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.

Tick findings from subterranean environments in the Central German Uplands and Luxembourg reveal a predominance of male Ixodes hexagonus.

Questing ticks are usually collected by flagging or dragging. Mostly exophilic tick species are caught, such as Ixodes ricinus, the most common tick in Central Europe. In the present study, ticks collected from underground environments in the Grand Duchy of Luxembourg and in the Central German Uplands (Federal States of Hesse, Bavaria, Thuringia, Baden-Wuerttemberg, Rhineland-Palatinate, Saarland and Northrhine-Westphalia) were investigated. Six tick species were revealed among the 396 analyzed specimens: Ixodes ariadnae, Ixodes canisuga, Ixodes hexagonus, I. ricinus, Ixodes trianguliceps, and Dermacentor marginatus. Adults and immatures of I. hexagonus dominated the findings (57% of all specimens), especially in shelters acting as potential resting places of main hosts. Ixodes canisuga and I. trianguliceps were for the first time recorded in Luxembourg, and one nymph of the bat tick I. ariadnae represents only the second report for Germany. Collecting ticks in subterranean environments turned out to be a useful approach to increase knowledge about the occurrence of relatively rare tick species, including those that spend most of their lifetime on their hosts, but detach in such environmental settings.

Atlas of ticks (Acari: Argasidae, Ixodidae) in Germany: 1st data update.

The first data update of the atlas of ticks in Germany published in 2021 is presented here. This atlas provides maps based on georeferenced tick locations of 21 species endemic in Germany as well as three tick species that are regularly imported to Germany. The data update includes the following numbers of newly georeferenced tick locations: 17 Argas reflexus, 79 Carios vespertilionis, 2 Dermacentor marginatus, 43 Dermacentor reticulatus, 4 Haemaphysalis concinna, 3 Haemaphysalis punctata, 3 Hyalomma rufipes, 3 Ixodes apronophorus, 9 Ixodes arboricola, 1 Ixodes ariadnae, 30 Ixodes canisuga, 3 Ixodes frontalis, 80 Ixodes hexagonus, 3 Ixodes lividus, 497 Ixodes ricinus/inopinatus, 1 Ixodes rugicollis, 17 Ixodes trianguliceps, 14 Ixodes vespertilionis, and 45 Rhipicephalus sanguineus sensu lato. Old and new tick findings were mapped, such as the northernmost occurrence of D. marginatus in Germany observed in 2021, but also the historical records from the first descriptions of I. apronophorus and I. arboricola, which were georeferenced here for the first time. The digital dataset of tick locations available for Germany is supplemented by 854 new tick locations. These records increase the number of tick species mapped in the federal states Bavaria, Brandenburg and Mecklenburg Western Pomerania by five each, those in Berlin and Schleswig-Holstein by four each, those in Hamburg by three, those in Baden-Wuerttemberg, Bremen, Lower Saxony, Northrhine-Westphalia, Rhineland Palatinate and Thuringia by two each, and those in Hesse, Saxony and Saxony-Anhalt by one each. Thus, the first data update of the tick atlas in Germany and the underlying digital dataset significantly improve our knowledge of the distribution of these tick species and helps to investigate the effects of climate change and habitat changes on them.

Quantitative monitoring of diverse fish communities on a large scale combining eDNA metabarcoding and qPCR.

Environmental DNA (eDNA) metabarcoding is an effective method for studying fish communities but allows only an estimation of relative species abundance (density/biomass). Here, we combine metabarcoding with an estimation of the total abundance of eDNA amplified by our universal marker (teleo) using a quantitative (q)PCR approach to infer the absolute abundance of fish species. We carried out a 2850-km eDNA survey within the Danube catchment using a spatial integrative sampling protocol coupled with traditional electrofishing for fish biomass and density estimation. Total fish eDNA concentrations and total fish abundance were highly correlated. The correlation between eDNA concentrations per taxon and absolute specific abundance was of comparable strength when all sites were pooled and remained significant when the sites were considered separately. Furthermore, a nonlinear mixed model showed that species richness was underestimated when the amount of teleo-DNA extracted from a sample was below a threshold of 0.65 × 106 copies of eDNA. This result, combined with the decrease in teleo-DNA concentration by several orders of magnitude with river size, highlights the need to increase sampling effort in large rivers. Our results provide a comprehensive description of longitudinal changes in fish communities and underline our combined metabarcoding/qPCR approach for biomonitoring and bioassessment surveys when a rough estimate of absolute species abundance is sufficient.

