USGS Science⌕ Search

SEARCH · USGS Science

Results for “Genes”

Search indexed USGS publications on groundwater, aquifers, geologic maps, mineral resources and earthquakes. Explore source records by subject and place.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 901 records · Page 50Linked to original sources

Molecular detection of bacteria in the families Rickettsiaceae and Anaplasmataceae in northern crested caracaras ( Caracara cheriway )

Bacterial pathogens of the families Anaplasmataceae and Rickettsiaceae are often spread to humans or other animals from bites from infected arthropod hosts. Recently, an increasing number of studies have implicated migratory birds in the circulation of these pathogens through the spread of arthropod vectors. However, few studies have examined the potential for resident bird populations to serve as reservoirs for these zoonoses. In this study, we used nested PCRs of the GroESL and 17 kDa genes to screen for Anaplasmataceae and Rickettsiaceae , respectively, in a resident population of the northern crested caracara ( Caracara cheriway ) from Florida ( n = 55). Additionally, a small number ( n = 6) of captive individuals from Texas were included. We identified one individual (1.64%) positive for Rickettsia felis and one (1.64%) positive for Ehrlichia chaffeensis; both these individuals were from Florida. Presence of these pathogens demonstrates that these birds are potential hosts; however, the low prevalence of infections suggests that these populations likely do not function as an ecological reservoir.

Ticks and Tick-borne Diseases↗

Infectivity of attenuated poxvirus vaccine vectors and immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian Free-tailed bat ( Tadarida brasiliensis )

Bats (Order Chiroptera) are an abundant group of mammals with tremendous ecological value as insectivores and plant dispersers, but their role as reservoirs of zoonotic diseases has received more attention in the last decade. With the goal of managing disease in free-ranging bats, we tested modified vaccinia Ankara (MVA) and raccoon poxvirus (RCN) as potential vaccine vectors in the Brazilian Free-tailed bat ( Tadarida brasiliensis ), using biophotonic in vivo imaging and immunogenicity studies. Animals were administered recombinant poxviral vectors expressing the luciferase gene (MVA- luc , RCN- luc ) through oronasal (ON) or intramuscular (IM) routes and subsequently monitored for bioluminescent signal indicative of viral infection. No clinical illness was noted after exposure to any of the vectors, and limited luciferase expression was observed. Higher and longer levels of expression were observed with the RCN- luc construct. When given IM, luciferase expression was limited to the site of injection, while ON exposure led to initial expression in the oral cavity, often followed by secondary replication at another location, likely the gastric mucosa or gastric associated lymphatic tissue. Viral DNA was detected in oral swabs up to 7 and 9 days post infection (dpi) for MVA and RCN, respectively. While no live virus was detected in oral swabs from MVA-infected bats, titers up to 3.88 x 10 4 PFU/ml were recovered from oral swabs of RCN-infected bats. Viral DNA was also detected in fecal samples from two bats inoculated IM with RCN, but no live virus was recovered. Finally, we examined the immunogenicity of a RCN based rabies vaccine (RCN-G) following ON administration. Significant rabies neutralizing antibody titers were detected in the serum of immunized bats using the rapid fluorescence focus inhibition test (RFFIT). These studies highlight the safety and immunogenicity of attenuated poxviruses and their potential use as vaccine vectors in bats.

Vaccine↗

Comparative analysis of Edwardsiella isolates from fish in the eastern United States identifies two distinct genetic taxa amongst organisms phenotypically classified as E. tarda

