Decoding Lepidopteran Biodiversity through DNA Barcodes

Amphibians constitute a diverse yet still incompletely characterized clade of vertebrates, in which new species are still being discovered and described at a high rate. Amphibians are also increasingly endangered, due in part to disease-driven threats of extinctions. As an emergency response, conservationists have begun ex situ assurance colonies for priority species. The abundance of cryptic amphibian diversity, however, may cause problems for ex situ conservation. In this study we used a DNA barcoding approach to survey mitochondrial DNA (mtDNA) variation in captive populations of 10 species of Neotropical amphibians maintained in an ex situ assurance programme at El Valle Amphibian Conservation Center (EVACC) in the Republic of Panama. We combined these mtDNA sequences with genetic data from presumably conspecific wild populations sampled from across Panama, and applied genetic distance-based and character-based analyses to identify cryptic lineages. We found that three of ten species harboured substantial cryptic genetic diversity within EVACC, and an additional three species harboured cryptic diversity among wild populations, but not in captivity. Ex situ conservation efforts focused on amphibians are therefore vulnerable to an incomplete taxonomy leading to misidentification among cryptic species. DNA barcoding may therefore provide a simple, standardized protocol to identify cryptic diversity readily applicable to any amphibian community.

Geographical scale might be expected to impact significantly the efficiency of DNA barcoding as spatially comprehensive sampling provides opportunities to uncover intricate relationships among closely related species and to detect cryptic diversity for widespread taxa. Here, we present a DNA barcoding study on a Xencyprididae subfamily (Culterinae) involving the production of 998 newly generated DNA barcodes from East Asian drainages (80 localities). Together with 513 barcodes mined from BOLD and GenBank, a reference library consisting of 1511 DNA barcodes (116 localities) for 42 species was assembled, accounting for 66% of known Culterinae species. Intraspecific genetic distances are positively correlated to geographical scale, while a negative correlation is detected between interspecific genetic distances and geographical scale. The present study demonstrates that geographical scale influences the efficiency of DNA barcoding by narrowing the width of the barcoding gap. DNA‐based species delimitation analyses delimited 44 molecular operational taxonomic units (MOTUs). Rampant cryptic diversity is detected within eight species with multiple MOTUs, whereas 25 species present mismatch between morphological and molecular delimitations. A total of 18 species are lumped into nine MOTUs due to low interspecific divergence and/or mixed lineages. Several MOTU divergences are hypothesized to relate to known biogeographical barriers and geological events during the Pliocene and Pleistocene. This study provides new insights into the taxonomy and phylogeography of the subfamily Culterinae.

DNA barcoding has gained increased recognition as a molecular tool for species identification in various groups of organisms. In this preliminary study, we tested the efficacy of a 615-bp fragment of the cytochrome c oxidase I (COI) as a DNA barcode in the medically important family Simuliidae, or black flies. A total of 65 (25%) morphologically distinct species and sibling species in species complexes of the 255 recognized Nearctic black fly species were used to create a preliminary barcode profile for the family. Genetic divergence among congeners averaged 14.93% (range 2.83-15.33%), whereas intraspecific genetic divergence between morphologically distinct species averaged 0.72% (range 0-3.84%). DNA barcodes correctly identified nearly 100% of the morphologically distinct species (87% of the total sampled taxa), whereas in species complexes (13% of the sampled taxa) maximum values of divergence were comparatively higher (max. 4.58-6.5%), indicating cryptic diversity. The existence of sibling species in Prosimulium travisi and P. neomacropyga was also demonstrated, thus confirming previous cytological evidence about the existence of such cryptic diversity in these two taxa. We conclude that DNA barcoding is an effective method for species identification and discovery of cryptic diversity in black flies.