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.

Molecular analyses of groundwater amphipods (Crustacea: Niphargidae) from Luxembourg: new species reveal limitations of morphology-based checklists.

Niphargus amphipods were collected from 2007 to 2018 at 98 sites comprising artificial caverns, springs and interstitial waters in the Grand Duchy of Luxembourg. Opportunistic sampling was combined with passive trapping. Specimen identification was achieved using morphological keys and molecular data. Initial morphological determination and literature data suggested five species, whereas sequencing of fragments of the mitochondrial cytochrome c oxidase subunit 1 gene and nuclear 28S rDNA marker supported the presence of seven species: Niphargus schellenbergi, Niphargus puteanus, Niphargus fontanus, one species of the Niphargus kochianus complex, and three species of the Niphargus aquilex complex. Niphargus schellenbergi was by far the most abundant and widespread species. Limited overlap was observed between literature-based records, our initial morphological determinations based on classical taxonomic characters, and genetic sequence data. In general, the combination of phenotypically variable taxa, such as N. schellenbergi, and cryptic or near-cryptic species, as in the N. aquilex complex, renders morphological identification of niphargids from Luxembourg a challenging or even impossible task. DNA taxonomy will therefore have to be used in future studies of the fauna of this region.

First systematic inventory of the jumping plant lice of Luxembourg (Hemiptera, Sternorrhyncha, Psylloidea).

Psyllids (superfamily Psylloidea), also known as jumping plant lice, are a group of plant-sap sucking Hemiptera having significant pest status for crops, forest trees and ornamental plants. Only seven species of psyllids have been recorded in Luxembourg so far. An additional group of seven species has been recorded exclusively, based on the findings of their galls or specific plant deformations; but no mention exists in literature on the actual collection of the inducing insect in Luxembourg. To fill this knowledge gap, field collections were carried out during the years 2019-2020. In addition, samples from 1999-2000 stored in the wet collection of the Musée National d'Histoire Naturelle de Luxembourg were studied. This research, in combination with information coming from literature, allowed us to list 48 species of the families Aphalaridae (5 species), Liviidae (5), Psyllidae (24) and Triozidae (14), though the presence of one species within the last family (Triozarhamni) needs to be confirmed. Brief information on geographical distribution, biology and (if available) illustrations of diagnostic characters are provided on the psyllid species detected in Luxembourg so far.

Mosquitoes (Diptera: Culicidae) in the Dark-Highlighting the Importance of Genetically Identifying Mosquito Populations in Subterranean Environments of Central Europe.

The common house mosquito, Culex pipiens s. l. is part of the morphologically hardly or non-distinguishable Culex pipiens complex. Upcoming molecular methods allowed us to identify members of mosquito populations that are characterized by differences in behavior, physiology, host and habitat preferences and thereof resulting in varying pathogen load and vector potential to deal with. In the last years, urban and surrounding periurban areas were of special interest due to the higher transmission risk of pathogens of medical and veterinary importance. Recently, surveys of underground habitats were performed to fully evaluate the spatial distribution of rare members of the Cx. pipiens complex in Europe. Subterranean environments and their contribution to mosquito-borne pathogen transmission are virtually unknown. Herein, we review the underground community structures of this species complex in Europe, add new data to Germany and provide the first reports of the Cx. pipiens complex and usually rarely found mosquito taxa in underground areas of Luxembourg. Furthermore, we report the first finding of Culiseta glaphyroptera in Luxembourg. Our results highlight the need for molecular specimen identifications to correctly and most comprehensively characterize subterranean mosquito community structures.

Updates to the checklist of the wild bee fauna of Luxembourg as inferred from revised natural history collection data and fieldwork.

