Species Composition and Structure of Beetle Associations in Caves of the Cz ˛estochowa Upland, Poland

: This paper presents the study’s results on beetles (Insecta: Coleoptera) inhabiting caves in the Cz˛estochowa Upland, southern Poland. During two years of research, 2084 specimens, representing 105 species from 19 beetle families, were collected. The obtained results indicate that many beetle species choose to inhabit caves despite lacking speciﬁc adaptations for living in such environments. The cave entrance zone is the most attractive place for surface species to inhabit because its climatic conditions are more stable than outside the cave, some sunlight is present, and the availability of organic matter is high. In the deeper parts of the studied caves, the number of occurring species rapidly decreased. Three species were recognised as troglobionts, four as troglophiles and 32 as subtroglophiles. Speonomus normandi hydrophilus , Choleva lederiana gracilenta and Quedius mesomelinus mesomelinus were identiﬁed as the most frequent in the studied caves. Speonomus normandi hydrophilus is endemic to the Arize massif in the Central Pyrenees (France) and was experimentally introduced into the Dzwonnica cave (Poland) in 1982, while Ch . lederiana gracilenta is one of two troglobitic beetles native to Polish caves and Q . imesomelinus mesomelinus is a widespread, very common troglophile.

Beetles also dwell in caves in Poland, and several species are known from previous research conducted in recent years [11,12]. However, a lack of comprehensive data about beetles inhabiting the subterranean environment in Poland was the main impetus for the authors to undertake this research. In Poland, caves occur in several of its southern regions, such as the Sudety Mountains, Tatra Mountains, Kraków-Częstochowa Upland, Carpathian Flysch Belt, Nidziańska Basin andŚwiętokrzyskie Mountains. The total number of caves is over 5000; the majority (about 1500) are located in the Kraków-Częstochowa Upland [13].
The purpose of the present research was to observe the species communities of beetles from caves of the Częstochowa Upland, which is a part of the Kraków-Częstochowa Upland.
The purpose of the present research was to observe the species communities of beetles from caves of the Częstochowa Upland, which is a part of the Kraków-Częstochowa Upland. Special attention was paid to the degree of certain species' relations with the subterranean habitat. Moreover, the particular beetle association structure was also analysed.

Study Area
Six caves in the Częstochowa Upland were investigated ( Figure 1); these were previously described in detail by Kocot-Zalewska and Woźnica [14]. Graphic diagrams of the studied caves have been taken from the public website of the Polish Geological Institute-National Research Institute [15]. All these caves are horizontal, with similar lengths and altitude a.s.l., but with slight height differences [16,17]. The size and exposure of the entrance, shape, configuration of passages and vegetation around the cave entrance vary for each cave (Figures 2-4). The caves are situated in forest zones except for the Towarna cave, which lies in an area with xerothermic grassland. Five of the six studied caves have one entrance; only the Kroczycka cave has two, but both are much smaller than the entrances of other caves.

Sampling Design
The specimens were sampled from December 2014 to February 2017 using 5 cmdiameter Barber's traps with 30% propylene glycol (as a preservative liquid). From December 2014 to January 2016, the material was sampled constantly (12 samples from every site per year). The following year, samples were gathered every two months (thus, six times per year). The direct-search method was also used for collecting the insects. However, individuals sampled in such a way were used only for the qualitative analysis and were not included in the quantitative evaluation.
The traps were always situated in the same area in each cave. The first was placed in the entrance zone of the cave (S1), the second in the middle of the passage (S2) and the third at the end of the passage (S3) ( Figure 3). Additionally, one trap was always placed in the direct surroundings of each cave entrance (S0) (Figure 3).
Because of the enormous entrance of the Pod Sokolą cave, the entrance zone (ca 3-4 m inside the cave) was not characterised by specific features. Therefore, an additional trap was used there. In this cave, for the statistical analyses, the trap in S2 was treated as the entrance zone trap, the trap in S3 as a transition zone trap, and that in S4 as a deep zone trap ( Figure 4A).
In the Kroczycka cave, four traps were also used, due to the cave morphology resembling a rectangle with two entrances ( Figure 4B). Traps S1 and S4 were located near the entrances, and, in the statistical analyses, they were used collectively as the entrance zone traps. Trap S5 was placed approximately in the middle of the cave length, and was thus treated as a trap appropriate for the transition zone. Trap S3 was placed at the end of the cave and represented a deeper part of the cave. Trap S2 marked a microclimate measurement point.

