Biogeography of larval trematodes in the freshwater snail, Semisulcospira libertina: a comparison of the morphological and molecular approaches.

While biogeographic patterns of free-living organisms are well documented, the biogeography of parasitic fauna remains largely unclear. Due to morphological similarities, parasites are often difficult to identify without the aid of molecular genetics, further complicating the interpretation of their biogeographic patterns. We investigated trematode parasites infecting the East Asian freshwater snail Semisulcospira libertina to understand their biogeography and to evaluate how molecular approaches influence the interpretation of biogeographic patterns of the trematode fauna. We identified 46 genetically delimited species from 19 morphologically distinguishable trematodes infecting S. libertina and found that their species richness was negatively correlated to latitude. We also found that potential definitive host (fishes) richness and host body size were positively correlated with trematode species richness, suggesting that host attributes are essential factors shaping the biogeographic pattern in trematodes. These trends were observed irrespective of species identification methods, demonstrating that classical morphological identification can also effectively identify the latitudinal gradient pattern in trematodes. We further detected the distance decay of similarity in trematode communities, although this trend was only detectable in the biogeographic dataset based on molecular identification. Our study showed that morphological identification sufficiently reflects the latitudinal richness gradient while molecular identification is essential to estimate accurate local species richness and increase the resolution of the large-scale pattern of population similarities in the trematode communities.

Facing the infinity: tackling large samples of challenging Chironomidae (Diptera) with an integrative approach.

Background: Integrative taxonomy is becoming ever more significant in biodiversity research as scientists are tackling increasingly taxonomically challenging groups. Implementing a combined approach not only guarantees more accurate species identification, but also helps overcome limitations that each method presents when applied on its own. In this study, we present one application of integrative taxonomy for the highly abundant and particularly diverse fly taxon Chironomidae (Diptera). Although non-biting midges are key organisms in merolimnic systems, they are often cast aside in ecological surveys because they are very challenging to identify and extremely abundant. Methods: Here, we demonstrate one way of applying integrative methods to tackle this highly diverse taxon. We present a three-level subsampling method to drastically reduce the workload of bulk sample processing, then apply morphological and molecular identification methods in parallel to evaluate species diversity and to examine inconsistencies across methods. Results: Our results suggest that using our subsampling approach, identifying less than 10% of a sample's contents can reliably detect >90% of its diversity. However, despite reducing the processing workload drastically, the performance of our taxonomist was affected by mistakes, caused by large amounts of material. We conducted misidentifications for 9% of vouchers, which may not have been recovered had we not applied a second identification method. On the other hand, we were able to provide species information in cases where molecular methods could not, which was the case for 14% of vouchers. Therefore, we conclude that when wanting to implement non-biting midges into ecological frameworks, it is imperative to use an integrative approach.

Diversity and Phylogeny of Cattle Ixodid Ticks and Associated Spotted Fever Group Rickettsia spp. in Tunisia.