Aphids (family Aphididae) are among the most species-rich groups of Sternorrhyncha in the order Hemiptera, and have a complex life cycle that can include several different phenotypes that are perfectly adapted to specific ecological niches. However, because aphids have a small body size, indistinct appearance, and cryptic adult behavior, their species level identification is often difficult and may be time-consuming. To overcome these limitations, DNA barcoding has been employed as an effective tool for species identification. In this study, we conducted a DNA barcoding test based on 566 specimens of Korean Aphididae, representing 125 morphospecies. Based on intraspecific genetic divergence, a threshold of 2% was estimated to efficiently differentiate the morphospecies. Only 87 morphospecies (69.6%) identified across four species delimitation methods (namely, automatic barcode gap discovery, assemble species by automatic partitioning, Poisson-tree-processes or PTP, and Bayesian implementation of the PTP) were consistent with the morphological identifications of the species. This indicates the presence of many cases of cryptic diversity among the other morphospecies, except the abovementioned 87 species. Careful morphological examination of morphospecies exceeding 2.0% intraspecific variability revealed cryptic diversity in three species (Eriosoma yangi, Tuberculatus kuricola, and Greenidea kuwanai). Two morphospecies, Sitobion avenae and Aphis craccivora, also exhibited high intraspecific divergence and comprised a single molecular operational taxonomic unit. Overall, our findings indicate that DNA barcoding can be a powerful tool for identifying species belonging to the family Aphididae, while also revealing cases of cryptic diversity.

Marine biodiversity is a valuable gift of nature, as the marine environment is exceptionally complicated, diverse and of utmost economic value. There is a desperate need to evaluate and protect this treasure because of its multi facet uses and its richness in species composition. Biodiversity protection particularly emphasizes on the ability of quantifying and tracking changes in the marine ecosystem. Phytoplankton and Algal biodiversity too comes under this category because of their diverse species population, vast habitat and most of them having microscopic structure. Thereby both marine phytoplankton and algae play a significant role in marine biodiversity and their taxonomic identification remain a big challenge. With all the merits and limitations of DNA barcoding and amongst the furious debate of researchers in context of its comparison with alpha taxonomic identification, DNA barcoding certainly is a promising tool of future in the field of marine species identification, biodiversity assessment and conservation.

DNA barcoding and metabarcoding are now widely used to advance species discovery and biodiversity assessments. High-throughput sequencing (HTS) has expanded the volume and scope of these analyses, but elevated error rates introduce noise into sequence records that can inflate estimates of biodiversity. Denoising -the separation of biological signal from instrument (technical) noise-of barcode and metabarcode data currently employs abundance-based methods which do not capitalize on the highly conserved structure of the cytochrome c oxidase subunit I (COI) region employed as the animal barcode. This manuscript introduces debar, an R package that utilizes a profile hidden Markov model to denoise indel errors in COI sequences introduced by instrument error. In silico studies demonstrated that debar recognized 95% of artificially introduced indels in COI sequences. When applied to real-world data, debar reduced indel errors in circular consensus sequences obtained with the Sequel platform by 75%, and those generated on the Ion Torrent S5 by 94%. The false correction rate was less than 0.1%, indicating that debar is receptive to the majority of true COI variation in the animal kingdom. In conclusion, the debar package improves DNA barcode and metabarcode workflows by aiding the generation of more accurate sequences aiding the characterization of species diversity.

Ticks are of medical and veterinary concern due to their role as vectors of a wide range of pathogens, including viruses, protozoa, and bacteria. Accurate species identification is essential during tick surveillance and disease control programmes, as certain pathogens are associated with particular tick species. In this study, genetic variation of a partial sequence of the cytochrome c oxidase I ( COI ) gene was used for molecular identification of tick species to corroborate morphological identification. This also enabled investigation of cryptic diversity within tick species. Thirty species belonging to the genera Amblyomma, Argas, Carios, Dermacentor, Haemaphysalis, Hyalomma, Ixodes, Ornithodoros , and Rhipicephalu s were assessed. Tree-topology analysis confirmed discrete clustering of specimens according to species for the majority of taxa. Intraspecific genetic divergence ranged from 0 to 6.08%. In taxa where species complexes are known, separation of discrete groups were found. Several species yielded >2% intraspecific genetic divergence when compared to other taxa, suggesting potential cryptic diversity. DNA barcoding was an effective approach for the morphological identification of UK and non-native ticks, and for the detection of cryptic diversity within species.

