Well-spread samples with dynamic sample sizes.

A spatial sampling design determines where sample locations are placed in a study area so that population parameters can be estimated with relatively high precision. If the response variable has spatial trends, spatially balanced or well-spread designs give precise results for commonly used estimators. This article proposes a new method that draws well-spread samples over arbitrary auxiliary spaces and can be used for master sampling applications. All we require is a measure of the distance between population units. Numerical results show that the method generates well-spread samples and compares favorably with existing designs. We provide an example application using several auxiliary variables to estimate total aboveground biomass over a large study area in Eastern Amazonia, Brazil. Multipurpose surveys are also considered, where the totals of aboveground biomass, primary production, and clay content (3 responses) are estimated from a single well-spread sample over the auxiliary space.

Evaluation of sponge wipe surface sampling for collection of potential surrogates for non-spore-forming bioterrorism agents.

AIM: Evaluate the efficacy of sponge wipe sampling at recovering potential bacterial surrogates for Category A and B non-spore-forming bacterial bioterrorism agents from hard, nonporous surfaces. METHODS: A literature survey identified seven nonpathogenic bacteria as potential surrogates for selected Category A and B non-spore-forming bacterial agents. Small (2 × 4 cm) and large (35.6 × 35.6 cm) coupons made from either stainless steel, plastic, or glass, were inoculated and utilized to assess persistence and surface sampling efficiency, respectively. Three commercially available premoistened sponge wipes (3M™, Sani-Stick®, and Solar-Cult®) were evaluated. RESULTS: Mean recoveries from persistence testing indicated that three microorganisms (Yersinia ruckeri, Escherichia coli, and Serratia marcescens) demonstrated sufficient persistence across all tested material types. Sampling of large inoculated (≥107 CFU per sample) coupons resulted in mean recoveries ranging from 6.6 to 3.4 Log10 CFU per sample. Mean recoveries for the Solar-Cult®, 3M™ sponge wipes, and Sani-Sticks® across all test organisms and all material types were ≥5.7, ≥3.7, and ≥3.4 Log10 CFU per sample, respectively. Mean recoveries for glass, stainless steel, and ABS plastic across all test organisms and all sponge types were ≥3.8, ≥3.7, and ≥3.4 Log10 CFU per sample, respectively. CONCLUSIONS: Recovery results suggest that sponge wipe sampling can effectively be used to recover non-spore-forming bacterial cells from hard, nonporous surfaces such as stainless steel, ABS plastic, and glass.

Nosocomial outbreak caused by disinfectant-resistant Serratia marcescens in an adult intensive care unit, Hungary, February to March 2022.

In 2022, an outbreak with severe bloodstream infections caused by Serratia marcescens occurred in an adult intensive care unit (ICU) in Hungary. Eight cases, five of whom died, were detected. Initial control measures could not stop the outbreak. We conducted a matched case-control study. In univariable analysis, the cases were more likely to be located around one sink in the ICU and had more medical procedures and medications than the controls, however, the multivariable analysis was not conclusive. Isolates from blood cultures of the cases and the ICU environment were closely related by whole genome sequencing and resistant or tolerant against the quaternary ammonium compound surface disinfectant used in the ICU. Thus, S. marcescens was able to survive in the environment despite regular cleaning and disinfection. The hospital replaced the disinfectant with another one, tightened the cleaning protocol and strengthened hand hygiene compliance among the healthcare workers. Together, these control measures have proved effective to prevent new cases. Our results highlight the importance of multidisciplinary outbreak investigations, including environmental sampling, molecular typing and testing for disinfectant resistance.

Path to autonomous soil sampling and analysis by ground-based robots.

Good site characterization is essential for the selection of remediation alternatives for impacted soils. The value of site characterization is critically dependent on the quality and quantity of the data collected. Current methods for characterizing impacted soils rely on expensive manual sample collection and off-site analysis. However, recent advances in terrestrial robotics and artificial intelligence offer a potentially revolutionary set of tools and methods that will help to autonomously explore natural environments, select sample locations with the highest value of information, extract samples, and analyze the data in real-time without exposing humans to potentially hazardous conditions. A fundamental challenge to realizing this potential is determining how to design an autonomous system for a given investigation with many, and often conflicting design criteria. This work presents a novel design methodology to navigate these criteria. Specifically, this methodology breaks the system into four components - sensing, sampling, mobility, and autonomy - and connects design variables to the investigation objectives and constraints. These connections are established for each component through a survey of existing technology, discussion of key technical challenges, and highlighting conditions where generality can promote multi-application deployment. An illustrative example of this design process is presented for the development and deployment of a robotic platform characterizing salt-impacted oil & gas reserve pits. After calibration, the relationship between the in situ robot chloride measurements and laboratory-based chloride measurements had a good linear relationship (R2-value = 0.861) and statistical significance (p-value = 0.003).

