Mitochondrial Barcode Research Articles

Description and Molecular Characterization of Richtersius ziemowiti sp. nov. (Richtersiidae) from Nepal (Asia) with Evidence of Heterozygous Point Mutation Events in the 28S rRNA

Applying an integrative taxonomy approach, we describe a new tardigrade species from Nepal. Light and scanning electron microscopy are used for morphological as well as morphometric analysis of the new species. Furthermore, multilocus molecular data (nuclear markers, i.e., 18S rRNA, 28S rRNA, ITS-2 and mitochondrial COI barcode sequences) were analysed to confirm the novelty of species. The p–distances enabled an independent verification of Richtersius ziemowitisp. nov. as species new to science and molecular data allowed us to determine the phylogenetic position of the new species. Morphologically, Richtersius ziemowitisp. nov. differs from nominal Ric. coronifer mainly by lower pores density on cuticle, smaller eggs and lower number of processes on the egg circumference. What is more, we identified double (overlapping) peaks in mutated loci in the 28S rRNA chromatograms and obtained heterozygous sequences.

Revision of Eudorylas Aczél, 1940 (Diptera, Pipunculidae) in the Middle East, with the description of four new species.

BackgroundThe Middle Eastern species of Eudorylas Aczél, 1940 are revised through an integrative taxonomic approach by combining morphological and sequence data from the mitochondrial COI barcoding gene. Four new species of the genus Eudorylas are described, males and females of three species are associated, DNA sequence data of 11 Middle Eastern Eudorylas species are provided and 15 additional species are discussed. To facilitate their recognition, we provide diagnoses, descriptions, an identification key and distributional maps for all species.New informationThe following new species are described from the Middle East: E.avis Motamedinia & Skevington sp. n., E.bihamatus Motamedinia & Skevington sp. n., E.corniculans Motamedinia & Skevington sp. n., E.nasicus Motamedinia & Skevington sp. n.

Canthigaster aziz, a new deep-dwelling toby fish (Tetraodontiformes: Tetraodontidae) from the Red Sea.

A new species of toby fish, Canthigaster aziz, is described based on a single specimen collected from the northern Red Sea off Saudi Arabia. The holotype was trawled from a depth of 315 m, the second deepest record for the genus. The new species is distinguished from other species of the genus by the following combination of characters: 8 dorsal-fin rays; 8 anal-fin rays; 15 pectoral-fin rays; dorsal-fin origin opposite to anal-fin origin; five diffuse, saddle-like, black blotches along dark yellowish dorsal edge of body between nape and dorsal-fin origin; dorsal half of body light brown with concentrated dark pigments just behind eye and with a longitudinal, irregular, pale golden stripe running from area just behind eye to dorsal side of caudal peduncle; ventral half of posterior part of body pinkish with tiny subcutaneous black spots; head and ventral half of body before anus white; and all fins uniformly pale grey. A phylogenetic analysis of the mitochondrial COI barcoding region resulted in a new and unique evolutionary lineage for the new species that is sister to a clade composed of C. leoparda, C. pygmaea and C. valentini. It also shows C. aziz to be evolutionary deeply divergent from its closest congeners. In addition to the description of the new species, comparisons with congeners and a revised key to the Indo-Pacific species are provided.

How consistent is RAD-seq divergence with DNA-barcode based clustering in insects?

Promoted by the barcoding approach, mitochondrial DNA is more than ever used as a molecular marker to identify species boundaries. Yet, it has been repeatedly argued that it may be poorly suited for this purpose, especially in insects where mitochondria are often associated with invasive intracellular bacteria that may promote their introgression. Here, we inform this debate by assessing how divergent nuclear genomes can be when mitochondrial barcodes indicate very high proximity. To this end, we obtained RAD-seq data from 92 barcode-based species-like units (operational taxonomic units [OTUs]) spanning four insect orders. In 100% of the cases, the observed median nuclear divergence was lower than 2%, a value that was recently estimated as one below which nuclear gene flow is not uncommon. These results suggest that although mitochondria may occasionally leak between species, this process is rare enough in insects to make DNA barcoding a reliable tool for clustering specimens into species-like units.

