Morphological Identities of Two Different Marine Stramenopile Environmental Sequence Clades: Bicosoeca kenaiensis (Hilliard, 1971) and Cantina marsupialis (Larsen and Patterson, 1990) gen. nov., comb. nov.

Although environmental DNA surveys improve our understanding of biodiversity, interpretation of unidentified lineages is limited by the absence of associated morphological traits and living cultures. Unidentified lineages of marine stramenopiles are called “MAST clades”. Twenty‐five MAST clades have been recognized: MAST‐1 through MAST‐25; seven of these have been subsequently discarded because the sequences representing those clades were found to either (1) be chimeric or (2) affiliate within previously described taxonomic groups. Eighteen MAST clades remain without a cellular identity. Moreover, the discarded “MAST‐13” has been used in different studies to refer to two different environmental sequence clades. After establishing four cultures representing two different species of heterotrophic stramenopiles and then characterizing their morphology and molecular phylogenetic positions, we determined that the two different species represented the two different MAST‐13 clades: (1) a lorica‐bearing Bicosoeca kenaiensis and (2) a microaerophilic flagellate previously named “Cafeteria marsupialis”. Both species were previously described with only light microscopy; no cultures, ultrastructural data or DNA sequences were available from these species prior to this study. The molecular phylogenetic position of three different “C. marsupialis” isolates was not closely related to the type species of Cafeteria; therefore, we established a new genus for these isolates, Cantina gen. nov.     

An ITS-based phylogenetic framework for the genus Vorticella: finding the molecular and morphological gaps in a taxonomically difficult group

Vorticella includes more than 100 currently recognized species and represents one of the most taxonomically challenging genera of ciliates. Molecular phylogenetic analysis of Vorticella has been performed so far with only sequences coding for small subunit ribosomal RNA (SSU rRNA); only a few of its species have been investigated using other genetic markers owing to a lack of similar sequences for comparison. Consequently, phylogenetic relationships within the genus remain unclear, and molecular discrimination between morphospecies is often difficult because most regions of the SSU rRNA gene are too highly conserved to be helpful. In this paper, we move molecular systematics for this group of ciliates to the infrageneric level by sequencing additional molecular markers—fast-evolving internal transcribed spacer (ITS) regions—in a broad sample of 66 individual samples of 28 morphospecies of Vorticella collected from Asia, North America and Europe. Our phylogenies all featured two strongly supported, highly divergent, paraphyletic clades (I, II) comprising the morphologically defined genus Vorticella. Three major lineages made up clade I, with a relatively well-resolved branching order in each one. The marked divergence of clade II from clade I confirms that the former should be recognized as a separate taxonomic unit as indicated by SSU rRNA phylogenies. We made the first attempt to elucidate relationships between species in clade II using both morphological and multi-gene approaches, and our data supported a close relationship between some morphospecies of Vorticella and Opisthonecta, indicating that relationships between species in the clade are far more complex than would be expected from their morphology. Different patterns of helix III of ITS2 secondary structure were clearly specific to clades and subclades of Vorticella and, therefore, may prove useful for resolving phylogenetic relationships in other groups of ciliates.     

Systematics of Cladophora spp. (Chlorophyta) from North Carolina,USA, based upon morphology and DNA sequence data with a description of Cladophora subtilissima sp. nov.

