Barcoding Markers Research Articles

Molecular Identification of Fungal Endophytes of Medicinal Plant Citrullus colocynthis (L.) Schrad as a Medicinal Plant: Role of Tissue Type and Sampling Location on the Diversity.

Endophytic fungi are an important group of organisms in association with plants which are able to colonize all plant internal tissues and improve their fitness. The present research aims to isolate and identify endophytic fungi of Citrullus colocynthis plant and then investigate the effects of sampling location and tissue type on the fungal endophyte diversity of this plant. To do so, a sampling program was done in 11 geographically isolated C. colocynthis growing areas of Hormozgan province, Iran. For molecular identification of endophytic fungi of C. colocynthis, the internal transcribed spacer region (ITS1-5.8S-ITS4), as a universal DNA barcode marker for fungi, was amplified using primer sets. Totally, 12 taxa (Alternaria solani, Cladosporium halotolerans, Setosphaeria rostrata, Aspergillus niger, A. allahabadii, A. terreus, A. occultus, A. cristatus, Penicillium chrysogenum, Talaromyces purpureogenus, Fusarium sp., and Pseudozyma flocculosa) were isolated. Our findings also showed that the diversity of fungal endophytes isolated from C. colocynthis was affected by the tissue type and sampling site. Accordingly, the leaves and seeds were found to have the highest and lowest rates of endophyte colonization and richness in all sampling seasons, respectively. Simpson's diversity index of 0.8165 in root tissue indicated the high diversity of endophytes in this organ. In addition, Shannon's diversity index in the root (1.846) was higher than that in the other organs. The highest Shannon's and Simpson's indices were observed in Khoon Sorkh and Minab regions. Generally, at least two factors (region and type of tissue) played the most important roles in determining the composition of fungal endophytes in C. colocynthis.

Cryptic, sibling or neither of the two? Integrative species delimitation ofPsylliodesflea beetles with overlapping ranges

AbstractSpecies identification and delimitation are particularly challenging for morphologically similar and geographically overlapping species, such as in the case of Western Palaearctic flea beetle speciesPsylliodes kiesenwetteriKutschera, 1864 andPsylliodes ruffoiLeonardi, 1975. In this study, we implemented an integrative taxonomic approach based on a comprehensive geographic assessment of morphological and genetic variation, including 142 adult specimens from 15 sympatric and allopatric populations. Results of species delimitation methods using the barcode marker COI show that molecular identification and delimitation betweenP. kiesenwetteriandP. ruffoiare straightforward. Single‐locus and multi‐locus phylogenetic analyses indicate these two species have a large genetic divergence and belong to two distinct clades of thePsylliodes gibbosaspecies group. Morphological identification based on qualitative characters shows great differences in identification success depending on the character used. Characters of male and female genitalia perform very well, but can only be assessed on fully sclerified individuals, whereas the colour of antennae was discovered as a new reliable diagnostic character both for teneral and fully sclerified individuals. Morphological identification is particularly sharp when multiple characters are used in combination or when a morphometric approach is performed. In conclusion, despite overall morphological similarity,P. kiesenwetteriandP. ruffoiare not cryptic species neither they are sibling species, as they belong to distinct clades within thegibbosaspecies group, and they can be reliably distinguished by morphological characters. This study substantiates the relevance of a range‐wide assessment of morphological and genetic variation, including individuals from the type locality, and both sympatric and allopatric populations, for taxonomic assessment of morphologically similar species with overlapping ranges.

Plastid DNA Barcoding and RtActin cDNA Fragment Isolation of Reutealis Trisperma: A Promising Bioresource for Biodiesel Production.

