Of sea, rivers and symbiosis: Diversity, systematics, biogeography and evolution of the deeply diverging florideophycean order Hildenbrandiales (Rhodophyta).

The Hildenbrandiales, a typically saxicolous red algal order, is an early diverging florideophycean group with global significance in marine and freshwater ecosystems across diverse temperature zones. To comprehensively elucidate the diversity, phylogeny, biogeography, and evolution of this order, we conducted a thorough re-examination employing molecular data derived from nearly 700 specimens. Employing a species delimitation method, we identified Evolutionary Species Units (ESUs) within the Hildenbrandiales aiming to enhance our understanding of species diversity and generate the first time-calibrated tree and ancestral area reconstruction for this order. Mitochondrial cox1 and chloroplast rbcL markers were used to infer species boundaries, and subsequent phylogenetic reconstructions involved concatenated sequences of cox1, rbcL, and 18S rDNA. Time calibration of the resulting phylogenetic tree used a fossil record from a Triassic purportedly freshwater Hildenbrandia species and three secondary time points from the literature. Our species delimitation analysis revealed an astounding 97 distinct ESUs, quintupling the known diversity within this order. Our time-calibration analysis placed the origin of Hildenbrandiales (crown age) in the Ediacaran period, with freshwater species emerging as a monophyletic group during the later Permian to early Triassic. Phylogenetic reconstructions identified seven major clades, experiencing early diversification during the Silurian to Carboniferous period. Two major evolutionary events-colonization of freshwater habitats and obligate systemic symbiosis with a marine fungus-marked this order, leading to significant morphological alterations without a commensurate increase in species diversification. Despite the remarkable newly discovered diversity, the extant taxon diversity appears relatively constrained when viewed against an evolutionary timeline spanning over 800 million years. This limitation may stem from restricted geographic sampling or the prevalence of asexual reproduction. However, species richness estimation and rarefaction analyses suggest a substantially larger diversity yet to be uncovered-potentially four times greater. These findings drastically reshape our understanding of the deeply diverging florideophycean order Hildenbrandiales species diversity, and contribute valuable insights into this order's evolutionary history and ecological adaptations. Supported by phylogenetic, ecological and morphological evidence, we established the genus Riverina gen. nov. to accommodate freshwater species of Hildenbrandiales, which form a monophyletic clade in our analyses. This marks the first step toward refining the taxonomy of the Hildenbrandiales, an order demanding thorough revisions, notably with the creation of several genera to address the polyphyletic status of Hildenbrandia. However, the limited diagnostic features pose a challenge, necessitating a fresh approach to defining genera. A potential solution lies in embracing a molecular systematic perspective, which can offer precise delineations of taxonomic boundaries.

Evaluation of the paired-Cas9 nickase and RNA-guided FokI genome editing tools in precise integration of an anti-CD52 bicistronic monoclonal antibody expression construct at Chinese hamster ovary cells 18S rDNA locus.

INTRODUCTION: The aim of this study was to compare two CRISPR/Cas9-based orthogonal strategies, paired-Cas9 nickase (paired-Cas9n) and RNA-guided FokI (RFN), in targeting 18S rDNA locus in Chinese hamster ovary (CHO) cells and precisely integrating a bicistronic anti-CD52 monoclonal antibody (mAb) expression cassette into this locus. METHODS: T7E1 and high-resolution melt (HRM) assays were used to compare the ability of mentioned systems in inducing double-strand break (DSB) at the target site. Moreover, 5'- and 3'-junction polymerase chain reactions (PCR) were used to verify the accuracy of the targeted integration of the mAb expression cassette into the 18S rDNA locus. Finally, anti-CD52 mAb gene copy number was measured and, its expression was analyzed using ELISA and western blot assays. RESULTS: Our results indicated that both paired-Cas9n and RFN induced DSB at the target site albeit RFN performance was slightly more efficient in HRM analysis. We also confirmed that the anti-CD52 mAb cassette was accurately integrated at the 18S rDNA locus and the mAb was expressed successfully in CHO cells. CONCLUSION: Taken together, our findings elucidated that both paired-Cas9n and RFN genome editing tools are promising in targeting the 18S rDNA locus. Site specific integration of the bicistronic anti-CD52 mAb expression cassette at this locus in the CHO-K1 cells was obtained, using RFN. Moreover, proper expression of the anti-CD52 mAb at the 18S rDNA target site can be achieved using the bicistronic internal ribosome entry site (IRES)-based vector system.

