Panax L., renowned as ginseng genus, is a famous medicinal group of family Araliaceae. Within this genus, the taxa of Panax bipinnatifidus complex are mainly distributed in Himalayas and Hengduan Mountain areas. Due to the complex evolutionary history and short-term rapid radiation, the relationships among species within the complex have not been clearly resolved, and the taxa identification is difficult due to the intermediate morphological traits. This study aimed to use the available restriction-site associated DNA sequence data from 29 individuals of P. bipinnatifidus complex to mine high-polymorphic simple sequence repeat (SSR) markers, with the goal of evaluating their utility in taxa identification. Eleven polymorphic SSR loci were ultimately selected and validated through polymerase chain reactions amplifying across 63 individuals of P. bipinnatifidus complex and 13 individuals of three outgroup species. The subsequent genetic diversity analysis uncovered 76 alleles in total, ranging from 5 to 15 per locus. Observed heterozygosity spanned 0.241–0.512, while expected heterozygosity ranged between 0.345 and 0.644. The genetic kinship analysis revealed a sister relationship between Panax zingiberensis and Panax vietnamensis. The analysis result also supported the classification of samples from Hunan and Hubei provinces into a single genetic unit within the P. bipinnatifidus complex. These newly developed SSR markers will facilitate the identification of wild ginseng plants.

The Cyathocotylidae Mühling, 1898 is a family of primitive diplostomoid trematodes important for understanding the evolution of the superfamily Diplostomoidea. However, cyathocotylids remain poorly studied with the use of molecular techniques. In this study we sequenced the 5.8S + ITS2 region, 28S rRNA, and cox1 genes of two cyathocotylid species and obtained new morphological data on them. We propose Georduboisia nom. nov. instead of the preoccupied name Duboisia Szidat, 1936 (junior homonym of Duboisia Stremme, 1911). Adults of Georduboisia cf. teganuma (Ishii, 1935) and Paracoenogonimus ovatus Katsurada, 1914 were collected from fish-eating birds in the south of the European part of Russia. Georduboisia cf. teganuma was very similar to G. teganuma but differed from it in the shape of the testes. The 28S rRNA gene dataset provided the best-resolved phylogeny of the Cyathocotylidae to date. In the phylogram based on partial sequences of this gene, P. ovatus was close to members of Holostephanoides Dubois, 1983, Neogogatea Chandler & Rausch, 1947 and Gogatea Szidat, 1936. Georduboisia cf. teganuma clustered with members of Cyathocotyle Mühling, 1896 and Holostephanus Szidat, 1936. Phylogenetic analysis based on the 5.8S + ITS2 dataset showed that adults of P. ovatus examined in our study were conspecific with the metacercariae from the musculature of fish collected in Hungary and Italy. It also revealed probable misidentifications of larvae and adults of cyathocotylids whose sequences are deposited in GenBank NCBI.

iNaturalist is a widely-utilized platform for data collection and sharing among non-professional volunteers and is widely employed in citizen science. This platform's data are also used in scientific studies for a wide range of purposes, including tracking changes in species distribution, monitoring the spread of alien-invasive species, and assessing the impacts of urbanization and land-use change on biodiversity. Lichens, due to their year-round presence on trees, soil and rocks, and their diverse shapes and colours, have captured the attention of iNaturalist users, and lichen records are widely represented on the platform. However, due to the complexity of lichen identification, the use of data collected by untrained, or poorly trained volunteers in scientific investigation poses concerns among lichenologists. To address these concerns, this study assessed the reliability of lichen identification by iNaturalist users by comparing records on the platform with identifications carried out by experts (experienced lichenologists) in three cities where citizen science projects were developed. Results of this study caution against the use of unchecked data obtained from the platform in lichenology, demonstrating substantial inconsistency between results gathered by iNaturalist users and experts.

In this research communication we describe a straightforward triplex-PCR protocol able to differentiate the origin of milk from three closely related species (goat, sheep and cow) in Halloumi, a cheese with Protected Designation of Origin (PDO), and yogurts. Halloumi must contain at least 51% sheep or goat milk, therefore, the fraudulent adulteration of this cheese with excess of cow milk must be routinely tested. The assay employs one universal forward primer and three species-specific reverse primers giving rise to 287 bp (cow), 313 bp (goat), and 336 bp (sheep) amplicons, under the same amplification conditions. This protocol, when used to test a small number of Cyprus commercial products, correctly detected mislabeling in Halloumi (2 out of 6 samples were adulterated) and yogurt brands (1 out of 4 was adulterated). The suggested protocol is a reliable tool for identifying the origin of milk in Halloumi cheeses and yogurts and can be used in any laboratory equipped with a thermocycler and an agarose gel electrophoresis apparatus.

