Spermophilus major is known as a fine biological species according to a number of criteria—morphological, ecological, and behavioral, respectively. Nevertheless, a large body of evidence has accumulated on the hybridization of S. major with four neighboring Spermophilus species. Our goal was to identify their contribution to the nuclear and mitochondrial genome of S. major, and to propose a hypothesis describing the hybridization history in space and time. We found that 36% of S. major individuals had extraneous alleles, and every contacting species contributed to S. major’s genetic variability. Our data suggested at least five independent hybridization events that were associated with shifts in the species ranges due to paleoclimate changes. Two of them were potentially accompanied by mitochondrial captures with replacement of the mitochondrial genome of one of the hybridizing species. We highlight the potential threat to the existence of S. major as a species under the current conditions of population decline along with the simultaneous influx of extraneous genes. It is also particularly important to emphasize the necessity of protecting this inhabitant of unique steppe communities.The role of hybridization in biological evolution and cases of hybridogenic speciation is one of the most complicated but also interesting and actively studied topics. Russet ground squirrel Spermophilus major, whose range has repeatedly changed due to climatic fluctuations and now borders the ranges of four related species, is of great interest. The main aims of this study were to determine the direction and intensity of gene introgression, the spatial depth of the infiltration of extraneous genes into the S. major range, and to refine the hypothesis of the hybridogenic replacement of mitochondrial genomes in the studied group. Using phylogenetic analysis of the variability of mitochondrial  and nuclear  markers, we determined the contribution of neighboring species to the S. major genome. We showed that 36% of S. major individuals had extraneous alleles. All peripheral species that were in contact with S. major contributed towards its genetic variability. We also proposed a hypothesis for the sequence and localization of serial hybridization events. Our assessment of the S. major genome implications of introgression highlights the importance of implementing conservation measures to protect this species.Among the numerous described cases of hybridization in mammals, the most intriguing are (a) cases of introgressive hybridization deeply affecting the evolutionary history of species, and (b) models involving not a pair of species but a multi-species complex. Therefore, the hybridization history of the russet ground squirrel  Numerous studies examining gene flow between species in various groups of animals have shown that hybridization in secondary contact zones is a more frequent event then the occasional crossing of reproductive barriers ,5,6,7,8.Hybridization in mammals is no longer considered a rare phenomenon ,25,26,27In the case of introgressive hybridization, on the contrary, the haplotypes of the donor species are integrated into the gene pool of the recipient species , and traHowever, in addition to the spatial aspect of hybridization, there is also a temporal aspect. With repeated occurrences of secondary contacts between closely related species, multiple separated in time cases of hybridization may occur as a result. Moreover, each hybridization event can contribute to the genomes of the contacting species ,38,39. TSpermophilus Cuvier, 1825 are a relatively young group of ground squirrels. A few species of this taxa were formed under allopatry and are poorly differentiated [Spermophilus are colonial hibernating inhabitants of the steppe and meadow ecosystems of open landscapes in most parts of Eurasia [Spermophilus species hybridize with each other in different combinations [Spermophilus major Pallas, 1778, a species that interbreeds with the largest number of other Spermophilus species  in nature [S. major genome is of undoubted interest for understanding the ways of expanding intra-specific variability, and the mechanisms of maintaining the species identity under the influence of hybridization.Palearctic ground squirrels of the genus entiated . Fourtee Eurasia . Seven Sinations ,44. Suchn nature ,44. Neven nature ,45,46. TS. fulvus) and little (S. pygmaeus) ground squirrels on the left bank of the Volga River [S. suslicus) on the right bank [S. major × S. suslicus is determined by the time of the transition of the russet ground squirrel to the right bank of the Volga River, which hardly exceeds a hundred years [S. major × S. suslicus have only been found in the contact zone on the right bank of the Volga River [S. suslicus cannot significantly affect the genome of S. major in the main range (to the east of the Volga River). Therefore, we excluded the consideration of the hybridization of this pair of species from our study. Population genetic analysis revealed a significant introgression of the mitochondrial haplotypes of S. fulvus and S. pygmaeus in the Volga populations of S. major, located not only in the sympatry zones, but also at a considerable distance from them, both on the right and on the left bank of the Volga River [The russet ground squirrel has an extended range ,47,48 paga River , and speght bank . Meanwhied years . Traces ga River , indicatga River ,49,52. Mga River ,53. Actiga River  were onlS. major range, its hybridization with ground squirrels of the heptneri form [Colobotis group, bordering on S. major in the east, is disputable. For a long time, all forms of red-cheeked ground squirrels living in Western Kazakhstan and Siberia were considered as sub-species of the red-cheeked ground squirrel S. erythrogenys [S. erythrogenys and the Brandt’s ground squirrel S. brevicauda [heptneri form was assigned as a sub-species to S. major [S. erythrogenys [heptneri form and S. brevicauda are phylogenetically close [S. brevicauda and S. erythrogenys and considered heptneri as a sub-species of the Brandt’s ground squirrel S. b. heptneri. Accordingly, in the east, S. major formed a wide zone of hybridization with S. brevicauda.In the eastern part of the eri form  was founeri form ,59. It shrogenys ,60. At tevicauda ,47,61, wevicauda . The hepS. major  or to S.hrogenys . Allozymly close . In thisly close  on the iS. major’s own specific mitochondrial genome. Based on the sequencing of cytochrome b (cytb) and a fragment of the first subunit of the cytochrome oxidase (COI) gene of mtDNA, a wide introgression of the mt genome of S. brevicauda into the genome of S. major leading to the replacement of the mt genome in S. major was suggested [S. major within the entire range made it possible to determine the species-specific mt genome of S. major, which was differentiated to varying degrees from the genomes of other Spermophilus species [S. major not only in the contact zones, but also within the entire distribution area of the species, has hardly been studied. Active research was conducted mainly in the sympatric zones or at the boundaries of the range. It can be assumed that the events of genetic exchange involving S. major and other Spermophilus species in contact with it could occur repeatedly. This was supported by the wide geography of the hybrid zones and the alleged recent rapid expansion of the russet ground squirrel to the west from the Trans-Ural refugium [Thus, we observed a wide introgression of extraneous mitochondrial haplotypes in the west and active hybridization in the east. This became the basis for the question of the existence of uggested ,63. A ph species . Howeverrefugium .S. major and four neighboring species that were identified previously with the phenotypic and mtDNA data using genotyping with a set of nuclear and mitochondrial molecular markers with materials from all parts of the species’ range. The main goals of our work were to determine the direction and intensity of gene introgression, the spatial depth of the infiltration of extraneous haplotypes into the range of S. major, and to clarify the hypothesis of the hybridogenic substitution of mitochondrial genomes in the study group. We present a hypothesis of a sequence of spatiotemporal hybridization events that left a trace on the genomes of the studied Spermophilus species.In our study, we confirmed the occurrence of introgressive hybridization between S. major, 51 S. brevicauda, 14 S. fulvus, 4 S. erythrogenys, and 23 S. pygmaeus samples were analyzed, respectively. Specimens defined by phenotype as hybrids of S. major × S. pygmaeus  and S. major × S. brevicauda  were included in S. major. The characteristics and localities of the materials used are shown in S. major mtDNA control region (CR) haplotype sequences (MW149931-MW150003) [cytb sequences of S. b. iliensis (AF157863), S. b. intermedius, and S. b. brevicauda  that were deposited in the GenBank database were also used.The material included tissue samples  obtained from ground squirrels deposited in the large-scale research facility “Collection of Wildlife tissues for genetic research” of the Core Centrum of the Koltzov Institute of Developmental Biology, Russian Academy of Sciences (CWT IDB RAS), state registration number 3579666, and the Collection of Penza State University (PSU). Most of the material was collected by the authors. A total of 243 phenotypic W150003)  and cytbAnimal work was performed in accordance with the established international protocols as recommended in the Guidelines for Humane Endpoints for Animals Used in Biomedical Research. All experimental procedures were approved by the Ethics Committee for Animal Research of the Koltzov Institute of Developmental Biology RAS in accordance with the Regulations for Laboratory Practice in the Russian Federation; the most recently accepted protocol was numbered 37-25.06.2020. Every effort was made to treat animals as humanely as possible and to minimize their suffering.Genomic DNA was isolated by either saline  or phenocytb mtDNA were used for species differentiation and the analysis of mitochondrial introgression. To assess the effect of hybridization on the Spermophilus nuclear genome, we assessed the genetic markers of the fragments of the male-specific histocompatibility antigen SmcY, containing intron 8 and partial cds [BGN) [i13BCR) [MGF), beta-acid glucosidase (GBA) [PRKCI) [THY) [c-myc) [i6p53) [i6p53, were sequenced for 20 randomly selected samples of S. major from the entire range and from 2 (for S. erythrogenys) to 43 samples of the other species. Individual samples of S. brevicauda, S. erythrogenys, and S. pygmaeus were selectively sequenced to determine the i6p53 ID repeat structure. The i6p53 nucleotide sequences of S. major and S. fulvus were obtained from the publication of Titov et al. [S. major and other species. For further analysis, we selected five markers that reliably differentiate the russet ground squirrel from most of the neighboring species, as well as have the least haplotypic diversity in the S. major sample: SmcY, BGN, PRKCI, c-myc, and the i6p53 ID repeat, which differs in sequence length in most of the species that were evaluated in this work [PRKCI, which gave an additional short product of ~200 bp in the samples of little ground squirrel. The analyzed c-myc gene sequence included 192 bp of 2 introns and 683 bp of 3 exons, respectively.CR and tial cds , and bigds [BGN) , localiz[i13BCR) , mechanose (GBA) , protein [PRKCI) , thyrotrI) [THY) , proto-o [c-myc) , and the [i6p53) . For thiv et al. . The moshis work . The gen® BigDye™ Terminator v. 3.1 Kit , the NovaDye Terminator Cycle Sequencing Kit 3.1 , the AB 3500 Genetic Analyzer , and the Nanofor-05 Genetic Analyzer  at the Core Centrum of the Koltzov Institute of Developmental Biology, Russian Academy of Sciences.Automated sequencing was performed using the ABI PRISMChromatograms were reviewed and manually edited using the Lasergene 11 SeqMan package . Due to the presence of a short PCR side product, the end sections of the PRKCI sequence were read from the reverse primer. DNA