Nuclear rDNA Sequences Research Articles

A new species of the Trichia botrytis complex from the cloud forest in Ecuador

The paper describes a new myxomycete species, Trichia tuberculata, from the Ecuadorian cloud forest. The phylogeny constructed with nuclear 18S rDNA and mitochondrial 17S rDNA sequences indicates that the taxon is closely related to the recently described species T. acetocorticola, T. nubila, and T. pinicola, all within the T. botrytis complex. The morphology of the inner peridium surface and capillitium also aligns T. tuberculata with these species. However, the distinctive tuberculate peridium, short elater tips, and perhaps the distribution range (so far found in the equatorial zone only) distinguish T. tuberculata from related taxa.

Freshwater ‘microcroissants’ shed light on a novel higher-level clade within the Trebouxiophyceae and reveal the genus Chlorolobion to be a trebouxiophyte

ABSTRACT The Trebouxiophyceae is a widespread and species-rich green algal class encompassing mostly coccoid algae with a simple spherical, ovoid or ellipsoidal outline. However, some poorly sampled lineages have evolved more elaborate shapes or even complex thalli, adding to the morphological diversity of the class. By investigating new and previously established strains, this study expands the range of morphologies exhibited by the class members by uncovering a clade of croissant-like trebouxiophytes. Phylogenetic analyses inferred from nuclear 18S rDNA and chloroplast rbcL sequences confirmed the monophyly of the ‘microcroissant’ clade, which we propose to be classified as a new family, Ragelichloridaceae. This family includes two novel genera, Ragelichloris and Navichloris, and the previously described Thorsmoerkia. The position of Ragelichloridaceae within Trebouxiophyceae stayed unresolved but chloroplast phylogenomics showed that the family belongs to the broader incertae sedis group that also includes Xylochloris and Leptosira. In addition, our study showed that the similar morphotype-bearing genus Chlorolobion, previously classified within Chlorophyceae, is a genuine trebouxiophyte, potentially related to Ragelichloridaceae.

Morphological and phylogenetic analyses reveal four species of myxomycetes new to China.

Four species of myxomycetes (Arcyria pseudodenudata, Diderma europaeum, Lycogala irregulare, and Trichia armillata) new to China were observed via light microscope and scanning electron microscope, and detailed descriptions and illustrations are provided, along with comparisons with related species. Among them, A. pseudodenudata was discovered for the first time outside of the type locality, D. europaeum was discovered for the first time outside of Europe, and L. irregulare and T. armillata were reported again after being named. Phylogenetic analyses based on nuclear 18S rDNA and elongation factor-1 alpha sequences or nuclear 18S rDNA and cytochrome oxidase subunit I sequences was performed to provide a molecular basis for morphological identification. These specimens were deposited in the Herbarium of Fungi of Nanjing Normal University.

Taxonomy and fatty acid profile of Picochlorum geojense sp. nov. (Chlorophyta, Trebouxiophyceae) from Korean coastal waters

ABSTRACT The microalgal genus known as Picochlorum, belonging to the green algae family Trebouxiophyceae, is recognized for its rapid growth and high lipid productivity. A Picochlorum strain (GJ210610) was recently established from the waters off Geoje, South Sea of Korea. Picochlorum sp. GJ210610 was a single, green ellipsoidal cell with an average size ranging from 1 to 2 µm. Its reproduction occurred through autosporulation. A phylogenetic tree based on nuclear small-subunit rDNA sequences revealed that this strain formed a unique branch, distinct from other Picochlorum species. Furthermore, the secondary structure of the ITS2 sequences was revealed for the first time within Picochlorum. The newly discovered Picochlorum sp. grew between 15°C and 25°C, with survival ceasing at 30°C. The maximum specific growth rate was measured at 0.91 d−1 at 25°C. The fatty acids of Picochlorum sp. GJ210610 were palmitic acid, oleic acid, linoleic acid, and alpha-linolenic acid. Based on morphological and phylogenetic analyses, we propose Picochlorum geojense sp. nov. (GJ210610) as a new species. The results of the present study may enhance our knowledge of the taxonomy and ecological characteristics of Picochlorum.

