Centre for Biodiversity Genomics

Traditional morphological diagnoses of taxonomic status remain widely used while an increasing number of studies show that one morphospecies might hide cryptic diversity, i.e. lineages with unexpectedly high molecular divergence. This hidden diversity can reach even tens of lineages, i.e. hyper cryptic diversity. Even well-studied model-organisms may exhibit overlooked cryptic diversity. Such is the case of the freshwater crustacean amphipod model taxon Gammarus fossarum. It is extensively used in both applied and basic types of research, including biodiversity assessments, ecotoxicology and evolutionary ecology. Based on COI barcodes of 4926 individuals from 498 sampling sites in 19 European countries, the present paper shows (1) hyper cryptic diversity, ranging from 84 to 152 Molecular Operational Taxonomic Units, (2) ancient diversification starting already 26 Mya in the Oligocene, and (3) high level of lineage syntopy. Even if hyper cryptic diversity was already documented in G. fossarum, the present study increases its extent fourfold, providing a first continental-scale insight into its geographical distribution and establishes several diversification hotspots, notably south-eastern and central Europe. The challenges of recording hyper cryptic diversity in the future are also discussed.

Here we elucidate and justify a DNA barcode approach to insect species description that can be applied to name tens of thousands of species of Ichneumonoidea and many other species-rich taxa. Each description consists of a lateral habitus image of the specimen, a COI barcode diagnosis, and the holotype specimen information required by the International Code of Zoological Nomenclature. We believe this approach, or a slight modification of it, will be useful for many other underdescribed hyperdiverse taxa, especially in the tropics. Due to the extreme species-richness of the Ichneumonoidea, the very low percentage of described species, and the lack of detailed biological information for most described species, the standard taxonomic approach is inefficient and overwhelmingly time consuming. A DNA barcode-based approach to initial description will provide a solid foundation of species hypotheses from which more comprehensive descriptions can be developed as other data, time, and budgets permit. Here we elucidate this view and detailed methodology that can generally be applied to species-rich underdescribed taxa. A real example is given by describing species in two genera, Hemichoma and Zelomorpha , reared from the Área de Conservación Guanacaste in northwestern Costa Rica. The generic type species Zelomorphaarizonensis is given a DNA barcode diagnosis and the following new species are described: Zelomorphaangelsolisi , Zelomorphabobandersoni , Zelomorphadanjohnsoni , Zelomorphadonwindsori , Zelomorphaeffugia , Zelomorphajohnchemsaki , Zelomorphakellyanneae , Zelomorphalarrykirkendalli , Zelomorphamariyavladmirovnae , Zelomorphamikeiviei , Zelomorphamyricagaleae , Zelomorphanoahjaneae , Zelomorphapaulgoldsteini , Zelomorphaterryerwini , Zelomorphawillsflowersi , Hemichomadonwhiteheadi , Hemichomafrankhovorei , and Hemichomajohnkingsolveri .

Fecal contamination of recreational waters (i.e. lakes, rivers, beaches) poses an on-going problem for environmental and public health. Heavy rainfall can exacerbate existing problems with fecal contamination. As there could be variable sources of fecal contamination, identifying the source is critical for remediation efforts. This study utilized microbial source tracking (MST), chemical source tracking (CST) markers and environmental DNA (eDNA) metabarcoding to profile sampling areas and identify sources of fecal contamination in creek, stormwater outfall and beach sites in the Etobicoke Creek watershed (Toronto, ON). Water samples were collected before and immediately following an extreme rain event. MST and CST identified stormwater outfalls as an important source of human fecal contamination during dry and wet conditions. eDNA metabarcoding allowed for potential identification of additional sources of fecal contamination and provided additional evidence of human fecal contamination. The extreme rainfall event altered the eDNA profiles, causing creek and beach sites to reflect a greater diversity of mammal and bird eDNA sequences. The profiles provided by eDNA metabarcoding provide a proof of concept suggesting that eDNA metabarcoding can be a useful tool to complement MST and CST methods for profiling sources of fecal contamination and studying impacts of extreme rain events.

