Large Subunit Ribosomal RNA Research Articles

New morphological and genetic data of Gigantorhynchus echinodiscus (Diesing, 1851) (Acanthocephala: Archiacanthocephala) in the giant anteater Myrmecophaga tridactyla Linnaeus, 1758 (Pilosa: Myrmecophagidae).

Gigantorhynchus echinodiscus (Diesing, 1851) is a parasite of anteaters in South America. Although described by Diesing in 1851, there is still a lack of taxonomic and phylogenetic information regarding this species. In the present study, we redescribe G. echinodiscus collected from a giant anteater, Myrmecophaga tridactyla Linnaeus, 1758, from the Brazilian Cerrado (Savannah) in the State of São Paulo by light and scanning electron microscopy. In addition, phylogenies were inferred from partial DNA gene sequence of the nuclear large subunit ribosomal RNA gene (28S rRNA). We provide for the first time details of the proboscis with a crown having 18 large hooks and numerous small hooks, a lateral papilla at the base of the proboscis, a ringed pseudo-segmented body, large testes, cemented glands in pairs, and a non-segmented region in the posterior end of the body, which contributed to the diagnosis of the species. Molecular phylogenetic analyses recovered G. echinodiscus forming a well-supported monophyletic group with Mediorhynchus sp., which was congruent with morphological studies that allocate both genera within the family Gigantorhynchidae. In conclusion, the present work adds new morphological and molecular information, emphasizing the importance of adopting integrative taxonomic approaches in studies of Acanthocephala.

Molecular phylogeny of the parasitic dinoflagellate Syltodinium listii (Gymnodiniales, Dinophyceae) and generic transfer of Syltodinium undulans comb. nov. (= Gyrodinium undulans)

The parasitic dinoflagellate Syltodinium listii was investigated from the open waters of the English Channel, southern North Sea and the NW Mediterranean Sea. Syltodinium listii has been unreported since its original description in the North Sea. Cells of S. listii were able to infect copepod eggs of different species, and even nauplii, and after each infection formed up to 32 cells embedded in a mucous envelope. Infection of the same host by more than one dinoflagellate was frequent; although overall, the progeny were reduced in number. Molecular phylogeny based on the small subunit ribosomal RNA (SSU rRNA) gene revealed that S. listii clusters with a group of environmental sequences from the cold North Atlantic region as a sister group of Gymnodinium aureolum. The large subunit ribosomal RNA (LSU rRNA) gene sequences of S. listii from the English Channel and cf. Gyrodinium undulans from the Mediterranean Sea were identical. Thus, we propose Syltodinium undulans comb. nov. for Gyrodinium undulans. The first internal transcribed spacer (ITS) and complete SSU rRNA gene sequences of Dissodinium pseudolunula are provided. The parasitic species of Chytriodinium, Dissodinium and Syltodinium cluster together within the family Chytriodiniaceae, including the free-living species Gymnodinium aureolum, G. corollarium and G. plasticum.

Spencerozyma acididurans sp. nov., an acid-tolerant basidiomycetous yeast species isolated from acid mine drainage

Strain SYSU-17, representing a novel acid-tolerant yeast species which can grow at pH 2.0 weakly, was isolated from acid mine drainage collected in a tailing impoundment of the Fankou Lead/Zinc Mine, Guangdong Province, PR China. Phylogenetic analysis of strain SYSU-17 based on the internal transcribed spacer (ITS) region and the D1/D2 domains of the large subunit ribosomal RNA (LSU rRNA) gene suggested that strain SYSU-17 was a novel species belonging to the genus Spencerozyma (class Microbotryomycetes, subphylum Pucciniomycotina). It differed from the type strain of the closest related species, Spencerozyma crocea CBS 2029T, by 0.7 % sequence divergence (three gaps and one nucleotide substitution out of 594 bp) in the D1/D2 domains of the LSU rRNA gene and 7.6 % sequence divergence (32 gaps and 22 nucleotide substitutions out of 714 bp) in the ITS region. In contrast to the physiological properties of S. crocea, the novel yeast species was unable to assimilate galactose, d-ribose, xylitol, succinate, d-xylose, ethanol, nitrate and nitrite. The name Spencerozyma acididurans sp. nov. is proposed and SYSU-17 is designated as the holotype.

Heterocephalacria mucosa sp. nov., a new basidiomycetous yeast species isolated from a mangrove forest in Thailand.