Uncovering further diversity of Ochoterenatrema Caballero, 1943 (Digenea: Lecithodendriidae) in South American bats

Ochoterenatrema Caballero, 1943 is a genus of lecithodendriid digeneans that prior to this study included 8 species parasitic in bats in the Western Hemisphere. Species of Ochoterenatrema possess a unique morphological feature in form of the pseudogonotyl on the sinistral side of the ventral sucker. In this study, we describe 2 new species of Ochoterenatrema from bats in Ecuador. The new species are readily differentiated from their congeners by a combination of morphological characters, including the distribution of vitelline follicles, length of oesophagus, sucker ratio and the body shape, among other features. We have generated partial nuclear 28S rDNA and mitochondrial cox1 gene DNA sequences from both new species. The newly obtained sequences were used to differentiate among species and study the phylogenetic interrelationships among Ochoterenatrema spp. The internal topology of the clade was weakly supported, although the cox1 tree was much better resolved than the 28S tree. Comparison of sequences revealed 0–1.2% interspecific divergence in 28S and 3.3–20.5% interspecific divergence in cox1 among Ochoterenatrema spp. The new findings demonstrate that bats in South America likely harbor multiple additional undescribed species of Ochoterenatrema. More extensive sampling from broader geographic and host ranges, especially in North America, should allow for a better understanding of the evolution of host associations and morphological traits of this lineage of lecithodendriid digeneans.

Five new species of Pseudosperma (Inocybaceae, Agaricales) from Benin and Turkey based on morphological characteristics and phylogenetic evidence

Species of Pseudosperma (Inocybaceae) are widely distributed from temperate to tropical regions. In this study, we describe and illustrate five new species of Pseudosperma: P. beninense, P. cremeo-ochraceum, P. squarrosofulvum, P. stramineum, and P. tiliae, based on comprehensive analyses of morphological and molecular data derived from specimens collected in Benin (West Africa) and Turkey (Western Eurasia). These new species have been found in forests with Isoberlinia spp. and other ectomycorrhizal tree species in Benin and in association with Tilia platyphyllos in Turkey. The phylogenetic relationships of the new species were inferred through analyses of nuclear rDNA sequences, encompassing the internal transcribed spacer (ITS), 28S rDNA, and RNA polymerase II second largest subunit (RPB2) region. Phylogenetic analyses revealed that P. beninense, P. cremeo-ochraceum, P. squarrosofulvum, and P. stramineum from Benin cluster with species from Australia, China, and India within a clade formed exclusively by species known from the palaeotropics and Australia, whereas P. tiliae from Turkey clustered with P. mediterraneum from Italy. Detailed descriptions are provided, supplemented by illustrations and line drawings of key micromorphological features. In addition, a comparative analysis with morphologically similar and phylogenetically closely related species is presented and discussed in detail.

DNA Barcoding of Nematode Parasites Infecting Ompok bimaculatus and Nemacheilus anguilla From Barvi Reservoir, Maharashtra

In the present study, the DNA barcoding of the nematode parasite infecting Ompok bimaculatus and Nemacheilus anguilla fish species was carried out in Barvi Reservoir, Maharashtra. To ascertain the taxonomic status of these nematode parasites, an 18S gene marker was used. Accurate identification of fish parasites is essential to formulate preventive strategies and to study host–environment relations. The present study did barcoding of the nematode parasites of the fishes caught from the Barvi Reservoir using the nuclear 18S rDNA (SSU) sequence. The nuclear 18S rDNA (SSU) was amplified into two overlapping amplicons and sequenced to identify the species based on the sequence similarity with the NCBI GenBank database. The present study sequences (both fragments) showed 98% similarity with the species of Eustrongylides. The average genetic distance value between the present study sample and species of Eustrongylides was 0.003. In the phylogenetic tree also, the sequence was clustered with the species of Eustrongylides with significant bootstrap values. The present study identified the nematode parasite of the fish caught from the Barvi Reservoir, as species of Eustrongylides. The species‐level identification could not be possible due to the insufficient/lack of reference sequences in the database. It indicates the knowledge gap concerning the species‐specific molecular markers for nematode parasites of the fish.