Non-Steady-State Fickian Diffusion Models Decrease the Estimated Gel Layer Diffusion Coefficient Uncertainty for Diffusive Gradients in Thin-Films Passive Samplers.

Mass transport in diffusive gradients in thin-film passive samplers is restricted to diffusion through a gel layer of agarose or agarose cross-linked polyacrylamide (APA). The gel layer diffusion coefficient, DGel, is typically determined using a standard analysis (SA) based on Fick's first law from two-compartment diffusion cell (D-Cell) tests. The SA assumes pseudo-steady-state flux, characterized by linear sink mass accumulation-time profiles with a typical threshold R2 ≥ 0.97. In 72 D-Cell tests with nitrate, 63 met this threshold, but the SA-determined DGel ranged from 10.1 to 15.8 × 10-6 cm2·s-1 (agarose) and 9.5 to 14.7 × 10-6 cm2·s-1 (APA). A regression model developed with the SA to account for the diffusive boundary layer had 95% confidence intervals (CIs) on DGel of 13 to 18 × 10-6 cm2·s-1 (agarose) and 12 to 19 × 10-6 cm2·s-1 (APA) at 500 rpm. A finite difference model (FDM) developed based on Fick's second law with non-steady-state (N-SS) flux decreased uncertainty in DGel tenfold. The FDM-captured decreasing source compartment concentrations and N-SS flux in the D-Cell tests and, at 500 rpm, the FDM-determined DGel ± 95% CIs were 14.5 ± 0.2 × 10-6 cm2·s-1 (agarose) and 14.0 ± 0.3 × 10-6 cm2·s-1 (APA), respectively.

Design-based properties of the nearest neighbor spatial interpolator and its bootstrap mean squared error estimator.

Nearest neighbor spatial interpolation for mapping continuous populations and finite populations of areas or units is approached from a design-based perspective, that is, populations are fixed, and uncertainty stems from the sampling scheme adopted to select locations. We derive conditions for design-based pointwise and uniform consistency of the nearest neighbor interpolators. We prove that consistency holds under certain schemes that are widely applied in environmental and forest surveys. Furthermore, we propose a pseudopopulation bootstrap estimator of the root mean squared errors of the interpolated values. Finally, a simulation study is performed to assess the theoretical results.

Toward the robust resolution of taxonomic ambiguity within Lepocinclis (Euglenida) based on DNA sequencing and morphology.

DNA sequences were analyzed for three groups of species from the Lepocinclis genus (L. acus-like, L. oxyuris-like, and L. tripteris-like) along with cellular morphology. Phylogenetic analyses were based on nuclear SSU rDNA, LSU rDNA, and plastid-encoded LSU rDNA. DNA sequences were obtained from species available in culture collections (L. acus SAG 1224-1a and UTEX 1316) and those isolated directly from the environment in Poland (48 isolates), resulting in 79 new sequences. The obtained phylogenetic tree of Lepocinclis included 27 taxa, five of which are presented for the first time (L. convoluta, L. gracillimoides, L. longissima, L. pseudospiroides, and L. torta) and nine taxonomically verified and described. Based on morphology, literature data, and phylogenetic analyses, the following species were distinguished: in the L. acus-like group, L. longissima and L. acus; in the L. tripteris-like group, L. pseudospiroides, L. torta, and L. tripteris; in the L. oxyuris-like group, L. gracillimoides, L. oxyuris var. oxyuris, and L. oxyuris var. helicoidea. For all verified species, diagnostic descriptions were emended, nomenclatural adjustments were made, and epitypes were designated.

Detection and isolation of airborne SARS-CoV-2 in a hospital setting.

