Large Subunit Ribosomal DNA Sequences Research Articles

Establishment of a multiplex polymerase chain reaction detection assay for three common harmful microalgae in the East China Sea.

It is urgent to develop techniques that can simultaneously detect multiple microalgae, due to the diversity of harmful algal blooms (HABs)-forming algal species. The target algae species in this study are Heterosigma akashiwo, Prorocentrum donghaiense and Karenia mikimotoi. These algae are the dominant species that cause HABs in the East China Sea, and the multiple detection technique focusing on these three algae is not common. Therefore, this study established a multiplex polymerase chain reaction(mPCR) to diagnose the three algae, which is simple and low cost. First, the corresponding specific primers were designed based on the D1-D2 region of the large subunit (LSU) ribosomal DNA sequence. Then, mPCR was established and the reaction conditions were optimized. And the specificity, sensitivity, and stability of mPCR were evaluated. The result of specificity test showed that the established mPCR had good specificity for the target microalgae and did not cross-react with eighteen non-target microalgae. The sensitivity of experiment was 3.3 × 10-1ng μL-1, and the established mPCR was not affected by the interfering microalgae. Moreover, the practicability evaluation of mPCR by using the simulated natural water samples showed that the detection limit of target microalgae was 100 cells mL-1, which could meet the demand for early warning of HABs. In summary, the established mPCR is characterized by strong specificity, good stability, and multiple analysis to detect H. akashiwo, P. donghaiense, and K. mikimotoi.

Morphological and Molecular Characterization of Filenchus multistriatus n. sp. (Tylenchomorpha: Tylenchidae) and Data on a Known Species of the Genus from Bushehr Province, Southern Iran.

During a nematological survey in southern Iran, a population belonging to the family Tylenchidae was recovered from a tomato field in Bushehr province. The recovered population belongs to the genus Filenchus, was described and illustrated herein as F. multistriatus n. sp. It is mainly characterized by having a wide and low annulated lip region continuous with adjacent body; amphidial openings confined to the labial plate; four lines in lateral fields forming three bands, with the two outer bands broken by transverse, and the inner one broken by both transverse and longitudinal lines; and median bulb oval with visible valve and elongate-conoid tail uniformly and gradually narrowing toward the distal region, ending in a widely rounded tip. Its morphological and morphometric differences with three closely similar species were discussed. The phylogenetic relationships of the new species with other relevant genera and species were reconstructed using partial sequences of small, and large subunit ribosomal DNA (SSU and LSU rDNA) sequences. Morphometric and morphological data were also provided for an Iranian population of F. sandneri recovered from Bushehr province. Both populations were characterised using SEM data.

Molecular phylogeny and morphological characterization of Paramicrothyrium bambusae sp. nov. and Tumidispora thailandica sp. nov. from leaf litter

During our ongoing studies of fungal diversity on leaf litter, two exciting species were collected from Thailand. Morphological examination coupled with phylogenetic analyses revealed that our species belongs to Paramicrothyrium and Tumidispora in Microthyriaceae. Maximum likelihood, maximum parsimony, and Bayesian analyses of combined internal transcribed spacer (ITS) and large nuclear subunit ribosomal DNA (LSU) sequence data were performed to clarify the phylogenetic affinities of the species. Based on their distinctive morphological traits and phylogenetic support, the new species Paramicrothyrium bambusae and Tumidispora thailandica were introduced. In addition, the new species were compared with phylogenetically and morphologically closely related species, and detailed descriptions, micrographs, and phylogenetic trees of the new species are provided.

