Widespread infection, diversification and old host associations of Nosema Microsporidia in European freshwater gammarids (Amphipoda).

The microsporidian genus Nosema is primarily known to infect insects of economic importance stimulating high research interest, while other hosts remain understudied. Nosema granulosis is one of the formally described Nosema species infecting amphipod crustaceans, being known to infect only two host species. Our first aim was to characterize Nosema spp. infections in different amphipod species from various European localities using the small subunit ribosomal DNA (SSU) marker. Second, we aimed to assess the phylogenetic diversity, host specificity and to explore the evolutionary history that may explain the diversity of gammarid-infecting Nosema lineages by performing a phylogenetic reconstruction based on RNA polymerase II subunit B1 (RPB1) gene sequences. For the host species Gammarus balcanicus, we also analyzed whether parasites were in excess in females to test for sex ratio distortion in relation with Nosema infection. We identified Nosema spp. in 316 individuals from nine amphipod species being widespread in Europe. The RPB1-based phylogenetic reconstruction using newly reported sequences and available data from other invertebrates identified 39 haplogroups being associated with amphipods. These haplogroups clustered into five clades (A-E) that did not form a single amphipod-infecting monophyletic group. Closely related sister clades C and D correspond to Nosema granulosis. Clades A, B and E might represent unknown Nosema species infecting amphipods. Host specificity seemed to be variable with some clades being restricted to single hosts, and some that could be found in several host species. We show that Nosema parasite richness in gammarid hosts is much higher than expected, illustrating the advantage of the use of RPB1 marker over SSU. Finally, we found no hint of sex ratio distortion in Nosema clade A infecting G. balcanicus. This study shows that Nosema spp. are abundant, widespread and diverse in European gammarids. Thus, Nosema is as diverse in aquatic as in terrestrial hosts.

Microsporidian diversity in the aquatic isopod Asellus aquaticus.

We conducted a molecular survey on microsporidian diversity in different lineages (operational taxonomic units = OTUs) of Asellus aquaticus from 30 sites throughout Europe. Host body length was determined, and DNA was extracted from host tissue excluding the intestine and amplified by microsporidian-specific primers. In total, 247 A. aquaticus specimens were analysed from which 26.7% were PCR-positive for microsporidians, with significantly more infections in larger individuals. Prevalence ranged between 10 and 90%. At 9 sites, no microsporidians were detected. A significant relationship was found between the frequency of infected individuals and habitat type, as well as host OTU. The lowest proportion of infected individuals was detected in spring-habitats (8.7%, n = 46) and the highest in ponds (37.7%, n = 53). Proportion of infected individuals among host OTUs A, D and J was 31.7, 21.7 and 32.1%, respectively. No infections were detected in OTU F. Our results are, however, accompanied by a partially low sample size, as only a minimum of 5 individuals was available at a few locations. Overall, 17 different microsporidian molecular taxonomic units (MICMOTUs) were distinguished with 5 abundant isolates (found in 4­17 host individuals) while the remaining 12 MICMOTUs were "rare" and found only in 1­3 host individuals. No obvious spatio-genetic pattern could be observed. The MICMOTUs predominantly belonged to Nosematida and Enterocytozoonida. The present study shows that microsporidians in A. aquaticus are abundant and diverse but do not show obvious patterns related to host genetic lineages or geography.

First records raise questions: DNA barcoding of Odonata in the middle of the Mediterranean.

We present the results of the first-ever DNA barcoding study of odonates from the Maltese Islands. In total, 10 morphologically identified species were collected during a two-week long expedition in 2018. Eighty cytochrome c oxidase subunit I (COI) barcodes were obtained from the collected specimens. Intra- and interspecific distances ranged from 0.00% to 2.24% and 0.48% to 17.62%, respectively. Successful species identification based on ascribing a single morphological species to a single Barcode Index Number (BIN) was achieved for eight species (80%). In the case of two species, Ischnura genei and Anax parthenope, BINs were shared with other closely related species. The taxonomic status of I. genei is questionable and the phylogenetic relationship between A. imperator/parthenope is not clear. Further studies involving a series of adult specimens collected in a wide spatial range and nuclear markers are necessary to resolve these cases. Therefore, this dataset serves as an initial DNA barcode reference library for Maltese odonates, within a larger project: Aquatic Macroinvertebrates DNA Barcode Library of Malta.