Identification of Cladophora species is challenging due to conservation of gross morphology, few discrete autapomorphies, and environmental influences on morphology. Twelve species of marine Cladophora were reported from North Carolina waters. Cladophora specimens were collected from inshore and offshore marine waters for DNA sequence and morphological analyses. The nuclear‐encoded rRNA internal transcribed spacer regions (ITS) were sequenced for 105 specimens and used in molecular assisted identification. The ITS1 and ITS2 region was highly variable, and sequences were sorted into ITS Sets of Alignable Sequences (SASs). Sequencing of short hyper‐variable ITS1 sections from Cladophora type specimens was used to positively identify species represented by SASs when the types were made available. Secondary structures for the ITS1 locus were also predicted for each specimen and compared to predicted structures from Cladophora sequences available in GenBank. Nine ITS SASs were identified and representative specimens chosen for phylogenetic analyses of 18S and 28S rRNA gene sequences to reveal relationships with other Cladophora species. Phylogenetic analyses indicated that marine Cladophorales were polyphyletic and separated into two clades, the Cladophora clade and the “Siphonocladales” clade. Morphological analyses were performed to assess the consistency of character states within species, and complement the DNA sequence analyses. These analyses revealed intra‐ and interspecific character state variation, and that combined molecular and morphological analyses were required for the identification of species. One new report, Cladophora dotyana, and one new species Cladophora subtilissima sp. nov., were revealed, and increased the biodiversity of North Carolina marine Cladophora to 14 species.     

Interrelationships of the Gastrotricha and their place among the Metazoa inferred from 18S rRNA genes

The phylum Gastrotricha includes about 700 species. They are small worm‐like organisms abundant among marine and freshwater meiobenthos. In spite of their ubiquity, diversity and relative abundance, phylogenetic relationships of these animals remain enigmatic due to the conflicting results of morphological and molecular cladistic analyses. Also unclear are the alliances within the phylum. In order to best estimate the position of Gastrotricha among the Metazoa and to shed some light on the ingroup phylogenetic relationships, small subunit (SSU) ribosomal DNA (rDNA) from 15 species of Chaetonotida (eight genera) and 28 species of Macrodasyida (26 genera) were included in an alignment of 50 metazoan taxa representing 26 phyla. Of the gastrotrich SSU rDNA sequences, eight are new and, along with published sequences represent eight families, including the five marine most speciose. Gastrotricha were resolved within a monophyletic Lophotrochozoa as part of a clade including Micrognathozoa, Rotifera and Cycliophora. The Gnathostomulida were sister to this clade. Nodal support was low for all of these relationships except the grouping of the Micrognathozoa, Rotifera and Cycliophora. Bayesian inference resolved the Gastrotricha as monophyletic with weak nodal support; the Macrodasyida were resolved as paraphyletic with many basal nodes poorly supported. Within the Chaetonotida, the monotypic Multitubulatina Neodasys was found in alliance with the macrodasyidan Urodasys while all the Paucitubulatina were found to form a single, well‐supported clade, with Musellifer as the most basal member. Among the more densely sampled Macrodasyida the Lepidodasyidae and Macrodasyidae were each found to be polyphyletic while monophyly was well supported for the Turbanellidae and Thaumastodermatidae. The congruence of our results with those of the cladistic analysis based on morphological traits provides confidence about the value of each dataset, and calls for widening of the research to include additional taxa of particular phylogenetic significance such as the Dactylopodolidae, Diuronotus, Heteroxenotrichula and Draculiciteria. The study highlights the problems in working with small species, the need for voucher specimens and the confused taxonomic status and membership of various gastrotrich families.     