Unveiling mesophotic diversity in Hawai‘i: two new species in the genera <italic>Halopeltis</italic> and <italic>Leptofauchea</italic> (Rhodymeniales, Rhodophyta)

Two genera of the Rhodymeniales, Halopeltis and Leptofauchea, are here reported for the first time from the Hawaiian Islands and represent the deepest records for both genera. Molecular phylogenetic analyses of cytochrome oxidase subunit I (COI), rbcL, and large subunit ribosomal DNA (LSU) sequences for Hawaiian specimens of Leptofauchea revealed one well-supported clade of Hawaiian specimens and three additional lineages. One of these clades is described here as Leptofauchea huawelau sp. nov., and is thus far known only from mesophotic depths at Penguin Bank in the Main Hawaiian Islands. L. huawelau sp. nov. is up to 21 cm, and is the largest known species. An additional lineage identified in the LSU and rbcL analyses corresponds to the recently described L. lucida from Western Australia, and is a new record for Hawai‘i. Hawaiian Halopeltis formed a well-supported clade along with H. adnata from Korea, the recently described H. tanakae from mesophotic depths in Japan, and H. willisii from North Carolina, and is here described as Halopeltis nuahilihilia sp. nov. H. nuahilihilia sp. nov. has a distinctive morphology of narrow vegetative axes that harbor constrictions along their length. The current distribution of H. nuahilihilia includes mesophotic depths around W. Maui, W. Moloka‘i, and the island of Hawai‘i in the Main Hawaiian Islands. Few reproductive characters were observed because of the small number of specimens available; however, both species are distinct based on phylogeny and morphology. These descriptions further emphasize the Hawaiian mesophotic zone as a location harboring many undescribed species of marine macroalgae.

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.

Two New Species of Backusella (Mucorales, Mucoromycota) from Soil in an Upland Forest in Northeastern Brazil with an Identification Key of Backusella from the Americas.

During a survey of Mucorales from a forest located in Pernambuco state, Brazil, two new Backusella species were discovered and described based on morphological and molecular data (internal transcribed spacer and large subunit ribosomal DNA sequences). Both species were characterized as unbranched sporangiophores and sporangia with columellae of varied shapes forming. Multispored sporangiola were frequent, whereas unispored sporangiola were rare. URM 8395 forms sporangiophores that may support hyaline, slightly curved or circinate pedicels with multispored sporangiola at their apical portion, and abundant giant cells and chlamydospores. Columellae of sporangia are hyaline, conical (majority), or ellipsoidal with a truncate base, globose to subglobose or subglobose to conical, and, rarely, with slight medial constriction. URM 8427 does not form sporangiola from pedicels, giant cells are not observed, and columellae of sporangia are globose to subglobose, cylindrical with a truncate base, some with a slight constriction, applanate, obovoid, ellipsoidal, or, rarely, conical. Some columellae may have one side more swollen than the other and some are arranged obliquely on the sporangiophores. Sterile sporangia may or may not be formed on short sporophores. The detailed description and illustration of both novel species as well as an identification key for Backusella from the Americas are provided.

Threat of cercarial dermatitis in Hungary: A first report of Trichobilharzia franki from the mallard (Anas platyrhynchos) and European ear snail (Radix auricularia) using molecular methods.

Cercarial dermatitis in humans is a re-emerging zoonotic disease caused by infectious larvae of avian blood flukes within the Schistosomatidae family. Upon water contact, these avian schistosome larvae directly penetrate human skin and cause irritation. Between September 2018 and September 2020, carcasses of 94 mallards (Anas platyrhynchos), two green-winged teals (Anas crecca) along with one ferruginous duck (Aythya nyroca) were examined. Birds were collected within 12 regions of Hungary, representative of 9 different counties. Inspecting both morphological characters and molecular data, the cytochrome oxidase subunit I (COI) and large subunit ribosomal DNA (LSU rDNA) sequences, Bilharziella polonica and Dendritobilharzia pulverulenta were each identified. Most importantly, Trichobilharzia franki was found for the first time in Hungary and in 5 dispersed counties in conjunction with of 3 counties where other avian schistosomes were found. Of note, these avian blood flukes were predominately encountered in wild mallards (∼50%) and not in birds reared for hunting (∼1%). In total, 245 European ear snails (Radix auricularia), a known intermediate host of Trichobilharzia spp., were collected from an urban pond in Eger, Hungary. Five snails (∼2%) consistently shed numerous furcocercariae of T. franki that were confirmed by molecular methods. Our findings help to pinpoint a contemporary life cycle of this avian schistosome within an urban environment, a location regularly visited by various wild waterfowl and the possible species of avian schistosomes responsible for cercarial dermatitis in Hungary. Taken as a whole, we demonstrate the actual and potential risk zone for cercarial dermatitis, particularly with reference to R. auricularia distributions, within Hungary and across Central Europe.