Phylogenetic position and morphological descriptions ofChitonspecies from the south-eastern Pacific

AbstractIn the south-eastern Pacific Ocean (SEP), six species of the genus Chiton have been recorded. However, the taxonomic status and geographic distribution of some of these species is controversial. In this study, we compare and describe Chiton species in the SEP. More than 3000 specimens of six species from 49 localities were analysed for morphological features. Among these, 27 specimens of the six species from the SEP were sequenced for the mitochondrial barcode region (COI) and ribosomal 16S and 18S to infer phylogenetic relationships. Phylogenetic reconstruction indicates that the genus Chiton in the SEP represents a polyphyletic group and confirms the placement of C. barnesii in Chiton and not in Radsia, as previously suggested. Among Chiton s.s., C. cumingsii is the sister-species of C. magnificus, while C. granosus is a sister-species of C. barnesii. This analysis also indicates that C. bowenii is a junior synonym of C. magnificus. Our results improved the knowledge of systematics and distribution of Chiton species in the SEP, suggesting the presence of five valid species of the genus. In this study, we designate a lectotype for C. cumingsii and a neotype for C. granosus.

Taxonomic determination of the cryptogenic red alga,Chondria tumulosasp. nov.,(Rhodomelaceae, Rhodophyta) fromPapahānaumokuākea Marine National Monument, Hawai'i, USA: A new species displaying invasive characteristics.

Survey cruises by the National Oceanic and Atmospheric Administration (NOAA) in 2016 and 2019 yielded specimens of an undetermined red alga that rapidly attained alarming levels of benthic coverage at Pearl and Hermes Atoll, Papahānaumokuākea Marine National Monument, Hawaiʻi. By 2019 the seaweed had covered large expanses on the northeast side of the atoll with mat-like, extensive growth of entangled thalli. Specimens were analyzed using light microscopy and molecular analysis, and were compared to morphological descriptions in the literature for closely related taxa. Light microscopy demonstrated that the specimens likely belonged to the rhodomelacean genus Chondria, yet comparisons to taxonomic literature revealed no morphological match. DNA sequence analyses of the mitochondrial COI barcode marker, the plastidial rbcL gene, and the nuclear SSU gene confirmed its genus-level placement and demonstrated that this alga was unique compared to all other available sequences. Based on these data, this cryptogenic seaweed is here proposed as a new species: Chondria tumulosa A.R.Sherwood & J.M.Huisman sp. nov. Chondria tumulosa is distinct from all other species of Chondria based on its large, robust thalli, a mat-forming tendency, large axial diameter in mature branches (which decreases in diameter with subsequent orders of branching), terete axes, and bluntly rounded apices. Although C. tumulosa does not meet the criteria for the definition of an invasive species given that it has not been confirmed as introduced to Pearl and Hermes Atoll, this seaweed is not closely related to any known Hawaiian native species and is of particular concern given its sudden appearance and rapid increase in abundance in the Papahānaumokuākea Marine National Monument; an uninhabited, remote, and pristine island chain to the northwest of the Main Hawaiian Islands.

Character-based identification system of scombrids from Indian waters for authentication and conservation purposes

Scombrids are the important component of pelagic fishery resources which include 54 species under 15 genera commonly known as mackerels, bonitos, and tunas. Due to the high commercial value attained, there are real chances of fraudulent substitution by species of inferior value. DNA based species identification methods can be applied to detect product adulteration, as well as to better contribute to the conservation and management of these species by providing accurate species identification independently of the age of the individuals or the tissue processed. In this study, a total of 15 commercially important scombrid species from Indian waters were analyzed. Due to the inadequacy of mitochondrial COI barcoding gene in discriminating between some Thunnus species, cytochrome b sequences were used instead. For all the 15 species, we propose a DNA character-based keys which uses a diagnostic combination of nucleotides and respective probes, including the first character-based keys and probes to differentiate between Thunnus albacares and T. obsesus.

Molecular diversity of freshwater fishes of Bangladesh assessed by DNA barcoding

This study represents the comprehensive molecular identification of freshwater fishes of Bangladesh based on a fragment of the cytochrome c oxidase subunit I (COI) gene in the mitochondrial genome. A total of 315 mitochondrial COI barcode sequences were obtained from 153 species,114 genera, 49 families and 16 orders of fishes. The mean length of the sequences was 652 base pairs. For all the samples, %G was significantly lower compared to the other nucleotides and %GC was lower compared to %AT (p-value ˂ 0.05). Also, a significantly lower %GC content was observed in second and third codon position compared to the first one in all the samples (1st>2nd>3rd, p-value˂ 0.05). The average K2P distances within species, genera, families and orders were 0.38%, 7.02%, 12.75% and 18.68%, respectively. The mean interspecific distance was 18-fold higher than the mean intraspecific distance. The K2P neighbor-joining (NJ) trees based on the sequences generally clustered species according to their taxonomic position. A total of 12 species were newly recorded in Bangladesh. High efficiency in species identification were demonstrated in the present study by DNA barcoding, and concluded that COI sequencing can be used as an authentic identification marker for freshwater fish species.
 Bangladesh J. Zool. 48(1): 1-19, 2020