Edwardsiella tarda, a Gram-negative member of the family Enterobacteriaceae, has been implicated in significant losses in aquaculture facilities worldwide. Here, we assessed the intra-specific variability of E. tarda isolates from 4 different fish species in the eastern United States. Repetitive sequence mediated PCR (rep-PCR) using 4 different primer sets (ERIC I & II, ERIC II, BOX, and GTG 5 ) and multi-locus sequence analysis of 16S SSU rDNA, groEl, gyrA, gyrB, pho, pgi, pgm, and rpoA gene fragments identified two distinct genotypes of E. tarda (DNA group I; DNA group II). Isolates that fell into DNA group II demonstrated more similarity to E. ictaluri than DNA group I, which contained the reference E. tarda strain (ATCC #15947). Conventional PCR analysis using published E. tarda-specific primer sets yielded variable results, with several primer sets producing no observable amplification of target DNA from some isolates. Fluorometric determination of G + C content demonstrated 56.4% G + C content for DNA group I, 60.2% for DNA group II, and 58.4% for E. ictaluri. Surprisingly, these isolates were indistinguishable using conventional biochemical techniques, with all isolates demonstrating phenotypic characteristics consistent with E. tarda. Analysis using two commercial test kits identified multiple phenotypes, although no single metabolic characteristic could reliably discriminate between genetic groups. Additionally, anti-microbial susceptibility and fatty acid profiles did not demonstrate remarkable differences between groups. The significant genetic variation (<90% similarity at gyrA, gyrB, pho, phi and pgm; <40% similarity by rep-PCR) between these groups suggests organisms from DNA group II may represent an unrecognized, genetically distinct taxa of Edwardsiella that is phenotypically indistinguishable from E. tarda.

Veterinary Microbiology↗

Molecular identification of erythrocytic necrosis virus (ENV) from the blood of Pacific herring ( Clupea pallasii )

Viral erythrocytic necrosis (VEN) is a condition affecting the red blood cells of more than 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. Among populations of Pacific herring ( Clupea pallasii ) on the west coast of North America the disease causes anemia and elevated mortality in periodic epizootics. Presently, VEN is diagnosed by observation of typical cytoplasmic inclusion bodies in stained blood smears from infected fish. The causative agent, erythrocytic necrosis virus (ENV), is unculturable and a presumed iridovirus by electron microscopy. In vivo amplification of the virus in pathogen-free laboratory stocks of Pacific herring with subsequent virus concentration, purification, DNA extraction, and high-throughput sequencing were used to obtain genomic ENV sequences. Fragments with the highest sequence identity to the family Iridoviridae were used to design four sets of ENV-specific polymerase chain reaction (PCR) primers. Testing of blood and tissue samples from experimentally and wild infected Pacific herring as well as DNA extracted from other amphibian and piscine iridoviruses verified the assays were specific to ENV with a limit of detection of 0.0003 ng. Preliminary phylogenetic analyses of a 1448 bp fragment of the putative DNA polymerase gene supported inclusion of ENV in a proposed sixth genus of the family Iridoviridae that contains other erythrocytic viruses from ectothermic hosts. This study provides the first molecular evidence of ENV's inclusion within the Iridoviridae family and offers conventional PCR assays as a means of rapidly surveying the ENV-status of wild and propagated Pacific herring stocks.

Veterinary Microbiology↗

An update on Toxoplasma gondii infections in northern sea otters (Enhydra lutris kenyoni) from Washington State, USA

Toxoplasmosis in marine mammals is epidemiologically and clinically important. Toxoplasma gondii antibodies (by modified agglutination test, cut-off ≥1:25) were detected in serum of 65 of 70 (92.9%) northern sea otters ( Enhydra lutris kenyoni ) from Washington State, USA. Brains and/or muscles of 44 sea otters were bioassayed in mice (INF-γ knock-out [KO], Swiss Webster outbred [SW]) and viable T. gondii was isolated from 22 of 44 (50%); T. gondii strains were lethal to KO mice but not SW mice. These T. gondii isolates were further propagated in cell culture. Multi-locus PCR-RFLP genotyping of cell culture-derived tachyzoites revealed four different genotypes among 22 isolates including ToxoDB PCR-RFLP genotype #5 (14 isolates), #1 (three isolates), #3 (four isolates), and #167 (one isolate). PCR-DNA sequencing based genotyping using polymorphic gene GRA6 revealed one of four different alleles. Among the 14 RFLP genotype #5 strains, 10 have GRA6 sequences that match with the Type A, one match with the Type X, two strains did not generate sequence data, and one strain had double peaks at known polymorphic sites indicating a mixed infection. The seven strains belong to genotypes #1 and #3, all have identical sequences to T. gondii Type II reference isolate ME49. Genotype #167 strain has identical sequence to Type I reference strain. In summary, we observed high seroprevalence, and high rate of isolation of T. gondii from northern sea otters and predominant genotype #5 that has been previously reported a dominant and widespread strain among terrestrial wildlife in North America. GRA6 sequence analysis of the genotype #5 isolates indicated the dominance of Type A lineage in sea otters in Washington State.