sequences were aligned using the MUSCLE algorithm  in MEGA BGN, PRKCI, and c-myc sequences, species-specific substitutions were determined for which restriction enzymes were selected. These restriction enzymes were selected to produce fragments that distinguished S. major from the species that were in contact with it  in the presence of the intercalating dye iridium bromide. Visual detection in the ultraviolet spectrum was performed using the Bio-Rad ChemiDoc MP System gel-documenting system . The SmcY sequence was obtained in all males in a total sample of all the analyzed ground squirrel species.After alignment in the  with it . Speciescytb: OR088804 - OR088840, SmcY: OR088841 - OR088846, BGN: OR088847 - OR088859, c-myc: OQ929560 - OQ929573, PRKCI: OQ954111 - OQ954121.All newly sequenced nucleotide sequences of the haplotypes were deposited in GenBank with access numbers CR: OR088768-OR088803, cytb was performed with the ModelTest package in the MEGA X software [S. major specimens, a phylogenetic analysis (ML) of cytb variation was performed using the Tamura-Nei+G nucleotide evolution model in the MEGA X software. Sequences of geographically distant sub-species of S. b brevicauda and S. b. intermedius were added while constructing the phylogeny of cytb. All trees were rooted using S. pygmaeus as the external group, being the most distant taxon. Genetic differences were assessed by pairwise distances .Phylogenetic analysis based on the haplotype variability of the mitochondrial markers was performed for the entire set of samples studied. Selection of the optimal nucleotide substitution model for CR and software  using thsoftware . Based oH), total number of polymorphic loci (S), total number of mutations (η), haplotype (h), and nucleotide (π) diversity, along with the average number of pairwise differences (k). Spatial expansion tests were also performed using Fu’s (Fs) and Tajima’s (D) models. Frequency analysis of species-specific alleles of the SmcY, BGN, PRKCI, and c-myc genes of the entire S. major sample was performed to assess the introgression of extraneous nDNA. Since the aims of the study did not include the assessment of intra-specific variability for the studied markers, most of the samples were analyzed based on restriction analysis of the studied fragments without determining their nucleotide composition.CR genetic variability was analyzed using Arlequin v. 3.5.2.2 , with caS. major haplotypes, haplotypic networks were constructed for the BGN, PRKCI, and c-myc genes using the HaplowebMaker software (https://eeg-ebe.github.io/HaplowebMaker/ (accessed on 15 July 2022)) [In addition to the frequency analysis of the y 2022)) . Haplotyy 2022))  “allele y 2022)) ,76,77,78S. major, thirty-nine—S. brevicauda (thirty-eight—S. b. heptneri and one—S. b. iliensis), thirteen—S. fulvus, three—S. erythrogenys, and twenty-three—S. pygmaeus, respectively. The complete nucleotide sequences (1140 bp) of cytb were also determined for fifty-eight specimens of S. major: fifteen of S. brevicauda (eight of S. b. heptneri and seven of S. b. iliensis), five of S. fulvus, one of S. erythrogenys, and four of S. pygmaeus, respectively.We sequenced one-hundred and fourteen complete nucleotide sequences (1005–1008 bp) of the mtDNA control region: thirty-six—S. major specimens. Sixty-six of these haplotypes formed a separate clade on the ML tree (“major”). In addition, 10 haplotypes of 29 individuals of S. b. heptneri were included in the same clade. Four S. b. heptneri specimens had the S. major haplotypes h10, h18, and h21. Haplotype hb05 was found in three specimens of S. b. heptneri from population 6b, which was clustered separately from S. major, and basally to the haplotypes of S. b. iliensis. Three haplotypes found in phenotypic S. major (thirteen specimens) clustered with the species-specific haplotypes of S. fulvus and three haplotypes (six specimens) clustered with S. pygmaeus (cytb haplotypes were found in fifty-eight specimens of S. major. Two haplotypes found in phenotypic specimens of S. major (ten samples) clustered with the species-specific haplotypes of S. fulvus and two haplotypes (five samples) clustered with S. pygmaeus, respectively. The topology of the phylogenetic tree (ML) constructed on the basis of 100 cytb sequences of five Spermophilus species which we newly obtained and took from the GenBank mainly corresponded to the branching CR tree, but haplotype c25 found in S. b. heptneri individuals with hb05 CR were included in the “major” clade  included individuals with only the S. fulvus haplotypes , and for a sample of individuals with only species-specific haplotypes for S. major (B) (“major”). The results of the analysis are shown in h) in the samples was not determined to be statistically different, but the level of nucleotide variability (k) and nucleotide diversity (π) of sample A was found to be two times greater than in sample B. The values of the tests for spatial expansion were found to be negative in the analysis of both samples, but for sample A the Fs-test was deemed to be unreliable, and for sample B the Tajima’s test was determined to be unreliable.An analysis of the population-genetic variability of CR was performed separately for the entire sample of phenotypic S. major haplotypes and most of their contacting species by both cytb and CR. However, the genetic distances between S. major, S. fulvus, S. b. iliensis, and S. b. intermedius were within the dp~0.03 (3%), which is below the 5% empirical potential species level and above the 2% level of intra-specific variation suggested by Baker and Bradley [p-distances of cytb between the S. major and most S. b. heptneri specimens were at the level of the intra-specific variability of S. major. The mean pairwise genetic distances between the cytb haplotypes of S. major in the sample without extraneous haplotypes , clustered by CR separately from S. major and basally to S. b. iliensis  were selected for further analysis  from eighty-six S. major males, thirteen S. brevicauda males (twelve S. b. heptneri and one S. b. iliensis), five S. fulvus, and six S. pygmaeus, respectively (SmcY haplotypes were found in the S. major sample. One of them (m2) was unique, found in two individuals, and differs from the haplotypes of all neighboring species. This marker differentiates both detected haplotypes of S. major from S. erythrogenys by three substitutions, S. major from S. fulvus by five substitutions, and S. major from S. pygmaeus by six substitutions, respectively, which corresponds to the previously described [S. major and S. brevicauda. All individuals analyzed for this marker carried only species-specific haplotypes. The specimen from locality 30 phenotypically determined as a hybrid of S. major × S. pygmaeus had a S. pygmaeus-specific haplotype.We sequenced ectively . Two Smcescribed   detected in S. major differs from the haplotypes of other species by the substitution of A/G in position 289 and T/C in position 505, respectively. The last substitution formed the recognition site of Bso31 I restrictase in all haplotypes except for haplotype m. Restrictase analysis showed that all individuals of S. major had the species-specific haplotype m. Three of the four S. major × S. brevicauda hybrids  had the specific haplotype brevicauda (b1) in homozygote, while the fourth had haplotype major (m) in homozygote. In the hybrid S. major × S. pygmaeus a haplotype specific to S. pygmaeus was found. A haplotype network (“haploweb”) constructed from BGN gene sequences . Of these, thirty-seven were S. major, forty-three were S. brevicauda (eight were S. b. iliensis and thirty-five were S. b. heptneri), six were S. fulvus, four were S. erythrogenys, and seven were S. pygmaeus, respectively. Two PRKCI haplotypes were found in S. major, differing from the haplotypes of all other species by the A/G transition at the position 343  was found to be common to the S. fulvus and the S. b. heptneri forms, which may be a manifestation of ancestral polymorphism. Two species-specific haplotypes were also found in S. erythrogenys and one species-specific haplotype in S. pygmaeus, respectively.We obtained 97 sequences of tion 343 c. This sS. major (92.6%). In 13 individuals of phenotypic S. major from the Trans-Ural populations . Haplotype m1 was found to be more common than haplotype m2 in individuals of S. major heterozygous for this marker  in the analysis of 135 sequences obtained  was found in S. major, which differed from the haplotypes of the other species by the G/A transition in position 635. This substitution formed the recognition site of restrictase BstDS I in haplotype m. Three species-specific haplotypes were found in S. fulvus. Another c-myc haplotype was found to be common to S. fulvus and S. b. heptneri (c), which may be a manifestation of the ancestral polymorphism as in the case of PRKCI. Furthermore, two species-specific haplotypes were found in S. brevicauda, and one species-specific haplotype was detected in S. erythrogenys, respectively. In the sample of S. pygmaeus, four species-specific haplotypes were detected. The p5 haplotype, which is close to S. pygmaeus, was also found in the S. major sample, but was not detected in S. pygmaeus. We identified this haplotype as belonging to S. pygmaeus due to their similarity in nucleotide composition in the conserved region of this sequence.We detected 13 obtained d. Of theS. major. In addition, individuals carrying haplotypes specific for S. pygmaeus (p1-5) and the haplotype common to S. fulvus and S. b. heptneri (c) were found both in heterozygous  and in homozygous (2.1% each) states. The haplotype erythrogenys was found in the heterozygote with major and brevicauda in 2.1% and 1.25% of individuals, respectively. Two of the four hybrids of S. major × S. brevicauda had haplotype c in homozygote, with one of these hybrids having the haplotypes c and b1, and the other being heterozygous for haplotypes c and e. The hybrid S. major × S. pygmaeus had haplotypes of both species.Restriction analysis showed the predominance of individuals with the species-specific haplotype in the homozygote (72.9%) in the entire sample of c-myc gene region  contained the p5 haplotype found only in the S. major populations (The haplotype network based on the sequences of the e region c showed ulations d.i6p53 sequences between 100 and 173 bp in length (including primers) were sequenced. Of these, seven were S. brevicauda, one was S. erythrogenys, and four were S. pygmaeus, respectively. The primary structure of the i6p53 ID repeats differed in all the species studied, except for S. major and S. b. heptneri  based on the combined sequence of CI genes  showed aS. major was performed. Only species-specific haplotypes of the studied markers were found in 63.7% of the russet ground squirrels. Among the ninety S. major individuals with extraneous haplotypes, eleven (12.2%) were hybrid in two or more markers, four of which were phenotypically defined hybrids. More than half of the S. major individuals with extraneous haplotypes (52.2%) had S. fulvus and/or S. brevicauda haplotypes, 43.3% had S. pygmaeus haplotypes, and 8.9% had S. erythrogenys haplotypes, respectively. In addition, two S. major individuals in the west of the range had both S. fulvus and S. pygmaeus haplotypes, and two individuals in the east had both S. erythrogenys and S. brevicauda haplotypes.Based on the data on the allelic composition of the studied molecular markers, genotyping of all individuals in the total sample of S. fulvus haplotypes were found in populations 18, 19, and 69 located in the center of the western part of the range, whereas S. pygmaeus haplotypes were only found in the Volga region (populations 70–72) , half of which were haplotypes specific to S. pygmaeus. Among all markers analyzed in S. major, 5.6% of haplotypes of the other species were found (The s 70–72) a. The mtre found .S. major, both nuclear and mitochondrial contains borrowed haplotypes, thus reflecting the history of its hybridization with neighboring species. Extraneous haplotypes are widespread in many parts of the range of the