The microcotylid Sciaenacotyle pancerii is a pathogenic monogenean infecting Argyrosomus regius, a candidate for species diversification in the Mediterranean aquaculture. Life-history stages of S. pancerii commonly co-occur in field infections, but to date, morphological data have only been provided for oncomiracidia and adults although identifying life-history stages can be useful in infection management. A total of 114 specimens of S. pancerii were analysed to characterize the developmental events and to assess morphological and morphometric variations before and after maturity. The post-larval development of S. pancerii is characterized by: expansion and bifurcation of the gut, loss of the larval haptor, protandrous development of the genitalia and vitellaria formation. The size variability of larval hooks, hamuli and germanium of S. pancerii is firstly reported and dimensional ranges of parasite body, haptor, testes, posteriormost clamps and eggs are widened. The size of most of the diagnostic features of S. pancerii significantly increases after parasite maturity and therefore, only those specimens with more than 116 clamps should be considered for minimising development-related variability in size. The high number of clamps, their fast development and the asymmetry in their size and arrangement suggest that S. pancerii may use a mixed attachment strategy between the closely related microcotylids and heteraxinids. This combination of features may be host related and linked to the gill structure of the sciaenid fish and the phylogenetic position of the genus Sciaenacotyle; distant from other microcotylids while close to heteraxinid species.

The European (Melolontha melolontha L.) and Forest (M. hippocastani F.) cockchafer are widespread pests throughout Central Europe. Both species exhibit a 3–5-year life cycle and occur in temporally shifted populations, which have been monitored and documented for more than 100 years. Visual identification of adults and larvae belonging to these morphologically similar species requires expertise and, particularly in the case of larvae, is challenging and equivocal. The goal of the study was the development of an efficient and fast molecular genetic tool for the identification and discrimination of M. melolontha and M. hippocastani. We established a collection of both species from Switzerland, Austria and Northern Italy in 2016, 2017 and 2018. An approximately 1550 bp long fragment of the cytochrome c oxidase subunit 1 (CO1) mitochondrial gene was amplified and sequenced in 13 M. melolontha and 13 M. hippocastani beetles. Alignment of the new sequences with reference sequences (NCBI GenBank and BOLDSYSTEMS databases) and subsequent phylogenetic analysis revealed consistent clustering of the two species. After the identification of M. melolontha and M. hippocastani species-specific single nucleotide polymorphisms (SNPs) in the CO1 alignment, we developed an effective SNP tool based on the ABI PRISM® SNaPshot™ Multiplex Kit for the rapid and accurate species discrimination of adults and larvae.

With the rapid rise in the prevalence of non-tuberculous mycobacteria (NTM) diseases across the world, the microbiological diagnosis of NTM isolates is becoming increasingly important for the diagnosis and treatment of NTM disease. In this study, the clinical presentation, species distribution and drug susceptibility of patients with NTM disease visiting the Chongqing Public Health Medical Centre during March 2016–April 2019 were retrospectively analysed. Among the 146 patients with NTM disease, eight NTM species (complex) were identified. The predominant NTM species in these patients were identified to be Mycobacterium abscessus complex (53, 36.3%), M. intracellulare (38, 26%) and M. fortuitum (17, 11.7%). In addition, two or more species were isolated from 7.5% of the patients. Pulmonary NTM disease (142, 97.3%) showed the highest prevalence among the patients. It was observed that 40.1% of the patients with pulmonary NTM disease had chronic pulmonary obstructive disease and bronchiectasis, while 22.5% had prior tuberculosis. Male patients showed more association with the conditions of cough and haemoptysis than the female patients. In an in vitro antimicrobial susceptibility testing, most of the species showed susceptibility to linezolid, amikacin and clarithromycin, while M. fortuitum exhibited low susceptibility to tobramycin. In conclusion, the prevalence of NTM disease, especially that of the pulmonary NTM disease, is common in Southwest China. Species identification and drug susceptibility testing are thus extremely important to ensure appropriate treatment regimens for patient care and management.