New molecular evidence on the members of the genus Ortholinea (Cnidaria, Myxozoa) and the description of Ortholinea hamsiensis n. sp. infecting the urinary bladder of European anchovy Engraulis engrasicolus in the Black Sea.

Members of the genus Ortholinea are among the worldwide distributed myxozoan parasites that mainly infect marine fish. In this study, a new myxosporean species, Ortholinea hamsiensis n. sp., was isolated from the urinary bladder of European anchovy Engraulis engrasicolus collected from the Sinop coasts of the Black Sea. The prevalence and density values of infection were 1.4% and 1­5 individuals in the field of view (1 + ), respectively. Mature myxospores are subspherical with slight tapering down to the less pronounced tip in the frontal view and subspherical in the sutural view. Myxospores measured 9.1 ± 0.25 (8.8­9.9) µm in length, 9.2 ± 0.11 (8.9­9.4) µm in thickness, and 8.4 ± 0.33 (8.2-9.1) µm in width. Two polar capsules equal in size measured 3.1 ± 0.11 (3.0­3.3) µm in length and 2.7 ± 0.11 (2.6­2.9) µm in width. The polar tubule had 3­4 coils. Along with morphological peculiarities, the results of the 18S rDNA also revealed it to be a new species for science compared to the other species of the genus. In this study, another myxosporean species O. gobiusi was also detected in round goby Neogobius melanostomus with a prevalence of infection value of 4.8% and a density of 1­5 individuals in the field of view (1 + ). The present study also provided the first data of 18S rDNA of O. gobiusi from N. melanostomus and type species of the genus O. divergens from Gobius niger and the phylogenetic relationships of these species with other Ortholinea species have been revealed.

Description of an intramonocytic haemoparasite, Hepatozoon lainsoni sp. nov. (Apicomplexa: Adeleorina: Hepatozoidae), infecting Ameiva ameiva lizard (Reptilia: Squamata: Teiidae) in northern Brazil.

Haemogregarine (Apicomplexa: Adeleorina) parasites are considered to be the most common and widespread haemoparasites in reptiles. The genus Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae) can be found parasitizing a broad range of species and, in reptiles, they infect mainly peripheral blood erythrocytes. The present study detected and characterized a haemogregarine isolated from the lizard species, Ameiva ameiva, collected from the municipality of Capanema, Pará state, north Brazil. Blood smears and imprints from lungs, brain, heart, kidney, liver, bone marrow and spleen were observed using light microscopy and the parasite was genetically identified by molecular analysis. Morphological, morphometric and molecular data were obtained. Parasite gamonts were found in 49.5% (55/111) of the blood smears from A. ameiva, and were characterized as oval, averaging 12.0 ± 0.8 × 5.9 ± 0.6 µm2 in size, which displaced the nuclei of parasitized monocytes laterally. Parasite forms resembling immature gamonts were observed in the spleen and bone marrow of the lizards. Furthermore, phylogenetic analyses of 18S rRNA sequences did not reveal gene similarity with other Hepatozoon spp. sequences from reptiles. Thus, morphological and molecular analyses have identified a new species of Hepatozoon parasite, Hepatozoon lainsoni sp. nov., which infects monocytes of the A. ameiva lizard.

Harmonized coexistence of intragenomic variations in diatom Skeletonema strains.

