Barcoding Species Research Articles

Taxonomy of Diamesa steinboecki group (Diptera: Chironomidae: Diamesinae), with description and DNA barcoding of known species. II. Subgroups davisi, leona and loeffleri.

Illustrated redescription of the adult males Diamesa alpina Tokunaga from the Russian Far East and North America, D. amplexivirilia Hansen from Arctic and the Russian Far East, D. saetheri Willassen from Chukotka Region and Kolyma River basin, D. lupus Willassen from Alaska, D. serratosioi Willassen from Norway and Russian Far East, D. leoniella Hansen from Alaska, D. leona Roback from Eurasia, D. japonica Tokunaga from Japan and Russian Far East, D. khumbugelida Saether et Willassen and D. loeffleri Reiss from Himalayas are provided. Morphological data and DNA sequences of the mitochondrial cytochrome c oxidase subunit I gene (COI) were used to delimit of seven species from subgroups davisi (D. alpina, D. amplexivirilia, D. serratosioi), leona (D. japonica, D. leona) and loeffleri (D. khumbugelida, D. loeffleri). Taxonomic remarks with data on geographical distribution of the investigated species are given.

Mitochondrial cytochrome oxidase 1 (CO1) and morphology of Penja fish (Sicyopterus spp.) in Budong-Budong River, West Sulawesi, Indonesia

Abstract. Astuti I, Fadjar M, Nurdiani R, Sulistiyani TD. 2022. Mitochondrial cytochrome oxidase 1 (CO1) and morphology of Penja fish (Sicyopterus spp.) in Budong-Budong River, West Sulawesi, Indonesia. Biodiversitas 23: 4724-4729. Penja fish (local name) is a fish that is often found in West Sulawesi. Penja fish belong to the Goobidae family, which has a habit of migrating from the sea to the river. Penja fish species have not been widely published or in the Gen bank. The objective of this study was to analyze the Penja fish species found in the Budong-Budong river, West Sulawesi, Indonesia. The method used was a measurement of fish morphology and analysis of mitochondrial cytochrome oxidase I (COI) using a species barcode (~700). Samples were taken from the catch of fishermen at the mouth of the Budong-Budong river. The result of morphological measurements obtained an average total length of 25-38 mm, standard length 22-35 mm, head length 2-5 mm, tail length 3-5 mm, pelvic fins 0.5-2 mm, pectoral fins 1-3 mm, dorsal fins 1-3 mm and anal fin 0.1-1 and body width 3-6 mm. Electrophoretic analysis on Penja fish samples showed that the DNA between P1 and P2 samples was not much different and has very small genetic variation in 696 bp fragment length. The analysis on the Basic Local Alignment Search Tool (BLAST) showed the identity percentage reached 99.85%. The Penja fish identified in the Budong-Budong river is of the family Gobiidae, genus Sicyopterus with the species name Sicyopterus pugnans, which is classified as post larva.

Mitochondrial DNA barcoding of mosquito species (Diptera: Culicidae) in Thailand

The correct identification of mosquito species is important for effective mosquito vector control. However, the standard morphological identification of mosquito species based on the available keys is not easy with specimens in the field due to missing or damaged morphological features during mosquito collections, often leading to the misidentification of morphologically indistinguishable. To resolve this problem, we collected mosquito species across Thailand to gather genetic information, and evaluated the DNA barcoding efficacy for mosquito species identification in Thailand. A total of 310 mosquito samples, representing 73 mosquito species, were amplified using mitochondrial cytochrome c oxidase subunit I (COI) primers. The average maximum intraspecific genetic variation of the 73 mosquito species was 1% ranged from 0-5.7%. While, average minimum interspecific genetic variation (the distance to the nearest neighbour) of the 73 mosquito species was 7% ranged from 0.3-12.9%. The identification of success rates based on the "Best Match," "Best Close Match," and "All Species Barcodes" methods were 97.7%, 91.6%, and 81%, respectively. Phylogenetic analyses of Anopheles COI sequences demonstrated a clear separation between almost all species (except for those between An. baimaii and An. dirus), with high bootstrap support values (97%-99%). Furthermore, phylogenetic analyses revealed potential sibling species of An. annularis, An. tessellatus, and An. subpictus in Thailand. Our results indicated that DNA barcoding is an effective molecular approach for the accurate identification of mosquitoes in Thailand.

