Life in the dark: metagenomic evidence that a microbial slime community is driven by inorganic nitrogen metabolism

Beneath Australia''s large, dry Nullarbor Plain lies an extensive underwater cave system, where dense microbial communities known as ‘slime curtains'' are found. These communities exist in isolation from photosynthetically derived carbon and are presumed to be chemoautotrophic. Earlier work found high levels of nitrite and nitrate in the cave waters and a high relative abundance of Nitrospirae in bacterial 16S rRNA clone libraries. This suggested that these communities may be supported by nitrite oxidation, however, details of the inorganic nitrogen cycling in these communities remained unclear. Here we report analysis of 16S rRNA amplicon and metagenomic sequence data from the Weebubbie cave slime curtain community. The microbial community is comprised of a diverse assortment of bacterial and archaeal genera, including an abundant population of Thaumarchaeota. Sufficient thaumarchaeotal sequence was recovered to enable a partial genome sequence to be assembled, which showed considerable synteny with the corresponding regions in the genome of the autotrophic ammonia oxidiser Nitrosopumilus maritimus SCM1. This partial genome sequence, contained regions with high sequence identity to the ammonia mono-oxygenase operon and carbon fixing 3-hydroxypropionate/4-hydroxybutyrate cycle genes of N. maritimus SCM1. Additionally, the community, as a whole, included genes encoding key enzymes for inorganic nitrogen transformations, including nitrification and denitrification. We propose that the Weebubbie slime curtain community represents a distinctive microbial ecosystem, in which primary productivity is due to the combined activity of archaeal ammonia-oxidisers and bacterial nitrite oxidisers.     

Isolation and metagenomic characterization of bacteria associated with calcium carbonate and struvite precipitation in a pure moving bed biofilm reactor-membrane bioreactor

A bench-scale pure moving bed bioreactor-membrane bioreactor (MBBR-MBR) used for the treatment of urban wastewater was analyzed for the identification of bacterial strains with the potential capacity for calcium carbonate and struvite biomineral formation. Isolation of mineral-forming strains on calcium carbonate and struvite media revealed six major colonies with a carbonate or struvite precipitation capacity in the biofouling on the membrane surface and showed that heterotrophic bacteria with the ability to precipitate calcium carbonate and struvite constituted ~7.5% of the total platable bacteria. These belonged to the genera Lysinibacillus, Trichococcus, Comamomas and Bacillus. Pyrosequencing analysis of the microbial communities in the suspended cells and membrane biofouling showed a high degree of similarity in all the samples collected with respect to bacterial assemblage. The study of operational taxonomic units (OTUs) identified through pyrosequencing suggested that ~21% of the total bacterial community identified in the biofouling could potentially form calcium carbonate or struvite crystals in the pure MBBR-MBR system used for the treatment of urban wastewater.     

Studies in cave life evolution: a rationale for future theoretical developments using phylogenetic inference

As in every field of comparative biology, phylogeny provides an independent reference system in studies on cave life evolution to test current theoretical proposals. Using phylogeny, sound hypotheses on the ancestral states of characters and their subsequent changes can be made by polarizing the characters between related taxa. Hypotheses on evolutionary processes can also be tested by comparing the patterns they imply with independently inferred phylogenetic patterns. The power of the tests relies upon the independence of phylogenetic patterns (built with cladistics using Wagner parsimony) and the theoretical proposals under study. Classical assumptions on the evolution of troglobitic life are analysed with this methodology. The following points are discussed: what is a troglobitic taxon? Are there features characteristic of troglobitic taxa? Is troglobitic life an evolutionary dead end? What circumstances favour troglobitic evolution? Using phylogenetic analysis, the presence or absence of so-called troglomorphic features were inferred in troglobitic taxa. In fact these taxa can be characterized only by their behavioural ecology. Pre-adaptations (exaptations) can also be precisely defined. Cave living does not appear to be an evolutionary dead end. Two patterns subsequent to cave life appearance have been documented: speciation of troglobitic taxa in the subterranean environment, and reversal to an epigean habitat. Troglobitic life thus turns out to be one step in the diversification of clades. Troglobitic life is usually explained as an evolution under the pressure of unfavourable environmental conditions, or the conquest of a new resource, or the result of biological interactions (competition, predation). Phylogenetic analyses show that none of these hypotheses propose clear alternatives on cave life evolution. Moreover most of their a priori statements cannot easily be falsified. As such they have only limited explanatory power.     