Washington↗

The Ozobranchus leech is a candidate mechanical vector for the fibropapilloma-associated turtle herpesvirus found latently infecting skin tumors on Hawaiian green turtles (Chelonia mydas)

Fibropapillomatosis (FP) of marine turtles is a neoplastic disease of ecological concern. A fibropapilloma-associated turtle herpesvirus (FPTHV) is consistently present, usually at loads exceeding one virus copy per tumor cell. DNA from an array of parasites of green turtles ( Chelonia mydas ) was examined with quantitative PCR (qPCR) to determine whether any carried viral loads are sufficient to implicate them as vectors for FPTHV. Marine leeches ( Ozobranchus spp.) were found to carry high viral DNA loads; some samples approached 10 million copies per leech. Isopycnic sucrose density gradient/qPCR analysis confirmed that some of these copies were associated with particles of the density of enveloped viruses. The data implicate the marine leech Ozobranchus as a mechanical vector for FPTHV. Quantitative RT-PCR analysis of FPTHV gene expression indicated that most of the FPTHV copies in a fibropapilloma have restricted DNA polymerase expression, suggestive of latent infection.

Florida, Hawai'i↗

Attenuation of monkeypox virus by deletion of genomic regions

Monkeypox virus (MPXV) is an emerging pathogen from Africa that causes disease similar to smallpox. Two clades with different geographic distributions and virulence have been described. Here, we utilized bioinformatic tools to identify genomic regions in MPXV containing multiple virulence genes and explored their roles in pathogenicity; two selected regions were then deleted singularly or in combination. In vitro and in vivo studies indicated that these regions play a significant role in MPXV replication, tissue spread, and mortality in mice. Interestingly, while deletion of either region led to decreased virulence in mice, one region had no effect on in vitro replication. Deletion of both regions simultaneously also reduced cell culture replication and significantly increased the attenuation in vivo over either single deletion. Attenuated MPXV with genomic deletions present a safe and efficacious tool in the study of MPX pathogenesis and in the identification of genetic factors associated with virulence.

Virology↗

SARS-CoV-2 utilization of ACE2 from different bat species allows for virus entry and replication in vitro

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is believed to have a zoonotic origin with bats suspected as a natural host. In this work, we individually express the ACE2 of seven bat species including, little brown, great roundleaf, Pearson's horseshoe, greater horseshoe, Brazilian free-tailed, Egyptian rousette, and Chinese rufous horseshoe in DF1 cells and determine their ability to support attachment and replication of SARS-CoV-2 viruses. We demonstrate that the ACE2 receptor of all seven species made DF1 cells permissible to SARS-CoV-2. The level of virus replication differed between bat species and variants tested. The Wuhan lineage SARS-CoV-2 virus replicated to higher titers than either variant virus tested. All viruses tested grew to higher titers in cells expressing the human ACE2 gene compared to a bat ACE2. This study provides a practical in vitro method for further testing of animal species for potential susceptibility to current and emerging SARS-CoV-2 viruses.