Mitochondrial Genomes, Phylogenetic Associations, and SNP Recovery for the Key Invasive Ponto-Caspian Amphipods in Europe.

The Ponto-Caspian region is the main donor of invasive amphipods to freshwater ecosystems, with at least 13 species successfully established in European inland waters. Dikerogammarus spp. and Pontogammarus robustoides are among the most successful, due to their strong invasive impact on local biota. However, genomic knowledge about these invaders is scarce, while phylogeography and population genetics have been based on short fragments of mitochondrial markers or nuclear microsatellites. In this study, we provide: (i) a reconstruction of six mitogenomes for four invasive gammarids (D. villosus, D. haemobaphes, D. bispinosus, and P. robustoides); (ii) a comparison between the structure of the newly obtained mitogenomes and those from the literature; (iii) SNP calling rates for individual D. villosus and D. haemobaphes from different invasion sites across Europe; and (iv) the first time-calibrated full mitogenome phylogeny reconstruction of several Ponto-Caspian taxa. We found that, in comparison to other gammarids, the mitogenomes of Ponto-Caspian species show a translocation between the tRNA-E and tRNA-R positions. Phylogenetic reconstruction using the mitogenomes identified that Ponto-Caspian gammarids form a well-supported group that originated in the Miocene. Our study supports paraphyly in the family Gammaridae. These provided mitogenomes will serve as vital genetic resources for the development of new markers for PCR-based identification methods and demographic studies.

The Use of Yeast Mixed Cultures for Deacidification and Improvement of the Composition of Cold Climate Grape Wines.

Interest in the use of non-Saccharomyces yeast in mixed cultures is increasing due to the perceived improvement in the quality and complexity of the resulting wines. The aim of the study was to determine the ability of monocultures and mixed yeast cultures for deacidification and improvement of the composition of cold climate grape wines. Fermentation of grape musts with increased total acidity was carried out with the use of monocultures of Saccharomyces cerevisiae MH020215 (Sc), Zygosaccharomyces bailii 749 (Zb) and Metschnikowia pulcherrima MG970690 (Mp), and their mixed cultures, inoculated simultaneously and sequentially. Oenological parameters, organic acids and volatile compounds profiles of obtained wines were characterized. The fermentation kinetics and analytical profiles of the obtained wines showed that the use of mixed yeast cultures contributed to the reduction of volatile acidity and acetic acid content in the wines, as well as obtaining a favorable aromatic profile of the wines. The dominant higher alcohols in all wines were 2-methyl-1-propanol, 3-methyl-1-butanol and 2-methyl-1-butanol. Significantly higher amounts of the first two compounds were found in wines obtained with M. pulcherrima MG070690, both in monoculture and in mixed cultures. The monocultures of M. pulcherrima MG070690 (Mp) compared with Z. bailli 749 (Zb) synthesized higher levels of esters in wines, including ethyl acetate, ethyl propionate, isobutyl acetate, ethyl pyroracemate and isoamyl acetate.

Dictyocoela microsporidia diversity and co-diversification with their host, a gammarid species complex (Crustacea, Amphipoda) with an old history of divergence and high endemic diversity.