Molecular data and the evolutionary history of dinoflagellates

We have sequenced small-subunit (SSU) ribosomal RNA (rRNA) genes from 16 dinoflagellates, produced phylogenetic trees of the group containing 105 taxa, and combined small- and partial large-subunit (LSU) rRNA data to produce new phylogenetic trees. We compare phylogenetic trees based on dinoflagellate rRNA and protein genes with established hypotheses of dinoflagellate evolution based on morphological data. Protein-gene trees have too few species for meaningful in-group phylogenetic analyses, but provide important insights on the phylogenetic position of dinoflagellates as a whole, on the identity of their close relatives, and on specific questions of evolutionary history. Phylogenetic trees obtained from dinoflagellate SSU rRNA genes are generally poorly resolved, but include by far the most species and some well-supported clades. Combined analyses of SSU and LSU somewhat improve support for several nodes, but are still weakly resolved. All analyses agree on the placement of dinoflagellates with ciliates and apicomplexans (=Sporozoa) in a well-supported clade, the alveolates. The closest relatives to dinokaryotic dinoflagellates appear to be apicomplexans, Perkinsus, Parvilucifera, syndinians and Oxyrrhis. The position of Noctiluca scintillans is unstable, while Blastodiniales as currently circumscribed seems polyphyletic. The same is true for Gymnodiniales: all phylogenetic trees examined (SSU and LSU-based) suggest that thecal plates have been lost repeatedly during dinoflagellate evolution. It is unclear whether any gymnodinialean clades originated before the theca. Peridiniales appear to be a paraphyletic group from which other dinoflagellate orders like Prorocentrales, Dinophysiales, most Gymnodiniales, and possibly also Gonyaulacales originated. Dinophysiales and Suessiales are strongly supported holophyletic groups, as is Gonyaulacales, although with more modest support. Prorocentrales is a monophyletic group only in some LSU-based trees. Within Gonyaulacales, molecular data broadly agree with classificatory schemes based on morphology. Implications of this taxonomic scheme for the evolution of selected dinoflagellate features (the nucleus, mitosis, flagella and photosynthesis) are discussed.     

Description of Paratetrahymena parawassi n. sp. using Morphological and Molecular Evidence and a Phylogenetic Analysis of Paratetrahymena and Other Taxonomically Ambiguous Genera in the Order Loxocephalida (Ciliophora,Oligohymenophorea)

ABSTRACT. The marine scuticociliate Paratetrahymena parawassi n. sp. is described on the basis of morphology, especially infraciliature, and the sequence of its small subunit (SSU) rRNA gene to become the second known member of its genus. Paratetrahymena and other ciliates in the order Loxocephalida possess a mixture of morphological and morphogenetic features characteristic of the subclasses Hymenostomatia and Scuticociliatia. Accordingly, we used SSU rRNA sequences to analyze the phylogeny of Paratetrahymena and three other loxocephalid genera. Paratetrahymena and Cardiostomatella vermiformis formed a moderately well‐supported clade that diverged at a deep level from all other scuticociliates, supporting separation of loxocephalids from other scuticociliates as a suprafamilial taxon. Sathrophilus holtae was a sister taxon to Paratetrahymena and Cardiostomatella in a poorly supported, unresolved relationship; nevertheless, association of all three genera into a single clade was supported by an approximately unbiased (AU) test. Any association of these genera singly or as a group with the Hymenostomatia was rejected decisively by AU tests and by a complete absence in the loxocephalids of the unique nucleotide identities that distinguish hymenostomes. Therefore, the morphological and morphogenetic similarities of loxocephalids to hymenostomes may be plesiomorphies, and the conflicting mix of scuticociliate and hymenostome characteristics seen in loxocephalids may result from differing rates of character evolution. Dexiotrichides pangi and Urocentrum, which is currently classified as a peniculid, formed a small clade that associated with hymenostomes and peritrichs. Monophyly of the Loxocephalida with Dexiotrichides and/or Urocentrum included was not rejected by AU; however, inclusion of Urocentrum in the Peniculia was rejected by AU tests. A hypothesis is offered to explain the lack of resolution of loxocephalid ciliates and Urocentrum in phylogenetic trees, namely that their phylogenetic positions are influenced by a combination of heterogeneous data and long‐branch attraction caused by poor representation of taxa in analyses. The well‐known genus Cyclidium, a member of the order Pleuronematida, was revealed to be polyphyletic as a byproduct of our analyses of loxocephalids. In particular, Cyclidium porcatum appears to fall outside the clade containing typical members of the subclass Scuticociliatia and thus invites investigation as a possible member of the order Loxocephalida.     

