Nuclear Small Subunit Ribosomal RNA Research Articles

Molecular Identification and Survey of Cyclospora spp. in Cattle in Shanxi Province, North China

To date, more than 20 species in the genus Cyclospora have been reported. Among them, Cyclospora cayetanensis has been recognized as the causative agent of human cyclosporiasis, which is characterized by severe intestinal injury and prolonged diarrhea in patients with immune dysfunction. The presence of C. cayetanensis in cattle has been confirmed. To date, however, no surveillance data are available on the occurrence and prevalence of Cyclospora spp. in cattle in Shanxi Province, North China. In the present study, a total of 761 fecal samples collected from cattle in three representative counties (Qi, Jishan, and Shanyin) in this Province were examined for Cyclospora spp. by using a polymerase-chain-reaction–restriction-fragment-length polymorphism (PCR–RFLP) test based on the nuclear small subunit ribosomal RNA (SSU rRNA) gene. The prevalence of Cyclospora spp. in cattle was 2.1%, and region, age, sex, and breed were not identified to be risk factors. Molecular evolutionary analysis based on the SSU rRNA sequences revealed that all 12 of the isolates were relatively distant from the human pathogen C. cayetanensis; seven isolates were grouped with Cyclospora colobi, whereas the others were grouped with cattle Cyclospora spp. reported previously. Though C. cayetanensis was not detected in cattle in the present study, more investigations should be performed in human populations, other animal species, or cattle from other regions of Shanxi Province and other environmental sources from the One Health perspective.

Trichuris Globulosa Von Linstow, 1901 from one-humped camel (Camelus dromedarius) in Egypt: prevalence, morphological and molecular study

BackgroundTrichuris spp. (whipworms) are soil-transmitted helminths distributed worldwide, parasitizing several mammalian hosts such as ruminants, primates, and rodents. Trichuris spp. is one of the most common intestinal parasites affecting both humans and animals, and it can spread directly through the fecal-oral route, resulting in severe illness and financial loss. So, this work aims to detect the frequency of Trichuris spp. in camels in Beheira Governorate, Egypt, and to identify Trichuris spp. through morphometrical studies, molecular analysis, and phylogenetic analysis.ResultsA total of 35 dromedaries out of 127 investigated had Trichuris spp. infection, meaning that the overall prevalence was 27.56%. The age of the camel affected the infection rate, older animals (> 5 years) having a higher prevalence of infection (24%) than animals of ages (< 3 years) (20%) than animals of ages (3–5 years) (19.14%). According to season: Trichuris spp. showed a unique pattern in camels in different seasons: summer (31.25%) > autumn (28.13%) > spring (25.8%) > winter (25%) indicating year-round infection. T. globulosa was identified morphometrically from camels in Beheira Governorate, Egypt. The BLAST analysis revealed the presence of T. globulosa isolate from camels using the Genbank database depending on nuclear small subunit ribosomal RNA (18s) and cytochrome b (Cytb) genes.ConclusionA high prevalence of T. globulosa was found in camels in Beheira Governorate, Egypt. This is the first report to confirm the identification of T. globulosa from camel based on morphometrical studies and molecular and phylogenetic analysis in Egypt. More thorough studies on the incidence, molecular, and genetic analysis of Trichuris spp. in Egypt are required in addition to camel control programs.

Cryptosporidium occultus in disguise

As data accumulate in GenBank, the difficulties of delineating species of Cryptosporidium based on nuclear small subunit ribosomal RNA (ssu rRNA) gene information alone becomes increasingly evident. Here, we summarize currently available evidence suggesting that several ssu rDNA sequences primarily referred to as Cryptosporidium suis (some of them from non-suid hosts) should be considered Cryptosporidium occultus.

Novel Species and Records of Dictyosporiaceae from Freshwater Habitats in China and Thailand.