Ancient mitochondrial genomes from Chinese cave hyenas provide insights into the evolutionary history of the genus Crocuta

Cave hyenas (genus Crocuta) are extinct bone-cracking carnivores from the family Hyaenidae and are generally split into two taxa that correspond to a European/Eurasian and an (East) Asian lineage. They are close relatives of the extant African spotted hyenas, the only extant member of the genus Crocuta. Cave hyenas inhabited a wide range across Eurasia during the Pleistocene, but became extinct at the end of the Late Pleistocene. Using genetic and genomic datasets, previous studies have proposed different scenarios about the evolutionary history of Crocuta. However, causes of the extinction of cave hyenas are widely speculative and samples from China are severely understudied. In this study, we assembled near-complete mitochondrial genomes from two cave hyenas from northeastern China dating to 20 240 and 20 253 calBP, representing the youngest directly dated fossils of Crocuta in Asia. Phylogenetic analyses suggest a monophyletic clade of these two samples within a deeply diverging mitochondrial haplogroup of Crocuta. Bayesian analyses suggest that the split of this Asian cave hyena mitochondrial lineage from their European and African relatives occurred approximately 1.85 Ma (95% CI 1.62–2.09 Ma), which is broadly concordant with the earliest Eurasian Crocuta fossil dating to approximately 2 Ma. Comparisons of mean genetic distance indicate that cave hyenas harboured higher genetic diversity than extant spotted hyenas, brown hyenas and aardwolves, but this is probably at least partially due to the fact that their mitochondrial lineages do not represent a monophyletic group, although this is also true for extant spotted hyenas. Moreover, the joint female effective population size of Crocuta (both cave hyenas and extant spotted hyenas) has sustained two declines during the Late Pleistocene. Combining this mitochondrial phylogeny, previous nuclear findings and fossil records, we discuss the possible relationship of fossil Crocuta in China and the extinction of cave hyenas.     

Life in leaf litter: novel insights into community dynamics of bacteria and fungi during litter decomposition

Microorganisms play a crucial role in the biological decomposition of plant litter in terrestrial ecosystems. Due to the permanently changing litter quality during decomposition, studies of both fungi and bacteria at a fine taxonomic resolution are required during the whole process. Here we investigated microbial community succession in decomposing leaf litter of temperate beech forest using pyrotag sequencing of the bacterial 16S and the fungal internal transcribed spacer (ITS) rRNA genes. Our results reveal that both communities underwent rapid changes. Proteobacteria, Actinobacteria and Bacteroidetes dominated over the entire study period, but their taxonomic composition and abundances changed markedly among sampling dates. The fungal community also changed dynamically as decomposition progressed, with ascomycete fungi being increasingly replaced by basidiomycetes. We found a consistent and highly significant correlation between bacterial richness and fungal richness (R = 0.76, P < 0.001) and community structure (R_Mantel = 0.85, P < 0.001), providing evidence of coupled dynamics in the fungal and bacterial communities. A network analysis highlighted nonrandom co‐occurrences among bacterial and fungal taxa as well as a shift in the cross‐kingdom co‐occurrence pattern of their communities from the early to the later stages of decomposition. During this process, macronutrients, micronutrients, C:N ratio and pH were significantly correlated with the fungal and bacterial communities, while bacterial richness positively correlated with three hydrolytic enzymes important for C, N and P acquisition. Overall, we provide evidence that the complex litter decay is the result of a dynamic cross‐kingdom functional succession.     

宏基因组及培养组学技术在粪菌移植中的应用

粪菌移植技术（fecal microbiota transplantation,FMT）是利用健康人群的粪便或经过处理的粪便中的微生物来治疗消化、代谢等系统诸多疾病的一项古老而又新兴的技术。宏基因组、培养组等肠道微生物组前沿研究技术的飞速发展,为粪菌移植治疗疾病提供了强有力的研究和临床实践武器。宏基因组技术可全面揭示健康及疾病状态下肠道微生态的组成及功能变化,而培养组学技术则可以用来分离和鉴定人类肠道中的诸多在常规培养条件下未可培养菌,两项技术联用不仅可以使我们更为深入地理解粪菌移植在临床实践中的因果规律,还将有力推动粪菌移植技术在未来的应用与发展。基于此,本文综述了宏基因组及培养组学技术在粪菌移植中的应用及未来发展趋势。     

