Reliable Species Identification Research Articles

Identification of three Asian otter species (Aonyx cinereus, Lutra sumatrana, and Lutrogale perspicillata) using a novel noninvasive PCR-RFLP analysis.

Four species of otters occur in tropical Asia, and all face multiple threats to their survival. Studies of distribution and population trends of these otter species in Asia, where they occur sympatrically, are complicated by their elusive nature and difficulties with reliable identification of species in field surveys. In Malaysia, only three species, the smooth-coated otter, Asian small-clawed otter, and hairy-nosed otter have been reliably reported as residents. We designed a replicable and cost-efficient PCR-RFLP protocol to identify these three species. Using published reference sequences of mitochondrial regions, we designed and tested three PCR-RFLP protocols on DNA extracted from reference samples and 33 spraints of wild otters collected along the North Central Selangor Coast of Malaysia. We amplified and sequenced two fragments (450 and 200 bp) of the mt D-loop region and a 300-bp fragment of the mt ND4 gene using primer sets TanaD, TanaD-Mod, and OTR-ND4, respectively. Amplification products were digested with restriction enzymes to generate species-specific RFLP profiles. We analyzed the costs of all three protocols and compared these with the costs of sequencing for species identification. Amplification success was highest for the smallest PCR product, with the TanaD-Mod primer amplifying DNA from all 33 spraints. TanaD and OTR-ND4 primers amplified DNA from 60.6% and 63.6% spraints, respectively. PCR products of TanaD-Mod provided the expected species-specific RFLP profile for 32 (97%) of the spraints. PCR products of OTR-ND4 provided the expected RFLP profile for all 21 samples that amplified, but TanaD produced spurious bands and inconsistent RFLP profiles. The OTR-ND4 primer-enzyme protocol was the least expensive (437 USD) for processing 100 samples, followed by TanaD-Mod (455 USD). We suggest the use of both OTR-ND4 and TanaD-Mod protocols that show potential for highly efficient and reliable species identification from noninvasive genetic sampling of three Asian otter species. We expect our novel noninvasive PCR-RFLP analysis methods to facilitate population monitoring, ecological and behavioral studies on otters in tropical and subtropical Asia.

An application of morphological analysis and DNA barcoding to identify Ipnops from the Clarion-Clipperton Zone (CCZ) as I. meadi Nielsen, 1966 with notes on other species of the genus (Aulopiformes: Ipnopidae)

Although Ipnops specimens are relatively common in the Clarion-Clipperton Fracture Zone (CCZ), an area targeted for potential future deep-sea mining, a reliable species identification has not yet been possible due to the lack of a captured specimen. In April 2012, an Ipnops specimen was caught for the first time from the eastern CCZ during an exploration cruise of the BGR. Species identification of this specimen was performed using a comparative application of morphological analysis and DNA barcoding and resulted in its clear assignment to Ipnops meadi Nielsen, 1966. Of the 23 compared morphological characters, 22 are inside the ranges available for I. meadi. Molecular analyses show a sequence distance of 0.76% divergence to an Ipnops specimen collected off Hawaii, close to the CCZ and also within the known geographical distribution range of I. meadi. The additional study of five specimens of I. meadi from the Arabian Sea has extended the previously known range of the following morphological characters of this species: gill rakers on anterior arch (17–21), head length (17.6–24.0 % SL), upper jaw length (10.7–14.0 % SL), maximum width of eye-plates (7.8–9.8 % SL), preanal length (58.8–79.0 % SL), and predorsal length (34.5–40.5 % SL). Ipnops specimens deriving from Australian waters could not be clearly assigned with confidence to one of the valid Ipnops species based on current morphological and molecular analyses. It seems possible that at least one previously undescribed Ipnops species occurs in Australian waters and further work is required on the genus to resolve uncertainties.

Geographical Variation of Cuticular Hydrocarbon Profiles of Adult Flies and Empty Puparia Amongst Three Populations of Calliphora vicina (Diptera: Calliphoridae).

