The shallow underground of rock debris is a unique animal refuge. Nevertheless, the research of this habitat lags far behind the study of caves and soil, due to technical and time-consuming demands. Data on Myriapoda in scree habitat from eleven localities in seven different geomorphological units of the Czech and Slovak Republics were processed. Based on previous studies, as well as knowledge of cave and soil fauna, it was hypothesised that the occurrence of a varied and peculiar fauna would show a pattern of depth distribution with variations due to local specificities. From 2005–2016 (at least one year on each site), macrofauna was collected via sets of three long-term exposed subterranean traps consisting of 110 cm long perforated tube, with ten cups located in a gradient at 5–95 cm below the soil surface. In total, 14 symphylans (not identified to species level), 271 centipedes (23 spp.) and 572 millipedes (32 spp.) were sampled. The overall depth distribution of centipedes and millipedes appeared to have relatively similar pattern, with both groups being found at all depth levels. Nevertheless, this pattern depends on locations. The depth distribution trend lines are mostly in the form of an asymmetric ‘U’, with decreased abundance until the middle of the gradient, followed by increase in the deepest levels. Epigeic species were sporadically distributed along the whole depth gradient, but concentrated at the soil surface, while some subterranean species, such as the centipede Lithobius lucifugus and the millipedes Geoglomeris subterranea, Cibiniulus slovacus and Archiboreoiulus pallidus, were recorded in the deepest parts of the gradient. This characterises the debris community as a mixture of soil and subterranean species with an absence of species exclusively found in caves. The use of different fixation methods in traps had a significant and selective impact on samples; millipedes were either attracted by ethylene glycol or repelled by formaldehyde. Centipedes were also captured more frequently in ethylene glycol; however, the species composition varied in each of the fixatives. Depth distribution of myriapods was similar in both fixative solutions. Traps with these fixatives could be recommended for similar ecological studies.

Chilopoda, Diplopoda, Myriapoda, MSS, subterranean traps

The paper is dedicated to Christian Juberthie (12 Mar 1931–7 Nov 2019), the author of the concept of MSS (milieu souterrain superficiel) and the doyen of modern biospeleology

Forested scree slopes (slope deposits) represent a unique type of shallow subterranean domain, which are frequently labelled in literature as shallow subterranean habitat (SSH; Culver and Pipan 2014) or meso-void shallow substratum (milieu souterrain superficiel, MSS of Juberthie et al. 1980; preferred abbreviation for this paper). The main condition for the existence of MSS is the presence of stone deposits forming in the underground environment. Weathered rock fragments and sediments accumulate in several layers, which are subsequently covered by the soil. Due to this isolation from the surface, MSS serves as a stable habitat with conditions similar to those in caves (Juberthie 2000; Giachino and Vailati 2010). Cracks and air-filled voids create a network of interconnected corridors that provide suitable shelter for various groups of invertebrates, but also serve as a refugium for relic fauna (Růžička and Klimeš 2005; Zacharda et al. 2005; Mock et al. 2015; Kováč et al. 2016; Růžička et al. 2016; Nitzu 2016; Tuf et al. 2017; Nitzu et al. 2018b; Mammola 2018). Shallow subterranean habitats can be defined as a habitat on a transition zone with the ongoing adaptation of organisms to underground life (Pipan et al. 2011; Ortuño et al. 2013; Nitzu et al. 2014; Mammola et al. 2017). The presence of invertebrate life forms possessing convergent morphological adaptations (depigmentation, eye absence, reduction of size, etc.) to subterranean life is one of the main criteria for distinguishing surface from underground habitats.