Morphological and molecular studies of a rare mucoralean species causing flower rot in Hylocereus polyrhizus

AbstractThe order Mucorales typically includes fast‐growing saprotrophic and plant pathogenic fungi. Flower rot of red‐fleshed dragon fruit (Hylocereus polyrhizus) caused by Mucor‐like fungi was observed in a field in Phatthalung province, southern Thailand. Agglomeration of sporangiophores and sporangia on the wet rotten tissues of the red‐fleshed dragon fruit flower was detected. Fungal isolates obtained from symptomatic tissues were cultured on potato dextrose agar for macroscopic and microscopic observations. Based on the growth rate and morphology of sporangia and sporangiospores, the fungus was identified as Gilbertella persicaria, which was further confirmed using phylogenetic analyses of the internal transcribed spacer and nuclear large subunit ribosomal DNA sequences. Pathogenicity tests revealed that the fungal isolate G. persicaria caused flower rot, similar to that observed in the field. This study is the first report of flower rot disease in H. polyrhizus caused by G. persicaria in Thailand.

A Novel Parasitoid of Marine Dinoflagellates, Pararosarium dinoexitiosum gen. et sp. nov. (Perkinsozoa, Alveolata), Showing Characteristic Beaded Sporocytes

The phylum Perkinsozoa is known as an exclusively parasitic group within alveolates and is widely distributed in various aquatic environments from marine to freshwater environments. Nonetheless, their morphology, life cycle, the identity of the host, and physiological characteristics remain still poorly understood. During intensive sampling along the west coast of Korea in October and November 2017, a new parasitoid, which shares several characteristics with the extant families Perkinsidae and Parviluciferaceae, was discovered and three strains of the new parasitoid were successfully established in cultures. Cross-infection experiments showed that among the examined planktonic groups, only dinoflagellates were susceptible to the new parasitoid, with infections observed in species belonging to eight genera. Even though the new parasitoid shared many morphological and developmental characteristics with other Perkinsozoan parasites, it differed from them by its densely packed trophocyte structure without a large vacuole or hyaline material during the growth stage. These characteristics are common among Parviluciferaceae members. Furthermore, through palintomic extracellular sporogenesis, it produced characteristic interconnected sporocytes resembling a string of beads. Phylogenetic analyses based on the small subunit and large subunit ribosomal DNA sequences revealed that the new parasitoid was distantly related to the family Parviluciferaceae and was more closely related to the families Perkinsidae and Xcellidae. Morphological, ultrastructural, and molecular data on the new parasitoid raised the need to erect a new family, i.e., Pararosariidae, within the phylum Perkinsozoa with Pararosarium dinoexitiosum gen. et sp. nov. as the type species. The isolation and establishment in culture of the new parasitoid outside the family Parviluciferaceae in the present study would contribute to the better understanding of the diversity of Perkinsozoan parasites and provide useful material for comparisons to other parasite species in the further study.

Fulvifomes nonggangensis and F. tubogeneratus (Hymenochaetales, Basidiomycota): Two New Species from Southern China Based on Morphological and Molecular Evidences

Two new species of Fulvifomes are described from specimens collected in rainforests of Nonggang Nature Reserve of southern China, based on morphological characteristics and molecular phylogenetic analysis of the internal transcribed spacer (ITS) and nuclear large subunit ribosomal DNA (nLSU) sequences. Fulvifomes nonggangensis sp. nov. is characterized by perennial, sessile and solitary basidiocarps, applanate pileus, small cystidioles of 9.9–15.4 × 2.9–3.5 μm, large pores of 5–6 per mm, a dimitic hyphal system, and broadly ellipsoid basidiospores of 4.3–5.3 × 3.3–4.2 μm. F. tubogeneratus sp. nov. is characterized by perennial, sessile, and imbricate basidiocarps, a duplex context, small pores of 7–8 per mm, a dimitic hyphal system, and ovoid to subglobose basidiospores of 5.72 × 5.00 μm.