Blowflies (Diptera: Calliphoridae) are of great importance in forensic entomology and in determining the minimum post-mortem interval, as they may be the first group of insects to colonize decomposing remains. Reliable species identification is an essential prerequisite. Classically, morphological characters or DNA sequences are used for this purpose. However, depending on the species and the condition of the specimen, this can be difficult, e.g., in the case of empty fly puparia. Recent studies have shown that cuticular hydrocarbon (CHC) profiles are species-specific in necrophagous taxa and represent another promising tool for identification. However, the population-specific variability of these substances as a function of e.g., local climatic parameters has not yet been sufficiently investigated. The aim of this study was to determine the geographical variation of CHC profiles of the blowfly Calliphora vicina (Robineau-Desvoidy, 1830) depending on different countries of origin. Flies were reared in the United Kingdom, Germany, and Turkey in common garden experiments under ambient conditions. CHC profiles of the resulting adult flies and their empty puparia were analyzed using gas chromatography-mass spectrometry. Data were visualized by principal component analysis and clustered by population. The populations of the United Kingdom and Germany, both having similar climates and being geographically close to each other, showed greater similarities in CHC profiles. However, the CHC profile of the Turkish population, whose climate is significantly different from the other two populations, was very different. Our study confirms the high potential of CHC analysis in forensic entomology but highlights the need to investigate geographical variability in chemical profiles.

Animal species identification utilising DNAs extracted from traditionally manufactured gelatin (Wanikawa)

Gelatin, sourced from collagen, is an acid-, alkali- or enzymatically hydrolysed product obtained from animal skins and bones. Gelatin has been widely used for the manufacture of various cultural objects, e.g. as a water-soluble binder for dissolving pigments, and as a glue for musical instruments and traditional crafts along with human history. The identification of animal species in gelatin, hence, could provide a critical clue for understanding human history including lifestyles, the culture and the technologies. However, there has been no valid method established to date for identifying the animal species from traditional gelatins. We herein report that the nucleic acids contents (dsDNA, ssDNA and miRNA) from commercially-available gelatins manufactured according to classical procedures (wanikawa) exhibited much higher (about 10 times) than those from modern gelatins made through an industrialised process (yonikawa), suggesting that DNA analysis using the gelatins from cultural assets could be substantially feasible. Moreover, targeting not only commercially available niwaka but also Ukiyo-e, Japanese classical art manufactured through woodblock printings, we here illustrate partial successes in the animal species identification coupled with DNA barcoding technique, hopefully paving the way for scientifically more reliable animal species identifications of archaeological specimens made with a gelatin component.

Molecular Phylogeny of Dermestidae (Coleoptera) Reveals the Polyphyletic Nature ofTrogodermaLatreille and the Taxonomic Placement of the Khapra BeetleTrogoderma granariumEverts

AbstractThe hide, larder, and carpet beetles (Coleoptera: Dermestidae) are a family of mainly scavenger beetles, with numerous species such as the khapra beetle (Trogoderma granarium Everts, 1898), the black carpet beetle [Attagenus unicolor (Brahm, 1791)] and the hide beetle (Dermestes maculatus De Geer, 1774) being widely recognized as serious economic pests of stored products and museum collections. A stable classification and reliable identification of genera and species of these pests and their 1,700 relatives are of great relevance for trade restrictions, biosecurity, pest management, forensics, and biodiversity surveys. In this study, we examined and sequenced mitochondrial genomes of 477 dermestid specimens, representing all subfamilies and 90% of the globally recognized tribes and subtribes. Our study provides the most comprehensive, taxonomically verified, and vouchered resource of mitochondrial reference sequences linked to specimen images and occurrence records of pests and their relatives, enabling eDNA surveys, metabarcoding and molecular species identification. It also reconstructs the phylogeny of Dermestidae based on molecular and morphological data for the first time, thereby providing robust phylogenetic hypotheses for a stable classification system from family to genus-level. Accordingly, a revised classification of Dermestidae with formal nomenclatural changes is proposed, recognizing six subfamilies: Orphilinae, Trinodinae, Trogoparvinae subfam. nov. (type genus TrogoparvusHáva, 2001), Dermestinae, Attageninae, and Megatominae. Trinodinae is recovered towards the base of Dermestidae with three tribes: Trinodini (=Trinoparvini syn. nov.), Thylodriini, and Trichelodini. Dermestinae is the only subfamily with adults lacking a median ocellus, and it includes the tribes Thorictini stat. nov. (that is downgraded from Thorictinae), Marioutini, and Dermestini. The endemic Australian genus Derbyana Lawrence and Ślipiński was recovered within Holarctic Dermestes Linnaeus. Attageninae is strongly supported and includes the monogeneric Adelaidiini and polygenic Attagenini. Former subgenera of Attagenus Latreille, i.e., Lanorus Mulsant and Rey (= Paranovelsis Casey syn. nov.), Telopes Redtenbacher and Aethriostoma Motschulsky, are elevated to generic level. The largest clade, Megatominae, is confirmed as monophyletic and is divided into three tribes: Anthrenini, Ctesiini, and Megatomini. Megatomini is divided into three subtribes: Megatomina, Orphinina subtribe nov. (type genus Orphinus Motschulsky), and Trogodermina. Within the economically important lineage Trogodermina, Trogoderma Latreille is delimited to contain only Holarctic species including the Khapra beetle T. granarium Everts, while a Southern Hemisphere clade is here recognized as Eurhopalus Solier in Gay, 1849 (= AnthrenocerusArrow, 1915; Myrmeanthrenus Armstrong, 1945; Neoanthrenus Armstrong, 1941; SodaliatomaHáva, 2013; Reesa Beal, 1967 syn. nov.). A revised classification of the extant genera of Dermestidae is also provided.

