Pathotype Identification and Host Resistance Evaluation of Clubroot in Zhejiang Province, China.

Clubroot, caused by Plasmodiophora brassicae, is a globally destructive soil-borne disease affecting cruciferous plants. Here, the predominant pathotypes of P. brassicae in six cities within Zhejiang Province were identified using the Williams and European Clubroot Differential (ECD) systems. A phylogenetic analysis of P. brassicae isolates infecting cruciferous crops worldwide was conducted using MEGA, and their ITS2 secondary structures were predicted through the ITS2 database. Accessions of B. rapa, B. oleracea, B. juncea, and Eruca sativa Mill. were employed to assess clubroot resistance. The results revealed that the prevalent pathotypes in Zhejiang Province were pathotype 1, ECD20/31/12 and ECD24/16/30; pathotype 3, ECD20/15/4; pathotype 8, ECD16/0/0 and ECD24/0/0; and pathotype 2, ECD16/15/15. Isolates from distinct genera of Brassicaceae formed separate branches in the evolutionary tree. Moreover, isolates of Brassica crops from Zhejiang Province exhibited homology with those from other global regions, a finding corroborated by their ITS2 secondary structure. Approximately 80% and 95% of B. rapa and B. juncea crops displayed susceptible phenotypes for pathotype 8, ECD16/0/0, whereas approximately 60% of B. oleracea crops exhibited resistance. Furthermore, three Brassica crop accessions showed significant variation in resistance to the pathogen, both among morphological and geographical origin groups. This study contributes to understanding the distribution of diverse P. brassicae pathotypes in different regions of Zhejiang Province and facilitates the identification of Brassica crops with potential disease resistance suitable for cultivation in the province.

Morphopathological and Molecular Morphometric Characterization of Waitea circinata var. prodigus Causing a Novel Sheath Spot Disease of Maize in India.

Maize brown sheath spot (MBSS), a new disease of maize, was discovered while surveying for maize leaf and sheath blight diseases in the Indian states of Assam, Jharkhand, Meghalaya, Manipur, and Odisha. Maize is the third most important cereal after rice and wheat in India. Unlike banded leaf and sheath blight disease caused by Rhizoctonia solani, MBSS symptoms on maize were discrete and limited to sheaths only. Symptoms of MBSS in the field were initially water-soaked necrotic lesions of 1 to 2 cm in diameter on the lowermost leaf sheaths, which then progressed to the upper sheaths. Lesions coalesced and covered approximately 2 to 5% of the sheath area. Infected dried lower leaves were shed, whereas infected upper leaves remained on the stem. The pathogen was isolated, characterized morphologically, pathologically, and molecularly, and identified as Waitea circinata var. prodigus, a basidiomycete known to cause basal leaf blight of seashore paspalum. The internal transcribed spacer (ITS) sequence 2 (ITS2) of rDNA from MBSS isolates formed a well supported clade with known W. circinata var. prodigus isolates. Molecular morphometric analysis of the ITS2 regions of the five known varieties of W. circinata detected distinguishing variations in GC content, compensatory base changes (CBCs), hemi- CBCs, indels, and altered base-pairing of helices. Variation in these characteristics may indicate that varieties are distinct biological species within W. circinata sensu lato. The geographical distribution and potential impacts of MBSS on the maize crop in India necessitate further investigations of pathogen identification and disease management.

Delimitation of five astome ciliate species isolated from the digestive tube of three ecologically different groups of lumbricid earthworms, using the internal transcribed spacer region and the hypervariable D1/D2 region of the 28S rRNA gene.