Laimaphelenchus africanus n. sp. (Tylenchomorpha: Aphelenchoididae) from South Africa, a morphological and molecular phylogenetic study, with an update to the diagnostics of the genus.

A newly recovered population of the genus Laimaphelenchus from a dead maritime pine wood sample in Potchefstroom, South Africa, representing a new species, named L. africanus n. sp., is herein described and illustrated based on morphological and molecular data. The new species is mainly characterized by the following: 750–987 µm long females; a cephalic region with no disc and six cephalic lobs not divided by ribs; a 10.0–12.5 µm long stylet; four incisures in the lateral field; secretory-excretory pore (SE-pore) at slightly posterior to the nerve ring; vulva with a well-developed anterior flap, vagina with two well-developed sclerotized pieces; post-vulval uterine sac (PUS) 63–125 µm long; tail conical, 30–44 µm long, ventrally curved with a subventral stalk in terminus, lacking tubercles, with six to nine small projections at the tip in scanning electron microscopy (SEM); and rare males with 17 μm long spicules. The new species was morphologically compared to those species of the genus with a stalk in tail terminus, lacking tubercles, a vulval flap and four incisures in the lateral field viz., L. liaoningensis, L. preissii, L. simlaensis, L. sinensis, L. spiflatus, and L. unituberculus. Phylogenetically, the new species was placed into the major Laimaphelenchus clade using partial large subunit ribosomal DNA (LSU rDNA D2-D3) sequences. An overall literature review corroborated the presence of the stalk (currently with two main groups) at the tail end is the main characteristic trait delimiting the genus. A compendium based on the characters of the stalk, presence/absence of a vulval flap in females and number of the lateral lines was also established.

An interesting rare tylenchid species, Antarctenchus urmiensis n. sp. (Tylenchomorpha; Psilenchidae) from Urmia Lake islands, northwest Iran, with a discussion on the taxonomy of related genera

Abstract A population of an amphidelphic tylenchid nematode was recovered from Urmia Lake islands in association with native shrubs of the region. It had a Tylenchidae-type cloacal bursa in male and looked morphologically similar to the genera under the family Psilenchidae sensu Siddiqi, representing a new species, described and illustrated in present study as Antarctenchus urmiensis n. sp. It is characterized by the slender body at anterior region, gradually narrowing toward posterior end. Four lines in lateral fields in extremities, increasing to six at mid-body. Deirids at secretory-excretory pore (S-E pore) level and phasmids at 38–62% of the tail. Low-expanded cephalic region having a moderately sclerotized cephalic framework, continuous with body, and not flattened dorso-ventrally. Finely annulated cuticle. Amphidial apertures pore-like. Short (13–15 µm long) stylet, its conus 35–42% of the total length and three small tear-drop like knobs. Pharynx with ellipsoid metacorpus and small valve, small saccate pharyngeal bulb offset from intestine. Reproductive system didelphic-amphidelphic, vulva with no membrane or epyptigma. Tail conical, not elongate or filiform, usually slightly ventrally bent, with a sharp or blunt tip and a small indentation at dorsal side close to tip. Males with similar anterior body region and tail to those of female, tylenchoid spicules and small cloacal bursa. In molecular phylogenetic analyses using partial small and large subunit ribosomal DNA (SSU and LSU rDNA D2-D3) sequences, the new species appeared as an independent lineage between the clades of Hoplolaimina sensu Siddiqi and the clades of Atetylenchus spp. + Psilenchus spp. in the SSU phylogeny. In LSU phylogeny, it placed in a clade including representatives of Atetylenchus, Psilenchus, Amplimerlinius, Pratylenchoides, Nagelus, and Geocenamus. The currently available phylogenies and taxonomic frameworks were re-evaluated for the placement of the didelphic Tylenchidae-like genera, the overall results showing the best resolution is achieved with the family concept of Psilenchidae under Dolichodoroidea.