Reutealis trisperma belonging to the family Euphorbiaceae is currently used for biodiesel production, and rapid development in plant-based biofuel production has led to its increasing demand. However, massive utilization of bio-industrial plants has led to conservation issues. Moreover, genetic information on R trisperma is still limited, which is crucial for developmental, physiological, and molecular studies. Studying gene expression is essential to explain plant physiological processes. Nonetheless, this technique requires sensitive and precise measurement of messenger RNA (mRNA). In addition, the presence of internal control genes is important to avoid bias. Therefore, collecting and preserving genetic data for R trisperma is indispensable. In this study, we aimed to evaluate the application of plastid loci, rbcL, and matK, to the DNA barcode of R trisperma for use in conservation programs. In addition, we isolated and cloned the RtActin (RtACT) gene fragment for use in gene expression studies. Sequence information was analyzed in silico by comparison with other Euphorbiaceae plants. For actin fragment isolation, reverse-transcription polymerase chain reaction was used. Molecular cloning of RtActin was performed using the pTA2 plasmid before sequencing. We successfully isolated and cloned 592 and 840 bp of RtrbcL and RtmatK fragment genes, respectively. The RtrbcL barcoding marker, rather than the RtmatK plastidial marker, provided discriminative molecular phylogenetic data for R Trisperma. We also isolated 986 bp of RtACT gene fragments. Our phylogenetic analysis demonstrated that R trisperma is closely related to the Vernicia fordii Actin gene (97% identity). Our results suggest that RtrbcL could be further developed and used as a barcoding marker for R trisperma. Moreover, the RtACT gene could be further investigated for use in gene expression studies of plant.

Morphological and Molecular Evidence for the Identity of Two Land Hermit Crabs Coenobita longitarsis De Man, 1902 and C. pseudorugosus Nakasone, 1988 (Crustacea: Decapoda: Anomura: Coenobitidae).

Two species of land hermit crabs, Coenobita longitarsis De Man, 1902 and C. pseudorugosus Nakasone, 1988 were described based on female specimens from Maluku, Indonesia and specimens from Cebu, the Philippines, respectively. However, no confirmed records of either species have been reported since their original descriptions. In this study, we examined specimens with typical morphological characters of C. longitarsis from Papua New Guinea and C. pseudorugosus from the Philippines and Indonesia, further supported by the analyses of the DNA barcoding marker, cytochrome oxidase subunit I (COI). The characters of male C. longitarsis are provided for the first time, with coxae of male fifth pereiopods subequal, without sexual tubes developed. Their slender morphology is suggested to be an adaptation to utilize native terrestrial snail shells in inland forests, which may also explain its rarity. Coenobita pseudorugosus is very similar to C. rugosus H. Milne Edwards, 1837, but can be distinguished by the adult sizes, as well as the morphology of sexual tubes of male fifth pereiopods and propodus of left third pereiopod. Morphological variation and the fresh coloration of C. longitarsis and C. pseudorugosus are also provided in this study.

Notes on the use of genus Hyloicus Hübner, 1819, with the description of a new species of the “H. pinastri complex” from Corsica (Lepidoptera, Sphingidae)

The authors briefly discuss the merits of separating the genus Hyloicus Hübner, 1819, from the genus Sphinx Linnaeus, 1758, a treatment that has been debated for more than forty years. They provide a brief history of the development of the taxonomy of the members (all Palaearctic) of the “H. pinastri complex”. Then they report the unexpected finding that the representative of this complex on the island of Corsica represents a new species, described herein as Hyloicus corsica n. sp. It can be distinguished from its continental relatives, H. pinastri (Linnaeus, 1758) and H. maurorum Jordan, 1931, by its larger size, characters of the male genitalia and consistent genetic divergence of the standard DNA barcode marker (a fragment of COI gene). Only males have been observed so far, and the species is currently known as a Corsican endemic found in several localities in both southern and northern parts of the island.