Microclimate Measurements
The temperature and humidity were measured from December 2014 to February 2017 in the studied caves. The Assmann psychrometer was set up monthly at the same points The traps were always situated in the same area in each cave. The first was placed in the entrance zone of the cave (S1), the second in the middle of the passage (S2) and the third at the end of the passage (S3) (Figure 3). Additionally, one trap was always placed in the direct surroundings of each cave entrance (S0) (Figure 3).
Because of the enormous entrance of the Pod Sokolą cave, the entrance zone (ca 3-4 m inside the cave) was not characterised by specific features. Therefore, an additional trap was used there. In this cave, for the statistical analyses, the trap in S2 was treated as the entrance zone trap, the trap in S3 as a transition zone trap, and that in S4 as a deep zone trap ( Figure 4A).
In the Kroczycka cave, four traps were also used, due to the cave morphology resembling a rectangle with two entrances ( Figure 4B). Traps S1 and S4 were located near the entrances, and, in the statistical analyses, they were used collectively as the entrance zone traps. Trap S5 was placed approximately in the middle of the cave length, and was thus treated as a trap appropriate for the transition zone. Trap S3 was placed at the end of the cave and represented a deeper part of the cave. Trap S2 marked a microclimate measurement point.

Microclimate Measurements
The temperature and humidity were measured from December 2014 to February 2017 in the studied caves. The Assmann psychrometer was set up monthly at the same points (S1 to S3 in every cave, and S4 in the Pod Sokolą cave). The psychrometer consisted of two thermometers (wet and dry) and an electric fan. In situ, the temperature was measured on both thermometers, and then, using psychrometric tables, the relative humidity was estimated. It was not possible to use automatic data loggers.

Data Analysis
In order to identify species associated with the subterranean environment, the dominance was calculated, and habitat and food preferences were analysed. Additionally, species richness for each cave was estimated using Hill numbers ( q D, sensu Jost [18]). It was parameterised with q values of 1 and 2 to obtain the Shannon-Wiener diversity (q = 1) and Simpson diversity (q = 2) [19,20] (Appendix A, Formulas (A1) and (A2)). Both formulas are modified versions of the Shannon-Wiener and Simpson indices, respectively [18][19][20]. However, modification of the first index enables the elimination of favouritism of rare or common species, while Simpson diversity puts more weight on the frequencies of species and discounts rare species [19,20]. The analyses were performed using the PAST3 software ver. 4.03 [21].
The analyses of species habitat and food preferences were based on the literature data . According to the habitat, species were assigned to the proper environment groups, e.g., species living in the forest, grasslands, MSS (inhabiting mammal burrows and nests, living under moss, stones, in cave entrances) and others. Regarding diet, the studied beetle species were divided into five groups: herbivores, predators, fungivores, detritivores and omnivores. The group of detritivores includes all the phytosaprophages, zoosaprophages, coprophages and necrophages.
For subterranean species, we used four categories of ecological classification based on Sket's concept [55], i.e., the group of troglobionts consists of species exclusively found in caves and, in the literature, considered as this form. The group of eutroglophiles is formed by species consistently found in caves throughout the year. However, they are generally epigean species. The group of subtroglophiles is composed of species that temporarily occur in caves; however, they were collected several times in many caves. Those species prefer living in mesovoid shallow substratum. The trogloxenes group includes accidental species for which caves and other subterranean shelters are not a typical habitat.
Moreover, a different group found in shallow subterranean habitats-MSS-was recognised [5][6][7]. In order to assign each of the analysed beetle species to the appropriate ecological category, we used previously published data [56][57][58][59][60][61]. All usable information was involved, i.e., the presence of species throughout the year, food and habitat preference, the area of the cave in which the species was most frequently caught.
Additionally, we compared the frequency of particular species collected from caves with results obtained from the traps outside the studied caves (especially those species considered eutroglophiles and troglobionts). None of the troglobionts and eutroglophiles, was sampled outside the cave, with one exception. Several specimens of Catops picipes (Fabricius, 1787) were collected from traps located outside the caves. However, sample sites S0 were still situated among rocks and debris near the cave entrances. The presence of the beetles inside the caves varied and depended on the zones within the caves ( Figure 5). Many collected specimens for each family were also correlated with the cave zones ( Figure 6). When certain zones of the caves were considered, the entrance zone (S1) was the most numerous in species (10 to 35 species). Five to eleven species were collected in the middle of the passages (S2), whereas at the end of the caves (S3), one to eleven species were recorded. The results presenting the number of recorded species in particular parts of each cave are provided in Table 1, and the total number of species in each cave is presented in Table 2.