DNA Barcode of Red Junglefowl Gallus gallus L, 1958 (Aves: Phasianidae) of Sumatra Based on Mitochondrial COI DNA Gene

Genetic data of red junglefowl (Gallus gallus) from southern Sumatra is valuable for conservation efforts in Indonesia. A mitochondrial COI DNA gene sequencing was performed to elucidate its genetic character, single nucleotide polymorphism, genetic distance, and phylogeny. Blood samples (±0.5 ml) from 20 individuals of Gallus gallus were taken from the living collections of the people of Bengkulu Province (Central Bengkulu Regency and Seluma Regency) and South Sumatra Province (North Musi Rawas Regency) from May to November 2021. Total DNA isolation followed the procedure of The Spin-Column Protocol Kit uses the Dneasy® Blood and Tissue Kit, Qiagen. DNA replication using the Polymerase Chain Reaction technique with primers (COIGG_F and COIGG_R) using MEGA 10.0 software and Bioedit for data analysis. The results revealed716 conserved sites, 16 variable sites, 9 parsimony sites, and 6 singleton sites from the 732 bp nucleotide sequence. Six specific sites (SNPs) as barcodes for Sumatran Junglefowl were found at sequences 51, 273, 327, 721, 729, and 732. The mean genetic distance between individuals was 0.1%, between populations was 0.8%, between species was 7.4%, and between genera was 15.5%. The red junglefowlof South Sumatra Province and Bengkulu Province are closely related with 98% bootstrapping and separated from other Gallus in the same group (ingroup) with 100% bootstrap. The Gallus-gallus group is quite far apart from the outgroup species in the Phasianidae family with 47-100% bootstrap. Red junglefowl from southern Sumatra has genetic differences from other chickens in the world and these differences can be used as a species barcode and as origin identification the widely traded red jungle fowl.

CUREs

Course-based undergraduate research experiences (CUREs) are one way instructors can increase engagement and learning of material. One of the goals in the report Vision and Change in Undergraduate Biology Education: A Call to Action is to increase active learning activities. By implementing a CURE or CURE-type model, instructors provide students with the opportunity to develop a better understanding of science content, to apply what they have learned, and make an impact in real-world science. Our classes replicated a subset of the work being completed in Gorongosa National Park in Mozambique. We had students complete biodiversity surveys through collection and classification of ant species using field and lab techniques. DNA barcoding analyses are commonly used techniques in biology labs worldwide. Polymerase chain reaction and cycle sequencing will be taught to illustrate how the extracted DNA can be amplified at different markers and used to identify species. We utilized the CURE model to have students complete a biodiversity survey of both a southern intermountain-west and a southeastern state through collection, classification, and genotyping and barcoding of ant species.

DNA barcoding of insects from India: Current status and future perspectives.

Insect fauna occupy the largest proportion of animal biodiversity on earth, but the assessment or quantification in terms of species diversity is far from complete. Several recent studies have demonstrated the rapid pace at which insect population decline is occurring. There is an urgent need to document and quantify the diversity of insect fauna for a proper understanding of terrestrial ecosystems. This can be achieved by using modern technology to identify species much faster than relying on traditional methods alone. In line with this, the molecular approach through DNA barcoding coupled with morphological identification needs to be focused and accelerated. The present paper describes the current status of barcoding of insect species in India along with the gaps that need to be remedied. This analysis shows that barcoded specimens cover a very meagre proportion of less than 3.73% of the known taxa/described species and the most represented orders are Lepidoptera and Hemiptera followed by Diptera and Coleoptera. There is a need to expedite insect species discovery and documentation in a collaborative mode between traditional taxonomists and molecular biologists, to accomplish the DNA barcoding of all known insect taxa from India.Supplementary informationThe online version contains supplementary material available at 10.1007/s11033-022-07628-2.