A molecular phylogenetic reappraisal of Richtersiidae Kreis, 1929 (Desmodorida: Desmodoroidea), with two new species of intertidal nematodes from the Persian Gulf

Summary Synonchium iranicum sp. n. and Richtersia bispinata sp. n. are described from the Persian Gulf and their relationships investigated using SSU phylogenetic analyses. Synonchium iranicum sp. n. is characterised by a buccal cavity with three equally sized mandibles, each with five small teeth, a rounded tail with three distinct caudal glands and distinct duct, males without gubernaculum and supplements, and short spicules. Richtersia bispinata sp. n. is characterised by multispiral amphids with 3.0-3.5 turns in males and 2.0-2.5 turns in females, cuticle with numerous, irregularly arranged, simple spines in the cervical region, 20-25 longitudinal rows of double spines in the mid-body region, 15-25 longitudinal rows of simple spines in the tail region, and the left spicule markedly longer than the right one. We provided the first near-full-length small subunit ribosomal DNA (SSU rDNA) sequences for both newly described species, as well as cytochrome c oxidase I (COI), and provided D2-D3 expansion segments of large subunit ribosomal DNA (LSU rDNA) sequence for the new species of Richtersia. Our SSU phylogenetic analyses confirmed the placement of Synonchium iranicum sp. n. within the Selachinematidae, and suggested that the selachinematids may form a distinct clade from other chromadorids Our phylogenetic analyses indicated that Richtersia should not be classified within the Selachinematidae as suggested by some authors. Instead, we propose that Richtersia should be placed within the Desmodorida. Although Richtersia differs from Desmodorida in the arrangement of cephalic sensilla, it shares the golden body colouration characteristic of desmodorids in glycerin preparations, the same structure of genital branches in both males and females, and similar cuticular ornamentation to some desmodorid genera (notably Desmodorella).

Taxonomic studies of Cryptomonas lundii clade (Cryptophyta: Cryptophyceae) with description of a new species from Vietnam

A new species from Vietnam, Cryptomonas vietnamica sp. nov., is described based on morphological and molecular data. Six strains from different habitats were analyzed. Cryptomonas vietnamica is characterized by large cells up to 54 µm long, 30 µm wide and 20 µm thick, flattened in dorso-ventral plane and often twisted. Phylogenetic relationships inferred from nuclear small and large subunit ribosomal DNA sequences show that the new species forms a clade with Cryptomonas lundii, a rare taxon, described from Europe. Cryptomonas lundii is reported for the first time from Russia and second time since its initial description.

Parvilucifera multicavata sp. nov. (Alveolata, Perkinsozoa), a New Parasitoid Infecting Marine Dinoflagellates Having Abundant Apertures on the Sporangium

The phylum Perkinsozoa is known as an exclusively parasitic group including the parasites of shellfish, fish, dinoflagellates, cryptophytes, and tadpoles and at present comprises seven genera across three families (Parviluciferaceae, Perkinsidae, and Xcellidae), with the genus Parvilucifera having the most abundant species in the family Parviluciferaceae. During intensive sampling along the Korean coast in August and September 2017, a new species of the genus Parvilucifera was discovered and successfully established in cultures. Morphological and ultrastructural observations revealed that the new parasitoid shares almost all known diagnostic characters with other species of Parvilucifera, except that its sporangium has a higher number of apertures although with smaller diameters than those in P. infectans. Molecular phylogenetic trees based on both nuclear small subunit (SSU) and concatenated SSU and large subunit (LSU) ribosomal DNA (rDNA) sequences revealed that the new parasitoid was nested within the family Parviluciferaceae and had a sister relationship with P. infectans. Based on morphological, ultrastructural, and molecular data, we propose to erect a new species, P. multicavata sp. nov., for the new parasitoid found in this study.