This study examines the utility of morphology and DNA barcoding in species identification of freshwater fishes from north‐central Nigeria. We compared molecular data (mitochondrial cytochrome c oxidase subunit I (COI) sequences) of 136 de novo samples from 53 morphologically identified species alongside others in GenBank and BOLD databases. Using DNA sequence similarity‐based (≥97% cutoff) identification technique, 50 (94.30%) and 24 (45.30%) species were identified to species level using GenBank and BOLD databases, respectively. Furthermore, we identified cases of taxonomic problems in 26 (49.00%) morphologically identified species. There were also four (7.10%) cases of mismatch in DNA barcoding in which our query sequence in GenBank and BOLD showed a sequence match with different species names. Using DNA barcode reference data, we also identified four unknown fish samples collected from fishermen to species level. Our Neighbor‐joining (NJ) tree analysis recovers several intraspecific species clusters with strong bootstrap support (≥95%). Analysis uncovers two well‐supported lineages within Schilbe intermedius. The Bayesian phylogenetic analyses of Nigerian S. intermedius with others from GenBank recover four lineages. Evidence of genetic structuring is consistent with geographic regions of sub‐Saharan Africa. Thus, cryptic lineage diversity may illustrate species’ adaptive responses to local environmental conditions. Finally, our study underscores the importance of incorporating morphology and DNA barcoding in species identification. Although developing a complete DNA barcode reference library for Nigerian ichthyofauna will facilitate species identification and diversity studies, taxonomic revisions of DNA sequences submitted in databases alongside voucher specimens are necessary for a reliable taxonomic and diversity inventory.

There is an ongoing campaign to DNA barcode the world's >20 000 bee species. Recent revisions of Lasioglossum (Dialictus) (Hymenoptera: Halictidae) for Canada and the eastern United States were completed using integrative taxonomy. DNA barcode data from 110 species of L. (Dialictus) are examined for their value in identification and discovering additional taxonomic diversity. Specimen identification success was estimated using the best close match method. Error rates were 20% relative to current taxonomic understanding. Barcode Index Numbers (BINs) assigned using Refined Single Linkage Analysis (RESL) and barcode gaps using the Automatic Barcode Gap Discovery (ABGD) method were also assessed. RESL was incongruent for 44.5% of species, although some cryptic diversity may exist. Forty-three of 110 species were part of merged BINs with multiple species. The barcode gap is non-existent for the data set as a whole and ABGD showed levels of discordance similar to the RESL. The viridatum species-group is particularly problematic, so that DNA barcodes alone would be misleading for species delimitation and specimen identification. Character-based methods using fixed nucleotide substitutions could improve specimen identification success in some cases. The use of DNA barcoding for species discovery for standard taxonomic practice in the absence of a well-defined barcode gap is discussed.

Delineating species boundaries using an iterative taxonomic approach: The case of soldierless termites (Isoptera, Termitidae, Apicotermitinae)