Virology↗

H5N1 highly pathogenic avian influenza clade 2.3.4.4b in wild and domestic birds: Introductions into the United States and reassortments, December 2021–April 2022

Highly pathogenic avian influenza viruses (HPAIVs) of the A/goose/Guangdong/1/1996 lineage H5 clade 2.3.4.4b continue to have a devastating effect on domestic and wild birds. Full genome sequence analyses using 1369 H5N1 HPAIVs detected in the United States (U.S.) in wild birds, commercial poultry, and backyard flocks from December 2021 to April 2022, showed three phylogenetically distinct H5N1 virus introductions in the U.S. by wild birds. Unreassorted Eurasian genotypes A1 and A2 entered the Northeast Atlantic states, whereas a genetically distinct A3 genotype was detected in Alaska. The A1 genotype spread westward via wild bird migration and reassorted with North American wild bird avian influenza viruses. Reassortments of up to five internal genes generated a total of 21 distinct clusters; of these, six genotypes represented 92% of the HPAIVs examined. By phylodynamic analyses, most detections in domestic birds were shown to be point-source transmissions from wild birds, with limited farm-to-farm spread.

Virology↗

Replication and shedding kinetics of infectious hematopoietic necrosis virus in juvenile rainbow trout

Viral replication and shedding are key components of transmission and fitness, the kinetics of which are heavily dependent on virus, host, and environmental factors. To date, no studies have quantified the shedding kinetics of infectious hematopoietic necrosis virus (IHNV) in rainbow trout ( Oncorhynchus mykiss ), or how they are associated with replication, making it difficult to ascertain the transmission dynamics of this pathogen of high agricultural and conservation importance. Here, the replication and shedding kinetics of two M genogroup IHNV genotypes were examined in their naturally co-evolved rainbow trout host. Within host virus replication began rapidly, approaching maximum values by day 3 post-infection, after which viral load was maintained or gradually dropped through day 7. Host innate immune response measured as stimulation of Mx-1 gene expression generally followed within host viral loads. Shedding also began very quickly and peaked within 2 days, defining a generally uniform early peak period of shedding from 1 to 4 days after exposure to virus. This was followed by a post-peak period where shedding declined, such that the majority of fish were no longer shedding by day 12 post-infection. Despite similar kinetics, the average shedding rate over the course of infection was significantly lower in mixed compared to single genotype infections, suggesting a competition effect, however, this did not significantly impact the total amount of virus shed. The data also indicated that the duration of shedding, rather than peak amount of virus shed, was correlated with fish mortality. Generally, the majority of virus produced during infection appeared to be shed into the environment rather than maintained in the host, although there was more retention of within host virus during the post-peak period. Viral virulence was correlated with shedding, such that the more virulent of the two genotypes shed more total virus. This fundamental understanding of IHNV shedding kinetics and variation at the individual fish level could assist with management decisions about how to respond to disease outbreaks when they occur.

Virus Research↗

Multi-laboratory evaluations of the performance of Catellicoccus marimammalium PCR assays developed to target gull fecal sources

Here we report results from a multi-laboratory ( n = 11) evaluation of four different PCR methods targeting the 16S rRNA gene of Catellicoccus marimammalium originally developed to detect gull fecal contamination in coastal environments. The methods included a conventional end-point PCR method, a SYBR ® Green qPCR method, and two TaqMan ® qPCR methods. Different techniques for data normalization and analysis were tested. Data analysis methods had a pronounced impact on assay sensitivity and specificity calculations. Across-laboratory standardization of metrics including the lower limit of quantification (LLOQ), target detected but not quantifiable (DNQ), and target not detected (ND) significantly improved results compared to results submitted by individual laboratories prior to definition standardization. The unit of measure used for data normalization also had a pronounced effect on measured assay performance. Data normalization to DNA mass improved quantitative method performance as compared to enterococcus normalization. The MST methods tested here were originally designed for gulls but were found in this study to also detect feces from other birds, particularly feces composited from pigeons. Sequencing efforts showed that some pigeon feces from California contained sequences similar to C. marimammalium found in gull feces. These data suggest that the prevalence, geographic scope, and ecology of C. marimammalium in host birds other than gulls require further investigation. This study represents an important first step in the multi-laboratory assessment of these methods and highlights the need to broaden and standardize additional evaluations, including environmentally relevant target concentrations in ambient waters from diverse geographic regions.

