Research Article |
Corresponding author: Najla Baković ( najla.bakovic@gmail.com ) Academic editor: Rosaura Mayén-Estrada
© 2022 Najla Baković, Renata Matoničkin Kepčija, Ferry J. Siemensma.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Baković N, Matoničkin Kepčija R, Siemensma FJ (2022) Transitional and small aquatic cave habitats diversification based on protist assemblages in the Veternica cave (Medvednica Mt., Croatia). Subterranean Biology 42: 43-60. https://doi.org/10.3897/subtbiol.42.78037
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Protists in caves are scarcely researched. Most cave studies address the diversity of protists, but very little is known about their habitats and spatio-temporal dynamics. The aim of this study was to investigate the diversity and abundance of protists in the Veternica cave in the Medvednica mountain in Croatia on hygropetric and sinter and clay pools during six months. During this study, 47 protists taxa were distinguished belonging to the groups of heterotrophic flagellates, ciliates, heliozoans, stramenopiles and testate and naked amoebae. The highest taxon richness was found at the sinter pool site richest in bats guano (36 taxa). Most diverse were testate amoebae and ciliates. The number of taxa and their abundance differed significantly between the sampling sites. The prospected habitats supported different protist assemblages, proved by ordination analysis. The most distinctive habitat was the hygropetric. Habitat heterogeneity could be attributed to the presence of inorganic and organic sediments at the sites and habitat microhydrology (lotic or lentic system). Kendall’s concordance coefficient showed a good synchronicity between the habitats in the Veternica cave, based on taxon richness and abundance of protists indicating similar seasonal trends. Seasonality in the studied habitats is attributed to the hydro-meteorological conditions in the Veternica cave drainage area. This study is one of the few studies of spatio-temporal diversity and abundance of protists in caves. Despite the similar appearance of small transitional and aquatic habitats in caves, an example of this study showed specific habitat diversification.
Bat’s guano, cave protists, clay pools, habitat heterogeneity, heliozoan, hygropetric, seasonality in cave, sinter pools, spatio-temporal dynamics, testate amoebae
Freshwater karst caves are formed in soluble rock such as limestone or dolomites by dissolution processes induced by rainwater that is slightly acidic by dissolved CO2 from the atmosphere and also biogenically produced within soil layers (
The majority of current cave research was focused on animals that are often highly endemic and exhibit morphological, behavioural and physiological adaptations to their environment (
Nevertheless, many researchers have made contributions to our knowledge of cave protists, even though these are mostly based on on-off sampling. To date, several hundred species of cave protists from all continents have been discovered. In their comprehensive work,
Most protists species found in caves have been previously registered in non-cave habitats, and many of them were allocated as euribiotic species (
The ecology of cave protists has very scarcely been investigated. The main gaps in studies on the subterranean protist ecology is the lack of quantitative methods, as well as precise and thus comparable habitat characterisation (e.g.
This research represents the continuation of the study of protists in the Veternica cave in the Medvednica mountain in Croatia. The impact of hydro-meteorological conditions on the protist assemblages in this cave was discussed by
The aim of this study was to investigate the spatio-temporal taxon richness and abundance of protists present in the Veternica cave. We also compared various habitats in order to test how habitat heterogeneity within this cave affects protist assemblages. This research is, to our knowledge, one of the few studies of spatio-temporal changes in taxon richness and abundance of protist taxa in freshwater karst caves.
The Veternica cave is a complex speleological object located in the southwestern part of the Medvednica mountain (45°50'27.22"N, 15°52'24.75"E) at an altitude of 330 m above sea level (Fig.
The research was conducted from October – December 2011 and February – April 2012 at six cave sites with three habitat types: sinter pools (site SP-1, SP-2), clay pools (site CP-1, CP-2) and hygropetric (site HP-1, HP-2). Sampling at sites SP-1 and CP-1 was not performed in October 2011 due to technical difficulties. All sampling sites were out of reach of sunlight. During the research, pre-hibernation and hibernation colonies of bats (mostly Rhinolophus spp.) were present.