Description of a New Freshwater Ciliate Epistylis wuhanensis n. sp. (Ciliophora,Peritrichia) from China,with a Focus on Phylogenetic Relationships within Family Epistylididae

Two populations of Epistylis wuhanensis n. sp., a new freshwater peritrich ciliate, were isolated from different freshwater ponds located in Hubei, China. Their morphological characteristics were investigated using live observation, protargol impregnation, and scanning electron microscopy (SEM). Specimens from the two populations showed identical arrangement of the infraciliature and identical small subunit ribosomal RNA (SSU rRNA) gene and ITS1‐5.8S‐ITS2 sequences. The zooids present bell‐shaped and 90–175 × 27–54 μm in vivo. Macronucleus is variable in shape and located in the middle of cell. Pellicle is usually smooth with 139–154 and 97–105 striations above and below the trochal band, respectively. SSU rRNA gene and ITS1‐5.8S‐ITS2 sequences of E. wuhanensis n. sp. did not match any available sequences in GenBank. Phylogenetically, E. wuhanensis n. sp. clusters with the other Epistylis within the family Epistylididae, but is distinct from the major clades of Epistylis. Above all, the morphological characteristics and molecular analyses support that the present Epistylis is a new species. Expanded phylogenetic analyses of sessilids based on both SSU rRNA gene sequences and ITS1‐5.8S‐ITS2 sequences reveal that the genus Epistylis consists of Epistylis morphospecies and taxonomic revision of the genus is needed.     

UPDATING THE GENUS DICTYOSPHAERIUM AND DESCRIPTION OF MUCIDOSPHAERIUM GEN. NOV. (TREBOUXIOPHYCEAE) BASED ON MORPHOLOGICAL AND MOLECULAR DATA1

Recent molecular analyses of Dictyosphaerium strains revealed a polyphyletic origin of this morphotype within the Chlorellaceae. The type species Dictyosphaerium ehrenbergianum Nägeli formed an independent lineage within the Parachlorella clade, assigning the genus to this clade. Our study focused on three different Dictyosphaerium species to resolve the phylogenetic position of remaining species. We used combined analyses of morphology; molecular data based on SSU and internally transcribed spacer region (ITS) rRNA sequences; and the comparison of the secondary structure of the SSU, ITS‐1, and ITS‐2 for species and generic delineation. The phylogenetic analyses revealed two lineages without generic assignment and two distinct clades of Dictyosphaerium‐like strains within the Parachlorella clade. One clade comprises the lineages with the epitype strain of D. ehrenbergianum Nägeli and two additional lineages that are described as new species (Dictyosphaerium libertatis sp. nov. and Dictyosphaerium lacustre sp. nov.). An emendation of the genus Dictyosphaerium is proposed. The second clade comprises the species Dictyosphaerium sphagnale Hindák and Dictyosphaerium pulchellum H. C. Wood. On the basis of phylogenetic analyses, complementary base changes, and morphology, we describe Mucidosphaerium gen. nov with the four species Mucidosphaerium sphagnale comb. nov., Mucidosphaerium pulchellum comb. nov., Mucidosphaerium palustre sp. nov., and Mucidosphaerium planctonicum sp. nov.     

MITOCHONDRIAL DNA PHYLOGENY OF THE SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM,DINOPHYTA)1

Symbiotic dinoflagellates belonging to the genus Symbiodinium (Freudenthal) are found worldwide in association with shallow‐water tropical and subtropical marine invertebrates. Most phylogenetic studies of Symbiodinium have used nuclear rRNA (nrDNA) genes to infer relationships among members of the genus. In this report, we present the first phylogeny of Symbiodinium based on DNA sequences from a mitochondrial protein‐coding gene (cytochrome oxidase subunit I [cox1]). Two principal groups, one comprised of Symbiodinium clade A and the second encompassing Symbiodinium clades B/C/D/E/F, are strongly supported in the cox1 phylogeny. Relationships within Symbiodinium clades B/C/D/E/F, however, are less well resolved compared with phylogenies inferred from nrDNA and chloroplast large subunit (cp23S)‐rDNA genes. Statistical tests between alternative tree topologies verified, with an exception being the position of one controversial member of Symbiodinium clade D, that relationships inferred from cox1 are congruent with those inferred from nrDNA and cp23S‐rDNA. Taken together, the relationships between the major Symbiodinium clades are robust, and there appears to be no evidence of hybridization or differential introgression of nuclear and plastid genomes between clades.     