The present study provides a DNA barcode library for the world Zygaenidae (Lepidoptera). This study reports 1031 sequence data of the COI gene DNA barcodes for more than 240 species in four of the five subfamilies of the family Zygaenidae. This is about 20% of the world Zygaenidae species. Our results demonstrate the specificity of the COI gene sequences at the species level in most of the studied Zygaenidae and agree with already established taxonomic opinions. The study confirms the effectiveness of DNA barcoding as a tool for determination of most Zygaenidae species. However, some of the results are contradictory. Some cases of shared barcodes have been found, as well as cases of deep intraspecific sequence divergence in species that are well separated by morphological and biological characters. These cases are discussed in detail. Overall, when combined with morphological and biochemical data, as well as biological and ecological observations, DNA barcoding results can be a useful support for taxonomic decisions.

Sundaland constitutes one of the largest and most threatened biodiversity hotspots; however, our understanding of its biodiversity is afflicted by knowledge gaps in taxonomy and distribution patterns. The subfamily Rasborinae is the most diversified group of freshwater fishes in Sundaland. Uncertainties in their taxonomy and systematics have constrained its use as a model in evolutionary studies. Here, we established a DNA barcode reference library of the Rasborinae in Sundaland to examine species boundaries and range distributions through DNA-based species delimitation methods. A checklist of the Rasborinae of Sundaland was compiled based on online catalogs and used to estimate the taxonomic coverage of the present study. We generated a total of 991 DNA barcodes from 189 sampling sites in Sundaland. Together with 106 previously published sequences, we subsequently assembled a reference library of 1097 sequences that covers 65 taxa, including 61 of the 79 known Rasborinae species of Sundaland. Our library indicates that Rasborinae species are defined by distinct molecular lineages that are captured by species delimitation methods. A large overlap between intraspecific and interspecific genetic distance is observed that can be explained by the large amounts of cryptic diversity as evidenced by the 166 Operational Taxonomic Units detected. Implications for the evolutionary dynamics of species diversification are discussed.

Bread wheat is a major crop that has long been the focus of basic and breeding research. Assembly of its genome has been difficult because of its large size and allohexaploid nature (AABBDD genome). Following the first reported assembly of the genome of the experimental strain Chinese Spring (CS), the 10+ Wheat Genomes Project was launched to produce multiple assemblies of worldwide modern cultivars. The only Asian cultivar in the project is Norin 61, a representative Japanese cultivar adapted to grow across a broad latitudinal range, mostly characterized by a wet climate and a short growing season. Here, we characterize the key aspects of its chromosome-scale genome assembly spanning 15 Gb with a raw scaffold N50 of 22 Mb. Analysis of the repetitive elements identified chromosomal regions unique to Norin 61 that encompass a tandem array of the pathogenesis-related 13 family. We report novel copy-number variations in the B homeolog of the florigen gene FT1/VRN3, pseudogenization of its D homeolog and the association of its A homeologous alleles with the spring/winter growth habit. Furthermore, the Norin 61 genome carries typical East Asian functional variants different from CS, ranging from a single nucleotide to multi-Mb scale. Examples of such variation are the Fhb1 locus, which confers Fusarium head-blight resistance, Ppd-D1a, which confers early flowering, Glu-D1f for Asian noodle quality and Rht-D1b, which introduced semi-dwarfism during the green revolution. The adoption of Norin 61 as a reference assembly for functional and evolutionary studies will enable comprehensive characterization of the underexploited Asian bread wheat diversity.

Gammarus roeselii Gervais, 1835 is a morphospecies with a wide distribution range in Europe. The Balkan Peninsula is known as an area of pre-Pleistocene cryptic diversification within this taxon, resulting in at least 13 Molecular Operational Taxonomic Units (MOTUs). The morphospecies diversified there during Neogene and has probably invaded other parts of the continent very recently, in postglacial or even historical times. Thus, the detailed goals of our study were to (1) identify which lineage(s) colonized Central-Western Europe (CWE), (2) determine their possible geographical origin, (3) verify, whether the colonisation was associated with demographic changes. In total, 663 individuals were sequenced for the cytochrome oxidase I (COI) barcoding fragment and 137 individuals for the internal transcribed spacer II (ITS2). We identified two MOTUs in the study area with contrasting Barcode Index Number and haplotype diversities. The Pannonian Basin (PB) appeared to be a potential ice age refugium for the species, while CWE was colonised by a single lineage (also present in PB), displaying low genetic diversity. Our results suggest that G. roeselii is a relatively recent coloniser in CWE, starting demographic expansion around 10 kya.