Diamond frogs forever: a new species of Rhombophryne Boettger, 1880 (Microhylidae, Cophylinae) from Montagne d’Ambre National Park, northern Madagascar

Although taxonomic progress on the frogs of Madagascar is currently proceeding at an unprecedented pace, the goal of completing the amphibian inventory of this hyper-diverse island is still far off. In part this is because more new species continue to be discovered at a high rate, in some cases within well-studied areas. Here, I describeRhombophryne ellaesp. nov., a new species of diamond frog discovered in Montagne d’Ambre National Park in northern Madagascar in 2017. This new species is highly distinctive in having orange flash-markings on its hindlimbs (not known from any described species ofRhombophryne), and large, black inguinal spots (larger than in all other describedRhombophrynespecies). It is separated from all named species ofRhombophryneby a substantial uncorrected pairwise distance in the 16S rRNA mitochondrial barcode marker (> 7%) and is most closely related to an undescribed candidate species from Tsaratanana in northern Madagascar.Rhombophryne ellaesp. nov.adds another taxon to the growing list of cophyline microhylids that have red to orange flash-markings, the function of which remains unknown and which has clearly evolved repeatedly in this radiation. The discovery of such a distinctive species within a comparatively well-studied park points toward the low detectability of semi-fossorial frogs and the role of inclement weather in increasing that detectability.

The puzzling mitochondrial phylogeography of the black soldier fly (Hermetia illucens), the commercially most important insect protein species

BackgroundThe black soldier fly (Diptera: Stratiomyidae, Hermetia illucens) is renowned for its bioconversion ability of organic matter, and is the worldwide most widely used source of insect protein. Despite varying extensively in morphology, it is widely assumed that all black soldier flies belong to the same species, Hermetia illucens. We here screened about 600 field-collected and cultured flies from 39 countries and six biogeographic regions to test this assumption based on data for three genes (mitochondrial COI, nuclear ITS2 & 28S rDNA) and in order to gain insights into the phylogeography of the species.ResultsOur study reveals a surprisingly high level of intraspecific genetic diversity for the mitochondrial barcoding gene COI (divergences up to 4.9%). This level of variability is often associated with the presence of multiple species, but tested nuclear markers (ITS2 and 28S rDNA) were invariant and fly strain hybridization experiments under laboratory conditions revealed reproductive compatibility. COI haplotype diversity is not only very high in all biogeographic regions (56 distinct haplotypes in total), but also in breeding facilities and research centers from six continents (10 haplotypes: divergences up to 4.3%). The high genetic diversity in fly-breeding facilities is mostly likely due to many independent acquisitions of cultures via sharing and/or establishing new colonies from field-collected flies. However, explaining some of the observed diversity in several biogeographic regions is difficult given that the origin of the species is considered to be New World (32 distinct haplotypes) and one would expect severely reduced genetic diversity in the putatively non-native populations in the remaining biogeographic regions. However, distinct, private haplotypes are known from the Australasian (N = 1), Oriental (N = 4), and the Eastern Palearctic (N = 4) populations. We reviewed museum specimen records and conclude that the evidence for introductions is strong for the Western Palearctic and Afrotropical regions which lack distinct, private haplotypes.ConclusionsBased on the results of this paper, we urge the black soldier fly community to apply molecular characterization (genotyping) of the fly strains used in artificial fly-breeding and share these data in research publications as well as when sharing cultures. In addition, fast-evolving nuclear markers should be used to reconstruct the recent invasion history of the species.