BACKGROUND: Although the processes of co-evolution between parasites and their hosts are well known, evidence of co-speciation remains scarce. Microsporidian intracellular parasites, due to intimate relationships with their hosts and mixed mode of transmission (horizontal but also vertical, from mother to offspring), may represent an interesting biological model for investigating co-speciation. Amphipod crustaceans, especially gammarids, are regular hosts of microsporidian parasites, in particular the Dictyocoela spp., which have so far been found limited to these amphipods and are known to use a vertical mode of transmission. The amphipod genus Gammarus has a diversification history spanning the last 50-60 Mya and an extensive cryptic diversity in most of the nominal species. Here, we investigated the degree of co-diversification between Dictyocoela and Gammarus balcanicus, an amphipod with high degrees of ancient cryptic diversification and lineage endemism, by examining the genetic diversity of these parasites over the entire geographic range of the host. We hypothesised that the strong host diversification and vertical transmission of Dictyocoela would promote co-diversification. RESULTS: Using the parasite SSU rDNA as a molecular marker, analyzing 2225 host specimens from 88 sites covering whole host range, we found 31 haplogroups of Dictyocoela, 30 of which were novel, belonging to four Dictyocoela species already known to infect other Gammarus spp. The relationships between Dictyocoela and gammarids is therefore ancient, with the speciation in parasites preceding those of the hosts. Each novel haplogroup was nevertheless specific to G. balcanicus, leaving the possibility for subsequent co-diversification process during host diversification. A Procrustean Approach to Co-phylogeny (PACo) analysis revealed that diversification of Dictyocoela was not random with respect to that of the host. We found high degrees of congruence between the diversification of G. balcanicus and that of Dictyocoela roeselum and D. muelleri. However, we also found some incongruences between host and Dictyocoela phylogenies, e.g. in D. duebenum, probably due to host shifts between different G. balcanicus cryptic lineages. CONCLUSION: The evolutionary history of Dictyocoela and Gammarus balcanicus represents an example of an overall host-parasite co-diversification, including cases of host shifts.

Environmental factors affecting water mite assemblages along eucrenon-hypocrenon gradients in Mediterranean karstic springs.

Springs are often recognized as biodiversity hotspots on the regional scale but at the same time they are among the most endangered freshwater habitats. Water mites are among the aquatic animal groups with highest share of crenobiotic (= spring-dwelling) species and, therefore, are possibly the best indicators of the ecological status of spring habitats. We studied water mites and environmental factors correlated with their distribution pattern along a eucrenon-hypocrenon gradient. The sampling was conducted in 14 karstic springs located in the Mediterranean part of Montenegro. We collected 17 water mite species of which four species were crenobiotic. We did not find significant differences between the water mite assemblages from the source and springbrook. Similarly, there were no significant differences in number of species and abundance between the studied spring sections, neither for crenobiotic taxa nor for non-crenobiotic taxa. We found that the number of non-crenobiotic taxa was predicted mainly by water depth, whereas the abundance of crenobionts was most strongly associated with temperature. No significant predictors for the number of crenobiotic species in spring habitats were found. Our results revealed also that distance from the nearby water body was the main driver of the crenobiotic species abundance in eucrenon suggesting the large effects of the local flooding events on crenobiotic species. Water mites may help in assessing the response of crenobiotic assemblages in those spring habitats which likely to be flooded in future as the results of ongoing climatic changes.

Evidence for interactions between homocysteine and genistein: insights into stroke risk and potential treatment.

Elevated plasma homocysteine (2-amino-4-sulfanylbutanoic acid) level is a risk factor for stroke. Moreover, it has been suggested that high levels of homocysteine in the acute phase of an ischemic stroke can predict mortality, especially in stroke patients with the large-vessel atherosclerosis subtype. In clinical studies, supplementation with genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one) decreased plasma homocysteine levels considerably. Therefore, genistein could be considered as a potential drug for prevention and/or treatment of stroke. However, the mechanism of the effect of genistein on homocysteine level remains to be elucidated. In this report, direct functional interactions between homocysteine and genistein are demonstrated in in vitro experimental systems for determination of methylenetetrahydrofolate reductase (MetF) and glutathione peroxidase (GPx) activities, reconstructed with purified compounds, and in a simple in vivo system, based on measurement of growth rate of Vibrio harveyi and Bacillus subtilis cultures. Results of molecular modelling indicated that homocysteine can directly interact with genistein. Therefore, genistein-mediated decrease in plasma levels of homocysteine, and alleviation of biochemical and physiological effects of one of these compounds by another, might be ascribed to formation of homocysteine-genistein complexes in which biological activities of these molecules are abolished or alleviated.

The legacy of a vanished sea: a high level of diversification within a European freshwater amphipod species complex driven by 15 My of Paratethys regression.