BACKGROUND: Various ecological groups of earthworms very likely constitute sharply isolated niches that might permit speciation of their symbiotic ciliates, even though no distinct morphological features appear to be recognizable among ciliates originating from different host groups. The nuclear highly variable ITS1-5.8S-ITS2 region and the hypervariable D1/D2 region of the 28S rRNA gene have proven to be useful tools for the delimitation of species boundaries in closely related free-living ciliate taxa. In the present study, the power of these molecular markers as well as of the secondary structure of the ITS2 molecule were tested for the first time in order to discriminate the species of endosymbiotic ciliates that were isolated from the gastrointestinal tract of three ecologically different groups of lumbricid earthworms. RESULTS: Nineteen new ITS1-5.8S-ITS2 region and D1/D2-28S rRNA gene sequences were obtained from five astome species (Anoplophrya lumbrici, A. vulgaris, Metaradiophrya lumbrici, M. varians, and Subanoplophrya nodulata comb. n.), which were living in the digestive tube of three ecological groups of earthworms. Phylogenetic analyses of the rRNA locus and secondary structure analyses of the ITS2 molecule robustly resolved their phylogenetic relationships and supported the distinctness of all five species, although previous multivariate morphometric analyses were not able to separate congeners in the genera Anoplophrya and Metaradiophrya. The occurrence of all five taxa, as delimited by molecular analyses, was perfectly correlated with the ecological groups of their host earthworms. CONCLUSIONS: The present study indicates that morphology-based taxonomy of astome ciliates needs to be tested in the light of molecular and ecological data as well. The use of morphological identification alone is likely to miss species that are well delimited based on molecular markers and ecological traits and can lead to the underestimation of diversity and overestimation of host range. An integrative approach along with distinctly increased taxon sampling would be helpful to assess the consistency of the eco-evolutionary trend in astome ciliates.

Evolutionary Origin and Host Range of Plagiotoma lumbrici (Ciliophora, Hypotrichia), an Obligate Gut Symbiont of Lumbricid Earthworms.

Four common earthworm species, the anecic Lumbricus terrestris, the endogeic Octolasion tyrteum as well as the epigeic Eisenia fetida and Dendrobaena veneta, were examined for the presence of the microbial gut symbiont Plagiotoma lumbrici. The evolutionary origin of this endobiotic microbe was reconstructed, using the 18S rRNA gene, the ITS1-5.8S-ITS2 region, and the first two domains of the 28S rRNA gene. Plagiotoma lumbrici was exclusively detected in the anecic Lumbricus terrestris. Multigene analyses and the ITS2 secondary structure robustly determined the phylogenetic home of Plagiotoma lumbrici populations within the oxytrichid Dorsomarginalia (Spirotrichea: Hypotrichia) as a sister taxon of the free-living Hemiurosomoida longa. This indicates that earthworms obtained their gut endosymbiont by ingesting soil/leaf litter containing oxytrichine ciliates that became adapted to the intestinal tract of earthworms. Interestingly, according to the literature data, Plagiotoma lumbrici was detected in multiple anecic and some epigeic but never in endogeic earthworms. These observations suggest that Plagiotoma lumbrici might be adapted to certain gut conditions and the lifestyle of anecic Lumbricidae, such as Lumbricus, Aporrectodea, and Scherotheca, as well as of some co-occurring epigeic Lumbricus species.

Chlorocladiella gen. nov. (Pithophoraceae, Cladophorales, Chlorophyta), Including Four New Species From Various Freshwater Habitats in China.

Samples of Pithophoraceae, collected in diverse freshwater and damp terrestrial habitats from tropical and subtropical China, were characterized morphologically and ultrastructurally, and their phylogenetic position was determined based on nuclear ribosomal DNA sequences. Our phylogenetic analysis resolved a novel lineage of Pithophoraceae, sister to Aegagropilopsis. Based on our phylogenetic results, morphological observations and comparative rDNA ITS2 secondary structure analysis, we propose Chlorocladiella gen. nov., characterized by a well-developed system of prostrate filaments, and describe four new species, C. cochlea sp. nov., C. erecta sp. nov., C. medogensis sp. nov., and C. pisformis sp. nov. Two species were found growing on damp soil, which is an unusual habitat for cladophoralean green algae, indicating that the diversity of Cladophorales in terrestrial habitats may be greater than currently recognized.

Untangling a mess of worms: Species delimitations reveal morphological crypsis and variability in Southeast Asian semi-aquatic earthworms (Almidae, Glyphidrilus).