Tick-borne rickettsioses are mainly caused by obligate intracellular bacteria belonging to the spotted fever group (SFG) of the Rickettsia genus. So far, the causative agents of SFG rickettsioses have not been detected in cattle ticks from Tunisia. Therefore, the aim of this study was to investigate the diversity and phylogeny of ticks associated with cattle from northern Tunisia and their associated Rickettsia species. Adult ticks (n = 338) were collected from cattle in northern Tunisia. The obtained ticks were identified as Hyalomma excavatum (n = 129), Rhipicephalus sanguineus sensu lato (n = 111), Hyalomma marginatum (n = 84), Hyalomma scupense (n = 12) and Hyalomma rufipes (n = 2). After DNA extraction from the ticks, 83 PCR products based on the mitochondrial 16S rRNA gene were sequenced and a total of four genotypes for Rh. sanguineus s.l., two for Hy. marginatum and Hy. excavatum and only one for Hy. scupense and Hy. rufipes were recorded, with the occurrence of one, two and three novel genotypes, respectively, for Hy. marginatum, Hy. excavatum and Rh. sanguineus s.l. mitochondrial 16S rRNA partial sequences. The tick DNA was tested for the presence of Rickettsia spp. by using PCR measurements and sequencing targeting three different genes (ompB, ompA and gltA). Of the 338 analyzed ticks, 90 (26.6%), including 38 (34.2%) Rh. sanguineus s.l., 26 (20.1%) Hy. excavatum, 25 (29.8%) Hy. marginatum and one (50%) Hy. rufipes tick, were positive for Rickettsia spp. Based on 104 partial sequences of the three analyzed genes, the BLAST analysis and phylogenetic study showed the infection of Hy. excavatum, Hy. marginatum and Rh. sanguineus s.l. tick specimens with R. massiliae, R. aeschlimannii and R. sibirica subsp. mongolitimonae and one Hy. rufipes tick specimen with R. aeschlimannii. In addition, coinfection with R. massiliae and R. aeschlimannii was reported in one Hy. marginatum and one Rh. sanguineus s.l. tick specimen, while a coinfection with R. massiliae and R. sibirica subsp. mongolitimonae was recorded in one Rh. sanguineus s.l. tick specimen. In conclusion, our study reports, for the first time in Tunisia, the infection of cattle ticks belonging to Hyalomma and Rhipicephalus genera with zoonotic Rickettsia species belonging to the SFG group.

Application Progress of High-Throughput Sequencing Technology in Forensic Diatom Detection.

Diatom detection is an important method for identifying drowning and throwing corpses after death and inferring the drowning sites in forensic examination of corpses in water. In recent years,high-throughput sequencing technology has achieved rapid development and has been widely used in research related to diatom taxonomic investigations. This paper reviews the research status and prospects of high-throughput sequencing technology and its application in forensic diatom detection.

Application Progress of High-Throughput Sequencing Technology in Forensic Diatom Detection / 法医学杂志

Diatom detection is an important method for identifying drowning and throwing corpses after death and inferring the drowning sites in forensic examination of corpses in water. In recent years,high-throughput sequencing technology has achieved rapid development and has been widely used in research related to diatom taxonomic investigations. This paper reviews the research status and prospects of high-throughput sequencing technology and its application in forensic diatom detection.

Phenotypic, molecular and pathogenic characterization of Colletotrichum scovillei infecting Capsicum species in Rio de Janeiro, Brazil.

Anthracnose is a disease caused by Colletotrichum spp., one of the world's most damaging sweet and chili pepper pathogens, especially in tropical and subtropical regions. In the state of Rio de Janeiro, anthracnose is one of the main obstacles for pepper crops. However, to date no research has focused on the identification and characterization of the pathogen, which is fundamental to understand the scope of the disease in the state. Thus, the correct identification of the fungal species and pathogenicity studies can provide important support for disease management and control, apart from identifying possible resistance sources for exploitation in peppers breeding programs. In this study, 11 Colletotrichum isolates were collected from peppers with typical symptoms in the Rio de Janeiro state. These isolates were characterized based on morpho-cultural characteristics and sequencing data from five regions (ITS, ACT, CAL, ß-TUB and GAPDH), and the genetic variability was estimated by AFLP markers. Simultaneously, microscopy images of the colonization by the fungal species on unripe Capsicum annuum fruits were taken. Pathogenicity was tested and resistance sources were sought by means of infection of ripe and unripe fruits of 50 Capsicum baccatum accessions. The resulting data showed that all isolates belong to Colletotrichum scovillei specie. About the pathogenicity of Capsicum baccatum, differentiated, stage-specific responses, with higher resistance of ripe fruits were recorded. In addition, four possible sources of Colletotrichum scovillei resistance were detected among the tested accessions. The combination of these data can contribute to future studies on the interaction of Colletotrichum scovillei-Capsicum spp., a research line that is still unexploited in the main areas of this anthracnose fungus.

DNA mini-barcoding of leporids using noninvasive fecal DNA samples and its significance for monitoring an invasive species.