Let’s talk about the (lady)birds and the bees: how insects can whisper a multitude of stories

If you have watched A Bug’s Life, you would have seen that insects come in an assortment of colours, shapes, and sizes. They are the perfect organism that could be used to describe the myriad diversity of all life on earth. These six-legged creatures are one of the most diverse groups of species, accounting for more than 80% of all documented living animals Ødegaard 2008. This huge diversity also makes it extremely time- and labour-intensive to carry out large-scale monitoring of insects. High-throughput sequencing technologies and the use of DNA barcodes for species identification have paved the way for the rapid biomonitoring of insects Hebert et al. 2003. However, millions of insect species are not well-represented in DNA reference databases, making species-level identification challenging for molecular research. Projects such as the Darwin Tree of Life (DToL) aim to cover this gap and generate DNA barcodes for all eukaryotic species found in the UK Blaxter et al. 2022. At the Wellcome Sanger Institute, the BIOSCAN UK for Flying Insects project has two main aims; Documenting the diversity of UK flying insects Discovering insect-cobiont interactions Documenting the diversity of UK flying insects Discovering insect-cobiont interactions To meet these aims, we will be documenting the diversity of one million malaise-caught insects from 100 sites across the UK in the next five years. We will be using a non-destructive DNA extraction technique to preserve insect specimen integrity for museum collections or educational purposes Korlević et al. 2021. COI barcoding will be carried out using ONT and/or PacBio long-read technology to identify each insect specimen. To tease apart insect conbiont interactions, we will carry out mini barcoding using primers targeting microbes, parasites, vertebrates, invertebrates, and plants Fig. 1. Together, this molecular dataset consisting of one million specimens collected over space and time in the next five years will allow us to discover how insects interact with the ecosystem and advance insect biomonitoring research in the UK.

Karyotype Evolution of Talking Thorny Catfishes Anadoras (Doradidae, Astrodoradinae): A Process Mediated by Structural Rearrangements and Intense Reorganization of Repetitive DNAs

Anadoras is a thorny catfish genus widespread through the Amazon and Paraguay river basins. It includes 2 nominal species, A. grypus and A. weddellii, plus Anadoras sp. “araguaia,” an undescribed species only recognized morphologically. Since Anadoras occupies a basal position within the Astrodoradinae phylogeny, it is crucial to identify its cytogenetic features to comprehend the mechanisms involved in the chromosomal diversification of this subfamily. Therefore, we performed a comparative cytogenetic analysis including all species of Anadoras. Furthermore, we applied a species delimitation analysis based on 600 bp of the mitochondrial cytochrome oxidase subunit 1 (CO1) gene to investigate the taxonomic status of the species. Cytogenetic markers revealed a high degree of similarity among Anadoras weddellii and Anadoras sp. “araguaia,” both have 2n = 56 chromosomes (24m + 10sm + 22st/a), single NOR sites on acrocentric pair 28, and 5S rDNA sites on submetacentric pair 15. A. grypus has the most divergent chromosomal characteristics because, even though it also has 2n = 56 chromosomes, it exhibits several differences in the chromosome formula, heterochromatin distribution, and number/position of the rDNA sites. In sum, we believe that the chromosome diversification of Anadoras is due to 4 mechanisms: centric fusion, pericentric/paracentric inversions, nonreciprocal translocations, and activity of transposable elements. Additionally, our phylogenetic tree revealed well-supported clades and, by barcode species delimitation analysis, confirmed the existence of 3 molecular operational taxonomic units, including the putative new species Anadoras sp. “araguaia.”

DNA barcoding the Lake Edward basin: high taxonomic coverage of a tropical freshwater ichthyofauna

We present an extensive DNA barcoding study of the fish species of the Lake Edward system, including information on intraspecific variation. The DNA barcode gene, cytochrome c oxidase I (COI), failed to discriminate the 35 species of the genus Haplochromis analysed. For the non-Haplochromis species, the reference library had a near complete coverage of 91.2%, with 31 out of the 34 known species of the system sequenced. With the recent morphology-based review of the ichthyofauna of the system as a backbone, the DNA barcoding library proved to be very effective for future species identifications. High identification successes were obtained based on Best Match (98.2%), Best Close Match (97.9%), and All Species Barcode (95.0%) criteria. Surprisingly, Laciris pelagicus and Micropanchax vitschumbaensis, two morphologically distinct species, had very similar and even identical COI haplotypes, whilst in Enteromius the existence of possible cryptic species was revealed. Few species occurring in the Lake Edward system were already represented in the global database Genbank. For some widespread species, regional genetic differences were found, highlighting the value of basin-specific information in reference databases. The provided reference library of DNA barcodes is a valuable tool for future conservation actions, which is certainly relevant in a system where the fisheries are under pressure.