russet ground squirrel . A comparable contribution (2.2%) to the genetic diversity of S. major was made by S. pygmaeus, despite the fact that it was the most phylogenetically distant among the species hybridizing with S. major. Despite the wide distribution of extraneous haplotypes in S. major populations, their cumulative contributions to the genetic diversity of the species was only ~6%. However, in S. major individuals, PRKCI amplification detected an additional PCR product characteristic only of S. pygmaeus, suggesting a greater contribution of this and other species to the S. major genome. Our data indicate repeated substitutions of the mitochondrial genome in S. major and S. brevicauda. The clustering of the mitochondrial trees (S. major with the forms of S. b. heptneri and S. b. iliensis (or their common ancestor). It is therefore likely that originally, as a result of introgressive hybridization, the mt genome of the ancestral form of S. b. heptneri/iliensis was replaced by the mt genome of S. major. This was evident by the fact that two forms of S. brevicauda, S. b. brevicauda, and S. b. intermedius formed a separate branch on the mt-trees, while S. b. iliensis was clustered together with S. major. The position of S. major on the mt phylogeny as the youngest species of the Colobotis group was consistent with the revealed variability of the nuclear markers. The haplotypes of S. major by three independent nuclear markers were characterized by the absence of synapomorphies uniting all other Spermophilus species and show relatively low variability  at a distance of about 200 km from the Volga . In addition, the hybridization of S. major × S. fulvus apparently occurs not only in the Volga region, but also in other parts of the Cis-Ural overlapping zone of their ranges, as evidenced by the detection of the S. fulvus mt haplotype introgression in the south of the S. major range in the Poduralsky Plateau (population 29). In contrast to mtDNA, there was no extensive introgression of the S. fulvus nDNA haplotypes in the Cis-Ural populations of the russet ground squirrel. This is consistent with the finding of limited introgression of the nuclear haplotypes in only the mixed populations of S. major and S. fulvus in the Volga region, along with the single case of SmcY haplotype introgression of S. fulvus in the S. major population south of Aktobe [Further dispersal of distant hybrids eastward could also have occurred along the right tributaries of the Volga–Kama basin. This was supported by the finding of mt haplotypes of he Volga a. The fof Aktobe ,53. The S. major × S. pygmaeus in the southern Volga Region leads to the introgression of S. pygmaeus mtDNA northward along both banks of the Volga River [S. pygmaeus to the introgression of the mt haplotypes , we didcordance ,82 sincecordance .c-myc haplotypes of the S. pygmaeus were common throughout the Cis-Ural part of the S. major range, as well as in the eastern macroslope of the Southern Urals . “The system of now degraded old runoff troughs ... testifies to a threefold large-scale watering of the plains of the Southern Turgai and Northern Aral Sea region” [S. superciliosus, which is considered an ancestral form of S. major and morphologically almost indistinguishable from it, are known from the Middle Pleistocene [Paleogeographic events that determined the fragmentation of the ranges of the steppe species and the following secondary contacts of the diverging phyletic lineages had both common features and peculiarities in the Trans-Ural and Cis-Ural territories. The Trans-Ural part of the modern  region” . Around  region” ,86. Step region” . Fossilsistocene ,88. The S. major and S. erythrogenys in Western Siberia, which left c-myc haplotypes of S. erythrogenys in the S. major genome. This could have occurred if the range of S. erythrogenys extended to the left bank of the Irtysh River. However, the paucity of the paleontological records and the poor study of ground squirrels in the territory of the Tobol–Irtysh interfluve do not allow us to determine the paleo-range boundaries of these species. The contact between S. major and S. erythrogenys may have been interrupted during the middle Pleistocene period as a result of the formation of one of the early Turgai spillway [Apparently, one of the earliest hybridization events, traces of which we found, was the contact between spillway .S. brevicauda sensu lato, which led to the complete replacement of the mtDNA of one of the species. Equal genetic distance between all species of the Colobotis group (excluding the more distant S. erythrogenys) suggests the simultaneous radiation of S. fulvus, two forms of Brandt’s ground squirrel—S. b. brevicauda, S. b. intermedius, and the S. major—S. b. heptneri/iliensis group. Hybridization of S. major × S. brevicauda sensu lato apparently occurred after the division of S. brevicauda into the S. b. brevicauda, S. b. intermedius, and S. b. heptneri/iliensis lineages, respectively, as indicated by the different genetic distances between these forms and S. major. However, this hybridization event apparently preceded the separation of S. b. heptneri/iliensis into two separate forms, which is supported by the proximity of the CR haplotypes of S. b. iliensis and the presumably discovered ancestral haplotype of S. b. heptneri  and is dization .S. major in the Trans-Ural part of its range can be considered as the hybridization of S. major × S. b. heptneri, which resulted in an almost complete replacement of the S. b. heptneri mt genome by the S. major ones. This may have occurred in the case of hybridization, when the short-tailed ground squirrel moved into the territory of the russet ground squirrel, resulting in the subsequent displacement of the last [The most recent evolutionarily significant event for the last . To dateS. major with S. pygmaeus and S. fulvus was probably formed more recently in the south of the Volga Region [S. major range from the Volga River to the east, active hybridization was only observed in a small contact zone located in the Saratov Region. This was probably due to the presence of ecological niches in this region that were suitable for all three species, thereby contributing to the formation of joint settlements and hybridization.At the same time, a hybrid zone of a Region . In spitOchotona) [Modern approaches to the study of inter-specific interactions allow us to operate not only with a pair of hybridizing species but also consider entire species complexes. The gene flow and direction of allele introgression may differ in different species complexes. For example, asymmetric mtDNA introgression from several species into one universal mtDNA recipient species in a group has been achieved with a complex of western North American chipmunk species . Howeverchotona) , which wSpermophilus ground squirrel species that we studied also form a complex of hybridizing species formed around S. major. All peripheral species in contact with S. major contribute to its genetic variability. This admixture is not limited to the introgressive hybridization of the closely related species of Colobotis, which diverged relatively recently as a result of radiation from a common ancestor. The relatively evolutionarily distant little ground squirrel has also made a significant contribution to the general gene pool of the russet ground squirrel. The inclusion of a relatively small number of extraneous mtDNA haplotypes into the gene pool of the russet ground squirrel increases its nucleotide diversity by an order of magnitude  having aberrant acoustic signals. The level of genetic mitochondrial differentiation within this group of species could not be a meaningful indicator of species differences due to past mitochondrial captures. The true level of genetic divergence in this group may be revealed based on a whole nuclear-genome phylogenetic study.The agnitude . In this species ,53. All c signal , which ic signal . The onlheptneri , and oneS. major and S. brevicauda apparently led to the formation of an independent haplogroup iliensis within S. brevicauda as a result of subsequent genetic divergence, as in Odocoileus deer [heptneri form that continues to the present day. The hybrid nature of the heptneri genome seems to be related to the intermediate character of the morphological variability of this form and is consistent with the assumption of Gromov et al. [The first mitochondrial capture during the late Pleistocene hybridization of eus deer . A seconv et al.  on its hv et al. .S. major is experiencing a deep depression: its abundance is rapidly declining, population sizes are shrinking, and small colonies are disappearing [In current times, the russet ground squirrel ppearing . Under t


Lygus lineolaris, is a major pest in eastern Canada strawberry fields. The objective of the present study was to evaluate the potential of two omnivorous predators of the pest: the damsel bug, Nabis americoferus, and the minute pirate bug, Orius insidiosus. The results in the laboratory showed that N. americoferus attacked all stages of the tarnished plant bug and, in the field, reduced its population for several weeks. Additionally, the impact was significant for every tested release period. Conversely, O. insidiosus only attacked smaller nymphs, and its effect was marginal. These results lead the way for an effective biological control strategy based on the use of N. americoferus against the tarnished plant bug.The tarnished plant bug, Lygus lineolaris, is a major strawberry pest. Only marginally effective control methods exist to manage this pest. Various predators attack L. lineolaris, but their potential is overlooked. In this study, we explore the potential of two omnivorous predators of the tarnished plant bug: the damsel bug, Nabis americoferus, and the minute pirate bug, Orius insidiosus. Firstly, the predation rate of these predators was measured in laboratory tests. Secondly, their potential release rates and release periods were determined in the field using strawberry plants. The results show that N. americoferus feeds on all nymphal stages and adults of the tarnished plant bug, while O. insidiosus attacks only smaller nymphs (up to the N2 stage). In the field, all tested densities of N. americoferus  reduced the population of the tarnished plant bug for several weeks compared with the control treatment, but the effect of O. insidiosus alone was marginal. Additionally, for all the release periods tested, Nabis americoferus was efficient in reducing the pest population. These results demonstrate the potential of N. americoferus to control the tarnished plant bug in strawberry fields. We discuss the possible application of these results for establishing an effective and economically viable biological control strategy.The tarnished plant bug,  Lygus lineolaris  (Hemiptera: Miridae), is a major pest in strawberry fields ) times higher than in the plots with  = 0.04) .N. americoferus at the third release period (observation of second-generation TPB adults) significantly reduced the TPB population   . The  = 0.04) . On aver = 0.12) .N. americoferus (with or without O. insidiosus) negatively impacted TPB’s population  , 3.0 , and 2.3  higher in the control treatment than in the treatments with singly O. insidiosus, O. insidiosus, and N. americoferus and singly with N. americoferus, respectively.Both treatments with  = 0.04) . HoweverN. americoferus on all the tested stages of TPB demonstrate its potential as a biological control agent against this pest. Each stage of N. americoferus can kill the equivalent or younger stages of TPB. This indicates that most N. americoferus stages can contribute to the control of TPB. Additionally, our laboratory results show that O. insidiosus has the potential to contribute to the control of TPB at the N2 nymphal stage and presumably  also to younger ones (N1). This potential of N. americoferus was confirmed in field experiments on strawberries, but the effect of O. insidiosus on the TPB population was marginal and not significant. Then, in a biological control strategy against TPB, N. americoferus may