BACKGROUND: Museums and other institutions curating natural history collections (NHCs) are fundamental entities to many scientific disciplines, as they house data and reference material for varied research projects. As such, biological specimens preserved in NHCs represent accessible physical records of the living world's history. They provide useful information regarding the presence and distribution of different taxonomic groups through space and time. Despite the importance of biological museum specimens, their potential to answer scientific questions, pertinent to the necessities of our current historical context, is often under-explored.The currently-known wild bee fauna of Luxembourg comprises 341 registered species distributed amongst 38 different genera. However, specimens stored in the archives of local NHCs represent an untapped resource to update taxonomic lists, including potentially overlooked findings relevant to the development of national conservation strategies. NEW INFORMATION: We re-investigated the wild bee collection of the Zoology Department of the National Museum of Natural History Luxembourg by using morphotaxonomy and DNA barcoding. The collection revision led to the discovery of four species so far not described for the country: Andrena lagopus (Latreille, 1809), Nomada furva (Panzer, 1798), Hoplitis papaveris (Latreille, 1799) and Sphecodes majalis (Pérez, 1903). Additionally, the presence of Nomada sexfasciata (Panzer, 1799), which inexplicably had been omitted by the most current species list, can be re-confirmed. Altogether, our findings increase the number of recorded wild bee species in Luxembourg to 346. Moreover, the results highlight the crucial role of NHCs as repositories of our knowledge of the natural world.

Small-scale phenotypic differentiation along complex stream gradients in a non-native amphipod.

BACKGROUND: Selective landscapes in rivers are made up by an array of selective forces that vary from source to downstream regions or between seasons, and local/temporal variation in fitness maxima can result in gradual spatio-temporal variation of phenotypic traits. This study aimed at establishing freshwater amphipods as future model organisms to study adaptive phenotypic diversification (evolutionary divergence and/or adaptive plasticity) along stream gradients. METHODS: We collected Gammarus roeselii from 16 sampling sites in the Rhine catchment during two consecutive seasons (summer and winter). Altogether, we dissected n = 1648 individuals and quantified key parameters related to morphological and life-history diversification, including naturally selected (e.g., gill surface areas) as well as primarily sexually selected traits (e.g., male antennae). Acknowledging the complexity of selective regimes in streams and the interrelated nature of selection factors, we assessed several abiotic (e.g., temperature, flow velocity) and biotic ecological parameters (e.g., conspecific densities, sex ratios) and condensed them into four principal components (PCs). RESULTS: Generalized least squares models revealed pronounced phenotypic differentiation in most of the traits investigated herein, and components of the stream gradient (PCs) explained parts of the observed differences. Depending on the trait under investigation, phenotypic differentiation could be ascribed to variation in abiotic conditions, anthropogenic disturbance (influx of thermally polluted water), or population parameters. For example, female fecundity showed altitudinal variation and decreased with increasing conspecific densities, while sexual dimorphism in the length of male antennae-used for mate finding and assessment-increased with increasing population densities and towards female-biased sex ratios. CONCLUSIONS: We provide a comprehensive protocol for comparative analyses of intraspecific variation in life history traits in amphipods. Whether the observed phenotypic differentiation over small geographical distances reflects evolutionary divergence or plasticity (or both) remains to be investigated in future studies. Independent of the mechanisms involved, variation in several traits is likely to have consequences for ecosystem functions. For example, leaf-shredding in G. roeselii strongly depends on body size, which varied in dependence of several ecological parameters.

DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work.

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.

The future of biotic indices in the ecogenomic era: Integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems.

The bioassessment of aquatic ecosystems is currently based on various biotic indices that use the occurrence and/or abundance of selected taxonomic groups to define ecological status. These conventional indices have some limitations, often related to difficulties in morphological identification of bioindicator taxa. Recent development of DNA barcoding and metabarcoding could potentially alleviate some of these limitations, by using DNA sequences instead of morphology to identify organisms and to characterize a given ecosystem. In this paper, we review the structure of conventional biotic indices, and we present the results of pilot metabarcoding studies using environmental DNA to infer biotic indices. We discuss the main advantages and pitfalls of metabarcoding approaches to assess parameters such as richness, abundance, taxonomic composition and species ecological values, to be used for calculation of biotic indices. We present some future developments to fully exploit the potential of metabarcoding data and improve the accuracy and precision of their analysis. We also propose some recommendations for the future integration of DNA metabarcoding to routine biomonitoring programs.