Recent technical advances combined with novel computational approaches have promised the acceleration of our understanding of the tree of life. However, when it comes to hyperdiverse and poorly known groups of invertebrates, studies are still scarce. As published phylogenies will be rarely challenged by future taxonomists, careful attention must be paid to potential analytical bias. We present the first molecular phylogenetic hypothesis for the family Chalcididae, a group of parasitoid wasps, with a representative sampling (144 ingroups and seven outgroups) that covers all described subfamilies and tribes, and 82% of the known genera. Analyses of 538 Ultra-Conserved Elements (UCEs) with supermatrix (RAxML and IQTREE) and gene tree reconciliation approaches (ASTRAL, ASTRID) resulted in highly supported topologies in overall agreement with morphology but reveal conflicting topologies for some of the deepest nodes. To resolve these conflicts, we explored the phylogenetic tree space with clustering and gene genealogy interrogation methods, analyzed marker and taxon properties that could bias inferences and performed a thorough morphological analysis (130 characters encoded for 40 taxa representative of the diversity). This joint analysis reveals that UCEs enable attainment of resolution between ancestry and convergent/divergent evolution when morphology is not informative enough, but also shows that a systematic exploration of bias with different analytical methods and a careful analysis of morphological features is required to prevent publication of artifactual results. We highlight a GC content bias for maximum-likelihood approaches, an artifactual mid-point rooting of the ASTRAL tree and a deleterious effect of high percentage of missing data (>85% missing UCEs) on gene tree reconciliation methods. Based on the results we propose a new classification of the family into eight subfamilies and ten tribes that lay the foundation for future studies on the evolutionary history of Chalcididae.

The regions of the Black, Caspian, and Azov seas are known for being both (i) the place of extensive crustacean radiation dated to the times of Paratethys and Sarmatian basins, and (ii) present donors of alien and invasive taxa to many areas worldwide. One amphipod morphospecies, Dikerogammarus haemobaphes , is known both as native to rivers draining to the Black and Caspian seas as well as a successful invader (nicknamed demon shrimp) in Central and Western European rivers. Based on mitochondrial (COI and 16S) and nuclear (28S) datasets and 41 sampling sites, representing both the native (19) and the invaded (22) range, we assessed cryptic diversity, phylogeography and population genetics of this taxon. First, we revealed the presence of two divergent lineages supported by all markers and all species delimitation methods. The divergence between the lineages was high (18.3% Kimura 2-parameter distance for COI) and old (ca. 5.1 Ma), suggesting the presence of two cryptic species within D. haemobaphes . Lineage A was found only in a few localities in the native range, while lineage B was widespread both in the native and in the invaded range. Although genetic divergence within lineage B was shallow, geographic distribution of 16S and COI haplotypes was highly heterogeneous, leading us to the definition of four Geo-Demographic Units (GDUs). Two GDUs were restricted to the native range: GDU-B1 was endemic for the Durugöl (aka Duruşu) Liman in Turkey, whereas GDU-B2 occurred only in the Dniester River. GDU-B3 was both present in several localities in the native range in the Black Sea drainage area and widespread in Central and Western Europe. The GDU-B4 was found exclusively in the Moskva River in Russia. Extended Bayesian Skyline Plot indicated steady growth of GDU-B3 population size since 30 ka, pointing to the rather old history of its expansion, first in the late Pleistocene in the native range and nowadays in Central and Western Europe. The analysis of haplotype distribution across the present distribution range clearly showed two invasion routes to Central and Western Europe. The first one, originating from the lower Dnieper, allowed the demon shrimp to colonize Polish rivers and the Mittellandkanal in Germany. The second one, originating from the Danube delta, allowed to colonize the water bodies in the upper Danube basin. The UK population has originated from the Central Corridor, as only a haplotype found exclusively along this route was recorded in the UK. Population genetics analysis showed that the invasion of the demon shrimp along the Central Corridor was not associated with the loss of genetic diversity, which might contribute to the success of this invader in the newly colonized areas.