China and Thailand are rich in fungal diversity with abundant freshwater resources that are favorable for numerous fungal encounters. Resulting from this, the majority of the Dictyosporiaceae species reported were from these two countries. During the investigation on the diversity of lignicolous freshwater fungi in the Greater Mekong Subregion, eleven collections of cheirosporous species on submerged wood were collected from lentic and lotic habitats in China and Thailand. Phylogenetic analysis that combined nuclear small-subunit ribosomal RNA (SSU), internal transcribed spacer region (ITS), nuclear large subunit ribosomal RNA (LSU) and translation elongation factor 1α (tef 1-α) loci revealed six new species: Dictyocheirospora chiangmaiensis, D. multiappendiculata, D. suae, Digitodesmium aquaticum, Vikalpa grandispora and V. sphaerica. In addition, four known species were also identified and reported based on morphological and phylogenetic evidence. The detailed descriptions and illustrations of these taxa are provided with an updated phylogenetic tree of Dictyosporiaceae.

Diophrys pauciciliata n. sp. (Ciliophora, Euplotida), a new marine spirotrich ciliate with highly static morphometrics

A new marine ciliate was discovered from intertidal mudflat from South Korea. Based on the observation of living cells, protargol-impregnated specimens and SEM, we describe it as new species belonging to the genus Diophrys Dujardin, 1841, namely Diophrys pauciciliata n. sp. Its nuclear small subunit ribosomal RNA gene also supports the taxonomic assignment. Diophrys pauciciliata n. sp. is one of the smallest species in the genus. Besides, the bipartite adoral zone and oligomerized dorsal bristles easily discriminate the new species from other congeners. Interestingly, D. pauciciliata n. sp. showed highly static states in all morphometrics (<8.5 % of coefficient of variation) and in particular, the number of frontal adoral membranelles and the counts of dorsal ciliature (e.g., number of dorsal kineties and numbers of dikinetids within each kinety) are invariable. Considering morphometric data of previous studies in the genus Diophrys, the narrow range of character states is unexpected because cell length is usually positively correlated with the numbers of adoral membranelles and dorsal dikinetids.

Insights into the phylogeny of the family Deviatidae (Protozoa, Ciliophora, Hypotrichia) based on multi-gene, morphological and ontogenetic information, with the establishment of a new species Deviata multilineae n. sp.

Hitherto, the phylogeny of ciliated protists, an important group of model organisms in many fields, has been mainly based on a single marker gene (SSU rDNA, nuclear small subunit ribosomal RNA gene). However, there is increasing evidence showing this is insufficient to provide robust phylogenies and has resulted in confusing systematics in many ciliates groups. Among these, the phylogenies within family Deviatidae (Spirotrichea, Hypotrichia) are ambiguous due to the dependence on SSU rDNA and undersampling. Here, we provide eight new sequences and conduct phylogenetic analyses based on both multi-gene and single-gene to clarify evolutionary relationships among all deviatids for which gene sequence are available. The results reveal that: (1) the monophyly of Deviatidae is well-supported by both single-gene and concatenated data; (2) the presence of fine cirri and relatively wide spacing of these cirri within all rows are plesiomorphies of Deviatidae; (3) Pseudosincirra longicirrata is closely related to Deviata rositae, which is supported by their shared possession of dorsomarginal kineties; (4) phylogenetic analyses and approximately unbiased test based on multi-gene support a close relationship among taxa lacking dorsomarginal kineties (D. parabacilliformis, D. multilineae nov. spec., D. abbrevescens, D. brasiliensis and Perisincirra paucicirrata); (5) Deviatidae shows a close relationship with Dorsomarginalia and Strongylidium-Hemiamphisiella-Pseudouroleptus assemblage, suggesting the presence/absence of dorsomarginal kineties is phylogenetically informative in this family and presence of them may be a plesiomorphy. Based on the morphological, morphogenetic and phylogenetic data, the evolutionary relationships within Deviatidae are hypothesized, and a new ciliate, Deviata multilineae nov. spec., collected from China, is investigated.

New contributions to the Cyrtophoria ciliates (Protista, Ciliophora): Establishment of new taxa and phylogenetic analyses using two ribosomal genes.