Some Barcoding DNA Sequence Analysis of Sphagnum nepalense H. Suzuki, a Bryophyte Species Endemic to East Nepal

Sphagnum nepalense is a bryophyte endemic to Nepal. The objective of the present study is to analyze DNA barcoding markers useful for delineating the Sphagnum species. Here, a specimen of Sphagnum nepalense collected from the bank of Maipokhari lake, Ilam (2107 m asl) was used. Three chloroplast loci from the sample viz. rbcL, psbA-trnH and trnF-trnL, the latter two being intergenic spacers, were amplified and sequenced. Four accessions of plastome sequences of S. junghuhnianum, S. multifibrosum, S. palustre and S. subsecundum were retrieved from the National Center for Biotechnology Information (NCBI). Evolutionary analysis was performed following the Maximum Likelihood approach using MEGA X. The result showed that the evolutionary tree generated with single locus trnF-trnL and combined sequences of trnF-trnL and psbA-trnH was better compared to that generated with the sequence of other single locus and even the combined sequence of rbcL, psbA-trnH and trnF-trnL. The sequence data generated in this study for Sphagnum nepalense are novel to the scientific community.

Building a local reference library for metabarcoding survey of lake macrobenthos: oligochaetes and chironomids from Lake Maggiore

This study represents a first reference database of genetic diversity of macroinvertebrates for a barcoding marker for Lake Maggiore, focusing on the two dominant groups of the littoral benthic fauna (chironomids and oligochaetes), commonly used for biological monitoring of freshwater lakes. Sediment samples were sorted at the stereomicroscope and single animals were cut in two pieces, one piece to be used for morphological identification and one piece for DNA extraction. This study allowed us to collect and identify 427 organisms: 309 oligochaetes belonging to 27 identifiable taxa and 118 chironomid larvae belonging to 26 identifiable taxa. Four families of oligochaetes: Naididae, Lumbricidae, Lumbriculidae, and Enchytraeidae and five subfamilies of Chironomidae: Chironominae, Tanypodinae, Orthocladiinae, Diamesinae, and Prodiamesinae were found. The extraction and amplification of the DNA covered a total of 10 oligochaete taxa. For 7 of them (Ophidonais serpentina, Uncinais uncinata, Vejdovskyella intermedia, Psammoryctides barbatus, Limnodrilus hoffmeisteri, Tubifex tubifex, and Bothrioneurum vejdovskyanum), we found other sequences in GenBank to compare genetic similarities with available data. For the other taxa (Lumbriculidae, and Enchytraeidae, and Nais sp.) no best hits were found in GenBank. The extraction and amplification of the DNA covered a total of 21 chironomid taxa. For ten species (Cladotanytarsus mancus, Cladotanytarsus atridorsum, Polypedilum scalaenum, Polypedilum nubeculosum, Benthalia carbonaria, Phaenopsectra flavipes, Clinotanypus nervosus, Paracladopelma laminatum, Cryptochironomus rostratus and Parakiefferiella finnmarkica) sequences were available in GenBank to compare genetic similarities. For the other taxa (Cryptochironomus sp., Demicryptochironomus vulneratus, Chironomus sp., Stictochironomus sp., Orthocladius sp., Cricotopus sp., Eukiefferiella sp., Procladius sp., Diamesa sp., Potthastia sp., and Monodiamesa bathyphila) no best hits were found in GenBank. For chironomids, DNA taxonomy revealed the existence of several species complexes. Covering more populations and more genetic markers for those taxa within a rationale of integrative taxonomy could solve the taxonomic problems and provide a reliable description of diversity.

Analysis of Genetic Diversity and Phylogenetic Relationships of Wheat (Triticum aestivum L.) Genotypes Using Phenological, Molecular and DNA Barcoding Markers.