Introduced in South America at the end of the 19th century, the European hare population has expanded dramatically and now represents a risk to native Brazilian forest rabbits. Monitoring the invasive Lepus europaeus and its coexistence with native Sylvilagus brasiliensis is a challenge that can be efficiently addressed by the use of molecular tools. This work describes a set of primers useful for amplifying three mini-barcodes for the molecular identification of both invasive and native leporid species using degraded fecal DNA. In addition, tests in silico indicate that these mini-barcodes can successfully amplify the DNA sequences of a number of leporids. These mini-barcodes constitute a powerful tool for the monitoring and management of the invasive L. europaeus and the conservation of native rabbits.

Redescription and DNA barcoding of diurnal moth Athroolopha latimargoRothschild, 1914 bona sp., stat. rev. from the southern Iberian Peninsula (Lepidoptera: Geometridae: Ennominae).

The rare and diurnal geometrid moth Athroolopha latimargo Rothschild, 1914 bona sp., stat. rev. is re-discovered and redescribed from the furthest point of the south of the Iberian Peninsula, for the first time since its original description as a subspecies of Athroolopha chrysitaria (Hübner, 1813) from North Africa. The range of this taxon is questioned. A mitochondrial COI barcode sequence was generated for the specimens and compared with Iberian and Sicilian Athroolopha species.

Rapid mitochondrial genome sequencing based on Oxford Nanopore Sequencing and a proxy for vertebrate species identification.

Molecular information is crucial for species identification when facing challenging morphology-based specimen identifications. The use of DNA barcodes partially solves this problem, but in some cases when PCR is not an option (i.e., primers are not available, problems in reaction standardization), amplification-free approaches could be an optimal alternative. Recent advances in DNA sequencing, like the MinION device from Oxford Nanopore Technologies (ONT), allow to obtain genomic data with low laboratory and technical requirements, and at a relatively low cost. In this study, we explore ONT sequencing for molecular species identification from a total DNA sample obtained from a neotropical rodent and we also test the technology for complete mitochondrial genome reconstruction via genome skimming. We were able to obtain "de novo" the complete mitogenome of a specimen from the genus Melanomys (Cricetidae: Sigmodontinae) with average depth coverage of 78X using ONT-only data and by combining multiple assembly routines. Our pipeline for an automated species identification was able to identify the sample using unassembled sequence data (raw) in a reasonable computing time, which was substantially reduced when a priori information related to the organism identity was known. Our findings suggest ONT sequencing as a suitable candidate to solve species identification problems in metazoan nonmodel organisms and generate complete mtDNA datasets.

Comparison of morphological, DNA barcoding, and metabarcoding characterizations of freshwater nematode communities.

Biomonitoring approaches and investigations of many ecological questions require assessments of the biodiversity of a given habitat. Small organisms, ranging from protozoans to metazoans, are of great ecological importance and comprise a major share of the planet's biodiversity but they are extremely difficult to identify, due to their minute body sizes and indistinct structures. Thus, most biodiversity studies that include small organisms draw on several methods for species delimitation, ranging from traditional microscopy to molecular techniques. In this study, we compared the efficiency of these methods by analyzing a community of nematodes. Specifically, we evaluated the performances of traditional morphological identification, single-specimen barcoding (Sanger sequencing), and metabarcoding in the identification of 1500 nematodes from sediment samples. The molecular approaches were based on the analysis of the 28S ribosomal large and 18S small subunits (LSU and SSU). The morphological analysis resulted in the determination of 22 nematode species. Barcoding identified a comparable number of operational taxonomic units (OTUs) based on 28S rDNA (n = 20) and fewer OTUs based on 18S rDNA (n = 12). Metabarcoding identified a higher OTU number but fewer amplicon sequence variants (AVSs) (n = 48 OTUs, n = 17 ASVs for 28S rDNA, and n = 31 OTUs, n = 6 ASVs for 18S rDNA). Between the three approaches (morphology, barcoding, and metabarcoding), only three species (13.6%) were shared. This lack of taxonomic resolution hinders reliable community identifications to the species level. Further database curation will ensure the effective use of molecular species identification.