Strains of yeast were isolated under a nitrogen-depleted culture condition from decaying tree bark (strain N-12.1) and from mangrove forest water (strain 1-7W.1) sampled at different locations within a mangrove forest site in Ranong province, Thailand. They were found to be genetically and phenotypically different from any currently recognised yeast species. Phylogenetic analysis of nucleotide sequence of three genes, the internal transcribed spacer region 1 and 2 plus 5.8S ribosomal RNA gene (ITS), D1/D2 domain of the large subunit ribosomal RNA gene (LSU D1/D2) and the small subunit of the ribosomal RNA gene (SSU), revealed that these two strains were related to but distinguished from Heterocephalacriaarrabidensis. Several distinct physiological characteristics of these two strains were detected, namely inability to assimilate glycerol, dl-lactic acid, succinic acid, citric acid, d-gluconic acid, and ability to grow well at 25 °C, which were different from those of H. arrabidensis. Accordingly, the name Heterocephalacria mucosa sp. nov., is proposed to accommodate this novel species. The MycoBank number is MB 828624.

Use of Markers to Determine Cryptosporidium Genotypes for Epidemiology Tracking and Detection.

Polymerase chain reaction (PCR) enables amplification of specific DNA fragments for the detection and tracking of Cryptosporidium spp. Newly obtained DNA are compared to an ever-growing database of Cryptosporidium sequences with uncertain or outdated metadata in primary public repositories (i.e., EMBL/DDBJ/GenBank). Here, we describe standard operating procedures to obtain DNA sequences from Cryptosporidium spp. marker genes. Small-subunit ribosomal RNA gene, large-subunit ribosomal RNA gene, and glycoprotein 60 are amplified using conventional PCR. Amplified and sequenced genes are compared to a reference library of up-to-date curated gene sequences to identify Cryptosporidium species and variants.

Complete mitogenome for the ber fruit fly, Carpomya vesuviana (Diptera: Tephritidae) from Toksun, Northwest China and its phylogenetic relationship within family Tephritidae

Carpomya vesuviana (Diptera: Tephritidae), commonly known as the ber fruit fly, is the most destructive insect pest of 'ber' (jujube) throughout Asia and Western Europe. Complete sequence of the mitogenome of C. vesuviana has been determined in this study. The circular genome is 15,267 bp long and contains a standard gene complement, that is, the large and small ribosomal RNA subunits, 22 transfer RNA genes, 13 genes encoding mitochondrial proteins, and a non-coding A + T-rich control region. The phylogeny showed that C. vesuviana of subfamily Trypetinae was monophyletic and clearly separated from both Dacinae and Tephritinae with maximum support (p = 1).

Occurrence of leaf spot disease caused by Neopestalotiopsis clavispora on Taxus chinensis in China

AbstractTaxus chinensis is a valuable tree with uses for timber, medicinal, ornamental and landscape purposes. Most importantly, it is a source of the anti‐cancer drug paclitaxel. In 2016, needle spot disease was observed on T. chinensis in Fujian Province of China. Five isolates of similar morphology were isolated from diseased samples. The pathogen was identified as Neopestalotiopsis clavispora based on morphological and cultural characteristics and confirmed by sequence analysis of the internal transcribed spacer region (ITS) and the large subunit ribosomal RNA gene (LSU). The pathogenicity of the isolates was confirmed by fulfilling Koch's postulates. To the best of our knowledge, this is the first report to indicate that the causal agent of needle spot disease on Chinese yew in China is N. clavispora.

First Report of Laurel Wilt Disease Caused by Raffaelea lauricola on Swamp Bay in Louisiana