Periphytic ciliates play a vital role in the material cycle and energy flow of microbial food web, however, their taxonomy and biodiversity are inadequately studied given their high species richness. Two new and one little known species, viz. Derouxella lembodes gen. et sp. nov., Cyrtophoron multivacuolatum sp. nov., and Cyrtophoron apsheronica Aliev, 1991, collected from coastal waters of China, were investigated using modern methods. Derouxella gen. nov. can be recognized by having dorsoventrally flattened body, a podite, one fragmented preoral kinety, two parallel circumoral kineties, and somatic kineties progressively shortened from right to left. Morphological classification and phylogenetic analyses based on nuclear small subunit ribosomal RNA (nSSU rRNA) and mitochondrial small subunit ribosomal RNA (mtSSU rRNA) gene sequence data inferred that Derouxella gen. nov. occupies an intermediate position between Hartmannulidae and Dysteriidae. Cyrtophoron multivacuolatum sp. nov. is characterized by large body size, the numbers of somatic kineties and nematodesmal rods, and having numerous contractile vacuoles. The genus Cyrtophoron and the poorly known species C. apsheronica were redefined. Even with the addition of newly obtained nSSU rRNA and mtSSU rRNA gene sequencesof Cyrtophoron, the family Chlamydodontidae was still recovered as a monophyletic group, the monophyly of Cyrtophoron was supported too.

Molecular characterisation and genetic affinities of Cyclophyllidean cestodes infecting wild rats in Peninsular Malaysia.

Cestode infections is widely reported in rodents, however species identification remains problematic due to the genetic or interspecies variation. Therefore, this study was aimed to verify the Cyclophyllidean parasites recovered from wild rats captured from different forest types using molecular based methods. Maximum-likelihood (ML) and neighbour-joining (NJ) trees were constructed inferred from 18 small subunit nuclear ribosomal RNA gene (18SrDNA) and mitochondrial cytochrome c oxidase subunit one gene (COX1) sequences of cestode worms recovered from 124 individuals from four rat species. Sequences obtained from both Hymenolepis diminuta and Hydatigera parva represents the first records in Malaysia. All the sequences were successfully amplified with product with total length of 205 and 1202 base pairs (bp), respectively. Three cestode species from the Family Hymenolepididae (Hymenolepis diminuta) and Family Taeniidae (Hydatigera parva; Hydatigera taeniaeformis) were successfully characterized using phylogenetic analyses and haplotype networking. Phylogenetic analysis showed that H. diminuta, Hydatigera parva (Hy. parva) and Hydatigera taeniaeformis (Hy. taeniaeformis) formed its own monophyletic clade in 18SrDNA analyses. Results also showed that Hy. taeniaeformis shared the same haplotype group with Hy. taeniaeformis from China (COX1) and linked with Hy. taeniaeformis from Japan (18SrDNA) while the Malaysian H. diminuta clearly formed a separate haplotype and networked with other regions. The Malaysian Hy. parva isolation, on the other hand, appeared to be genetically distinct from the European Hy. parva (Spain) strain, but closely linked to the local isolates. Molecular methods employed successfully improved in the detection of complex species in this group. The findings showed that molecular data can be useful to deeply study intra-specific variation in other cestode worms.

Stigmatomyces aff. limnophorae on dipteran hosts in Peninsular Malaysia

Flies parasitized by Laboulbeniales, captured in Selangor state, were examined and identified as Boettcherisca javanica, Boettcherisca sp ., and Hypopygiopsis violacea. The fungus was identified as Stigmatomyces aff. limnophorae based on morphology and phylogenetic analysis of sequences of the partial nuclear small and large subunit ribosomal RNA genes. This fungus represents a new record for Malaysia; and these are the first reports of Boettcherisca and Hypopygiopsis as hosts for any laboulbenialean species.

Species diversity, systematic revision and molecular phylogeny of Ganodermataceae (Polyporales, Basidiomycota) with an emphasis on Chinese collections.