Metabarcoding analysis has been demonstrated to be an effective technology for monitoring diversity and dynamics of phytoplankton including Skeletonema species. Although molecular diversity uncovered in metabarcoding projects has generally been interpreted as sum of interspecies diversity and intraspecies diversity, accumulating evidence suggests that it also harbors unprecedentedly high levels of intra-genomic variations (IGVs). As up to thousands of amplicon sequence variants (ASVs) identified in a typical metabarcoding project can be annotated to be Skeletonema species, we hypothesize that substantial portions of these ASVs are contributed by IGVs. Here, the nature of IGVs in Skeletonema species was quantitatively analyzed by carrying out single-strain metabarcoding analysis of 18S rDNA V4 in 49 strains belonging to seven Skeletonema species. Results showed that each Skeletonema strain harbored a high level of IGVs as expected. While many Skeletonema strains each contained one dominant ASV and a substantial number of ASVs displaying much lower relative abundance, other Skeletonema strains each contained multiple ASVs with comparable or nearly equally abundances. Thus the co-existence of multiple dominant ASVs in a single cell indicated a tug-of-war of these variants in evolution, which may eventually result in harmonized coexistence of multiple dominant ASVs. A total of nine dominant ASVs and 652 non-dominant ASVs were found in 49 strains of seven Skeletonema species, indicating rich interspecies and intraspecies variations, and complex evolution of IGVs in genus of Skeletonema. The results confirmed that the extensive degree of IGVs was the main contributor to the high molecular diversity revealed by metabarcoding analysis. This study highlights the importance of quantitative characterization of IGVs in Skeletonema species for accurate interpretation of species diversity in metabarcoding analysis.

Characterization of novel aurantiactinomyxon types (Cnidaria, Myxosporea) from the oligochaete Ilyodrilus templetoni (Southern, 1909), with a comprehensive phylogeny of the collective group.

Three new aurantiactinomyxon types are described from the oligochaete Ilyodrilus templetoni (Southern, 1909) (Naididae) collected from a northern Portuguese estuary, based on light microscopy and sequencing of the 18S rDNA. The addition of I. templetoni to the group of freshwater annelids known to be permissive for aurantiactinomyxon development reinforces the crucial role of naidids in the evolution and settlement of myxozoans in estuarine environments. Maximum likelihood and Bayesian inference analyses of a comprehensive 18S rDNA dataset placed the novel types within the Paramyxidium clade. This positioning suggests them as probable life cycle counterparts to Paramyxidium spp. that most likely infect the European eel Anguilla anguilla, as the sole representative of Elopomorpha in Portuguese rivers. Although distance estimation revealed a genetic difference of only 0.4 % between Aurantiactinomyxon types 1 and 3, this value was determined to be representative of interspecific variability based on the consistent matching of both genotypes with distinct actinospore morphologies, and potential richness of closely related species of Paramyxidium infecting the European eel in Portuguese waters. The clustering of aurantiactinomyxon types within distinct myxosporean lineages, representative of the suborders Variisporina and Platysporina, demonstrates that the aurantiactinomyxon morphotype is highly functional in promoting myxozoan infections in estuarine environments.

Metarhabditis amsactae: A potential biopesticide isolated from Punjab (India) with potent insecticidal activity and immunomodulatory effects against Galleria mellonella (Lepidoptera: Pyralidae).

A survey was undertaken to isolate entomopathogenic nematodes from Amritsar district of Punjab, India. Out of 20 soil samples collected, two were found positive for the presence of nematodes. 18S and ITS rDNA gene sequencing revealed their identity as Metarhabditis amsactae. To assess its biocontrol potential, Galleria mellonella larvae were treated with concentrations of 20, 40, 80 and 160 IJs/L (infective juveniles/larva) and mortality was recorded from 24 h up to 96 h of nematode exposure. Distilled water without nematodes was used as an untreated control. M. amsactae showed potent larvicidal activity against G. mellonella that was found to be concentration and time dependent. Nematode infection caused 93.33 % larval mortality at 80 IJs/L after 72 h of treatment. 100 % mortality was observed after 96 h. No mortality was observed in control. To evaluate the immunomodulatory effects of M. amsactae, G. mellonella larvae were infected with 100 IJs/L and activities of antioxidant and detoxifying enzymes viz., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX), phenol oxidase (PO), glutathione-S-transferase (GST) and acetylcholine esterase (AChE) were appraised after 12, 24, 36 and 48 h of nematode exposure. Malondialdehyde content was also determined. The results obtained demonstrated a significant elevation in all the enzyme activities at all time intervals in treated larvae when compared with untreated control. MDA levels were also enhanced in response to nematode infection. Thus, the present study revealed high insecticidal potential and immunomodulatory effects of M. amsactae on G. mellonella that should be further explored on other insect pests as well.