In the past two decades, laurel wilt disease has significantly affected members of the Lauraceae in the southeast United States, causing widespread mortality of native redbay (Persea borbonia (L.) Spreng), and incidence of infections in avocado (Persea americana Mill.), sassafras (Sassafras albidum L.) and swamp bay (Persea palustris [Raf.] Sarg.) (Fraedrich et al., 2008, 2015, Olatinwo, et al. 2019). Laurel wilt is a vascular disease caused by Raffaelea lauricola (T.C. Harr., Fraedrich & Aghayeva), a fungus vectored by a non-native ambrosia beetle Xyleborus glabratus Eichhoff (Fraedrich et al. 2008). In August 2020, we investigated the mortality of a spicebush shrub (Lindera benzoin L.) (3.8 cm diameter at root collar, two m height) located ca. 17 mi northeast of Colfax, Grant Parish, Louisiana (31.750263° N, -92.643694° W). Evaluation of the dead shrub revealed brown, persistent foliage, and black vascular discoloration of the sapwood, typical symptoms of laurel wilt (Fig. S1). Although, beetle holes were observed on the sapwood, no beetle was found in galleries at the time. In the laboratory, a fungus consistently isolated from surface-sterilized sapwood tissues plated on potato dextrose agar (PDA) was identified as R. lauricola based on the morphological characteristics of the isolate (i.e., mucoid growth, conidiophores, and oblong/ovoid shape conidia [Harrington et al. 2008]). The fungal isolate was denoted as SB1. The identity of the fungus was confirmed by positive PCR amplification of the large subunit ribosomal RNA gene region using species-specific primers; rab-lsu-rl_F: CCCTCGCGGCGTATTATAG and rab-lsu-rl_R: GCGGGGCTCCTACTCAAA (Olatinwo, unpublished). The sequence of the isolate SB1 (GenBank Accession no. MW207371) showed 100% homology to the R. lauricola strain CBS 127349 sequence (GenBank Accession no. MH877933). The pathogenicity of SB1 on spicebush was evaluated on four healthy shrubs (average: 1 m height and 40 mm in diameter) at the location from which the original detection was made. Stems of two spicebush shrubs were inoculated with SB1 agar plugs from a 14-day old culture on PDA, while plain PDA plugs were used on the remaining two shrubs as non-inoculated controls. Agar plugs were placed in 5 mm (0.2 in) diameter hole punched on the bark with cork-borer as described by Mayfield et al (2008). After six weeks, the R. lauricola inoculated shrubs were wilted with noticeable blackened tissue discoloration in the sapwood, while the control trees remained healthy (Fig. S2). Raffaelea lauricola was re-isolated from tissue of the two inoculated, symptomatic shrubs, but not from the control trees. The sequence of the re-isolated R. lauricola isolate, denoted as SB3 (GenBank Accession no. MW207372), showed 100% homology to the R. lauricola strain CBS 127349 and isolate SB1. This first documentation of laurel wilt on spicebush in Louisiana is significant because, spicebush berries, leaves, and twigs are food sources for forest animals, birds, and insects including whitetail deer and spicebush swallowtail (Papilio troilus L.). Since its first report on sassafras in 2014 (Fraedrich et al. 2015), laurel wilt has spread across Louisiana on sassafras and swamp bay (Olatinwo et al. 2019) and has been confirmed in14 parishes. This report shows the relentless nature of the disease, as the pathogen moves from one vulnerable host to the next, expanding into new locations and threatening forest ecosystems across the southern United States.

New evidence of nematode-endosymbiont bacteria coevolution based on one new and one known dagger nematode species of Xiphinema americanum-group (Nematoda, Longidoridae).

Three populations of Xiphinema primum n. sp. and two populations of X. pachtaicum were recovered from natural forests and cultural regions of northern Iran. Both species belong to the X. americanum-group and were characterized by their morphological, morphometric and molecular data. The new species, which was recovered in three locations, belongs to the X. brevicolle-complex and is characterized by 2124–2981 μm long females with a widely rounded lip region separated from the rest of the body by a depression, 103–125 μm long odontostyle, two equally developed genital branches with endosymbiont bacteria inside the ovary, which are visible under light microscope (LM), vulva located at 51.8–58.0%, the tail is 26–37 μm long with a bluntly rounded end and four juvenile developmental stages. It was morphologically compared with nine similar species viz. X. brevicolle, X. diffusum, X. incognitum, X. himalayense, X. luci, X. parabrevicolle, X. paramonovi, X. parataylori and X. taylori. The second species, X. pachtaicum, was recovered in two geographically distant points close to city of Amol. Molecular phylogenetic studies of the new species were performed using partial sequences of the D2-D3 expansion segments of the large subunit ribosomal RNA gene (LSU rDNA D2-D3), the internal-transcribed spacer rDNA (ITS = ITS1+5.8S+ITS2), and the mitochondrial cytochrome c oxidase I gene (COI mtDNA) regions. The Iranian population of X. pachtaicum was also phylogenetically studied based upon its LSU rDNA D2-D3 sequences. Both species were also inspected for their putative endosymbiont bacteria. Candidatus Xiphinematobacter sp. was detected from two examined populations of the new species, whereas the second endosymbiont bacterium, detected from three examined isolates of X. pachtaicum, was related to the plant and fungal endosymbionts of the family Burkholderiaceae. The phylogenetic analyses of the two endosymbiont bacteria were performed using partial sequences of 16S rDNA. In cophylogenetic analyses, significant levels of cophylogenetic signal were observed using both LSU rDNA D2-D3 and COI mtDNA markers of the host nematodes and 16S rDNA marker of the endosymbiont bacteria.

