DNA Barcode Sequences Research Articles

Prey-associated genetic differentiation in two species of silver fly (Diptera: Chamaemyiidae), Leucotaraxis argenticollis and L. piniperda

Abstract Sympatric host-associated genetic differentiation is a prominent pattern that could lead to speciation. In insects, there are numerous examples of host-associated differentiation among herbivores that prefer different plants, and parasitoids that prefer different hosts, but few examples for specialist predators. We developed new microsatellite loci for two species of silver fly, Leucotaraxis argenticollis (Zetterstedt) and L. piniperda (Malloch) (Diptera: Chamaemyiidae), being evaluated as biological control agents for the hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), in eastern North America where it is a nonnative pest. We obtained DNA from specimens of both fly species feeding on native A. tsugae in western North America, as well as on other western and eastern adelgid species. We performed population genetic analyses using the new loci and DNA barcode sequences. Our results confirmed east–west allopatric divergence and uncovered nested genetic differentiation associated with different adelgid prey species and their host plants in western North America for both species of silver flies. For both species, there is also evidence for a longer history of diversification in the west, with ancestral specialization of feeding on pine adelgids, which was retained after range expansion to the east. More recently, divergence to feeding on new adelgid prey species occurred in the west. Our findings are consistent with the hypothesis that host-alternating life cycles in Adelgidae may provide temporary escape from specialist predators. We discuss the implications for biological control efficacy and potential for lineage hybridization as western flies are released in the east to control A. tsugae.

Phylogenetic analysis based on the ITS, matK and rbcL DNA barcodes and comparison of chemical contents of twelve Paeonia taxa in Türkiye

Twelve taxa of herbaceous Paeonia species were recorded in Türkiye. All definitions were performed morphologically and/or anatomically and there is no study based on DNA barcode sequences. Three barcode regions were sequenced to determine the phylogenetic relationships of Turkish Paeonia taxa. The chemical comparison of roots was also investigated. The taxons were collected between May and June 2021 from nine cities. Leaf materials were used for DNA isolation and ITS, matK and rbcL regions were amplified and sequenced. There was no difference among taxa in terms of rbcL sequences. But the ITS and matK regions distinguished 12 taxa and structured them in two groups. ITS region distinguished P. peregrina, P. arietina, and P. tenuifolia from other taxa, while matK region distinguished P. arietina and P. witmanniana from other taxa. Both barcode sequences actually showed that the registration of P. mascula subsp. arasicola was actually 100% similar to P. arietina. ITS was the most polymorphic region (n = 54) followed by matK (n = 9). These sequences could successfully discriminate Paoenia species from each other and diploid P. tenuifolia. The methanolic root (100 gr) extracts were examined for total phenolic and flavonoid content, and antioxidant activities. Significant variation was found for polyphenolic content, and antioxidant properties (TPC from 204.23 to 2343.89mg, TFC from 7.73 to 66.16mg, and FRAP from 523.81 to 4338.62mg). SC50 values of ABTS and DPPH were ranged from 115.08 to 1115.52μg/ml and 73.83 to 963.59μg/ml, respectively. It was concluded that 11 of 12 taxa had differences in terms of ITS and matK sequences and these region must be used for the correct identification of Turkish Paeonia.

Morphology and DNA marker for distinguishing Paphiopedilum hangianum and Paphiopedilum emersonii from Vietnam

Genus Paphiopedilum has species having lovely flowers which are incredibly attractive to everyone. Their ornamental and commercial value caused over-collection and illegal poaching and trade. Due to these reasons, nowadays, the Venus slipper orchids are facing to deplete in nature. Therefore, it is important to consider these species conservation. Mainly, it is necessary to prioritize the identification and phylogenetic analysis methods of the genus Paphiopedilum which includes many species with similar morphological characteristics. Consequently, it isn't easy to distinguish the identical species of this genus when the plants are young or not yet fully flowering. Therefore, this study aimed to distinguish two Paphiopedilum species, i.e. P. hangianum and P. emersonii, which have similar morphological characteristics, through comparative morphological analysis and differences in DNA barcoding sequences. To solve the problem associated with species identifications, a morphological comparison table was created with the four DNA sequence markers matK, rbcL, rpoC1 and trnH-psbA. The results of the morphological analysis showed that P. hangianum and P. emersonii are significantly different from each other in the flower's characteristics. While the difference in leaf morphology of both selected species is found very little, it is also distinguishable upon careful comparison. Moreover, the DNA barcoding indicator gave accurate and rapid distinctions between the two species, even when the plants are young or without flowers. Furthermore, this DNA barcoding can establish an evolutionary relationship between the two selected species and the other species of the genus Paphiopedilum. The results of this study also suggested that the indicator trnH-psbA is a suitable marker for distinguishing these two species and can be applied for the phylogenetic analysis of the genus Paphiopedilum in Vietnam.