Hexokinase (HK) is a core glycolytic enzyme of Microsporidia which regulates host cell metabolic processes. The goal of the present study was to test for the utility of HK for molecular phylogenetics, species identification and molecular detection of microsporidia in infected insects. HK sequence-based reconstructions were essentially similar to those based upon largest subunit RNA polymerase (RPB1) gene sequences, as well as previously published rRNA gene and genome-based trees. Comparing HK sequences allowed clear differentiation of closely related taxa, such as Nosema bombycis and Nosema pyrausta. In Nosema ceranae, unique SNPs were found for an isolate from wild colonies of the Burzyan dark honey bee as compared with the isolates from domesticated European honey bee. Similarly, in Encephalitozoon cuniculi, HK was as effective as RPB1 for discrimination of isolates belonging to different ITS genotypes. Amplification using species-specific primers flanking short fragments at the 3′-end of HK gene showed the presence of infection in insect tissues infected with N. pyrausta, Nosema ceranae and Paranosema (Antonospora) locustae. For the latter parasite species, HK expression was also demonstrated at early stages of infection using total mRNA extracts of locust larvae. These results indicate the suitability of HK as a novel tool for molecular genetic studies of Microsporidia.

The genus Acacia Miller is species-rich, and species discrimination is challenging owing to morphological similarities between closely related species. Naming of specimens is particularly difficult in the Middle East, where confusion in taxonomic identification exists within the context of a wider international debate on the generic systematics of Acacia sensu lato. At least five segregate genera for Acacia s.l. have been advocated: Acacia sensu stricto, Vachellia, Senegalia, Acaciella and Mariosousa. Furthermore, identification to species of the only remaining native Acacia s.l. tree in Kuwait is still a matter of controversy. The present study used multilocus chloroplast DNA sequence data analyses following maximum likelihood (ML) and Bayesian approaches to: 1) test the species concepts of Vachellia pachyceras (≡ Acacia pachyceras O.Schwartz) from the Middle East, and Vachellia tortilis (Forssk.) Galasso & Banfi (≡ Acacia tortilis (Forssk.) Hayne) and Vachellia gerrardii (Benth.) P.J.H.Hurter (≡ Acacia gerrardii Benth.) from Kenya, as well as to investigate species divergence times; and 2) identify the only remaining native Acacia s.l. tree in Kuwait (known as the Lonely Tree), as well as other unidentified Acacia s.l. specimens in cultivation. The Bayesian and ML topologies clearly differentiated Vachellia pachyceras, V. tortilis and V. gerrardii, and demonstrated that the three species are distinct. Divergence time estimates using the ML topology suggested that Vachellia gerrardii diverged from a common ancestor no later than the early Pliocene (3.3 Mya), whereas V. pachyceras originated at least 2.0 Mya (Pliocene). The unknown remaining native Acacia s.l. tree in Kuwait and other specimens collected from the nursery were identified as Vachellia pachyceras. These results stress the need to use plastid DNA barcodes complemented by population genetics approaches to address systematic issues in this complex of Acacia s.l. species in the Middle East and the Arabian Peninsula.

Pseudapocryptes elongatus is one of the oxudercine gobies, which show varying degrees of amphibious behaviour and capacities to breathe air. There is little information on the early life history of P. elongatus, particularly of their morphology and larval habitat and duration. This study focused on the life history of larval and juvenile stages of P. elongatus investigated by genetic species identification, morphological observation and otolith analyses using specimens collected in June and October 2012 from estuaries in Bac Liêu Province, southern Vietnam (09°14′N 105°43′E). Genetically identified juvenile P. elongatus were characterized by (1) a slender body form, (2) the anterior edges of both the anal and second dorsal fins located at the midpoint along the body axis, (3) scarce chromatophores over the body surface, (4) melanophores in the parietal region between the eyes, and (5) a single row of melanophores along the base of the anal fin. Mean age at recruitment to estuaries was 38.0 ± 4.1 days, and otolith Sr/Ca ratios ranged from 8.9 to 9.9 mmol mol−1, suggesting that larval migration from their spawning sites requires more than 1 month in saline environments.