Wheat (Triticum aestivum L.) is a key food crop, accounting for approximately 765 million tons produced worldwide. The present study evaluated 16 wheat genotypes using 19 morphological and phenological traits, 16 molecular markers (Inter Simple Sequence Repeats and Start Codon Targeted; ISSR and SCoT) and rbcL and matK plastid gene barcoding. The 16 wheat genotypes showed significant genetic variation using the markers assayed. Cell plot of phenological parameters revealed significant differences among the 16-day-old seedlings of wheat genotypes at Z1.1 growth stage. Collectively, W2 genotype had the lowest shoot length (SL), length of first internodes (LFI) and leaf area (LA) values, while W8 genotype had the highest diameter of first internode (DFI) and LA values. Furthermore, W7 genotype had the maximum plant biomass (PB) and leaf width (LW) values. Geometric models grouped wheat kernels into "rounded" and "nearly elongated". Estimates of heritability (H2) for these morphological characters ranged from 4.93 to 100%. The highest H2 values were recorded for root number (RN) (100%) followed by SL (88.72%), LFI (88.30%), LA (87.76%) and Feret diameter (86.68%), while the lowest H2 value was recorded for DFI (4.93%). Furthermore, highly significant genotypic and phenotypic correlations were also observed among those traits. Reproducible fingerprinting profiles and high levels of polymorphism (PPB%) of SCoT (95.46%) and ISSR (82.41%) were recorded, indicating that they are effective tools for detecting genetic variation levels among wheat genotypes. The informativeness of markers were measured through estimation of polymorphic information content (PIC), resolving power (RP) and marker index (MI). The RP and PPB% of SCoT were significantly higher compared to those of ISSR. Comparatively, the two molecular markers were effective for studying genetic diversity among wheat genotypes, but SCoT markers were more informative. Moreover, based on the two chloroplast DNA regions (rbcL and matK), MatK was found to be more reliable for differentiating among T. aestivum genotypes. Taken together, using all the studied attributes, a clear taxonomic relationship can be used to identify T. aestivum species and improve their pragmatic production and development.

High-Throughput DNA Metabarcoding as an Approach for Ichthyoplankton Survey in Oujiang River Estuary, China

High-throughput DNA metabarcoding of mitochondrial 12S rRNA and Cyt b gene sequences was coupled with a morphology-based identification tool to assess ichthyoplankton community structure in Oujiang River Estuary, China. The performances of 12S and Cyt b barcoding markers were compared in terms of taxonomic resolution, detection and coverage, and their suitability was established for use as a quick and powerful ichthyoplankton assessment tool. A total of 30,138 ichthyoplankton (2462 eggs and 27,676 larvae) samples were collected from April to August 2015 and identified to 145 taxa belonging to 57 families and 105 genera. June and July were the main spawning months. Ichthyoplankton were more abundant around Lingkun and Qidu Islands and the upper parts of Oujiang River Estuary. The 12S gene marker presented higher species coverage and detection rate than Cyt b. DNA metabarcoding exhibited more representative species identification power than morphology. The findings reported in this study provided a key attempt towards the development of time-efficient and cost-effective ichthyoplankton identification and assessment tool.

Differentiation of Lingxiaohua and Yangjinhua by chloroplast genome sequencing and DNA barcoding markers

Lingxiaohua (Campsis Flos, Campsis grandiflora (Thunb.) K. Schum) is a medicinal herb used for promoting diuresis and treating blood-related disorders by the promotion of blood circulation. It also possesses anti-inflammatory and antioxidative properties. This non-poisonous plant is frequently confused with poisonous Yangjinhua (Daturae Metelis Flos, Datura metel Linnaeus) in the market, resulting in serious anticholinergic poisoning. The confusion of these two herbs is due to the similarity in their appearances. In our study, we compared the complete chloroplast genomes of the two plants and found that they are very different in terms of their gene content and gene arrangement. There were also significant differences in the number and repeating motifs of microsatellites and complex repeats. We used universal primers for the amplification of rbcL, matK, psbA-trnH, and ITS2 regions and successfully differentiated the two plants. Furthermore, we designed two pairs of primers based on the nucleotide differences in chloroplast genomes at the rps14 and rpoC1 regions to provide additional authentication markers. The universal primers and specific primers when used together can accurately discriminate Lingxiaohua and Yangjinhua.

Quality control of fighting fish nucleotide sequences in public repositories reveals a dark matter of systematic taxonomic implication.

