South Western Italian Alps are rich in natural caves of diverse origins and at different altitudes. The range of cave temperature is varied, with cold caves yielding temperatures lower than 8 °C, mainly at high altitude, and more mesophilic caves with average temperature at around 9–13 °C. This area is also rich in artificial cavities of interest for hypogean life study (Fig. 1).

In this area, six hypogean species of the E. spelaea species-complex have been reported (Balestra et al. 2019; Christian et al. 2014; Isaia et al. 2011; Pantini and Isaia 2019): E. spelaea, E. strinatii Condé, 1977, E. bonadonai (Condé, 1979), E. roscia Christian, 2014, E. lanai Christian, 2014, and an undescribed species of Eukoenenia. (E. Christian in litteris).

The investigated cavities are listed in Table 1 and reported in Fig. 1. Their entrances are located at altitudes between 554 and 2163 m a.s.l. Two of them, Grotta della Mottera and Carsena di Piaggia Bella, are cold caves while the others 18 have an average temperature ranging between 9 and 13 °C. Miniera superiore di Monfieis is an artificial cavity.

The majority of the data reported in this paper were collected from the Bossea cave, the first Italian show cave, that is composed by different environments, and from four other cavities where Eukoenenia strinatii have been already reported (Balestra et al. 2019).

Pre-evaluations based on literature research, biogeographic and field observations allowed the selection of 20 different caves where biotic and abiotic factors that can influence palpigrade life as temperature (T °C), relative humidity (RH%), light intensity (LI lux), type of substrate, speleothems, presence, and abundance of decomposing organic matter and presence of water and fractures were monitored.

Random observations of each cave were performed in different periods of the year, with a minimum number of 2 days per cave in two different seasons. One up to six observation areas were defined for each cave at different distances from the cave entrance (ranging from few meters from the entrance to great depths) depending on the cave structure and the presence of peculiar microhabitat (Table 1). These areas were coded according to the cave name (three letters) and a number, corresponding with the inner sampling sites. The observations for each cave area lasted 20 minutes. Visual encountered surveys, supported with the acquisition of macrophotographs were performed. This is a non-invasive method, however, it required a priori biological knowledge of the taxa observed for the identification and it does not allow always a correct determination at a specific level of the taxa. Palpigrades are tiny arachnids and often close related species have very uniform general morphology, consequently, specimens for species identification were collected manually, using a metal spatula made by one of the authors (E.L.) which is more effective in collecting palpigrades, and placed directly in 70–96% ethyl alcohol in sampling tubes. Identification was performed by specialists, as listed in the acknowledgments.

Macrophotography has several advantages, such as highlighting details not visible at naked eye or reviewing behaviour of the observed individuals, however, also different disadvantages, especially in cave where bringing photographic equipment can be complex due to habitat impediments (see Balestra et al. 2021; Mammola et al. 2021a). Photographs of the specimens were taken using a Canon EOS 550D and 760D reflex cameras equipped with MP-E 65 mm Macro lenses and MT-24EX Macro flash and Canon EOS 70D reflex camera equipped with EF 100 mm Macro lens 1:2.8 USM and integrated flash. For the environmental photos, a Canon EOS 70D reflex camera equipped with EFS 18–55 mm lens and a Canon Power Shot D30 camera were used.

Environmental parameters were recorded in the presence of living animals. An HD 2101.1 Delta Ohm Thermohygrometer with a combined probe HP 472AC %RH and temperature Pt100 were used for environmental temperature and relative humidity measurements (Thermohygrometer HD 2101.1 Delta Ohm: Temperature: -200 / +650 °C, Relative humidity: 0.0 / 100.0%. HP 472AC %RH and temperature Pt100 combined probe: Area of use: -20 / +80 °C, 0 / 100% RH; accuracy: ±2% (5 to 95% RH), ±3% (95 to 99% RH), ±0.3 °C (-20 to + 80 °C)). Due to the use of a 2.5 m long extension for the termohygrometer probe, all parameters were recorded without close human presence. pH was measured using litmus paper (Vetrotecnica 08.3000.00 pH 1 / 11).

114 surveys in caves were performed finding at least one Eukoenenia specimen in 65.79% of the cases. No specimens were observed in cold caves (Grotta della Mottera and Carsena di Piaggia Bella) or in Grotta occidentale del Bandito, despite a previous record from this cave (Brignoli 1976). A total of 143 individuals in 17 different caves of the Western Italian Alps were observed (Table 2). 110 individuals were observed in the last 4 years of monitoring. The number of specimens observed in a sampling site in a day ranged from 0 to 5 (Fig. 2), with an average of 0.93 individuals/sampling site/day, and the number of specimens in a cave ranged from 0 to 8 per day.