Ganodermataceae is one of the main families of macrofungi since species in the family are both ecologically and economically important. The double-walled basidiospores with ornamented endospore walls are the characteristic features of Ganodermataceae. It is a large and complex family; although many studies have focused on Ganodermataceae, the global diversity, geographic distribution, taxonomy and molecular phylogeny of Ganodermataceae still remained incompletely understood. In this work, taxonomic and phylogenetic studies on worldwide species of Ganodermataceae were carried out by morphological examination and molecular phylogenetic analyses inferred from six gene loci including the internal transcribed spacer regions (ITS), the large subunit of nuclear ribosomal RNA gene (nLSU), the second largest subunit of RNA polymerase II gene (rpb2), the translation elongation factor 1-α gene (tef1), the small subunit mitochondrial rRNA gene (mtSSU) and the small subunit nuclear ribosomal RNA gene (nSSU). A total of 1 382 sequences were used in the phylogenetic analyses, of which 817 were newly generated, including 132 sequences of ITS, 139 sequences of nLSU, 83 sequences of rpb2, 124 sequences of tef1, 150 sequences of mtSSU and 189 sequences of nSSU. The combined six-gene dataset included sequences from 391 specimens representing 146 taxa from Ganodermataceae. Based on morphological and phylogenetic analyses, 14 genera were confirmed in Ganodermataceae: Amauroderma, Amaurodermellus, Cristataspora, Foraminispora, Furtadoella, Ganoderma, Haddowia, Humphreya, Magoderna, Neoganoderma, Sanguinoderma, Sinoganoderma, Tomophagus and Trachydermella. Among these genera, Neoganoderma gen. nov. is proposed for Ganoderma neurosporum; Sinoganoderma gen. nov. is proposed for Ganoderma shandongense; Furtadoella gen. nov. is proposed to include taxa previously belonging to Furtadoa since Furtadoa is a homonym of a plant genus in the Araceae; Trachydermella gen. nov. is proposed to include Trachyderma tsunodae since Trachyderma is a homonym of a lichen genus in the Pannariaceae. Twenty-three new species, viz., Ganoderma acaciicola, G. acontextum, G. alpinum, G. bubalinomarginatum, G. castaneum, G. chuxiongense, G. cocoicola, G. fallax, G. guangxiense, G. puerense, G. subangustisporum, G. subellipsoideum, G. subflexipes, G. sublobatum, G. tongshanense, G. yunlingense, Haddowia macropora, Sanguinoderma guangdongense, Sa. infundibulare, Sa. longistipitum, Sa. melanocarpum, Sa. microsporum and Sa. tricolor are described. In addition, another 33 known species are also described in detail for comparison. Scanning electron micrographs of basidiospores of 10 genera in Ganodermataceae are provided. A key to the accepted genera of Ganodermataceae and keys to the accepted species of Ganoderma, Haddowia, Humphreya, Magoderna, Sanguinoderma and Tomophagus are also provided. In total, 278 species are accepted as members of Ganodermataceae including 59 species distributed in China. Taxonomic novelties: New genera: Furtadoella B.K. Cui & Y.F. Sun, Neoganoderma B.K. Cui & Y.F. Sun, Sinoganoderma B.K. Cui, J.H. Xing & Y.F. Sun and Trachydermella B.K. Cui & Y.F. Sun; New species: Ganoderma acaciicola B.K. Cui, J.H. Xing & Y.F. Sun, G. acontextum B.K. Cui, J.H. Xing & Vlasák, G. alpinum B.K. Cui, J.H. Xing & Y.F. Sun, G. bubalinomarginatum B.K. Cui, J.H. Xing & Y.F. Sun, G. castaneum B.K. Cui, J.H. Xing & Y.F. Sun, G. chuxiongense B.K. Cui, J.H. Xing & Y.F. Sun, G. cocoicola B.K. Cui, J.H. Xing & Y.F. Sun, G. fallax B.K. Cui, J.H. Xing & Vlasák, G. guangxiense B.K. Cui, J.H. Xing & Y.F. Sun, G. puerense B.K. Cui, J.H. Xing & Y.F. Sun, G. subangustisporum B.K. Cui, J.H. Xing & Y.F. Sun, G. subellipsoideum B.K. Cui, J.H. Xing & Y.F. Sun, G. subflexipes B.K. Cui, J.H. Xing & Y.F. Sun, G. sublobatum B.K. Cui, J.H. Xing & Y.F. Sun, G. tongshanense B.K. Cui, J.H. Xing & Y.F. Sun, G. yunlingense B.K. Cui, J.H. Xing & Y.F. Sun, Haddowia macropora B.K. Cui, Vlasák & Y.F. Sun, Sanguinoderma guangdongense B.K. Cui & Y.F. Sun, Sa. infundibulare B.K. Cui & Y.F. Sun, Sa. longistipitum B.K. Cui & Y.F. Sun, Sa. melanocarpum B.K. Cui & Y.F. Sun, Sa. microsporum B.K. Cui & Y.F. Sun and Sa. tricolor B.K. Cui & Y.F. Sun; New combinations: Furtadoella biseptata (Costa-Rezende et al.) B.K. Cui & Y.F. Sun, Fu. brasiliensis (Singer) B.K. Cui & Y.F. Sun, Fu. corneri (Gulaid & Ryvarden) B.K. Cui & Y.F. Sun, Neoganoderma neurosporum (J.S. Furtado) B.K. Cui & Y.F. Sun, Sinoganoderma shandongense (J.D. Zhao & L.W. Xu) B.K. Cui, J.H. Xing & Y.F. Sun and Trachydermella tsunodae (Yasuda ex Lloyd) B.K. Cui & Y.F. Sun. Citation: Sun Y-F, Xing J-H, He X-L, Wu D-M, Song C-G, Liu S, Vlasák J, Gates G, Gibertoni TB, Cui B-K (2022). Species diversity, systematic revision and molecular phylogeny of Ganodermataceae (Polyporales, Basidiomycota) with an emphasis on Chinese collections. Studies in Mycology 101: 287-415. doi: 10.3114/sim.2022.101.05.