Aphids are among pests of economic importance throughout the world. Together with transmitting plant viruses, aphids are capable of inflicting severe crop production losses. They also excrete honeydew that favours the growth of sooty mold which reduces the quality of vegetables and fruits and hence their market values. Rapid and accurate identification of aphids to the species level is a critical component in effective pest management and plant quarantine systems. Even though morphological taxonomy has made a tremendous impact on species-level identifications, polymorphism, morphological plasticity and immature stages are among the many challenges to accurate identification. In addition, their small size, presence of cryptic species and damaged specimens dictate the need for a strategy that will ensure timely and accurate identification. In this study, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based on mitochondrial cytochrome c oxidase subunit I gene and DNA barcoding were applied to identify different aphid species collected from different agro-ecological zones of Kenya. Three restriction enzymes RsaI, AluI and Hinf1 produced patterns that allowed unambiguous identification of the species except Aphis craccivora and Aphis fabae. Analyses of the barcode region indicated intraspecific and interspecific sequence divergences of 0.08 and 6.63%, respectively. DNA barcoding identified all species, including the morphologically indistinguishable A. craccivora and A. fabae and separated two subspecies of A. fabae. Based on these results, both PCR-RFLPs and DNA barcoding could provide quick and accurate tools for identification of aphid species within Aphididae subsequently aiding in effective pest management programmes and enhance plant quarantine systems.

Sea cucumbers are important due to their rotation of key nutrients and high medicinal values. With this in mind and considering the lack of scientific knowledge about them, the purpose of this research was to identify the species of sea cucumber occurring in the intertidal habitats of Hengam Island. Field sampling and surveys were carried out along the coastline based on the geomorphic and habitat characteristics of eight sampling stations in the inter-tidal zone. The samples that were collected from the stations were transferred to the laboratory and identified using morphological keys and a review of their ossicles. By reviewing the literature on the distribution of sea cucumbers, it was determined that a new observation had been made of Holothuria arenicola and Holothuria bacilla from Hengam Island (Persian Gulf).

The anguillid eels found in Peninsular Malaysia were identified using a morphological analysis and that identification was further validated as Anguilla bengalensis bengalensis and Anguilla bicolor bicolor by an analysis of the eels' mitochondrial cytochrome oxidase subunit I (COI) sequences. Because of the difficulty of accurately identifying tropical eels solely on morphological analyses, previous studies had reported the occurrence of the tropical eel species Anguilla marmorata in Peninsular Malaysia. This study suggests the occurrence of Anguilla bengalensis bengalensis in Malaysian waters, confirmed by both morphological and molecular genetic analyses. Anguilla bicolor is further confirmed as the subspecies of Anguilla bicolor bicolor by molecular genetic analyses. The present study also suggests that accurate tropical eel species identification requires validation by molecular genetic analysis after a morphological observation.

A real-time polymerase chain reaction (PCR) assay was developed for the identification and quantification of two oyster species: Ostrea edulis and Crassostrea gigas. Two sets of primers and TaqMan-MGB probes were designed, based on partial sequences of the 16S rRNA gene. An amplification positive control system was also located in the 18S rRNA gene sequences. Closely related species of oysters and other bivalves, known to co-occur with the target species in European waters, were used to test the assay for cross-reactivity. The assay designed was specific for the target species and no signal or no significant signal was detected for all non-target species tested. The high sensitivity of this method was demonstrated since it is possible to detect just one larva (150–200 μm size) of each species even when it is present with others. Furthermore, this assay provided an acceptable quantification of the number of spiked larvae (1, 10 and 100 larvae) in plankton samples employing a standard curve for larvae.

Sea cucumbers have many important and useful properties known for human health. In this respect and with the lack of scientific knowledge about them, this study has been conducted to identify the present species in the northern part of the Persian Gulf. Two species of sea cucumber (one belonging to the genus Holothuria and the other to Stichopus) were collected in the subtidal zone of the northern part of Qeshm Island (Persian Gulf) via SCUBA diving. The literature review on the distribution was determined to be the first report of H. sacabra and S. hermanni from Qeshm Island (Persian Gulf). The species identification was accomplished through morphological keys and review of their ossicles.