Cryptic species and their utilization of indigenous and non-indigenous intermediate hosts in the acanthocephalan Polymorphus minutus sensu lato (Polymorphidae).

The bird-infecting acanthocephalan Polymorphus minutus has been suggested to comprise different lineages or even cryptic species using different intermediate hosts. To clarify this open question, we investigated Polymorphus cf. minutus cystacanths originating from amphipod intermediate hosts from 27 sites in Germany and France. Parasites and hosts were identified using integrated datasets (COI and/or morphology for hosts and COI + ITS1-5.8S-ITS2 for parasites).Mitochondrial and nuclear data (ITS1) strongly support the existence of three cryptic species in Polymorphus cf. minutus (type 1-3). These three types reveal a high degree of intermediate host specificity, with Polymorphus type 1 only encountered in Gammarus fossarum type B, Polymorphus type 2 in Echinogammarus sp. and Echinogammarus berilloni, and Polymorphus type 3 in Gammarus pulex and Gammarus roeselii. Our results point to a so far neglected cryptic diversity of the genus Polymorphus in Central Europe. Furthermore, Polymorphus type 2 is most likely a non-native parasite in Germany that co-invaded with E. berilloni from the Mediterranean area. Potentially, type 3 originates from South-East Europe and migrated to Germany by G. roeselii, where it might have captured G. pulex as an intermediate host. Therefore, our findings can be seen in the context of ecological globalization in terms of the anthropogenic displacement of intermediate hosts and its impact on the genetic divergence of the parasites.

Genome-wide single-nucleotide polymorphism data reveal cryptic species within cryptic freshwater snail species-The case of the Ancylus fluviatilis species complex.

DNA barcoding utilizes short standardized DNA sequences to identify species and is increasingly used in biodiversity assessments. The technique has unveiled an unforeseeably high number of morphologically cryptic species. However, if speciation has occurred relatively recently and rapidly, the use of single gene markers, and especially the exclusive use of mitochondrial markers, will presumably fail in delimitating species. Therefore, the true number of biological species might be even higher. One mechanism that can result in rapid speciation is hybridization of different species in combination with polyploidization, that is, allopolyploid speciation. In this study, we analyzed the population genetic structure of the polyploid freshwater snail Ancylus fluviatilis, for which allopolyploidization was postulated as a speciation mechanism. DNA barcoding has already revealed four cryptic species within A. fluviatilis (i.e., A. fluviatilis s. str., Ancylus sp. A-C), but early allozyme data even hint at the presence of additional cryptic lineages in Central Europe. We combined COI sequencing with high-resolution genome-wide SNP data (ddRAD data) to analyze the genetic structure of A. fluviatilis populations in a Central German low mountain range (Sauerland). The ddRAD data results indicate the presence of three cryptic species within A. fluviatilis s. str. occurring in sympatry and even syntopy, whereas mitochondrial sequence data only support the existence of one species, with shared haplotypes between species. Our study hence points to the limitations of DNA barcoding when dealing with organismal groups where speciation is assumed to have occurred rapidly, for example, through the process of allopolyploidization. We therefore emphasize that single marker DNA barcoding can underestimate the true species diversity and argue in strong favor of using genome-wide data for species delimitation in such groups.

A new cave centipede from Croatia, Eupolybothrus liburnicus sp. n., with notes on the subgenus Schizopolybothrus Verhoeff, 1934 (Chilopoda, Lithobiomorpha, Lithobiidae).