play a main role in rapidly (from two weeks after release) and sustainably reducing the populations of the pest, while O. insidiosus may play a role as a secondary (and complementary) biological control agent since its contribution is limited.The predation rates of N. americoferus and O. insidiosus. Therefore, synchronization between the developmental stages of N. americoferus and TPB will be decisive for the effectiveness of biological control by the predator, given that in this study, N. americoferus was shown to be able to affect prey that were at most at about the same stage of development. Under Canadian conditions, overwintering TPB species become active in April and/or the first weeks of May [N. americoferus species are found active about a week or two after TPB . Hence, the natural life cycle of N. americoferus seems to be slightly behind that of TPB. Moreover, at a temperature of 27 °C, the nymphal development of N. americoferus takes an average of 24 days to be completed [N. americoferus and TPB in spring may favor the prey over the predator. Consequently, the period of the year when N. americoferus effectively and naturally regulates the populations of TPB could be limited to some weeks during summer and fall (most N. americoferus are adults at that time). As a result, N. americoferus would not naturally protect strawberry plants during the whole production season (mainly June for summer-bearing strawberry plants and from midsummer to early autumn for everbearing plants). The natural regulation of TPB is certainly not to be overlooked, especially since the TPB and N. americoferus use the same autumnal hosts  [N. americoferus to TPB’s biological control during both summer and fall remains to be determined. Finally, the effectiveness of biological control by N. americoferus can vary from year to year depending on weather conditions and demographic factors of the prey population. Since our study was only conducted over one summer, future studies will increase our knowledge of this system.The size ratio between predators and their prey is decisive in the outcome of a predator–prey encounter . Our labs of May , while oompleted , compareompleted . This su plants) . HoweverN. americoferus to the TPB cycle could constitute an effective augmentative biocontrol strategy. From an augmentative biocontrol perspective, such as our field experiment, the predators were released depending on the developmental stage of the TPB. Nabis americoferus adults were efficient in reducing the TPB population regardless of the developmental stage of the TPB. Therefore, the window for controlling the TPB is wide when N. americoferus is released at the adult stage, and the releases of N. americoferus could be synchronized with the period of vulnerability of strawberries. In our experiment, N. americoferus was shown as a promising biological control agent of the TPB when it was released at either 0.25, 0.5, or 0.75 individuals per strawberry plant. We released mainly adults and measured their local effect on the TPB population. However, these N. americoferus may have dispersed shortly after their release. The release of N. americoferus nymphs could extend the local effect, as these nymphs have a more limited dispersal capacity. Thus, when young nymphal stages (N1 and N2) of the TPB are first observed in the field, the release of N3–N4 nymphs of N. americoferus could have an optimal impact on the pest’s population.Thus, well-synchronized releases of Orius insidiosus feeds on the TPB, but it is not an effective biological control agent against this pest. Hagler et al. [O. tristicolor in strawberry fields had TPB in their gut, a rate comparable to what they determined for G. punctipes (18.2%) and lower than what they determined for N. alternatus (33.3%). That level seems not to be enough for the effective control of the TPB population. Moreover, our results show that N. americoferus and O. insidiosus did not have a synergetic effect on TPB’s population. The expected facilitation effect between the active searching O. insidiosus and ambush predator N. americoferus was not observed. However, O. insidiosus was not harmful to N. americoferus, which maintained its effectiveness even in the presence of Orius. Thus, a management strategy favorable to O. insidiosus and other predators, from the perspective of biological control through conservation, could be an interesting strategy. Theoretically, the TPB could respond to predation risk and modulate feeding and egg-laying behavior [Our results indicate that r et al.  observedbehavior . The antN. americoferus over the entire area of a strawberry field is a strategy that seems economically unviable. Thus, biological control based on N. americoferus would require a combination of strategies that reduce the extent of the surface to be treated. First, TPB has been shown to aggregate in trap crops, where they can be targeted by repressive treatments [N. americoferus to be released. Moreover, Hagler et al. [N. alternatus was higher in the alfalfa trap crops than in the strawberry plants. This increased performance of N. alternatus in alfalfa could be explained by its type of predation. Nabis spp. are ambush predators waiting for their prey. In alfalfa plots, where L. hesperus populations were denser, the rate of the encounter between N. alternatus and its prey was certainly higher [N. alternatus was increased. Hence, the combination of trap crop and predator release may have a synergetic effect.Massive releases of eatments ,35,36,37r et al.  observedy higher . As a re


Aphaenogaster species from the subterranea group in the western Mediterranean: A.ichnusa Santschi, 1925, from south-western Europe, and A.subterranea , also occurring in central and eastern Europe. Historically, the two species have been widely misunderstood: A.ichnusa was long considered a Sardinian endemic subspecies of A.subterranea, while its continental populations were misidentified as A.subterranea s. str. Recently, A.ichnusa was elevated to species rank and its worker caste was redescribed with that of A.subterranea, allowing for their correct identification. Yet their distribution was documented in detail only for France and Sardinia. Furthermore, no morphological characters were described to distinguish the males and queens of the two species. By investigating private and museum collections, 276 new records of A.ichnusa are provided here and 154 of A.subterranea from the western Mediterranean. Additionally, qualitative and quantitative morphological characters were combined to identify their males and queens. We present the new southernmost, easternmost, and westernmost distribution limits for A.ichnusa. Based on our results, this species is widely distributed in Italy and Catalonia (Spain), also occurring on several Mediterranean islands, avoiding areas with continental climate and high altitudes. Sicily is the only island to host the less thermophilous A.subterranea, which otherwise extends westward to Galicia (Spain). Sympatric occurrence is not rare along the contact zone. Additional natural history observations are reported regarding foraging habits, associated myrmecophiles, habitat preferences, and colony structure in the two species.There are only two  Aphaenogaster Mayr, 1853 belongs to the myrmicine tribe Stenammini Ashmead, 1905 , A.holtzi , A.ichnusa Santschi, 1925, A.kurdica Ruzsky, 1905, A.lesbica Forel, 1913, A.maculifrons Kiran & Aktaç, 2008, A.subcostata Viehmeyer, 1922, A.subterranea ], mostly residing in the eastern Mediterranean and Caucasus region, where several putative undescribed species also exist  is in fact A.strioloides  (terra typica: mainland France) and A.ichnusa Santschi, 1925 (terra typica: Sardinia). The first was described using the spelling “subteranea” . Until recent years A.ichnusa was considered as a Sardinian endemic and a subspecies of A.subterranea. Their separation as two distinct species was only recently demonstrated based on worker morphology and mitochondrial DNA . A complete list of the investigated material and of the morphological data is provided in the Suppl. material A.subterranea, A.ichnusa is characterized by a different propodeal shape with relatively well-developed dentiform spines, as opposed to slight edges. This difference can be appreciated both in lateral view  and in dorsal view (as the spines are wider than the remaining propodeum). Consequently, the SPWI/PrW ratio was found to be very different between the two species, with no overlap: 1.36 ± 0.14  in A.ichnusa, 0.911 ± 0.06  in A.subterranea  that significantly protrude out of the head capsule, well-distanced from three large ocelli. Mandibles dentate, ~ ½ length of the head capsule, their external side mostly straight for more than ¾. Clypeus medially slightly emarginated. Mesosoma clearly wider than the head, characterized by a well-developed anterior gibbosity. Propodeum much shorter than the remainder of the mesosoma. The horizontal section of the propodeal profile is shorter than its two vertical sections (anterior and posterior), which both form angles of ~ 90° with it. A ventral cuticular process protrudes from the metasternum. Long pedunculate petiole, globose postpetiole, the petiole and postpetiole have a similar heigh, while the postpetiole is wider than the petiole. The legs are long, with hind femurs approximately the same length as the entire mesosoma. The antennae comprise of 11 flagellomeres, the scapi are short, measuring ca. twice the pedicel, and approximately extending slightly beyond the eyes if aligned perpendicularly to the head length axis. Body color pale to dark brown, appendages whitish. Surface sculpturing mostly weak, most of the ant is smooth and shiny, with at most isolated stickman-like units, very feeble rugae or reticulate microsculpture near the sutures, and feeble reticulate microsculpture on the head. Sparse erect and suberect setae mostly occur dorsally over the head, mesosoma and nodes, and both dorsally and ventrally on the first gastral segment and on the distalmost margins of the remaining segments, while shorter suberect or appressed setae cover the appendages.The following key is meant to facilitate this distinction in the examined region. However, the intraspecific morphological variation of several species is still little known, so we recommend a careful approach in its use.A.ichnusa and A.subterranea are much more difficult to tell apart based on their general shape as compared to workers and males. However, a subtle but very reliable character was found in the surface microsculpture of the dorsal mesosoma: in A.ichnusa, stickman-like reticulations occur sparsely and are modestly developed, while in A.subterranea they are visibly more developed and sometimes single stickman-like complexes may connect to each other covering very large areas. MCMssctm ranges do not overlap between the two species, while A.subterranea shows a high variation in the upper range: 4.33 ± 0.78  in A.ichnusa, 24.68 ± 22.97  in A.subterranea  slightly protruding out of the head capsule. The distance between the central among the three ocelli and the level of the compound eyes is ca. the length of the ocellus itself. Mandibles dentate, ~ ½ length of the head capsule, their external side mostly straight for over ¾. Clypeus medially slightly emarginated. Massive mesosoma, slightly wider than the head. Propodeal spines well developed and straight, in profile view the of the propodeum is steep, with the section above the spines slightly inclined and the section below entirely vertical. Long pedunculate petiole, globose postpetiole, the petiole and postpetiole have a similar heigh, while the postpetiole is wider than the petiole. The antennae comprise ten flagellomeres, the scapi are moderately long, surpassing the posterior margin of the head if aligned parallel to the head length axis, while the antennal club consists of four flagellomeres and the flagellomeres 3–8 are only slightly longer than wide. Body color normally not uniform, ferruginous to very dark brown, with blackish patches usually occurring on the frons and paler orangish