Natural history collections are a valuable resource for molecular taxonomic studies and for examining patterns of evolutionary diversification, particularly in the case of rare or extinct species. However, the recovery of sequence information is often complicated by DNA degradation. This article describes use of the Sequel platform (Pacific Biosciences) to recover the 658 bp barcode region of the mitochondrial cytochrome c oxidase I (COI) gene from 380 butterflies with an average age of 50 years. Nested multiplex PCR was employed for library preparation to facilitate sequence recovery from extracts with low concentrations of highly degraded DNA. By employing circular consensus sequencing (CCS) of short amplicons (circa 150 bp), full-length barcodes could be assembled without a reference sequence, an important advance from earlier protocols which required reference sequences to guide contig assembly. The Sequel protocol recovered COI sequences (499 bp on average) from 318 of 380 specimens (84%), much higher than for Sanger sequencing (26%). Because each read derives from a single molecule, it was also possible to quantify the incidence of substitutions arising from DNA damage. In agreement with past work on sequence changes induced by DNA degradation, the transition C/G → T/A was the most prevalent category of change, but its rate of occurrence (4.58E−4) was so low that it did not impede the recovery of reliable sequences. Because the current protocol recovers COI sequence from most museum specimens, and because sequence fidelity is unaffected by nucleotide misincorporations, large-scale sequence characterization of museum specimens is feasible.

The interplay between geographic barriers and climatic oscillations over the past 2.6 million years structured genetic variation at the continental scale. The genetic legacy of the Quaternary ice ages (GLQ) hypothesis outlines this phenomenon for Europe, but a comprehensive data-driven assessment is lacking. Using innovative genetic landscape methods, we model the GLQ in the West Palearctic based on 31,653 Cytochrome c oxidase subunit 1 (COI) sequences from 494 butterfly species and three functional traits. Seven distinct bioregions with varying levels of genetic endemicity emerge, revealing a latitudinal gradient in variation that confirms the "southern richness, northern purity" hypothesis. Through shift from case studies to a comparative approach, we objectively identify the main glacial refugia, colonization routes, and barriers to dispersal. Our findings offer a quantitative model of the GLQ across Europe, North Africa, and neighboring Asia, with broader applicability to other taxa and potentially scalable to encompass life on Earth.

We present the results of the first-ever DNA barcoding study of odonates from the Maltese Islands. In total, 10 morphologically identified species were collected during a two-week long expedition in 2018. Eighty cytochrome c oxidase subunit I (COI) barcodes were obtained from the collected specimens. Intra- and interspecific distances ranged from 0.00% to 2.24% and 0.48% to 17.62%, respectively. Successful species identification based on ascribing a single morphological species to a single Barcode Index Number (BIN) was achieved for eight species (80%). In the case of two species, Ischnura genei and Anax parthenope, BINs were shared with other closely related species. The taxonomic status of I. genei is questionable and the phylogenetic relationship between A. imperator/parthenope is not clear. Further studies involving a series of adult specimens collected in a wide spatial range and nuclear markers are necessary to resolve these cases. Therefore, this dataset serves as an initial DNA barcode reference library for Maltese odonates, within a larger project: Aquatic Macroinvertebrates DNA Barcode Library of Malta.

Biodiversity loss has accelerated over the past century and freshwater species overall are among those experiencing greatest declines. Genetic resources have the potential to help evaluate the full magnitude of this loss and represent a key tool to effectively allocate conservation resources and monitor the success of restoration efforts. The full power of genetic resources will be realized when the daunting task of referencing all DNA sequences of freshwater organisms is complete. Here, we quantified the availability and distribution of barcode and genome data for freshwater macroscopic organisms in Canada, a country rich in inland water resources and thus particularly vulnerable to aquatic species losses. Impressively, most inland water species (86 %) were represented by barcodes recorded in the BOLD Systems database, while very few had full genomes available (<4 %) in the NCBI database. We identified barcode data deficiencies in northern regions and for taxa assessed as most at risk or without sufficient information for conservation status classification. As expected, the speciose insect group had a lower-than-average number of records per species and a high proportion of data deficient species without adequate barcode coverage. This study highlights where future sequencing resources should be prioritized within initiatives such as the Canada BioGenome Project and BIOSCAN Canada and provides a workflow that could be applied internationally to inform conservation management plans and to mitigate biodiversity loss.

