In the present paper, we report data from a CMR study on an Eastern Herzegovinian population of P. anguinus . Since in situ sexing methods are not yet feasible and we found the marking of juveniles to be too risky, we focussed solely on adults. We were interested in their spatial strategy and general movement patterns. Based on previous experience (Gergely Balázs pers. obs.), we knew that there are certain places where P. anguinus can be regularly seen in the cave systems. However, without individual marking, it was unknown whether these observations represented preferred spots by all individuals or the same individuals holding to the same area. At any rate, the species is capable of effective swimming, moving over tens of metres easily during a single escape burst (Balázs G. pers. obs.), so there is no intrinsic barrier to use large areas within the habitat. Still, based on published laboratory experiments (Parzefall, 1976 ; Guillaume, 2000 ), we hypothesized that adult P. anguinus are not moving randomly in their intuitively simple and homogeneous habitat, but have distinct home ranges.

Amphibian movement patterns are well studied. The field is complex, since it includes several different types of movement‐related topics, like migration from hibernating sites to breeding sites, home range during breeding in the aquatic habitat, home range after breeding in the terrestrial habitat, homing behaviour or breeding site fidelity across successive years. Studies reviewing amphibian movement patterns (e.g. Smith & Green, 2005 ; Wells 2007 ; Sinsch, 2014 ) report high variation including species with very limited movements as well as species where individuals move over large distances. Further, high variation within species is also common (Wells, 2007 ). There are records available from species that can inhabit caves but are not restricted to cave environments. For instance, by studying a Speleomantes ambrosii population in an artificial tunnel, Salvidio et al. ( 1994 ) reported mean home range size of 6 m 2 , while Fenolino et al. ( 2014 ) reported that the aquatic larvae of Eurycea spelaea moved approximately four times more than the terrestrial adults between recaptures. However, the number of studies regarding obligate cave‐dwelling species is limited, not at least because the number of such species is very low. Therefore, understanding P. anguinus spatial strategy would not only give further data to the biology of the species, but also increase our knowledge on amphibian behaviour in general.

In Europe, obligate cave‐dwelling vertebrate species are surprisingly rare, even though subterranean diversity hotspots are abundant. The emblematic European species is the olm ( Proteus anguinus ), which is the only member of its genus, the only troglobiont amphibian in the Palearctic and the largest troglobiont vertebrate in the World. The family Proteidae split from Rhyacotritonidae more than 124 million years ago, while the genus Proteus splits from the only other recent genus Necturus (found in North America) more than 87 million years ago (Zhang & Wake, 2009 ). Proteus anguinus colonized caves approximately 8.8–20 million years ago (Bulog, 1994 ; Trontelj et al. , 2007 ). It is also the troglobiont species with the longest recorded scientific history (see e.g. Zois, 1807 ). Detailed descriptions of its general biology, ecology and behaviour were published long ago (Brieglieb, 1962 , 1963 ). The species is optically blind, all but one of its known populations have lost their pigmentation (Sket & Arntzen, 1994 ); they are neotenic, showing high tolerance for low levels of dissolved oxygen (Issartel et al. , 2009 ) and exhibit an array of non‐visual sensory systems like underwater hearing, rheotaxis and magnetic or olfactory sensing (e.g. Parzefall, 1976 ; Durand & Parzefall, 1987 ; Guillaume, 2002 ; Schlegel, Steinfartz & Bulog, 2009 ). Proteus anguinus is also known for a number of extreme life‐history adaptations, such as high longevity (possibly over 100 years), sparse reproduction (females reproduce ca. once in every 12.5 years) and resistance to starvation (Hervant, Mathieu, & Durand, 2001 ; Issartel et al. , 2009 ; Speakman & Selman, 2011 ; Voituron et al. , 2011 ). To date, ecological or behavioural data on the species typically came from observations made in wild‐caught individuals or captive populations held in laboratory or in semi‐natural conditions. While such research forms the cornerstone of our current knowledge, ex situ results need to be verified under natural conditions. The lack of field studies on P. anguinus is easy to understand; their characteristic habitats are hard to access for researchers and in situ observations are challenging to perform. Previously, we developed a method for underwater tagging of aquatic amphibians with minimal manipulation and without even temporary removal from the site of capture (Balázs, Lewarne & Herczeg, 2015 ), which opened up the possibilities for long‐term CMR studies on P. anguinus .