Morphology and phylogenetic relationships of two Antarctic strains within the genera Carolibrandtia and Chlorella (Chlorellaceae, Trebouxiophyceae)

The genera Carolibrandtia and Chlorella have been described as small green algae with spherical cell shapes that inhabit various environments. Species of these genera are often difficult to identify because of their simple morphology and high phenotypic plasticity. We investigated two small coccoid strains from Antarctica based on morphology, molecular phylogeny by two alignment methods which have been applied to previous phylogenetic studies of the genus Chlorella, and comparison of the secondary structures of nuclear small subunit (SSU) and internal transcribed spacer (ITS) rDNA sequences. Light microscopy of two strains revealed spherical cells containing chloroplasts with pyrenoids, and the morphological characteristics of the strains were nearly identical to those of other Chlorella species. However, based on the phylogenetic analyses of nuclear SSU and ITS rDNA sequences, it was determined that the Antarctic microalgal strains belonged to two genera, as the Chlorella and Carolibrandtia. In addition, the secondary structures of the SSU and ITS2 sequences were analyzed to detect compensatory base changes (CBCs) that were used to identify and describe the two strains. A unique CBC in the SSU rDNA gene was decisive for distinguishing strain CCAP 211/45. The ITS2 rDNA sequences for each strain were compared to those obtained previously from other closely related species. Following the comparison of morphological and molecular characteristics, we propose KSF0092 as a new species, Chlorella terrestris sp. nov., and the reassignment of the strain Chlorella antarctica CCAP 211/45 into Carolibrandtia antarctica comb. nov.

Resurrection of genus Phocanema Myers, 1959, as a genus independent from Pseudoterranova Mozgovoĭ, 1953, for nematode species (Anisakidae) parasitic in pinnipeds and cetaceans, respectively.

Species of the genus Pseudoterranova, infect kogiid cetaceans and pinnipeds. However, there is mounting molecular evidence that those from cetaceans and pinnipeds are not congeneric. Here, we provide further evidence of the non-monophyly of members of Pseudoterranova from phylogenetic analyses of the conserved nuclear LSU rDNA gene, entire ITS rDNA region and mtDNA cox2 gene, and identify morphological characters that may be used to distinguish the members of the two clades. We propose the resurrection of the genus Phocanema, with Ph. decipiens (sensu stricto) as the type species, to encompass Ph. decipiens, Ph. azarasi, Ph. bulbosa, Ph. cattani and Ph. krabbei, all parasites of pinnipeds. We propose to restrict the conception of genus Pseudoterranova, which now harbours two species infecting kogiid whales; Ps. kogiae (type species) and Ps. ceticola. Members of the genera Phocanema and Pseudoterranova differ by the shape and orientation of the lips, relative tail lengths, adult size, type of final host (pinniped vs. cetacean) and phylogenetic placement based on nuclear rDNA and mtDNA cox2 sequences.

Biecheleria donggangensis sp. nov. (Suessiaceae, Dinophyceae), a new marine woloszynskioid species germinated from coastal sediment of the Yellow Sea, China

ABSTRACT A new marine woloszynskioid dinoflagellate, Biecheleria donggangensis sp. nov., germinated from the sediments of Rizhao coastal area, China, was examined by light and scanning electron microscopy. Its phylogenetic position was inferred from nuclear rDNA (partial LSU, SSU and ITS1–5.8S–ITS2) sequences. Cells were spherical to ellipsoidal (8.5‒19.2 μm long and 5.9‒11.5 μm wide) with numerous small and discoid chloroplasts, and a centrally located nucleus. Scanning electron microscopy showed an apical furrow area consisting of a single elongated apical vesicle (EAV) with 20 to 34 small globular knobs at the top of the epicone, and 13–15 latitudinal series of amphiesmal vesicles (AVs). Biecheleria donggangensis can be distinguished from other congeneric species by the configuration of the AVs bordering the EAV, the number of latitudinal AVs, and the trichocyst pore (TP) structures around the EAV. Thick-walled and spherical (or oval) cysts were 16.1‒23.5 μm in diameter and decorated with needle-like processes, each with a distal swelling. Molecular phylogenies using maximum likelihood (ML) were examined by three statistical methods (bootstrap, approximate Bayes and the Shimodaira-Hasegawa-like approximate likelihood-ratio test), which clearly demonstrated that the isolate belonged to the genus Biecheleria but formed a clade distinct from all previously described species. Genetic distance analyses justified describing the isolate as a new species rather than a new ribotype of B. cincta.