Towards understanding insect species introduction and establishment: A community-level barcoding approach using island beetles.

Since Darwin put forward his opposing hypotheses to explain the successful establishment of species in areas outside their native ranges, the preadaptation and competition-relatedness hypotheses, known as Darwin's naturalization conundrum, numerous studies have sought to understand the relative importance of each. Here, we take advantage of well-characterized beetle communities across laurel forests of the Canary Islands for a first evaluation of the relative support for Darwin's two hypotheses within arthropods. We generated a mitogenome backbone tree comprising nearly half of the beetle genera recorded within the Canary Islands for the phylogenetic placement of native and introduced species sampled in laurel forests, using cytochrome c oxidase I (COI) sequences. For comparative purposes, we also assembled and phylogenetically placed a data set of COI sequences for introduced beetle species that were not sampled within laurel forests. Our results suggest a stronger effect of species preadaptation over resource competition, while also revealing an underappreciated shortfall in arthropod biodiversity data-knowledge of species as being native or introduced. We name this the Humboldtean shortfall and suggest that similar studies using arthropods should incorporate DNA barcode sequencing to mitigate this problem.

Enhancing DNA barcode reference libraries by harvesting terrestrial arthropods at the Smithsonian's National Museum of Natural History.

The use of DNA barcoding has revolutionised biodiversity science, but its application depends on the existence of comprehensive and reliable reference libraries. For many poorly known taxa, such reference sequences are missing even at higher-level taxonomic scales. We harvested the collections of the Smithsonian's National Museum of Natural History (USNM) to generate DNA barcoding sequences for genera of terrestrial arthropods previously not recorded in one or more major public sequence databases. Our workflow used a mix of Sanger and Next-Generation Sequencing (NGS) approaches to maximise sequence recovery while ensuring affordable cost. In total, COI sequences were obtained for 5,686 specimens belonging to 3,737 determined species in 3,886 genera and 205 families distributed in 137 countries. Success rates varied widely according to collection data and focal taxon. NGS helped recover sequences of specimens that failed a previous run of Sanger sequencing. Success rates and the optimal balance between Sanger and NGS are the most important drivers to maximise output and minimise cost in future projects. The corresponding sequence and taxonomic data can be accessed through the Barcode of Life Data System, GenBank, the Global Biodiversity Information Facility, the Global Genome Biodiversity Network Data Portal and the NMNH data portal.

Description of a New Species of Parathalestris Brady & Robertson, 1873 (Copepoda: Harpacticoida: Thalestridae) from China, with a Key to Its Affiliated Species Group

A new species belonging to the genus Parathalestris Brady & Robertson, 1873 was identified and described here based on samples recently collected from a survey on fouling organisms at the Zhonggang wharf in Qingdao, China. The new species differs from its congeners in the following characteristics: caudal rami 2.0 times longer than wide, with bulbous setae II and setae III of caudal ramus issuing terminally and laterodistally, respectively; exopod of antenna two-segmented, with two setae on first segment and four setae on second one;maxilla with two bipinnate setae on proximal endite, and three spinulose setae on middle and distal endites; basis of maxilliped with concave inner edge; P1 exopod and endopod approximately same length, enp-1 about 6.7 times as long as greatest width, with inner seta reaching proximal 2/3 of segment; baseoendopod and exopod of female P5 with transversal rows of spinules on its surface, exopod bearing six marginal setae; male P1 carrying an inner curved spine on basis; exopod of male P5 bearing six elements. The DNA barcode (COI) sequence of the new species was obtained and submitted to GenBank. This is the first report of Parathalestris from the China Seas.

Description of two new species of Dicranomyia (Erostrata) crane fly (Diptera, Limoniidae) from Korea, with remarks on DNA barcoding and updated taxonomic key.