Hybrid between Lemna minor and L. turionifera (L. × japonica, Lemnaceae) in East Europe is more frequent than parental species and poorly distinguishable from them

Biodiversity studies and practical use of duckweeds are impossible without reliable species identification. Despite of clear genetic differentiation of L. minor, L. turionifera and their hybrid L. × japonica, their morphological discrimination is challenging even for professional botanists; consequently, the distribution of the two latter taxa is not precisely known. Although numerous determination keys exist, none of them is based on quantitative analysis of the morphological variation observed in nature. Thus, we aimed to study variation of morphological characteristics most frequently proposed for discrimination between L. minor, L. turionifera and L. × japonica (frond shape and pigmentation, papule presence along the median nerve) on specimens collected in the wild, which were taxonomically identified using molecular tools (intron length polymorphism in the β-tubulin gene). We focused on samples from the poorly studied East Europe, including some from the Russian Far East for comparison. Lemna × japonica appeared to be quite abundant in different regions of East Europe. The Far Eastern occurrences of L. minor and L. × japonica should be carefully verified. Even our limited sampling demonstrated that morphological characters conventionally used for delimitation of L. minor, L. turionifera and L. × japonica do not work in wild populations. Additional studies should be performed for verification of diagnostic value of generative characters. Large-scale genetic screening of Lemna populations in nature is also indispensable to clarify distribution of taxa from the L. minor complex.

Internal Transcribed Spacer (ITS) Region as Molecular Marker for Parasite Identification in Aquaculture: A Review

Molecular technologies may be quicker and more accurate than the culture, serology, and histology procedures that have historically been employed to identify fish parasites. By using molecular diagnostic methods, parasites can be found in asymptomatic fish, allowing disease outbreaks to be stopped. This review discusses the use of Internal Transcribed Spacer (ITS) markers to detect parasite in aquaculture and their potential applications are discussed. It was found that it is a more reliable marker and species identification in combination with other DNA markers. ITS region are highly polymorphic non - coding region with sufficient taxonomic units. Thus, sequences can be separated at the species level. though ribosomal ITS as a universal barcode marker for parasite identification is still hampered by few limitations, the ITS will remain as the key choice for parasite identification. It remains the most important option for parasite identification. Many studies have been carried out using ITS markers for parasites identification such as cestode, nematodes, Pseudoterranova spp., Anisakis type I, Diplostomum, Gyrodactylids Hysterothylacium sp, Glochidia larvae. This identification of parasites are taken from the species Rainbow trout (Oncorhynchus mykiss), Argentinesea bass, (Acanthistius patachonicus), and other samples from various places.

DNA Barcoding of Stingless Bees (Hymenoptera: Meliponini) in Northern Peruvian Forests: A Plea for Integrative Taxonomy