Species Richness
The number of species in particular caves differed ( Table 2). The highest numbers were observed in the Pod Sokolą cave, while the lowest were observed in the Psia and Kroczycka caves. The highest species richness was calculated for the Zegar and Kroczycka caves, and the lowest in the Towarna cave (Table 2). 5). Many collected specimens for each family were also correlated with the cave zones ( Figure 6). When certain zones of the caves were considered, the entrance zone (S1) was the most numerous in species (10 to 35 species). Five to eleven species were collected in the middle of the passages (S2), whereas at the end of the caves (S3), one to eleven species were recorded. The results presenting the number of recorded species in particular parts of each cave are provided in Table 1, and the total number of species in each cave is presented in Table 2.

Species Dominance
Speonomus normandi hydrophilus (Jeannel, 1907), representing the family Leiodidae, was the most abundant among collected specimens (a total of 1120 individuals, 53% of all sampled specimens). Despite its abundance, all its specimens were exclusively found in the Towarna cave. Choleva lederiana gracilenta Szymczakowski, 1957, also from Leiodidae, was the second most abundant beetle, comprising 11% of the total collected specimens. As with S. normandi hydrophilus, all its individuals were exclusively sampled in one cave, namely, the Pod Sokolą cave. The third most abundant species found (representing 3.8% of the assemblage) was the staphylinid Quedius mesomelinus mesomelinus Marsham, 1802, which was found in each of the studied caves. Moreover, each cave had its own different set of eudominants and dominants (Table 3).
Unfortunately, for all other species (especially those representing the family Staphylinidae), these data were impossible to collect due to inadequate knowledge regarding their biology.

Microclimatic Analyses
The temperature in all studied caves fluctuated, but the greatest temperature changes were detected in the entrance zone (S1). Additionally, it was observed to decrease towards the deeper parts of the cave. The most stable temperatures were observed at the terminal parts of the caves (Figure 7). Humidity in the studied caves was generally high. However, it fluctuated similarly to temperature, especially in the entrance zone (S1). Notably, humidity fluctuations during the summer and winter periods were recorded even in the deeper parts of the caves (Figure 8).  (Table S2).

Microclimatic Analyses
The temperature in all studied caves fluctuated, but the greatest temperature changes were detected in the entrance zone (S1). Additionally, it was observed to decrease towards the deeper parts of the cave. The most stable temperatures were observed at the terminal parts of the caves (Figure 7). Humidity in the studied caves was generally high. However, it fluctuated similarly to temperature, especially in the entrance zone (S1). Notably, humidity fluctuations during the summer and winter periods were recorded even in the deeper parts of the caves (Figure 8).

Species Richness
Altogether, 105 Coleoptera species were detected in the studied caves (Table S2). The beetle species number provided in the present study is twice larger than that reported in all previous studies in Polish caves [11,12].
The highest species frequencies were observed in Leiodidae, Staphylinidae and Cryptophagidae. The occurrence of round fungus beetles (Leiodidae) in the caves was not surprising, because many species of this family are highly associated with subterranean environments [66]. Many rove beetles (Staphylinidae) are also known from subterranean areas [8,66]. However, silken-fungus beetles (Cryptophagidae) are not usually associated with subterranean environments, being closely associated with forest ecosystems and often considered within the ranks of saproxylic and xylobiont Coleoptera [67]. Therefore, the high species frequency of silken-fungus beetles in the studied caves was slightly unexpected. However, most of the Cryptophagidae specimens were collected from the entrance zone of one cave (the W Zielonej Górze cave), and only a few occurred in its deeper zones and in the other caves. Thus, despite collecting about 200 specimens, only two species were classified as subtroglophiles, and five were classified as trogloxenes.
The highest species richness among the studied caves was recorded for the Zegar cave (15.95863401 and 1.051524711 in Shannon diversity and Simpson diversity indices, respectively) ( Table 2). The high frequency of those species among the samples may explain such a result. The Towarna cave represented the lowest value of the Shannon