Simple SummarySerpusia is a wingless grasshopper genus that is restricted to African Rainforests. Serpusia species exhibit largely external resemblance to species of several related genera, including Aresceutica, Pseudophialosphera, Segellia, Auloserpusia, Pteropera, Coenona, Serpusilla, and Veseyacris. The taxonomic status of Serpusia remains challenging, and its classification is unstable. Despite the alarming ongoing habitat loss in the African Rainforests, this genus has never been revised using modern molecular techniques. In this study, we used both morphological and molecular approaches to provide insights into the current diversity of this genus. We sequenced two mitochondrial markers (COI-5P and 16S rDNA) and constructed the phylogenetic relationships among species of Serpusia and closely related genera. It appeared that Serpusia is more diverse than what is currently known, encompassing five species, four of which we described in this study. In addition, one of the previously known species of Serpusia, the so-called S. succursor, is genetically very distinct from the true Serpusia and also encompasses hidden species. We then established a new genus, Paraserpusia, to accommodate S. succursor and its hidden species, which were formally described in this study. Overall, this study highlights the importance of combining molecular and morphological data to uncover and formally describe previously underestimated groups.Background/Objectives: Despite their ecological significance, DNA barcoding data for African rainforest Orthoptera remain underrepresented globally, limiting progress in species discovery, biodiversity assessment, and conservation. This study aimed to generate molecular data for morphologically identified Serpusia Karsch, 1891 species to evaluate their taxonomic status. Methods: Specimens were collected from multiple sites in Cameroon and analyzed using DNA barcoding with COI-5P and 16S rDNA markers. Species delimitation was performed with Automatic Barcode Gap Discovery, and phylogenetic relationships were inferred using Maximum Likelihood and Bayesian Inference. Additionally, external morphology and the male phallic complex were examined. Results: Molecular analyses delineated 19 MOTUs, five corresponding to Serpusia opacula, seven to Serpusia succursor and the remainder to outgroups. Similarity-based assignments matched these MOTUs to 19 BINs. Phylogenetic reconstruction revealed S. opacula and S. succursor as two genetically distinct clades, with the S. opacula group more closely related to Aresceutica Karsch, 1896 than to the S. succursor group. Accordingly, we established a new genus, Paraserpusia gen. nov., to accommodate S. succursor. Within the S. opacula group, five species are recognized: one previously described (S. opacula) and four new species (S. kennei sp. nov., S. missoupi sp. nov., S. seinoi sp. nov., and S. verhaaghi sp. nov.). The former S. succursor, now Paraserpusia succursor, is divided into six well-supported lineages, five of which are formally described here (P. hoeferi sp. nov., P. husemanni sp. nov., P. kekeunoui sp. nov., P. tamessei sp. nov., and P. tindoi sp. nov.). A haplotype network based on COI-5P sequences corroborates three major clades corresponding to the S. opacula group, the S. succursor group, and Aresceutica. Diagnostic morphological differences between Serpusia and Paraserpusia are consistently supported across characters. Conclusions: This integrative approach reveals substantial hidden diversity within Serpusia and highlights the importance of combining molecular and morphological data to uncover and formally describe previously overlooked taxa.

Wild bees are essential pollinators and therefore play a key role in both natural and agricultural ecosystems. However, bees have often been neglected in conservation studies and policies worldwide, which is surprising given their ecological importance. As a result, little is known on the conservation status of the vast majority of wild bee species in Europe, and even less worldwide. Limited surveys suggest important declines in the abundance and diversity of most wild bee communities worldwide. It is therefore urgent to implement targeted measures for the conservation of these keystone species. Once implemented, the effectiveness of these measures must be evaluated using adequate monitoring programs. To date, wild bee surveys are entirely based on morphological identification, which is both labor intensive and time consuming. Consequently, an affordable, high-throughput identification method is needed to reduce costs and improve bee monitoring. The objective of this thesis was to evaluate novel genetic techniques based on Next Generation Sequencing (NGS) methods for facilitating surveys of wild bees. NGS tools were mainly investigated for bridging two important impediments to wild bee conservation efforts, i.e., the cost of biodiversity assessment schemes and taxonomic incompleteness. With the development of NGS techniques, DNA barcoding has gained enormous momentum, enabling cost-effective, fast and accurate identifications. Before these methods can be routinely used in monitoring programs, there are however still important knowledge gaps to fill. These gaps mainly concern the detection of rare species and the acquisition of accurate quantitative data on species abundance; more generally the cost and labour effectiveness of these methods need to be evaluated. To provide a comprehensive presentation of the advantages and weaknesses of different NGS-based identification methods, we assessed three of the most promising ones, namely metabarcoding, mitogenomics and NGS barcoding. Using a regular monitoring data, we found that NGS barcoding performed best for both species’ presence/absence and abundance data, producing only few false positives and no false negatives. The other methods investigated were less reliable in term of species detection and inference of abundance data, and partly led to erroneous ecological conclusions. In terms of workload and cost, we showed that NGS techniques were more expensive than morphological identification with our dataset, although these techniques would become slightly more economical in large-scale monitoring programs. A second aim of this thesis was to provide an easy and robust genomic solution to alleviate taxonomical incompleteness, one of the major impediments to the effective conservation of many insect taxa. For conservation purposes, having stable and well-delimited species hypotheses is essential. Currently, most species are delimitated based on morphology and/or DNA barcoding. These methods are however associated with important limitations, and it is widely accepted that species delimitation should rely on multi-locus genomic markers. To overcome these limitations, ultraconserved elements (UCEs) were tested as a fast and robust approach using different species-complexes harbouring cryptic diversity, mitochondrial introgression, or mitochondrial paraphyly. Phylogenetic analyses of UCEs were highly conclusive and yielded meaningful species delimitation hypotheses in all cases. These results provide strong evidence for the potential of UCEs as a fast method for delimiting species even in cases of recently diverged lineages. Advantages and limitations of UCEs for shallow phylogenetic studies are further discussed.