Molecular and morphological characterization of a poorly known marine ciliate, Myoschiston duplicatum precht 1935: implications for phylogenetic relationships between three morphologically similar genera -- Zoothamnium, Myoschiston, and Zoothamnopsis (Ciliophora, Peritrichia, Zoothamniidae)

We studied the morphology and molecular phylogeny of Myoschiston duplicatum, a peritrich ciliate that has been recorded as an epibiont of crustaceans, but which we also identified on marine algae from Korea. The important morphological characteristics revealed by silver staining of Myoschiston species have not been described because they are rarely collected. Using morphological methods, we redescribed the type species of the genus, Myoschiston duplicatum, and provided an improved diagnosis of Myoschiston. In addition, the coding regions for nuclear small subunit (SSU) rRNA and internal transcribed spacer 1‐5.8S‐internal transcribed spacer 2 sequences were sequenced. Phylogenetic analyses that included available SSU rDNA sequences of peritrichs from GenBank strongly supported a position of M. duplicatum within the family Zoothamniidae. In addition, phylogenetic analyses were performed with single datasets (ITS1‐5.8S‐ITS2) and combined datasets (SSU rDNA + ITS1‐5.8S‐ITS2) to explore further the phylogenetic relationship in the family Zoothamniidae between the three morphologically similar genera—Zoothamnium, Myoschiston, and Zoothamnopsis.     

Molecular diversity of the syndinean genus Euduboscquella based on single-cell PCR analysis

The genus Euduboscquella is one of a few described genera within the syndinean dinoflagellates, an enigmatic lineage with abundant diversity in marine environmental clone libraries based on small subunit (SSU) rRNA. The region composed of the SSU through to the partial large subunit (LSU) rRNA was determined from 40 individual tintinnid ciliate loricae infected with Euduboscquella sampled from eight surface water sites in the Northern Hemisphere, producing seven distinct SSU sequences. The corresponding host SSU rRNA region was also amplified from eight host species. The SSU tree of Euduboscquella and syndinean group I sequences from environmental clones had seven well-supported clades and one poorly supported clade across data sets from 57 to 692 total sequences. The genus Euduboscquella consistently formed a supported monophyletic clade within a single subclade of group I sequences. For most parasites with identical SSU sequences, the more variable internal transcribed spacer (ITS) to LSU rRNA regions were polymorphic at 3 to 10 sites. However, in E. cachoni there was variation between ITS to LSU copies at up to 20 sites within an individual, while in a parasite of Tintinnopsis spp., variation between different individuals ranged up to 19 polymorphic sites. However, applying the compensatory base change model to the ITS2 sequences suggested no compensatory changes within or between individuals with the same SSU sequence, while one to four compensatory changes between individuals with similar but not identical SSU sequences were found. Comparisons between host and parasite phylogenies do not suggest a simple pattern of host or parasite specificity.     

Distribution patterns of marine planktonic cyanobacterial assemblages in transitional marine habitats using 16S rRNA phylogeny