Hydropodia silvae-nipponicae (Porotheleaceae), a new species from evergreen broad-leaved forests in Japan.

Hydropodia silvae-nipponicae, a new agaric species was described based on the specimens collected from evergreen broad-leaved forests in Japan. The present new species is distinct from H. subalpina, the type species of the genus by morphological characteristics and nuclear rDNA sequences. Hydropodia silvae-nipponicae is characterized by its nodulose cystidium-like terminal elements of pileipellis, cylindrical to clavate pileocystidia with irregular excrescences, and dimorphic pleurocystidia. It is the second species belonging to the genus Hydropodia.

Colletotrichum galinsogae sp. nov. Anthracnose Pathogen of Galinsoga parviflora

Galinsoga parviflora is an herbaceous dicotyledonous plant in the Asteraceae that is common in disturbed habitats and agricultural areas across various temperate and subtropical regions of the world. In this study, several pathogenic strains were isolated from this host, and further morphological and phylogenetic analysis of DNA sequences of nuclear rDNA ITS1-5.8S-ITS2 (ITS barcode) and five other gene regions (act, chs-1, gapdh, his3, and tub2) revealed a new species, described here as Colletotrichum galinsogae sp. nov. The pathogenicity of C. galinsogae sp. nov. was also tested and confirmed on leaf segments and seedlings of G. parviflora.

Intraspecific heterogeneity, multiple allelism and morphological divergence between morphotypes of Thalassiosira allenii (Bacillariophyta) from the Sea of Japan

ABSTRACT Thalassiosira allenii is reported for the first time from marine phytoplankton of the Russian far East. Five strains representing three morphological variants of T. allenii, from three locations in the Sea of Japan, were investigated. The D8–D10 domain of LSU rDNA and rbcL of T. allenii were sequenced for the first time. Three morphological forms of T. allenii showed completely identical rbcL sequences and distinctly different nuclear rDNA sequences. The genetic differences between morphotypes were determined by the number of allelic variants of LSU rDNA, one of which was common to all studied strains. Two different types of the D1–D3 domain and three different types of the D8–D10 divergent domain were detected in the cultured strains of T. allenii. The three morphotypes are described in detail and compared. One allele of D1–D3 and one allele of D8–D10 were identified in the strains of T. allenii from two stations in Ussuri Bay. Two and three alleles of the D8–D10 domain were identified in the strains with an ambiguous morphotype and in T. allenii var. striata, respectively, from one station in Amur Bay. Subdivision of T. allenii into morphotypes based on morphology matched that based on molecular-genetic analyses using D8–D10 LSU rDNA. Caution is recommended in using LSU rDNA sequences and rbcL divergence as the principal criterion for delimiting and metabarcoding T. allenii varieties/morphotypes. The lack of genetic differentiation in rbcL, and the presence of clear intra-specific genetic differentiation among morphotypes in two regions of LSU rDNA suggest a link between morphological differences and the nuclear genome. The D8–D10 domain of LSU rDNA appears more suitable for discriminating morphotypes within T. allenii. The rbcL is an effective marker for recognizing T. allenii at species level, but not at infraspecific level.

PHYLOGENETIC ANALYSIS OF THE LUNGWORMS (NEMATODA: METASTRONGYLOIDEA) INFERRED USING NUCLEAR RIBOSOMAL AND MITOCHONDRIAL DNA SEQUENCES.

