshwater systems in the Arctic and Antarctic (Ohtsuka, Kudoh, Imura, & Ohtani, 2006;Rautio & Vincent, 2007), and many studies have identified such biofilm as an important carbon source for invertebrates (Hall & Meyer, 1998;Hansson & Travik, 2003;Rautio & Vincent, 2007). The Cyanobacteria genus Oscillatoria forms algal mats, which are known to grow around artesian springs at the bottom of Lake Þingvallavatn where nitrogen-rich water is discharged. As long as the nitrogen availability is sufficient, Cyanobacteria of this genus can photosynthesize under very low light conditions (Jónsson, Gunnarsson, & Jónasson, 2011). Cyanobacteria seem to cope well within caverns and in narrow fissures where light penetration may be limited. Since such biofilm mats cover the entire substrate in fissures, they may continue to provide suitable habitat and be a food source for invertebrates regardless of substrate type.
During the project, a new fissure was discovered in Þingvellir National park and was named Huldugjá (The Hidden fissure). This fissure is underground and is approximately 80 m long and 2 m wide and reaches a depth of 40 m making it among the deepest accessible fissures in Iceland. Five samples for qualitative analysis were collected in Huldugjá fissure, and two species were found: Copepod Megacyclops viridis, which was also common in the open water fissures, and a groundwater amphipod Crangonyx islandicus that is endemic to Iceland (Svavarsson & Kristjánsson, 2006). C. islandicus was seen moving around and looked healthy, while the M. viridis specimen appeared less healthy and had indications of deterioration of the tissues. This specimen may have been swept into the cave although there is also a slight possibility that sampling equipment was contaminated from earlier sampling procedures. As this is so far the only known fissure in Iceland that is completely underground, it may prove to be an interesting study site for future research on groundwater ecosystems and cave populations.
Benthic invertebrates provide essential ecosystem services by transferring energy through trophic levels and by mixing sediments (Covich et al., 1999). Furthermore, invertebrates play a key role in nutrient cycling along with fungi and bacteria (Vanni, 2002). Scientists are still working on unraveling the complex connections within ecosystems and the role of organisms and biodiversity in their functioning. Meanwhile, there are signs that we are losing many species before we ever get a chance to study them (Covich et al., 1999;Singh, 2002). Far too often, freshwater invertebrate assemblages are overlooked and their importance for ecosystem services is not realized until unforeseen changes occur (Covich et al., 1999). Invertebrates make up most of the fauna in groundwater fissures in Iceland.

| CON CLUS IONS
Fissures that form on a divergent plate boundary can be accessed in very few places in the world as these are ordinarily suboceanic   (Bonilla, 2005;Hall & Meyer, 1998;Hansson & Travik, 2003;Rautio & Vincent, 2007). Therefore, the health of invertebrate communities in fissures is likely to be related to the health of the biofilm mats.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interests.