Sinter pools (SP) in this investigation represented small cascade pools on speleothems filled with water. They were created by gradual precipitation and dissolution of calcium carbonate from seeping water. At this specific location, cascades of sinter pools were of very small depth, ranging from a few millimetres to several centimetres. Slowly dripping water from the speleothems above this habitat contributes to the constant presence of water. Samples were taken at Koncertna dvorana, located in the main cave channel, at a distance of 90 m from the cave entrance; the distance between SP-1 and SP-2 is about 1.5 m.
Clay pools (CP) are small pools in cave clay deposits filled with water. They are created by the constant dripping of water from the speleothems onto clay deposits. The depth of these pools varies from a few millimetres to several centimetres. Site CP-1 was located in a small side channel named Separe (about 1.8 m from stations HP-1 and HP-2), the distance to the cave entrance is about 250 m. Site CP-2 was located in a hall named Kalvarija, at a distance of about 380 m from the cave entrance. The distance between site CP-1 and CP-2 was about 100 m.
Hygropetric (HP) is a habitat type characterized by a thin layer of water (~1 mm) constantly seeping onto the surface of speleothems. Location in the cave: a small side channel named Separe, distance to the cave entrance is about 250 m; distance between sites HP-1 and HP-2 is 1.2 m.
Cave air temperature and cave relative air humidity were measured using the Kestrel 3000 instrument.
Plastic containers were filled with 40 ml of tap water. Water and bottom sediment from sinter and clay pools were transferred into the prepared containers using graduated plastic pipettes. Additionally, solid surfaces of sinter pools were rubbed with plastic brushes and the collected sediment was afterwards transported into the plastic containers by repeated “washing” the brush in the collected water in the container. Samples from the cave hygropetric were collected by rubbing its surface with plastic brushes and repeated “washing” the brush in the water in the container.
The collected samples contained water, mineral particles and bats guano (if present in the habitat). Samples were maintained at temperatures of 4–10 °C and prospected within 48 hours after sampling. After decantation, a mixture of sediment and water was taken and a triplet of 0.2 ml (total 0.6 ml) samples were prospected and data on diversity of protist taxa were noted, together with the taxon richness of the accompanying meiofauna. The number of individuals of each protist taxa in prospected volume (total of 0.6 ml) was counted. The abundance of protist taxa in 1 ml was estimated based on the data of the abundance in 0.6 ml of prospected volume. Diversity and abundance were estimated using a Carl Zeiss Primostar light microscope. The presence of guano in the collected sample was estimated by using insects leftovers as indicators (exoskeleton fragments, butterfly wings scales) and their aggregations on the microscopy slide (present in fresh guano deposits rich also in non-chitin material). It was rated according to the following scale: 0 – absent (no traces of insects leftovers), 1 – low quantity (up to ten insects leftovers in the sample, leftovers are not aggregated), 2 – medium quantity (eleven or more insects leftovers present, leftovers are not aggregated), 3 – high quantity (aggregation of insects guano is present regardless of number of insects leftovers).
The samples were scanned for protists using a Nikon Diaphot inverted light microscope and a Carl Zeiss Primostar. Selected cells were examined in detail with the Primostar in higher magnifications or with an Olympus BX51 light microscope with differential interference contrast (DIC) and Phase Contrast optics.
Additional samples were collected in July 2020 to confirm the identification of heliozoans from the sinter pools (SP). Drops of this material were pipetted onto light microscope slides and covered with a slip. These slides were kept in a humidity chamber for several days and observed for heliozoans and naked amoebae. When heliozoans had established their populations after several days, the slip was gently removed and the slide was left to dry. This air-dried material, containing plate and spine-scales of heliozoans, was photographed with FEM-REM Zeiss Neon by Steffen Clauß in Chemnitz (Germany) to facilitate their identification.
Specimens were identified by genus and, if possible, by species level, mainly based on the following references: ciliates:
Data about the taxon richness and abundance between the sampling sites were analysed using descriptive statistics in Statistica software and MS Office Excel.