The study of deeper layers of forested scree slopes reveals that refugia of rare fauna add another dimension of environmental heterogeneity affecting overall biodiversity. Rather numerous studies on the ecology of various groups of fauna in MSS in Europe were conducted (reviewed in Mammola et al. 2018), some of them focusing on scree slopes of Slovakia and the Czech Republic (Růžička and Klimeš 2005; Laška et al. 2011; Rendoš et al. 2012, 2014, 2016a; Mock et al. 2015; Růžička and Dolanský 2016; Rudy et al. 2018; Jakšová et al. 2019; Jureková et al. 2019). Subterranean diversity of Myriapoda inhabiting underground of scree slopes has been investigated as well (Nitzu et al. 1999; Ilie 2003a, 2003b; Rendoš et al. 2012, 2016a; Jiménez-Valverde et al. 2015; Mock et al. 2015; Tuf et al. 2017); some studies even have described new myriapod taxa (e.g., Stoev 2008; Gilgado et al. 2015; Akkari et al. 2018). Attention should also be paid to the results of studies testing the reliability of the use of a set of subterranean traps inserted to the substrate through plastic pipe and other methodological aspects. The type of fixative solution and trap exposure duration can also affect species assemblages (Mammola et al. 2016; Jureková et al. 2019). Additionally, environmental variables affect the distribution pattern of invertebrates below the surface of stone debris (Rendoš et al. 2016b; Nitzu et al. 2018a). Our contribution completes the knowledge from a number of little or unstudied sites of Slovak and the Czech republics. The aims of our study were to (1) analyse the structure and diversity of myriapods dwelling in forested scree slopes, and (2) to generalise the distribution of myriapods along the depth gradient at selected locations in Central Europe.

Myriapoda were recorded at all eleven study sites and were one of the less frequent groups of arthropods. In total, 857 individuals were identified to 55 species. Diplopoda, unlike to ever-present Chilopoda, were missing at study site 11; however, it was the richest represented class of myriapods, regarding both individuals and species. Symphyla were represented only by 14 individuals at four study sites. The fourth myriapod class, Pauropoda, was not documented at any of the study sites. Species diversity and depth distribution showed geographical differences, but overall, two dominant groups demonstrated similar indicators. Alternation of two fixative solutions in traps resulted in significant differences in both the number of individuals and the species composition, with Diplopoda and Chilopoda responding differently to the type of fixation.

Representatives of Symphyla were captured at four of the study sites. All collected individuals (not identified to species level) were distributed unevenly along the depth gradient, present at almost every depth of the top half of the gradient. One third of all captured symphylans were present in the bottom half of the gradient, at depths of 65 and 95 cm. Almost two-thirds of Symphyla specimens were captured in traps with formaldehyde.

Centipedes

Overall, 271 specimens of Chilopoda were sampled, belonging to 23 species and five families (See Suppl. material 1: Table S1). The number of centipede species sampled on individual study sites varied from 1–8 species, with an average of 5.2 species sampled per site. The highest value of Shannon’s diversity index (H’ = 1.58) was recorded on the scree slope at the edge of the Bečva River (site 7), while the highest value of Pielou’s evenness index (J’ = 1) was recorded at the scree slope near Silická ľadnica Ice Cave (site 1) (Fig. 2B). At the scree slope next to the quarry of the village of Hněvětice (site 11), only one species, Lamyctes emarginatus, was caught in the traps. Among collected Chilopoda, two species can be classified as eudominant, Lithobius forficatus (D = 47%) and Lithobius lucifugus (D = 14%); Lithobius forficatus represented the species with the highest constancy (C = 82%), occurring at nine of eleven sites.

Figure 2. 

A Overall depth distribution of centipede individuals and species B values of Shannon’s diversity index and Pielou’s evenness index, calculated for centipedes, at each of the study sites C mean values of Shannon’s diversity index (±SD) calculated for centipedes, at each depth of the gradient (summarised data from all localities) D overall depth distribution of millipede individuals and species E values of Shannon’s diversity index and Pielou’s evenness index, calculated for millipedes, at each of the study sites F mean values of Shannon’s diversity index (±SD) calculated for millipedes, at each depth of the gradient.