Semi-aquatic freshwater earthworms in the genus Glyphidrilus from Southeast Asia are characterized by both an extreme morphological crypsis among divergent phylogenetic lineages and a high morphological variability within the same phylogenetic lineages. The present study provides a new taxonomic framework for this problematic genus in SE Asia by integrating DNA sequence and morphological data. When single-locus and multilocus multispecies coalescent-based (MSC) species delimitation methods were applied to DNA sequence data, they usually yielded highly incongruent results compared to morphology-based species identifications. This suggested the presence of several cryptic species and high levels of intraspecific morphological variation. Applying reciprocal monophyly to the cytochrome c oxidase subunit 1 (COI) gene tree allowed us to propose the existence of 33 monophyletic species. Yet, often substantially more molecular operational taxonomic units (MOTUs) were obtained when species delimitation was based on COI and 16S rRNA sequences. In contrast, the ITS1 and ITS2 sequences suggested fewer MOTUs and did not recover most of the monophyletic species from the Mekong basin. However, several of these latter taxa were better supported when MSC species delimitation methods were applied to the combined mtDNA and ITS datasets. The ITS2 secondary structure retrieved one unnamed Mekong basin species that was not uncovered by the other methods when applied to ITS2 sequences. In conclusion, based on an integrative taxonomic workflow, 26 Glyphidrilus candidate species were retained and two remained to be confirmed. As such, this study provides evidence to suggest nine species new to science and to synonymize 12 nominal morphospecies. It also illustrates that the uncritical use of COI as a universal DNA barcode may overestimate species diversity because COI may be unable to distinguish between divergent conspecific lineages and different candidate species.

Morphology and molecular phylogeny of coccoid green algae Coelastrella sensu lato (Scenedesmaceae, Sphaeropeales), including the description of three new species and two new varieties.

The genus Coelastrella was established by Chodat (Bull. Soc. Bot. Geneve, 13 [1922] 66), and was characterized as being unicellular or in few-celled aggregations with many longitudinal ribs on the cell wall. Many species of this genus showed strong ability to accumulate carotenoids and oils, so they have recently attracted much attention from researchers due to its potential applicability in the energy and food industries. In this study, a total of 23 strains of Coelastrella were sampled from China, and three new species and two new varieties were described: C. thermophila sp. nov., C. yingshanensis sp. nov., C. tenuitheca sp. nov., C. thermophila var. globulina var. nov., C. rubescens var. oocystiformis var. nov. Besides 18S rDNA and ITS2 sequences, we have newly sequenced the tufA gene marker for this taxon. Phylogenetic analysis combined with morphological studies revealed four morphotypes within the Coelastrella sensu lato clade, which contained the morphotype of original Coelastrella, original Scotiellopsis, Asterarcys, and morphotype of C. vacuolata and C. tenuitheca sp. nov. The relationships between morphological differences and phylogenic diversity based on different markers were discussed. Our results support that 18S rDNA was too conserved to be used a species-specific or even a genus-specific marker in this clade. The topology of tufA gene-based phylogenetic tree had a better match with the morphological findings.

[Application of ITS2 secondary structure phylogenetic information in DNA barcode identification of Chrysanthemum indicum and its related plants].

By exploring additional phylogenetic information hidden in ITS2 secondary structure,the possibility of identifying Chrysanthemum indicum and its related species with DNA barcode of ITS2 nucleic acid sequence and its structure information were discussed.The genomic DNA was extracted from 12 samples. The ITS2 fragments were amplified by PCR and sequenced bidirectionally to obtain ITS2 sequence information. 28 sequences of related species for Ch. indicum were downloaded from Gen Bank. Until all 40 ITS2 sequences were aligned,ITS2 secondary structure prediction and structure comparison were finished. Then ITS2 secondary structure information was coded. After comparing ITS2 structure information and nucleic acid information,MP phylogenetic trees were built. The results showed that the secondary structures of ITS2 shared the same structure model--a four-fingered hand. They not only have the common characteristics of ITS2 secondary structures in plants,but also have many other conservative sequences,and their overall conservativeness is high. Among all species used in this study,their ITS2 secondary structures had obvious difference. In addition,the number of mutation sites in the joint matrix compared with the nucleic acid sequences increased by nearly 90%,which greatly enriched the number of mutation sites. This method of information analysis distinguished Ch. indicum from its related species. At the same time,the support rate of the branches of evolutionary trees and the identification rate of species were significantly improved. Although there was no distinction between Ch. zawadskii and Ch. morifolium,it effectively distinguished the three species,namely,Ch. hypargyrum,Ch.oreastrum,and Ch. dichrum. Therefore,the authors suggest that the ITS2 sequence combined with its structural data information should be applied to the identification of Ch. indicum and its related species,and be widely applied to DNA barcode research.

ITS2 sequence-structure phylogeny reveals diverse endophytic Pseudocercospora fungi on poplars.