Morphological and Phylogenetic Analyses Reveal a New Species of Ceratocystiopsis (Ophiostomataceae, Ophiostomatales) Associated with Ips subelongatus in Inner Mongolia (China) with Weak Host Pathogenicity

Ophiostomatoid fungi are known for their associations with bark beetles, and some species are important sources of tree diseases. Ceratocystiopsis is a genus of the ophiostomatoid fungi in order Ophiostomatales. The shortage of DNA barcodes for many species in this genus has resulted in the presence of many unnamed cryptic species. In this study, Ceratocystiopsis subelongati sp. nov. associated with Ips subelongatus infesting Pinus sylvestris var. mongolica in Inner Mongolia, China, was identified and described based on phylogenetic inference of multi-gene DNA sequences and morphological characteristics. The species is characterized by a hyalorhinocladiella- to sporothrix-like asexual state and an optimal growth temperature of 30 °C. Artificial inoculation tests in the field showed that it is mildly pathogenic to five-year-old larch trees, the main host of I. subelongatus. It is also the first described Ceratocystiopsis species associated with I. subelongatus in China. This discovery should provide new avenues for studying the symbiosis between bark beetles and ophiostomatoid fungi.

Evaluation of Arabian Vascular Plant Barcodes (rbcL and matK): Precision of Unsupervised and Supervised Learning Methods towards Accurate Identification

Arabia is the largest peninsula in the world, with >3000 species of vascular plants. Not much effort has been made to generate a multi-locus marker barcode library to identify and discriminate the recorded plant species. This study aimed to determine the reliability of the available Arabian plant barcodes (>1500; rbcL and matK) at the public repository (NCBI GenBank) using the unsupervised and supervised methods. Comparative analysis was carried out with the standard dataset (FINBOL) to assess the methods and markers’ reliability. Our analysis suggests that from the unsupervised method, TaxonDNA’s All Species Barcode criterion (ASB) exhibits the highest accuracy for rbcL barcodes, followed by the matK barcodes using the aligned dataset (FINBOL). However, for the Arabian plant barcode dataset (GBMA), the supervised method performed better than the unsupervised method, where the Random Forest and K-Nearest Neighbor (gappy kernel) classifiers were robust enough. These classifiers successfully recognized true species from both barcode markers belonging to the aligned and alignment-free datasets, respectively. The multi-class classifier showed high species resolution following the two classifiers, though its performance declined when employed to recognize true species. Similar results were observed for the FINBOL dataset through the supervised learning approach; overall, matK marker showed higher accuracy than rbcL. However, the lower rate of species identification in matK in GBMA data could be due to the higher evolutionary rate or gaps and missing data, as observed for the ASB criterion in the FINBOL dataset. Further, a lower number of sequences and singletons could also affect the rate of species resolution, as observed in the GBMA dataset. The GBMA dataset lacks sufficient species membership. We would encourage the taxonomists from the Arabian Peninsula to join our campaign on the Arabian Barcode of Life at the Barcode of Life Data (BOLD) systems. Our efforts together could help improve the rate of species identification for the Arabian Vascular plants.

GENETIC DIVERSITY OF CYSTODISCUS SPECIES IN AMPHIBIANS IN THE SOUTHERN UNITED STATES.

Myxosporean species in the genus Cystodiscus are parasites of amphibians and have been reported from several continents. Typically used for the identification of myxozoans, the spores produced by these species are similar to one another, possessing 2 polar capsules and being ovoid. The number of transverse depressions on the spore can be useful for delineating species, but these can sometimes be difficult to distinguish. In North America, Cystodiscus serotinus and Cystodiscus melleni have been described, and for C. serotinus in particular, numerous reports and a wide range of hosts have been associated with this species. Given the challenges of identifying some of these species, we questioned whether all encounters of Cystodiscus species can be attributed to these 2 described species, or if there may be additional undescribed species or cryptic species. Over 7 yr, 383 amphibians representing 13 species of toads, frogs, and salamanders were collected from sites in Oklahoma and Arkansas. Cystodiscus infections were found in 56 individuals (14.6%). Tissues from these infected individuals were preserved in alcohol for genetic analysis. The small subunit (SSU) and large subunit (LSU) ribosomal RNA genes were partially sequenced and analyzed phylogenetically. Nine distinct SSU sequence types and 7 distinct LSU sequence types were identified. Phylogenetically, sequence types were attributable to C. serotinus, C. melleni, Cystodiscus axonis, and an undescribed species. For the previously described species, there were multiple SSU sequence types: 4 for C. serotinus and 2 for both C. melleni and C. axonis. Phylogenetic patterns were similar for the LSU sequence analysis using a shorter sequence than the SSU, and we propose that the LSU is useful for initial barcoding of Cystodiscus species in any future surveys. In our qualitative assessment of sequence types compared to geography and host species, SSU types C1 and C2 (C. axonis) were only found in Union County, Arkansas, and McCurtain County, Oklahoma, respectively. Also, salamanders were only infected with SSU types B or D (C. melleni), and type B was only found in salamanders. Our finding of C. axonis in North America is notable because this species was described in Australia and is associated with host pathology. Our work reveals that there are cryptic species of Cystodiscus in the United States, one of which may be a pathogen, highlighting the importance of genetic analysis for future surveys of these species.