areas near the sutures of the mesosoma. Appendages pale orange to ferruginous. Rugose sculpture on most of the head, partly on the sides of the mesosoma including the entire propodeum, and on the nodes. The remainder of the body is smooth and shiny, except for stickman-like microsculpture. Abundant erect or suberect setae cover the head, the dorsum of the mesosoma and nodes, the first gastral segment, and the distalmost margins of the remaining segments, while shorter suberect or appressed setae cover the appendages.The following key is meant to facilitate this distinction in the examined region. However, the intraspecific morphological variation of several species is still little known, so we recommend a careful approach in its use.A.cardenai and A.strioloides are currently unknown and, therefore, they are not included in this key. The first only occurs in Iberia and, considering that its workers are unique among West Palearctic Aphaenogaster and that queens usually retain a number of characters of workers, they are highly unlikely to be confusable with species of the subterranea group  occupied by A.ichnusa by 145% (103 new compared to 71 from literature) and of those occupied by A.subterranea by 64% (130/202). The two species showed a parapatric distribution, with only 4.5% of the cells (22/484) occupied by both species , the easternmost at 18.46 longitude , the southernmost at 36.94 latitude  and the northernmost at 44.48 latitude (mainland France). For A.subterranea, the westernmost record is at -8.46 longitude  and the southernmost at 37.64 latitude .We recovered a total of 274 new records of A.ichnusa for the first time for several islands and archipelagos: some of the Aeolian Islands , the Balearic Islands , the Maddalena Archipelago (Caprera), the Phlegrean Islands , Tavolara island, the Tremiti islands (San Domino), and the Tuscan Archipelago .We report subterranea group species in the region is relatively easy . We show that A.ichnusa is frequent across a vast region of the western Mediterranean . Aphaenogastersubterranea may have been temporarily introduced to Madagascar , oribatid mites, silverfish, leaf beetle larvae (Chrysomelidae: Clytrinae), and planthoppers nymphs (Cixiidae) in A.ichnusa nests, and isopods (Platyarthridae in A.subterranea nests, in which also Cixiidae were reported by A.ichnusa mostly started to forage outside the nest at dusk. Perhaps the foraging activity is often conducted within the leaf litter or in endogean microspaces, but none of these species is truly subterranean (However, despite their ecological success and wide distribution, still relatively little is known ca. the habits of these species besides anecdotal reports. They are probably mostly predators and scavengers, but at least erranean .A.subterranea at least. Furthermore, A.subterranea workers are known to use tools: they drop small debris into liquid food and transport food-soaked tools back into their nest (A.ichnusa and A.subterranea, suggesting at least occasional polygyny in both species.Some of us also observed cooperative food transport in eir nest . This beeir nest . NotablyA.subterranea and A.ichnusa seemingly overlap in late summer/early autumn. However, the relatively large size of the contact zone compared to the distribution range of A.ichnusa, and the consistent morphological differentiation from A.subterranea, suggest some adaptation to counter hybridization. At the same time, the data we are providing could facilitate future studies focused on the ecology and niche partitioning of these species.An integrative approach is needed to clarify whether hybridization or introgression between the two species have a significant role as observed in other ants . Nuptial


Boon. During the revision of the manuscript, the addition of C.J.F. Boon was discussed in writing between the first and last author, but due to a misunderstanding, C.J.F. Boon was not added as a coauthor. C.J.F. Boon made significant contributions to the revised manuscript by being responsible for patient materials and information, and C.J.F. Boon has now been added to the author list in the online version of the manuscript. Note that there are no familial or other relationships between C.J.F. Boon and N. Boon.Additionally, in the originally published version of the supplemental figures that accompanied our manuscript, the majority of the legend for Figure S2 was accidentally removed. We have since corrected the supplemental figures and the complete legend is now contained therein. We apologize for any confusion.


Plateumaris species regarded as valid three were described in English, the remaining seven in Latin, French, or German: P.amurensis Weise, 1898, P.bracata , P.consimilis , P.roscida Weise, 1912, P.rustica , P.sericea , and P.weisei . These seven non-English original descriptions and their translations into English are presented here. Additionally, the translations of the first descriptions of the genus Plateumaris and of its 19 synonyms  are given.Many original descriptions of beetles were published in Latin with specific idioms and technical terms, which are sometimes difficult to understand. The exact meaning of these descriptions is necessary for taxonomic and systematic research. Of the ten Palaearctic  Furthermore, knowledge of the Latin language is in rapid decline. It is no longer part of the education curriculum of grammar schools, but was mandatory in Europe for centuries. Nowadays, English is the worldwide communication language used in science. The second author is a classicist and an entomologist with profound knowledge in systematics who translated numerous original Latin descriptions of Donaciinae taxa in the last years for systematic studies by the first author, such as Donaciaclavareaui Jacobson, 1906  of the genus Plateumaris, and, additionally, of 19 taxa which are regarded as synonyms. These taxa are discussed in detail in Plateumaris species.In the genus Plateumaris taxa are arranged alphabetically, regardless of whether they are now synonyms or names of a valid species. The Palaearctic species P.akiensis Tominaga & Katsura, 1984, P.constricticollis Jacoby, 1885, and P.shirahatai Kimoto, 1971 are not included here because they were originally described in English.All original descriptions published in Latin were translated into German by the second author. Then the first author translated them into English. There also exist some original descriptions in German, which were translated into English by the first author. Some original descriptions were published in French, Russian, and Serbian. They were first translated into English by Google translate. Then the translations of French texts were revised together with Gloria Geiser. Translations of texts in a Slavic language were checked with Irmgard Geiser. Some original descriptions are multilingual. They begin in Latin, but often more details are explained in the author’s native language. The original orthography of the description is printed here, even though it is now outdated; the translation into English is as accurate and faithful as possible to the original text. The names of the Text in square brackets contains additions by the authors and is not part of the original description.Described on page 154.Donaciaaffinis on page 37. Synonym of P.rustica.Described as Described on page 179. The German part of the text is shown in the original but the now-outdated orthography.P.roscida.Described on page 41. Synonym of Lepturaassimilis on page 156. Synonym of P.consimilis.Described as Prionusbracatus on page 100.Described as Because this original description was very short, P.sericea.Described on page 111. Synonym of Lepturaconsimilis on page 155.Described as Donaciadiscolor on page 216. Synonym of P.sericea.Described as Donacianigra (synonym of Plateumarisbracata) on page 265.Described as variation of P.sericea.Described on page 239. Synonym of Donacialacordairii on page 300. Synonym of P.sericea.Described as Donacia (subgenusPlateumaris) mongolica on page 267. Synonym of P.weisei.Described as Donacianigra on page 117. Synonym of P.bracata.Described as P.shirahatai.Described on page 243. Probable synonym of P.weisei.Described as subspecies on page 49. Synonym of Donaciapallipes on page 35. Synonym of P.rustica.Described as Plateumarisrustica on page 180. Synonym of P.rustica.Described as variation of Donaciaplanicollis on page 34. Synonym of P.rustica.Described as Described on page 77.Donaciarustica on page 31.Described as P.weisei.Described on page 876. Synonym of Lepturasericea on page 397. First description in Described as Description of additional characters on page 196.Remark: “Solsky, 1872” is wrong. For details see Donaciasibirica on page 245. Synonym of P.sericea.Described as P.rustica.Described on page 267. Synonym of Plateumaris (Juliusina) tenuicornis on page 128. Synonym of P.consimilis.Described as Plateumaristenuicornis Balthasar looks the same as P.consimilis. This article was ignored for decades, perhaps because it was printed in Czech and Latin. Here the part of that article that deals with P.tenuicornis in Latin is copied and translated into English.Donacia (Plateumaris) weisei on page cxvi [116].Described as Plateumaris species regarded as valid: three of them were described in English , six in Latin , and one (P.weisei) was originally described in French. In general, most of the original descriptions of Plateumaris taxa are in Latin or began with a Latin diagnosis at least, and further explanations were then added in German, Russian, or French in most cases.There are ten Palaearctic Plateumaris species. We intend to continue publishing translations of original descriptions of Donaciinae taxa. Furthermore, we encourage other colleagues to do the same in their areas of expertise.In addition to the first descriptions of the valid species in the Palaearctic, another 19 original descriptions are presented here with their translations and the species names published therein are now regarded as synonyms. Mostly, these are names which were synonymized or their synonymisation was confirmed in Plateumaris established by Donaciinae species were assigned to the genus Leptura by Donacia, but did not change the genus name of Lepturasericea. Later, some authors described Plateumaris species with Donacia as the genus name, even after Thomson had established the name Plateumaris. Some authors regarded Plateumaris only as a subgenus of Donacia, and these opinions are reflected in the first descriptions. For more details see The Latin first description of the genus


Cortinarius  were described from the alpine zone of the Alps during the second half of the XX century, by the Swiss mycologist Jules Favre, and by the French mycologists Denise Lamoure and Marcel Bon. Notoriously difficult to identify by macro- and microscopical characters, most of these species, which belong to subgen. Telamonia, have been thoroughly revised in global publications based on type sequencing. Recent surveys in the alpine areas of France (Savoie) and Italy (Lombardy), as well as the sequencing of D. Lamoure’s collections, identified three new species that are here described and illustrated: C. dryadophilus in sect. Castanei, C. infidus in sect. Verni, and C. saniosopygmaeus in sect. Saniosi. The holotypes of C. caesionigrellus Lamoure and C. paleifer var. brachyspermus Lamoure could be sequenced. A recent collection of the former is described and illustrated here for the first time, and based on available data, the latter name is recombined as Cortinarius flexipes var. brachyspermus comb. nov. Lastly, C. argenteolilacinus var. dovrensis is reported from the alpine zone for the first time and a new combination, Thaxterogaster dovrensis comb. & stat. nov. is introduced in the present work.Thirty-one alpine species of  Cortinarius is the most species-rich fungal species, even after its revision and emendation by Liimatainen et al. ” was published , a breakushrooms  but showd genera ), LiimatThe collection illustrated here is the first French record for this species, as well as its first published observation in the alpine zone.