The community composition of marine planktonic cyanobacteria in transitional marine habitats can influence its overall contribution to aquatic primary production. To understand distribution patterns of marine planktonic cyanobacterial assemblages, phylogenetic and statistical analyses were undertaken on planktonic cyanobacterial 16S rRNA gene sequences from four transitional marine habitats [Baltic Sea (BL), Monterey Bay (MB), South China Sea (SCS) and Sundarbans (SB)]. Out of 3255 sequences analyzed, only 546 sequences were found to be planktonic cyanobacteria and were considered in this study. Among these, 338 sequences representative of Sundarbans, the world's largest mangrove were generated based on Sanger and Illumina sequencing approaches. Based on 16S rRNA phylogeny, four major taxonomic orders of marine planktonic cyanobacteria were recovered in varying proportions with several novel 16S rRNA sequences in each of the four targeted sites. Members of the order Synechococcales were dominant in all the sites (?94% sequences) while the orders Chroococcales and Oscillatoriales were only detected in SB and SCS sites, respectively. In the phylogenetic tree, sequences representing the major marine picocyanobacterial genus Synechococcus showed overwhelming dominance in SB and they were found in three other sites. Prochlorococcus ‐like sequences were found in sizeable number in MB and SCS but were absent in SB and coastal BL. Synechococcus ‐like sequences were represented by three major marine clusters (5.1, 5.2, and 5.3). Three novel clades as part of Synechococcus cluster were detected only in SB and one novel clade in BL. The majority of OTUs were found to be exclusive to each site, whereas some were shared by two or more sites as revealed by beta‐diversity analysis.     

EVIDENCE FOR LATERAL TRANSFER OF AN IE INTRON BETWEEN FUNGAL AND RED ALGAL SMALL SUBUNIT RRNA GENES1

A previous study of the North American biogeography of the red algal genus Hildenbrandia noted the presence of group I introns in the nuclear small subunit (SSU) rRNA gene of the marine species H. rubra (Sommerf.) Menegh. Group IC1 introns have been previously reported at positions 516 and 1506 in the nuclear SSU RNA genes in the Bangiales and Hildenbrandiales. However, the presence of an unclassified intron at position 989 in a collection of H. rubra from British Columbia was noted. This intron is a member of the IE subclass and is the first report of this intron type in the red algae. Phylogenetic analyses of the intron sequences revealed a close relationship between this IE intron inserted at position 989 and similar fungal IE introns in positions 989 and 1199. The 989 IE introns formed a moderately to well‐supported clade, whereas the 1199 IE introns are weakly supported. Unique structural helices in the P13 domain of the 989 and 1199 IE introns also point to a close relationship between these two clades and provide further evidence for the value of secondary structural characteristics in identifying homologous introns in evolutionarily divergent organisms. The absence of the 989 IE intron in all other red algal nuclear SSU rRNA genes suggests that it is unlikely that this intron was vertically inherited from the common ancestor of the red algal and fungal lineages but rather is the result of lateral transfer between fungal and red algal nuclear SSU rRNA genes.     

MOLECULAR PHYLOGENY OF SELECTED MEMBERS OF THE ORDER THALASSIOSIRALES (BACILLARIOPHYTA) AND EVOLUTION OF THE FULTOPORTULA1

Recent phylogenetic studies of the diatoms indicate that members of the order Thalassiosirales occupy an interesting position in the diatom evolutionary tree. Despite their radial morphology and scaly auxospores, they are consistently recovered in molecular analyses as a member of subdivision Bacillariophytina and a sister clade to non‐fultoportulate and non‐radial lithodesmioids. This study included 46 species from nine traditionally accepted extant genera, and analyzed 43 nuclear small subunit (SSU) rRNA sequences in parallel with a survey of the variation in fultoportula structure. Three possible scenarios leading to the evolution of the fultoportula are discussed in the context of molecular and morphological similarities between the examined Thalassiosirales and their SSU rRNA sister clade Lithodesmiales. We speculate that the fultoportula might be derived by a modification of either a cribrum in an areola (fultoportula within an areola), or structures similar to marginal ridges now seen in lithodesmioids around a cluster of poroids (fultoportula in a tube), or finally, that the central fultoportula may have an origin different from the marginal fultoportulae. Our data confirm that fultoportula‐bearing diatoms constitute a natural phylogenetic group. The families Thalassiosiraceae, Skeletonemaceae, and Stephanodiscaceae and the genus Thalassiosira Cleve were unexpectedly found to be paraphyletic. Further, Cyclotella Kutz. and Stephanodiscus Ehr. may not be closely related and some species of these genera are more closely allied to other species of Thalassiosira. The generitype, T. nordenskioeldii, is embedded within a large poorly structured cluster of species that includes several members of Thalassiosira, Planktoniella sol, Minidiscus trioculatus, and two members of Stephanodiscus. An emendment of the order Lithodesmiales and the family Lauderiaceae are proposed.     