The number of nucleotide sequences in public repositories has exploded recently. However, the data contain errors, leading to incorrect species identification. Several fighting fish (Betta spp.) are poorly described, with unresolved cryptic species complexes masking undescribed species. Here, DNA barcoding was used to detect erroneous sequences in public repositories. This study reflects the current quantitative and qualitative status of DNA barcoding in fighting fish and provides a rapid and reliable identification tool. A total of 1034 barcode sequences were analyzed from mitochondrial cytochrome c oxidase I (COI) and cytochrome b (Cytb) genes from 71 fighting fish species. The nearest neighbor test showed the highest percentage of intraspecific nearest neighbors at 93.41% for COI and 91.67% for Cytb, which can be used as reference barcodes for certain taxa. Intraspecific variation was usually less than 13%, while most species differed by more than 54%. The barcoding gap, calculated from the difference between inter- and intraspecific sequence divergences, was negative in the COI data set indicating overlapping intra- and interspecific sequence divergence. Sequence saturation was observed in the Cytb data set but not in the COI data set. The COI gene should thus be used as the main barcoding marker for fighting fish.

To be or not to be… Integrative taxonomy and species delimitation in the daddy long-legs spiders of the genus Physocyclus (Araneae, Pholcidae) using DNA barcoding and morphology.

Integrative taxonomy is crucial for discovery, recognition, and species delimitation, especially in underestimated species complex or cryptic species, by incorporating different sources of evidence to construct rigorous species hypotheses. The spider genus Physocyclus Simon, 1893 (Pholcidae, Arteminae) is composed of 37 species, mainly from North America. In this study, traditional morphology was compared with three DNA barcoding markers regarding their utility in species delimitation within the genus: 1) Cytochrome c Oxidase subunit 1 (CO1), 2) Internal Transcribed Spacer 2 (ITS2), and 3) Ribosomal large subunit (28S). The molecular species delimitation analyses were carried out using four methods under the corrected p-distances Neighbor-Joining (NJ) criteria: 1) Automatic Barcode Gap Discovery (ABGD), 2) Assemble Species by Automatic Partitioning (ASAP), 3) General Mixed Yule Coalescent model (GMYC), and 4) Bayesian Poisson Tree Processes (bPTP). The analyses incorporated 75 terminals from 22 putative species of Physocyclus. The average intraspecific genetic distance (p-distance) was found to be < 2%, whereas the average interspecific genetic distance was 20.6%. The ABGD, ASAP, and GMYC methods were the most congruent, delimiting 26 or 27 species, while the bPTP method delimited 33 species. The use of traditional morphology for species delimitation was congruent with most molecular methods, with the male palp, male chelicerae, and female genitalia shown to be robust characters that support species-level identification. The barcoding with CO1 and 28S had better resolution for species delimitation in comparison with ITS2. The concatenated matrix and traditional morphology were found to be more robust and informative for species delimitation within Physocyclus.

DNA Barcoding and ITS2 Secondary Structure Predictions in Taro (Colocasia esculenta L. Schott) from the North Eastern Hill Region of India.

Taro (Colocasia esculenta L. Schott, Araceae), an ancient root and tuber crop, is highly polygenic, polyphyletic, and polygeographic in nature, which leads to its rapid genetic erosion. To prevent the perceived loss of taro diversity, species discrimination and genetic conservation of promising taro genotypes need special attention. Reports on genetic discrimination of taro at its center of origin are still untapped. We performed DNA barcoding of twenty promising genotypes of taro indigenous to the northeastern hill region of India, deploying two chloroplast-plastid genes, matK and rbcL, and the ribosomal nuclear gene ITS2. The secondary structure of ITS2 was determined and molecular phylogeny was performed to assess genetic discrimination among the taro genotypes. The matK and rbcL genes were highly efficient (&gt;90%) in amplification and sequencing. However, the ITS2 barcode region achieved significant discrimination among the tested taro genotypes. All the taro genotypes displayed most similar sequences at the conserved matK and rbcL loci. However, distinct sequence lengths were observed in the ITS2 barcode region, revealing accurate discriminations among the genotypes. Multiple barcode markers are unrelated to one another and change independently, providing different estimations of heritable traits and genetic lineages; thus, they are advantageous over a single locus in genetic discrimination studies. A dynamic programming algorithm that used base-pairing interactions within a single nucleic acid polymer or between two polymers transformed the secondary structures into the symbol code data to predict seven different minimum free energy secondary structures. Our analysis strengthens the potential of the ITS2 gene as a potent DNA barcode candidate in the prediction of a valuable secondary structure that would help in genetic discrimination between the genotypes while augmenting future breeding strategies in taro.