Stingless bees (Hymenoptera: Meliponini) are among the most important pollinators of tropical forests. Peru is considered a hotspot of biodiversity of Meliponini, but many areas of this country (e.g., Peruvian Amazon) remain unexplored. We aimed to produce a first inventory of stingless bee species dwelling in humid and seasonally dry forests of northern Peru by combining traditional (morphologically-based) taxonomy and DNA barcoding. Specimens were collected in 2020 at five sites located in San Martin and Piura regions. We identified 12 genera of Meliponini. Among those, Trigona and Plebeia were the most abundant (45.9% and 12.8% respectively), whereas Nannotrigona and Scaura were the least represented ones (2.3%). We assigned a reliable species identification to about 30% of specimens (Trigona amazonensis, T. muzoensis, T. williana, Partamona testacea, Scaura tenuis, Tetragona goettei, and Tetragonisca angustula). Yet, more than a half of the specimens received a provisional identification (e.g., Geotrigona cf. fulvohirta, T. cf. amalthea, T. cf. fuscipennis, T. cf. hypogea, Melipona cf. cramptoni, Partamona cf. epiphytophila, Ptilotrigona cf. perenae, Scaura cf. latitarsis, Tetragona cf. clavipes, Trigonisca cf. atomaria). We also highlighted an extensive polyphyly that affected a number of currently recognized species (e.g., T. fulviventris, T. guianae, Plebeia franki, P. frontalis, M. eburnea, M. illota), whose members were split into various clades. Finally, 16% of individuals failed to be identified at the species level (Trigona sp. 1, T. sp. 2, Nannotrigona sp., Partamona sp., Scaptotrigona sp. 1, S. sp. 2, Trigonisca sp. 1, and Trigonisca sp. 2). We discuss our findings according to the current faunistic and biogeographic knowledge of Meliponini in Peru and the Neotropical region. We also remark on the importance of conducting a taxonomic revision of stingless bees and improving both their morphology-based identification keys and BOLD repository. Finally, we claim that integrative taxonomy shall be strongly implemented to truly assess the biodiversity of Neotropical stingless bees, allowing conserving these important pollinators and the associated traditional meliponiculture in an effective manner.

Superiority of two-dimensional correlation spectroscopy combined with ResNet in species identification of bolete

Food safety is an important topic of social concern. There are many species of bolete on the market, and shoddy products often occur. This phenomenon not only disrupts the market order, infringes on the rights and interests of consumers, and even endangers the lives of consumers. Therefore, it is of great significance to find a comprehensive, efficient and modern technology to identify and evaluate it in order to ensure its quality and safety. Spectroscopy analysis is fast, nondestructive and green. In our research, a fast and reliable bolete species identification method was established by combining Fourier transform near infrared spectroscopy (FT-NIR) with random forest (RF) method. In order to adapt to the development of the times, a practical method beyond traditional spectral analysis is established. Therefore, we establish a residual convolution neural network model (ResNet). The results show that the classification accuracy of the model is low and the out of bag error (OOB) error is high. After data preprocessing, the accuracy of RF model can be significantly improved. ResNet model has absolute advantages in bolete species identification. It is hardly affected by factors such as data type and sample size. Its overall recognition ability is obviously better than RF model. By comparing the identification accuracy and market application prospect of the two models, this research believes that ResNet model is more suitable for the identification of bolete species. In addition, the method can also be extended to the further study of other food, medicinal plants and agricultural products.

Molecular Methods to Study Great Cormorant Feeding Ecology

The feeding ecology of the Great Cormorant Phalacrocorax carbo has been the subject of many studies in which the hard parts of fish, contained in dietary samples such as regurgitated pellets, were identified using morphological characteristics. However, morphological prey identification does not necessarily permit the reliable identification of all fish species due to digestion eroding diagnostic characters and/or morphologically indiscernible hard parts in a range of fish species. Molecular methods have the potential to overcome these obstacles by allowing the detection and identification of minute quantities of prey DNA present in pellets, faeces, and stomach samples. Moreover, DNA of the consumer (i.e. the Cormorant) is also present in dietary samples and can thus be employed for ecological studies too. Here, we present a methodological overview of two molecular approaches commonly used to study trophic interactions, namely diagnostic PCR and next generation sequencing, along with their main advantages and disadvantages. Regarding the use of consumer DNA contained in dietary samples, molecular sexing, i.e. the non-invasive sex determination of the sample-producing bird, is presented. We exemplify the potential of DNA-based methods for future research via a case study on pellets collected at Chiemsee (Germany), which were subjected to molecular and morphological prey identification as well as to molecular sexing. Compared to morphological prey identification, molecular analysis led to a 53% increase in prey species and genera, mainly caused by eight additionally detected cyprinid taxa. For 79% of the pellets, the sex of the pellet-producing Cormorant could be successfully determined via molecular sexing. Our findings highlight the exciting possibilities molecular methods offer for future studies on Cormorant feeding ecology, especially regarding evaluations of prey spectra and the non-invasive assessment of sex-specific differences.

Rapid Detection of Carbapenem-Resistant Klebsiella pneumoniae Using Machine Learning and MALDI-TOF MS Platform.