Water Research↗

Interaction of bacterial communities and indicators of water quality in shoreline sand, sediment, and water of Lake Michigan

Shoreline sand harbors high concentrations of fecal indicator bacteria (FIB) that may be resuspended into the water column through washing and resuspension. Studies have explored coastal processes that influence this sand-water flux for FIB, but little is known about how microbial markers of contamination or the bacterial community interact in the sand-water interface. In this study, we take a three-tiered approach to explore the relationship between bacteria in sand, sediment, and overlying water at three shoreline sites and two associated rivers along an extended freshwater shoreline. Samples were collected over two years and analyzed for FIB, two microbial source tracking (MST) markers ( Catellicoccus marimammalium, Gull2; Bacteroides HF183), and targeted metagenomic 16S rRNA gene analysis. FIB was much higher in sand than in water at all three sites. Gull2 marker was abundant in shoreline sand and water while HF183 marker was mostly present in rivers. Overall bacterial communities were dissimilar between sand/sediment and water, indicating little interaction. Sediment composition was generally unfavorable to bacterial resuspension. Results show that FIB and MST markers were effective estimates of short-term conditions at these locations, and bacterial communities in sand and sediment reflected longer-term conditions. Findings are useful for locating contamination sources and targeting restoration by evaluating scope of shoreline degradation.

Lake Michigan↗

Sources and risk factors for nitrate, pathogens, and fecal contamination of private wells in rural southwestern Wisconsin, USA

Household well water can be degraded by contaminants from the land's surface, but private well owners lack means to protect the source water from neighboring disturbances. Rural residents of southwestern Wisconsin, USA, rely on private well water, and the combination of land use and fractured carbonate bedrock makes groundwater vulnerable to contamination. To identify the extent, sources, and risk factors of private well contamination, randomly selected wells sampled during two-day periods in fall (n = 301) and spring (n = 529) were analyzed for nitrate and indicator bacteria, and a subset (n = 138) was sampled across four seasonal events for analysis of pathogens and microbial source tracking markers by quantitative polymerase chain reaction. Risk factors representing land use, hydrology, geology, and well construction were analyzed for associations with contamination in multivariable models. The importance of risk factors varied by contaminant, illustrating the multifaceted nature of rural groundwater quality. Nitrate contamination was associated with agricultural land use, and wells with casings that extended below a shale aquitard accessed less contaminated water than those drawing water from above it. Human fecal microbes were detected in 64 wells (46%), and rainfall was the key risk factor for contamination, indicating that wastewater from septic systems was available to contaminate wells when transport conditions were favorable. Manure microbes from cattle/ruminants and pigs were detected in 33 and 13 wells, respectively, and concentrations increased with the hectarage of cultivated land near wells. Pathogen genes for viruses, bacteria, and protozoa were detected in 66 wells (48%), including more detections of zoonotic than human-specific pathogens, and human Bacteroides , an indicator of wastewater, was an equivocal predictor of pathogen presence in private wells. Characterizing important elements of the setting, like geology, and identifying sources and risk factors for contaminants can inform landscape-level policies to protect groundwater quality.