Regarding depth distribution of centipedes, the highest numbers of individuals and species were captured near the surface; however, the overall distribution curve resembled U-shape with decreasing abundances in upper part of profile replaced by opposite pattern in deepest layers (Fig. 2A). More than half of the species were sampled only in the first half of the depth profile (5–45 cm), while four species were collected exclusively in the uppermost levels (5–15 cm). None of the species were observed solely in the deeper half of the gradient (Table 2). Shannon’s diversity index (H’) was highest at a depth of 95 cm; however, at 5 cm depth, the value was really similar (Fig. 2C). Lithobius forficatus and L. lucifugus were the only two Chilopoda species with an occurrence at all layers of the depth profile; L. forficatus showed a significant affinity to the surface (F = 17.2, p < 0.01), while none of the species showed significant positive relation to deepest layers of the depth gradient (Fig. 3A).

Table 2.

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A summary overview of the centipede depth distribution in the eleven scree slopes of Slovakia and the Czech Republic.

Depth (cm)	5	15	25	35	45	55	65	75	85	95	Σ
Clinopodes flavidus	2	–	–	1	–	–	–	–	–	–	3
Cryptops parisi	1	1	–	1	2	2	–	1	2	1	11
Geophilus electricus	1	–	–	–	–	–	–	–	–	–	1
Geophilus insculptus	–	1	–	–	–	1	–	–	–	–	2
Geophilus flavus	1	–	1	1	–	3	1	–	–	–	7
Harpolithobius anodus	4	–	–	–	1	–	–	–	–	1	6
Lamyctes emarginatus	3	3	–	–	–	–	–	–	–	–	6
Lithobius agilis	–	2	–	1	–	1	–	–	–	–	4
Lithobius austriacus	–	1	–	–	–	–	–	–	–	1	2
Lithobius cyrtopus	–	1	–	–	–	–	–	–	–	–	1
Lithobius dentatus	–	–	–	–	1	–	–	–	–	–	1
Lithobius forficatus	29	25	14	9	8	8	5	9	14	5	126
Lithobius lucifugus	9	4	4	3	2	2	5	1	3	4	37
Lithobius macilentus	–	–	–	–	2	–	–	–	–	1	3
Lithobius microps	–	–	–	–	1	–	–	–	–	–	1
Lithobius mutabilis	2	5	1	–	–	–	1	–	–	–	9
Lithobius muticus	6	–	–	–	1	–	–	1	–	–	8
Lithobius nodulipes	2	1	–	1	–	–	–	–	–	–	4
Lithobius tenebrosus	–	–	–	1	–	–	–	–	–	–	1
Lithobius t. fennoscandius	1	–	1	–	1	–	1	2	1	1	8
Strigamia acuminata	4	1	1	–	2	–	–	1	–	5	14
Strigamia crassipes	–	–	1	–	–	–	–	–	–	1	2
Strigamia transsilvanica	4	1	3	–	–	–	2	1	1	2	14
Σ	69	46	26	18	21	17	15	16	21	22	271

Figure 3. 

Generalised Additive Models of depth distribution pattern of A centipedes and B millipedes. Only species with significant pattern are illustrated. (F-values, * p < 0.05, ** p < 0.01): A Lamyctes emarginatus (13.1**), Lithobius forficatus (17.2**), Lithobius lucifugus (5.0*), Lithobius nodulipes (9.7**) B Archiboreoiulus pallidus (22.7**), Cylindroiulus nitidus (5.4*), Glomeris connexa (5.4*), Hylebainosoma tatranum (5.4*), Leptoiulus proximus (7.3*), Mastigona bosniensis (10.5**), Megaphyllum projectum (5.4*), Melogona transsylvanica (15.2**), Polydesmus complanatus (13.1**), Trachysphaera acutula (5.4*), Unciger foetidus (4.9*).

Effects of fixative solutions on diversity and activity of myriapods along the depth gradient of screes

At five of the study sites (sites 1–5; all situated in Slovakia), formaldehyde (two trap sets) and ethylene glycol (one trap set) were used in parallel. At all of these five study sites, much greater numbers of individuals and species of myriapods were recorded in subterranean traps with ethylene glycol than in the traps with formaldehyde; however, each of the systematic groups of Myriapoda responded specifically to the type of fixation.