Two new crane fly species, Dicranomyia (Erostrata) jejuensissp. nov. and D. (E.) koreanasp. nov., from Korea are described on the basis of morphology and mitochondrial COI sequences. DNA barcode sequences for other four D. (Erostrata) species from Korea are also provided for the first time. The identification key for all known D. (Erostrata) species is presented.

Abstract 1062: A comprehensive CRISPR-enabled functional genomics profiling platform in acute myeloid leukemia (AML): Pilot study and validation of Fx Heme

Abstract AML is the most common acute leukemia in adults over 50 years of age. Current therapeutic approaches are focused on high or low dose chemotherapy in the front line setting, with use of molecularly targeted agents in unfit patients or the relapsed/refractory setting. However, routine genomic profiling for common molecular lesions (e.g. FLT3 mutation) incompletely stratifies the target patient population, and more personalized and comprehensive approaches could be of significant clinical benefit. To this end, we have developed an in vitro functional genomics platform for the systematic characterization of gene dependency on drug targets using CRISPR/Cas9 technology. Methods: Retrospective pre-therapy samples were obtained for a cohort of AML patients who subsequently went onto therapy with a combination of the tyrosine kinase inhibitor sorafenib plus chemotherapy (e.g. GCLAM). Unsorted primary tumor cells from these samples were transduced with lentivirus harboring Cas9 enzyme and an sgRNA library. This customized sgRNA library (Fx Heme) was designed to inhibit expression of genes encoding the targets of all FDA-approved oncology drugs, as well as internal positive and negative references. Transduced cells were harvested at multiple timepoints, and sgRNA distribution was assessed by amplicon sequencing of DNA barcodes. Changes in barcode abundance were quantitated and aggregated to calculate gene-level phenotype scores, enabling identification of gene dependencies. Dependency on targets of sorafenib was compared to clinical outcome (complete response (CR) without minimal residual disease (MRD), incomplete response, CR with MRD, or refractory) following treatment with sorafenib plus chemotherapy. Results: Among the 22 AML patients analyzed, 14 reached CR and 8 were classified as incomplete responders (refractory or MRD). Gene dependency on sorafenib targets including FLT3, KIT, PDGFR, and all RAF isoforms was frequently observed (12/22 cases). Among cases indicating any sorafenib target gene dependency, 5/12 (42%) demonstrated dependence on 2 or more sorafenib target genes. Overall, Fx Heme achieved 78.6% sensitivity, 87.5% specificity, and 91.7% PPV for prediction of observed clinical outcome. In contrast, stratification based on FLT3 mutation (internal tandem duplication or tyrosine kinase domain mutation) demonstrated 64.3% sensitivity, 50% specificity, 69.2% PPV. In summary, our results establish feasibility of CRISPR-based Fx Heme comprehensive functional genomic profiling for AML precision medicine, and validate this approach in a retrospective study cohort. Functional genomics may contribute to more effective personalization of AML treatment by uncovering drug target dependencies not readily identified by conventional genomic profiling, in an unbiased, comprehensive and patient-specific fashion. Citation Format: Stephane Corbel, Karl Hodel, Kathleen Tran, Maci Meyers, Jing Tong, Michele Baltay, Peter Kilfeather, Yali Li, Christian Schmedt, Srinath Sampath, Srihari Sampath. A comprehensive CRISPR-enabled functional genomics profiling platform in acute myeloid leukemia (AML): Pilot study and validation of Fx Heme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1062.

A new record family Neopseustidae (Insecta: Lepidoptera) from Chongqing of China, with the first description of the Neopseustis archiphenax female adult

In 2022, the insect inventories organized by Prof. Zhi-Sheng Zhang of Southwest University were constructed in Yintiaoling Nature Reserve, Chongqing Municipality, China. The neopseustid moth from Chongqing based on three specimens of Neopseustis archiphenax by light trapping were reported in this paper. The photos of the male and female adults, genitalia, and abdominal special structures are presented with the female described firstly. Also, the DNA barcoding sequence data is provided, and the key to the Neopseustis species is given.