Transmission mechanisms for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are incompletely understood. In particular, aerosol transmission remains unclear, with viral detection in air and demonstration of its infection potential being actively investigated. To this end, we employed a novel electrostatic collector to sample air from rooms occupied by COVID-19 patients in a major Swedish hospital. Electrostatic air sampling in conjunction with extraction-free, reverse-transcriptase polymerase chain reaction (hid-RT-PCR) enabled detection of SARS-CoV-2 in air from patient rooms (9/22; 41%) and adjoining anterooms (10/22; 45%). Detection with hid-RT-PCR was concomitant with viral RNA presence on the surface of exhaust ventilation channels in patients and anterooms more than 2 m from the COVID-19 patient. Importantly, it was possible to detect active SARS-CoV-2 particles from room air, with a total of 496 plaque-forming units (PFUs) being isolated, establishing the presence of infectious, airborne SARS-CoV-2 in rooms occupied by COVID-19 patients. Our results support circulation of SARS-CoV-2 via aerosols and urge the revision of existing infection control frameworks to include airborne transmission.

Application of Target Enrichment Sequencing for Population Genetic Analyses of the Obligate Plant Pathogens Pseudoperonospora cubensis and P. humuli in Michigan.

Technological advances in genome sequencing have improved our ability to catalog genomic variation and have led to an expansion of the scope and scale of genetic studies over the past decade. Yet, for agronomically important plant pathogens such as the downy mildews (Peronosporaceae), the scale of genetic studies remains limited. This is, in part, due to the difficulties associated with maintaining obligate pathogens and the logistical constraints involved in the genotyping of these species (e.g., obtaining DNA of sufficient quantity and quality). To gain an evolutionary and ecological perspective of downy mildews, adaptable methods for the genotyping of their populations are required. Here, we describe a targeted enrichment (TE) protocol to genotype isolates from two Pseudoperonospora species (P. cubensis and P. humuli), using less than 50 ng of mixed pathogen and plant DNA for library preparation. We were able to enrich 830 target genes across 128 samples and identified 2,514 high-quality single nucleotide polymorphism (SNP) variants. Using these SNPs, we detected significant genetic differentiation (analysis of molecular variance [AMOVA], P = 0.01) between P. cubensis subpopulations from Cucurbita moschata (clade I) and Cucumis sativus (clade II) in the state of Michigan. No evidence of location-based differentiation was detected within the P. cubensis (clade II) subpopulation in Michigan. However, a significant effect of location on the genetic variation of the P. humuli subpopulation was detected in the state (AMOVA, P = 0.01). Mantel tests found evidence that the genetic distance among P. humuli samples was associated with the physical distance of the hop yards from which the samples were collected (P = 0.005). The differences in the distribution of genetic variation of the Michigan P. humuli and P. cubensis subpopulations suggest differences in the dispersal of these two species. The TE protocol described here provides an additional tool for genotyping obligate biotrophic plant pathogens and the execution of new genetic studies.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Persistence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Virus and Viral RNA in Relation to Surface Type and Contamination Concentration.

The transmission of SARS-CoV-2 is likely to occur through a number of routes, including contact with contaminated surfaces. Many studies have used reverse transcription-PCR (RT-PCR) analysis to detect SARS-CoV-2 RNA on surfaces, but seldom has viable virus been detected. This paper investigates the viability over time of SARS-CoV-2 dried onto a range of materials and compares viability of the virus to RNA copies recovered and whether virus viability is concentration dependent. Viable virus persisted for the longest time on surgical mask material and stainless steel, with a 99.9% reduction in viability by 122 and 114 h, respectively. Viability of SARS-CoV-2 reduced the fastest on a polyester shirt, with a 99.9% reduction within 2.5 h. Viability on the bank note was reduced second fastest, with 99.9% reduction in 75 h. RNA on all surfaces exhibited a 1-log reduction in genome copy number recovery over 21 days. The findings show that SARS-CoV-2 is most stable on nonporous hydrophobic surfaces. RNA is highly stable when dried on surfaces, with only 1-log reduction in recovery over 3 weeks. In comparison, SARS-CoV-2 viability reduced more rapidly, but this loss in viability was found to be independent of starting concentration. Expected levels of SARS-CoV-2 viable environmental surface contamination would lead to undetectable levels within 2 days. Therefore, when RNA is detected on surfaces, it does not directly indicate the presence of viable virus, even at low cycle threshold values. IMPORTANCE This study shows the impact of material type on the viability of SARS-CoV-2 on surfaces. It demonstrates that the decay rate of viable SARS-CoV-2 is independent of starting concentration. However, RNA shows high stability on surfaces over extended periods. This has implications for interpretation of surface sampling results using RT-PCR to determine the possibility of viable virus from a surface, where RT-PCR is not an appropriate technique to determine viable virus. Unless sampled immediately after contamination, it is difficult to align RNA copy numbers to quantity of viable virus on a surface.