Ektaphelenchoides shiroodensis n. sp. (Rhabditida: Ektaphelenchinae) a new member of the genus

Summary Ektaphelenchoides shiroodensis n. sp. is described and illustrated based upon morphological, morphometric and molecular data. It was recovered from the bark samples of a dead alder tree (Alnus sp.) from countryside around Shirood city, Mazandaran province, in the north of Iran. The new species is characterised by 768-985 μm long females, its lip region separated from the body contour by constriction, lateral field with three barely visible lines, forming two weak bands, 26-29 μm long stylet with wide lumen without conophore and basal swellings, excretory pore always at the level with median bulb, post-vulval uterine sac (PUS) 43-76 μm long, elongate conoid posterior body region ending to a long filiform part with pointed tip, males common with dorsally convex conical tail ending to a short narrower region with pointed tip, seven precloacal + cloacal + caudal papillae and arcuate spicules with wide condylus, blunt rostrum and simple end. Based upon the similar posterior body region (‘tail’) and comparable PUS length, the new species was compared to three known species, viz., E. attenuata, E. hunti and E. pini. Comparisons with the aforementioned species and similar species under the genus Seinura are discussed. The phylogenetic affinities of the new species, based upon two partial small and large subunit ribosomal DNA (SSU and LSU rDNA D2-D3) sequences, are discussed. Furthermore, several ektaphelenchid and seinurid species previously described from Iran were recovered in the present study, sequenced for their aforementioned genomic markers, and included in the phylogenetic analyses.

An assessment on the intrapopulational and intraindividual genetic diversity in LSU rDNA in the harmful algal blooms-forming dinoflagellate Margalefidinium (= Cochlodinium) fulvescens based on clonal cultures and bloom samples from Jiaozhou Bay, China

Large subunit ribosomal DNA (LSU rDNA) sequences have been increasingly used to infer the phylogeny and species identity of organisms, a few previous studies, however, have observed high intraspecific and even intraindividual variability in LSU rDNA in some dinoflagellate species due to, assumably, large copy numbers of rDNA in dinoflagellates. Since the copy number of LSU rDNA varies tremendously among dinoflagellate species, the intraspecific and intraindividual diversity for a species of particular interest thus needs to be investigated individually. As a toxic and HABs-forming dinoflagellate, Margalefidinium (= Cochlodinium) fulvescens has been observed to approach blooming density in Jiaozhou Bay, China since 2015 after numerous blooms having been reported from other countries. In trying to identify the source of this newly observed HABs-forming species in China by sequencing the LSU rDNA for both field samples and clonal cultures, we noticed and thus further investigated high intrapopulational and intraindividual genetic diversities of the dinoflagellate. The D1–D6 region of the LSU rDNA (1,435 bases) was amplified from 7 field samples (pooled cells) and 11 clonal cultures, cloned, sequenced, and analyzed phylogenetically for 2,341 sequences obtained. All the numbers of sequences obtained from each clonal culture were far less than the estimated rDNA copy number in M. fulvescens. In the clone library, only one unique sequence was contained in all samples as the most dominant sequence. We found high intrapopulational and intraindividual genetic diversity in M. fulvescens as reflected in the number of polymorphic sites and unique sequences in the clone library for different field samples and clonal cultures in comparison to other species. The mean number of nucleotide differences of each sequence from different field samples and clonal cultures were 6.43 and 4.42 bases, respectively, with the highest being 132 bases, nearly 10%. The sequences with highest variability may be easily annotated as different species if they were obtained from environmental genomic studies because sequence-based species identification in meta-barcoding studies often use "97% identity" threshold. Based on that the mean and overall intrapopulational genetic diversity calculated for 7 field samples was equivalent to the mean and overall intraindividual variability for 11 clonal cultures in indices of genetic diversity, together with the result of AMOVA analysis, we infer that the variability within individual cells (i.e. variability among LSU rDNA polymorphic copies) caused both the intraindividual and intrapopulational genetic diversities observed in the M. fulvescens population, and a higher interpopulational diversity may exist among different geographic populations. The results provide an insightful basis for such a comprehensive interpopulational comparison and important implications for identifying species and establishing new taxa based on the similarity comparison to reference sequences deposited in databases.