For centipedes, ethylene glycol appears to be more attractive (or less repellent) than formaldehyde. Higher numbers of individuals were collected in traps with ethylene glycol. However, regarding species composition, the effect of both fixatives seems to be complementary, as each of the solutions contained some species exclusive for particular fixative too (Fig. 4A). Vertical distribution of centipedes along the depth gradient at five scree slopes was characterised by strong decline and later small increase in deepest layers in the number of individuals in formaldehyde as well as ethylene glycol fixative solution, resembling its general U-shape curve of abundances (cf. Figs 2A, 5A). On the other hand, and contrary to ethylene glycol fixative, a simple decreasing trend in number of species was recorded in traps with formaldehyde fixative (Fig. 5B).

Regarding millipedes, ethylene glycol showed significantly higher activity and species diversity compared to formaldehyde. All collected species preferred traps with ethylene glycol; almost half of the millipede species occurred exclusively in this type of fixative solutions (Fig. 4B). Number of species and individuals decreased with depth in ethylene glycol fixation (down to 35 cm) and increased again at depths of 75–95 cm. Vertical distributions of millipedes in formaldehyde showed no particular trend (Fig. 5C). Similar patterns were recognized for number of species too (Fig. 5D).

Figure 4. 

Graphical presentation of myriapod community characteristics in different fixative solutions (N = number of individuals). A Formaldehyde to ethylene glycol ratio of sampled centipede species from all study sites, where both fixating solutions were used B formaldehyde to ethylene glycol ratio of sampled millipede species from all study sites, where both fixating solutions were used.

Figure 5. 

Vertical distribution of myriapods along the depth gradient in different fixative solutions (data recalculated for the same number of traps). Trend line: dashed = formaldehyde, dotted = ethylene glycol. A Vertical distribution of Chilopoda specimens along the depth gradient (5–95 cm) at five scree slopes in different fixative solutions B vertical distribution of centipede species along the depth gradient at five scree slopes in different fixative solutions C vertical distribution of Diplopoda specimens along the depth gradient (5–95 cm) at five scree slopes in different fixative solutions D vertical distribution of millipede species along the depth gradient at five scree slopes in different fixative solutions.

Scree habitats serve as a prospective source of information on species habitat preference, diversity and potential migration between the shallow underground environment and the cave environment. Interspaces of forested scree slopes are inhabited by various groups of invertebrates, predominantly arthropods, represented by both edaphic and subterranean species (Juberthie 2000; Nitzu et al. 2014; Jiménez-Valverde et al. 2015). Our study confirmed that Central European myriapods colonise belowground zones in scree habitats covered with soil. It also indicates that there are some differences in diversity and distribution of centipede and millipede assemblages along depth profiles. The majority of myriapod species captured using subterranean traps represent common epigeic or edaphic fauna, occurring predominantly in the nutrient-rich uppermost layers of soil and litter. Some subterranean forms were captured, as well as species preferring scree slope habitats, including Archiboreoiulus pallidus and Cibiniulus slovacus (Antić et al. 2015; Mock et al 2015); these species are also cave-dwellers. However, some cave dwelling species known from the study region are absent in scree samples (Kováč et al. 2014).

The distribution pattern was characterised by typical sharp decline at the beginning of the measured depth gradient culminated near the middle zone, and increased occurrence and diversity of myriapods in the deeper parts of the gradient. Such decrease in abundance of the studied groups has been confirmed by several studies in different invertebrates (e.g., Laška et al. 2011; Rendoš et al. 2012, 2016a). Among other factors, the decline in upper layers may be due to the decreasing availability of organic resources as well as available spaces up to 50 cm (Gers 1998; Rendoš et al. 2012; Nitzu et al. 2014). On the other hand, from the depth of 70 cm we can see numerous communities of subterranean species that do not move to the upper layers. Some of the studies (Růžička et al. 1990; Růžička 1999; Laška et al. 2008) highlight the effect of substrate porosity, layer depth and vegetation on vertical distribution in soil. Regional surface species congregate deeper underground in screes similarly to caves especially in territories with rare obligate subterranean dwellers (Rendoš et al. 2016a; Fernandes et al. 2019).