Amplicon sequencing reveals the arbuscular mycorrhizal fungal community composition in Cryptomeria japonica at one local site

ABSTRACT Arbuscular mycorrhizal fungi (AMF) colonize the plant root endosphere and extend the host’s resource network. However, little is known about the method to characterize the intraradical AMF communities of a specific tree species in forests. Therefore, we aimed at testing a method for characterizing and comparing the extra- and intraradical AMF communities of Cryptomeria japonica (Cupressaceae). In February 2020, we tracked and sampled roots from 5 target trees and the surrounding soil and confirmed their identity using plan DNA barcoding methods. After a microscopic analysis of mycorrhizal root colonization, we used Illumina amplicon sequencing to characterize the corresponding AMF communities. The C. japonica roots were colonized by AMF, displaying the Paris- and Arum-types of mycorrhizae. At 100% sequence identity, amplicon sequences of the small-subunit nuclear ribosomal RNA gene clustered into 155 operational taxonomic units (OTUs), 33 and 139 in the intra- and extraradical AMF communities, respectively, corresponding to three families and five genera. Nevertheless, only Acaulospora, Glomus, and Rhizophagus were detected in the root samples. The AMF communities did not significantly differ between the root and the surrounding soil, but we observed a significant nestedness pattern in the community. To avoid erroneous conclusions, combining plant DNA barcoding with amplicon sequencing is a reliable approach, which we recommend for future studies to describe the intraradical AMF community of a specific tree species in forests.

Morphological and molecular characterization of Eustrongylides excisus larvae (Nematoda: Dioctophymatidae) in Sander lucioperca (L.) from Northern Turkey.

The genus Eustrongylides Jägerskiöld, 1909 includes parasitic nematodes (Dioctophymatidae) affecting various fish species and piscivorous birds of freshwater ecosystems. Currently, there is little information on the molecular characterization of E. excisus based on nuclear ribosomal internal transcribed spacer (ITS) rDNA regions. However, before the present study, there had been no reports of characterizing the E. excisus using nuclear small subunit ribosomal RNA (SSU rRNA) and mitochondrial cytochrome c oxidase subunit I (COI) genes sequences. In the present study, Eustrongylides spp. larvae were collected from pike-perch Sander lucioperca (L.) in Northern Turkey, and characterized by sequencing of ITS regions, SSU rRNA, and COI markers. Larvae herein morphologically identified as the fourth stage of Eustrongylides spp. were genetically identified as E. excisus based on the ITS sequence analysis. This study is the first record of SSU rRNA and COI sequences for E. excisus in GenBank. This is also a molecular characterization of E. excisus for the first time in Turkey. The ITS, SSU rRNA, and COI sequences of E. excisus can be used to establish the phylogenetic relationships of Eustrongylides species from Turkey and worldwide for further studies.