Nanopore sequencing of DNA barcodes to unveil the diversity of fungal mock communities

Background: The fungal component of the gut microbiome has been underrepresented in most gut microbiome studies. While next-generation sequencing (NGS) approaches have been used to assess the diversity and role of the gut mycobiome, differences in experimental design and downstream analyses may induce bias and differential outcomes. This study assessed the capacity of nanopore sequencing to retrieve the microbial profile and relative abundance of a mock fungal community comprised of Candida glabrata, Meyerozyma guilliermondii, Pichia kudriavzevii, Clavispora lusitaniae and Candida parapsilosis. Methods: The approach was implemented using the MinION system and involved the analysis of sequencing libraries made from equimolar mixtures of the PCR-amplified internal transcriber spacer genomic regions of the target species. Results: Nanopore sequencing successfully retrieved the composition of the fungal mock community in terms of the different taxa present. However, the approach was unable to correctly assess the expected relative abundances of each species in the same community, showing some yet undetermined bias that may be related to the size of the respective target DNA fragments. Clavispora lusitaniae was consistently overrepresented in the mixtures, while C. glabrata was underrepresented. The remaining three species showed relative abundances more aligned with the expected values of an equimolar mixture. Conclusions: Although not yielding the expected results for the relative abundances, the values obtained from independent sequencing runs were similar for all species, suggesting a good reliability but questionable accuracy in this sequencing approach.

Hannaella floricola sp. nov., a novel basidiomycetous yeast species isolated from a flower of Lantana camara in Portugal.

During a survey of floricolous yeasts in Portugal, a basidiomycetous yeast representing a novel species in the genus Hannaella was isolated in Portugal from the flower of Lantana camara, an ornamental exotic species native to Central and South America. A combination of phylogenetic analyses of DNA barcode sequences used in yeast molecular systematics, namely the D1/D2 domain and the complete internal transcribed spacer (ITS) region supported the recognition of a new species of Hannaella, that we designate Hannaella floricola sp. nov. (ex-type strain PYCC 9191T=CBS 18097T). Although the assignment of the new species to the genus Hannaella was evident, the detection of its closest relatives appeared more problematic. Nevertheless, our analyses suggested that H. floricola sp. nov. belongs a clade that also includes H. coprosmae, H. oryzae and H. surugaensis, together four candidate novel species. In addition we provide the molecular identification of several unidentified strains whose D1/D2 and ITS sequences are available from GenBank.

Phylogenetic of red snapper (Lutjanidae) in Yapen Island Waters, Papua, Indonesia

Abstract. Sala R, Kusuma AB, Pranata B. 2022. Phylogenetic of red snapper (Lutjanidae) in Yapen Island Waters, Papua, Indonesia. Biodiversitas 23: 6428-6436. Red snappers are an economically valuable fishery resource. Most of these snapper species are inhabitants of coral reef ecosystems. A variety of red snapper species can be found in the northern waters of Papua, particularly in the Yapen Regency. However, information regarding the biological and ecological aspects of these fish is virtually unavailable. On the other hand, the utilization intensity of red snapper as a source of community income continues to increase. By using DNA barcode sequences, this study attempted to examine species diversity and relationships among Lutjanidae family species. The study was carried out from June to August 2022 in the waters of Yapen Regency by taking fish samples from the fish caught by local hand-lining fishers. Red snapper samples were identified morphologically and molecularly. According to molecular analyses, it was identified nine red snapper species from the genera of Lutjanus, Aphareus and Pristipomoides. The morphological characteristics of the species from the genus of Aphareus are similar to those from the genus of Pristipomoides yet different from those of the species from the genus of Lutjanus. The phylogenetic tree consisted of four clades with significant bootstrap values ranging from 98 to 99%. Clades 1, 2 and 3 comprise the species from the genus of Lutjanus, while clade 4 contains species from the genera of Aphareus and Pristipomoides. The greatest genetic distance was found between Lutjanus fulvus and Pristipomoides multidens, while the smallest genetic distance was found between Lutjanus vitta and Lutjanus ehrenbergii. Based on the study results, some management implications are discussed. For example, information on species biodiversity maps is needed to determine the target stock of a managed species so that management objectives are more focused on the species level and not on the genus or family level.

The identity of Argyrialacteella (Fabricius, 1794) (Lepidoptera, Pyraloidea, Crambinae), synonyms, and related species revealed by morphology and DNA capture in type specimens.