Basidiobolus haptosporus-like fungus as a causal agent of gastrointestinal basidiobolomycosis.

Basidiobolus species were isolated from colonic biopsy samples of patients with gastrointestinal basidiobolomycosis (GIB) in southern Saudi Arabia. Isolated fungi were initially identified using classical mycological tools and confirmed by sequence analysis of the large subunit ribosomal RNA gene. Phenotypic tests revealed zygomycete-like fungi which conform to those of Basidiobolus species. Five sequenced strains formed a monophyletic clade in the 28S ribosomal RNA gene phylogenetic tree. They shared 99.97% similarity with B. haptosporus and 99.97% with B. haptosporus var. minor, and relatively lower similarity with B. ranarum (99.925%). The study suggests a new and a serious causal agent of GIB related to Basidiobolus haptosporus. These isolates are not related to B. ranarum, which is commonly linked to this disease.

Genetic diversity and metapopulation structures of two intertidal species along the coast of Zhejiang, China: implications for conservation

To priority areas for conservation in the coast of Zhejiang, this study attempts to map the spatial patterns of the intra-specific genetic diversity of two intertidal species, Septifer virgatus and Tetraclita japonica, using the sequences of mitochondrial DNA cytochrome oxidase subunit I (COI) and large subunit ribosomal RNA (16S rRNA) genes. In total, 129 and 112 specimens were collected from 12 and 11 populations for S. virgatus and T. japonica, respectively. These two species revealed congruent population historical signals, including (1) high levels of haplotype diversity and low values of nucleotide diversity, (2) post-LGM expansion, (3) the lack of population genetic structure, sharing haplotypes over all sampling populations, (4) gene flow connections, and (5) migration directions. Largely, all results supported that all sampling populations could be divided into two main metapopulations, exhibiting a northern and southern distribution. The north metapopulation could be considered as a marine protected area. The genetic patterns of the two species should be considered in the management and conservation in the islands along the Zhejiang, China.

Mitochondrial Gene Heterogeneity and Population Genetics of Haemaphysalis longicornis (Acari: Ixodidae) in China.

Haemaphysalis longicornis is an important ectoparasite of domestic and wild animals that can transmit many pathogens including viruses, fungi, bacteria and protozoa. In this study, we examined genetic variation and population genetics in three mitochondrial (mt) genes [cox1 (cytochrome c subunit 1), rrnL (large subunit ribosomal RNA) and nad5 (NADH dehydrogenase 5)] among four H. longicornis populations from China. The sizes of the partial sequences of cox1, rrnL and nad5 were 776bp, 409bp, 510bp, respectively. Among the obtained sequences, we identified 22 haplotypes for cox1, 2 haplotypes for rrnL and 17 haplotypes for nad5. Low gene flow and significant genetic differentiation (66.2%) were detected among H. longicornis populations. There was no rapid expansion event in the demographic history of four H. longicornis populations in China. In addition, phylogenetic analyses confirmed that all the Haemaphysalis isolates were H. longicornis which were segregated into two major clades. The mt DNA genes provide a potential novel genetic marker for molecular epidemiology of H. longicornis and assist in the control of tick and tick-borne diseases in humans and animals.

Genetic Characterization of Fungi Isolated from Environmental Samples: A Molecular Surveillance Study of Public Health Importance.