Phylogeny,Evidence for a Cryptic Plastid,and Distribution of Chytriodinium Parasites (Dinophyceae) Infecting Copepods

Spores of the dinoflagellate Chytriodinium are known to infest copepod eggs causing their lethality. Despite the potential to control the population of such an ecologically important host, knowledge about Chytriodinium parasites is limited: we know little about phylogeny, parasitism, abundance, or geographical distribution. We carried out genome sequence surveys on four manually isolated sporocytes from the same sporangium, which seemed to be attached to a copepod nauplius, to analyze the phylogenetic position of Chytriodinium based on SSU and concatenated SSU/LSU rRNA gene sequences, and also characterize two genes related to the plastidial heme pathway, hemL and hemY. The results suggest the presence of a cryptic plastid in Chytriodinium and a photosynthetic ancestral state of the parasitic Chytriodinium/Dissodinium clade. Finally, by mapping Tara Oceans V9 SSU amplicon data to the recovered SSU rRNA gene sequences from the sporocytes, we show that globally, Chytriodinium parasites are most abundant within the pico/nano‐ and mesoplankton of the surface ocean and almost absent within microplankton, a distribution indicating that they generally exist either as free‐living spores or host‐associated sporangia.     

Cultivation and Characterisation of New Species of Apusomonads (the Sister Group to Opisthokonts), Including Close Relatives of Thecamonas (Chelonemonas n. gen.)

Apusomonads comprise an understudied and undersampled group of heterotrophic flagellates that is closely related to opisthokonts, the supergroup containing animals and fungi. We cultured representatives of a new clade of apusomonads, Chelonemonas n. gen., which is sister to marine forms of Thecamonas in SSU rRNA gene phylogenies. Scanning electron microscopy shows that members of Chelonemonas have a hexagonal patterning to their submembranous pellicle, which is not known to exist in other apusomonads. We propose that the subfamily Thecamonadinae refer to the marine Thecamonas/Chelonomonas clade. We also report two new strains of Multimonas, one of which is genetically divergent from previously described strains, and here described as a new species, Multimonas koreensis. Both strains of Multimonas have appendages on their dorsal surface that could be extrusomes, and a frilled appearance to the border of their pellicle. Explorations of taxon sampling in SSU rRNA gene phylogenies confirm the new strains' evolutionary affinities, but do not resolve relationships among the five main apusomonad clades. These phylogenies also separate the freshwater species “Thecamonas” oxoniensis from the marine members of the genus Thecamonas. The new strains described here may provide valuable genetic and morphological data for evaluating the relationships and evolution of apusomonads.     

Evolutionary Relationships Among Old World Fruitbats (Megachiroptera: Pteropodidae) Based on 12S rRNA, tRNA Valine, and 16S rRNA Gene Sequences

Old World fruitbats were divided into the cynopterine, epomophorine, rousettine, eonycterine, and notopterine sections by Knud Andersen (1912). Among these, the eonycterine and notopterine sections together comprise the subfamily Macroglossinae, which includes forms with specializations for nectarivory. Single-copy DNA hybridization data argue against the monophyly of four of Andersen's sections and further suggest paraphyly or polyphyly of the Macroglossinae. DNA hybridization data provide support for an endemic African clade that includes Megaloglossus (an eonycterine), Epomophorus (an epomophorine), and Lissonycteris (a rousettine). Analyses of mitochondrial 12S rRNA-tRNA valine gene sequences corroborate the African clade but provide less resolution than hybridization data for most branches on the pteropodid tree. Here, we report 11 new 16S rRNA sequences and analyze a mitochondrial data set that includes 12S rRNA, tRNA valine, and 16S rRNA for 18 pteropodid genera. Parsimony, minimum evolution, and maximum likelihood were all employed in phylogenetic analyses. The addition of 16S rRNA sequences to the mitochondrial data set resulted in increased support for several clades, including Macroglossus + Syconycteris, Cynopterus + Thoopterus, Rousettus + the endemic African clade, and Eonycteris + Rousettus + the endemic African clade. Statistical tests suggest that another endemic African genus, Eidolon, is dissociated from the African clade and represents an independent invasion into Africa. We constructed a molecular phylogenetic framework that incorporated clades that were strongly supported by both single-copy DNA hybridization and 12S rRNA-tRNA valine-16S rRNA sequences. Using this framework as a backbone phylogenetic constraint, we then analyzed a morphological data matrix for 34 pteropodid genera with parsimony. Results of this analysis suggest that other epomophorines and Myonycteris (a cynopterine) are also part of the endemic African clade.     