Assessing candidate DNA barcodes for Chinese and internationally traded timber species.

Accurate identification of species from timber is an essential step to help control illegal logging and forest loss. However, current approaches to timber identification based on morphological and anatomical characteristics have limited species resolution. DNA barcoding is a proven tool for plant species identification, but there is a need to build reliable reference data across broad taxonomic and spatial scales. Here, we construct a species barcoding library consisting of 1550 taxonomically diverse timber species from 656 genera and 124 families, representing a comprehensive genetic reference data set for Chinese timber species and international commercial traded timber species, using four barcodes (rbcL, matK, trnH-psbA, and ITS2). The ITS2 fragment was found to be the most efficient locus for Chinese timber species identification among the four barcodes tested, both at the species and genus level, despite its low recovery rate. Nevertheless, the barcode combination matK+trnH-psbA+ITS2 was required as a complementary barcode to distinguish closely related species in complex data sets involving internationally traded timber species. Comparative analyses of family-level discrimination and species/genus ratios indicated that the inclusion of closely related species is an important factor affecting the resolution ability of barcodes for timber species verification. Our study indicates that although nuclear ITS2 is the most efficient single barcode for timber species authentication in China, complementary combinations like matK+trnH-psbA+ITS2 are required to provide broader discrimination power. These newly-generated sequences enrich the existing publicly available databases, especially for tropical and subtropical evergreen timber trees and this current timber species barcode reference library can serve as an important genetic resource for forestry monitoring, illegal logging prosecution and biodiversity projects.

CURRENT PERSPECTIVES OF DNA BARCODING IN BOSNIA AND HERZEGOVINA THROUGH BOLD DATABASE

UDK: 577.212:574]:007.5(497.6)
 DNA barcoding is a method designed to provide rapid and precise species identifications by using one or more of short gene sequences called barcodes. In most plant and fungi studies, the standard barcodes of choice are three plastid (rbcL, matK and trnH-psbA) and one nuclear (ITS) gene regions. The relatively high, but comparatively conserved rate of sequence evolution of mtDNA has made COI the marker of choice in animals. BOLD is a freely available cloud-based data storage and analysis platform developed with the aim to advance biodiversity science through DNA barcoding species identification. To date, over 6 million barcodes have been deposited in BOLD with 196,000 animal species, 68,000 plant species and 22,000 species of fungi and other organism entries. In this database, there are currently 447 entries for organisms from Bosnia and Herzegovina, which makes 0.0067% of the total number of BOLD entries. According to BOLD statistics, only 1.11% of all organism entries from B&H were submitted by B&H institutions.
 Despite the fact that Bosnia and Herzegovina has valuable natural resources with a high percentage of endemic and autochthonous species, BOLD statistics elucidated the lack of coordinated and systematic DNA barcoding research so far. It is necessary to establish continuous progress of molecular-genetic characterization of these resources in the future. It is up to B&H institutions to decide if they want to continue the practice of submitting the data sporadically or if they will animate the research community to actively participate in this global project.

