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Massive fish introductions have taken place throughout much of the world, mostly over the last 70 years, and present a major threat to the genetic diversity of native fishes. Introductions have been reported for European Phoxinus, a ubiquitous small cyprinid that populates a wide variety of habitats. Species delineation in European Phoxinus has proven difficult with one reason being ranges of distribution that often traverse drainage boundaries. The present study combines recent samples with museum samples to better understand the current distribution of Phoxinus species and their distributions prior to the massive introductions of fishes in Europe, and to evaluate the use of museum specimens for species distribution studies. For these purposes, genetic lineages from sites collected prior to 1900 (n = 14), and between 1900 and 1950 (n = 8), were analysed using two mitochondrial and nuclear markers. Although possible fish introductions were detected, our results show that the distribution of genetic lineages of museum samples is comparable to that of the extant lineages of European Phoxinus present in those areas. These observations suggest that in the studied ranges the distribution of Phoxinus lineages has been driven by natural processes.

ABSTRACT The Dinaric Karst, a biodiversity hotspot, features complex surface and subterranean hydrological networks that influence aquatic species distribution. This study investigates how karst hydrology shapes the genetic structure of the surface‐dwelling minnow Phoxinus lumaireul, examining both large‐scale and small‐scale population patterns. Using mitochondrial DNA and genome‐wide single nucleotide polymorphism (SNP) data of 827 specimens of P. lumaireul, three hypotheses were tested: (1) karst underground water connections facilitate genetic connectivity within and across river systems, whereas non‐karst rivers exhibit genetic connectivity mostly within the same system; (2) historical and occasional hydrological connections have shaped present‐day population structure, leaving genetic signatures of relatedness where no contemporary hydrological links exist; and (3) genomic approaches provide additional insights into biologically relevant connections that may not be captured by classical tracing tests. The large‐scale analyses confirmed three main genetic groups (1a–c), whose structure was likely shaped by Pleistocene glaciations and associated microrefugia rather than by karst hydrology. Small‐scale structure analyses revealed that while karst hydrology facilitated gene flow within specific areas, connectivity was uneven and influenced by local hydrological dynamics and historical admixture events. Furthermore, some underground pathways identified by classical tracing tests lacked evidence of genetic connectivity, underscoring the limitations of traditional methods and the added value of genomic data in indirectly detecting biologically relevant hydrological connections. These findings highlight the influence of both historical processes and contemporary karst hydrology on P. lumaireul populations, emphasizing their vulnerability in karst ecosystems and the need for targeted conservation efforts.

Harboring a large number of endemic species, the Alps and the Western Carpathians are considered as major centers of biodiversity. Nonetheless, the general opinion until the turn of the millennium was that both Central European mountain regions did not provide suitable habitat during the Last Glacial Maximum, but were colonized later from southern refuges. However, recent molecular genetic studies provide new evidence for peripheral Alpine refuges. We studied the phylogeography of the calciphilous land snail O. dolium across its distribution in the Alps and the Western Carpathians to assess the amount of intraspecific differentiation and to detect potential glacial refuges. A partial sequence of the mitochondrial COI was analyzed in 373 specimens from 135 sampling sites, and for a subset of individuals, partial sequences of the mitochondrial 16S and the nuclear histone H3 and H4 were sequenced. A molecular clock analysis was combined with a reconstruction of the species' geographic range history to estimate how its lineages spread in the course of time. In order to obtain further information on the species' past distribution, we also screened its extensive Pleistocene fossil record. The reconstruction of geographic range history suggests that O. dolium is of Western Carpathian origin and diversified already around the Miocene-Pliocene boundary. The fossil record supports the species' presence at more than 40 sites during the last glacial and earlier cold periods, most of them in the Western Carpathians and the Pannonian Basin. The populations of O. dolium display a high genetic diversity with maximum intraspecific p-distances of 18.4% (COI) and 14.4% (16S). The existence of various diverged clades suggests the survival in several geographically separated refuges. Moreover, the sequence patterns provide evidence of multiple migrations between the Alps and the Western Carpathians. The results indicate that the Southern Calcareous Alps were probably colonized only during the Holocene.

Background Selecting high-quality habitat and the optimal time to reproduce can increase individual fitness and is a strong evolutionary factor shaping animal populations. However, few studies have investigated the interplay between land cover heterogeneity, limitation in food resources, individual quality and spatial variation in fitness parameters. Here, we explore how individuals of different quality respond to possible mismatches between a cue for prey availability (land cover heterogeneity) and the actual fluctuating prey abundance. Results We analyse timing of breeding and reproductive success in a migratory population of Eurasian kestrels ( Falco tinnunculus ) breeding in nest-boxes, over a full three-year abundance cycle of main prey (voles), and consider several components of individual quality, including body condition, blood parasite infection, and genetic diversity ( n  = 448 adults) that act on different time scales. Older individuals, and kestrel parents in higher body condition started egg-laying earlier than younger birds and those in lower body condition. Additionally, egg-laying was initiated earlier during the increase and decrease phases (2011 and 2012) than during the low phase of the vole cycle (2013). Nestling survival (ratio of eggs that fledged successfully) was higher in early nests and in heterogeneous landscapes (i.e., mosaic of different habitat types), which was evident during the increase and decrease phases of the vole cycle, but not during the low vole year. Conclusions We found a strong positive effect of landscape heterogeneity on nestling survival, but only when voles were relatively abundant, whereas a difference in the timing of breeding related to territory landscape heterogeneity was not evident. Therefore, landscape heterogeneity appeared as the main driver of high reproductive performance under favourable food conditions. Our results show that landscape homogenization linked to agricultural intensification disrupts the expected positive effect of vole abundance on reproductive success of kestrels.