The rainbowfishes of the family Melanotaeniidae represent one of the largest radiations of freshwater fishes from the Indo-Australian archipelago. A total of 75 nominal species have been described, among which several have become very popular among tropical fish hobbyists because of their tendency to form large schools of colourful individuals. Facing habitat loss and competition or predation by introduced species, this group has become a priority in the conservation of ornamental fishes in Indonesia. In this context, several expeditions have been conducted between 2007 and 2010 in Indonesian Papua with the aim to initiate a large-scale survey of the genetic resources in this group. We assessed the diversity of the Papua rainbowfishes with DNA barcoding. We sequenced the mitochondrial COI gene for 350 specimens belonging to 53 nominal species throughout the Indo-Australian archipelago. Unexpected levels of cryptic diversity and endemism were detected since additional cryptic lineages were detected in several watersheds from the Vogelkop and the Lengguru massif. DNA barcoding supports the presence of nearly 30 evolutionary lineages among the 15 nominal species sampled in the Vogelkop and all these lineages are endemic to a single lake or watershed. This result highlights that the diversity of the family has been largely underestimated and urges for the identification of conservation priorities in Papua.

This thesis examines the phylogenetic diversity patterns of beetle (Coleoptera) communities across tropical forests by developing and implementing molecular methodologies. A novel protocol for authenticating amplicon sequence variants was designed using 10,395 specimens representing 98 families from 11 countries, integrating abundance-based filtering with phylogenetic placement to achieve an authentication success rate of 82.34%. Multivariate analysis identified preservation methodology (χ² = 145.00, p < 0.001) and sequencing protocols (χ² = 287.45, p < 0.001) as primary determinants of authentication outcomes. Machine learning frameworks were implemented to automate sequence authentication, with DNA barcoding and metabarcoding pipelines achieving an accuracy of 99.80%. Field applications in Thailand's protected areas yielded 1,800 specimens (71 families) processed via mitogenomics (187 mitogenomes), DNA barcoding (584 sequences), and metabarcoding (608 operational taxonomic units, or OTUs). Phylogenetic analysis revealed that Thai specimens (1.54% of the global dataset) contributed disproportionately to total phylogenetic diversity (3.23%, p<0.01). Community structure analysis revealed significant phylogenetic clustering at Khao Yai National Park (NRI = 1.875207, p < 0.001) and overdispersion at Sakaerat Environmental Research Station (NRI =-0.82255, p < 0.01). Comprehensive phylogenetic analysis integrated 12,320 mitochondrial genome sequences, establishing sequential infraorder diversification in Polyphaga spanning approximately 56 million years (254.3-198.5 Ma). The lineages-through-time analysis identified an inflexion point at approximately 120 MYA, coinciding with angiosperm radiation. This research advances the understanding of beetle diversity while establishing methodological frameworks for molecular biodiversity assessment in hyperdiverse taxa.

Mask Use in Community Settings in the Context of COVID-19: A Systematic Review of Ecological Data