Background: A multistate fungal meningitis outbreak began in September 2012 that affected 751 individuals who received contaminated spinal injections across 20 states in the United States, which led to 64 deaths. In our previous study, we examined 26 environmental swab samples collected from various locations of the manufacturing premises of the compounding company to determine the possible cause of this outbreak and identified 14 novel species of fungi. Objective: In this follow-up study, a total of 198 environmental surveillance samples were collected and analyzed to detect pathogenic fungal species from other compounding company premises located in three regions of the United States. Methods: DNA sequencing was performed at the large subunit ribosomal RNA (LSU rRNA) and internal transcribed spacer (ITS) regions on the 25 positive fungal isolates. Results: Sequence analysis of the ITS1, the ITS2, and the LSU rRNA regions confirmed the presence of the following fungal species in the samples analyzed: (1) Pestalotiopsis cocculi from the region I; (2) Epicoccum nigrum and Trichaptum biforme from the region I; (3) Nigrospora sphaerica and Fusarium sp. from the region II; and (4) Curvularia sp., Fusarium sp., Penicillium sp., and Preussia sp. from the region III. Conclusions: Our results suggest that the LSU and ITS regions are good genetic markers to perform fungal typing. Highlights: DNA sequencing technology can be used in the implementation of effective environmental monitoring programs of public health importance.

First Report of Alternaria alternata Causing Leaf Spot on Mungbean (Vigna radiata) in the Republic of Korea

Mungbean, Vigna radiata (L.) R. Wilczek, is an important legume crop in many countries, providing protein and other essential nutrients. In October 2017, at the Seoul National University Suwon Experimental Farm (Suwon, Republic of Korea), we observed several individual plants across various mungbean accessions with dark-brown leaf spots. Spots were most prominent on the underside of the leaf and were circular to irregular in shape. On average, the diameter of spots was approximately 2 mm. A standard tissue isolation method was used, and a total of seven fungal isolates were made. The produced conidia were beaked or beakless, had transverse and sometimes latitudinal septa, ellipsoid or obclavate, with an average width of 11 µm and an average length of 22 µm, similar to the description of Alternaria alternata by Woudenberg et al. (2015). Genomic DNA of the fungi from two out of seven isolates was extracted using GeneAll Exgene Plant SV mini (Seoul, Republic of Korea). Using each DNA as a template, we amplified the internal transcribed ribosomal spacer region (ITS) and the 28s large subunit ribosomal RNA D1/D2 region using ITS1/ITS4 primers (White et al. 1990) and NL1/NL4 primers (Reynolds and Taylor 1993), respectively. BLAST analysis of these amplicon sequences showed 100% identity match to those of A. alternata, and the sequence was deposited in GenBank (no. MH633697-700). Furthermore, we compared ITS and D1/D2 sequences to the A. alternata reference sequence from Woudenberg et al. (2015) by aligning the sequences using ClustalW (BioEdit, version 7.0.5.3). Both of the isolates had >99.99% sequence identity with the reference. Based on both sequencing and morphological information, we concluded that the isolates were A. alternata. To fulfill Koch’s postulates, we carried out both in vivo and in vitro inoculation tests. For in vitro test, we harvested the conidia by flooding the fungal plate with 0.1% Tween 20. The concentration of spores was adjusted to 1 × 10⁵ conidia/ml by further diluting with 0.1% Tween 20. Detached leaves from 3-week-old mungbean plants were sprayed with the conidia suspension until there was surface run-off, and the leaves were placed in a humidity chamber. The control leaves were sprayed with 0.1% Tween 20. After 5 days, we observed brown spots on the surface of the leaves, but no symptoms appeared on the control plants. For the in vivo test, 1-month-old potted mungbean plants were inoculated with conidia suspended in sterile distilled water adjusted to 1 × 10⁴ conidia/ml. Each plant was sprayed with 5 ml of solution. The in vivo test showed a similar symptom to the in vitro test, producing brown leaf spots. From the brown leaf spots, we again isolated the fungi and sequenced the 28s large subunit ribosomal RNA D1/D2 region. The sequencing result showed 100% similarity of the reisolate with the original inoculum, fulfilling Koch’s postulates. To our knowledge, this is the first report that A. alternata causes leaf spot disease in mungbean in Korea. This report helps to develop mungbean cultivars resistant to biotic stresses affecting mungbean yield.

Emergence of Leaf Spot Disease on Leafy Vegetable and Ornamental Crops Caused by Paramyrothecium and Albifimbria Species.