SARS-CoV-2 in hospital indoor environments is predominantly non-infectious.

BACKGROUND: The ongoing SARS-CoV-2 pandemic has spread rapidly worldwide and disease prevention is more important than ever. In the absence of a vaccine, knowledge of the transmission routes and risk areas of infection remain the most important existing tools to prevent further spread. METHODS: Here we investigated the presence of the SARS-CoV-2 virus in the hospital environment at the Uppsala University Hospital Infectious Disease ward by RT-qPCR and determined the infectivity of the detected virus in vitro on Vero E6 cells. RESULTS: SARS-CoV-2 RNA was detected in several areas, although attempts to infect Vero E6 cells with positive samples were unsuccessful. However, RNase A treatment of positive samples prior to RNA extraction did not degrade viral RNA, indicating the presence of SARS-CoV-2 nucleocapsids or complete virus particles protecting the RNA as opposed to free viral RNA. CONCLUSION: Our results show that even in places where a moderate concentration (Ct values between 30 and 38) of SARS-CoV-2 RNA was found; no infectious virus could be detected. This suggests that the SARS-CoV-2 virus in the hospital environment subsides in two states; as infectious and as non-infectious. Future work should investigate the reasons for the non-infectivity of SARS-CoV-2 virions.

Paenibacillus tianjinensis sp. nov., isolated from corridor air.

A Gram-stain-positive, facultatively anaerobic, non-motile, endospore-forming and rod-shaped bacterium, occurring singly or in pairs, designated TB2019T, was isolated from environmental monitoring samples of corridor air collected at the Tianjin Institute for Drug Control, Tianjin Province (PR China). The isolate was able to grow at 15-40 °C (optimum growth at 37 °C), pH 6.0-8.0 (pH 7.0) and in the presence of 0-2% (w/v) NaCl (0% NaCl). Comparison of 16S rRNA gene sequences indicated that TB2019T was most closely related to Paenibacillus typhae CGMCC 1.11012T (98.63%), Paenibacillus albidus Q4-3T (98.19%), Paenibacillus borealis DSM 13188T (97.55%), Paenibacillus helianthi P26ET (97.33%) and Paenibacillus odorifer DSM 15391T (97.19%). The digital DNA-DNA hybridization and the average nucleotide identity values between TB2019T and the five type strains mentioned above ranged from 20.7 to 25.0% and 75.2 to 81.3%, respectively, and the genomic DNA G+C content was 49.52 mol%. The diagnostic cell-wall sugar was ribose, and the diagnostic amino acid was meso-diaminopimelic acid. The polar lipids of TB2019T included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unidentified aminophospholipids and one unidentified phospholipid. MK-7 was the predominant menaquinone, and anteiso-C15:0 (30.6%) was the major fatty acid. Based on the polyphasic taxonomic data, strain TB2019T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus tianjinensis sp. nov. is proposed. The type strain is TB2019T (=CICC 25065T=JCM 34610T).

Longitudinal Effects of a Sanitation Intervention on Environmental Fecal Contamination in a Cluster-Randomized Controlled Trial in Rural Bangladesh.