Differences in the number of taxa and the abundance between the sites were analysed using Friedman’s ANOVA, a nonparametric alternative of one-way repeated measures ANOVA. The analyses were done in Statistica 13 (TIBCO Software Inc.). Similarities between protist assemblages were tested using the Bray-Curtis similarity, which was used for nMDS analysis. The latter analysis was done in PRIMER6 (PRIMER-e Ltd).
The largest variability of the microclimate in the Veternica Cave was recorded in Koncertna dvorana – the air temperature ranged from 4.1 °C (Feb 2012) to 10.5 °C (Oct 2011), while the relative air humidity ranged from 70.7% (Mar 2012) to 91.5% (April 2012). The air temperature in Separe ranged from 8.1 °C (Dec 2011) to 10.7 °C (Oct 2011), while the relative air humidity ranged from 77.7% (Feb 2012) to 81.6% (Nov 2011). The air temperature in Kalvarija ranged from 7.5 °C (Feb 2012) to 11.2 °C (Oct 2011), while the relative air humidity ranged from 82.2% (Oct 2011) to 90.8% (April 2012).
A total of 47 taxa of protists belonging to heterotrophic flagellates, ciliates, heliozoans (Fig.
Taxa | Research sites | |||||
SP-1 | SP-2 | CP-1 | CP-2 | HP-1 | HP-2 | |
Testate amoebae | ||||||
Arcella artocrea Leidy, 1876 | - | + | - | - | - | - |
Arcella rotundata Playfair, 1918 | - | - | - | - | - | + |
Centropyxis aerophila Deflandre, 1929 | + | - | - | - | + | + |
Centropyxis bipilata Baković, Siemensma, Baković, Rubinić, 2019 | - | + | - | - | + | - |
Centropyxis laevigata Penard, 1890 | - | + | - | - | - | - |
Cryptodifflugia oviformis Penard, 1902 | + | + | + | - | - | - |
Cyclopyxis sp. | + | - | - | - | + | + |
Cyphoderia ampulla Ehrenberg, 1840 | - | + | - | + | - | - |
Difflugia oblonga Ehrenberg, 1838 | - | + | - | - | - | - |
Difflugia sp. | - | + | - | - | - | - |
Euglypha laevis (Ehrenberg, 1832) Perty, 1849 | + | + | + | + | + | + |
Euglypha rotunda Wailes, 1915 | - | + | - | - | - | - |
Euglypha sp. | + | - | - | - | - | + |
Euglypha tuberculata Dujardin, 1841 | - | + | - | + | - | - |
Tracheleuglypha dentata Deflandre, 1928 | - | + | - | + | - | - |
Trinema lineare Penard, 1890 | + | + | + | + | + | - |
Heliozoa | ||||||
Acanthocystis myriospina Penard, 1890, emend. Dürrschmidt, 1985* | - | + | - | - | - | - |
Raphidocystis marginata (Siemensma, 1981) Zlatogursky, 2018* | + | + | + | + | - | - |
Ciliophora | ||||||
Ciliophora 1 | - | + | - | - | - | - |
Ciliophora 2 | - | + | - | - | - | - |
Ciliophora 3 | - | - | + | - | - | - |
Cinetochilum margaritaceum Perty, 1849 | + | + | + | + | - | - |
Colpoda sp. | - | - | + | - | - | - |
Colpoda steini Maupas, 1883 | - | + | + | + | - | - |
Cyclidium glaucoma O.F.M., 1786 | + | + | + | - | - | - |
Cyrtophoryda | - | + | - | - | - | - |
Glaucoma sp. | + | + | + | + | - | - |
Hymenostomata 1 | - | + | + | + | - | - |
Hymenostomata 2 | - | - | - | - | + | + |
Litonotus lamella Schewiakoff, 1896 | - | + | - | - | - | - |
Nassulida 1 | + | + | + | + | - | + |
Nassulida 2 | - | - | - | - | - | + |
Pleuronema sp. | + | + | + | + | - | - |
Sphatidium sp. | - | - | + | - | - | - |
Stramenopiles | ||||||
Actinophrys sol Ehrenberg, 1830 | + | + | + | - | - | - |
Heterotrophic flagellates | ||||||
Nanoflagellata (five taxa) | + | + | + | + | + | + |
Peranema trichophorum (Ehrenberg 1838) Stein 1878 | + | + | - | - | - | - |
Naked amoebae | ||||||
Amoebozoa 1 | - | + | - | - | - | - |
Amoebozoa 2 | + | - | - | - | - | - |
Korotnevella sp. | - | + | - | - | - | - |
Mayorella sp. | - | + | - | - | - | - |
Rhizamoeba sp. | + | - | + | - | - | - |
Vahlkampfia sp. | - | + | - | - | - | - |
After six months of research, the sites with the highest taxa number were SP-2 (36 taxa), SP-1 and CP-1 (both 20 taxa). A somewhat lower taxon richness was recorded at site CP-2 (17 taxa).