Correction to: Takeuchi et al. Respir Res (2021) 22:3510.1186/s12931-021-01629-8In the originally published version of the article , there wIn the Funding the information of and the International Community Health Research Fund was missing. In the Acknowledgements, the authors missed to express their thanks to for sincere and active involvement in the data collection of this study. The authors missed to express their thanks to Dr. Takahashi J, Dr. Yunus M, Dr. Chowdhury HR, Dr. Arifeen SE, Dr. Baqui A, Dr. Wakai S, Dr. Tariq Anwar, Dr. J. Chakraborty, Dr. Mahbubur Rahman, Professor David Sack, Dr. Yukiko Wagatsuma, and Professor Kanehisa Morimoto, for the active involvement in the creation of the dataset of the article with which this study was accomplished. They also missed erroneously to express their thanks to the study participants, the field research staff and Dr Ipshita Hamid Trisha who were sincerely involved in the data collection of the previous article (Reference 27). We have corrected the statement in the Acknowledgement. Below is the updated statement of the “Acknowledgement.”This work was funded by a Grant-in-Aid for Scientific Research (Basic Research (A), 26257507) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, spanning the years 2014-2016, and the International Community Health Research Fund. It was also funded for unwavering support by the core donors of The International Centre for Diarrhoeal Disease Research, Bangladesh  - The governments of Bangladesh, Canada, Sweden, and the UK.The authors gratefully acknowledge all participating children who generously provided their invaluable blood and stool samples and their guardians who accompanied them. The authors gratefully acknowledge Prof. Shinji Nakahara also for statistical analysis. Moreover, the authors sincerely thank the field research staff and Dr. Ipsita Hamid Trisha for the rigorous data collection. The authors also acknowledge the valuable contributions of Dr. Junkichi Takahashi, Dr. Md Yunus, Dr. Hafizur Rahman Chowdhury, Dr. Shams EL Arifeen, Dr. Abdullah Baqui, and Susumu Wakai, who actively participated in creating the dataset referenced in this study (Reference 27), enabling its successful completion. Finally, the authors would like to express their appreciation to Prof. David Sack and Prof. Kanehisa Morimoto, Dr. Tariq Anwar, Dr. J. Chakraborty, Dr. Mahbubur Rahman, Dr. Yukiko Wagatsuma, for their kind assistance in supporting the creation of the dataset used in this study.We apologize for the error.


This article has been retracted: The authors have been unable to replicate their published results and are therefore no longer confident in the conclusions of their research. In light of this, and with the agreement of all authors, Oncotarget has decided to retract this paper.11621-11628. https://doi.org/10.18632/oncotarget.14598Original article: Oncotarget. 2017; 8:11621–11628.


This position statement represents a consensus of an expert committee composed by the Italian Academy of General Dentistry  and Italian Academy of Legal and Forensic Dentistry  on the appropriate use of cone beam computed tomography (C.B.C.T.) in dentistry. This paper analyzes the use of C.B.C.T. in light of the rapid evolution of volumetric technologies, with the new low- and ultra-low-dose exposure programs. These upgrades are determining an improvement in the precision and safety of this methodology; therefore, the need of a guideline revision of the use of C.B.C.T. for treatment planning is mandatory. It appears necessary to develop a new model of use, which, in compliance with the principle of justification and as low as reasonably achievable (ALARA) and as low as diagnostically acceptable (ALADA), can allow a functional “Dedicated C.B.C.T.” exam optimized for the individuality of the patient. The evolution of volumetric technologies in dentistry is determining the need for a revision of the objective indications, in the diagnostic and operative fields, on the use of cone beam computer tomography (C.B.C.T.) ,2,3. TheIn the dental field, the ever-increasing demand for three-dimensional investigations determines the need for an adaptation, based on updated scientific evidence, of the recommendations for performing the C.B.C.T. examination .In fact, technological progress has led to a substantial decrease in irradiation, which corresponds to a better quality of information to perform the diagnostic task ,2,3. ThiLow-dose and ultra-low-dose cone beam computed tomography (C.B.C.T.) have several advantages compared to conventional 3D radiological methods. The primary advantage is the lower radiation exposure to the patient, which is particularly important for younger patients and those who require multiple scans. Traditional C.B.C.T. scanners can expose patients to radiation doses that are 10–50 times higher than conventional 2D radiological methods. Low-dose and ultra-low-dose C.B.C.T. scanners, on the other hand, can reduce the radiation dose by up to 87% compared to traditional scanners , if usedAnother advantage of low-dose and ultra-low-dose C.B.C.T. is that they provide high-quality, accurate 3D images of the oral and maxillofacial region. This is particularly useful for orthodontic, implant and maxillofacial surgery planning ,5 see S, in periThe advantages of low and ultra-low-dose protocols in the use of C.B.C.T. have been evidenced in the literature, where the effectiveness of the volumetric examination for the vertical evaluation and the thickness of the bone adjacent to the anterior teeth has been evaluated as an important anatomical parameter to plan and/or monitor the effects of orthodontic or periodontal treatment. The doses and image quality were compared ex vivo with the diagnostic task of three different acquisition protocols and parameters, (low- and high-resolution) two low-dose C.B.C.T. and one normal-dose C.B.C.T. The absorbed dose was calculated using the product dose area (DAP). The Ultra low-dose C.B.C.T. protocol and the Low Dose C.B.C.T. protocol used, ejected a dose (dap 69 and 87 mGycm2) which is lower than that of panoramic views (Dap 88 mGycm2) . Thus, with a lower absorbed dose, a higher quantity and quality of diagnostic information was obtained than those using traditional two-dimensional radiodiagnostic examinations. See . The C.BLow-dose and ultra-low-dose C.B.C.T. scanners can capture images in a matter of seconds or minutes, compared to several minutes or hours required by traditional scanners ,9. These machines use wider cone angles, faster rotating gantries, and optimized image acquisition protocols that can minimize patient motion and improve image registration. Additionally, the images produced by low-dose and ultra-low-dose C.B.C.T. can be easily shared and viewed by multiple practitioners, enabling more efficient communication and collaboration among specialists. Low-dose and ultra-low-dose C.B.C.T. images can be easily processed and analyzed using various software tools, such as virtual implant planning, airway analysis, and bone density measurement, that can help clinicians make more accurate and informed treatment decisions. Overall, the use of low-dose and ultra-low-dose C.B.C.T. provides many technical advantages over traditional 3D radiological methods, making it a preferred option for many dental and maxillofacial imaging needs ,9.Moreover, the use of digital technology has made the acquisition, storage, and transfer of C.B.C.T. images much easier and more efficient. Digital images can be easily retrieved, manipulated, and shared between dental professionals, allowing for improved collaboration and more informed treatment decisions. Furthermore, the use of digital technology has significantly reduced the need for the physical storage of radiographic images, freeing up valuable office space and reducing environmental impact.Although the use of C.B.C.T. remains a diagnostic study that can only be used on the basis of specific criteria, its use appears justified in all those cases in which the limitations of the two-dimensional examination become clear ,12,13,14In endodontics, for example, the bidimensional intraoral examination is the main radiological support in the diagnosis, planning, and execution of treatments and in the subsequent evaluation of the results . HoweverThe precise evaluation, before treatment, of the dental/alveolar complex to be recovered and of the surrounding tissues, is essential to define both the prognosis and the most suitable treatment strategy. The physical limitations of two-dimensional radiodiagnostic examination are, essentially, as follows: ,13,14. Geometric alterations, which cause disparallelisms, and consequently, image distortions;Anatomical overlays that lead to the failure to identify the actual number of root canals and their morphological configuration;Attenuation of the beam induced by the anatomical thickness of the tissues crossed.It is essential to be aware of these limits as a radioprotection measure to prevent un-necessary repetitions of 2D intraoral examinations and to be twice as precise in identifying both the lesions and in defining the outcomes of root canal therapy, compared to the traditional intraoral examination . It is pMoreover, the diagnostic limitations of two-dimensional radiographic examination are particularly relevant in certain categories of patients with cardiovascular and systemic diseases. Cardiac patients with structural problems of valves, device carriers, and/or heart prostheses are more exposed to the risk of developing infectious endocarditis (E.I.) . InterveNowadays, these patients can benefit, thanks to the C.B.C.T., from better diagnostic and treatment effectiveness before surgery or interventional cardiology. In addition, in these patients, the execution of a dedicated C.B.C.T. is indicated for diagnostic purposes and at the beginning of the follow-up after the cardio/valvular intervention .In 2021, the American Association of Endodontists (AAE) and the American Academy of Oral and Maxillofacial Radiology (AAOMR) reached the joint conclusion that C.B.C.T. must be the reference exam in root canal retreatment and in the initial endodontic treatment of complex cases. ,14,17 FuDiagnosis of periapical disease in the presence of inconsistent clinical signs or symptoms;Need for diagnostic confirmation in non-odontogenic pathologies;Presence of extremely complex root canal anatomy prior to orthograde treatment;Evaluation of endodontic failures before retrograde treatment;Diagnosis of external and internal resorption;Evaluation of oromaxillofacial traumas and their treatmentPatel et al. state that the 3D survey is indicated in the case of the following:A further indication for the use of C.B.C.T. is represented by the follow-up in the cases of root canal retreatments in order to objectively evaluate the effectiveness of root canal treatment and proceed, if necessary, with a timely re-operation ,18. Low-The therapeutic advantages are not limited to a single branch of dentistry, but extended to other specialties such as surgery, prosthetics, and the planning of prosthetic and implant-prosthetic treatment ; orthodoFurthermore, it must be considered that one of the fields of the greatest therapeutic evolution in dentistry is represented by the introduction, in clinical practice, of both static and dynamic guided therapies and patient virtualization procedures. During the diagnostic phase, these systems enable efficient, minimally invasive, rapid, precise, and effective programming, even in complex cases ,24,25.The result is obtained thanks to the convergence of data, derived from 3D investigations of the face , of the oral cavity and teeth , and of the skeletal bases (C.B.C.T.) ,25.A further aspect, which is essential for defining the problem of using 3D radiological methods in their entirety, is represented by the need for knowledge that allows for the right balance between biological and environmental costs and the benefit of the patient .Human decision-making ability and related expertise are necessary for the appropriate use of the available technology, as they are the only means of identifying the appropriate areas of use and justifications from a radiodiagnostic perspective in each individual clinical case ,27,28.Low-dose and ultra-low-dose protocols have been included by