DNA metabarcoding of zooplankton communities: species diversity and seasonal variation revealed by 18S rRNA and COI.

BackgroundZooplankton is an important component of aquatic organisms and has important biological and economical significance in freshwater ecosystems. However, traditional methods that rely on morphology to classify zooplankton require expert taxonomic skills. Moreover, traditional classification methods are time-consuming and labor-intensive, which is not practical for the design of conservation measures and ecological management tools based on zooplankton diversity assessment.MethodsWe used DNA metabarcoding technology with two different markers: the nuclear small subunit ribosomal RNA (18S rRNA) and mitochondrial cytochrome c oxidase (COI), to analyze 72 zooplankton samples collected in 4 seasons and 9 locations from the Sanmenxia Reservoir. We investigated seasonal changes in the zooplankton community and their relationship with water environmental factors.ResultsA total of 190 species of zooplankton were found, belonging to 12 phyla, 24 classes, 61 orders, 111 families, and 174 genera. Protozoa, especially ciliates, were the most diverse taxa. Richness and relative abundance of zooplankton showed significant seasonal changes. Both alpha and beta diversity showed seasonal trends: the diversity in summer and autumn was higher than that in winter and spring. The zooplankton diversity was most similar in winter and spring. By correlating metabarcoding data and water environmental factors, we proved that water temperature, chemical oxygen demand, total nitrogen and ammoniacal nitrogen were the main environmental factors driving the seasonal changes in zooplankton in the Sanmenxia Reservoir. Water temperature, followed by total nitrogen, were the most influential factors. This study highlights the advantages and some limitations of zooplankton molecular biodiversity assessment using two molecular markers.

Anteholosticha foissneri n. sp., a marine hypotrich ciliate (Ciliophora: Spirotrichea) from Vietnam: Morphology, morphogenesis, and molecular phylogeny

In a study of marine ciliate diversity, we collected an Anteholosticha monilata-like population from Vietnam. To identify this population, we analyzed its morphology, some morphogenetic stages, and molecular phylogeny. Based on these data, we conclude that the Vietnamese population is new to science. Anteholosticha foissneri n. sp. resembles Anteholosticha monilata-like species considering (1) the number and arrangement of macronuclear nodules and micronuclei; (2) the presence of cortical granules; and (3) the saline habitat. However, the new species can be easily distinguished from these species by the arrangement, color, and shape of the cortical granules. The divisional morphogenesis commences with the de novo proliferation of basal bodies as a single longitudinal patch left of the posteriormost midventral cirral pair. This character state has not been reported before in Anteholosticha (based on check of the available data) and probably reflects a distinct clade within the nuclear small subunit ribosomal RNA gene tree.

Species diversity, taxonomy and phylogeny of Polyporaceae (Basidiomycota) in China