Functional endogenous viral elements in the genome of the parasitoid wasp Cotesia congregata: insights into the evolutionary dynamics of bracoviruses

Bracoviruses represent the most complex endogenous viral elements (EVEs) described to date. Nudiviral genes have been hosted within parasitoid wasp genomes since approximately 100 Ma. They play a crucial role in the wasp life cycle as they produce bracovirus particles, which are injected into parasitized lepidopteran hosts during wasp oviposition. Bracovirus particles encapsidate multiple dsDNA circles encoding virulence genes. Their expression in parasitized caterpillars is essential for wasp parasitism success. Here, we report on the genomic organization of the proviral segments (i.e. master sequences used to produce the encapsidated dsDNA circles) present in the Cotesia congregata parasitoid wasp genome. The provirus is composed of a macrolocus, comprising two-thirds of the proviral segments and of seven dispersed loci, each containing one to three segments. Comparative genomic analyses with closely related species gave insights into the evolutionary dynamics of bracovirus genomes. Conserved synteny in the different wasp genomes showed the orthology of the proviral macrolocus across different species. The nudiviral gene odv-e66-like1 is conserved within the macrolocus, suggesting an ancient co-localization of the nudiviral genome and bracovirus proviral segments. By contrast, the evolution of proviral segments within the macrolocus has involved a series of lineage-specific duplications.     

Metagenomic insights into strategies of carbon conservation and unusual sulfur biogeochemistry in a hypersaline Antarctic lake

Organic Lake is a shallow, marine-derived hypersaline lake in the Vestfold Hills, Antarctica that has the highest reported concentration of dimethylsulfide (DMS) in a natural body of water. To determine the composition and functional potential of the microbial community and learn about the unusual sulfur chemistry in Organic Lake, shotgun metagenomics was performed on size-fractionated samples collected along a depth profile. Eucaryal phytoflagellates were the main photosynthetic organisms. Bacteria were dominated by the globally distributed heterotrophic taxa Marinobacter, Roseovarius and Psychroflexus. The dominance of heterotrophic degradation, coupled with low fixation potential, indicates possible net carbon loss. However, abundant marker genes for aerobic anoxygenic phototrophy, sulfur oxidation, rhodopsins and CO oxidation were also linked to the dominant heterotrophic bacteria, and indicate the use of photo- and lithoheterotrophy as mechanisms for conserving organic carbon. Similarly, a high genetic potential for the recycling of nitrogen compounds likely functions to retain fixed nitrogen in the lake. Dimethylsulfoniopropionate (DMSP) lyase genes were abundant, indicating that DMSP is a significant carbon and energy source. Unlike marine environments, DMSP demethylases were less abundant, indicating that DMSP cleavage is the likely source of high DMS concentration. DMSP cleavage, carbon mixotrophy (photoheterotrophy and lithoheterotrophy) and nitrogen remineralization by dominant Organic Lake bacteria are potentially important adaptations to nutrient constraints. In particular, carbon mixotrophy relieves the extent of carbon oxidation for energy production, allowing more carbon to be used for biosynthetic processes. The study sheds light on how the microbial community has adapted to this unique Antarctic lake environment.     

Description of Triplophysa luochengensis sp. nov. (Teleostei: Nemacheilidae) from a karst cave in Guangxi,China

A new cave‐dwelling fish Triplophysa luochengensis is described based on specimens collected from a karst cave in Guangxi Zhuang Autonomous Region, China, that is interconnected to the Hongshui River drainage. The species can be distinguished from its congeners by a combination of characters: eyes degenerated, anal fin with six branched rays, caudal fin with 16–17 branched rays, pectoral‐fin length 72·4–95·8% of the distance between pectoral‐fin origin and pelvic‐fin origin, lateral head length 26·2–28·2% of standard length (L_S), eye diameter 7·5–8·6 of L_S, body covered by sparse scales, lateral line complete and 7–8 pre‐operculo‐mandibular pores. Dark pigments irregularly present on dorsum of head, dorsum and flank.     