The genera Paramyrothecium and Albifimbria have been established from the former genus Myrothecium and they generally comprise common soil-inhabiting and saprophytic fungi. Within these genera, only two fungi have been recognized as phytopathogenic thus far: P. roridum and A. verrucaria, both of which cause necrotic leaf spots and plant collapse. Severe leaf necrosis and plant decay have been observed in Northern and Southern Italy on leafy vegetable crops. Thirty-six strains of Paramyrothecium- and Albifimbria-like fungi were isolated from affected plants belonging to eight different species. Based on morphological characteristics, 19 strains were assigned to A. verrucaria, whereas the remaining strains, which mostly resembled Paramyrothecium-like fungi, could not be identified precisely. Molecular characterization of six loci (internal transcribed spacer [ITS], β-tubulin [tub2], calmodulin [cmdA], translation elongation factor 1-alpha [tef1], large subunit ribosomal RNA [LSU], and mitochondrial ATP 6synthase 6 [ATP6]) of the 36 new isolates and three previously ITS-characterized isolates assigned all strains to four species: A. verrucaria, P. roridum, P. foliicola, and P. nigrum. Single and concatenated phylogenetic analyses were conducted, and they clearly distinguished the isolated fungi into four different groups. A. verrucaria, P. roridum, P. foliicola, and P. nigrum were able to induce leaf necrosis singly, and they were confirmed to be the causal agents of the leaf spot disease through pathogenicity assays. The involvement of fungi previously considered saprophytic (i.e., P. foliicola and P. nigrum) in the development of plant disease for the first time deserves particular attention because of the possibility of their transmission by seeds and the limited knowledge of their management with chemicals.

Two new taxa of the Auriscalpium vulgare species complex with substrate preferences

Basidiomes of the cone-inhabiting Auriscalpium species, usually regarded as A. vulgare Gray, are widely distributed across the Northern Hemisphere and easily recognized because of their unique macro-morphology. However, phylogenetic diversity patterns among different geographic populations and different substrates are completely unknown. In this study, samples of A. vulgare s.l. in different areas of China were studied phylogenetically, morphologically, and ecologically. For comparison, European collections of A. vulgare were included. Our phylogenetic analyses, inferred from partial nucleotide sequences including the internal transcribed spacers (ITS) 1 and 2 with the 5.8S nrDNA, the large subunit of nuclear ribosomal RNA (nrLSU), and the region between the conserved domain 6 and 7 of the gene for the second largest subunit of RNA polymerase II (RPB2), indicated that three phylogenetic species can be recognized, which correspond well with morphological and ecological evidence. Therefore, three species, having distinct substrate preferences, are documented here, namely the well-known A. vulgare, and the newly described species A. orientale and A. microsporum. Auriscalpium vulgare is widely distributed throughout the northern temperate Eurasia and North America, while the new species have been known mainly from subtropical East Asia and northeastern India to date.

A new species of Alpestrisphaeria (Dothideomycetes) with monodictys-like anamorph and revision of three Monodictys species

Endophytic fungal strains isolated from the coastal plant Vitex rotundifolia in Taiwan were accommodated in Alpestrisphaeria according to an analysis of internal transcribed spacer (ITS) and large subunit ribosomal RNA gene sequences. Based on this analysis and on a unique monodictys-like anamorph, the species, A. monodictyoides, is proposed as new. Type specimens of three species of Monodictys were investigated. A new name is proposed for the invalid M. indica S.M. Singh & Barde (non M. indica K. Chowdhery & Rehill); the invalid M. saudii is considered sclerotium-like structures of a sterile mycelium and excluded from any taxonomic naming; M. torulosa is synonymized with M. fluctuata. Early stages of conidium ontogeny are suggested as potential phylogenetically significant markers for a future revision of the polyphyletic genus Monodictys.

Taxonomy and toxicity of Prorocentrum from Perhentian Islands (Malaysia), with a description of a non-toxigenic species Prorocentrum malayense sp. nov. (Dinophyceae)