Species Richness
Altogether, 105 Coleoptera species were detected in the studied caves (Table S2). The beetle species number provided in the present study is twice larger than that reported in all previous studies in Polish caves [11,12].
The highest species frequencies were observed in Leiodidae, Staphylinidae and Cryptophagidae. The occurrence of round fungus beetles (Leiodidae) in the caves was not surprising, because many species of this family are highly associated with subterranean environments [66]. Many rove beetles (Staphylinidae) are also known from subterranean areas [8,66]. However, silken-fungus beetles (Cryptophagidae) are not usually associated with subterranean environments, being closely associated with forest ecosystems and often considered within the ranks of saproxylic and xylobiont Coleoptera [67]. Therefore, the high species frequency of silken-fungus beetles in the studied caves was slightly unexpected. However, most of the Cryptophagidae specimens were collected from the entrance zone of one cave (the W Zielonej Górze cave), and only a few occurred in its deeper zones and in the other caves. Thus, despite collecting about 200 specimens, only two species were classified as subtroglophiles, and five were classified as trogloxenes.
The highest species richness among the studied caves was recorded for the Zegar cave (15.95863401 and 1.051524711 in Shannon diversity and Simpson diversity indices, respectively) ( Table 2). The high frequency of those species among the samples may explain such a result. The Towarna cave represented the lowest value of the Shannon diversity (1.447734615), but the highest of the Simpson diversity (7.692307692). It can be explained by the extremal domination of one species (among 1206 specimens in the collection, 1120 belonged to Speonomus normandi hydrophilus). The Psia cave represented rather high values of the abovementioned indices (8.758284041 and 2.949852507, respectively). It was surprising due to the generally poor habitat quality of the cave, which was also confirmed by the low frequency of specimens representing other arthropod groups compared to other studied caves. In addition, we examined the contents of organic carbon in every sampling place of the studied caves only once. The Psia cave is characterised by twice the lower value of organic carbon than other caves. However, this parameter was not elaborated on further due to the small amount of data.
In terms of cave zonation, the highest values of the analysed indices were associated with the entrance zones. This was an expected result because the entrance zones were always characterised by toned temperature fluctuation (in comparison to the surface), a lack of violent weather phenomena, a high accumulation of organic matter, and the presence of direct or indirect sunlight. All these conditions significantly impact cave-dwelling by invertebrate fauna, including beetles. Stable temperatures, high relative humidity and extreme organic matter accumulation were associated with the deepest zones. Despite this, the lowest values of species richness indices resulted from complete darkness, which is not conducive to penetration by surface fauna.

Species Dominance
The analyses of species dominance indicated the overwhelming contribution of accidental species in the entrance zone (S1), especially in the caves with large entrances. This was evident, for instance, in the Pod Sokolą, Zegar and W Zielonej Górze caves, where the accidental species reached 60% to 80% of the total amount of collected species (Table 3).
Moreover, each studied cave had a different set of eudominant or dominant species (Table 3). In the W Zielonej Górze cave, two species (Cryptophagus distinguendus J. Sturm, 1845 and C. punctipennis Ch.N.F. Brisout de Barneville, 1863) represented the eudominant class. Speonomus normandi hydrophilus, in the Towarna cave, and Choleva lederiana gracilenta and Catops tristis infernus, in the Pod Sokolą cave, exhibited a spectacular dominance. This reached 98% in the deepest part of Towarna (S3) for S. normandi hydrophilus and 87.5% and 20% in the deepest part of Pod Sokolą (S4) for the latter two beetles, respectively.
In the caves of the central and southern part of the investigated area-i.e., the Kroczycka, Psia and Zegar caves-none of the analysed species achieved a high dominance value.