Cave-dwelling taxa often share the same phenotypic modifications like absence of eyes and pigmentation. These “troglomorphic characters” are expressed in the populations of Garra barreimiae from the Al Hoota Cave and nearby Hoti Pit in Northern Oman. Surface morphotypes of this cyprinid species are common throughout the distribution area. Very rarely individuals with intermediate phenotypes can be found. In the present study, potential gene flow between cave and surface populations was tested and population structure within five sampling sites was assessed. Overall, 213 individuals were genotyped at 18 microsatellite loci. We found that the cave populations have lower genetic diversity and are clearly isolated from the surface populations, which seem to be sporadically in contact with each other. The results indicate a recent genetic bottleneck in the cave populations. Thus, it can be assumed that during climatic changes the connection between cave and surface water bodies was disjoined, leaving a subpopulation trapped inside. Nevertheless, occasional gene flow between the morphotypes is detectable, but hybridisation seems only possible in cave habitat with permanent connection to surface water. Individuals from surface sites bearing intermediate phenotypes but cave genotypes imply that phenotypic plasticity might play a role in the development of the phenotype.

Reef-building corals have a variety of green fluorescent protein (GFP)-like proteins, also known as fluorescent proteins (FPs). These proteins have broad spectral properties covering most of the visible spectrum, with fluorophores fluorescing from cyan to red. However, the role of FPs is still a topic of debate and requires further investigation, particularly in the direction of mapping these FPs within the coral tissue and describing their cell- and tissue-level distributions. This study applied confocal laser scanning microscopy (CLSM) to investigate species-specific differences in the distribution of FPs in three coral taxa ( Stylophora sp., Acropora sp., Echinopora sp.), combined with their photoacclimation response and that of associated symbiotic algae to light gradients in a controlled aquarium experiment. CLSM produced high-resolution images that enabled the identification of different FPs, their tissue distribution and quantification of their fluorescence intensity, as well as quantification of symbiont chlorophyll a (chl- a ) fluorescence. Emission scans revealed three emission peaks between 490 - 501 nm (cyan, CFPs), 510 - 515 nm (green, GFPs), and 679 nm (chl- a fluorescence signal; Fchl) shared by all three studied species. The distribution of GFPs in Stylophora was concentrated in the intermesenterial muscle bands of the polyp, whereas CFPs were typically located at the tips of the tentacles. In contrast, Acropora and Echinopora exhibited agglomeration of CFPs and GFPs primarily in the epidermis. In general, species-specific differences in FP distribution remained unaltered during the experiment. However, linear regression models showed a significant negative relationship between CFP fluorescence intensity and light irradiance in Stylophora , whereas Echinopora exhibited a negative relation between chlorophyll fluorescence (Fchl) and light. In summary, the CLSM methodology provided a high-resolution tool to study coral FP patterns and symbiont response to irradiance, revealing ecophysiological differences among coral species at the tissue and cellular levels. CLSM has the potential to elucidate the intricacies of coral photobiology within the natural environment and to discern their adaptive responses in situ .

The Kalkalpen National Park is situated in Upper Austria and contains more than 800 springs. The international importance of this Park is, from the perspective of nature conservation directives, highly significant (European Nature Reserve Natura 2000, recognised wetland of the Ramsar convention). In the current study, the hydrobioid fauna (‘spring snails’) of the Kalkalpen National Park was evaluated. These tiny snails are difficult to determine; however, their investigation is especially desirable, as several species are threatened and as they are important for water quality assessment. Snails collected in 39 selected springs were examined with classical morphological methods (shell and genital anatomy) and, subsequently, by DNA analysis. For this task, the DNA barcode, a partial sequence of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene (length of the sequence 658-682 bp), was PCR amplified and sequenced. From 107 specimens, the DNA barcoding sequence could be obtained and compared with already existing DNA sequences. The (sub)endemic species Bythinella conica , Hauffenia kerschneri , Hauffenia wienerwaldensis and Belgrandiella aulaei could be clearly identified. For Bythiospeum nocki , despite the ambitious collecting effort, only empty shells were found in four springs (including the locus typicus spring) in the Park and its surroundings. The genus Bythinella was detected in 36 springs. From 25 of these localities, DNA barcodes could be created, which matches those of Bythinella conica (comparison data from ABOL). It is, therefore, concluded that the species occurs widely in the Kalkalpen National Park. The genus Hauffenia was sampled from 16 springs. From one, the haplotype of Hauffenia wienerwaldensis could be identified (spring is 5 km outside the Park) and from six, the haplotype of Hauffenia kerschneri . Belgrandiella aulaei was found in three springs, which all lie outside the boundaries and are, therefore, not included in the protection measures of the National Park. The data and analyses obtained contribute to the assessment of the taxonomic status of the species studied. The present study gives a good baseline for further monitoring of the hydrobioids in the Kalkalpen National Park, which is important to evaluate current as well as to decide on future protection measures for this group.