Thirteen isolates of Prorocentrum species were established from the coral reefs of Perhentian Islands Marine Park, Malaysia and underwent morphological observations and molecular characterization. Six species were found: P. caipirignum, P. concavum, P. cf. emarginatum, P. lima, P. mexicanum and a new morphotype, herein designated as P. malayense sp. nov. Prorocentrum malayense, a species closely related to P. leve, P. cf. foraminosum, P. sp. aff. foraminossum, and P. concavum (Clade A sensu Chomérat et al. 2018), is distinguished from its congeners as having larger thecal pore size and a more deeply excavated V-shaped periflagellar area. Platelet arrangement in the periflagellar area of P. malayense is unique, with the presence of platelet 1a and 1b, platelet 2 being the most anterior platelet, and a broad calabash-shaped platelet 3. The species exhibits consistent genetic sequence divergences for the nuclear-encoded large subunit ribosomal RNA gene (LSU rDNA) and the second internal transcribed spacer (ITS2). The phylogenetic inferences further confirmed that it represents an independent lineage, closely related to species in Clade A sensu Chomérat et al. Pairwise comparison of ITS2 transcripts with its closest relatives revealed the presence of compensatory base changes (CBCs). Toxicity analysis showed detectable levels of okadaic acid in P. lima (1.0–1.6 pg cell˗1) and P. caipirignum (3.1 pg cell˗1); this is the first report of toxigenic P. caipirignum in the Southeast Asian region. Other Prorocentrum species tested, including the new species, however, were below the detection limit.

Next generation sequencing from Hepatozoon canis (Apicomplexa: Coccidia: Adeleorina): Complete apicoplast genome and multiple mitochondrion-associated sequences

Extrachromosomal genomes of the adeleorinid parasite Hepatozoon canis infecting an Israeli dog were investigated using next-generation and standard sequencing technologies. A complete apicoplast genome and several mitochondrion-associated sequences were generated. The apicoplast genome (31,869 bp) possessed two copies of both large subunit (23S) and small subunit (16S) ribosomal RNA genes (rDNA) within an inverted repeat region, as well as 22 protein-coding sequences, 25 transfer RNA genes (tDNA) and seven open reading frames of unknown function. Although circular-mapping, the apicoplast genome was physically linear according to next-generation data. Unlike other apicoplast genomes, genes encoding ribosomal protein S19 and tDNAs for alanine, aspartic acid, histidine, threonine and valine were not identified. No complete mitochondrial genome was recovered using next-generation data or directed PCR amplifications. Eight mitochondrion-associated (215–3523 bp) contigs assembled from next-generation data encoded a complete cytochrome c oxidase subunit I coding sequence, a complete cytochrome c oxidase subunit III coding sequence, two complete cytochrome B coding sequences, a non-coding, pseudogene for cytochrome B and multiple fragmented mitochondrial rDNA genes (SSUA, SSUB, SSUD, LSUC, LSUG, RNA6, RNA10, RNA14, RNA18). The paucity of NGS reads generating each of the mitochondrion-like sequences suggested that a complete mitochondrial genome at typically high copy number was absent in H. canis. In contrast, the complete nuclear rDNA unit sequence of H. canis (18S rDNA to 28S rDNA, 6977 bp) had >1000-fold next-generation coverage. Multiple divergent (from 93.6% to 99.9% pairwise identities) nuclear 18S rDNA contigs were generated (three types with 10 subtypes total). To our knowledge this is the first apicoplast genome sequenced from any adeleorinid coccidium and the first mitochondrion-associated sequences from this serious pathogen of wild and domestic canids. These newly generated sequences may provide useful genetic loci for high-resolution species-level genotyping that is currently impossible using existing nuclear rDNA targets.

More pieces to a huge puzzle: Two new Escovopsis species from fungus gardens of attine ants.

Escovopsis (Ascomycota: Hypocreales, Hypocreaceae) is the only known parasite of the mutualistic fungi cultivated by fungus-growing ants (Formicidae: Myrmicinae: Attini: Attina, the “attines”). Despite its ecological role, the taxonomy and systematics of Escovopsis have been poorly addressed. Here, based on morphological and phylogenetic analyses with three molecular markers (internal transcribed spacer, large subunit ribosomal RNA and the translation elongation factor 1-alpha), we describe Escovopsisclavatus and E.multiformis as new species isolated from fungus gardens of Apterostigma ant species. Our analysis shows that E.clavatus and E.multiformis belong to the most derived Escovopsis clade, whose main character is the presence of conidiophores with vesicles. Nevertheless, the most outstanding feature of both new species is the presence of a swollen region in the central hypha of the conidiophore named swollen cell, which is absent in all previously described Escovopsis species. The less derived Escovopsis clades lack vesicles and their phylogenetic position within the Hypocreaceae still remains unclear. Considering the high genetic diversity in Escovopsis, the description of these new species adds barely two pieces to a huge taxonomic puzzle; however, this discovery is an important piece for building the systematics of this group of fungi.