The river Danube is the backbone of the ‘southern invasion corridor’, one of the most important passages for the spread of Ponto-Caspian invaders in Europe. However, not all of these species used the passive or active upstream movement in the main channel to reach the upper sections and tributaries, some found detours. Mass occurrences of the Ponto-Caspian peracarid, Pontogammarus robustoides (Sars, 1894) were recorded at 17 sites along the entire Hungarian section of the River Maros, for the first time in the River Tisza catchment and also in Hungary. Those populations are found ca. 707 km upstream from the closest known and confirmed locality in the lower Danube section. We confirmed their identity by DNA barcoding and showed that all individuals fit in with the lower Danube population, thus identifying the source of this introduction. The most likely vector allowing the jump dispersal of the species is fish stocking in the Romanian section of the River Maros, which − combined with downstream drift to the Serbian Danube section and the relatively busy ship traffic between Belgrade and Vienna − might provide the opportunity to bypass the dispersal barrier represented by the unregulated Middle Danube and open the way towards Western Europe.

Biodiversity hotspots often suffer from a lack of taxonomic knowledge, particularly those in tropical regions. However, accurate taxonomic knowledge is needed to support sustainable management of biodiversity, especially when it is harvested for human sustenance. Sundaland, the biodiversity hotspot encompassing the islands of Java, Sumatra, Borneo, and Peninsular Malaysia, is one of those. With more than 900 species, its freshwater ichthyofauna includes a large number of medium- to large-size species, which are targeted by inland fisheries. Stock assessment requires accurate taxonomy; however, several species groups targeted by inland fisheries are still poorly known. One of those cases is the cyprinid genus Barbonymus. For this study, we assembled a consolidated DNA barcode reference library for Barbonymus spp. of Sundaland, consisting of mined sequences from BOLD, as well as newly generated sequences for hitherto under-sampled islands such as Borneo. A total of 173 sequences were analyzed using several DNA-based species delimitation methods. We unambiguously detected a total of 6 Molecular Operational Taxonomic Units (MOTUs) and were able to resolve several conflicting assignments to the species level. Furthermore, we clarified the identity of MOTUs occurring in Java.

Anguillid eels are widely acknowledged for their ecological and socio-economic value in many countries. Yet, knowledge regarding their biodiversity, distribution and abundance remains superficial—particularly in tropical countries such as Indonesia, where demand for anguillid eels is steadily increasing along with the threat imposed by river infrastructure developments. We investigated the diversity of anguillid eels on the western Indonesian islands of Sumatra and Java using automated molecular classification and genetic species delimitation methods to explore temporal patterns of glass eel cohorts entering inland waters. A total of 278 glass eels were collected from monthly samplings along the west coast of Sumatra and the south coast of Java between March 2017 and February 2018. An automated, DNA-based glass eel identification was performed using a DNA barcode reference library consisting of 64 newly generated DNA barcodes and 117 DNA barcodes retrieved from BOLD for all nine Anguilla species known to occur in Indonesia. Species delimitation methods converged in delineating eight Molecular Operational Taxonomic Units (MOTUs), with A. nebolusa and A. bengalensis being undistinguishable by DNA barcodes. A total of four MOTUs were detected within the glass eel samples, corresponding to Anguilla bicolor, A. interioris, A. marmorata, and A. nebulosa/A. bengalensis. Monthly captures indicated that glass eel recruitment peaks in June, during the onset of the dry season, and that A. bicolor is the most prevalent species. Comparing indices of mitochondrial genetic diversity between yellow/silver eels, originating from several sites across the species range distribution, and glass eels, collected in West Sumatra and Java, indicated a marked difference. Glass eels displayed a much lower diversity than yellow/silver eels. Implications for the management of glass eel fisheries and species conservation are discussed.

We report the results of DNA barcoding week-long Malaise trap catches from 11 sites in three Thai conservation areas, concentrating on the parasitoid Hymenoptera, particularly the superfamily Ichneumonoidea. From a total of 15,338 parasitoid wasp sequences, 13,473 were barcode compliant and could be assigned to a family based on morphology and sequence data. These collectively represented 4917 unique BINs (putative species) in 46 families, with the Scelionidae, Ichneumonidae, Eulophidae, Braconidae and Platygastridae being, by far, the most abundant. Spatial proximity had a strong positive effect on the numbers of BINs shared between traps.