A, B Raphidocystis marginata A LM micrograph of a living cell B REM micrograph of plate-scales and spine-scales C, D Acanthocystis myriospina C LM micrograph of a living cell D REM micrograph of plate-scales. Scale bars: 20 µm (A, C); 2 µm (B, D); LM, light microscopy; REM, raster electron microscopy.
Occasionally, resting stages (cysts) morphologically typical of amoeboid protists were found at sites SP-1, SP-2 and CP-1. Some of them were present in testate amoebae shells.
Meiofauna was occasionally registered in all habitats: SP (Rotifera, Nematoda, Turbellaria, Copepoda, Oligochaeta, Hydrachnidia, insect larvae), CP (Rotifera, Nematoda, Hydrachnidia, Oligochaeta, insect larvae) and HP (Nematoda, Hydrachnidia, Copepoda).
Taxon richness and abundance of protists were higher in habitats SP and CP, compared to HP (Fig.
Date | Protist taxa abundance (number of individuals in 1 ml) on research sites | |||||
---|---|---|---|---|---|---|
SP-1 | SP-2 | CP-1 | CP-2 | HP-1 | HP-2 | |
October 2011 | n/a | 270.00 | 10.00 | n/a | 8.33 | 3.33 |
November 2011 | 91.67 | 220.00 | 256.67 | 3.33 | 5.00 | 18.33 |
December 2011 | 288.33 | 351.67 | 278.33 | 131.67 | 5.00 | 0 |
February 2012 | 75.00 | 191.67 | 1.67 | 80.00 | 0 | 20.00 |
March 2012 | 13.33 | 246.67 | 30.00 | 41.67 | 6.67 | 8.33 |
April 2012 | 358.33 | 513.33 | 226.67 | 173.33 | 3.33 | 6.67 |
Number of samples | 5 | 6 | 6 | 5 | 6 | 6 |
Minimum | 13.33 | 191.67 | 1.67 | 3.33 | 0 | 0 |
Maximum | 358.33 | 513.33 | 278.33 | 173.33 | 8.33 | 20.00 |
Average | 165.33 | 298.89 | 133.89 | 86.00 | 4.72 | 9.44 |
Median | 91.67 | 258.33 | 128.33 | 80.00 | 5.00 | 7.50 |
The composition of protist assemblages (Table
In habitat HP testate amoebae were the dominant group with 63.6% (HP-1) and 50% (HP-2) of taxa, followed by the flagellates and ciliates.
Non-metric MDS plot, based on Bray-Curtis similarity between protist assemblages, suggested possible differentiation within prospected habitats (Fig.
Guano was absent in the hygropetric, while in other habitats it was present in variable quantities (Fig.
In this research, 47 taxa of protists (Table
Protists found in the Veternica cave are mostly taxa that have been registered by other researchers in non-cave habitats (i.e.