Temperature (T °C) and relative humidity (RH%) data related to the presence of 43 living individuals in eight different caves for different species have been recorded (Table 2, Fig. 3A, B). Eukoenenia species in Western Italian Alps resulted to be present in a temperature range between 9.0 and 13.7 °C and in a range of RH% between 81 and 100%.

A negative correlation between temperature and relative humidity is observed for the presence of Eukoenenia strinatii (r: -0.9285) (Fig. 3B). For this species, observation sites at low temperature have a higher RH% compared to the sites at higher temperature. Correlation between the two parameters was not calculated for the other species due to the paucity of data despite a potential trend, shown in Fig. 3A.

The pH of the water pools where 52 specimens were observed was close to neutral (pH 7). This result was obtained from 32 sampling sites, of which13 measurements were repetitions from the same site performed in different seasons confirming no seasonal variation (Table 2). The only exception was recorded from the Buranco di Bardineto cave, where the pH was nearly 6 in a single pool which was particularly rich in organic substance.

A total of 143 Eukoenenia individuals were observed on different microhabitat with the majority of them from water surfaces: one on wet wood, one under a stone, three near water, ten on the cave ground and 128 on the surface of pools that had calm water or a weak current (Table 2, Fig. 4, 5), especially in the rimstone dams, also called gours (Fig. 5A) – a particular type of speleothems (cave formation) in the form of a stone dam (Hill and Forti 1997). Moreover 16 dead individuals were observed but not considered for the ecological interpretation of the taxon and it has not been possible to define the way in which they reach the place they were observed.

A significant association between Eukoenenia individuals and environments rich in organic matter was observed (Yates’ Chi square: 62.41, p < 0.000), in fact, in the rimstone dams, where 112 living Eukoenenia individuals were sampled, trophic resources were abundant. Cave rafts, that are mainly calcite crystals, are common on the surface of quiescent waterbody such as the rimstone dams (Hill and Forti 1997) (Fig. 5E) and they often “trap” dead animals, fungal hyphae and organic remains (Fig. 5F).

Eukoenenia individuals were observed in different sampling areas ranging from a few meters to more than 500 m from the entrance. However, due to the sampling method and the structure of the investigated caves, no conclusion can be drawn about the effect of the distance from the entrance to the taxon distribution.

In addition, it is worth mentioning that E. strinatii was also observed in 3 sampling areas in the touristic Bossea cave where light, even if not direct or continual, was present.

As previously mentioned, an association between the presence of living specimens and the presence of organic matter on the water surface was demonstrated in the sampling area. Individuals of Eukoenenia were also observed feeding on different species of dead springtails. In particular, in June 2016, an E. strinatii individual was observed and photographed for the first time feeding on dead springtail (Balestra et al. 2019; Lana et al. 2016) (Fig. 6). The observed specimen (observation made through Canon macro lens, 65 and 100mm, f/2.8) moved on the surface of the water in an unusual way, using the three pairs of hind legs and the palps on the liquid, keeping up the legs of the first pair stretched forward and the flagellum in a vertical position (Fig. 6A–D). The arachnid approached a dead springtail, Pseudosinella alpina Gisin, 1950, and began to suck its internal liquids having damaged the integument of the corpse in the abdomen with the chelicerae; a drop of exudate came out in which Eukoenenia immersed the chelicerae and approached the mouthparts (Fig. 6E–G). The drop of exudate was consumed in about 15 minutes and then the specimen moved away from its meal with a particular walking: with short steps, with the paws of the second and third pair and the palps aligned, the paws of the first pair raise and face forward and the abdomen is raised with the flagellum pointing upwards (Fig. 6H). In addition, in June 2018, in Rio Borgosozzo cave, a specimen of E. strinatii that carried a dead springtail, holding it with chelicerae, was photographed (Fig. 7). While moving, it held the same position of the E. strinatii as previously described from the Bossea cave after its meal.

In contrast with the feeding activity of Eukoenenia on dead springtail specimens and despite the observed proximity between specimens of Eukoenenia and living and floating entomobrid Collembola apart from rare phenomena of palpation with the paws of the first pair, no attacks by the palpigrades on the living springtails were observed, recorded and documented.

On the contrary, in one case we observed and documented a potential response of a springtail against Eukoenenia (Fig. 8). After several palpations by the palpigrade on two springtails (Fig. 8A, B), E. strinatii tried to climb on one of the springtails (Fig. 8C), which in reaction, potentially bit the arachnid in the lower part of the body. The springtail moved to another part of the gour and for a few moments E. strinatii remained lying on its side, touching the mesosoma with its chelicerae (Fig. 8D). Later the arachnid got up and started to walk again on the surface of water.