Habitat and Food Preferences
The examination of food preferences indicated that beetles mostly choose detritus or fungi, and some are predators. Phytophagous species represented only a tiny part of the collected material. Similar results were demonstrated for invertebrates collected in caves in Germany [68]. The analyses of habitat preferences showed that most of the sampled beetle species prefer living in a forest (under rocks, fallen leaves and rotten wood). Beetles preferring open spaces were less abundant (only four per cent of the identified species).
Despite Ch. lederiana gracilenta and C. tristis infernus not presenting particular adaptations for living underground, both species were described from caves [69], and thus far, they have never been found in the epigean environment. In turn, S. normandi hydrophilus is a highly specialised subterranean species; however, it can still be found in caves and the MSS environment, such as scree and rock debris [46].
While Q. mesomelinus mesomelinus and T. subterraneus are commonly accepted as (eu)troglophiles species, C. picipes and O. validum are not so obviously classified. C. picipes is a common, widely distributed European species. It is found in both epigean and hypogean environments. However, its preference to occur in subterranean environments and high frequency in samples from various caves in Europe influenced considering this species as an (eu)troglophile [61,70].
Similar results concern O. validum, which occurs primarily in mountain regions and lives in burrows, rock debris and caves [29,71]. The species was found not only in Polish caves but also in many caves in Czechia [72], Germany [61], and Slovakia [8]. Moreover, it is also known for living in the riparian-sylvan microhabitat [73]. It was considered an (eu)troglophile by both Zaenker et al. [61] and Rendoš et al. [8]. In our investigation, we observed it every season through both years of the study. Burakowski [29] indicated that faint pigmentation, small eyes and more extended feet are an adaptation for subterranean living.
It is worth noting that among all collected species, more than 30 were indicated as subtroglophiles or MSS species, which is approximately 30% of all collected species. It is an interesting result because it proves that many species penetrating caves prefer this habitat. The attractiveness of the MSS environment was already pointed out [8]. On the other hand, more than 50% of collected beetle species were trogloxenes. However, a majority of them were sampled only in the entrance zone. This is not entirely unexpected, since the high ratio of trogloxene diversity (3.560852562-5.640653908 Shannon diversity index value) in the entrance zone was also reported in a temperate marble cave in the Sierra Nevada Range, California, USA [74]. Moreover, the occurrence of many trogloxenes was also observed in the entrance zones of caves in Slovakia [59], Romania [73] and Italy [75], where it was mainly attributed to the contact between subterranean and surface environments and a high accumulation of decomposed organic matter.
The presence of very few species that could be classified as troglobionts and eutroglophiles most probably resulted from the environmental conditions peculiar to caves of the Częstochowa Upland. The temperatures inside the studied caves were unstable and varied throughout the year. Temperature fluctuations were higher in the entrance zones (about several dozen degrees Celsius) than in the depths of the caves (several degrees Celsius). Similarly, relative humidity was inconstant, especially in the entrance zone. Moreover, in some caves, the direct or diffracted light reached even a half-length of the cave passages. Because species strictly associated with subterranean environments prefer stable environmental factors, such as temperature and humidity [76,77], they cannot find such conditions in the investigated caves.
Nevertheless, despite conditions that are not very suitable for dwelling in the analysed caves, two strict troglobionts, Choleva lederiana gracilenta and Catops tristis infernus, have populations living in the Pod Sokolą cave. This was a slightly unexpected situation, since some diffracted light reaches even the very end of this cave, and a minor fluctuation in air temperature occurs there. In this case, however, stable humidity was a crucial factor for the presence of both these cave-obligated beetles.
In contrast, the deepest terminal part of the Psia cave boasts a very stable temperature (the yearly fluctuation was less than 1 • C), high and stable humidity, and complete darkness. Despite these preferable conditions, this cave zone had outstandingly poor fauna (Tables S1 and S2). Within a two-year study period, a single beetle specimen was sampled in this zone of the Psia cave.
However, there was also a cave in which the climatic conditions appeared to be very dynamic throughout the year, but it still possessed its troglobiont beetle, namely, Speonomus normandi hydrophilus. Moreover, this troglobiont was the most abundant beetle in this cave, being extremely dominant (Table 3). It is worth mentioning that S. n. hydrophilus is alien to the fauna of Poland. Endemic to the Pyrenees, it is a beetle that was intentionally introduced to the Dzwonnica cave in 1982 [78]. Within 40 years, this species colonised nearby caves (i.e., the Towarna and Cabanowa caves) [79,80]. This case of colonisation success proves that, in Poland, the environmental conditions in caves are sufficient for habitation by troglobionts, but for some reason, are only barely adequate or inadequate for highly specialised dwelling species. As suggested by some authors [56,58,64], this phenomenon can be explained by the geological history of Central Europe, where, during the Pleistocene, glaciations influenced the entire fauna to such an extent that it could not even survive in the caves, which are normally refugial environments.
There is one more reason for this hypogean environment to be vastly attractive for many beetles. Though the epigean surroundings have their own environmental and climatic conditions, they are not as stable as those in the cave entrances. Additionally, as was suggested in many previous studies, organic matter from the external environment can easily flow into the caves via physical or biological means [4,9,[81][82][83][84]. Most importantly, the entrance and transition zones of the studied caves exhibit conditions similar to the MSS environment to a greater extent than the deeper cave zones. The MSS, as a specific space [5,6], connects components originating from the underground and the surface environments. Hence, it seems to provide an additional explanation for why so many beetle species live in these caves, albeit being weakly associated with them ecologically.

1.
Many beetle species choose to dwell in caves, despite lacking specific adaptations for living in such environments.

2.
The entrance zone is the most attractive place for surface species to inhabit because the climatic conditions there are more stable than outside the cave (due to the presence of some light and high availability of organic matter).

3.
In the deeper parts of the studied caves, the number of occurring species rapidly decreased, though this zone sometimes had a stable environmental condition (namely, humidity).