In this study the aim was to resolve the taxonomy of several species of Argyria Hübner (Pyraloidea, Crambinae) with previously unrecognised morphological variation. By analysing the DNA barcode (COI-5P) in numerous specimens, the aim was to reconstruct phylogenetic relationships between species, to provide better evidence for synonymies, and to circumscribe their geographical distribution. Using an innovative DNA hybridisation capture protocol, the DNA barcode of the lectotype of Argyrialacteella (Fabricius, 1794) was partially recovered for comparison with the 229 DNA barcode sequences of Argyria specimens available in the Barcode of Life Datasystems, and this firmly establishes the identity of the species. The same protocol was used for the following type specimens: the Argyriaabronalis (Walker, 1859) holotype, thus confirming the synonymy of this name with A.lacteella, the holotype of A.lusella (Zeller, 1863), syn. rev., the holotype of A.multifacta Dyar, 1914, syn. nov. newly synonymised with A.lacteella, and a specimen of Argyriadiplomochalis Dyar, 1913, collected in 1992. In addition, nine specimens of A.lacteella, A.diplomochalis, A.centrifugens Dyar, 1914 and A.gonogramma Dyar, 1915, from North to South America were sampled using classical COI amplification and Sanger sequencing. Argyriagonogramma Dyar, described from Bermuda, is the name to be applied to the more widespread North American species formerly identified as A.lacteella. Following morphological study of its holotype, Argyriavestalis Butler, 1878, syn. nov. is also synonymised with A.lacteella. The name A.pusillalis Hübner, 1818, is considered a nomen dubium associated with A.gonogramma. The adult morphology is diagnosed and illustrated, and distributions are plotted for A.lacteella, A.diplomochalis, A.centrifugens, and A.gonogramma based on slightly more than 800 specimens. For the first time, DNA barcode sequences are provided for the Antillean A.diplomochalis. This work provides a modified, improved protocol for the efficient hybrid capture enrichment of DNA barcodes from 18th and 19th century type specimens in order to solve taxonomic issues in Lepidoptera.

Domestic Cattle in a National Park Restricting the Sika Deer Due to Diet Overlap.

Managers need to know the extent of the conflict between livestock and wild animals. Although many studies have reported the conflict between livestock and wild animals, few have checked the extent of the conflict. Cattle raising in the Northeast Tiger and Leopard National Park is considered one of the main driving forces behind the restricted distribution of sika deer. To understand whether foraging competition is contributing to avoidance patterns between sika deer and cattle, we investigated their feeding habits using DNA barcoding and high-throughput sequencing. Our study shows that although cattle are grazers in the traditional division of herbivores, their diet shifted to a predominance of dicotyledonous woody plants, and this diet shift resulted in a high degree of dietary overlap between sika deer and cattle. Moreover, compared to sika deer, cattle diets are more diverse at the species level with a wider ecological niche. Our results confirm that overlapping dietary niches and the superior competitive abilities of cattle contribute to the restricted distribution of the sika deer, which has critical implications for the conservation of their predators. Our study suggests that cattle grazing should be prohibited in the Park and effective measures should be taken for the benefit of sika deer.

DNA Barcoding of Morphologically Characterized Mosquitoes Belonging to the Genus Mansonia from the Atlantic Forest and Brazilian Savanna

The identification of mosquito species is necessary for determining the entomological components of disease transmission. However, identification can be difficult in species that are morphologically similar. The cytochrome c oxidase subunit I (COI) DNA barcode region is considered a valuable and reliable diagnostic tool for mosquito species recognition, including those that belong to species complexes. Mansonia mosquitoes are found in forests near swampy areas. They are nocturnal and are highly attracted to light. Hematophagous adult females exhibit aggressive biting behavior and can become infected with and transmit pathogens during their feeding, including some epizootic viruses and avian malaria. In Brazil, twelve Mansonia species have been reported. In a recent study from the São Paulo Zoo in Brazil, three morphologically distinct species were collected and identified, namely: Mansonia (Mansonia) indubitans, Ma. (Man.) pseudotitillans and Ma. (Man.) titillans. However, confirmation of these species by molecular identification was unsuccessful due to a lack of COI sequences in the GenBank database. Thus, this research aimed to describe the COI DNA barcode sequences of some morphologically characterized Mansonia (Man.) species from Brazil and to determine their utility in delimiting species collected from the Atlantic Forest and Brazilian Savanna. Accordingly, we provide tools for the genetic identification of species that play a significant role in pathogen transmission in wildlife and potentially humans. We show that the delimitation of Mansonia species via five different approaches based on COI DNA sequences (BI, NJ, ASAP, bPTP and GMYC) yield basically the same groups identified by traditional taxonomy, and we provide the identification of specimens that were previously identified only up to the subgenus level. We also provide COI sequences from two Mansonia species that were not previously available in sequence databases, Ma. wilsoni and Ma. pseudotitillans, and thus contribute to the ongoing global effort to standardize DNA barcoding as a molecular means of species identification.