Species delimitation analyses of NE Atlantic Chaetozone (Annelida, Cirratulidae) reveals hidden diversity among a common and abundant marine annelid

The polychaetes of the family Cirratulidae (Annelida) are common inhabitants in continental shelf benthic environments and considered an important group of organisms in environmental monitoring surveys. The family represents a taxonomic and systematic challenge, as monophyly of genera and evolutionary relationships within the family remain to be explored in a proper phylogenetic framework. Bitentaculate cirratulids, especially the genus Chaetozone, form one of the most species-diverse group of polychaetes worldwide. In this study, we aimed at evaluating the species diversity of the genus Chaetozonein benthic environments in the North East Atlantic by molecular means. We tested whether traditional morphological diagnostic characters are able to discriminate between the species hypothesis after species delimitation analyses, and assessed monophyly of the genera involved. Two DNA markers were sequenced from about 200 specimens belonging to Chaetozone, Aphelochaeta, Dodecaceria, Cirriformia and Cirratulus – the universal mitochondrial barcoding region COI, and the D1-D2 regions of the nuclear 28S rRNA – and analyzed with Bayesian inference, Maximum Likelihood and the species delimitation methods mPTP and GMYC. The first phylogeny of the family Cirratulidae is inferred and the genera Chaetozone, Dodecaceria and Cirratulus are recovered monophyletic. A total of 14 clusters of sequences – corresponding to species of Chaetozone – were found in the study area, and only one of them is here referred to a nominal species, Chaetozone setosa. Our results reveal several species complexes in the genus Chaetozone, that some of these independent lineages are unnamed and undescribed, and that morphological diagnostic features are in most cases unable to discriminate between the most similar species.

First record of the moorland clouded yellow Colias palaeno (Linnaeus, 1761) (Lepidoptera: Pieridae) in Altai Krai (Russia, West Siberia) with notes on its DNA barcode

Colias palaeno (Linnaeus, 1761) is reported from Altai Krai for the first time. The DNA barcode of the collected specimen was analysed and compared with molecular data on European populations of C. palaeno available in the public databases GenBank (http://www.ncbi.nlm.nih.gov) and BOLD (http://www.boldsystems.org). The molecular analysis has shown that the specimen from Altai Krai shares mitochondrial barcode with some specimens from mountain populations of the Alps and the Czech Republic, and differs significantly from lowland populations of C. palaeno from Central and Northern Europe.

Re-description and range extension of the Afrotropical mayfly Cloeon perkinsi (Ephemeroptera, Baetidae)

Cloeon perkinsi was described from South Africa in 1932 by Barnard. Despite being relatively common in Africa, it was mentioned in the literature quite rarely, and its known distribution to date includes most of sub-Saharan Africa. Material collected recently in Ethiopia, Israel, Saudi Arabia, and Yemen extends its distribution in East Africa, Arabian Peninsula and the Levant. We examined this material, and provide a re-description of adults (females and males) and nymphs of the species. It represents a much-needed urge mainly due to inconsistencies in literature reports regarding colouration, and sometimes incomplete morphological description of all stages. We demonstrate the intraspecific morphological variability that we have witnessed, and provide information regarding the range of habitats colonised by C. perkinsi. Based on geological and climatic history of the studied region, taken together with among countries genetic distances of the mitochondrial barcoding gene COI, we propose colonisation mechanisms for the north-easternmost limit of distribution. The fragmented distribution pattern of the species highlights the conservation importance of isolated aquatic habitats in the region, as well as current knowledge gaps.

Description of a new snake eel Ophichthus olivaceus (Teleostei: Anguilliformes, Ophichthidae) from the Red Sea.

A new species of snake eel Ophichthus olivaceus is described based on two specimens trawled from a depth of 35-63 m from a soft substratum off Jizan, Red Sea coast of southern Saudi Arabia. It differs from its congeners by the following combination of characters: vertebrae 141-145; tail moderately short (2.15 in TL); head short (9.6-11.1 in TL); uniserial teeth in jaws and on vomer; pectoral fins slightly elongate, not lanceolate, upper rays longer than the lower; dorsal-fin origin above middle of pectoral fin; and a generally uniform, dark tan body with an olivaceous hue shading to tan or pale orange ventrally, with two pale yellow blotches above pectoral-fin base, snout and lower jaw dark brown, and olivaceous median fins. Its divergence from other mitochondrial-analyzed species is shown by phylogenetic analysis of the mitochondrial COI barcoding region. A key to the Indian Ocean species is provided.

Contribution to the mayflies (Insecta, Ephemeroptera) of Israel and the Palestinian Authority

We here list four unreported mayfly species from Israel and the Palestinian Authority: Cloeon vanharteni Gattolliat & Sartori, 2008, Cheleocloeon soldani Gattolliat & Sartori, 2008, Oligoneuriella orontensis Koch, 1980 and Prosopistoma oronti Alouf, 1977. They are reported, morphologically distinguished from other local relatives, and sequenced for their mitochondrial COI barcoding segment. Two species, C. vanharteni and Ch. soldani, are known from the Arabian Peninsula, while O. orontensis and P. oronti are Levantine (with wide or more restricted ranges), emphasizing the importance of the Jordan River catchment as a biogeographic meeting-point.