Subterranean habitats are widespread throughout the world and have been attracting the attention of evolutionary biologists ever since Darwin ( 1859 ). Caves and related habitats are characterized by the absence of light, food scarcity and simplified communities and are strongly buffered against daily, seasonal and yearly environmental variations (Culver & Sket, 2000 ; Gibert & Deharveng, 2002 ; Culver & Pipan, 2009 ). However, the potential of caves in evolutionary ecology remained relatively unexploited. This is particularly true for aquatic caves, which can only be accessed by diving techniques. Aquatic cave ecosystems are important for evolutionary ecologists as an overlooked model system and for conservation biologists as a vulnerable and unique habitat, but we also need to improve our understanding of how these unique ecosystems perform ecological services that benefit ecosystems beyond cave systems, including human access to fresh water (Herman, Culver & Salzman, 2001 ; Bulog et al. , 2002 ). A common approach in gathering basic ecological data from natural populations is capture–mark–recapture (CMR), which can provide information on spatial strategy, movement patterns and dispersal, in addition to population parameters such as sex ratio, survival and recruitment. CMR studies on aquatic troglobionts are rare, restricted to easily accessible cave streams and typically involve the temporary removal of the study individuals (e.g. Knapp & Fong, 1999 ; Zakšek & Trontelj, 2017 ).

We were interested in whether the distance moved by individuals between subsequent recaptures (hereafter ‘distance’, measured in metres) depended on the time elapsed between subsequent recaptures (hereafter ‘time’, measured in days). To this end, we built generalized linear mixed models with Poisson distribution and log link and ran them on both the restricted and extended datasets. In the models, ‘distance’ was the dependent variable, ‘time’ was the fixed predictor variable, and we also added ‘individual’ as a random intercept to control for the non ‐ independence of the repeated observations of the same individuals.

Thirteen individuals out of the 19 were recaptured later. During our pilot study (Balázs et al. , 2015 ), we also tagged seven adults in 2010. Two individuals out of the seven were also recaptured during the 2016 tagging period and again afterwards; hence, these individuals were also included in the analyses. Therefore, we could analyse 15 adult P. anguinus individuals that were captured during the 2016 tagging period with 26 recaptures. We only caught two previously untagged adults in the study transect after the 2016 tagging period (on 03 May 2017), with one being recaptured on another later dive. We did not encounter any other untagged adult individuals after this.

Our study was carried out in the Vruljak 1 Cave (Trebinje, Gorica district, Bosnia and Herzegovina). This is a permanent flow spring cave with only mild floods thanks to the slow release of groundwater from grusified dolomite and dolomitic limestone (for more detail of the cave, see Lewarne, Balázs & Smith, 2010 ; Lewarne, 2016 ). The known passages of the cave are almost entirely under water, with occasional air chambers (i.e. the passage is only partly under water). The cave was chosen due to relative ease of access and the high density of P. anguinus it contains compared to other Herzegovinian sites we know. We monitored a ca. 350 m long section starting from the entrance, but tagged individuals only in the first 270 m. We used the last 80 m of the transect to monitor how far the tagged individuals can disperse. We captured and tagged 19 adult individuals between 07 and 11 May 2016. We note that separating sexually mature adults from immature subadults is ambiguous. However, we only tagged animals that are larger than 20 cm in total length due to a simple reason: this is the size limit for the quick and efficient tagging. Hence, we use the term ‘adult’ without actually knowing whether all tagged individuals reached sexual maturity. However, it is accepted as a rule of thumb to assume sexual maturity over 20 cm in the species (Fišer et al. , 2017 and references therein). These individuals were then followed until 17 September 2018 during four diving expeditions (15–17 September 2016; 03‐06 May 2017; 17–23 September 2017; 17. September 2018). During one expedition to the site, there were often several consecutive dives within few days. However, we only considered the first observation for every individual within a given expedition. All dives were performed in a similar fashion. Two divers were involved: one was always the first author, and the other was a co‐worker from the Caudata Hungarian Cave Research Team (Budapest, Hungary). The divers were always using the same equipment for lighting to standardize the conditions for observing individuals. The divers swam at a slow and steady pace and upon observing a tagged individual; the first author recorded its position, while upon observing an untagged individual, the co ‐ diver caught it, and the first author then performed the tagging. Since the mapping of the cave was done by the same team with the leadership of the first author, accurate positions of the animals could be recorded. In practice, all observed adult P. anguinus were caught. The animals released after tagging always remained in the area.