A new species of Eupolybothrus Verhoeff, 1907 discovered in caves of Velebit Mountain in Croatia is described. E. liburnicussp. n. exhibits a few morphological differences from its most similar congeners, all of which are attributed to the subgenus Schizopolybothrus Verhoeff, 1934, and two approaches to species delimitation using the COI barcode region identify it as distinct from the closely allied E. cavernicolus Stoev & Komericki, 2013. E. spiniger (Latzel, 1888) is redescribed and a lectotype is designated for it as well as E. caesar (Verhoeff, 1899) to stabilize their respective taxonomic status. The subspecies E. acherontis wardaranus Verhoeff, 1937, previously suspected to be a synonym of E. caesar (Verhoeff, 1899), is redescribed and its taxonomy revised after the study of type material whereas the identity of E. acherontis (Verhoeff, 1900) described from a female from southwest Trebinje (Bosnia and Herzegovina) remains unknown. Type material of E. stygis (Folkmanova, 1940) is confirmed to be lost and future designation of neotypes from topotypic specimens is necessary to stabilize its taxonomy. The importance of setal arrangement on the intermediate and 14th tergites and the sexual modifications on the male 15th prefemur for species identification is discussed in the light of present findings, and a review of the species of E. (Schizopolybothrus) that display these traits is also provided.

Three new species of Carychium O.F. Müller, 1773 from the Southeastern USA, Belize and Panama are described using computer tomography (CT) (Eupulmonata, Ellobioidea, Carychiidae).

Three new species of the genus Carychium O.F. Müller, 1773, Carychium hardiei Jochum & Weigand, sp. n., Carychium belizeense Jochum & Weigand, sp. n. and Carychium zarzaae Jochum & Weigand, sp. n. are described from the Southeastern United States, Belize and Panama, respectively. In two consecutive molecular phylogenetic studies of worldwide members of Carychiidae, the North and Central American morphospecies Carychium mexicanum Pilsbry, 1891 and Carychium costaricanum E. von Martens, 1898 were found to consist of several evolutionary lineages. Although the related lineages were found to be molecularly distinct from the two nominal species, the consequential morphological and taxonomic assessment of these lineages is still lacking. In the present paper, the shells of these uncovered Carychium lineages are assessed by comparing them with those of related species, using computer tomography for the first time for this genus. The interior diagnostic characters are emphasized, such as columellar configuration in conjunction with the columellar lamella and their relationship in context of the entire shell. These taxa are morphologically described and formally assigned their own names.

The complete mitochondrial genome of a cryptic amphipod species from the Gammarus fossarum complex.

The freshwater amphipod Gammarus fossarum is widely distributed throughout Europe and an important species for stream biomonitoring. It is known to consist of several cryptic species. We here report the complete mitochondrial genome of G. fossarum clade 11/type B with a length of 15,989 bp, encoding for 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. Protein-coding and ribosomal genes have a similar arrangement as in other gammarid amphipods. A phylogenetic analysis clarifies the placement of G. fossarum within the Gammaridae.

Positive selection on panpulmonate mitogenomes provide new clues on adaptations to terrestrial life.

BACKGROUND: Transitions from marine to intertidal and terrestrial habitats resulted in a significant adaptive radiation within the Panpulmonata (Gastropoda: Heterobranchia). This clade comprises several groups that invaded the land realm independently and in different time periods, e.g., Ellobioidea, Systellomatophora, and Stylommatophora. Thus, mitochondrial genomes of panpulmonate gastropods are promising to screen for adaptive molecular signatures related to land invasions. RESULTS: We obtained three complete mitochondrial genomes of terrestrial panpulmonates, i.e., the ellobiid Carychium tridentatum, and the stylommatophorans Arion rufus and Helicella itala. Our dataset consisted of 50 mitogenomes comprising almost all major panpulmonate lineages. The phylogenetic tree based on mitochondrial genes supports the monophyly of the clade Panpulmonata. Terrestrial lineages were sampled from Ellobioidea (1 sp.) and Stylommatophora (9 spp.). The branch-site test of positive selection detected significant non-synonymous changes in the terrestrial branches leading to Carychium (Ellobiodea) and Stylommatophora. These convergent changes occurred in the cob and nad5 genes (OXPHOS complex III and I, respectively). CONCLUSIONS: The convergence of the non-synonymous changes in cob and nad5 suggest possible ancient episodes of positive selection related to adaptations to non-marine habitats. The positively selected sites in our data are in agreement with previous results in vertebrates suggesting a general pattern of adaptation to the new metabolic requirements. The demand for energy due to the colonization of land (for example, to move and sustain the body mass in the new habitat) and the necessity to tolerate new conditions of abiotic stress may have changed the physiological constraints in the early terrestrial panpulmonates and triggered adaptations at the mitochondrial level.