Genetic variation of the freshwater snail Indoplanorbis exustus (Gastropoda: Planorbidae) in Thailand, inferred from 18S and 28S rDNA sequences.

Indoplanorbis exustus, a freshwater pulmonate snail, is widely distributed in tropical and subtropical zones and plays a significant role as an intermediate host for trematode parasites. Various genetic markers have been used for species identification and phylogenetic studies of this snail. However, there are limited studies about their molecular genetics based on nuclear ribosomal DNA (rDNA) genes. A genetic analysis of I. exustus in Thailand was conducted based on the nuclear 18S rDNA (339 bp) and 28S rDNA (1036 bp) genes. Indoplanorbis snails were collected from 29 localities in 21 provinces covering six regions of Thailand. Nucleotide sequences from 44 snails together with sequences from the GenBank database were examined for phylogenetic relationships and genetic diversity. All sequences of the selected nucleotide regions exhibited a high level of similarity (99%) to the sequences of I. exustus in the GenBank database. The maximum likelihood tree based on the 18S and 28S rDNA fragment sequences of I. exustus in Thailand revealed only one group with clear separation from another genus in the family Planorbidae. The I. exustus 28S rDNA sequences showed intraspecific genetic divergence ranging from 0 to 0.78% and were classified into 8 different haplotypes. Conversely, the 18S rDNA data showed lower variation than the 28S rDNA data and revealed a single haplotype and intraspecific distances of zero among all sampled individuals. The haplotype network of 28S rDNA sequences of I. exustus in Thailand revealed six unique haplotypes and two haplotypes shared by at least two regions. Overall, both markers were successful in the identification of I. exustus. However, these markers, particularly the 18S rDNA, may not be suitable for genetic analysis within the species, particularly for population genetic studies, due to their limited variation as seen in this study. In summary, this study not only enhances understanding of genetic variation in I. exustus but is also useful for the selection of molecular markers in future genetic research.

Ubiquitous arbuscular mycorrhizal fungi in the roots of herbaceous understory plants with hyphal degeneration in Colchicaceae and Gentianaceae.

Due to the loss of photosynthetic ability during evolution, some plant species rely on mycorrhizal fungi for their carbon source, and this nutritional strategy is known as mycoheterotrophy. Mycoheterotrophic plants forming Paris-type arbuscular mycorrhizas (AM) exhibit two distinctive mycorrhizal features: degeneration of fungal materials and specialization towards particular fungal lineages. To explore the possibility that some understory AM plants show partial mycoheterotrophy, i.e., both photosynthetic and mycoheterotrophic nutritional strategies, we investigated 13 green herbaceous plant species collected from five Japanese temperate forests. Following microscopic observation, degenerated hyphal coils were observed in four species: two Colchicaceae species, Disporum sessile and Disporum smilacinum, and two Gentianaceae species, Gentiana scabra and Swertia japonica. Through amplicon sequencing, however, we found that all examined plant species exhibited no specificity toward AM fungi. Several AM fungi were consistently found across most sites and all plant species studied. Because previous studies reported the detection of these AM fungi from various tree species in Japanese temperate forests, our findings suggest the presence of ubiquitous AM fungi in forest ecosystems. If the understory plants showing fungal degeneration exhibit partial mycoheterotrophy, they may obtain carbon compounds indirectly from a wide range of surrounding plants utilizing such ubiquitous AM fungi.

Description of Acromoldavicus xerophilus n. sp. (Nematoda, Rhabditida, Elaphonematidae) from the southern Iberian Peninsula, including a key to species of the genus.