The detailed methodology of tagging can be found in Balázs et al. ( 2015 ). Briefly, individuals were caught by hand and a black colour Visible Implant Elastomer (VIE, Northwest Marine Technology Inc., Shaw Island, WA, USA) was administered under their tail ‐ fin skin with a standard 29 gauge needle. The whole process took less than 2 min for one individual, and all individuals were released at the exact site of capture. We used a unique marking pattern for each individual that could be visually identified from a distance of approx. 1–2 m during diving.

Distribution, movement and number of observations of the tagged Proteus anguinus individuals along the 270 m long transect (extended dataset). Horizontal dashed lines represent the original polygon (fixed transect rope line followed by the divers, installed as close to the passage bottom midline as possible) of the cave mapping survey. Every individual's capture and recapture points are shown along a separate dashed line for illustrative purposes. Subsequent individuals are marked with different colours (black, grey) for clarity. Distribution is shown in two dimensions; distance scaling of the Y axis is similar to the X axis. When the number of dots for an individual is lower than the number of observations, the given individual was observed in the exact same spot more than once.

The largest distance moved was 38 m during 230 days. No tagged individual was seen out of the 270 m zone where the tagging took place during the entire study period, despite the fact that we observed several untagged individuals in the monitored 80 m after the tagging zone. We found no individuals in the first 50m from the entrance. Because the majority of recaptured individuals moved less than 10 m during several years, while being distributed throughout the study transect, non‐random occurrences within the transect were evident (Fig. 2 , for a realistic presentation within a detailed map, see Electronic Supplement Figure S1 ). Taken together, animals typically moved only short distances during long time periods and longer time periods between recaptures did not predict longer distances moved.

In the restricted dataset, the number of recaptures varied between one and three. There was a large variation in both distance and time between recaptures (distance: median = 5; quartiles = 1.25–11.75; minimum–maximum = 0–38; time: median = 357.5; quartiles = 130–499; minimum–maximum = 98–732). Distance and time were unrelated ( z = 1.60, P = 0.11; Fig. 1 a). This pattern did not change in the extended dataset ( z = −1.74, P = 0.08; Fig. 1 b), even though this dataset included four recapture times (all different individuals) over 1000 days (1480, 2074, 2197 and 2569 days). Interestingly, in three cases out of the latter four recaptures, the given individual was seen in the exact same spot as previously. Individuals did not differ in the restricted dataset ( chi 2 = 1.45; d.f. = 1; P = 0.23), while they did in the extended dataset ( chi 2 = 6.10; d.f. = 1; P = 0.01). However, because individuals were observed for different time periods (28 months vs. 8 years) within the extended dataset, we do not treat significant individual variation as an indicator of different individual movement strategies or animal personality ( sensu Gosling, 2001 ).

Among the 19 individuals tagged in 2016, 13 individuals were recaptured during the ca. 28 months long study period. Among the seven individuals tagged in 2010, five individuals were recaptured during the c. 8 years long study period. These can be considered as high recapture rates. Since no new untagged individuals were encountered in the last two expeditions, we think that the 26 adults seen in the 270 m long tagging zone is a good approximation of the number of adults living there.