Household latrine access generally is not associated with reduced fecal contamination in the environment, but its long-term effectiveness has not been measured. We conducted an environmental assessment nested within the WASH Benefits Bangladesh randomized controlled trial (NCT01590095). We quantified E. coli and fecal coliforms in samples of stored drinking water, child hands, mother hands, soil, and food among a random sample of households from the sanitation and control arms of the trial. Samples were collected during eight quarterly visits approximately 1-3.5 years after intervention initiation. Overall, there were no substantial differences in environmental fecal contamination between households enrolled in the sanitation and control arms. Statistically significant reductions were found in stored water and child hands after pooling across sampling rounds, but the effects were small and not consistent across rounds. In addition, we assessed potential effect modification of intervention effects by follow-up time, season, wealth, community-level latrine density and coverage, population density, and domestic animal ownership. While the intervention had statistically significant effects within some subgroups, there were no consistent patterns of effect modification. Our findings support a growing consensus that on-site latrines are insufficient to prevent fecal contamination in the rural household environment.

Description of Flexiglena gen. nov. and new members of Discoplastis and Euglenaformis (Euglenida).

Environmental sampling in Poland and the United States and phylogenetic analyses based on 567 sequences of four genes (155 sequences of nuclear SSU rDNA, 139 of nuclear LSU rDNA, 135 of plastid-encoded SSU rDNA, and 138 of plastid-encoded LSU rDNA) resulted in description of the new genus Flexiglena, which has been erected by accommodating Euglena variabilis, and enriching the Discoplastis and Euglenaformis genera with five new species. Four of them have joined the Discoplastis genus, currently consisting of six representatives: D. adunca, D. angusta (=Euglena angusta), D. constricta (=Lepocinclis constricta), D. excavata (=E. excavata), D. gasterosteus (=E. gasterosteus), and D. spathirhyncha. One of them has enriched the Euglenaformis genus, currently represented by two species: Euf. chlorophoenicea (= E. chlorophoenicea) and Euf. proxima. For most studied species, the diagnostic descriptions have been emended and epitypes were designated. Furthermore, the emending of Discoplastis and Euglenaformis diagnoses was performed.

Methods for interpolating missing data in aerobiological databases.

Missing data is a common problem in scientific research. The availability of extensive environmental time series is usually laborious and difficult, and sometimes unexpected failures are not detected until samples are processed. Consequently, environmental databases frequently have some gaps with missing data in it. Applying an interpolation method before starting the data analysis can be a good solution in order to complete this missing information. Nevertheless, there are several different approaches whose accuracy should be considered and compared. In this study, data from 6 aerobiological sampling stations were used as an example of environmental data series to assess the accuracy of different interpolation methods. For that, observed daily pollen/spore concentration data series were randomly removed, interpolated by using different methods and then, compared with the observed data to measure the errors produced. Different periods, gap sizes, interpolation methods and bioaerosols were considered in order to check their influence in the interpolation accuracy. The moving mean interpolation method obtained the highest success rate as average. By using this method, a success rate of the 70% was obtained when the risk classes used in the alert systems of the pollen information platforms were taken into account. In general, errors were mostly greater when there were high oscillations in the concentrations of biotic particles during consecutive days. That is the reason why the pre-peak and peak periods showed the highest interpolation errors. The errors were also higher when gaps longer than 5 days were considered. So, for completing long periods of missing data, it would be advisable to test other methodological approaches. A new Variation Index based on the behaviour of the pollen/spore season (measurement of the variability of the concentrations every 2 consecutive days) was elaborated, which allows to estimate the potential error before the interpolation is applied.

Successful Isavuconazole Salvage Therapy for a Cryptococcus deuterogattii (AFLP6/VGII) Disseminated Infection in a European Immunocompetent Patient.

Members of the Cryptococcus gattii species complex are notorious causes of cryptococcosis as they often cause severe, life-threatening infections. Here we describe a case of a severe disseminated C. deuterogattii infection in a previously healthy patient who was initially treated with amphotericin B, 5-fluorocytosine and fluconazole, which led to a good neurological response, but the infection in the lungs remained unaltered and was not completely resolved until switching the antifungal therapy to isavuconazole. The infection was likely acquired during a one-month stay at the Azores Islands, Portugal. Environmental sampling did not yield any cryptococcal isolate; therefore, the source of this apparent autochthonous case could not be determined. Molecular typing showed that the cultured C. deuterogattii isolates were closely related to the Vancouver Island outbreak-genotype.

Environmental sampling of volatile organic compounds during the 2018 Camp Fire in Northern California.

