The cave salamander Proteus represents a flagship and keystone genus of the subterranean ecosystems of the Dinaric Karst. Currently classified as “Vulnerable” by the International Union for Conservation of Nature, its long-term monitoring is essential for understanding subterranean ecosystem dynamics and for guiding evidence-based conservation strategies. Given its protected status and the strict permitting requirements for research and monitoring, the development of non-invasive protocols is of paramount importance. The present study aims to present a non-invasive, underwater-capable photographic tracking method for this highly vulnerable subterranean species that minimizes handling-related stress and potential impacts on animal welfare. Specifically, this work explores the application of UV photography as a novel tool for the detection and characterization of individual skin patterns, enabling the recognition of over 300 individual specimens based on unique and naturally occurring morphological features. By providing a reliable means of distinguishing individuals without the need for invasive or potentially stressful traditional marking techniques, such as physical tagging, UV-based imaging may represent a valuable alternative for the long-term monitoring of olm populations. This approach contributes to the development of scientifically grounded, welfare-centered monitoring protocols that effectively balance conservation requirements with the highest standards of animal care while improving the accuracy, repeatability, and ethical sustainability of individual-based ecological studies. Importantly, although recent evidence has revealed the presence of at least two distinct Proteus species within the study area, the present imaging-based approach remains effective for the individual identification of specimens across both species. This demonstrates that the method captures sufficient inter-individual variation to reliably distinguish individuals, irrespective of their species identity, highlighting its robustness and broad applicability in sympatric Proteus spp. populations.
Achalinus vanhoensis Ha et al., 2022 was previously known only from the adult male holotype collected in Van Ho, Son La Province, northwestern Vietnam. The second known specimen and first known female of the species are reported from Cuc Phuong National Park, Ninh Binh Province, northern Vietnam. Morphologically, the female differs from the male holotype mainly in having a proportionally shorter tail (tail length/total length ratio 0.192 vs. 0.264), more ventrals (202 vs. 176), and fewer subcaudals (74 vs. 84). These differences may reflect sexual dimorphism in A. vanhoensis . However, as the species is currently known from only one specimen of each sex, additional specimens are required to determine whether these differences represent consistent sex-related variation or individual variation. Molecular phylogenetic analyses showed that the newly collected specimen clustered with the holotype with moderate support and exhibited an uncorrected COI p -distance from it of 3.8%, supporting its identification as A. vanhoensis . The new record extends the known range of the species approximately 112 km southeastward and lowers its documented elevational limit from 1,121 to 440 m a.s.l. An updated identification key to the nine species of Achalinus recorded from Vietnam is also provided, and it is provisionally recommended that A. vanhoensis be assessed as Data Deficient (DD) under the IUCN Red List Categories and Criteria (IUCN Standards and Petitions Committee 2024).
The herpetofauna of the biodiverse Sakar Mountains and Dervent Heights (SE Bulgaria) remains understudied. The most recent review provided only limited distributional data for 8 amphibian and 20 reptile species (Stoev 2000), while subsequent records have been published either as isolated observations or as part of national-scale species mapping, typically at a resolution of 10×10 km. We analyzed georeferenced herpetological observations collected between 2003 and 2025 from four Special Areas of Conservation within the Natura 2000 ecological network: “Sakar” (BG0000212), “Zhdreloto na reka Tundzha” (BG0000217), “Derventski vazvishenia 1” (BG0000218), and “Derventski vazvishenia 2” (BG0000219). We compiled 10,649 records (150 from GBIF and 10,499 from SmartBirds.org), representing 34 taxa: 10 amphibians (3 caudates and 7 anurans) and 24 reptiles (4 chelonians and 20 squamates, including 9 lizards and 11 snakes). We mapped the observations on a 2×2-km grid to identify priority areas for future surveys and for conservation management. The occurrence records fall within 42 of the 46 (91%) 10-km MGRS grid cells encompassing the study area and 432 of the 716 (60%) 2-km grid cells. We confirmed the presence of all taxa expected to occur in the study area, including the first documented observation of Salamandra salamandra . We identified vehicular traffic, fires, and removal of shrubs from grasslands as major threats. Specifically, 3.7% of the records were of road-killed individuals, representing 19 of the 34 taxa. Our habitat analysis confirmed that grasses and shrubs are key for maintaining high reptile abundance and diversity. However, over the past 20 years, these habitats have been under strong anthropogenic pressure, highlighting the need for urgent conservation measures.
Determining the breeding frequencies and breeding periods of species that spend part or all of their lives underground is challenging because of limited population surveys. Environmental DNA (eDNA) analysis can be performed to address this issue. eDNA analysis and population surveys were combined to verify and compare the breeding frequencies and breeding periods of Onychodactylus koreanus and O. sillanus in mountain valley streams, which are their typical habitats in the Republic of Korea, between April 2024 and May 2025. Both species externally fertilize their eggs. Adult O. koreanus appeared at the sites during two periods, from April to June and from October to early December, whereas adult O. sillanus was not detected despite similar survey efforts. eDNA concentrations detected in the upper, middle, and lower streams differed; however, the major eDNA detection patterns were largely consistent. In O. koreanus , two major eDNA detections occurred in early May and early December, whereas in O. sillanus , eDNA detections occurred between January and June. These results suggest that the two species have different breeding frequencies and breeding periods. However, whether these differences are species- or habitat-specific needs to be further explored.
Reliable individual identification is fundamental to ecological and behavioural studies, yet many marking techniques are invasive and may affect animal behaviour or survival. Biometric identification approaches offer a non-invasive alternative for individual recognition using phenotypic traits. In amphibians, body patterns, especially colouration, have been used to identify individuals, but species lacking permanent body patterns require alternative traits. This study evaluated whether iris patterns could serve as a marker for individual recognition in the treefrog Osteocephalus oophagus . A capture-recapture study was conducted over three breeding seasons (2021–2023) in French Guiana. At each encounter, eye photographs were compared with an image library to identify individuals without handling. Manual identifications were independently verified using Wild-ID software. Across 392 capture events involving 103 individuals, iris patterns were highly distinctive among individuals and enabled repeated identification across encounters. Within-season recapture rates ranged from 56% to 80%, indicating that iris patterns remained sufficiently stable for short-term monitoring. However, only three individuals were re-identified across years, preventing a robust assessment of long-term pattern stability. As these individuals showed no substantial alteration in iris patterns, the low interannual recapture rate was more likely explained by population turnover or emigration than by changes in iris morphology. The findings suggest that iris recognition provides an effective, non-invasive method for short-term individual identification in O. oophagus , reducing the need for repeated handling and supporting ethical wildlife research. Further studies are needed to assess the long-term stability of iris patterns and evaluate the applicability of this method to other amphibian species.