Population dynamics of a phytophagous lady-beetle,Epilachna vigintioctopunctata (Fabricius), living in spatio-temporally heterogeneous habitats. III. Effects of habitat structure on population dynamics

Temporal changes in the population size of a phytophagous lady-beetle were analyzed to identify mechanisms affecting lady-beetle population dynamics at different spatial scales. The study area (15 ha) included 18 habitat patches. The major host plants were potato for first generation larvae and eggplant for second generation larvae. The habitat patches were classified into three groups according to the major host plants in each patch: P-E patches (both host plants available), P patches (potato only), and E patches (eggplant only). The winter disappearance of adults in the whole study area, and larval mortality in E patches were apparently the most important factors disturbing the overall population density. Density-dependent movement of females appeared to have the greatest stabilizing effect on the yearly fluctuation of population density. Rate of increase of female adults from the first to the second generation,R, was generally higher on eggplants in E patches than in P-E patches because the adult density of the first generation was much higher in P-E patches. The yearly fluctuation of adult density in each generation tended to be less in patches with all habitat components necessary for the full life cycle (P-E patches). However, such patches were not favorable for first generation females, as indicated by the lower rate of increase from the first to the second generation. The density and stability of lady-beetle populations is discussed in relation to habitat structure.     

Linking fish population dynamics to habitat conditions: insights from the application of a process-oriented approach to several Great Lakes species

One of the major challenges facing fishery scientists and managers today is determining how fish populations are influenced by habitat conditions. Many approaches have been explored to address this challenge, all of which involve modeling at one level or another. In this paper, we explore a process-oriented model approach whereby the critical population processes of birth and death rates are explicitly linked to habitat conditions. Application of this approach to five species of Great Lakes fishes including: walleye (Sander vitreus), lake trout (Salvelinus namaycush), smallmouth bass (Micropterus dolomieu), yellow perch (Perca flavescens), and rainbow trout (Onchorynchus mykiss), yielded a number of insights into the modeling process. One of the foremost insights is that processes determining movement and transport of fish are critical components of such models since these processes largely determine the habitats fish occupy. Because of the importance of fish location, an individual-based model appears to be a nearly inescapable modeling requirement. There is, however, a paucity of field-based data directly relating birth, death, and movement rates to habitat conditions experienced by individual fish. There is also a paucity of habitat information at a fine temporal and spatial scale for many important habitat variables. Finally, the general occurrence of strong ontogenetic changes in the response of different life stages to habitat conditions emphasizes the need for a modeling approach that considers all life stages in an integrated fashion.     

Diversity of microbial communities colonizing the walls of a Karstic cave in Slovenia

Karstic cave systems in Slovenia receive substantial amounts of organic input from adjacent forest and freshwater systems. These caves host microbial communities that consist of distinct small colonies differing in colour and shape. Visible to the naked eye, the colonies cover cave walls and are strewn with light-reflecting water droplets. In this study, the diversity of prokaryotes constituting these unusual microbial communities in Pajsarjeva jama cave was examined. A molecular survey based on small subunit rRNA diversity showed a high diversity within the Bacteria , while members of Archaea were not recovered. A total of eight bacterial phyla were detected. The application of various species richness estimators confirmed the diverse nature of the microbial community sample. Members of Gammaproteobacteria were most abundant in the clone libraries constructed and were followed in abundance by members of Actinobacteria and Nitrospira . In addition, members of Alphaproteobacteria, Betaproteobacteria and Deltaproteobacteria as well as Acidobacteria, Verrucomicrobia, Planctomycetes, Chloroflexi and Gemmatimonadetes were identified in clone libraries. The high number of clones most closely related to environmental 16S rRNA gene clones showed the broad spectrum of unknown and yet to be cultivated microorganisms inhabiting these cave systems.     