DDQR (dynamic DNA QR coding): An efficient algorithm to represent DNA barcode sequences.

A DNA barcode is a short piece of standard DNA sequence used for species determination and discrimination. Representation of DNA barcodes is essential for DNA barcodes' applications in the transportation and recognition of biological materials. Previously, we have compared different strategies for representing the DNA barcodes. In the present study, we have developed a compression algorithm based on binary coding or Huffman coding scheme, followed by converting the binary digits into Base64 digits. The combination of this compression algorithm and the QR representation leads to the dynamic DNA QR coding algorithm (DDQR). We tested the DDQR algorithm on simulated data and real DNA barcode sequences from the commonly used plant and animal DNA barcode markers: rbcL, matK, trnH-psbA, ITS2, and COI. We compared the compression efficiency of DDQR and another state-of-the-art DNA compression algorithm GeCo3 for sequences with various base compositions and lengths. We found that DDQR had a higher compression rate than GeCo3 for DNA sequences shorter than 800 bp, which is the typical size range for DNA barcodes. We also upgraded a web server (http://www.1kmpg.cn/ddqr) that provides three functions: retrieval of DNA barcode sequences, encoding DNA barcode sequences to DDQR codes, and decoding DDQR codes to DNA barcode sequences. The DDQR algorithm and the webserver will be invaluable to applying DNA barcode technology in the food and traditional medicine industries.

DNA barcoding as a tool to monitor the diversity of endangered spring snails in an Austrian National Park.

The Kalkalpen National Park is situated in Upper Austria and contains more than 800 springs. The international importance of this Park is, from the perspective of nature conservation directives, highly significant (European Nature Reserve Natura 2000, recognised wetland of the Ramsar convention). In the current study, the hydrobioid fauna ('spring snails') of the Kalkalpen National Park was evaluated. These tiny snails are difficult to determine; however, their investigation is especially desirable, as several species are threatened and as they are important for water quality assessment. Snails collected in 39 selected springs were examined with classical morphological methods (shell and genital anatomy) and, subsequently, by DNA analysis. For this task, the DNA barcode, a partial sequence of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene (length of the sequence 658-682 bp), was PCR amplified and sequenced. From 107 specimens, the DNA barcoding sequence could be obtained and compared with already existing DNA sequences. The (sub)endemic species Bythinellaconica, Hauffeniakerschneri, Hauffeniawienerwaldensis and Belgrandiellaaulaei could be clearly identified. For Bythiospeumnocki, despite the ambitious collecting effort, only empty shells were found in four springs (including the locus typicus spring) in the Park and its surroundings. The genus Bythinella was detected in 36 springs. From 25 of these localities, DNA barcodes could be created, which matches those of Bythinellaconica (comparison data from ABOL). It is, therefore, concluded that the species occurs widely in the Kalkalpen National Park. The genus Hauffenia was sampled from 16 springs. From one, the haplotype of Hauffeniawienerwaldensis could be identified (spring is 5 km outside the Park) and from six, the haplotype of Hauffeniakerschneri. Belgrandiellaaulaei was found in three springs, which all lie outside the boundaries and are, therefore, not included in the protection measures of the National Park. The data and analyses obtained contribute to the assessment of the taxonomic status of the species studied. The present study gives a good baseline for further monitoring of the hydrobioids in the Kalkalpen National Park, which is important to evaluate current as well as to decide on future protection measures for this group.