When barcoding fails: development of diagnostic nuclear markers for the sibling caddisfly species Sericostoma personatum (Spence in Kirby & Spence, 1826) and Sericostoma flavicorne Schneider, 1845.

The larval stages of the central European sibling caddisfly species Sericostoma personatum (Spence in Kirby and Spence, 1826) and S. flavicorne Schneider, 1845 are morphologically similar and can only be distinguished by differences in coloration in late larval instars. Identification using the mitochondrial barcoding gene, i.e., the Cytochrome c Oxidase 1, is impossible, as both species share the same highly differentiated haplotypes due to introgression. Nuclear gene markers obtained through double digest restriction site associate sequencing (ddRAD seq), however, can reliably distinguish both species, yet the method is expensive as well as time-consuming and therefore not practicable for species determination. To facilitate accurate species identification without sequencing genome-wide markers, we developed nine diagnostic nuclear RFLP markers based on ddRAD seq data. The markers were successfully tested on geographically distinct populations of the two Sericostoma species in western Germany, on known hybrids, and on another sericostomatid caddisfly species, Oecismus monedula (Hagen, 1859) that sometimes shares the habitat and can be morphologically confounded with Sericostoma. We describe a simple and fast protocol for reliable species identification of S. personatum and S. flavicorne independent of the life cycle stage of the specimens.

Distribution of Cereal Cyst Nematodes (Heterodera avenae and H. filipjevi) in Eastern Washington State.

Cereal cyst nematodes (CCN; Heterodera avenae and H. filipjevi), cause substantial worldwide yield loss in small grain cereals such as wheat, barley, and oat. H. avenae was first detected in the United States in western Oregon in 1974 and had spread to northeast Oregon by the mid-1980s. Although H. avenae was detected in eastern Washington in 1984, extensive infestations were not recognized until 2010. H. filipjevi, first detected in Oregon in 2008, was found in eastern Washington in 2014. To gain more information about the distribution of these two species, an extensive survey was undertaken in eastern Washington, and methods were developed to distinguish species using DNA sequencing of single cysts. In this study, we surveyed 356 wheat and barley fields in eastern Washington from 2007 to 2017. CCN from the infested locations were identified to species level by sequencing the ribosomal internal transcribed spacers (ITS) and/or 28S ribosomal RNA (rRNA) genes. The sequences were compared in the GenBank database in the National Center for Biotechnology Information (NCBI) to identify species. The results show that H. filipjevi is primarily confined to southern Whitman County, WA; and H. avenae has a wider distribution across the higher precipitation annual cropping area of eastern Whitman County. Knowledge of species identification is critical for deployment of host resistance as an effective means of management, since resistance genes for one species of CCN may not be effective against the other.

Utility of mitochondrial CO1 sequences for species discrimination of Spirotrichea ciliates (Protozoa, Ciliophora).

Ciliates are a diverse species group of the Protozoa, and nuclear and mitochondrial genes have been utilized to discover new species and discriminate closely related species. The mitochondrial cytochrome c oxidase subunit 1 (CO1) gene has been used to discriminate metazoan species and has also been applied for some groups in the phylum Ciliophora. However, it is difficult to produce a universal primer as a standard barcode, because unlike metazoans, mitochondrial DNA sequences of ciliates are long and highly variable. Therefore, to design the new primer set, we sequenced the mitochondrial genomes of two pseudokeronopsids in the class Spirotrichea using next-generation sequencing technology (HiSeq™ 2000). Based on putative CO1 gene fragments of the pseudokeronopsids, we designed the new primer set and successfully sequenced the CO1 of 69 populations representing 47 species (five orders, 14 families, and 27 genera). We found that CO1 showed higher resolution for separating congeneric species than did nuclear SSU rRNA gene sequences, and we identified some putative cryptic species.