BackgroundRapid detection of carbapenem-resistant Klebsiella pneumoniae (CRKP) is essential for specific antimicrobial therapy. Machine learning techniques combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be used as a rapid, reliable, sensitive, and low-cost species identification method.MethodsClinically collected K. pneumoniae were subjected to MALDI-TOF MS analysis. A random forest (RF) algorithm and non-linear support vector machine (SVM) were used to construct the RF, SVM, and dimension reduction (SVM-K) models, and their performance was assessed for accuracy, sensitivity, specificity, and area under the subject worker curve (AUC).ResultsThe RF, SVM and SVM-K models showed good classification performance with 0.88, 0.88, and 0.91 accuracy, 0.82, 0.85, and 0.89 sensitivity, 0.93, 0.92, and 0.94 specificity with an AUC of 0.9013, 0.9298, and 0.9356, respectively. For the SVM-K model, the optimal dimension reduction was 105 to 153, and the average accuracy was >0.9. The top 10 peak features of significance according to the RF algorithm with 6515 Da appeared in 56.8% of CRKP isolates and 5.3% of CSKP isolates, which indicated the best classification performance.ConclusionThe three RF, SVM, and SVM-K models showed excellent classification performance differentiating the CRKP from CSKP; the SVM-K model was the best. Data analysis with machine learning combined with MALDI-TOF MS can be employed as a rapid and inexpensive alternative to existing detection methods.

Can camera traps be used to differentiate species of North American flying squirrels?

AbstractCamera traps are becoming an increasingly important tool to survey wildlife populations. However, the application of camera trapping for reliable species identification between nondistinctive, morphologically similar sympatric species is untested for most small mammals, including North American flying squirrels (Glaucomys spp.). Camera traps are a successful monitoring technique where flying squirrel species are allopatric, however there are zones of sympatry between Humboldt's flying squirrel (HFS, G. oregonensis) and northern flying squirrel (NFS, G. sabrinus) in the Pacific Northwest and NFS and southern flying squirrels (SFS, G. volans) in eastern North America. We used camera trap data collected during flying squirrel surveys in 2013–2020 at 59 sites in California, North Carolina, and Virginia, USA, to determine if a reliable method could be used to differentiate the species. With a subset of 100 high‐quality, independent capture events per species (50 of dorsal views, 50 of lateral views), we used body measurements and pelage characteristics to differentiate species using random forest classification models. Our models predicted species identification accuracy rates of 90.9% for dorsal views and 68.2% for lateral views. Species misclassification rates between HFS and NFS were 23.5% for dorsal views and 26.5% for lateral views, whereas misclassification rates between NFS and SFS were 16.6% for dorsal views and 5.7% for lateral views. Although misclassification rates were lower than we expected between NFS and SFS, we are cautious about recommending camera trapping as opposed to ultrasonic acoustics for North American flying squirrel species identification, especially in areas of species sympatry due to potential false positive rates and high numbers of unusable images. However, camera trap settings and white‐flash photography should be explored to determine their potential for improving flying squirrel species classification rates and the percentage of usable photos.

The use of taxonomic families as biological surrogates of the diversity of the Amazonian stream fish

The reliable identification of the fish species found in Amazonian streams requires a considerable investment of both time and resources, which often hampers the biodiversity pattern mapping and conservation planning. This problem can be overcome using biological surrogates such taxonomic families, this would reduce the number of fish to be identified and thus facilitate the development of studies. The present study analyzes the potential of families as surrogates of the fish diversity of Amazonian streams. For this, we verified the degree of congruence between the distribution of species organized by family and the composition of the assemblage in two different spatial scenarios. At the biome scale, we verified the degree of congruence in the fish faunas of 54 streams located within six drainage basins in Brazilian Amazonia. At the regional scale, we compared the data from the basins located on the lowland floodplain and those at higher altitudes. The Cichlidae and Lebiasinidae were identified as potential biological surrogates at both spatial scenarios, demonstrating more than 80% similarity with species matrix in all the analytical approaches. The families Crenuchidae and Hypopomidae were the most congruent with the fish from the floodplain basins, both were more than 80% congruent with the species matrix. In upland basins, Characidae, Gymnotidae and Rivulidae were highly similar to the species matrix, in particular Gymnotidae and Rivulidae with more than 90% similarity. Overall, we can conclude that the biological surrogacy approach is a potentially valuable alternative for species diversity evaluation of Amazonian streams ecosystems, considering the accelerated loss of the diversity of these systems.