Phylogenetic relationships among the mammal-parasitic lungworms (Metastrongyloidea) were inferred using small- and large-subunit ribosomal DNA sequences together with 12S ribosomal mtDNA sequences. Maximum parsimony and Bayesian inference methods were used from optimal alignments and those filtered for alignment ambiguity. Analysis of 30 ingroup sequences using ribosomal DNA sequences yielded a single most parsimonious tree. Monophyly of the Metastrongyloidea was supported, but there was no support for monophyly of any of the 7 families as they have been traditionally defined. Parafilaroides decorus, an abursate lungworm of pinnipeds currently classified in the Filaroididae, was nested within a clade containing members of the Pseudaliidae, parasites of cetaceans. The tree also shows clades somewhat resembling the traditional familial divisions of the Metastrongyloidea, but in all groups, paraphyletic relationships were recovered. In a combined analysis of nuclear rDNA and 12S mtDNA, maximum parsimony and Bayesian analyses showed similar patterns to those observed with only nuclear rDNA sequences. Based on the phylogeny, the respiratory tract was inferred to be the ancestral predilection site for Metastrongyloidea, with multiple evolutionary invasions of extrapulmonary sites such as sinuses, circulatory system, and meninges. Similarly, the ancestral host was inferred to be a carnivore with subsequent colonization events into marsupial, rodent, artiodactyl, pinniped, and cetacean hosts.

Puccinia caricis-smilacis, a new caricicolous rust fungus producing spermogonial and aecial stages on Smilax china in Japan.

Puccinia caricis-smilacis, a new species of caricicolous rust fungus was described based on morphological characteristics and nuclear rDNA sequences from Japan. The heteroecious life cycle of P. caricis-smilacis was elucidated via field observations, inoculation experiments and molecular analyses. This rust fungus produces uredinial and telial stages on Carex fibrillosa, and spermogonial and aecial stages on Smilax china.

Phylogeny of Trachelomonas and Strombomonas (Euglenaceae) Based on Morphological and Molecular Data

The classification of Trachelomonas and Strombomonas is based on the morphology of loricae, which may not reflect phylogenetic relationships. There are different views on the relationship between the two genera. It is thus important for researchers to classify the two genera based on more characteristics besides loricae, such as protoplast and molecular data, and to establish a natural classification system. In this study, 29 strains (13 species) of Trachelomonas and 12 strains (eight species) of Strombomonas were collected from various locations in China. The morphological characteristics of Trachelomonas and Strombomonas were observed in the field and culture conditions, and the sequences of nuclear SSU rDNA, nuclear LSU rDNA, plastid-encoded LSU rDNA and plastid-encoded LSU rDNA of strains were obtained. We constructed a phylogenetic tree by combining four gene sequences with maximum likelihood and Bayesian methods. Based on the development of the loricae, it was found that the shape of the loricae and the presence or absence of the collar were relatively stable in culture, while the length of the collar presence or absence of spines and the color of the loricae changed. The phylogenetic tree showed that Trachelomonas and Strombomonas were sister branches, which supported Trachelomonas and Strombomonas as independent genera. Taxa in the genus Strombomonas sorted into two clades and Trachelomonas sorted into five strongly supported clades. Key morphological features could be attributed to each of the clades.

Phylogenetic relationships of Ochoterenatrema Caballero, 1943 (Digenea: Lecithodendriidae) with descriptions of two new species.