A new Stygonitocrella Petkovski (Copepoda: Harpacticoida) from a cave in Northern Mexico with comments on the taxonomy of the genus

The cave-dwelling harpacticoid copepod fauna of Mexico is still yet to be known. There are only a few records related to karstic caves, but most refer to epigean forms. The analysis of samples collected in a cave system in Northern Mexico yielded two female specimens of an unknown harpacticoid copepod species which is described herein. It was assigned to the genus Stygonitocrella Petkovski, 1976 mainly because of the presence of a single endopodal segment on legs 1–4; the new species, S. mexicana n. sp. differs from its congeners by a combination of characters related to the segmentation and armature of the swimming legs 1–4, the fifth leg of the female, and the strcture of the antennae. Based on the current limits of Stygonitocrella, two new subgenera are proposed to recognize different morphological patterns based on the presence or absence of an inner baseoendopodal lobe in the female fifth leg, a character not evaluated before. This lobe is absent in the new species and in other five species now contained in the subgenus Eustygonitocrella. A lobe is present in the new subgenus Fiersiella, also with six species. This is the second record of Stygonitocrella in North America, after S. (Fiersiella) sequoyahi Reid, Hunt & Stanley, 2003; it is the only North American cave-dwelling species of the genus. It is suggested that the influence of marine transgressions in Northern Mexico was related to the origin of the new species as part of a group of troglobitic forms originated from marine interstitial ancestors that colonized the freshwater caves. A key for the identification of all the known species of Stygonitocrellais also provided.     

Ecological characteristics of a cave‐dwelling moth species,Triphosa dubitata (Lepidoptera: Geometridae), in Baram and Ssang caves in Gangwon Province,Korea

This study was performed to clarify the ecological characteristics of a cave‐dwelling moth species, Triphosa dubitata Linnaeus (Lepidoptera: Geometridae), at Baram cave and at Ssang cave within Pyeongchang‐gun of Gangwon province in south Korea. We investigated T. dubitata population for 3 days from March 2013 to October 2013 (except September) and in January 2014. We used a section investigation method in the caves every month to determine their ecological characteristic. A nocturnal investigation using a high intensity discharge lamp adjacent to the entrance of the cave was conducted in parallel with a diurnal investigation using butterfly traps to investigate the movement of T. dubitata. In order to investigate the range of habitat temperature of moths, we used a dual laser targeting infrared thermometer to measure the cave temperatures. We caught individuals of T. dubitata L.: 8,574 individuals in Baram cave and 828 individuals in Ssang cave. As 9,095 individuals (97 %) appeared at a temperature of ?3°C ~ 8°C, it was verified that the adults were living at specific temperatures. In addition, T. dubitata adults were not active outside the caves; they entered the cave as soon as they developed from pupa into adults. These results showed that T. dubitata depended on food plants outside of the cave during the egg–larva–pupa stages, and they used the cave as a habitat for long‐term survival when they develop into adults.     

Introduction of the European minnow into a subalpine lake: habitat use and long-term changes in population dynamics

Minnows Phoxinus phoxinus , studied 30 years after the first record of the species in the subalpine Lake Øvre Heimdalsvatn, Norway, ≥55 mm L _T, were estimated to have densities of c . 4.7 kg ha⁻¹ (120 000 fish) in June 1999 and 2.1 kg ha⁻¹ (63 000 fish) in June 2000. The population was characterized by low individual growth, delayed age of maturity and lived longer when compared to values reported in a previous study in the early phase of its establishment, and other values reported in the literature. Most minnows reached sexual maturity at 4–5 years and >55 mm L _T. Although the estimated annual survival of minnows >55 mm was low ( S =0.2), ages up to 13 years were recorded. Despite a moderate increase in the population size during the last 20 years, the present reduction in individual growth, followed by delayed age of maturity, suggested the existence of density-dependent effects on the population dynamics of the minnows. The minnows were restricted to the littoral zone and near bottom areas. A vertical or horizontal expansion in habitat use was probably prevented by the presence of piscivorous brown trout Salmo trutta .     

Identification of a mitochondrial alcohol dehydrogenase in Schizosaccharomyces pombe: new insights into energy metabolism

In the present study we have shown that mitochondria isolated from Schizosaccharomyces pombe exhibit antimycin A-sensitive oxygen uptake activity that is exclusively dependent on ethanol and is inhibited by trifluoroethanol, a potent inhibitor of ADH (alcohol dehydrogenase). Ethanol-dependent respiratory activity has, to our knowledge, not been reported in S. pombe mitochondria to date, which is surprising as it has been concluded previously that only one ADH gene, encoding a cytosolic enzyme, occurs in this yeast. Spectrophotometric enzyme assays reveal that ADH activity in isolated mitochondria is increased approximately 16-fold by Triton X-100, which demonstrates that the enzyme is located in the matrix. Using genetic knockouts, we show conclusively that the novel mitochondrial ADH is encoded by adh4 and, as such, is unrelated to ADH isoenzymes found in mitochondria of other yeasts. By performing a modular-kinetic analysis of mitochondrial electron transfer, we furthermore show how ethanol-dependent respiratory activity (which involves oxidation of matrix-located NADH) compares with that observed when succinate or externally added NADH are used as substrates. This analysis reveals distinct kinetic differences between substrates which fully explain the lack of respiratory control generally observed during ethanol oxidation in yeast mitochondria.     