For matching the new fungal nomenclature to abolish pleomorphic names for a fungus, a genus Pseudocercospora s. str. was suggested to host holomorphic Pseudocercosproa fungi. But the Pseudocercosproa fungi need extra phylogenetic loci to clarify their taxonomy and diversity for their existing and coming species. Internal transcribed spacer 2 (ITS2) secondary structures have been promising in charactering species phylogeny in plants, animals and fungi. In present study, a conserved model of ITS2 secondary structures was confirmed on fungi in Pseudocercospora s. str. genus using RNAshape program. The model has a typical eukaryotic four-helix ITS2 secondary structure. But a single U base occurred in conserved motif of U-U mismatch in Helix 2, and a UG emerged in UGGU motif in Helix 3 to Pseudocercospora fungi. The phylogeny analyses based on the ITS2 sequence-secondary structures with compensatory base change characterizations are able to delimit more species for Pseudocercospora s. str. than phylogenic inferences of traditional multi-loci alignments do. The model was employed to explore the diversity of endophytic Pseudocercospora fungi in poplar trees. The analysis results also showed that endophytic Pseudocercospora fungi were diverse in species and evolved a specific lineage in poplar trees. This work suggested that ITS2 sequence-structures could become as additionally significant loci for species phylogenetic and taxonomic studies on Pseudocerospora fungi, and that Pseudocercospora endophytes could be important roles to Pseudocercospora fungi's evolution and function in ecology.

Morphological and genetic diversity of Beaufort Sea diatoms with high contributions from the Chaetoceros neogracilis species complex.

Seventy-five diatom strains isolated from the Beaufort Sea (Canadian Arctic) in the summer of 2009 were characterized by light and electron microscopy (SEM and TEM), as well as 18S and 28S rRNA gene sequencing. These strains group into 20 genotypes and 17 morphotypes and are affiliated with the genera Arcocellulus, Attheya, Chaetoceros, Cylindrotheca, Eucampia, Nitzschia, Porosira, Pseudo-nitzschia, Shionodiscus, Thalassiosira, and Synedropsis. Most of the species have a distribution confined to the northern/polar area. Chaetoceros neogracilis and Chaetoceros gelidus were the most represented taxa. Strains of C. neogracilis were morphologically similar and shared identical 18S rRNA gene sequences, but belonged to four distinct genetic clades based on 28S rRNA, ITS-1 and ITS-2 phylogenies. Secondary structure prediction revealed that these four clades differ in hemi-compensatory base changes (HCBCs) in paired positions of the ITS-2, suggesting their inability to interbreed. Reproductively isolated C. neogracilis genotypes can thus co-occur in summer phytoplankton communities in the Beaufort Sea. C. neogracilis generally occurred as single cells but also formed short colonies. It is phylogenetically distinct from an Antarctic species, erroneously identified in some previous studies as C. neogracilis, but named here as Chaetoceros sp. This work provides taxonomically validated sequences for 20 Arctic diatom taxa, which will facilitate future metabarcoding studies on phytoplankton in this region.

[Application of phylogenetic information of ITS2 secondary structure in DNA barcoding of Solanum medicinal plant].

Internal transcript spacer 2 (ITS2) is one of the broadly used standard core barcodes and also the only nuclear barcode in identification of Chinese traditional medicine. Although the DNA barcode method based on ITS2 is popular and has been used in Chinese Pharmacopoeia, its low discriminatory efficiency is still a problem to its extensive application. Therefore, further study is still necessary to explore its phylogenetic information for medicinal plants identification. In cells, ITS2 activity is based on its secondary structure. The secondary structures are particularly useful in phylogenetic analysis because they include information not found in the primary sequence. In this study ITS2 secondary structure of 40 samples from 26 species were predicted and used to explore their utility in addressing the identification problems of Chinese traditional medicine in Solanum. The secondary structures were predicted and aligned, and their consensus models were generated using the three different software of LocARNA, MASTR and PicXAA-R. RNAstat software was used to transform the secondary structures into 28 symbol code data for maximum parsimony (MP) analysis. The results showed that the phylogenetic information increased 88.57% after ITS2 secondary structure information has been added, and then the support values above 50%, 75% and 90% in the tree increased 19.05%, 66.67% and 66.67%, respectively, indicating that the identification of Solanum medical plants has been well resolved. Thus, our analysis suggests that ITS2 secondary structure information should be incorporated into the current DNA barcoding analysis as a beneficial supplement of phylogenetic information.

Molecular phylogeny and species delimitation within the ciliate genus Spirostomum (Ciliophora, Postciliodesmatophora, Heterotrichea), using the internal transcribed spacer region.