A new species of the genus Acromoldavicus is described from coastal sand dunes and sandy soil in the southeast of the Iberian Peninsula. Acromoldavicus xerophilus n. sp. is characterized by its 557-700 µm body length, cuticle tessellated, lip region with three pairs of expanded lips bearing a large labial expansion, primary axils bearing guard processes with two different morphology, secondary axils lacking guard processes, stoma short and tubular with prostegostom bearing prominent rhabdia directed towards the stoma lumen, female reproductive system monodelphic-prodelphic, post-vulval sac 0.6-0.9 times body diameter, rectum very large, female tail short with biacute terminus and males unknown. The description, light micrographs, scanning electron microscope images, illustrations, and molecular analyses are provided. Molecular analyses (based on 18S and 28S rDNA) revealed its relationship with some species of the genera Cephalobus (18S tree), Nothacrobeles, Paracrobeles, and Spinocephalus (28S tree). Keys to species identification of this genus are also included.

First report of a Kudoa lutjanus outbreak in farmed Chicken Grunts Parapristipoma trilineatum.

OBJECTIVE: As part of the National Disease Surveillance Program for Taiwanese Aquaculture, we investigated the causative agent of disease outbreaks in farmed Chicken Grunts Parapristipoma trilineatum. METHODS: In this study, outbreak cases on two separate farms were noticed in coastal Pingtung County, Taiwan. In total, 50 juvenile fish showing clinical signs (such as emaciation and erratic swimming behavior) and broodstock (two females and two males) from both farms were collected to perform gross lesion assessment, histopathological examination, and molecular identification of the pathogen. RESULT: Clinical symptoms were infected fish exhibited erratic swimming behavior, such as whirling and floating on the surface of the water. In the following months, cumulative mortality had reached 19% and 24%, respectively. The gross lesions in the infected fish included white oval cysts in the muscle, serosa of the internal organs, sclera of the eyes, and cerebral meninges. After conducting a wet mount examination of cysts using a light microscope, we observed a significant quantity of spores with morphological characteristics, suggesting their affiliation with the Myxosporea group. The spores were semiquadrate, with four tiny suture notches at the periphery; the mean spore length was 7.3 µm (SD = 0.5), and the mean spore width was 8.2 µm (SD = 0.6). The mean length and width of the pyriform polar capsules (nematocysts) were 3.6 µm (SD = 0.5) and 2.2 µm (SD = 0.5), respectively. The 18S and 28S ribosomal RNA sequences of these specimens were identical to those of Kudoa lutjanus. CONCLUSION: As this was the first time an outbreak of K. lutjanus in Chicken Grunts was confirmed, its reappearance with substantial mortality should serve as a warning to the aquaculture industry.

Metabarcoding of protozoa and helminth in black-necked cranes: a high prevalence of parasites and free-living amoebae.

Parasites and free-living amoebae (FLA) are common pathogens that pose threats to wildlife and humans. The black-necked crane (Grus nigricollis) is a near-threatened species and there is a shortage of research on its parasite diversity. Our study aimed to use noninvasive methods to detect intestinal parasites and pathogenic FLA in G. nigricollis using high-throughput sequencing (HTS) based on the 18S rDNA V9 region. A total of 38 fresh fecal samples were collected in Dashanbao, China, during the overwintering period (early-, middle I-, middle II-, and late-winter). Based on the 18S data, eight genera of parasites were identified, including three protozoan parasites: Eimeria sp. (92.1%) was the dominant parasite, followed by Tetratrichomonas sp. (36.8%) and Theileria sp. (2.6%). Five genera of helminths were found: Echinostoma sp. (100%), Posthodiplostomum sp. (50.0%), Euryhelmis sp. (26.3%), Eucoleus sp. (50.0%), and Halomonhystera sp. (2.6%). Additionally, eight genera of FLA were detected, including the known pathogens Acanthamoeba spp. (n = 13) and Allovahlkampfia spp. (n = 3). Specific PCRs were used to further identify the species of some parasites and FLA. Furthermore, the 18S data indicated significant changes in the relative abundance and genus diversity of the protozoan parasites and FLA among the four periods. These results underscore the importance of long-term monitoring of pathogens in black-necked cranes to protect this near-endangered species.