Protax-fungi: a web-based tool for probabilistic taxonomic placement of fungal internal transcribed spacer sequences.

Incompleteness of reference sequence databases and unresolved taxonomic relationships complicates taxonomic placement of fungal sequences. We developed Protax-fungi, a general tool for taxonomic placement of fungal internal transcribed spacer (ITS) sequences, and implemented it into the PlutoF platform of the UNITE database for molecular identification of fungi. With empirical data on root- and wood-associated fungi, Protax-fungi reliably identified (with at least 90% identification probability) the majority of sequences to the order level but only around one-fifth of them to the species level, reflecting the current limited coverage of the databases. Protax-fungi outperformed the Sintax and Rdb classifiers in terms of increased accuracy and decreased calibration error when applied to data on mock communities representing species groups with poor sequence database coverage. We applied Protax-fungi to examine the internal consistencies of the Index Fungorum and UNITE databases. This revealed inconsistencies in the taxonomy database as well as mislabelling and sequence quality problems in the reference database. The according improvements were implemented in both databases. Protax-fungi provides a robust tool for performing statistically reliable identifications of fungi in spite of the incompleteness of extant reference sequence databases and unresolved taxonomic relationships.

Sparing spiders: faeces as a non-invasive source of DNA.

INTRODUCTION: Spiders are important arthropod predators in many terrestrial ecosystems, and molecular tools have boosted our ability to investigate this taxon, which can be difficult to study with conventional methods. Nonetheless, it has typically been necessary to kill spiders to obtain their DNA for molecular applications, especially when studying their diet. RESULTS: We successfully tested the novel approach of employing spider faeces as a non-invasive source of DNA for species identification and diet analysis. Although the overall concentration of DNA in the samples was very low, consumer DNA, suitable for species identification, was amplified from 84% of the faecal pellets collected from lycosid spiders. Moreover, the most important prey types detected in the gut content of the lycosids were also amplified from the faecal samples. CONCLUSION: The ability to amplify DNA from spider faeces with specific and general primers suggests that this sample type can be used for diagnostic PCR and sequence-based species and prey identification such as DNA barcoding and next generation sequencing, respectively. These findings demonstrate that faeces provide a non-invasive alternative to full-body DNA extracts for molecular studies on spiders when killing or injuring the animal is not an option.

Molecular identification of the economically important freshwater mussels (Mollusca-Bivalvia-Unionoida) of Thailand: developing species-specific markers from AFLPs.

Shells of certain freshwater mussel (Unionoida) species are highly demanded and serve as raw material for a range of decorative and pharmaceutical products. In Thailand, most animals for this purpose are currently harvested from wild populations, with unionoid culture still being in its infancy. Whilst reliable species identification is a prerequisite for developing a large-scale industry, identification by morphological means is hampered by extensive phenotypic plasticity and poor knowledge of species delimitations. To facilitate alternative molecular identification, we developed species-specific markers for the three Thai unionoids with considerable economic potential (CEP): that is, Chamberlainia hainesiana, Hyriopsis desowitzi and Hyriopsis myersiana. For this purpose, amplified fragment length polymorphism (AFLP) fingerprints using 24 specific primer pairs were generated for eight samples of each CEP species and four samples of the closely related, non-CEP species Contradens contradens. Cloning and sequencing of 13 CEP species-specific AFLP bands revealed fragment collision at three occasions. In total, 16 species-specific primer pairs were designed and tested on 92 Thai specimens spanning seven species and four genera. Thereby, specificity of (1) three primers to C. hainesiana, (2) one primer to H. desowitzi + Hyriopsis bialata, (3) one primer to H. myersiana + H. bialata and (4) four primers to all three Hyriopsis species tested was confirmed. Respective multiplex PCR protocols are provided. The developed primers enable cheap, quick and reliable identification of the Thai CEP species by one to three PCRs and offer a tool for a range of additional applications within mussel culture and ecological and evolutionary research on these important organisms.