The formation of continental Europe in the Neogene was due to the regression of the Tethys Ocean and of the Paratethys Sea. The dynamic geology of the area and repetitious transitions between marine and freshwater conditions presented opportunities for the colonization of newly emerging hydrological networks and diversification of aquatic biota. Implementing mitochondrial and nuclear markers in conjunction with a large-scale sampling strategy, we investigated the impact of this spatiotemporal framework on the evolutionary history of a freshwater crustacean morphospecies. The Gammarus balcanicus species complex is widely distributed in the area previously occupied by the Paratethys Sea. Our results revealed its high diversification and polyphyly in relation to a number of other morphospecies. The distribution of the studied amphipod is generally characterized by very high local endemism and divergence. The Bayesian time-calibrated reconstruction of phylogeny and geographical distribution of ancestral nodes indicates that this species complex started to diversify in the Early Miocene in the central Balkans, partially in the shallow epicontinental sea. It is possible that there were several episodes of inland water colonization by local brackish water lineages. Subsequent diversification within clades and spread to new areas could have been induced by Alpine orogeny in the Miocene/Pliocene and, finally, by Pleistocene glaciations. The present distribution of clades, in many cases, still reflects Miocene palaeogeography of the area. Our results point out that investigations of the historical aspect of cryptic diversity in other taxa may help in a general understanding of the origins of freshwater invertebrate fauna of Europe.

The model homologue of the partially defective human 5,10-methylenetetrahydrofolate reductase, considered as a risk factor for stroke due to increased homocysteine level, can be protected and reactivated by heat shock proteins.

The A222 V substitution in the human MTHFR gene product (5,10-methylenetetrahydrofolate reductase) is responsible for a decreased activity of this enzyme. This may cause an increased homocysteine level, considered as a risk factor for arteriosclerosis and stroke. The bacterial homologue of the human enzyme, MetF, has been found to be a useful model in genetic and biochemical studies. The similarity of Escherichia coli MetF and human MTHFR proteins is so high that particular mutations in the corresponding human gene can be reflected by the bacterial mutants. For example, the A222 V substitution in MTHFR (caused by the C667T substitution in the MTHFR gene) can be ascribed to the A117 V substitution in MetF. Here, it is reported that a temperature-sensitive MetF117 (A117 V) protein can be partially protected from a thermal inactivation by the heat shock proteins from the Hsp70/100 systems. Moreover, activity of the thermally denatured enzyme can be partially restored by the same heat shock proteins. High temperature protein G (HtpG) had no effect on MetF117 activity in both experimental systems. The presented results indicate that functions of heat shock proteins may be required for maintenance of the MetF117 function. This may have implications for the mechanisms of arteriosclerosis and stroke, especially in the light of previous findings that the A222 V MTHFR polymorphism may be a risk factor for stroke, as well as recently published results which demonstrated the increased levels of antibodies against heat shock proteins in stroke patients.

Genistein inhibits activities of methylenetetrahydrofolate reductase and lactate dehydrogenase, enzymes which use NADH as a substrate.

Genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one) is a natural isoflavone revealing many biological activities. Thus, it is considered as a therapeutic compound in as various disorders as cancer, infections and genetic diseases. Here, we demonstrate for the first time that genistein inhibits activities of bacterial methylenetetrahydrofolate reductase (MetF) and lactate dehydrogenase (LDH). Both enzymes use NADH as a substrate, and results of biochemical as well as molecular modeling studies with MetF suggest that genistein may interfere with binding of this dinucleotide to the enzyme. These results have implications for our understanding of biological functions of genistein and its effects on cellular metabolism.

Isolation and characterization of 8 microsatellite loci for the "killer shrimp'', an invasive Ponto-Caspian amphipod Dikerogammarus villosus (Crustacea: Amphipoda).