Title: Métabarcoding des protozoaires et des helminthes chez les grues à cou noir : forte prévalence de parasites et d'amibes libres. Abstract: Les parasites et les amibes libres sont des agents pathogènes courants qui constituent une menace pour la faune et les humains. La grue à cou noir (Grus nigricollis) est une espèce quasi menacée et les recherches sur sa diversité parasitaire sont insuffisantes. Notre étude visait à utiliser des méthodes non invasives pour détecter les parasites intestinaux et les amibes libres pathogènes chez G. nigricollis en utilisant le séquençage à haut débit basé sur la région V9 de l'ADNr 18S. Au total, 38 échantillons de matières fécales fraîches ont été collectés à Dashanbao, en Chine, au cours de la période d'hivernage (début, milieu I, milieu II et fin de l'hiver). Sur la base des données 18S, huit genres de parasites ont été identifiés, dont trois parasites protozoaires : Eimeria sp. (92,1 %) était le parasite dominant, suivi de Tetratrichomonas sp. (36,8 %) et Theileria sp. (2,6 %). Cinq genres d'helminthes ont été trouvés : Echinostoma sp. (100 %), Posthodiplostomum sp. (50,0 %), Euryhelmis sp. (26,3 %), Eucoleus sp. (50,0 %) et Halomonhystera sp. (2,6 %). De plus, huit genres d'amibes libres ont été détectés, y compris les agents pathogènes connus Acanthamoeba spp. (n = 13) et Allovahlkampfia spp. (n = 3). Des PCR spécifiques ont été utilisées pour identifier davantage les espèces de certains parasites et amibes libres. En outre, les données 18S ont indiqué des changements significatifs dans l'abondance relative et la diversité des genres des parasites protozoaires et des amibes au cours des quatre périodes. Ces résultats soulignent l'importance de la surveillance à long terme des agents pathogènes chez les grues à cou noir pour protéger cette espèce quasi menacée.

18S rDNA sequence-structure phylogeny of the eukaryotes simultaneously inferred from sequences and their individual secondary structures.

OBJECTIVE: The eukaryotic tree of life has been subject of numerous studies ever since the nineteenth century, with more supergroups and their sister relations being decoded in the last years. In this study, we reconstructed the phylogeny of eukaryotes using complete 18S rDNA sequences and their individual secondary structures simultaneously. After the sequence-structure data was encoded, it was automatically aligned and analyzed using sequence-only as well as sequence-structure approaches. We present overall neighbor-joining trees of 211 eukaryotes as well as the respective profile neighbor-joining trees, which helped to resolve the basal branching pattern. A manually chosen subset was further inspected using neighbor-joining, maximum parsimony, and maximum likelihood analyses. Additionally, the 75 and 100 percent consensus structures of the subset were predicted. RESULTS: All sequence-structure approaches show improvements compared to the respective sequence-only approaches: the average bootstrap support per node of the sequence-structure profile neighbor-joining analyses with 90.3, was higher than the average bootstrap support of the sequence-only profile neighbor-joining analysis with 73.9. Also, the subset analyses using sequence-structure data were better supported. Furthermore, more subgroups of the supergroups were recovered as monophyletic and sister group relations were much more comparable to results as obtained by multi-marker analyses.

Morphology, morphogenesis, and molecular phylogeny of Aspidisca koreana n. sp. (Ciliophora, Euplotida) from South Korea.

The morphology, morphogenesis, and molecular phylogeny of a new ciliate, Aspidisca koreana n. sp., discovered in the eastern coast of South Korea, were investigated. The morphological description is based on the observation of living cells, 4'-6-diamidino-2-phenylindole (DAPI) and silver-stained specimens (e.g., protargol, silver nitrate), and scanning electron micrographs. The new species is characterized by having a small body size (17-25 × 15-18 µm in vivo), a distinct peristomial spur on the posterior portion of left margin, seven frontoventral cirri in "polystyla-arrangement", and the arrangement of the anterior portion of adoral zone of membranelles, i.e., anteriormost membranelle is distinctly separated from the other three membranelles. The morphogenesis follows the typical pattern of this genus. Phylogenetic analyses, using the 18S rDNA sequence, also support the establishment of a new species.