Evolution of cave living in Hawaiian Schrankia (Lepidoptera: Noctuidae) with description of a remarkable new cave species

Although temperate cave‐adapted fauna may evolve as a result of climatic change, tropical cave dwellers probably colonize caves through adaptive shifts to exploit new resources. The founding populations may have traits that make colonization of underground spaces even more likely. To investigate the process of cave adaptation and the number of times that flightlessness has evolved in a group of reportedly flightless Hawaiian cave moths, we tested the flight ability of 54 Schrankia individuals from seven caves on two islands. Several caves on one island were sampled because separate caves could have been colonized by underground connections after flightlessness had already evolved. A phylogeny based on approximately 1500 bp of mtDNA and nDNA showed that Schrankia howarthi sp. nov. invaded caves on two islands, Maui and Hawaii. Cave‐adapted adults are not consistently flightless but instead are polymorphic for flight ability. Although the new species appears well suited to underground living, some individuals were found living above ground as well. These individuals, which are capable of flight, suggest that this normally cave‐limited species is able to colonize other, geographically separated caves via above‐ground dispersal. This is the first example of an apparently cave‐adapted species that occurs in caves on two separate Hawaiian islands. A revision of the other Hawaiian Schrankia is presented, revealing that Schrankia simplex, Schrankia oxygramma, Schrankia sarothrura, and Schrankia arrhecta are all junior synonyms of Schrankia altivolans. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 156 , 114–139.     

Structural insights into the effect of hydration and ions on A-tract DNA: a molecular dynamics study

DNA structure is known to be sensitive to hydration and ionic environment. To explore the dynamics, hydration, and ion binding features of A-tract sequences, a 7-ns Molecular dynamics (MD) study has been performed on the dodecamer d(CGCAAATTTGCG)(2). The results suggest that the intrusion of Na(+) ion into the minor groove is a rare event and the structure of this dodecamer is not very sensitive to the location of the sodium ions. The prolonged MD simulation successfully leads to the formation of sequence dependent hydration patterns in the minor groove, often called spine of hydration near the A-rich region and ribbon of hydration near the GC regions. Such sequence dependent differences in the hydration patterns have been seen earlier in the high resolution crystal structure of the Drew-Dickerson sequence, but not reported for the medium resolution structures (2.0 approximately 3.0 A). Several water molecules are also seen in the major groove of the MD simulated structure, though they are not highly ordered over the extended MD. The characteristic narrowing of the minor groove in the A-tract region is seen to precede the formation of the spine of hydration. Finally, the occurrence of cross-strand C2-H2.O2 hydrogen bonds in the minor groove of A-tract sequences is confirmed. These are found to occur even before the narrowing of the minor groove, indicating that such interactions are an intrinsic feature of A-tract sequences.     

Chronological and Isotopic data support a revision for the timing of cave bear extinction in Mediterranean Europe

Abstract

The Cave Bear, Ursus spelaeus (sensu lato), was one of many megafaunal species that became extinct during the Late Pleistocene in Europe. With new data we revisit the debate about the extinction and paleoecology of this species by presenting new chronometric, isotopic and taphonomic evidence from two Palaeolithic cave bear sites in northeastern Italy: Paina Cave and Trene Cave. Two direct radiocarbon dates on well-preserved collagen have yielded ages around 24,200–23,500 cal yr BP, which make them the latest known representatives of the species in Europe. The carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopic values of bone collagen exhibit values similar to those of older cave bears from Swabian Jura and France, suggesting that the feedings preferences of cave bears remained unchanged until the disappearance of this species in Europe. Several bear remains preserved traces of human modification such as cut marks, which enables a reconstruction of the main steps of fur recovery and the butchering process. The broad range of plant types available and the favorable location of Berici Hills may have played an important role in the range expansion of cave bears and their interaction with the Paleolithic hunters settled the same area.