In surveillance of subterranean fauna, especially in the case of rare or elusive aquatic species, traditional techniques used for epigean species are often not feasible. We developed a non-invasive survey method based on environmental DNA (eDNA) to detect the presence of the red-listed cave-dwelling amphibian, Proteus anguinus, in the caves of the Dinaric Karst. We tested the method in fifteen caves in Croatia, from which the species was previously recorded or expected to occur. We successfully confirmed the presence of P. anguinus from ten caves and detected the species for the first time in five others. Using a hierarchical occupancy model we compared the availability and detection probability of eDNA of two water sampling methods, filtration and precipitation. The statistical analysis showed that both availability and detection probability depended on the method and estimates for both probabilities were higher using filter samples than for precipitation samples. Combining reliable field and laboratory methods with robust statistical modeling will give the best estimates of species occurrence.

21Biodiversity is declining at unprecedented rates, with species extinctions being an 22 important component of this process. Currently it is almost impossible to evaluate this loss since 23 biodiversity data is lacking even for emblematic species. Recent advances in eDNA marker 24 gene studies promise to deliver the necessary data, but these approaches have almost never 25 been evaluated in the tropics. Here we evaluate whether 1) eDNA metabarcoding may reliably 26 generate frog faunistic data in species-rich tropical areas, 2) the ecological signal in eDNA data 27 is compatible with data from visual and audio encounter survey (VAES), and 3) eDNA data 28 represent a financially viable solution to biodiversity data acquisition. We applied eDNA 29 metabarcoding to investigate frog species occurrence in five ponds in the Chiquitano dry forest 30 region in Bolivia and compared our data with a simultaneous VAES. We find that taxon lists 31 generated with eDNA and VAES correspond closely, and most deviations are attributable to 32 different species' life histories. The ecological signal in eDNA closely matches the signal from 33 VAES. We find that the cost efficiency of eDNA surveys is mostly influenced by the richness of 34 local fauna and the number of surveyed sites: VAES may be less costly in low-diversity regions, 35 but eDNA quickly becomes more cost-efficient in high-diversity regions, particularly with many 36 sites. The results underline that eDNA is suitable for large-scale biodiversity surveys in high-37 diversity areas if precautions in data generation and interpretation are taken and this projects 38 spatially extensive standardized biodiversity surveys in the close future. 39

Rapid environmental change in highly biodiverse tropical regions demands efficient biomonitoring programmes. While existing metrics of species diversity and community composition rely on encounter-based survey data, eDNA recently emerged as alternative approach. Costs and ecological value of eDNA-based methods have rarely been evaluated in tropical regions, where high species richness is accompanied by high functional diversity (e.g., the use of different microhabitats by different species and life stages). We first tested whether estimation of tropical frogs' community structure derived from eDNA data is compatible with expert field assessments. Next, we evaluated whether eDNA is a financially viable solution for biodiversity monitoring in tropical regions. We applied eDNA metabarcoding to investigate frog species occurrence in five ponds in the Chiquitano dry forest region in Bolivia and compared our data with a simultaneous visual and audio encounter survey (VAES). We found that taxon lists and community structure generated with eDNA and VAES correspond closely, and most deviations are attributable to different species' life histories. Cost efficiency of eDNA surveys was mostly influenced by the richness of local fauna and the number of surveyed sites: VAES may be less costly in low-diversity regions, but eDNA quickly becomes more cost-efficient in high-diversity regions with many sites sampled. The results highlight that eDNA is suitable for large-scale biodiversity surveys in high-diversity areas if life history is considered, and certain precautions in sampling, genetic analyses and data interpretation are taken. We anticipate that spatially extensive, standardized eDNA biodiversity surveys will quickly emerge in the future.