Some taxa, registered in the Veternica cave (Table
It should be noted that the presence or absence of certain species or taxa in the samples cannot always be reliably detected. Testate amoebae are still relatively easy to register and identify from their (usually empty) shells, although here too it is sometimes not always possible without observing living specimens for their pseudopodia (lobose or filose). Naked amoebae are much more difficult to detect, as they can die or encyst fairly quickly in the material being transported. For this group, wet slides kept in moist chambers for some time can reveal several species. The same goes for heliozoans. At present, heliozoans can only be reliably identified by SEM.
As the Veternica cave has a large number of visitors, they could also impact taxon richness. It is experimentally confirmed that visitors have strong impact on dispersion of microorganisms and seeding of species not typically found in caves (
Results of the nMDS analysis showed a specific grouping within the researched sites (Fig.
The differences between individual sites of habitats SP and CP can be explained by the temporal variability of bat guano (Fig.
The modifying effect of guano in the Veternica cave can have several consequences. Bat guano represents a rich food source for protists, thus it increases taxa abundance. This was also reported by some other researchers (e.g.
Except for the mentioned energy input to the habitats, the factors that are contributing to the distinction of the habitats showed by mMDS (Fig.
The differences between habitats can also be attributed to their microhydrology. SP and CP are actually small lentic habitats within caves with the ability to held sediments. Retention of sediments, especially bat guano, significantly increases the energy input to these habitats. As already mentioned, higher abundance and taxon richness of protists in habitats with bat guano and cricket excrements has already been observed in caves (
Good synchronicity between the different habitats in the Veternica cave indicates similar seasonal trends in diversity and abundance of protists. As the Veternica cave microclimate showed relatively small variations and considering the constant absence of light, the cause of similar seasonal trends could be a reflection of the hydro-meteorological conditions in the cave drainage area. The water coming from the surface transports organic matter to the cave ecosystems (
This research presents one of the rare studies of spatio-temporal taxon richness and abundance of protist assemblages in karst caves. Analysis, based on six months research, showed that statistically a significant difference between sinter and clay pools habitats and hygropetric habitats exist. This result is implying that cave habitats, holding very small quantities of water, are much more diverse than previously thought.
The recorded taxon richness of protists in the Veternica cave shows a high number of taxa, especially considering the small number of samples and prospected habitats. Thus, taxon richness of protists in caves could be much richer in comparison with diversity of cave animals. As protists are very scarcely researched in caves, future research should focus on investigating characteristics of small aquatic habitats in conditions where bat’s guano is absent in order to determine its impact on protist assemblages.
Research in the Veternica cave provided valuable information about the protists in caves that are an important component of subterranean food webs supporting stunning biodiversity of subterranean animals. Thus, further effort should be given to the research that could shed light on these trophic relations.
This research was performed as a student project for the Rector’s award of University of Zagreb and it was partially supported under project Implementation of the composition of aquatic fauna in evaluation of environment 119-0000000-1205 (University of Zagreb, Faculty of Science, project leader prof. dr. Biserka Primc, 2011–2012). We would like to express our gratitude to the colleagues who accompanied us for the field trips, provided us with valuable information, maps, support and advices during this research: R. Baković, P. Žvorc, J. Rubinić, S. Gottstein, P. Škuljević, I. Šimičić, M. Karlica, M. Uroić, J. Bedek, M. Lukić, S. Malić-Limari, D. Kovačić, T. Ban Ćurić and N. Fressl. Special thanks to the employees of Public Institution Nature Park Medvednica for their support. We thank Steffen Clauß from Germany for his willingness to take some SEM images of the scales of two heliozoans and Robert Baković for adaptation of Veternica cave map. This research was performed under the permission of the Croatian Ministry of Culture (Class: UP/I-612-07/11-33/0709, Ref. No.: 532-08-02-03/1-11-04, 15.6.2011) and Croatian Ministry of Nature and Environmental Protection (Class: UP/I-612-07/19- 48/38, Ref. No.: 517-05-1-1-19-5, 29.03.2019).