Wisconsin↗

An anti-steroidogenic inhibitory primer pheromone in male sea lamprey (Petromyzon marinus)

Reproductive functions can be modulated by both stimulatory and inhibitory primer pheromones released by conspecifics. Many stimulatory primer pheromones have been documented, but relatively few inhibitory primer pheromones have been reported in vertebrates. The sea lamprey male sex pheromone system presents an advantageous model to explore the stimulatory and inhibitory primer pheromone functions in vertebrates since several pheromone components have been identified. We hypothesized that a candidate sex pheromone component, 7&alpha;, 12&alpha;-dihydroxy-5&alpha;-cholan-3-one-24-oic acid (3 keto-allocholic acid or 3kACA), exerts priming effects through the hypothalamic-pituitary-gonadal (HPG) axis. To test this hypothesis, we measured the peptide concentrations and gene expressions of lamprey gonadotropin releasing hormones (lGnRH) and the HPG output in immature male sea lamprey exposed to waterborne 3kACA. Exposure to waterborne 3kACA altered neuronal activation markers such as jun and jun N-terminal kinase (JNK), and lGnRH mRNA levels in the brain. Waterborne 3kACA also increased lGnRH-III, but not lGnRH-I or -II, in the forebrain. In the plasma, 3kACA exposure decreased all three lGnRH peptide concentrations after 1 h exposure. After 2 h exposure, 3kACA increased lGnRHI and -III, but decreased lGnRH-II peptide concentrations in the plasma. Plasma lGnRH peptide concentrations showed differential phasic patterns. Group housing condition appeared to increase the averaged plasma lGnRH levels in male sea lamprey compared to isolated males. Interestingly, 15&alpha;-hydroxyprogesterone (15&alpha;-P) concentrations decreased after prolonged 3kACA exposure (at least 24 h). To our knowledge, this is the only known synthetic vertebrate pheromone component that inhibits steroidogenesis in males.

General and Comparative Endocrinology↗

Range-wide phylogeographic analysis of the spotted frog complex (Rana luteiventris and Rana pretiosa) in northwestern North America

The dynamic geological and climatic history of northwestern North America has made it a focal region for phylogeography. We conducted a range-wide phylogeographic analysis of the spotted frog complex (Rana luteiventris and Rana pretiosa) across its range in northwestern North America to understand its evolutionary history and the distribution of clades to inform conservation of R. pretiosa and Great Basin R. luteiventris, candidates for listing under the US Endangered Species Act. Mitochondrial DNA sequence data from a segment of the cytochrome b gene were obtained from 308 R. luteiventris and R. pretiosa from 96 sites. Phylogenetic analysis revealed one main R. pretiosa clade and three main R. luteiventris clades, two of which overlapped in southeastern Oregon. The three R. luteiventris clades were separated from each other by high levels of sequence divergence (average of 4.75-4.97%). Two divergent clades were also uncovered within the Great Basin. Low genetic variation in R. pretiosa and the southeastern Oregon clade of R. luteiventris suggests concern about their vulnerability to extinction. ?? 2008 Elsevier Inc.

Molecular Phylogenetics and Evolution↗

Molecular systematics of sturgeon nucleocytoplasmic large DNA viruses

Namao virus (NV) is a sturgeon nucleocytoplasmic large DNA virus (sNCLDV) that can cause a lethal disease of the integumentary system in lake sturgeon Acipenser fulvescens . As a group, the sNCLDV have not been assigned to any currently recognized taxonomic family of viruses. In this study, a data set of NV DNA sequences was generated and assembled as two non-overlapping contigs of 306,448 bp and then used to conduct a comprehensive systematics analysis using Bayesian inference of phylogeny for NV, other sNCLDV and representative members of six families of the NCLDV superfamily. The phylogeny of NV was reconstructed using protein homologues encoded by nine nucleocytoplasmic virus orthologous genes (NCVOGs): NCVOG0022 – mcp, NCVOG0038 – DNA polymerase B elongation subunit, NCVOG0076 – VV A18-type helicase, NCVOG0249 – VV A32-type ATPase, NCVOG0262 – AL2 VLTF3-like transcription factor, NCVOG0271 – RNA polymerase II subunit II, NCVOG0274 – RNA polymerase II subunit I, NCVOG0276 – ribonucleotide reductase small subunit and NCVOG1117 – mRNA capping enzyme. The accuracy of our phylogenetic method was evaluated using a combination of Bayesian statistical analysis and congruence analysis. Stable tree topologies were obtained with data sets differing in target molecule(s), sequence length and taxa. Congruent topologies were obtained in phylogenies constructed using individual protein data sets. The major capsid protein phylogeny inferred that ten representative sNCLDV form a monophyletic group comprised of four lineages within a polyphyletic Mimi-Phycodnaviridae group of taxa. Overall, the analyses revealed that Namao virus is a member of the Mimiviridae family with strong and consistent support for a clade containing NV and CroV as sister taxa.