Background Cylindrus obtusus is one of the most prominent endemic snail species of the Eastern Alps. It is restricted to alpine meadows and calcareous rocky habitats above 1500 m. Peculiar intraspecific differences have been observed in its genital tract in the eastern populations the two mucus glands associated with the love dart sac are highly variable, while almost no variation was observed in the western populations. This raises the question whether the mode and success of reproduction of the respective populations are different. To find out whether these anatomical differences reflect genetic differentiation, which might be an indication for distinct glacial refugia, we investigated a 650 bp fragment of the mitochondrial cytochrome oxidase subunit 1 gene ( COI ) (280 individuals) and 9 microsatellite loci from samples (487 individuals from 29 populations) covering the whole distribution range of the species. Results The COI sequences show a geographic differentiation between eastern, central and western populations. The westernmost localities, which were covered under ice sheets during glacial periods, are characterized by extreme low variability. Overall genetic distances among all individuals are small (max. 1.7% p -distance). The microsatellite analysis reveals a high differentiation between populations, implying restriction of gene flow. The highest genetic variability was found in the central populations. Remarkably, nearly all individuals from the eastern populations, which are more variable in their genital morphology, are homozygous in all microsatellite loci, although different alleles were found within populations. Conclusions The most peculiar outcome of the study is the strong evidence for selfing in C. obtusus as indicated by the microsatellite data in the easternmost populations. This finding is supported by the deformation of the mucus glands in the same populations. Since mucus glands play an important role in sexual reproduction, it seems plausible that in selfing organisms these structures are reduced. The phylogeographic structure revealed by COI sequences implies that the species has survived the ice ages within the Calcareous Alps. The small genetic distances among all individuals (max. 1.7%) suggest that C. obtusus has experienced severe bottlenecks in the past.

Background Phenotypic similarities among cave-dwelling animals displaying troglomorphic characters (e.g. reduced eyes and lack of pigmentation) have induced a long-term discussion about the forces driving convergent evolution. Here we introduce Garra barreimiae Fowler & Steinitz, 1956, as an interesting system to study the evolution of troglomorphic characters. The only hitherto known troglomorphic population of this species lives in Al Hoota Cave (Sultanate of Oman) close to a surface population. As a first approach, we assessed the genetic differentiation between the two morphotypes of G. barreimiae to determine whether gene flow still occurs. Results We analysed the mitochondrial control region (CR). In G. barreimiae the CR starts immediately downstream of the tRNA-Thr gene, while the tRNA-Pro gene is missing at this genomic location. Interestingly, a putative tRNA-Pro sequence is found within the CR. The phylogenetic analyses of the CR sequences yielded a tree divided into three clades: Clade 1 has a high genetic distance to the other clades and contains the individuals of three populations which are separated by a watershed from all the others. Clade 2 comprises the individuals from Wadi Bani Khalid, the geographically most remote population. Clade 3 comprises all other populations investigated including that of Al Hoota Cave. The latter forms a haplogroup which also includes individuals from the adjacent surface population. Conclusions Our data indicates that the troglomorphic cave population is of quite recent origin supporting the hypothesis that selection drives the fast evolution of troglomorphic traits. In this context pleiotropic effects might play an important role as it has been shown for Astyanax . There seems to be some gene flow from the cave population into the adjacent surface populations. One blind individual, found at a surface locality geographically distinct from Al Hoota Cave, is genetically differentiated from the other blind specimens indicating the probable existence of another cave population of G. barreimiae . The phylogeographic analyses show that while some of the surface populations are either still in contact or have been until recently, the population Wadi Bani Khalid is genetically separated. One group consisting of three populations is genetically highly differentiated questioning the conspecifity with G . barreimiae .

The complete sequence of the mitochondrial (mt) genome of Buteo buteo was determined. Its gene content and nucleotide composition are typical for avian genomes. Due to expanded noncoding sequences, Buteo possesses the longest mt genome sequenced so far (18,674 bp). The gene order comprising the control region and neighboring genes is identical to that of Falco peregrinus, suggesting that the corresponding rearrangement occurred before the falconid/accipitrid split. Phylogenetic analyses performed with the mt sequence of Buteo and nine other mt genomes suggest that for investigations at higher taxonomic levels (e.g., avian orders), concatenated rRNA and tRNA gene sequences are more informative than protein gene sequences with respect to resolution and bootstrap support. Phylogenetic analyses indicate an early split between Accipitridae and Falconidae, which, according to molecular dating of other avian divergence times, can be assumed to have taken place in the late Cretaceous 65–83 MYA.