various manufacturers within the settings function of C.B.C.T. to assist professionals in managing optimization. However, the search for the optimal balance between image quality and dose depends on the specific clinical requirements demanded by the diagnostic task that the professional must identify and perform each time. Pre-established C.B.C.T. protocols do not always meet this requirement, which is why it is essential to acquire adequate and specialized training in relation to the use of C.B.C.T. in dental practice, as indicated in the “European Academic of Dentomaxillofacial Radiology” (EADMFR) document ,28.The primary objective is to produce useful clinical information with the lowest possible dose by intervening on the physical factors of the exposure load and the qualitative balance of the image.Pipe current (mAs). This is the product of current intensity (mA) and exposure time (s). This value adjusts the sharpness and the lightness/darkness of the image. An increase in these values increases the exposure index and the dose ,27.Potential difference (kV) at the pipe outlet. This parameter quantifies the tissue penetration capacity and intervenes on the contrast. High kV values give rise to a more penetrating X-ray beam, with low contrast images and more details , while low values produce high contrast images .Voxel (µm). This is the constituent element of the three-dimensional physical image. Characteristic of C.B.C.T. technology is the isotropicity of the voxel. The size of the voxel can vary depending on the type of imaging exam, but typically ranges from 0.05 to 0.4 mm in size. The smaller the voxel size, the higher the resolution of the resulting 3D image, allowing for a more detailed representation of the patient’s anatomy. The size affects the spatial resolution of the image, meaning that the smaller the image, the higher the resolution. However, a decrease in the size also leads to an increase in image noise ,27There are large differences between the various C.B.C.T.s regarding the technologies and parameters for managing the dose load and image control . TherefoIt is useful to know as a further element of optimization that when smaller sized voxels are used, to increase the spatial resolution, an increase in the image noise is generated; to reduce this effect, many C.B.C.T. manufacturers associate, for example, the increase in the mAs, which determines an improvement of the sharpness, with an increase in the dose load. This aspect generates the false belief that the reduction in the voxel, in an isolated manner, determines an increase in the irradiation, but in reality, it is not the size of the voxel, but the increase in the current intensity that determines the increase in the radiation doses .4.. This is a term used in radiology to describe the portion of the patient’s anatomy that is captured in an imaging exam. The field of view (F.O.V.) is adjustable to capture a particular region of interest (ROI) within the volume acquisition area. The optimal F.O.V. size depends on the specific imaging exam and the clinical question being addressed [Field of View (F.O.V. cm)ddressed ,2,28. ThThe size of the F.O.V. does not directly affect the spatial resolution of the C.B.C.T. images unless related to other management parameters, including voxel and position . Since tAs a result, to limit and minimize the radiation, it is crucial to consider the position of the F.O.V. in addition to the width.Moreover, using narrow fields of vision with an appropriate voxel, even with low-dose protocols, is linked to higher spatial resolution in addition to dose containment, particularly when the priority is devoted to positioning the examination item as centrally as feasible within the field of view .The aspect related to the centralization of the tooth inside the F.O.V. is an essential element of further optimization related to the image quality, because it manages to improve the definition of diagnostic information, such as the possibility of identifying vertical fractures (VRF), even in the presence of channel filling ,20,29.On the other hand, the positioning at the periphery of the F.O.V. causes the loss of the homogeneous visual definition, increasing the presence of artifacts. It is important to underline how the accuracy of the positioning of the object at the center of the F.O.V. determines a greater sensitivity and diagnostic definition than the use of the algorithms of the reduction in metal artifacts (MAR) ,31.Proper training and recent C.B.C.T. devices with previsualization systems, for the correct placement of the F.O.V., help with this task .(A)The angle and rotation speed of the sensor assembly generator and exposure mode. Some equipment allows for partial or full rotations to be selected for specific acquisitions, allowing the modulation of both the patient dose and image quality. Full rotations result in a higher dose and a greater image definition, while semi-rotations reduce the dose but also the diagnostic quality. The scan speed can also affect the exposure time; higher speeds tend to reduce the dose. Additionally, most commercially available C.B.C.T.s emit pulsed exposure rather than continuous exposure, which allows for optimization by reducing the radiation ,15,27.(B)(Filters); additional filtration at the tube outlet eliminates low-energy radiation that is not useful for diagnostic purposes, helping to further reduce the dose to the patient .(C)(Flat panel); the detector is the heart of the C.B.C.T. Today, all equipment has digital detectors, with either indirect or direct conversion; various types are available on the market with varying characteristics and costs. The choice of the detector influences the image quality, and therefore, all parameters related to it .Furthermore, the optimal balance between the dose and the information required for diagnosis and therapy is influenced by specific technical parameters of the equipment used, which can be adjusted for some devices, such as the following:The new optimization possibilities linked to the evolution of radiological and information technology (IT) make it necessary to analyze and reassess the overall indications for the use of 3D C.B.C.T. in dentistry.The innovation speed, the frequent software update that further refines and improves the function and methodologies, and artificial intelligence (A.I.) can quickly make the scientific results reported in the literature obsolete, which must be continuously updated to provide solid support for the guidelines.Several studies show that part of the indications on the use of C.B.C.T. in certain areas of dentistry are based on opinions rather than on evidence or scientific evidence. The literature shows a lack and incompleteness of research papers on the parameter settings and clinical protocols ,22,27.The evaluation of more dedicated parameters, and therefore, a more appropriate use of C.B.C.T., appear to be evident when analyzing the literature and the C.B.C.T. use in different fields of dentistry.In orthodontics, the technological evolution and recent evidence show that the radiation of an ultra-low-dose C.B.C.T. (ULD-UL) with lateral cephalometric reconstruction (RLC) can deliver an effective dose with a reduction of 87% compared to standard C.B.C.T. protocols, resulting in an examination with 11–18 microSieverts (μSv).A combination of traditional 2D examinations, orthopantomography (PAN), and lateral cephalometric (SLC) results in a dose of 27–30 (μSv), which is therefore higher than the 11–18 (μSv) dose of the ULD-UL C.B.C.T. .In terms of radioprotection, it is possible to reduce the radiant impact suffered by the patient, improving the visibility and analysis of anatomical structures for diagnostic and treatment purposes, such as the management of impacted canines .This evaluation is in contrast to what is reported, for example, in the Italian text, “Recommendations for the correct use of volumetric TC equipment “Cone Beam”, regarding the amount of the effective dose expressed for C.B.C.T., PAN, and LC examinations currently in force . Contact proximity with nearby anatomical structures (roots of contiguous elements) can lead to root resorption. In certain circumstances, the European guidelines recommend the integration of 2D images with C.B.C.T. exams optimized with a reduced F.O.V. in order to allow an adequate and complete view of the anterior maxillary region and evaluate the impacted teeth with the surrounding structures, allowing an accurate diagnosis and surgical/orthodontic treatment ,6,10.It should be noted that missed diagnosis or late treatment can lead to root resorption of the permanent adjacent incisors in 48% of cases, often complicating the outcome of orthodontic treatment and the prognosis of the dental elements interested .2) and potentially combining minimal fields. As a result, effective doses can range widely for C.B.C.T., from 5 μSv to 1073 μSv [In implant dentistry, C.B.C.T. is recommended as a diagnostic tool for presurgical planning and oral implant-related diagnosis. Compared to 2D imaging, C.B.C.T. is more accurate in predicting implant dimensions and the need for bone grafting procedures. Low-dose C.B.C.T. (LD-C.B.C.T.) protocols produce acceptable 3D images while substantially reducing the patient’s radiation exposure ,26. The 1073 μSv .In oral surgery, in the surgical treatment of impacted teeth such as canines and in complex extraction procedures of third molars, the use and prescription of the C.B.C.T. is a commonly encountered event, representing a frequent indication; numerous evidences demonstrate how it is important and useful to significantly limit the irradiation to the patient, and to use adequate exposure parameters of F.O.V. as small as 4 × 4 or smaller ,17,32. TSome of the limitations linked to patient and environmental radioprotection criteria, applied as different standards for different states that regulate the use of C.B.C.T., are now based on outdated recommendations.Further research that takes into account the better management of exposure parameters and image quality linked to technological evolution is necessary to obtain objective data on the clinical use of 3D diagnostics technologies ,28.The primary objective is to produce useful clinical information with the lowest possible dose, by intervening on the physical factors of the exposure dose and the qualitative balance of the image . This reThe evolution of volumetric technologies in dentistry should lead to a revision of the objective indications for the use of C.B.C.T.The technology offers several advantages over traditional 2D imaging, including improved visualization of complex anatomy, improved diagnostic accuracy, and the ability to visualize 3D structures. However, the use of C.B.C.T. should always be guided by a careful assessment of the clinical requirements and a thorough understanding of the technology and its limitations ,27,28.This means that the imaging protocols should be tailored to the specific needs of each patient, taking into account factors such as their size, anatomy, and the type of treatment they are undergoing. This will result in a more efficient and effective exam that is optimized for the patient’s individual needs . AdequatALARA (as low as reasonably achievable) is a principle in medical imaging that aims to minimize a patient’s exposure to ionizing radiation by using the lowest effective radiation dose necessary to produce an accurate and diagnostic image. The goal of ALARA is to reduce the risk of adverse effects from radiation exposure while still obtaining the necessary diagnostic information. The concept of ALARA is a central principle of a general nature in radioprotection; it promotes and is essentially based on the importance of optimization processes and is based on the hypothesis of a linear dose-effect relationship without threshold  . It alreALADA  is a similar concept in medical imaging, but focuses on ensuring that the radiation dose used is low enough to minimize risks, but high enough to produce a diagnostic image. ALADA aims to express its optimization contribution exclusively to the medical/dental activity, linked to diagnostic acceptability. In other words, ALADA balances the need for diagnostic quality with the need for radiation safety .Both ALARA and ALADA are important principles in