Mealybugs (Hemiptera: Pseudococcidae) are common invasive pests in Europe, causing major problems on crops and ornamental plants. However, very few data are available concerning the mealybug fauna of southern Europe. This lack of data and the difficulty of identifying mealybugs morphologically by traditional techniques currently limit the perspectives for efficient specific pest management. The aim of this study was to provide multi-criterion characterization of mealybugs surveyed in eastern Spain in order to facilitate their routine identification through DNA sequencing or the use of derived species-specific molecular tools. We characterised 33 mealybug populations infesting crops and ornamental plants in eastern Spain, using a combination of molecular and morphological techniques, including the sequencing of the universal barcode DNA region cytochrome c oxidase subunit I (COI). This characterisation has led to the identification of ten species and provides sequence data for three previously unsequenced species, contributing to the phylogenetic knowledge of the family Pseudococcidae. In addition, the intraspecific variations found in the populations of five mealybug species provide insight into their invasion history.

We used a genetic distance approach in conjunction with molecular phylogeny to establish species boundaries and detect cryptic lineages in the Parmotrema reticulatum – P. pseudoreticulatum complex. The phylogeny of specimens from a broad geographic distribution was reconstructed from the internal transcribed spacer region. Pairwise genetic distances were calculated and compared to an intraspecific range defined for the parmelioid lichens to circumscribe species-level groups. Our results showed that P. reticulatum and P. pseudoreticulatum are polyphyletic, being comprised of at least seven well-supported lineages. In contrast, the genetic distance approach revealed ten cryptic lineages within the P. reticulatum – P. pseudoreticulatum complex. Neither morphology nor geography was conclusive in attempting to corroborate these genetic lineages. However FST indices suggest significant genetic differentiation between these lineages. Our results suggest that the morphology-based circumscriptions underestimated species in Parmotrema and that, in some cases, genetic distances may be used as an additional tool to determine species boundaries in morphologically cryptic species complexes. The most significant contribution of the present study is the application of a fast and accurate method to identify problematic groups and candidate species using the ITS locus with a genetic distances approach.

DNA barcoding is an effective technique to identify species and analyze phylogenesis and evolution. However, research on and application of DNA barcoding in Canis have not been carried out. In this study, we analyzed two species of Canis, Canis lupus (n = 115) and Canis latrans (n = 4), using the cytochrome c oxidase subunit I (COI) gene (1545 bp) and COI barcoding (648 bp DNA sequence of the COI gene). The results showed that the COI gene, as the moderate variant sequence, applied to the analysis of the phylogenesis of Canis members, and COI barcoding applied to species identification of Canis members. Phylogenetic trees and networks showed that domestic dogs had four maternal origins (A to D) and that the Tibetan Mastiff originated from Clade A; this result supports the theory of an East Asian origin of domestic dogs. Clustering analysis and networking revealed the presence of a closer relative between the Tibetan Mastiff and the Old English sheepdog, Newfoundland, Rottweiler and Saint Bernard, which confirms that many well-known large breed dogs in the world, such as the Old English sheepdog, may have the same blood lineage as that of the Tibetan Mastiff.

Syngnathus rostellatus is a nearshore pipefish species whose distributional range extends along the European Atlantic coast between Bergen (NO) and the Bay of Biscay (ES). Several recent articles suggest that this species has experienced a major range expansion of more than 4000 km into the eastern Mediterranean, but a critical review of these studies indicates that the majority of these reports are based on specimen misidentifications. Considering a reliable report of S. rostellatus from the Mediterranean coast near Gibraltar, it appears that the current distribution of this species is restricted to the north-eastern Atlantic Ocean and the southern Mediterranean coast of the Iberian Peninsula.

In French Polynesia, the black-lip pearl oyster Pinctada margaritifera has been farmed to produce pearls since the 1980s, forming the basis of a major industry. The sustainability of this activity relies on spat collection in the lagoons. However, pearl oyster spat can be difficult to identify for the evaluation of stock variations. It is especially hard to distinguish Pinctada spp. larvae at a very early stage of development. In the present study, a whole-mount in situ hybridisation (ISH) technique was developed to allow the discrimination of larvae of closely-related pearl oyster species found in the French Polynesian atolls. Using specific ribosomal 16S-DNA sequence data, we were able to successfully differentiate between Pinctada margaritifera and Pinctada maculata larvae from 5 to 13 days old. This is the first description of a non-destructive method allowing bivalve larvae discrimination between species within this genus. The method allowed us to successfully identify P. margaritifera larvae in natural plankton samples. This result is a key step needed to develop monitoring of P. margaritifera larval distribution in French Polynesian lagoons, a procedure which will increase spat collection efficiency and ensure sustainable development of pearl oyster farming.