Insights into chloroplast genome structure, intraspecific variation, and phylogeny of Cyclamen species (Myrsinoideae)

Species from the flowering plant genus Cyclamen are popular amongst consumers. In particular Cyclamen persicum Mill. has been significantly used commercially, and certain small flowering species such as Cyclamen hederifolium and Cyclamen coum are gradually growing in popularity in the potted flower market. Here, the chloroplast genomes of nine Cyclamen samples including four Cyclamen species and five varieties of C. hederifolium were sequenced for genome structure comparison, White green septal striped leaves related gene screening and DNA molecular markers were developed for phylogenetic analysis. In comparing Cyclamen species’ chloroplast genomes, gene content and gene order were found to be highly similar with the length of genomes ranging from 151,626 to 153,058 bp. The chloroplast genome of Cyclamen has 128 genes, including 84 protein-coding genes, 36 transfer RNA genes, and 8 ribosomal RNA genes. Based on intraspecific variation, seven hotspots, including three genes and four intergenic regions, were identified as variable markers for downstream species delimitation and interspecific relationship analyses. Moreover, a phylogenetic tree constructed with complete chloroplast genomes, revealed that Cyclamen are monophyletic with Lysimachia as the closest neighbor. Phylogenetic analyses of the 14 Cyclamen species with the seven variable regions showed five distinct clades within this genus. The highly supported topologies showed these seven regions may be used as candidate DNA barcode sequences to distinguish Cyclamen species. White green septal striped leaves is common in C. hederifolium, however the molecular mechanism of this has not yet been described. Here, we find that the intergenic region rps4-trnT-UGU seems related to white green septal striped leaves.

Our Planet Reviewed in Corsica 2019-2021: a large-scale survey of neglected biodiversity on a Mediterranean island

From 2019 to 2021, scientific field campaigns have been organised in Corsica by the Muséum national d’Histoire naturelle, the Office français de la biodiversité and the Collectivité de Corse as part of the “Our Planet Reviewed” naturalist exploration programme. This paper presents the context, the state of biogeographical and taxonomic knowledge prior to our expeditions, and the objectives, the methods and the first results obtained. The aim was to establish a modern survey of the species present in a selection of sites representative of different Corsican ecosystems, and to further develop the natural history collections through depositing new specimens and species with associated standard DNA barcodes, useful for their identification. Over a period of three years, nineteen sites were surveyed with a semi-standardised protocol and a large-scale trapping scheme was organised in three of these. Sampling efforts focused on forest habitats at higher altitudes and on coastal dune and lowland marshland habitats. A vast array of methods was used to collect invertebrates, with a specific effort on flightinterception traps and pan traps. A total of 34 experts participated to the field surveys and more than 80 further contributed to the study of the specimens. Occurrence data are available in the Inventaire national du patrimoine naturel (http://www.inpn.fr) and, for specimens processed through DNA barcoding, specimen and DNA sequencing data will be accessible in the Barcode of Life datasystems (BOLD : http://www.boldsystems.org). In early 2023, the assembled datasets included 31,100 occurrence data for 3,900 taxa of terrestrial arthropods, representing a 53% increase in publicly available data for the island. More than 6,800 DNA barcode sequences have been produced for arthropods, representing a 14-fold increase in available sequences of Corsican insects compared to those available before the start of the programme. So far, these efforts resulted in producing the first Corsican records for 148 species and in the description of 12 species new to science.

Applying a modified metabarcoding approach for the sequencing of macrofungal specimens from fungarium collections.

Fungaria are an underutilized resource for understanding fungal biodiversity. The effort and cost of producing DNA barcode sequence data for large numbers of fungal specimens can be prohibitive. This study applies a modified metabarcoding approach that provides a labor-efficient and cost-effective solution for sequencing the fungal DNA barcodes of hundreds of specimens at once. We applied a two-step PCR approach using nested, barcoded primers to sequence the fungal nrITS2 region of 766 macrofungal specimens using the Illumina platform. The specimens represent a broad taxonomic sampling of the Dikarya. Of these, 382 Lactarius specimens were analyzed to identify molecular operational taxonomic units (MOTUs) using a phylogenetic approach. The raw sequences were trimmed, filtered, assessed, and analyzed using the DADA2 amplicon de-noising toolkit and Biopython. The sequences were compared to the NCBI and UNITE databases and Sanger nrITS sequences from the same specimens. The taxonomic identities derived from the nrITS2 sequence data were >90% accurate across all specimens sampled. A phylogenetic analysis of the Lactarius sequences identified 20 MOTUs. The results demonstrate the capacity of these methods to produce nrITS2 sequences from large numbers of fungarium specimens. This provides an opportunity to more effectively use fungarium collections to advance fungal diversity identification and documentation.