Elucidation of the Lipid Composition of Hemp (Cannabis sativa L.) Products by Means of Gas Chromatography and Ultra-High Performance Liquid Chromatography Coupled to Mass Spectrometry Detection.

The growing demand in natural matrices that represent a source of dietary and nutraceutical molecules has led to an increasing interest in Cannabis sativa, considered to be a multipurpose, sustainable crop. Particularly, the considerable content in essential fatty acids (FAs) makes its derived-products useful food ingredients in the formulation of dietary supplements. In this research, the FA and triacylglycerol (TAG) composition of hempseed oils and flours were investigated using gas chromatography coupled to mass spectrometry and flame ionization detection as well as liquid chromatography coupled to mass spectrometry (LC-MS), respectively. Furthermore, a recently introduced linear retention index (LRI) approach in LC was successfully employed as a useful tool for the reliable identification of TAG species. A total of 30 FAs and 62 glycerolipids were positively identified in the investigated samples. Relative quantitative analyses confirmed linoleic acid as the most abundant component (50–55%). A favorable omega6/omega3 ratio was also measured in hemp-derived products, with the α-linolenic acid around 12–14%. Whereas, γ-linolenic acid was found to be higher than 1.70%. These results confirm the great value of Cannabis sativa as a source of valuable lipids, and the further improvement of the LRI system paves the way for the automatization of the identification process in LC.

RDNA-ITS2 characterization of Trichogramma species (Hymenoptera: Trichogrammatidae) in Turkey

Abstract Background ITS2 sequences can be used in systematic studies and proved to give reliable results in the distinguishing of Trichogramma species (Hymenoptera: Trichogrammatidae). Correct identification of natural enemies forms the basis of a biological control program. The present study aimed to compare sequences of rDNA-ITS2 of Trichogramma samples with those deposited in GenBank by using ITS2, as a barcode for reliable species identification and assessment of genetic diversity. Results Molecular identification methods were used to differentiate 2 Trichogramma species collected from Adana province of Turkey; Trichogramma brassicae (Bezdenko) and Trichogramma turkestanica Meyer (Hymenoptera: Trichogrammatidae). ITS2 sequences of samples ranged in size from 378 to 406 bp. The ITS2 sequences were aligned using Clustal W, genetic distances and phylogenetic tree were calculated using MEGA V7.0. rDNA-ITS2 sequences of 37 specimens of Trichogramma confirmed in GenBank in the study. Also, secondary structures of ITS2 sequences were predicted with the help of Mfold web server. All secondary structure constructions were performed at 37 °C using RNA version 2.3 default parameters. Conclusions A molecular marker can be used successfully to distinguish closely related groups if it is a rapidly evolving and highly conserved gene region. In the study, it was shown that ITS2 was a reliable molecular marker in distinguishing species. Therefore, with rDNA-ITS2 sequence analysis, Trichogramma spp., which is a very important natural enemy in biological control, has been identified.

Species identification of introduced veronicellid slugs in Japan.

Reliable identification of species is important for protecting native ecosystems against the invasion of non-native species. DNA barcoding using molecular markers, such as the mitochondrial cytochrome oxidase subunit 1 (COI) gene, helps researchers distinguish species. In this study, we focused on introduced veronicellid slugs in the Ryukyu Islands and some greenhouses on mainland Japan. Some veronicellids are medium-to-high risk pest species for humans. Identifying veronicellid species by their external morphology is difficult and unreliable because there is substantial overlap between intraspecific variation and interspecific differentiation. Therefore, internal morphologies such as male genitalia have been the primary traits used to distinguish veronicellids. To identify introduced veronicellid slugs in Japan to the species level, we used morphological assessment of male genitalia and DNA barcoding of the standard COI gene fragment. We also conducted species-delimitation analyses based on the genetic data. The results showed that five evolutionarily significant units, corresponding to four nominal species inhabit the Ryukyu Islands, of which two species were also found in the greenhouses of mainland Japan, including the first record of Sarasinula plebeia in Japan. The presence of non-native slug species could increase the transmission of parasites in Japan.