Taxonomic revision of Dasydorylas Skevington, 2001 (Diptera, Pipunculidae) in the Middle East.

Species of the distinctive and cosmopolitan genus Dasydorylas Skevington, 2001 in the Middle East are revised. Seven species are documented, and three new species, Dasydorylas dactylos sp. nov., D. forcipus sp. nov. and D. parazardouei sp. nov., are described, and one synonym, D. derafshani Motamedinia & Kehlmaier, 2017, syn. nov. is proposed, based on sequence information from the mitochondrial COI barcoding gene and morphological parameters. Diagnoses, illustrations and distributional data are provided for all studied species. Descriptions of new species as well as an identification key to all known species in the Middle East are also provided.

Acusicola margulisae n. sp. (Copepoda: Ergasilidae) from freshwater fishes in a Nicaraguan crater lake based on morphological and molecular evidence.

The ergasilid copepod Acusicola margulisae n. sp. is described based on material from three species of cichlid, Amphilophus citrinellus (Günther), Parachromis managuensis (Günther), and Oreochromis sp., and from the poecilid Poecilia mexicana (Steindachner), in the crater Lake Asososca León, Nicaragua. This constitutes the 15th species described in the genus Acusicola Cressey, 1970. The new species differs from all its congeners by the relatively longer first endopodal segment of leg 1, and the size and number of setae on second endopodal segment of leg 1. We provide the first gene sequence for a species of Acusicola. To examine the intraspecific genetic variation of the new species collected from different host species, sequences of the mitochondrial barcode region cox1 were generated. In addition, partial regions of the 18S and 28S ribosomal RNA genes were sequenced and used to infer the phylogenetic relationships of the genus Acusicola within the family Ergasilidae Burmeister, 1835. The phylogenetic trees yielded the isolates of Acusicola margulisae n. sp. as a reciprocally monophyletic lineage, and as the sister taxa of five genera of ergasilid copepods. The genus Ergasilus von Nordmann, 1832 was recovered as a paraphyletic group. These analyses indicate that phylogenetic relationships are not yet well resolved and more representative species and genera of the family are required to provide a robust classification of this highly diverse group of copepods.

Invasion History of Sirex noctilio Based on COI Sequence: The First Six Years in China.

Sirex noctilio F. (Hymenoptera: Siricidae: Siricinae), a new invasive species in China, is a significant international forestry pest which, transported via logs and related wood packing materials, has led to environmental damage and substantial economic loss in many countries around the world. It was first detected in China in 2013, and since then infestations have been found in 18 additional sites. Using a 322 bp fragment of the mitochondrial barcode gene COI, we studied the genetic diversity and structure of S. noctilio populations in both native and invaded ranges, with a specific focus in China. Twelve haplotypes were found across the native and invaded distribution of the pest, of which three were dominant; among these there were only one or two mutational steps between each pair of haplotypes. No obvious genetic structure was found other than in Chinese populations. China has a unique and dominant haplotype not found elsewhere, and compared with the rest of the world, the genetic structure of Chinese populations suggested a multiple invasion scenario.

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

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

DNA barcoding for identification of marine gastropod species from Hainan island, China

Morphological identification of gastropods can be difficult considering the larva of species and high similarity among the same genera. DNA barcoding has been widely used in species identification and biodiversity research. The purpose of this study was to explore the feasibility of using the cytochrome c oxidase subunit I (COI) gene for the identification of gastropod species and to construct a reliable DNA barcoding reference database of gastropods in Hainan island, China. A total of 306 mitochondrial COI barcode sequences were obtained from 120 species, 35 families and 7 orders of gastropods. The average length of the sequence was 640 base pairs. The average genetic distances based on Kimura two parameter (K2P) within species, genera, families, orders and classes were 0.9 %, 14.7 %, 18.9 %, 24.5 % and 28.6 %, respectively. Most of the gastropod species could be identified using COI sequences. Our results confirmed that the identification method combining morphology and DNA barcode greatly improved the efficiency of species identification. In this study, we found three new record species in China, namely Semiricinula tissoti (Petit de la Saussaye, 1852), Engina alveolata (Kiener, 1836) and Wallaconchis ater (Lesson, 1831). Overall, this study revealed that the identification of gastropods by DNA barcoding is efficient, and COI sequencing technology can be used for the identification of gastropod species and thereby can be used to manage fisheries and assess biodiversity.