radiology, and radiologists strive to achieve these goals in their practice by using appropriate imaging techniques, optimizing equipment settings, and taking other steps to minimize the patient’s exposure to ionizing radiation.For the purposes of correct radiological practice, it is also necessary to highlight how it is propaedeutic to preliminarily justify any radiological exposure for a specific diagnostic/therapeutic purpose, taking into account the individual characteristics, the patient, and the techniques which involve a minor exposure to ionizing radiation, as defined by the European directive 59/2013 art. 55 .It becomes necessary to ensure the patient the best diagnostic and therapeutic quality but also the best safety obtainable at the present moment. In Italy, the law in force on healthcare professional liability puts the safety of care and of the assisted person in first place .With reference to the European community regulations on radiation protection , any patient exposure to ionizing radiation for medical purposes must be subject to the principle of justification  and the principle of optimization (exposing the patient to the lowest level of radiation reasonably obtainable and compatible with the achievement of the required diagnostic information—art. 158) ,38.Furthermore, all new types of practices involving medical exposures must be preliminarily justified before being generally adopted and must be reviewed whenever new and relevant evidence about their efficacy is acquired ,38.It is therefore obvious that the justification process must be considered in continuous evolution, above all, in consideration of the diffusion of new technologies which involve an evolution of the modalities of access to diagnostic information, as in the case of the 3D C.B.C.T. The Italian ministerial “Recommendations for the correct use of Cone Beam volumetric CT equipment” date back to May 2010 , and theIt appears to be evident that there is a necessity to update the recommendations for the C.B.C.T use and to improve knowledge and training to fully exploit the potential and the benefits that the technological evolution has given to the clinician ,27. Cons


Acinetobacter lowffii, Bacillus megaterium, B. pumilus, Kocuria carniphila, K. palustris, K. rhizophila, Micrococcus flavus, M. luteus, Moraxella osloensis and Paracoccus yeei. The concentrations of Gram-negative bacteria in general and the species P. yeei were significantly associated with the season with the highest concentrations in spring. The concentrations of P. yeei, K. rhizophila and B. pumilus were associated positively with relative humidity (RH), and concentrations of K. rhizophila were associated negatively with temperature and air change rate (ACR). Micrococcus flavus concentrations were associated negatively with ACR. Overall, this study identified species which are commonly present in indoor air in homes, and that the concentrations of some species were associated with the factors: season, ACR and RH.The aim of this study is to obtain knowledge about which cultivable bacterial species are present in indoor air in homes, and whether the concentration and diversity of airborne bacteria are associated with different factors. Measurements have been performed for one whole year inside different rooms in five homes and once in 52 homes. Within homes, a room-to-room variation for concentrations of airborne bacteria was found, but an overlap in bacterial species was found across rooms. Eleven species were found very commonly and included:  Airborne bacteria in indoor environments are confirmed or presumed causal agents of various infectious diseases ,2. In adExposure assessment using personal samplers, for example, the Gesamtstaubprobenahme (GSP) , actively sampling airborne inhalable dust, is expected to be a good measure of personal exposure . HoweverAir is an important transmission route for bacteria and therefore it is important to obtain knowledge about which bacteria are present in the indoor air and which factors affect this presence. In one review paper it was suggested that outdoor bacteria from plants may enter the building through ventilation systems, doors, windows, attached to people, pets and other objects, and as a result, affects the indoor concentration level – but also that information on these factors is still not well understood .Staphylococcus is associated with the indoor air change rate (ACR) and area per occupant indicating that it might be possible to affect the presence of Staphylococcus in indoor air  across rooms and homes. A room-to-room variation within the five homes was found if data were analysed unaffected by home (P = 0.0089) with lowest bacterial concentrations in the cellars (P < 0.0001). In home 1, a high bacterial concentration was found in the bathroom. The temp and RH were different in the different room types. Thus, the temp was lower in the basements and bedrooms than in the other rooms while the RH was high in the bathrooms and basements  the species K. rhizophila was found in many samples and in high concentrations (Bacteria were identified in one sample per season in the living rooms of the five homes (Study A). Some species were observed frequently, but in low concentrations, for example, bacteria . Fourteere found . Of Gramtrations .To see whether there is an overlap in bacterial species as sampled using GSPs during 1 day each season (Study A) versus using EDC samplers for long-term sampling (Study B) the bacterial species found in the highest concentrations in the five homes are presented in a Venn diagram and an overlap in species is found . In addiTo get an impression of bacterial species in living rooms of Danish city homes in general, 52 samples taken using EDCs were screened for bacterial species (studies C and D), and the 25 species found in highest concentrations are presented in the Venn diagram. Eleven species were found in high concentrations in the living rooms of the five homes unaffected by the sampling method and in the 52 homes . A list P-values < 0.1 are considered significant. In Study A, the concentration of Gram-negative bacteria as measured using the GSP samplers was associated with the season (P = 0.0016) with lower concentrations in summer than in winter. The concentration of P. yeei tended to be associated negatively with increasing temperature and was associated with the season with the highest concentrations in spring. Micrococcus flavus was negatively associated with ACR and B. pumilus positively with RH. Kocuria rhizophila was positively associated with RH and negatively with temperature and ACR ; other associations were not significant. In the cross-sectional study (Study C), associations were found between area per occupant and K. palustris concentration , and temperature and M. flavus concentration .In Study B, K. palustris, K. rhizophila, M. luteus, M. flavus, B. pumilus, K. megaterium, K. carniphila, A. lowffii, M. osloensis and P. yeei are common in indoor air in homes in Greater Copenhagen, in addition to the previously found Staphylococcus species [In this study, we show that cultivable  species . These rP. yeei, which has caused, for example, keratitis and conjunctivitis [B. cereus, which is a food-poisoning agent [Aerococcus viridans, Acinetobacter lwoffii, Bacillus mycoides, Brevibacterium casei, Enterococcus casseliflavus and several Kocuria species. Some of the risk class 2 pathogens are normal skin-related bacteria such as B. casei and are expected to derive from the occupants. No risk class 3 pathogens were found. Some species are described as opportunistic pathogens, for example, M. osloensis  and Rhi patient ). An und patient ,32.P. yeei, B. megatarium and M. luteus, have previously been found on indoor surfaces [Bacillus species, and a high species richness of Bacillus has also been found in air samples from different occupational settings [Kocuria, Micrococcus, Bacillus and Paenibacillus were among the dominating ones, and these genera were also dominating in indoor air in Hong Kong and China .Corynebacterium xerosis and Dermacoccus sp. to the airborne bacteria in these rooms. However, it was only a tendency, and the lack of a general effect of room type for bacterial concentration is in accordance with what was found in Chinese homes [This study found a within-home variation (room-to-room) for concentrations of airborne bacteria. Thus, airborne bacterial concentrations were not uniform throughout the homes during the time of sampling, which is in agreement with a study in homes in the UK . In spitse homes . This inse homes .P. yeei was found in the highest concentrations in the spring. The habitat of P. yeei seems not to be well characterised, and in research papers it is mainly described concerning infections. Therefore, we do not know the source of exposure to this bacterium. Paracoccus yeei is a Gram-negative bacterium, and for Gram-negative bacteria in general lower concentrations were found in summer than in winter. This may be related to the impact of ultraviolet (UV) light on bacterial survival.The bacterial diversity in the living rooms differed between seasons with summer especially having another bacterial diversity, this might be caused by the high ACR in the summer. For some bacterial species, associations between concentrations in living rooms and seasons were found. Thus, B. pumilus and K. palustris were found in the highest concentrations in the autumn. These bacteria have previously been found in soil. At the genus level, we have previously observed that Kocuria is present in the lowest concentrations in summer [Kocuria palustris, K. rhizophila and M. flavus were associated negatively with indoor temperature. For the two Kocuria species, this is in accordance with what has previously been found for the genus and in contrast to what is found for the genus Staphylococcus in Danish homes [The two species n summer . Kocuriash homes  and bactsh homes .K. rhizophila and M. flavus were negatively associated with ACR and thus seem not to enter by open windows or ventilation systems. These species are not described as skin-related bacteria, but K. rhizophila has been found on the skin [K. rhizophila has been found on work clothes together with more than 200 different cultivable bacterial species [K. palustris concentration was negatively associated with area per occupant. Therefore, it may also have human or human activity as a source. The habitats of this species are not well described, but it has been isolated from very different environments including human skin [Bacillus and Acinetobacter but not Kocuria were among the most frequently found genera [Human skin is shed into the indoor air , and thethe skin . Another species ,44. In Sman skin , workersman skin , human nman skin  and mariman skin . In a std genera .Micrococcus luteus was very common in indoor air in this study, and it is described as a skin-related bacterium. Despite that, it was not significantly associated with ACR or area per occupant. Furthermore, the species did not show seasonality. The lack of association between the studied factors and concentrations of M. luteus might be because this species has several sources as it is found in soil, dust [il, dust , airwaysil, dust . It has il, dust  in the ail, dust .K. palustris, K. rhizophila, M. luteus, M. flavus, B. pumilus, B. megaterium, K. carniphila, A. lowffii, M. osloensis and P. yeei. Seasonality in bacterial diversity was found, and concentrations of P. yeei were significantly associated with the season. Bacterial concentrations were not uniform throughout the homes investigated, but no significant variation was observed for the general room type except for the lower concentration in the basements. The concentrations of P. yeei, K. rhizophila and B. pumilus were positively associated with RH, and concentrations of K. rhizophila were associated negatively with temp while K. rhizophila and M. flavus were negatively associated with ACR, and K. palustris negatively with area per occupant. Thus decreasing the RH, and increasing the ACR and area per occupant might be a strategy to reduce the exposure to some airborne bacterial species.Across homes and room types within homes, occupants are potentially exposed to some of the same cultivable bacterial species typically including: