Can the subterranean fauna be used as proxy for past environmental changes? – the example of the Carpathians cave fauna

Carpathian Mountains were one of the main refuge areas during the climate changes of the Pleistocene and the Holocene in Europe and one of the richest regions in the world in subterranean (caves and associated habitats) endemic species. Nevertheless, the Carpathian Mountains subterranean fauna importance is underestimated especially due to dispersed information on its diversity and the scarcity of molecular studies in the area. Here, we present a first general view of the cave fauna hotspot represented by the Romanian Carpathians and the geological and historical processes that shaped the patterns of subterranean distribution and diversity at regional scale. The Carpathians are an amalgam of various geological units with complex paleogeographical evolution that is reflected in completely different species assemblages dominated by unit specific fauna groups. Phylogeography of Coleoptera and environmental parameters are adding to the general view at regional scale and offer additional explanation for this exceptional subterranean diversification in a non-Mediterranean region. We also use the example of the Carpathians cave fauna as proxy for past environmental changes in the area. Troglobionts are endemic on small areas and by studying their present distributions and phylogeny, past processes of landscape evolution on the surface can be better understood.

Comparative phylogeography of two troglobitic Coleoptera (Leiodidae, Leptodirini) species from Romania based on mitochondrial DNA

About 50 species of cave-obligate Leptodirini (Leiodidae) beetles have been described so far in Romania, most of them populating caves in the Apuseni Mountains (north-western Romania) and the Southern Carpathians. In this contribution, we present the first molecular phylogeographic study of the two troglobiotic Pholeuon species from the Apuseni Mountains. The two species are Pholeuon (s.str.) leptodirum and Pholeuon (Parapholeuon) gracile, endemic to Bihorului Mountains and Pădurea Craiului Mountains, respectively. To examine the genetic divergence within and between the two species we sequenced 571 bp of the mitochondrial COI gene in a total of 145 specimens, 56 specimens of the first species (collected in five caves) and 89 of the second species (collected in eight caves) across their geographic ranges. We found very low genetic variation, four haplotypes in P. leptodirum and seven haplotypes in P. gracile, and a maximum of 0.7% and 0.9% intraspecific divergence, respectively. However, a significant genetic divergence of 6.55% was found between species. The results are consistent with previous definitions of the two species based on morphological characters, while caution should be taken in considering attributions to different subspecies. Our research contributes to the phylogeographic information of troglobitic beetles, providing a solid basis for future comparison with other terrestrial or aquatic cave adapted species

Comparative phylogeography of two troglobitic Coleoptera (Leiodidae, Leptodirini) species from Romania based on mitochondrial DNA

About 50 species of cave-obligate Leptodirini (Leiodidae) beetles have been described so far in Romania, most of them populating caves in the Apuseni Mountains (north-western Romania) and the Southern Carpathians. In this contribution, we present the first molecular phylogeographic study of the two troglobiotic Pholeuon species from the Apuseni Mountains. The two species are Pholeuon (s.str.) leptodirum and Pholeuon (Parapholeuon) gracile, endemic to Bihorului Mountains and Pădurea Craiului Mountains, respectively. To examine the genetic divergence within and between the two species we sequenced 571 bp of the mitochondrial COI gene in a total of 145 specimens, 56 specimens of the first species (collected in five caves) and 89 of the second species (collected in eight caves) across their geographic ranges. We found very low genetic variation, four haplotypes in P. leptodirum and seven haplotypes in P. gracile, and a maximum of 0.7% and 0.9% intraspecific divergence, respectively. However, a significant genetic divergence of 6.55% was found between species. The results are consistent with previous definitions of the two species based on morphological characters, while caution should be taken in considering attributions to different subspecies. Our research contributes to the phylogeographic information of troglobitic beetles, providing a solid basis for future comparison with other terrestrial or aquatic cave adapted species

Groundwater contamination and human health risk assessment in the main karst areas of Romania

Long-term monitoring reveals the temporal evolution of groundwater chemistry and the potential human health risk posed by the use of contaminated waters (Giri and Singh 2015). Groundwater aquifers are an important source of drinking water in Romania. This study was conducted to appraise the groundwater chemistry and the potential non-carcinogenic risks for human health associated with the groundwater consumption through oral and dermal pathways. In order to achieve this aim, 193 samples were collected from 29 groundwater sources in the main karst areas of Romania during 2019-2021. A total of 15 parameters were analyzed (F-, Cl-, SO42-, NO3, Ca, Na, K, Mg, Fe, Mn, Cu, Zn, Ni, Cr, and As) and compared with the World Health Organization standards for drinking water (WHO 2017). The heavy metals pollution index showed that no groundwater samples had detectable levels of metal contamination, while the heavy metal evaluation index revealed that 4 out of 29 groundwater sources were classified as contaminated: 3 sources with a medium level of pollution and 1 source with a high level of pollution, indicating a potential risk for human health. The human health risk for oral exposure indicated a potential non-carcinogenic risk only in the karst area from South Dobrogea. The non-carcinogenic risk posed by nitrates is higher than that posed by metals in the aquifers from South Dobrogea. Therefore, control and remedial actions should be taken to ensure appropriate water quality for the locals using the contaminated water sources

Database of Romanian cave invertebrates with a Red List of cave species and a list of hotspot/coldspot caves