The perspectives for DNA barcoding of Rhaponticum carthamoides (Willd.) Iljin using rbcL gene sequence

The methods of biological species identification using nucleotide sequences of short genome regions (DNA barcoding) are actively developed. The universal DNA barcode for plants remains to be discovered, and one of the leading candidates is the plastid gene of the large subunit of ribulose-bisphosphate carboxylase gene (rbcL). In our study, we estimated the part of rbcL gene as a possible marker for molecular identification of Rhaponticum carthamoides (Willd.) Iljin. Due to its officinal properties, the species is susceptible to uncontrolled and illegal harvesting from natural populations. Today, the species needs to be protected and therefore is included into the Red Data Books of the Russian Federation and certain regions. The study was carried out using plants from the natural populations sampled from the Khamar-Daban Ridge (South Siberia) and considering now as Rh. carthamoides var. chamarense (Peschkova) O S Zhirova. It was shown that rbcL gene can be used to identify Rh. carthamoides at least from the populations of the Khamar-Daban Ridge using a fragment of the maximum length or its 3’ region. Apparently, the 5’ region of the gene (rbcLa) most often used as DNA barcode for plants may be of lesser importance for Rh. carthamoides. The rbcL gene sequences can be also used for the development of approaches for Rh. carthamoides identification in the medicinal preparations and products containing dried tissues to prevent their falsification and illegal harvesting of this species. The combination of rbcL gene with additional markers seems to be highly desirable to create effective DNA barcodes for Rhaponticum species.

Barcoding of Some Plant Species Using rbcL Gene ‎in the Mediterranean Oolitic Sand Dunes West of Alexandria, Egypt

In the Mediterranean Basin, Oolitic sand dunes extend along the northeastern coast of Africa between Gulf of Gabes (Tunisia) and the Nile Delta (Egypt) attenuating coastal vulnerability to storms and saltwater intrusion and protecting the inland infrastructure. Psammophytes are the typical and distinctive plants for dunes habitat; they are the primary driver for dune growth and stabilization through trapping sand particles with their shoot and root systems. Anthropocene fingerprint may create an overall environment favorable for non-psammophytes invasion and subsequent alteration in the sand dunes habitat structure and functions. The authentication of sand dunes-inhabiting species should be a priority for conservation or restoration measures concerning sand dunes habitat. The present study is the first rbcL-based DNA barcoding documentation for 20 plant species inhabiting the vulnerable Egyptian oolitic sand dune habitats in Burg El-Arab area, west of Alexandria. The results highlighted the applicability of the rbcL locus sequence for species authentication and recognition of the examined plant taxa sharing the same habitat. The phylogenetic analysis of the rbcL barcodes polymorphism revealed the current taxonomic relationships between the assigned species. However, the ‎results add valuable information to the authentication of sand dune plants where it provided the first rbcL barcodes for species growing in the Egyptian oolitic sand dune-habitat in Egypt of which 6 were submitted for the first time to Gene Bank.

The InBIO Barcoding Initiative Database: DNA barcodes of Portuguese Hemiptera 01

BackgroundThe InBIO Barcoding Initiative (IBI) Hemiptera 01 dataset contains records of 131 specimens of Hemiptera. Most specimens have been morphologically identified to species or subspecies level and represent 88 species in total. The species of this dataset correspond to about 7.3% of continental Portuguese hemipteran species diversity. All specimens were collected in continental Portugal. Sampling took place from 2015 to 2019 and specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources.New informationThis dataset increases the knowledge on the DNA barcodes and distribution of 88 species of Hemiptera from Portugal. Six species, from five different families, were new additions to the Barcode of Life Data System (BOLD), with another twenty five species barcodes' added from under-represented taxa in BOLD. All specimens have their DNA barcodes publicly accessible through BOLD online database and the distribution data can be accessed through the Global Biodiversity Information Facility (GBIF). Eutettix variabilis and Fieberiella florii are recorded for the first time for Portugal and Siphanta acuta, an invasive species, previously reported from the Portuguese Azores archipelago, is recorded for the first time for continental Portugal.