Morphological and molecular delimitation of Spirostomum species is currently under debate. We addressed species boundaries within the genus Spirostomum, using the ITS1-5.8S-ITS2 region and the secondary structure of the ITS2 molecule, and 18S and 28S (D1D2) sequences additionally. The Spirostomum ITS region is among the shortest within the ciliates hitherto studied. The Spirostomum ITS2 molecule matches the "ring model", but exhibits only two helices radiating from a common loop. According to comparative analyses, they very likely correspond to helices II and III of other eukaryotes. Our phylogenetic analyses of the ITS region revealed a complex genealogical structure within the genus Spirostomum. However, boundaries among Spirostomum species could not be unambiguously determined either by phylogenetic trees, networks or sequence divergence cutoffs, because ITS2 sequences transcended species boundaries of the following morphospecies: S. ambiguum, S. minus, S. subtilis and S. teres. According to molecular diversity analysis, this is very likely caused by polymorphism in S. minus and S. teres, and by the lack of variability in S. ambiguum and S. subtilis. No compensatory base changes (CBCs) were detected in helices of the ITS2 molecule between different Spirostomum species, documenting that CBC analysis per se is not able to effectively discriminate Spirostomum species.

ITS2 secondary structure for species circumscription: case study in southern African Strychnos L. (Loganiaceae).

Recently developed computational tools in ITS2 sequence-structure phylogenetics are improving tree robustness by exploitation of the added information content of the secondary structure. Despite this strength, however, their adoption for species-level clarifications in angiosperms has been slow. We investigate the utility of combining ITS2 sequence and secondary structure to separate species of southern African Strychnos, and assess correlation between compensatory base changes (CBCs) and currently recognised species boundaries. Combined phylogenetic analysis of sequence and secondary structure datasets performed better, in terms of robustness and species resolution, than analysis involving primary sequences only, achieving 100 and 88.2 % taxa discriminations respectively. Further, the Strychnos madagascariensis complex is well-resolved by sequence-structure phylogenetic analysis. The 17 Strychnos species corresponded to 14 ITS2 CBC clades. Four of the five taxa in section Densiflorae belong to a single CBC clade, whose members tend to form natural hybrids. Our finding supports the application of ITS2 as a complementary barcoding marker for species identification. It also highlights the potential of comparative studies of ITS2 CBC features among prospective parental pairs in breeding experiments as a rapid proxy for cross compatibility assessment. This could save valuable time in crop improvement. Patterns of CBC evolution and species boundaries in Strychnos suggests a positive correlation. We conclude that the CBC pattern coupled with observed ITS2 sequence paraphyly in section Densiflorae points to a speciation work-in-progress.

Pseudo-nitzschia arctica sp. nov., a new cold-water cryptic Pseudo-nitzschia species within the P. pseudodelicatissima complex.

A new nontoxic Pseudo-nitzschia species belonging to the P. pseudodelicatissima complex, P. arctica, was isolated from different areas of the Arctic. The erection of P. arctica is mainly supported by molecular data, since the species shares identical ultrastructure with another species in the complex, P. fryxelliana, and represents a new case of crypticity within the genus. Despite their morphological similarity, the two species are not closely related in phylogenies based on LSU, ITS and rbcL. Interestingly, P. arctica is phylogenetically most closely related to P. granii and P. subcurvata, from which the species is, however, morphologically different. P. granii and P. subcurvata lack the central larger interspace which is one of the defining features of the P. pseudodelicatissima complex. The close genetic relationship between P. arctica and the two species P. granii and P. subcurvata is demonstrated by analysis of the secondary structure of ITS2 which revealed no compensatory base changes, two hemi-compensatory base changes, and two deletions in P. arctica with respect to the other two species. These findings emphasize that rates of morphological differentiation, molecular evolution and speciation are often incongruent for Pseudo-nitzschia species, resulting in a restricted phylogenetic value for taxonomic characters used to discriminate species. The description of a new cryptic species, widely distributed in the Arctic and potentially representing an endemic component of the Arctic diatom flora, reinforces the idea of the existence of noncosmopolitan Pseudo-nitzschia species and highlights the need for combined morphological and molecular analyses to assess the distributional patterns of phytoplankton species.

Gorgonia mariae and Antillogorgia bipinnata populations inferred from compensatory base change analysis of the internal transcribed spacer 2.