Discussion

Despite the wide relevance of P. anguinus for science, conservation and natural resource management, most of our knowledge of the species' biology came from captive populations or wild‐caught individuals held in laboratory. Besides early observations made by Brieglieb (1962, 1963), we are only aware of one study that addressed questions about P. anguinus behaviour studying a natural population in its natural habitat (Zakšek & Trontelj, 2017 showing preliminary data on genetic CMR). In the present paper, we provide data of its spatial strategy and general movement patterns based on CMR methods employed in a natural population. Our most salient finding was the extreme site fidelity of adult P. anguinus: recapture rate was high, we did not observe a single‐tagged individual within 80 m from the tagging area, and the recaptured individuals moved approximately 5 m during a year.

Little is known about the natural behaviour P. anguinus. The species lost its vision (but retained the ability to sense light), but its chemical, magnetic and acoustic senses work well (Parzefall, 1976; Schlegel et al., 2009). Hence, they have a number of means for communication and orientation. In captivity, sexually active individuals are solitary, the males being aggressive, while sexually inactive individuals form groups irrespective of gender (Parzefall, 1976; Guillaume, 2002). Apparently, P. anguinus scent‐marks the substrate it occupies and the mark is recognizable for days (Parzefall, 1976). In a somewhat small aquarium (80 × 30 cm, divided into three compartments for the two‐choice tests; study animals being approximately 20 cm long), Guillaume (2002) experimentally showed that sexually inactive P. anguinus could find their own shelter based on chemical cues even after the shelter was relocated, animals could discriminate between scent‐marks of their own and conspecifics, and they showed gregarious behaviour in a new environment. Guillaume (2000) concluded that P. anguinus prefers previously used shelters over unknown ones because they might be of better quality in terms of fighting currents and that they follow gregarious behaviour to aid searching for food.

In contrast to the laboratory observations, we did not observe signs of gregarious or hiding behaviour either in Vruljak 1 Cave during dozens of dives, or in several other Herzegovinian caves where we have monitored the species for more than a decade. The observed individuals were typically in the open, and even if some individuals were within metres of each other, they showed no other sign of grouping behaviour. In these caves, at least when diving is possible, currents seemingly cause no problem for P. anguinus and the lack of predation makes hiding pointless. On the other hand, the movement patterns revealed by the present study strengthen the previous observations that these animals are frequently associated with certain locations they know well. The surprisingly low movement activity revealed by recaptures adds another facet to the extreme lifestyle of the species. Out of 37 recaptures in the extended dataset, only 10 represented a longer than 10 m and only three longer than 20 m movement, with always more than 100 days having elapsed between sightings. One individual was found at the same location after 2569 days. But how can we invoke the already known adaptations of P. anguinus to explain the extreme site fidelity reported here? The species lives long, resistant to starvation or hypoxic water, reproduces sporadically, and while it lost its vision, it still has a number of well‐developed non‐visual sensory systems allowing efficient orientation (e.g. Parzefall, 1976; Durand & Parzefall, 1987; Guillaume, 2002; Schlegel et al., 2009). Further, looking at the observed spatial distribution we found (Fig. 2; Electronic Supplement Figure S1), there are no evident gaps that would indicate strong spatial avoidance within the studied area. Therefore, we cannot present any strong argument about the benefits of being sedentary or the costs/ risks of moving over larger areas in the studied environment, especially considering the lack of predators and interspecific competitors. We can only speculate that animals feeding on a very low food supply (and as consequence, resistant to starvation), reproducing sporadically (females reproducing on average once in 12.5 years, Voituron et al., 2011) and living for a century are very energy cautious and limit their movements to the minimum. It is noteworthy that genetic studies revealed very low genetic structuring along a 10 km long section of a subterranean river in the Postojna and Planina Caves, so gene flow seemed to be undisturbed (Zakšek et al., 2018). On the other hand, genetic CMR revealed very low individual movements (Zakšek & Trontelj, 2017). It was hypothesized that only juvenile animals are dispersing (Zakšek & Trontelj, 2017).