New Morphological and Molecular Data Reveal an Underestimation of Species Diversity of Mites of the Genus Geckobia (Acariformes: Pterygosomatidae) in India

Mites of the genus Geckobia (Acariformes: Pterygosomatidae) are permanent and highly specialised ectoparasites of geckos (Gekkota). We conducted a local study on Geckobia mites associated with the geckos of the family Gekkonidae found mainly in the territory of the Indian Institute of Science’s campus (Bangalore, India). In total, we examined 208 lizards belonging to two genera: Hemidactylus and Cnemaspis. We assessed the prevalence of the mites and identified the preferred site for their infestation. We extended the standard morphological identification of the mite species by using DNA barcode markers, partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene and nuclear ribosomal gene sequences: 18S rRNA and hypervariable region D2 of nuclear 28S rRNA. We checked the suitability of COI and nuclear (D2 of 28S rRNA) markers for species delimitations and identification purposes of the genus. The distance- and phylogeny-based approaches were applied: (i) to test the presence of a barcoding gap, we used the automated barcoding gap discovery tool (ABGD) and investigated intra- and interspecific genetic distances, and (ii) to reconstruct evolutionary relationships within the species, we performed maximum likelihood (ML) and Bayesian inference with Markov-Chain Monte Carlo (BI) analyses. As a result, we described five new species—Geckobia gigantea sp. n., G. treutleri sp. n., G. unica sp. n. and G. brevicephala sp. n.—from four Hemidactylus species: H. giganteus, H. treutleri, H. parvimaculatus and H. frenatus, respectively, and G. mysoriensis sp. n. from Cnemaspis mysoriensis. Additionally, we found three already described species: Geckobia indica Hirst, 1917 on H. treutleri (new host), Geckobia bataviensis Vitzhum, 1926 on H. parvimaculatus (new host) and H. frenatus (new locality) and Geckobia phillipinensis Lawrence, 1953 on H. frenatus (new locality). The diagnoses of G. indica and G. phillipinensis were improved and supplemented by descriptions of the males and juveniles. Both topologies of the BI and ML phylogenetic trees, as well as genetic distances, supported the species boundaries in the mite population shown by the morphological data. Hemidactylus frenatus was the most infected gecko species (61% prevalence), with the highest number of mite species (three spp.). The scale-mite richness was higher than expected; therefore, further research is required to evaluate the true diversity of Geckobia mites.

Three new species of the genus Perinereis (Annelida, Nereididae) from Egyptian coasts.

Despite being one of the most common groups of polychaetes on intertidal shores, the genus Perinereis (Nereididae) is comparatively poorly known taxonomically, with confusion still existing due to the lack of comprehensive systematic studies. The systematics of Perinereis species from the intertidal Egyptian coasts of the Red Sea, Gulf of Suez and Suez Canal have been investigated using morphology and the mitochondrial barcoding marker cytochrome oxidase subunit I (COI). New sequence data was obtained for 102 Perinereis specimens and analysis included all publicly available COI data from other Perinereis species. The COI data indicate that monophyly of the P.nuntia species group is doubtful, as specimens identified in this species group from south-eastern Asia and Australia form a monophyletic group exclusive of the three new species described in this study from the Red Sea region. A morphometric character set (26 characters) was used to identify and characterize each specimen in the study. Three distinct morphospecies belonging to the P.nuntia species group were found, each differentiated by the number and type of paragnaths on pharyngeal areas V and VI, relative sizes of parapodial lobes, type of notochaetae and neurochaetae, and form of the neurochaetal falciger blades. The three morphospecies were well supported by COI data: two of the three new species, Perinereissuezensis sp. nov. and Perinereisfayedensis sp. nov., are closely similar to P.nuntia sensu stricto, while the other, Perinereisdamietta sp. nov., is similar to P.heterodonta. The new species are described and illustrated, and bring the number of species in Perinereis to 97. The new species are compared and contrasted to the closely similar P.heterodonta, P.nuntia and other congeners from the region.