The systematics within some families of octocorals (Class Anthozoa: Sub-class Octocorallia) remains uncertain in terms of morphological variability and unresolved molecular phylogenies. Elucidating the phylogenetic relationships among octocorals can help for understanding the origin of its huge biodiversity and optimizing conservation plans. The phylogenetic relationships within Gorgoniidae family remain highly polyphyletic. This research explores Gorgonia mariae and Antillogorgia bipinnata populations from several study sites in the Caribbean sea using for the first time the compensatory base changes (CBCs) analysis of the internal transcribed spacer 2 (ITS2) as a molecular tool to elucidate the phylogenetic relationships within the gorgonians. In addition, the mitochondrial gene mtMutS was sequenced in order to complement the nuclear information. This research shows that the presence of CBCs and hemi-CBCs is useful as a complementary tool for providing insights between octocoral species and populations, as well as an approach to figure out the origin of the octocorals biodiversity.

Rhinomonas nottbecki n. sp. (cryptomonadales) and molecular phylogeny of the family Pyrenomonadaceae.

The cryptomonad Rhinomonas nottbecki n. sp., isolated from the Baltic Sea, is described from live and fixed cells studied by light, scanning, and transmission electron microscopy together with sequences of the partial nucleus- and nucleomorph-encoded 18S rRNA genes as well as the nucleus-encoded ITS1, 5.8S, ITS2, and the 5'-end of the 28S rRNA gene regions. The sequence analyses include comparison with 43 strains from the family Pyrenomonadaceae. Rhinomonas nottbecki cells are dorsoventrally flattened, obloid in shape; 10.0-17.2 µm long, 5.5-8.1 µm thick, and 4.4-8.8 µm wide. The inner periplast has roughly hexagonal plates. Rhinomonas nottbecki cells resemble those of Rhinomonas reticulata, but the nucleomorph 18S rRNA gene of R. nottbecki differs by 2% from that of R. reticulata, while the ITS region by 11%. The intraspecific variability in the ITS region of R. nottbecki is 5%. In addition, the predicted ITS2 secondary structures are different in R. nottbecki and R. reticulata. The family Pyrenomonadaceae includes three clades: Clade A, Clade B, and Clade C. All Rhinomonas sequences branched within the Clade C, while the genus Rhodomonas is paraphyletic. The analyses suggest that the genus Storeatula is an alternating morphotype of the genera Rhinomonas and Rhodomonas and that the family Pyrenomonadaceae includes some species that were described multiple times, as well as novel species.

Cryptic diversity in a cosmopolitan diatom known as Asterionellopsis glacialis (Fragilariaceae): Implications for ecology, biogeography, and taxonomy.

PREMISE OF THE STUDY: Diatoms have long been known as the most species-rich of algal groups, with a wide range of estimates for species number (20-200 k) due to differing species concepts. The fine valve structure in Asterionellopsis glacialis, a diatom believed cosmopolitan and eurytopic, has never been systematically examined using modern microscopy and is an excellent candidate to genetically test morphology-based conspecificity among its geographically distant culture isolates. METHODS: Isolates from the Atlantic and Pacific Oceans that were morphologically delineated as A. glacialis were genetically characterized using three nuclear DNA regions (two 18S rDNA fragments and ITS region) and one plastidal (rbcL) and one mitochondrial gene (cox1) and related to SEM-based morphometrics. KEY RESULTS: Five genetically distinct groupings were found, four of which are new to science. ITS2 RNA transcript secondary structure was species specific as were plastidal and mitochondrial genes, while the 18S gene fragments did not diverge sufficiently to segregate new species efficiently. We genetically circumscribed the A. glacialis epitype. CONCLUSIONS: The morphological diversification of the species examined in this study lags behind their genetic divergence. The currently accepted 2% cutoff level of operational taxonomic unit (OTU) clustering in 18S rDNA environmental sequencing is too high to recognize genetic diversity in Asterionellopsis and very likely in many other species. Our results support the notion that a considerable number of species and diversity remain to be discovered among diatoms and that species number may be more in line with higher estimates. Molecular signatures of the species discovered here will aid in their globally consistent identification and ultimate understanding of their ecology.

Findings on three endocommensal scuticociliates (Protista, Ciliophora) from freshwater mollusks, including their morphology and molecular phylogeny with descriptions of two new species.