Properly designed (randomized and/or balanced) experiments are standard in ecological research. Molecular methods are increasingly used in ecology, but studies generally do not report the detailed design of sample processing in the laboratory. This may strongly influence the interpretability of results if the laboratory procedures do not account for the confounding effects of unexpected laboratory events. We demonstrate this with a simple experiment where unexpected differences in laboratory processing of samples would have biased results if randomization in DNA extraction and PCR steps do not provide safeguards. We emphasize the need for proper experimental design and reporting of the laboratory phase of molecular ecology research to ensure the reliability and interpretability of results.

Dendrobaena byblica (Rosa, 1893) is a Circum-Mediterranean species complex composed of at least 17 nominal taxa. Regarding the most important species characters (clitellum and tubercles) the worms belonging to the genus Fitzingeria Zicsi, 1978 seem to be very close to the D. byblica species group. Here we provide the first molecular phylogenetic analysis of the byblica species group and the Fitzingeria species, and show that the genus Fitzingeria is polyphyletic and groups together with the Carpathian and Balkanic deeply pigmented byblica forms. The other main clade consists of the ‘classical’ byblica species. The morphological characteristics also support these results. On the basis of the molecular and morphological characters the species of the former Fitzingeria were relegated to Dendrobaena and four new species were described together with a new replacement name Dendrobaena carpathomontana nom. nov. for F. platyura montana (Černosvitov, 1932).

Molecular and morphological evolution of species may be decoupled which can lead to contrasting delimitation of taxonomic units based on morphological and genetic characters. The moss genus Sphagnum (peat mosses) provides a good example in which morphological and genetic characters often contradict each other. We investigated morphological and genetic variability within a highly polymorphic and taxonomically controversial species complex of the genus Sphagnum in order to reveal the ultimate causes of discordance between morphology and molecular evolution. Specifically, we aimed at addressing the following questions: (1) Are the three closely related European taxa (S. angustifolium, S. fallax, S. flexuosum) of the S. recurvum group morphologically and genetically distinct entities? (2) Are morphological and genetic groups concordant? (3) Is there any sign of hybridization among taxa that might complicate their morphological delimitation? We show that European specimens of the S. recurvum group form three distinct gene pools based on 11 polymorphic microsatellite markers. Individuals defined as morphotype “fallax” appear to be genetically clearly distinct from both morphotype “angustifolium” and “flexuosum”. However, for the latter two morphotypes, traditionally used morphological and genetic markers provide partly incongruent taxonomic delimitations. We show that in about 13% of the accessions 12 traditionally used morphological characters cannot fully distinguish the genetic groups revealed. Our genetic analysis also reveals that only a low proportion of the specimens (3%) is admixed suggesting that hybridization is unlikely to explain the taxonomical controversies observed. Therefore, we hypothesize that taxonomic controversies are likely due to the extreme phenotypic plasticity of morphological characters rather than to porous species boundaries.

Properly designed (randomized and/or balanced) experiments are standard in ecological research. Molecular methods are increasingly used in ecology, but studies generally do not report the detailed design of sample processing in the laboratory. This may strongly influence the interpretability of results if the laboratory procedures do not account for the confounding effects of unexpected laboratory events. We demonstrate this with a simple experiment where unexpected differences in laboratory processing of samples would have biased results if randomization in DNA extraction and PCR steps do not provide safeguards. We emphasize the need for proper experimental design and reporting of the laboratory phase of molecular ecology research to ensure the reliability and interpretability of results. K E Y W O R D Sbatch effect, bias, DNA extraction, environmental DNA, laboratory practice, lake community, metabarcoding, nondemonic intrusions, PCR, sediment

Abstract. The Allolobophora sturanyi Rosa, 1895 species group is revisited using DNA barcoding and morphology. Barcoding results corroborated the previous treatment of the Allolobophora sturanyi subspecies and furthermore proved that the morphologically similar Allolobophora gestroides Zicsi, 1970 species belong to this species group. Elaboration of new samples from the Apuseni Mts resulted in discovery of a new subspecies A. sturanyi biharica ssp. nov. from the summit of the Bihor range, and a new species A. zicsica from the Vladeasa range similar to A. gestroides described from Northern Hungary.