Use of PCR-DGGE-Based Molecular Methods to Analyze Nematode Community Diversity.

DGGE (denaturing gradient gel electrophoresis) is a nucleic acid separation technique applied to the evaluation of microbial biodiversity. This technique is quite rapid and cheap compared to other types of analysis. Here we describe the comparison of nematode communities inhabiting different ecosystems. After an ecologically representative sampling collection and the nematode extraction from soil, nematodes are centrifuged in Eppendorf tubes to facilitate DNA extraction. DNA from the whole community of each type of soil is extracted, amplified with primers for 18 S rDNA and used in DGGE analysis. The profiles of DGGE can be analyzed with appropriate software, and biodiversity indices can be estimated.

DNA barcodes reliably differentiate between nivicolous species of Diderma (Myxomycetes, Amoebozoa) and reveal regional differences within Eurasia.

The nivicolous species of the genus Diderma are challenging to identify, and there are several competing views on their delimitation. We analyzed 102 accessions of nivicolous Diderma spp. that were sequenced for two or three unlinked genes to determine which of the current taxonomic treatments is better supported by molecular species delimitation methods. The results of a haplotype web analysis, Bayesian species delimitation under a multispecies coalescent model, and phylogenetic analyses on concatenated alignments support a splitting approach that distinguishes six taxa: Diderma alpinum, D. europaeum, D. kamchaticum, D. meyerae, D. microcarpum and D. niveum. The first two approaches also support the separation of Diderma alpinum into two species with allopatric distribution. An extended dataset of 800 specimens (mainly from Europe) that were barcoded with 18S rDNA revealed only barcode variants similar to those in the species characterized by the first data set, and showed an uneven distribution of these species in the Northern Hemisphere: Diderma microcarpum and D. alpinum were the only species found in all seven intensively sampled mountain regions. Partial 18S rDNA sequences serving as DNA barcodes provided clear signatures that allowed for unambiguous identification of the nivicolous Diderma spp., including two putative species in D. alpinum.

Description, life cycle, and development of the myxozoan Myxobolus rasmusseni n. sp. in fathead minnows, Pimephales promelas: A possible emerging pathogen in southern Alberta, Canada.

Morphological, gene sequence, host tissue tropism, and life cycle characteristics were utilized to describe the myxozoan, Myxobolus rasmusseni n. sp. from fathead minnow, Pimephales promelas, collected from reservoirs in southern Alberta. Results from serial histological sections of whole heads showed that myxospores were contained within irregular-shaped and sized coelozoic capsules (=plasmodia). Clusters of membrane-bound, myxospore-filled plasmodia filled the head cavities of juvenile fathead minnows, leading to the development of large, white, disfiguring lesions in mid to late summer. Bilateral exopthalmia (pop-eye disease) was a common outcome of M. rasmusseni n. sp. development. BLASTn search of a 1974 bp sequence of the 18S rDNA gene isolated from myxospores indicated that M. rasmusseni n. sp. was distinct from other coelozoic and histozoic Myxobolus spp. cataloged in GenBank. 18S rDNA gene sequences from triactinomyxon spores released from the oligochaete Tubifex were 100% identical to sequences from myxospores collected from syntopic fathead minnows. Results from a longitudinal survey of the 2020 cohort of fathead minnows showed that young-of-the-year are exposed at 1-5 mo and that 60-90% of these had developed myxospore-filled lesions approximately one year later. Data regarding potential sources and timing of M. rasmusseni n. sp. emergence in fathead minnow populations are needed.

Integrative taxonomic study of mononchid nematodes from riparian habitats in Bulgaria. I. Genera Mononchus Bastian, 1865 and Coomansus Jairajpuri & Khan, 1977 with the description of Mononchuspseudoaquaticus sp. nov. and a key to the species of Mononchus.