Dikerogammarus villosus is a freshwater amphipod of the Ponto-Caspian origin recognized as one of the 100 worst alien species in Europe, having negative impact on biodiversity and functioning of the invaded aquatic ecosystems. The species has a wide ecophysiological tolerance and during the last 20 years it has rapidly spread throughout European inland waters. In consequence, it presents a major conservation management problem. We describe eight polymorphic microsatellite loci developed for D. villosus by combining a biotin-enrichment protocol and new generation 454GS-FLX Titanium pyrosequencing technology. When genotyped in 64 individuals from two locations, the loci exhibited a mean diversity of 4.87 alleles per locus (2-13). The mean observed and expected heterozygosities were, respectively, 0.439 (0.091-0.844) and 0.468 (0.089-0.843). Gametic disequilibrium was not detected for any pair of loci. The microsatellite markers will be a valuable tool in assessing the demographic processes associated with invasion of the killer shrimp from a genetic point of view.

Further records of Amphipoda from Baltic Eocene amber with first evidence of prae-copulatory behaviour in a fossil amphipod and remarks on the taxonomic position of Palaeogammarus Zaddach, 1864.

Two pieces of Baltic amber with amphipod inclusions were studied. One of them contained approximately twenty individuals identified as belonging to the extinct genus Palaeogammarus and described as P. debroyeri sp. nov. Interestingly, among the individuals there are two pairs preserved in an evident prae-copula position. This is the first finding of such mating behaviour in fossil amphipods. Based on this behavioural trait and on the observed morphological features, we conclude that the genus Palaeogammarus should be placed in Gammaridae and not in Crangonyctidae. The second amber piece contains two individuals identified as belonging to the still extant genus Synurella and described as S. aliciae sp. nov. 

Detailed DNA barcoding of mayflies in a small European country proved how far we are from having comprehensive barcode reference libraries.

Mayflies (Ephemeroptera) are among the crucial water and habitat quality bioindicators. However, despite their intensive long-term use in various studies, more reliable mayfly DNA barcode data have been produced in a negligible number of countries, and only ~40% of European species had been barcoded with less than 50% of families covered. Despite being carried out in a small area, our study presents the second-most species-rich DNA reference library of mayflies from Europe and the first comprehensive view from an important biodiversity hotspot such as the Western Carpathians. Within 1153 sequences, 76 morphologically determined species were recorded and added to the Barcode of Life Data System (BOLD) database. All obtained sequences were assigned to 97 BINs, 11 of which were unique and three represented species never barcoded before. Sequences of 16 species with high intraspecific variability were divided into 40 BINs, confirming the presence of cryptic lineages. Due to the low interspecific divergence and the non-existing barcoding gap, sequences of six species were assigned to three shared BINs. Delimitation analyses resulted in 79 and 107 putative species respectively. Bayesian and maximum-likelihood phylogenies confirmed the monophyly of almost all species and complexes of cryptic taxa and proved that DNA barcoding distinguishes almost all studied mayfly species. We have shown that it is still sufficient to thoroughly investigate the fauna of a small but geographically important area to enrich global databases greatly. In particular, the insights gained here transcend the local context and may have broader implications for advancing barcoding efforts.

A collaborative backbone resource for comparative studies of subterranean evolution: The World Asellidae database.

Transition to novel environments, such as groundwater colonization by surface organisms, provides an excellent research ground to study phenotypic evolution. However, interspecific comparative studies on evolution to groundwater life are few because of the challenge in assembling large ecological and molecular resources for species-rich taxa comprised of surface and subterranean species. Here, we make available to the scientific community an operational set of working tools and resources for the Asellidae, a family of freshwater isopods containing hundreds of surface and subterranean species. First, we release the World Asellidae database (WAD) and its web application, a sustainable and FAIR solution to producing and sharing data and biological material. WAD provides access to thousands of species occurrences, specimens, DNA extracts and DNA sequences with rich metadata ensuring full scientific traceability. Second, we perform a large-scale dated phylogenetic reconstruction of Asellidae to support phylogenetic comparative analyses. Of 424 terminal branches, we identify 34 pairs of surface and subterranean species representing independent replicates of the transition from surface water to groundwater. Third, we exemplify the usefulness of WAD for documenting phenotypic shifts associated with colonization of subterranean habitats. We provide the first phylogenetically controlled evidence that body size of males decreases relative to that of females upon groundwater colonization, suggesting competition for rare receptive females selects for smaller, more agile males in groundwater. By making these tools and resources widely accessible, we open up new opportunities for exploring how phenotypic traits evolve in response to changes in selective pressures and trade-offs during groundwater colonization.