The structure of centipede assemblage in scree habitats along the depth gradient has been described by various authors. In Querner and Gereben-Krenn (2005), the centipedes were distributed vertically only to a depth of 20 cm, while in our study, centipedes occurred at each depth of the gradient. Almost half of our collected centipedes were represented by Lithobius forficatus. The species is known for its wide ecological valency, characterised by Pan-European and Palearctic distribution. It has been documented from subterranean environment by Nitzu et al. (1999) from the caves of Romania (Ilie 2003a, 2003b) and caves of the Slovak Karst (Országh et al. 1994; Országh 2000). Lithobius forficatus is also known to penetrate deeper parts of the scree slope habitats (Rendoš et al. 2012, 2016a). The second-most abundant species of centipedes captured along the depth gradient was Lithobius lucifugus. This species was previously recorded at different karstic areas (e.g., Folkmanová 1951; Ilie 2003a, Ilie 2004) and is known to inhabit various underground habitats, including caves (Novák and Dányi 2011; Dvořák and Dvořáková 2015; Růžička et al. 2016; Tuf et al. 2017).

Relative abundance and diversity of millipedes captured using subterranean traps was higher in comparison to centipedes. Majority of the sampled species can be described as epigeic and edaphic. The most abundant species, collected only on the scree slopes of the Czech Republic, was Ochogona caroli. This species inhabits mainly higher altitudes of Central European mountains confirming its higher activity for the colder part of the year (Kocourek et al. 2017). Some important representatives of edaphic or subterranean forms were captured, including two species of family Blaniulidae, Archiboreoiulus pallidus and Cibiniulus slovacus; these species were captured in the deepest parts of depth gradient. Both species represent blind relict millipedes with similar ecological demands, known for their preference of shallow subterranean habitats. Archiboreoiulus pallidus, characterised by cryptic behaviour, has so far been documented only from the subterranean environment, for the studied area (Mock et al. 2015). Another blind relict species, Cibiniulus slovacus, was described only recently from caves in Slovakia (Antić et al. 2015); the species shows considerable affinity to forested scree habitats. The presence of similar species (morphotypes) that may coexist on a single site (e.g., chordeumatid diplopods of Ochogona caroli and Listrocheritium septentrionale) or that alternate similarly in the depth gradient (e.g., blind blaniulid millipedes and Blaniulus guttulatus, Archiboreoiulus. pallidus, and Cibiniulus slovacus) emphasises the need for precise taxonomic work. Another representative of subterranean millipede fauna captured in scree slopes is Geoglomeris subterranea. The species is morphologically well-adapted to subterranean environment and is known to inhabit various western-European caves (Gruber 1985; Kocourek et al. 2017). Its occurrence in soil has been documented (Wesener et al. 2019) even from artificial MSS habitat from the Czech Republic (Riedel et al. 2009). Apart from common European species, a few endemic species of millipedes were determined such as Cibiniulus slovacus, Hungarosoma bokori, Hylebainosoma tatranum, and Listrocheritium septentrionale. Some relict species with wider distribution include millipedes (Geoglomeris subterranea, Archiboreoiulus pallidus, Trachysphaera spp.) and centipedes (Harpolithobius anodus, Lithobius cyrtopus, Lithobius forficatus, Lithobius nodulipes) (Tuf and Tufová 2008; Kováč et al. 2014).

Nitzu et al. (2018a) emphasise, in addition to the geographical differences on the sites, the significant influence of the geological subsoil on the abundance and diversity of calciphile diplopods in favour of carbonate rocks. Although their study suggests a strong correlation of the calcareous geological substratum with an invertebrate species assemblage, our study found no statistically significant differences between assemblage abundance on limestone and non-limestone study sites either in Chilopoda or in Diplopoda (Chi-square test results).

Generalised additive models helped identify significant patterns of distribution, i.e., to find species, for which depth is useful predictor of its abundance. This analysis can record species with some preferences. A non-significant pattern of distribution could be evidence for either random distribution or equal distribution. Some species with equal depth distribution can be important and stable members of MSS, too. Such notable species in our material seem to be millipedes Cibiniulus slovacus, Haasea flavescens, Ochogona caroli, Polydesmus denticulatus, and Trachysphaera gibbula.