Drastic underestimation of amphipod biodiversity in the endangered Irano-Anatolian and Caucasus biodiversity hotspots.

Biodiversity hotspots are centers of biological diversity and particularly threatened by anthropogenic activities. Their true magnitude of species diversity and endemism, however, is still largely unknown as species diversity is traditionally assessed using morphological descriptions only, thereby ignoring cryptic species. This directly limits evidence-based monitoring and management strategies. Here we used molecular species delimitation methods to quantify cryptic diversity of the montane amphipods in the Irano-Anatolian and Caucasus biodiversity hotspots. Amphipods are ecosystem engineers in rivers and lakes. Species diversity was assessed by analysing two genetic markers (mitochondrial COI and nuclear 28S rDNA), compared with morphological assignments. Our results unambiguously demonstrate that species diversity and endemism is dramatically underestimated, with 42 genetically identified freshwater species in only five reported morphospecies. Over 90% of the newly recovered species cluster inside Gammarus komareki and G. lacustris; 69% of the recovered species comprise narrow range endemics. Amphipod biodiversity is drastically underestimated for the studied regions. Thus, the risk of biodiversity loss is significantly greater than currently inferred as most endangered species remain unrecognized and/or are only found locally. Integrative application of genetic assessments in monitoring programs will help to understand the true magnitude of biodiversity and accurately evaluate its threat status.

Invaders, natives and their enemies: distribution patterns of amphipods and their microsporidian parasites in the Ruhr Metropolis, Germany.

BACKGROUND: The amphipod and microsporidian diversity in freshwaters of a heterogeneous urban region in Germany was assessed. Indigenous and non-indigenous host species provide an ideal framework to test general hypotheses on potentially new host-parasite interactions, parasite spillback and spillover in recently invaded urban freshwater communities. METHODS: Amphipods were sampled in 17 smaller and larger streams belonging to catchments of the four major rivers in the Ruhr Metropolis (Emscher, Lippe, Ruhr, Rhine), including sites invaded and not invaded by non-indigenous amphipods. Species were identified morphologically (hosts only) and via DNA barcoding (hosts and parasites). Prevalence was obtained by newly designed parasite-specific PCR assays. RESULTS: Three indigenous and five non-indigenous amphipod species were detected. Gammarus pulex was further distinguished into three clades (C, D and E) and G. fossarum more precisely identified as type B. Ten microsporidian lineages were detected, including two new isolates (designated as Microsporidium sp. nov. RR1 and RR2). All microsporidians occurred in at least two different host clades or species. Seven genetically distinct microsporidians were present in non-invaded populations, six of those were also found in invaded assemblages. Only Cucumispora dikerogammari and Dictyocoela berillonum can be unambiguously considered as non-indigenous co-introduced parasites. Both were rare and were not observed in indigenous hosts. Overall, microsporidian prevalence ranged from 50% (in G. roeselii and G. pulex C) to 73% (G. fossarum) in indigenous and from 10% (Dikerogammarus villosus) to 100 % (Echinogammarus trichiatus) in non-indigenous amphipods. The most common microsporidians belonged to the Dictyocoela duebenum- /D. muelleri- complex, found in both indigenous and non-indigenous hosts. Some haplotype clades were inclusive for a certain host lineage. CONCLUSIONS: The Ruhr Metropolis harbours a high diversity of indigenous and non-indigenous amphipod and microsporidian species, and we found indications for an exchange of parasites between indigenous and non-indigenous hosts. No introduced microsporidians were found in indigenous hosts and prevalence of indigenous parasites in non-indigenous hosts was generally low. Therefore, no indication for parasite spillover or spillback was found. We conclude that non-indigenous microsporidians constitute only a minimal threat to the native amphipod fauna. However, this might change e.g. if C. dikerogammari adapts to indigenous amphipod species or if other hosts and parasites invade.