Taming the terminological tempest in invasion science.

Standardised terminology in science is important for clarity of interpretation and communication. In invasion science - a dynamic and rapidly evolving discipline - the proliferation of technical terminology has lacked a standardised framework for its development. The result is a convoluted and inconsistent usage of terminology, with various discrepancies in descriptions of damage and interventions. A standardised framework is therefore needed for a clear, universally applicable, and consistent terminology to promote more effective communication across researchers, stakeholders, and policymakers. Inconsistencies in terminology stem from the exponential increase in scientific publications on the patterns and processes of biological invasions authored by experts from various disciplines and countries since the 1990s, as well as publications by legislators and policymakers focusing on practical applications, regulations, and management of resources. Aligning and standardising terminology across stakeholders remains a challenge in invasion science. Here, we review and evaluate the multiple terms used in invasion science (e.g. 'non-native', 'alien', 'invasive' or 'invader', 'exotic', 'non-indigenous', 'naturalised', 'pest') to propose a more simplified and standardised terminology. The streamlined framework we propose and translate into 28 other languages is based on the terms (i) 'non-native', denoting species transported beyond their natural biogeographic range, (ii) 'established non-native', i.e. those non-native species that have established self-sustaining populations in their new location(s) in the wild, and (iii) 'invasive non-native' - populations of established non-native species that have recently spread or are spreading rapidly in their invaded range actively or passively with or without human mediation. We also highlight the importance of conceptualising 'spread' for classifying invasiveness and 'impact' for management. Finally, we propose a protocol for classifying populations based on (i) dispersal mechanism, (ii) species origin, (iii) population status, and (iv) impact. Collectively and without introducing new terminology, the framework that we present aims to facilitate effective communication and collaboration in invasion science and management of non-native species.

Cryptic Species in Ecotoxicology.

The advent of genetic methods has led to the discovery of an increasing number of species that previously could not be distinguished from each other on the basis of morphological characteristics. Even though there has been an exponential growth of publications on cryptic species, such species are rarely considered in ecotoxicology. Thus, the particular question of ecological differentiation and the sensitivity of closely related cryptic species is rarely addressed. Tackling this question, however, is of key importance for evolutionary ecology, conservation biology, and, in particular, regulatory ecotoxicology. At the same time, the use of species with (known or unknown) cryptic diversity might be a reason for the lack of reproducibility of ecotoxicological experiments and implies a false extrapolation of the findings. Our critical review includes a database and literature search through which we investigated how many of the species most frequently used in ecotoxicological assessments show evidence of cryptic diversity. We found a high proportion of reports indicating overlooked species diversity, especially in invertebrates. In terrestrial and aquatic realms, at least 67% and 54% of commonly used species, respectively, were identified as cryptic species complexes. The issue is less prominent in vertebrates, in which we found evidence for cryptic species complexes in 27% of aquatic and 6.7% of terrestrial vertebrates. We further exemplified why different evolutionary histories may significantly determine cryptic species' ecology and sensitivity to pollutants. This in turn may have a major impact on the results of ecotoxicological tests and, consequently, the outcome of environmental risk assessments. Finally, we provide a brief guideline on how to deal practically with cryptic diversity in ecotoxicological studies in general and its implementation in risk assessment procedures in particular. Environ Toxicol Chem 2023;42:1889-1914. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Cenozoic origins of the genus Calliarcys (Insecta, Ephemeroptera) revealed by Micro-CT, with DNA barcode gap analysis of Leptophlebiinae and Habrophlebiinae.