Molecular identification of Oncomelania hupensis lindoensis, snail intermediate hosts of Schistosoma japonicum from Central Sulawesi, Indonesia

Abstract. Sutrisnawati, Ramadhan A, Trianto M. 2022. Molecular identification of Oncomelania hupensis lindoensis, snail intermediate hosts of Schistosoma japonicum from Central Sulawesi, Indonesia. Biodiversitas 23: 5989-5994. Schistosomiasis is a zoonotic parasitic disease with Oncomelania hupensis lindoensis, intermediate snail hosts of Schistosoma japonicum. The spread of O. hupensis lindoensis snail habitat was found in the three areas of the Napu, Bada, and Lindu Highlands with an infection rate above 1%. Oncomelania hupensis lindoensis is primarily cryptic species that are morphologically difficult to identify and distinguish from other species. Consequently, it can be confused with the naming of species. One of the molecular approaches that can be used to identify the Oncomelania spp. quickly and accurately is DNA barcoding using the COI mitochondrial gene. However, the research on identifying Oncomelania spp. in Indonesia is still very limited. Therefore, this study aimed to identify six Oncomelania spp. from Napu and Lindu, Central Sulawesi using COI mitochondrial gene as a molecular marker for DNA barcoding. The method used in this study was a Polymerase Chain Reaction (PCR) method with universal primers, LCO-F and HCO-R. The data obtained were then analyzed using GeneStudio, DNASTAR, BLAST, Identification Engine, Mesquite, MEGAX, and BEAST. The analysis was conducted to obtain similarity, genetic distance and reconstruct a phylogenetic tree. The result revealed that all six samples of Oncomelania spp. collected from Napu and Lindu were identified in one species, namely Oncomelania hupensis lindoensis. This research is very important to be carried out regularly periodically so that it can be used as a basis for Schistosomiasis control program data and related sectors to eradicate snails effectively, efficiently, and on target.

Quantifying Trade-Offs in the Choice of Ribosomal Barcoding Markers for Fungal Amplicon Sequencing: a Case Study on the Grapevine Trunk Mycobiome.

The evolution of sequencing technology and multiplexing has rapidly expanded our ability to characterize fungal diversity in the environment. However, obtaining an unbiased assessment of the fungal community using ribosomal markers remains challenging. Longer amplicons were shown to improve taxonomic resolution and resolve ambiguities by reducing the risk of spurious operational taxonomic units. We examined the implications of barcoding strategies by amplifying and sequencing two ribosomal DNA fragments. We analyzed the performance of the full internal transcribed spacer (ITS) and a longer fragment including also a part of the 28S ribosomal subunit replicated on 60 grapevine trunk core samples. Grapevine trunks harbor highly diverse fungal communities with implications for disease development. Using identical handling, amplification, and sequencing procedures, we obtained higher sequencing depths for the shorter ITS amplicon. Despite the more limited access to polymorphism, the overall diversity in amplified sequence variants was higher for the shorter ITS amplicon. We detected no meaningful bias in the phylogenetic composition due to the amplicon choice across analyzed samples. Despite the increased resolution of the longer ITS-28S amplicon, the higher and more consistent yields of the shorter amplicons produced a clearer resolution of the fungal community of grapevine stem samples. Our study highlights that the choice of ribosomal amplicons should be carefully evaluated and adjusted according to specific goals. IMPORTANCE Surveying fungal communities is key to our understanding of ecological functions of diverse habitats. Fungal communities can inform about the resilience of agricultural ecosystems, risks to human health, and impacts of pathogens. Community compositions are typically analyzed using ribosomal DNA sequences. Due to technical limitations, most fungal community surveys were based on amplifying a short but highly variable fragment. Advances in sequencing technology enabled the use of longer fragments that can address some limitations of species identification. In this study, we examined the implications of choosing either a short or long ribosomal sequence fragment by replicating the analyses on 60 grapevine wood core samples. Using highly accurate long-read sequencing, we found that the shorter fragment produced substantially higher yields. The shorter fragment also revealed more sequence and species diversity. Our study highlights that the choice of ribosomal amplicons should be carefully evaluated and adjusted according to specific goals.