Milesina thailandica, a second rust fungus on an early diverged leptosporangiate fern genus, Lygodium, found in Thailand

A new rust fungus was found on Lygodium flexuosum at Doi Suthep-Pui National Park in Chiang Mai Province, Thailand. Puccinia lygodii was the only rust fungus currently known on Lygodium spp. (Lygodioideae, Schizaeaceae, Schizaeales) from southern North America through northern South America. The new fungus produced uredinia linearly between veins, causing yellowish streaks, on the abaxial frond surface. The sori were covered with a layer of thin-walled fungal cells beneath the host epidermis and surrounded by thin-walled paraphyses. The sori did not have a distinct central aperture with ostiolar cells but ruptured irregularly to release urediniospores. Urediniospores were obovoid, obovoid-ellipsoid, or pyriform. The wall was thin, colorless, and completely echinulate. Neither amphispores nor teliospores were observed. The sorus and spore morphology indicated the taxonomic affinity of the fungus to the fern rust genera Hyalopsora and Milesina. A molecular phylogenetic analysis using nuclear large subunit ribosomal DNA sequence revealed that the Lygodium rust fungus was grouped in a paraphyletic Milesina clade. From the putative host preference, morphology, and estimated phylogenetic relationships, it was concluded that the fungus was a new species of Milesina.

Description of Seinura italiensis n. sp. (Tylenchomorpha: Aphelenchoididae) found in the medium soil imported from Italy.

Seinura italiensis n. sp. isolated from the medium soil imported from Italy is described and illustrated using morphological and molecular data. The new species is characterized by having short body (477 (407-565) μm and 522 (469-590) μm for males and females, respectively), three lateral lines, stylet lacking swellings at the base, and excretory pore at the base or slightly anterior to base of metacorpus; females have 58.8 (51.1-69.3) μm long post-uterine sac (PUS), elongate conical tail with its anterior half conoid, dorsally convex, and ventrally slightly concave and the posterior half elongated, narrower, with finely rounded to pointed tip and males having seven caudal papillae and 14.1 (12.6-15.0) μm long spicules. Morphologically, the new species is similar to S. caverna, S. chertkovi, S. christiei, S. hyrcania, S. longicaudata, S. persica, S. steineri, and S. tenuicaudata. The differences of the new species with aforementioned species are discussed. In molecular phylogenetic analyses using near full-length small and large subunit ribosomal DNA (SSU and LSU rDNA D2-D3 expansion segments) sequences, the new species fell into a clade including three previously described/sequenced species of the genus in both SSU and LSU Bayesian phylogenetic trees.

Morphological and molecular characterization of Ektaphelenchoides pini (Massey, 1966) Baujard, 1984 (Aphelenchoididae; Ektaphelenchinae) from Iran, with morphological and taxonomic observations on some species

Ektaphelenchoides pini, the type species of the genus Ektaphelenchoides, was recovered from wood and bark samples of a dead broadleaf forest tree collected from the forests of Golestan province in north of Iran. The recovered population is mainly characterized by massive wide spicules of males with well-developed condylus marked by indentations at the apex and simple distal tip. It was further characterized by 756 to 947 μm long females having a cephalic region slightly separated from the rest body by a shallow depression, 20 to 23 μm stylet with wide lumen lacking conophore and knobs, excretory pore (E pore) at about one metacorpus length behind it, or 92 to 106 μm from anterior end and hemizonid just posterior to it, vagina anteriorly inclined, post uterine sac (PUS) ca 1.2 times vulval body width long, posterior body region elongate conoid, ending to a filiform tip, no functional rectum, a vestigial anus and common males with dorsally convex tail ending to an elongate spike and two pairs of precloacal (P2) and caudal (P3) large papillae at 5 to 6 μm distance anterior to cloacal opening and 30 to 41% of tail, respectively and lacking the single precloacal papilla (P1). In molecular phylogenetic analyses using small and large subunit ribosomal DNA (SSU, LSU rDNA) sequences, the Iranian population of E. pini fell in a clade including species of three genera Ektaphelenchus, Ektaphelenchoides, and Devibursaphelenchus in SSU, and a clade including species of Ektaphelenchus and Ektaphelenchoides in LSU tree, in close association with an isolate identified as E. pini in the latter phylogeny with high (0.99) Bayesian posterior probability (BPP). The comparisons with the type and French populations, as well as phylogenetic affinities of the species using ribosomal data, are discussed. This is the first report of E. pini from Iran, and its first simultaneous morphological and molecular phylogenetic study. New observations on some species of the genus were also presented and discussed.