Molecular phylogeny and detailed morphological analysis of two freshwater Rhizoclonium strains from contrasting spring types in Egypt and Italy

Two Rhizoclonium strains thriving in contrasting spring types (slightly hot spring/ambient, shaded pool spring) and biogeographic areas (El-Farafra Oasis in the Western Desert of Egypt/River-Po Plain in Northern Italy) were studied in depth based on field and cultured material, bright-field and fluorescence microscopy, and phylogenetic molecular analyses (SSU and LSU rRNA gene sequences). This polyphasic approach revealed that the two Rh. strains clearly belonged to the Rhizoclonium clade but differed in some of their key diagnostic features. The Egyptian Rhizoclonium strain, isolated from the spring-fed (Ain El-Balad) agricultural ditch in El-Farafra Oasis, was described with the working name ‘Rhizoclonium sp. 10.6 μm from a desert, slightly-hot spring’ based on its smaller cell diameter, ecological and phylogenetic molecular traits compared to allied morphospecies. Moreover, it was highlighted that the L/D ratio can be more than 3.0 within this group. The Italian Rhizoclonium strain, collected from the Fontanile Valle Re-shaded, pool ambient spring (Emilia-Romagna Region), was virtually identical to other strains identified as Rh. hieroglyphicum from China and Japan, due to its highly supported congruence in morphological characteristics and phylogenetic position. This study provided the first partial LSU and SSU rRNA gene sequences for European Rh. hieroglyphicum based on available literature. Phenotypic plasticity of rhizoid formation was also observed in both Rhizoclonium strains studied using culturing approaching techniques. Our investigation also confirms that a lot of work with a variety of approaches is still needed to assess the ecological preferences, morphological plasticity, and phylogenetics of freshwater Rhizoclonium taxa worldwide.     

Syndinean dinoflagellates of the genus Euduboscquella are paraphyletic

Syndinean dinoflagellates of the genus Euduboscquella infect marine ciliates and dinoflagellates. Euduboscquella species infecting dinoflagellates are understudied relative to congeners infecting ciliates and their molecular phylogeny remains uncertain. Morphology, development, and rRNA gene sequences of intracellular parasites infecting heterotrophic dinoflagellates from coastal waters of Busan, Republic of Korea in summer to fall of 2019–2021 indicate that Cucumeridinium coeruleum, Gyrodinium cf. ochraceum, and two unidentified species of Gyrodinium were each infected by a different Euduboscquella species. Morphological features including shield structure, shape and color of the mature trophont, and sporogenic process distinguished each of the four parasites from the 10 previously described species of Euduboscquella. Our molecular and phylogenetic analyses showed considerably greater genetic distance of SSU and ITS-LSU rRNA gene regions among Euduboscquella species infecting dinoflagellates than among those infecting ciliates. Rather than clustering as a group with Euduboscquella species infecting ciliates, SSU rRNA sequences of the four novel parasites spread out across the syndinean Group I phylogeny, occurring in two different clades and a new lineage. Placement of our novel parasites in multiple clades that encompass Ichythyodinium chabelardi strongly indicates that the genus Euduboscquella is paraphyletic.