Bat selfies: photographic surveys of flying bats

The recent pandemic and other environmental concerns have resulted in restrictions on research and surveys involving capture and handling bats. While acoustic surveys have been widely used as an alternative survey method, in this study, we show how photographic surveys can offer an important contribution to study and survey bats. We outline approaches, using high speed flash and automated trip beams to obtain photos of flying bats of sufficient quality for reliable identification of species. We show, through a series of examples of setups and photographs, that photography is effective for surveying bats at a variety of sites, where bats roost, drink, and forage. We note, however, that photographic surveys cannot replace capture in all situations. In addition, although photographing bats is less invasive than capturing them, it can involve disturbance, so we stress the importance of minimizing the impact of such operations on bats.

Machine learning for expert‐level image‐based identification of very similar species in the hyperdiverse plant bug family Miridae (Hemiptera: Heteroptera)

AbstractDeep learning algorithms and particularly convolutional neural networks are very successful in pattern recognition from images and are increasingly employed in biology. The development of automated systems for rapid and reliable species identification is vital for insect systematics and may revolutionize this field soon. In this study, we demonstrate the ability of a convolutional neural network to identify species based on habitus photographs with expert‐level accuracy in a taxonomically challenging group where a human‐based identification would require notorious genitalia dissections. Using the economically important and polymorphic plant bug genus Adelphocoris Reuter (Heteroptera: Miridae) as a model group, we explore the variability in the performance of 11 convolutional neural models most commonly used for image classification, test the role of class‐imbalance on the model performance assessment and visualize areas of interest using three interpretation algorithms. Classification performance in our experiments with collection‐based habitus photographs is high enough to identify very similar species from a large group with an expert‐level accuracy. The accuracy is getting lower only in the experiments with an additional dataset of Adelphocoris and other live plant bugs photographs taken from the Web. Our article demonstrates the importance of comprehensive institutional insect collections for bringing deep learning algorithms into service for systematic entomology using affordable equipment and methods.

Benefits and limitations of a new genome-based PCR-RFLP genotyping assay (GB-RFLP): A SNP-based detection method for identification of species in extremely young adaptive radiations.

High‐throughput DNA sequencing technologies make it possible now to sequence entire genomes relatively easily. Complete genomic information obtained by whole‐genome resequencing (WGS) can aid in identifying and delineating species even if they are extremely young, cryptic, or morphologically difficult to discern and closely related. Yet, for taxonomic or conservation biology purposes, WGS can remain cost‐prohibitive, too time‐consuming, and often constitute a “data overkill.” Rapid and reliable identification of species (and populations) that is also cost‐effective is made possible by species‐specific markers that can be discovered by WGS. Based on WGS data, we designed a PCR restriction fragment length polymorphism (PCR‐RFLP) assay for 19 Neotropical Midas cichlid populations (Amphilophus cf. citrinellus), that includes all 13 described species of this species complex. Our work illustrates that identification of species and populations (i.e., fish from different lakes) can be greatly improved by designing genetic markers using available “high resolution” genomic information. Yet, our work also shows that even in the best‐case scenario, when whole‐genome resequencing information is available, unequivocal assignments remain challenging when species or populations diverged very recently, or gene flow persists. In summary, we provide a comprehensive workflow on how to design RFPL markers based on genome resequencing data, how to test and evaluate their reliability, and discuss the benefits and pitfalls of our approach.

An Incomplete European Barcode Library Has a Strong Impact on the Identification Success of Lepidoptera from Greece

Species identification by means of DNA barcodes depends essentially on the scope and quality of a relevant reference library. The first analysis of a large number (about 600 morphospecies) of southern European Lepidoptera (Greece: Peloponnese) shows both the advantages and disadvantages with regard to a reliable identification of Mediterranean species. We determined 946 DNA barcode sequences from 47 families, of which 929 sequences from 46 families were successfully assigned to a Barcode Index Number (BIN) in the global Barcode of Life Data Systems (BOLD) database. A species level identification for 485 BINs representing 477 Linnaean names was successful. These taxa include 34 new records for Greece. However, 128 BINs (c. 20% of the inventory) could not be attached to a Linnaean name from referenced sequences available in BOLD. Of these BINs, 99 are new and hence represent unique records for BOLD. Intra- and inter-BIN divergences are presented and discussed. An initial and preliminary in-depth analysis of randomly selected species indicates an incomplete DNA barcode library in terms of Linnean taxa, in addition to a considerable number of probably undescribed species. It is therefore strongly recommended that the already advanced European barcode library of Lepidoptera should be supplemented with not-yet-sequenced taxa from the Mediterranean.