Polyporaceae is one of the most important families of Basidiomycota. Investigations on the species diversity, taxonomy and phylogeny of Polyporaceae in China are carried out. So far 217 species belonging to 42 genera are reported from China. Two new genera: Amylosporia gen. nov. and Murinicarpus gen. nov., twelve new species: Coriolopsis dendriformis sp. nov., C. hainanensis sp. nov., Funalia cystidiata sp. nov., Haploporus microsporus sp. nov., Perenniporia citrinoalba sp. nov., P. yinggelingensis sp. nov., Picipes hainanensis sp. nov., P. jiajinensis sp. nov., P. pseudovarius sp. nov., Trametes duplexa sp. nov., T. ellipsoidea sp. nov. and T. stiptica sp. nov., and six new combinations, Amylosporia hattorii comb. nov., Hornodermoporus latissimus comb. nov., Murinicarpus subadustus comb. nov., Picipes pumilus comb. nov., Vanderbylia delavayi comb. nov. and Vanderbylia robiniophila comb. nov., are proposed. All the species are described based on the Chinese collections. Keys to genera of Polyporaceae occurring in China and keys to species of each genus are provided. This monograph provides a revised classification of Polyporaceae in China according to the modern taxonomy. The phylogeny of Polyporaceae from China are reconstructed based on DNA sequences of multiple loci including the internal transcribed spacer (ITS) regions, the large subunit nuclear ribosomal RNA gene (nLSU), the small subunit nuclear ribosomal RNA gene (nSSU), the small subunit mitochondrial rRNA gene sequences (mtSSU), the translation elongation factor 1-α gene (TEF1), the β-tubulin gene (TBB1), the RNA polymerase II largest subunit (RPB1) and second largest subunit (RPB2) genes. In addition, full morphological descriptions, illustrations, color photographs, taxonomic notes, ecology and all the available sequences of Polyporaceae species found from China are provided.

Two new Neofomitella species (Polyporaceae, Basidiomycota) based on morphological and molecular evidence

Two new Neofomitella species, N. australiensis and N. guangxiensis, are described based on morphological and molecular characters. Neofomitella australiensis is characterized by white pore surface when fresh, dextrinoid skeletal and binding hyphae, and orange-red context near pileal surface when fresh. Neofomitella guangxiensis is characterized by round to angular pores (5–6 per mm), weakly dextrinoid skeletal and binding hyphae and large basidiospores (5.5–7.5 × 1.8–2.2 μm). Phylogenetic analyses were carried out based on sequences from the internal transcribed spacer (ITS), the large subunit nuclear ribosomal RNA gene (nLSU), the small subunit nuclear ribosomal RNA gene (nSSU), the small subunit mitochondrial rRNA gene (mtSSU), the translation elongation factor 1-α gene (EF1-α), the largest subunit of RNA polymerase II (RPB1) and the second largest subunit of RNA polymerase II (RPB2), and the result confirmed the affinities of the two new species within Neofomitella. An identification key to species of Neofomitella is provided.

Morphology and Molecular Phylogeny of Two New Terrestrial Ciliates, Australocirrus rubrus n. sp. and Notohymena gangwonensis n. sp. (Ciliophora: Oxytrichidae), from South Korea.

Two new soil oxytrichids, Australocirrus rubrus n. sp. and Notohymena gangwonensis n. sp., were discovered from South Korea. Morphologically, A.rubrus shares many features with A.australis, and these two species form a single clade in a molecular tree based on nuclear small subunit ribosomal RNA (SSU rRNA) gene sequences. Australocirrus rubrus mainly differs from A.australis in the color (citrine color vs. reddish) and distribution of the cortical granules. Additionally, we confirm that the genus Australocirrus is not a monophyletic group, as A.shii is separated from the clade comprising the other Australocirrus species, being clustered instead with other taxa. Notohymena gangwonensis n. sp. mainly differs from its congeners by the following combination of features: irregularly distributed cortical granules (vs. arranged in groups associated with cirri and dorsal kineties), variable four or five (usually four) transverse cirri (vs. invariable five), and the anteriormost pretransverse cirrus V/2 on 13.2-16.1% of cell length (vs. on or above 18.9% of cell length). Currently, there are no available gene sequences for members of the genus Notohymena, thus we provide SSU rRNA gene sequences from the new species of Notohymena, as well as detailed morphological descriptions of the novel species.