Molecular Phylogenetics and Evolution↗

Phylogeography and evolution of infectious hematopoietic necrosis virus in China

Infectious hematopoietic necrosis virus (IHNV) is a well-known rhabdoviral pathogen of salmonid fish. In this study, a comprehensive analysis of 40 IHNV viruses isolated from thirteen fish farms in nine geographically dispersed Chinese provinces during 2012 to 2017 is presented. Identity of nucleotide and amino acid sequences among all the complete glycoprotein (G) genes from Chinese isolates was 98.0–100% and 96.7–100%, respectively. Coalescent phylogenetic analyses revealed that all the Chinese IHN virus characterized in this study were in a monophyletic clade that had a most recent common ancestor with the J Nagano (JN) subgroup within the J genogroup of IHNV. Within the Chinese IHNV clade isolates obtained over successive years from the same salmon fish farm clustered in strongly supported subclades, suggesting maintenance and diversification of virus over time within individual farms. There was also evidence for regional virus transmission within provinces, and some cases of longer distance transmission between distant provinces, such as Gansu and Yunnan. The data demonstrated that IHNV has evolved into a new subgroup in salmon farm environments in China, and IHNV isolates are undergoing molecular evolution within fish farms. We suggest that Chinese IHNV comprises a separate JC subgroup within the J genogroup of IHNV.

Molecular Phylogenetics and Evolution↗

Integrative phylogenetic, phylogeographic and morphological characterisation of the Unio crassus species complex reveals cryptic diversity with important conservation implications

The global decline of freshwater mussels and their crucial ecological services highlight the need to understand their phylogeny, phylogeography and patterns of genetic diversity to guide conservation efforts. Such knowledge is urgently needed for Unio crassus , a highly imperilled species originally widespread throughout Europe and southwest Asia. Recent studies have resurrected several species from synonymy based on mitochondrial data, revealing U. crassus to be a complex of cryptic species. To address long-standing taxonomic uncertainties hindering effective conservation, we integrate morphometric, phylogenetic, and phylogeographic analyses to examine species diversity within the U. crassus complex across its entire range. Phylogenetic analyses were performed using cytochrome c oxidase subunit I (815 specimens from 182 populations) and, for selected specimens, whole mitogenome sequences and Anchored Hybrid Enrichment (AHE) data on ∼600 nuclear loci. Mito-nuclear discordance was detected, consistent with mitochondrial DNA gene flow between some species during the Pliocene and Pleistocene. Fossil-calibrated phylogenies based on AHE data support a Mediterranean origin for the U. crassus complex in the Early Miocene. The results of our integrative approach support 12 species in the group: the previously recognised Unio bruguierianus , Unio carneus, Unio crassus , Unio damascensis , Unio ionicus , Unio sesirmensis , and Unio tumidiformis , and the reinstatement of five nominal taxa: Unio desectus stat. rev. , Unio gontierii stat. rev. , Unio mardinensis stat. rev. , Unio nanus stat. rev. , and Unio vicarius stat. rev. Morphometric analyses of shell contours reveal important morphospace overlaps among these species, highlighting cryptic, but geographically structured, diversity. The distribution, taxonomy, phylogeography, and conservation of each species are succinctly described.

Molecular Phylogenetics and Evolution↗