We note that there are some potential weaknesses in our study. As the monitoring was done by humans diving in the cave passage, any Proteus individuals in inaccessible crevices or fissures were not observed. Our high recapture rate together with the low number of new individuals after the tagging period suggests that we were studying a particular subset of a population inhabiting the relatively wide passage, so we do not know whether animals in tight crevices followed a different movement strategy. We also note that diving in the cave was impossible during the winter flood season. However, when compared with many other locations inhabited by P. anguinus, the floods that do occur are not so intense in this cave because the year‐round permanent inflow supply originates from the slow release of groundwater from grusified dolomite and dolomitic limestone strata, resulting in a steadier base‐flow water supply. The floods only occur when the local, mid‐distance and distant ponors become occasionally active during periods of heavy rainfall, punctuating the pre‐existing base flow with extra inflows of water (Lewarne et al., 2010; Lewarne, 2016).

It is important to ask how extreme is the (in absolute terms) extreme site fidelity we report here compared to other members of Caudata. Looking at available reviews (Smith & Green, 2005; Wells 2007; Sinsch, 2014), one can see enormous variation in amphibian movement distances, dispersal potential and home range sizes. However, ecologically sound comparisons with aquatic cave‐dwelling amphibians are hard to make, because many studies focus on site fidelity regarding breeding pond use between years, movement patterns during migration to or from the breeding ponds, during or outside of the breeding season, during vastly different timescales, etc. Similar data on obligate cave‐dwelling and aquatic amphibians are missing. At any rate, extremely small home ranges and small distances moved are presented in several surface‐dwelling or facultative cave‐dwelling species. Hence, the extreme site fidelity in P. anguinus is not necessarily extreme among amphibians.

Finally, we do not know what happened with the individuals we could not recapture. We have never seen a dead individual; hence, we are confident that the tagging procedure induced mortality was negligible if not zero. We have never seen a tagged individual in the 80 m long passage section we monitored after the 270 m long tagging section; hence, dispersal away from the study area is also unlikely. These animals either remained accidently unseen during the dives following their tagging, or they dispersed into some of the side‐fissures of the main passage. During the monitoring in Vruljak 1 Cave since 2010 (several dives per year), we have observed a stable high density of adults and an abundance of juveniles every year. Hence, we think that this is a ‘healthy’ population and our results are representing natural patterns. However, we also note that our current population‐genetics research (G. Balázs, J. Vörös, B. Lewarne, G. Herczeg, unpublished data) has revealed zero genetic variation in 11 microsatellite loci, which is in contrast to results based on the same loci in four Croatian populations that were found to be highly variable in this respect (Vörös et al., 2019). Whether the background of the low genetic variation (e.g. recent bottleneck, founder effect or severe inbreeding) questions the generality of our results, can only be assessed after the movement patterns of populations with high genetic diversity are also studied.

Taken together, our ecological survey of P. anguinus in its natural habitat revealed extreme site fidelity for the species, with negligible relocations detected between years. It also proved that our tagging method (Balázs et al., 2015) not only works well, but is recommended over alternative approaches including the removal of individuals for tagging out of their natural habitat. The reported extreme site fidelity means that even a few hours of handling followed by a release at the approximate site of capture – that would be intuitively seen as an approach with negligible stress – might cause disturbance of an unknown magnitude for the studied population. This is particularly problematic for P. anguinus, because negative population‐level effects are not easy to detect in animals that live for 100 years, reproduce in every 12.5 years and can survive for several years without food. We hope that our study will stimulate researchers to study other P. anguinus populations, so we can see whether the extreme site fidelity reported in the present paper is a general behaviour throughout the species' geographical distribution or is special to our study population.