The increasing human impact in Romanian caves raises the urgency of publishing a correct database of the strictly-adapted cave fauna. Previous attempts at indexing cave fauna and classifying caves by using their fauna opened many questions regarding the use of an incomplete list of cave species and mixed lists of troglobionts/stygobionts with troglophiles/stygophiles for ranking caves with priority for protection. It has also become obvious that there is a need to publish a list of Romanian cave species that are under threat. Cave species in Romania (and elsewhere) are endemic on small ranges, are unique and must be considered as important units for conservation. A cave must be equally protected if it has one or more rare and strictly endemic cave species. Although not exhaustive, we here provide the first checklist of Romanian troglobionts/stygobionts developed in the framework of the DARKFOOD and GROUNDWATERISK projects, coordinated by the "Emil Racovita" Institute of Speleology, Cluj-Napoca, Romania. The GIS application was used to complement the checklist of cave species with data on caves and surface environments above the caves. Until complete data on species diversity and population sizes are made available for each cave, measures of conservation can be implemented, based on the presence/absence of cave species, while classifications of caves for protection, based on the number of species, must be avoided. We also propose a list of Romanian caves with fauna that are under threat and a tentative Red List of Romanian troglobiont/stygobionts.This is the first database with identified troglobiont and stygobiont species of Romania, with a critical analysis of their distribution inside the country. A list of caves that need protection for their rare and unique species and a tentative Red List of Romanian cave fauna are also added. A total of 173 species were identified, of which 77 troglobionts and 96 stygobionts are currently registered in 366 caves. The database is divided into two parts, one part with a list of troglobionts, their revised systematic position, cave name, cave code and geographic region; and the second part with the same information on stygobionts. The database represents the contribution of many active researchers, who are the authors of this paper and of review publications of many other authors of the "Emil Racoviță" Institute of Speleology

The microbiome of phosphate-rich deposits in Muierilor Cave, South-Western Carpathians

Muierilor Cave is one of the most important caves in Romania from paleontological, biological, and archaeological points of view. A newly discovered chamber, with unique yellow calcite crystals, fine-grained crusts, and black sediments, is connected to the upper levels that contains fossil bones and a large pile of guano. Samples were taken from this chamber and another passage to investigate the diversity of microorganisms related to the substrates and identify potential pathogenic taxa for humans and animals. Chemical, mineralogical, and whole community 16S rRNA gene-based metabarcoding analyses were undertaken, and the base of the guano deposit was radiocarbon dated. Metabarcoding of the analyzed samples found that ~16% of the identified bacteria are potentially pathogenic to humans. Moreover, more than 87% of the identified genera were not previously reported in caves. We identified bacteria involved in the phosphate cycle that can only originate from the organic deposits inside the cave, such as the bats' guano in the touristic, upper level, or the fossil bones. Our study indicate also the guano deposit as the possible source of potentially pathogenic microorganisms. The results on the microbiome of different deposit types unravel the diversity of microorganisms and indicate the potentially pathogenic taxa for humans and animals

Spatial Variation of Water Chemistry in Aries River Catchment, Western Romania

This study aims to investigate the quality and vulnerability of surface water (Aries River catchment) in order to identify the impact of past mining activities. For this purpose, the pollution and water quality indices, Piper and Durov plots, as well vulnerability modeling maps were used. The obtained results indicate that the water samples were contaminated with As, Fe, Mn, Pb and have relatively high concentrations of SO42−, HCO3−, TDS, Ca, K, Mg and high values for the electrical conductivity. Possible sources of the high content of chemicals could be the natural processes or the inputs of the mine drainage. Generally, according to the pollution indices, which were correlated to high concentrations of heavy metals, especially with Pb, Fe and Mn, the water samples were characterized by heavy metals pollution. The water quality index classified the studied water samples into five different classes of quality, namely: unsuitable for drinking, poor, medium, good and excellent quality. Similarly, medium, high and very high vulnerability classes were observed. The Durov and Piper plots classified the waters into Mg-HCO3− and Ca-Cl− types. The past and present mining activities clearly change the water chemistry and alter the quality of the Aries River, with the water requiring specific treatments before use

Potential for Natural Attenuation of Domestic and Agricultural Pollution in Karst Groundwater Environments

In karst areas, anthropogenic contaminants reach the subsurface with detrimental effects on the groundwater ecosystem and downstream springs, which often serve as drinking water sources for the local human communities. We analyzed the water chemistry and microbial community composition in upstream and downstream locations of five hydrokarst systems (HKS) during four seasons. Conductivity and nitrates were higher in the downstream springs than in the pre-karst waters, whereas the concentration of organic matter, considered here as a pollution indicator, was lower. The microbial community composition varied largely between upstream and downstream locations, with multiple species of potentially pathogenic bacteria decreasing in the HKS. Bacteria indicative of pollution decreased as well when passing through the HKS, but potential biodegraders increased. This suggests that the HKS can filter out part of the polluting organic matter and, with it, part of the associated microorganisms. Nevertheless, the water quality, including the presence of pathogens in downstream springs, must be further monitored to control whether the water is appropriate for consumption. In parallel, the human populations located upstream must be advised of the risks resulting from their daily activities, improper stocking of their various wastes and dumping of their refuse in surface streams