Ochoterenatrema is a small genus of lecithodendriid digeneans that includes six species parasitic in Neotropical bats in North and South America. Members of this genus are characterized by the presence of a pseudogonotyl formed by thickened tegument on the left side of the ventral sucker. We examined morphology of specimens belonging to five species of Ochoterenatrema from Brazil, Ecuador and the USA and describe two new Ochoterenatrema spp. (Ochoterenatrema sphaerula n. sp. and Ochoterenatrema gracilis n. sp.). These new species are readily differentiated from their congeners by a combination of morphological characters, including the distribution of vitelline follicles and the body shape. We have provided the first DNA sequences for Ochoterenatrema. Newly generated partial nuclear 28S rDNA and mitochondrial cox1 gene sequences were used to differentiate among species and study the phylogenetic affinities of Ochoterenatrema spp., including one of the new species, O. gracilis n. sp., as well as Ochoterenatrema diminutum, Ochoterenatrema fraternum and Ochoterenatrema cf. labda. The phylogeny of the Microphalloidea based on 28S had well-supported topology, particularly at the family level. The Ochoterenatrema clade was strongly supported; however, the internal topology of the clade was weakly supported. Comparison of sequences revealed 0.4-1.3% interspecific divergence in 28S and 9.1-19.7% interspecific divergence in cox1 among Ochoterenatrema spp. We hypothesize that extremely diverse fauna of bats in South and Central America harbors multiple undescribed species of Ochoterenatrema. Several lecithodendriid genera from bats and other hosts are yet to be included in future molecular phylogenetic analyses to test the monophyly of this extremely diverse digenean family and analyze evolutionary affinities of its constituent taxa.

Identification of fungi found on desiccated human remains in an arid outdoor environment.

Three fungi not previously reported on desiccated human remains were identified on cadavers at the Forensic Investigation Research Station (FIRS) in Whitewater, Colorado. The location of the FIRS provides the unique opportunity to observe the stages of decomposition in a high desert environment. The two cadavers used in the study were in the late stages of decomposition (PMI of approximately 1520 and 1820 days) to the point of desiccation and had developed an extensive black crust on the skin that remained. Skin samples of the two cadavers were taken and plated onto potato dextrose agar to determine whether fungi were present on the desiccated tissues. Three different fungi consistently dominated cultures grown from numerous samples taken from each cadaver. Based on morphological observations, nuclear rDNA sequence data, and phylogenetic analyses, two fungi were identified to species (Aureobasidium melanogenum and Didymella glomerata) and one fungus was identified to the genus level (Alternaria). These results will contribute to the understanding of the role that fungi might play in late-stage decomposition and the extended postmortem period.

The First Isospora Species (Apicomplexa: Eimeriidae) Described from the Northern Yellow-Shafted Flicker (Colaptes auratus luteus) in Ontario, Canada.

Members of the genus Isospora commonly infect a wide variety of wild birds. By combining morphometric and genetic data, we are able to differentiate and describe a new apicomplexan parasite, Isospora picoflavae n. sp., from the Northern Yellow-shafted Flicker (Colaptes auratus luteus) in Ontario, Canada. Unsporulated oocysts were observed in intestinal contents obtained during necropsy; these oocysts completed exogenous sporulation within 7days at room temperature. Complete nuclear 18S rDNA and complete mitochondrial genome sequences were obtained from this previously undescribed Isospora sp. to compare with related coccidia. Sporulated oocysts were subspherical in shape measuring an average of 22.7 × 21.7μm (n = 53; mean shape index 1.05) containing a near-spherical polar granule. Sporocysts were ovoidal to ellipsoidal in shape, measuring an average size of 17.0 × 10.5μm (n = 9; mean shape index 1.62). Sporocyst residuum was concentrated in an irregular, spherical mass in the middle of each sporocyst. The isolated oocysts differed morphologically from other Isospora species reported to infect members of the family Picidae. Based on phylogenetic analyses using either combined nuclear 18S rDNA and mitochondrial cytochrome c oxidase I (COI) sequences or complete mitochondrial genome sequences, this Isospora sp. n. isolated from the Northern Flicker grouped within a clade containing Isospora species described from various passeriform hosts. Based on combined morphological and molecular data, the oocysts found in the gastrointestinal contents of Colaptes auratus luteus represent a new species of Isospora named herein as Isospora picoflavae n. sp.

First Record of Bolbitius reticulatus (Agaricales: Bolbitiaceae) in Turkey, with Nuclear ITS and LSU rDNA Sequences Data

First Record of Bolbitius reticulatus (Agaricales: Bolbitiaceae) in Turkey, with Nuclear ITS and LSU rDNA Sequences Data