The species diversity of the genera Mononchus Bastian, 1865 and Coomansus Jairajpuri & Khan, 1977 was assessed in a study of the mononchid nematodes from a wide range of riparian habitats in Bulgaria. Four species were identified based on morphological and morphometric data: Coomansusparvus (de Man, 1880), Mononchustruncatus Bastian, 1865, Mononchuspseudoaquaticus sp. nov., and Mononchus sp. The first three species were characterised both morphologically and molecularly (18S and 28S rRNA gene sequences) and the integration of these data and phylogenetic analyses provided support for their distinct species status. This paper provides detailed descriptions, morphometric data for multiple species populations, drawings and photomicrographs, and the first taxonomically verified sequences for C.parvus (n = 6), M.truncatus (sensu stricto) (n = 4) and M.pseudoaquaticus sp. nov. (n = 3). Comparative sequence and phylogenetic analyses suggested that the utility of the 18S rRNA gene for species delimitation is rather limited at least for some species complexes within the genus Mononchus. At the generic and suprageneric level, the 18S and 28S rDNA phylogenies both recovered the three genera represented by two or more species (Mononchus, Mylonchulus, and Parkellus) as monophyletic with strong support, the Mononchidae as paraphyletic, the Anatonchidae as monophyletic, and there was no support for a sister-group relationship between Mylonchulus and Mononchus. A key to the species of Mononchus is provided to facilitate the identification of the currently recognised 31 species.

Biodiversity of autotrophic euglenids based on the group specific DNA metabarcoding approach.

This study reports a comprehensive analysis of photoautotrophic euglenids' distribution and biodiversity in 16 small water bodies of various types (including fish ponds, field ponds, rural ponds and park ponds) located in three regions of Poland: Masovia, Masuria and Pomerania during a period of three years. By employing a euglenid specific barcode marker and a curated database of V2 18S rDNA sequences it was possible to identify 97.7 % of euglenid reads at species level. A total of 152 species classified in 13 genera were identified. The number of euglenid species found in one pond varied from 40 to 102. The most common species were Euglena agilis and Euglenaria caudata, found in every analysed waterbody. The highest number of observed species belonged to Trachelomonas and Phacus. Certain species exhibited a tendency to coexist, suggesting the presence of distinct species assemblages. Among them, the most distinctive cluster was associated with water bodies located in the Masuria region, characterized also by the greatest species richness, including many very rare species: Euglenaformis chlorophoenicea, Lepocinclis autumnalis, L. marssonii, Trachelomonas eurystoma, T. manschurica, T. mucosa, T. zuberi, T. zuberi var. nepos.

18S and ITS2 rRNA gene sequence-structure phylogeny of the Phaeophyceae (SAR, Stramenopiles) with special reference to Laminariales.

The phylogeny of brown algae (Phaeophyceae) has undergone extensive changes in the recent past due to regular new scientific insights. We used nuclear 18S rDNA with an extensive dataset, aiming to increase the accuracy and robustness of the reconstructed phylogenetic trees using a simultaneous sequence-structure approach. Individual secondary structures were generated for all 18S rDNA sequences. The sequence-structure information was encoded and used for an automated simultaneous sequence-structure alignment. Neighbor-joining and profile neighbor-joining trees were calculated based on 186 phaeophycean sequence-structure pairs. Additionally, sequence-structure neighbor-joining, maximum parsimony and maximum likelihood trees were reconstructed on a representative subset. Using a similar approach, ITS2 rDNA sequence-structure information was used to reconstruct a neighbor-joining tree including 604 sequence-structure pairs of the Laminariales. Our study results are in significant agreement with previous single marker 18S and ITS2 rDNA analyses. Moreover, the 18S results are in wide agreement with recent multi-marker analyses. The bootstrap support was significantly higher for our sequence-structure analysis in comparison to sequence-only analyses in this study and the available literature. This study supports the simultaneous inclusion of sequence-structure data at least for 18S to obtain more accurate and robust phylogenetic trees compared to sequence-only analyses.