Mayflies (Ephemeroptera) are among the oldest pterygote insects, with the earliest fossils dating back to the Late Carboniferous. Within mayflies, Leptophlebiidae are a highly diverse and widespread group, with approximately 140 genera and 640 species. Whereas taxonomy, systematics, and phylogeny of extant Leptophlebiidae are in the focus of extensive studies, little is known about leptophlebiid fossil taxa. Because fossil remains of Ephemeroptera in sedimentary rocks are relatively rare, inclusions of mayflies in amber are a unique source of information on their evolution and diversity in the past. Leptophlebiidae found in Cenozoic resins mostly belong to the subfamilies Leptophlebiinae (in Eocene Baltic amber) and Atalophlebiinae (in Miocene Dominican and Mexican ambers). In the present contribution, we confirm the first finding of the genus Calliarcys from Eocene Baltic amber by using Micro-CT, which allowed confirming its generic placement by visualizing diagnostic key characters otherwise hidden by a cloud of turbidity. Additionally, we present first molecular data on the extant species Calliarcys humilis Eaton, 1881 from the Iberian Peninsula and the barcode gap analysis for Leptophlebiinae and Habrophlebiinae.

Identification and Spread of the Ghost Silverfish (Ctenolepisma calvum) among Museums and Homes in Europe.

Ctenolepisma calvum was first described in Sri Lanka (Ceylon) in 1910, and this island is probably the origin of this species. Later, it was also found in the Caribbean (Cuba and Trinidad and Tobago). Up until the present, it has only been identified within buildings (a synanthropic species), and its natural habitat is unknown. In 2007, it was discovered in Germany and was considered a neobiotic species of Lepismatidae in Europe. It has rapidly spread throughout Europe and beyond in recent years. This led us to analyze the available data of the first occurrences in Germany, Austria, and other European countries. Furthermore, we compared the spread inside of museums in Vienna (Austria) and Berlin (Germany). These museums have been monitored for a long period with sticky traps, representing the best source of information on the dispersion dynamics of Ctenolepisma calvum. We found a scattered occurrence of this species in 18 countries in Europe (including Russia and Ukraine). The first record for Poland has not previously been published; however, this species has been present there since 2014. Surprisingly, it was found in Hungary in 2003, but a record was only published online in 2021. Additionally, in Germany and Austria, where most data are available, the spread of the species does not follow any clear pattern. In museums in Berlin, the species has only been found in one location. In contrast, the species rapidly spread in museums in Vienna between 2014 and 2021, from four to 30 locations, and it is now a well-established species with occasional high abundance. We examined the spread of the species at three spatial scales: (i) Europe, (ii) national, and (iii) regional. Our observations indicate that it is possibly distributed with materials (packaging material, hygiene articles, paper, cardboard, and collection items). Little is yet known about the biology of this introduced pest. We describe its preferred habitat within buildings, its climate requirements, and its potential to act as a new museum pest in Central Europe. This species seems to thrive at room temperature in buildings. Further impact on the species due to climate change in the future is also discussed. We offer a simple morphological key and a detailed identification table to help correct species identification.

A DNA barcode reference library for endemic Ponto-Caspian amphipods.

The Ponto-Caspian region is an endemicity hotspot that harbours several crustacean radiations, among which amphipods are the most diverse. These poorly known species are severely threatened in their native range, while at the same time they are invading European inland waters with significant ecological consequences. A proper taxonomic knowledge of this fauna is paramount for its conservation within the native region and monitoring outside of it. Here, we assemble a DNA barcode reference library for nearly 60% of all known Ponto-Caspian amphipod species. We use several methods to define molecular operational taxonomic units (MOTUs), based on two mitochondrial markers (COI and 16S), and assess their congruence with current species-level taxonomy based on morphology. Depending on the method, we find that 54-69% of species had congruent morpho-molecular boundaries. The cases of incongruence resulted from lumping distinct morphospecies into a single MOTU (7-27%), splitting a morphospecies into several MOTUs (4-28%), or both (4-11%). MOTUs defined by distance-based methods without a priori divergence thresholds showed the highest congruence with morphological taxonomy. These results indicate that DNA barcoding is valuable for clarifying the diversity of Ponto-Caspian amphipods, but reveals that extensive work is needed to resolve taxonomic uncertainties. Our study advances the DNA barcode reference library for the European aquatic biota, paving the way towards improved taxonomic knowledge needed to enhance monitoring and conservation efforts.