Five of the study sites used of two types of fixative solutions in parallel and brought different results. For millipedes, traps with ethylene glycol show much higher efficiency in comparison to those with formaldehyde. This is consistent with other studies referring to the effectiveness of fixative solutions on some invertebrates in forested scree slopes (Rendoš et al. 2014, 2016a; Mock et al. 2015; Rudy et al. 2018; Jureková et al. 2019). This phenomenon seems to be caused by the repellent effect of formaldehyde solution on some species, observed by several authors (e.g., Renner 1982; Gerlach et al. 2009; Rendoš et al. 2016a). However, some fixative solutions could serve as attractants for different species (Skvarla et al. 2014). Regarding species composition, each of the millipede species preferred ethylene glycol, with some species collected exclusively in this fixative. Altogether, for millipedes there is no benefit to using formaldehyde in comparison to ethylene glycol. Regarding millipedes, it seems there is no sense in using formaldehyde as a fixative. On the contrary, ethylene glycol has been proved to be a suitable fixation. As an inexpensive alternative to ethylene glycol, propylene glycol can be used (Skvarla et al 2014). Propylene glycol is practically non-toxic and has the same preservation properties as ethylene glycol (Jud and Schmidt-Engling 2008; Aristophanous 2010). In addition, it is suitable for molecular studies, as the quality of DNA conservation in undiluted propylene glycol seems to be similar to DNA preserved in ethanol (Moreau et al. 2013).

Positive effects of ethylene-glycol were also observed in centipedes, with higher abundance in ethylene glycol traps documented at majority of the study sites with two fixative liquids used. Species composition, however, showed selective effects of both fixative solutions, as some of the centipede species were collected only in formaldehyde and others only in ethylene glycol. An attractive effect of formaldehyde was observed only in case of symphylans, with more individuals collected in traps using this fixative solution. Any small change in traps, including using different fixatives, can affect results. In our study, we generalise the main features, yet each of the study sites has specific characteristics. For a long-term depth gradient study, it would be advisable to use a completely neutral fixative solution (water); however, this is in principle impossible. After a short period of time, in any type of solution, the carcasses of captured animals accumulate and become attractant or repellent for other animals. The types of subterranean traps used in this study have limitations; however, it is effective for comparison of results to other methods of collecting of soil fauna and does not require excessive and time-consuming effort.

Subterranean diversity of Myriapoda inhabiting scree slopes has been investigated at various localities of mountainous Central Europe to the depth of one meter. Our study represents the first study with a larger number of sites dealing with the issue. Forested scree slopes in the region are usually lacking exclusively subterranean myriapod species and are largely colonised by surface-dwelling species of centipedes and millipedes. Deeper zones of screes are apparently parts of MSS and are clearly preferred only by two blind blaniulid millipedes, Archiboreoiulus pallidus and Cibiniulus slovacus. However, a relatively diverse community of relict myriapod fauna uses debris habitats as a climatic refugium. Although all studied locations and studied groups of invertebrates have their distinctive specifics, general depth distribution of Myriapoda has its pattern as well.

The design of subterranean traps had a significant effect on the findings. This was verified using two different fixative solutions in traps in parallel. The use of ethylene glycol in traps superimposed data obtained by formaldehyde; however, it did not provide a completely different picture of the depth distribution of myriapods. Any of the types of subterranean traps used in this study can only be recommended for any similar study.

The authors express their gratitude to Martina Červená, Vratislav Laška, Jan Mikula, and Tamara Šašková for collecting part of the material of myriapods. We would also like to thank Jana Tufová for identification of some millipedes and Peter Ľuptáčik, Vladimír Papáč, Nikola Jureková, Ľubomír Kováč, Peter Fenďa, Michal Krajňák, Michal Rendoš, and Maroš Dzurinka for their assistance during the field work. The study was supported by the grants VEGA 1/0346/18 and APVV–17–0477.