Abstract Background Pseudogenes are non-functional copies of protein coding genes that typically follow a different molecular evolutionary path as compared to functional genes. The inclusion of pseudogene sequences in DNA barcoding and metabarcoding analysis can lead to misleading results. None of the most widely used bioinformatic pipelines used to process marker gene (metabarcode) high throughput sequencing data specifically accounts for the presence of pseudogenes in protein-coding marker genes. The purpose of this study is to develop a method to screen for obvious pseudogenes in large COI metabarcode datasets. We do this by: 1) describing gene and pseudogene characteristics from a simulated DNA barcode dataset, 2) show the impact of two different pseudogene removal methods on mock metabarcode datasets with simulated pseudogenes, and 3) incorporate a pseudogene filtering step in a bioinformatic pipeline that can be used to process Illumina paired-end COI metabarcode sequences. Open reading frame length and sequence bit scores from hidden Markov model (HMM) profile were used to detect pseudogenes. Results Our simulations showed that it was more difficult to identify pseudogenes from shorter amplicon sequences such as those typically used in metabarcoding (∼300 bp) compared with full length DNA barcodes that are used in construction of barcode libraries (∼ 650 bp). It was also more difficult to identify pseudogenes in datasets where there is a high percentage of pseudogene sequences. We show that existing bioinformatic pipelines used to process metabarcode sequences already remove some apparent pseudogenes, especially in the rare sequence removal step, but the addition of a pseudogene filtering step can remove more. Conclusions The combination of open reading frame length and hidden Markov model profile analysis can be used to effectively screen out obvious pseudogenes from large datasets. There is more to learn from COI pseudogenes such as their frequency in DNA barcode and metabarcoding studies, their taxonomic distribution, and evolution. Thus, we encourage the submission of verified COI pseudogenes to public databases to facilitate future studies.

One of the main consequences of globalization is the intensification of biological introductions. Because of their negative impact on environments, the early detection and monitoring of introduced species through molecular approaches is gaining increased uptake. This study assembles 2,278 DNA barcode records to examine contemporary patterns of sequence variation in mitochondrial cytochrome c oxidase I (COI) in five butterfly species introduced to the Nearctic, with a focus on Pieris rapae Linnaeus (Lepidoptera: Pieridae) and Thymelicus lineola Ochsenheimer (Lepidoptera: Hesperiidae). Parameters of genetic diversity were low (i.e., h &lt; 0.606, &#960; &lt; 0.0039) for Nearctic populations of all analyzed species. Those of P. rapae and T. lineola showed marked genetic differentiation from their source populations in the Palearctic. Haplotype distributions in their Nearctic populations exposed a starburst pattern with a few common haplotypes known from Palearctic, and infrequent haplotypes diverging from them at only one or two nucleotide sites. Some uncommon haplotypes were only found in the Nearctic suggesting they originated after invasion, while others also occur in the Palearctic. This study provides an example of genetic paradox of invasion, where species often rapidly expand their distribution and become dominant in the new habitat despite their depleted levels of sequence variation.

A developing body of theory and empirical evidence suggest that feeding behaviour as measured by the functional response (FR) can assist researchers in assessing the relative potential, ecological impacts and competitive abilities of native and introduced species. Here, we explored the FRs of two land snails that occur in south-western Ontario, one native ( Mesodonthyroidus ) and one non-indigenous ( Cepaeanemoralis ) to Canada. The non-indigenous species appears to have low ecological impact and inferior competitive abilities. Consistent with theory, while both species conformed to Type II functional responses, the native species had a significantly higher attack rate (5.30 vs 0.41, respectively) and slightly lower handling time (0.020 vs 0.023), and hence a higher maximum feeding rate (50.0 vs 43.5). The non-indigenous species exhibited a significantly longer time to contact for a variety of food types, and appeared less discriminating of paper that was offered as a non-food type. The non-indigenous species also ate significantly less food when in mixed species trials with the native snail. These feeding patterns match the known low ecological impact of the introduced snail and are consistent with the view that it is an inferior competitor relative to the native species. However, field experimentation is required to clarify whether the largely microallopatric distributions of the two species in south-western Ontario reflect competitive dominance by the native species or other factors such as habitat preference, feeding preferences or predator avoidance. The relative patterns of feeding behaviour and ecological impact are, however, fully in line with recent functional response theory and application.