Trace analysis of volatile organic compounds (VOCs) during wildfires is imperative for environmental and health risk assessment. The use of gas sampling devices mounted on unmanned aerial vehicles (UAVs) to chemically sample air during wildfires is of great interest because these devices move freely about their environment, allowing for more representative air samples and the ability to sample areas dangerous or unreachable by humans. This work presents chemical data from air samples obtained in Davis, CA during the most destructive wildfire in California's history - the 2018 Camp Fire - as well as the deployment of our sampling device during a controlled experimental fire while fixed to a UAV. The sampling mechanism was an in-house manufactured micro-gas preconcentrator (µPC) embedded onto a compact battery-operated sampler that was returned to the laboratory for chemical analysis. Compounds commonly observed in wildfires were detected during the Camp Fire using gas chromatography mass spectrometry (GC-MS), including BTEX (benzene, toluene, ethylbenzene, m+p-xylene, and o-xylene), benzaldehyde, 1,4-dichlorobenzene, naphthalene, 1,2,3-trimethylbenzene and 1-ethyl-3-methylbenzene. Concentrations of BTEX were calculated and we observed that benzene and toluene were highest with average concentrations of 4.7 and 15.1 µg/m3, respectively. Numerous fire-related compounds including BTEX and aldehydes such as octanal and nonanal were detected upon experimental fire ignition, even at a much smaller sampling time compared to samples taken during the Camp Fire. Analysis of the air samples taken both stationary during the Camp Fire and mobile during an experimental fire show the successful operation of our sampler in a fire environment.

Environmental Sampling for Severe Acute Respiratory Syndrome Coronavirus 2 During a COVID-19 Outbreak on the Diamond Princess Cruise Ship.

During a COVID-19 outbreak on the Diamond Princess cruise ship we sampled environmental surfaces after passengers and crew vacated cabins. SARS-CoV-2 RNA was detected in 58 of 601 samples (10%) from case cabins 1-17 days after cabins were vacated but not from noncase cabins. There was no difference in detection proportion between cabins of symptomatic (15%, 28/189; cycle quantification [Cq], 29.79-38.86) and asymptomatic cases (21%, 28/131; Cq, 26.21-38.99). No SARS-CoV-2 virus was isolated from any of the samples. Transmission risk of SARS-CoV-2 from symptomatic and asymptomatic patients may be similar and surfaces could be involved in transmission.

Molecular and Morphological Delimitation of Species in the Group of Lepocinclis Ovum-like taxa (Euglenida).

Although Lepocinclis ovum is recognized as a cosmopolitan and common species, and Lepocinclis globulus is the type species of the genus Lepocinclis, their correct identification is nearly impossible. The reason is that over 30 morphologically similar taxa appear in the literature, but no good diagnostic features exist to distinguish amongst them. Using environmental sampling and nuclear SSU rDNA sequencing, we delimited species within the group of Lepocinclis ovum-like taxa. Morphological and molecular features were analyzed for taxa isolated from Poland and six cultured strains from algal collections. In the case of environmental sampling, DNA was obtained from a small number of cells (20-400) isolated with a micropipette without setting up laboratory cultures (52 isolates), and phylogenetic analyses were based on the variation in nSSU rDNA. Apart from L. ovum and L. globulus, 13 other species were distinguished and four taxa (Lepocinclis conica comb. nov., L. fominii comb. nov., L. gracilicauda comb. nov., and L. pseudofominii nom. nov.) had their taxonomic ranks changed. For all verified species, diagnostic descriptions were emended and epitypes designated. The only exception was L. ovum, for which the epitype was questioned and thus, a new candidate for the epitype was suggested for future adoption.

Taxon-rich phylogeny and taxonomy of the genus Phacus (Euglenida) based on morphological and molecular data.

Morphological and molecular features were analyzed for a species of Phacus to better understand the phylogenetic relationships among them and establish the taxonomy. Phylogenetic analyses were based on nSSU rDNA and the research resulted in 55 new sequences. The study included species available in algal collections and those isolated directly from the environment in Poland and the Czech Republic. As a result, the obtained phylogenetic tree of Phacus includes 50 species, out of which 7 are represented on a tree for the first time (Phacus anacoelus, P. anomalus, P. curvicauda, P. elegans, P. lismorensis, P. minutus and P. stokesii) and many have been taxonomically verified. For all verified species, diagnostic descriptions were amended, the naming was reordered and epitypes were designated.