Species of the ciliate genera Myxophyllum and Conchophthirus are found as endocommensals of terrestrial and freshwater mollusks, respectively. So far, there have been few studies of these genera and morphological data for most members are often incomplete. In the present work, two new species, Myxophyllum weishanense sp. nov. and Conchophthirus paracurtus sp. nov., and a known species, Conchophthirus lamellidens, were isolated from hosts in Lake Weishan Wetland, China. Taxonomic studies indicate that M. weishanense sp. nov. can be recognized mainly by the combination of about 60 somatic kineties on both ventral and dorsal sides and the presence of caudal cilia. Conchophthirus paracurtus sp. nov. differs from congeners in its body shape and size, having a glabrous area on the posterior right side, and having fewer somatic kineties. In addition, differences in their ITS2 (Internally Transcribed Spacer 2) secondary structures support the discrimination of the two new species from their highly similar congeners. An improved diagnosis for the poorly known species, C. lamellidens is also provided. Phylogenetic analyses reveal that members of the genus Myxophyllum belong to a fully supported clade that is sister to a large, poorly supported clade consisting of Hemispeiridae, Ancistridae, and several lineages of the nonmonophyletic Cyclidiidae. The Myxophyllum clade also includes Protophyra ovicola JQ956552, a possible misidentification. Sequences of the two new Conchophthirus species cluster with other congeners in a fully supported clade that is unrelated to either the 'typical' thigmotrichs or to pleuronematids, thus conflicting with the traditional classification, and may represent an orphan scuticociliate lineage. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00230-4.

The New Genus Caulinema Revealed New Insights into the Generic Relationship of the Order Ulotrichales (Ulvophyceae, Chlorophyta).

Traditionally, the order Ulotrichales comprised green algae of an unbranched, uniseriate, filamentous morphology. However, since the establishment of ultrastructural features, the circumscription of this order has dramatically changed. Some genera and species have been excluded from this order and others with different morphologies (sarcinoid, branched filaments or even parenchymatous taxa) have been included. Phylogenetic analyses have confirmed the monophyly of this order, but its differentiation from the Ulvales and Acrosiphoniales remains difficult because of the lack of synapomorphies at every level (morphology, molecular signatures). To demonstrate the difficulties of placement into genera and orders, we investigated two sarcinoid taxa with the absence of zoospore formation. SSU and ITS rDNA tree topology and the ITS-2/CBC approach revealed that both strains SAG 2661 and CCAP 312/1 belong to Ulosarcina terrestrica and the newly erected genus Caulinema, respectively. The species conception using this approach was evaluated by sequencing the plastid-coding gene tufA, a commonly used barcode marker for green algae. All three molecular markers resulted in similar topologies at the generic and species levels, which is consistent with the ITS-2/CBC approach and tufA for barcoding. The reevaluation of the ultrastructural features revealed that the presence of organic scales on the surfaces of motile cells is characteristic for the order Ulotrichales and can be used for separation from the closely related orders. As a consequence of our study, we propose the new genus Caulinema for strain CCAP 312/1.

Solotvynia, a New Coccoid Lineage among the Ulvophyceae (Chlorophyta).

Coccoid Ulvophyceae are often overlooked despite their wide distribution. They occur as epiphytes on marine seaweeds or grow on stones or on shells of mussels and corals. Most of the species are not easy to identify based solely on morphology. However, they form two groups based on the flagellated cells during asexual reproduction. The biflagellated coccoids are monophyletic and represent the genus Sykidion (Sykidiales). In contrast, the quadriflagellated taxa are polyphyletic and belong to different genera and orders. The newly investigated strains NIES-1838 and NIES-1839, originally identified as Halochlorococcum, belong to the genus Chlorocystis (C. john-westii) among the order Chlorocystidales. The unidentified strain CCMP 1293 had almost an identical SSU and ITS-2 sequence to Symbiochlorum hainanense (Ignatiales) but showed morphological differences (single chloroplast, quadriflagellated zoospores) compared with the original description of this species (multiple chloroplasts, aplanospores). Surprisingly, the strain SAG 2662 (= ULVO-129), together with the published sequence of MBIC 10461, formed a new monophyletic lineage among the Ulvophyceae, which is highly supported in all of the bootstrap and Bayesian analyses and approximately unbiased tests of user-defined trees. This strain is characterized by a spherical morphology and also form quadriflagellated zoospores, have a unique ITS-2 barcode, and can tolerate a high variation of salinities. Considering our results, we emend the diagnosis of Symbiochlorum and propose the new genus Solotvynia among the new order Solotvyniales.