DNA barcoding markers provide insight into species discrimination, genetic diversity and phylogenetic relationships of yam (Dioscorea spp.)

DNA barcoding markers provide insight into species discrimination, genetic diversity and phylogenetic relationships of yam (Dioscorea spp.)

New Records of the Cryptogenic Soft Coral Genus Stragulum (Tubiporidae) from the Eastern Caribbean and the Persian Gulf

The monotypic soft coral genus Stragulum van Ofwegen and Haddad, 2011 (Octocorallia: Malacalcyonacea: Tubiporidae) was originally described from Brazil, southwest Atlantic Ocean. Here, we report the first records of the genus from the eastern Caribbean and the Persian Gulf in the northwest Indian Ocean. We compare the morphological features of specimens, together with molecular data from three commonly used barcoding markers (COI, mtMutS, 28S rDNA) and 308 ultraconserved elements (UCE) and exon loci sequenced using a target-enrichment approach. The molecular and morphological data together suggest that specimens from all three localities are the same species, i.e., Stragulum bicolor van Ofwegen and Haddad, 2011. It is still not possible to establish the native range of the species or determine whether it may be an introduced species due to the limited number of specimens included in this study. However, the lack of historical records, its fouling abilities on artificial substrates, and a growing number of observations support the invasive nature of the species in Brazilian and Caribbean waters and therefore suggest that it may have been introduced into the Atlantic from elsewhere. Interestingly, the species has not shown any invasive behaviour in the Persian Gulf, where it has been found only on natural, rocky substrates. The aim of the present report is to create awareness of this taxon with the hope that this will lead to new records from other localities and help to establish its native range.

Complete chloroplast genome of Boesenbergia rotunda and a comparative analysis with members of the family Zingiberaceae.

Boesenbergia rotunda (L.) Mansf. is a medically important ginger species of the family Zingiberaceae but its genomic information on molecular phylogeny and identification is scarce. In this work, the chloroplast genome of B. rotunda was sequenced, characterized and compared to the other Zingiberaceae species to provide chloroplast genetic resources and to determine its phylogenetic position in the family. The chloroplast genome of B. rotunda was 163,817 bp in length and consisted of a large single-copy (LSC) region of 88,302 bp, a small single-copy (SSC) region of 16,023 bp and a pair of inverted repeats (IRA and IRB) of 29,746 bp each. The chloroplast genome contained 113 unique genes, including 79 protein-coding genes, 30 transfer RNA (tRNA) genes and four ribosomal RNA (rRNA) genes. Several genes had atypical start codons, while most amino acids exhibited biased usage of synonymous codons. Comparative analyses with various chloroplast genomes of Zingiberaceae taxa revealed several highly variable regions (psbK-psbI, trnT-GGU-psbD, rbcL-accD, ndhF-rpl32, and ycf1) in the LSC and SSC regions in the chloroplast genome of B. rotunda that could be utilized as molecular markers for DNA barcoding and species delimitation. Phylogenetic analyses based on shared protein-coding genes revealed that B. rotunda formed a distinct lineage with B. kingii Mood & L.M.Prince, in a subclade that also contained the genera Kaempferia and Zingiber. These findings constitute the first chloroplast genome information of B. rotunda that could be a reference for phylogenetic analysis and identification of genus Boesenbergia within the Zingiberaceae family. Supplementary InformationThe online version contains supplementary material available at 10.1007/s40415-022-00845-w.