Symbiochlorum hainanensis gen. et sp. nov. (Ulvophyceae, Chlorophyta) isolated from bleached corals living in the South China Sea

Light/scanning electron/transmission microscopy-based morphological analyses and multiple nucleotide sequences-based molecular phylogenetic analyses are used to identify and assess the phylogenetic position of a new unidentified green alga isolated from bleached corals living in the South China Sea. This new unidentified green alga is a unicellular marine alga and has uninucleate vegetative cells and multiple chloroplasts with a pyrenoid. It can form aplanosporangium covered by cell walls and reproduces by releasing autospore. These features differ substantially from those of the two genera Ignatius and Pseudocharacium. Those two genera have been accommodated in the Ignatius clade. Nucleotide sequences of the nuclear small subunit ribosomal RNA gene (18S rRNA), internal transcribed spacer 2 of ribosomal RNA gene (ITS2) and ribulose-1,5 bisphosphate carboxylase/oxygenase large subunit gene (rbcL, partial) are obtained and compared with published green algal sequences. The results from the morphology, ultrastructure, and multiple nucleotide sequences data support the placement of the new unidentified green alga in Ulvophyceae. This new unidentified isolate is described as Symbiochlorum hainanensis gen. et sp. nov., a new sister lineage to the Ignatius clade, Ulvophyceae, Chlorophyta.

DNA metabarcoding of littoral hard-bottom communities: high diversity and database gaps revealed by two molecular markers.

Biodiversity assessment of marine hard-bottom communities is hindered by the high diversity and size-ranges of the organisms present. We developed a DNA metabarcoding protocol for biodiversity characterization of structurally complex natural marine hard-bottom communities. We used two molecular markers: the “Leray fragment” of mitochondrial cytochrome c oxidase (COI), for which a novel primer set was developed, and the V7 region of the nuclear small subunit ribosomal RNA (18S). Eight different shallow marine littoral communities from two National Parks in Spain (one in the Atlantic Ocean and another in the Mediterranean Sea) were studied. Samples were sieved into three size fractions from where DNA was extracted separately. Bayesian clustering was used for delimiting molecular operational taxonomic units (MOTUs) and custom reference databases were constructed for taxonomic assignment. Despite applying stringent filters, we found high values for MOTU richness (2,510 and 9,679 MOTUs with 18S and COI, respectively), suggesting that these communities host a large amount of yet undescribed eukaryotic biodiversity. Significant gaps are still found in sequence reference databases, which currently prevent the complete taxonomic assignment of the detected sequences. In our dataset, 85% of 18S MOTUs and 64% of COI MOTUs could be identified to phylum or lower taxonomic level. Nevertheless, those unassigned were mostly rare MOTUs with low numbers of reads, and assigned MOTUs comprised over 90% of the total sequence reads. The identification rate might be significantly improved in the future, as reference databases are further completed. Our results show that marine metabarcoding, currently applied mostly to plankton or sediments, can be adapted to structurally complex hard bottom samples. Thus, eukaryotic metabarcoding emerges as a robust, fast, objective and affordable method to comprehensively characterize the diversity of marine benthic communities dominated by macroscopic seaweeds and colonial or modular sessile metazoans. The 18S marker lacks species-level resolution and thus cannot be recommended to assess the detailed taxonomic composition of these communities. Our new universal primers for COI can potentially be used for biodiversity assessment with high taxonomic resolution in a wide array of marine, terrestrial or freshwater eukaryotic communities.

Genetic and morphological characterization of Thysaniezia tapeworms from cattle and sheep in Senegal.

Genetic and morphological diversity of Thysaniezia tapeworms from cattle and sheep in Senegal was investigated using light and scanning microscopic observations and molecular analysis based on mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear small subunit ribosomal RNA gene (SSU rDNA). A total of 52 adult tapeworms were collected from sheep and cattle. Although the tapeworms of the two hosts were morphologically very close, phylogenetic analysis based on cox1 and SSU rDNA gene sequences showed that they were divided into two clades corresponding each to a host. The maximum pairwise divergence between the clades were 12.1% in cox1 and 2.9% in SSU rDNA, indicating they are distinct species. The tapeworms collected from sheep were morphologically identified as Thysaniezia ovilla, a cosmopolitan species in domestic ruminants. Detailed morphological observations revealed a consistent difference between the tapeworms obtained from sheep and those from cattle. The latter were identified as Thysaniezia connochaeti. The present study highlights presence of two species of Thysaniezia among domestic ruminants in Senegal: T. ovilla specific to sheep and T. connochaeti specific to cattle. Our work is the first report of T. connochaeti from domestic animals.