DNA barcode sheds light on species boundaries in the common morphologically variable rove beetle Quedius umbrinus-complex that puzzled taxonomists for more than a century (Coleoptera, Staphylinidae)

In the course of a long taxonomic history, Quedius umbrinus Erichson, a common West Palaearctic rove beetle, was gradually split into a dozen species before all of them, except Q. sigwalti Coiffait from Crete, were lumped back into a single species again. All these shifts were based on the intuitive evaluation of morphological variation only. We sequenced the barcoding fragment of the CO1 gene across specimens of Q. umbrinus-complex broadly covering its geographic range and performed several DNA barcode species delimitation analyses on this dataset: tree-based Bayesian inference and Maximum likelihood, as well as tree-independent character-based (PTP, GMYC) and distance-based (ABGD). Molecular clades were largely congruent among all analyses and revealed four candidate species lineages. Of them, only Q. sigwalti is easy to distinguish by morphology alone. Morphological differences between other candidate species are present, but they are weak and do not hold for all specimens. To reflect new data in the classification we reinstated Q. pseudoumbrinus Lohse (stat. res.) as a species even though it is not always distinct from Q. umbrinus Er. (stat. rev.) morphologically. Also, we described a new species Q. volkeri (sp. nov.) from the north-western Caucasus. Finally, we placed Q. angaricus Coiffat in synonymy with Q. pseudoumbrinus Lohse (syn. nov.). http://www.zoobank.org/urn:lsid:zoobank.org:act:B6E2ED11-2029-48E7-975F-060D9DA61648

DNA Barcodes Applied to a Rapid Baseline Construction in Biodiversity Monitoring for the Conservation of Aquatic Ecosystems in the Sian Ka’an Reserve (Mexico) and Adjacent Areas

This study is focused on the aquatic environments of the Sian Ka’an reserve, a World Heritage Site. We applied recently developed protocols for the rapid assessment of most animal taxa inhabiting any freshwater system using light traps and DNA barcodes, represented by the mitochondrial gene Cytochrome Oxidase I (COI). We DNA barcoded 1037 specimens comprising mites, crustaceans, insects, and fish larvae from 13 aquatic environments close or inside the reserve, with a success rate of 99.8%. In total, 167 barcode index numbers (BINs) were detected. From them, we identified 43 species. All others remain as a BIN. Besides, we applied the non-invasive method of environmental DNA (eDNA) to analyze the adult fish communities and identified the sequences obtained with the Barcode of Life Database (BOLD). All round, we found 25 fish species and other terrestrial vertebrates from this region. No alien species was found. After comparing the BINs from all systems, we found that each water body was unique with respect to the communities observed. The reference library presented here represents the first step for future programs to detect any change in these ecosystems, including invasive species, and to improve the knowledge of freshwater zooplankton, enhancing the task of compiling the species barcodes not yet stored in databases (such as BOLD or GenBank).

Genomic analysis of the lectotype specimens of European Ulva rigida and Ulva lacinulata (Ulvaceae, Chlorophyta) reveals the ongoing misapplication of names

ABSTRACT Species of Ulva Linnaeus are nearly impossible to identify using morpho-anatomy due to their simple thallus structure and phenotypic plasticity. The current solution to this problem is to sequence DNA from field-collected specimens and match these sequences to those available in public DNA databases. However, because type specimens of many species have not been sequenced, the accuracy of these identifications is highly doubtful. Ulva rigida C.Agardh is reported to be one of the most widespread and ecologically important Ulva species, but these records are based on either morpho-anatomy or, more recently, on DNA sequences. High-throughput sequencing (HTS) was performed on the lectotype specimen of U. rigida from Cádiz, Spain to determine the correct application of the name. The analysis yielded its complete plastid genome. rbcL, tufA and ITS sequences from the lectotype specimen differed at the species level from all U. rigida sequences deposited in public databases. Instead, the lectotype sequences of U. rigida were identical or very similar to sequences identified as U. rotundata Bliding (referred to by some as U. pseudorotundata Cormaci, G.Furnari & Alongi) from Ireland and Portugal, but not to the holotype of U. rotundata from Italy, which was identical to U. lactuca L. HTS of the lectotype of U. lacinulata (Kützing) Wittrock from Lesina, Croatia, a species morphologically similar to U. rigida with macroscopic marginal teeth, also yielded a complete plastid genome, with sequences identical or highly similar to GenBank U. armoricana Dion, Reviers & Coat, U. ‘laetevirens’, U. ‘rigida’ and U. scandinavica Bliding. Since U. lacinulata is the oldest validly published name, it is the correct one to apply to the globally distributed species that was previously but incorrectly known as U. rigida. Based on this genetic evidence, U. rigida is restricted to European waters and confirmed by DNA sequences from Ireland, Portugal and Spain. This analysis shows that many barcode species identifications and taxonomic conclusions in the genus Ulva are incorrect.