Molecular phylogeny of Pacific Archigregarines (Apicomplexa), including descriptions of Veloxidium leptosynaptae n. gen., n. sp., from the sea cucumber Leptosynapta clarki (Echinodermata), and two new species of Selenidium

Although archigregarines are poorly understood intestinal parasites of marine invertebrates, they are critical for understanding the earliest stages in the evolution of the Apicomplexa. Previous studies suggest that archigregarines are a paraphyletic stem group from which other lineages of gregarines, and possibly all other groups of apicomplexans, evolved. However, substantiating this inference is difficult because molecular phylogenetic data from archigregarines, in particular, and other gregarines, in general, are severely limited. In an attempt to help fill gaps in our knowledge of archigregarine diversity and phylogeny, we set out to discover and characterize novel lineages of archigregarines with high-resolution light and scanning electron microscopy and analyses of small subunit (SSU) rDNA sequences derived from single-cell (SC) PCR techniques. Here, we describe two novel species of Selenidium, namely Selenidium idanthyrsae n. sp. and S. boccardiellae n. sp., and demonstrate the surface morphology and molecular phylogenetic position of the previously reported species S. cf. mesnili. We also describe a novel genus of archigregarine, Veloxidium leptosynaptae n. gen., n. sp., which branches with an environmental sequence and, together, forms the nearest sister lineage to a diverse clade of marine eugregarines (i.e. lecudinids and urosporids). This molecular phylogenetic result is consistent with the hypothesis that archigregarines are deeply paraphyletic within apicomplexans, and suggests that convergent evolution played an important role in shaping the diversity of eugregarine trophozoites.     

Morphology and Molecular Phylogeny of Two New Marine Euplotids,Pseudodiophrys nigricans n. g., n. sp., and Paradiophrys zhangi n. sp. (Ciliophora: Euplotida)

ABSTRACT. The morphology, infraciliature, and molecular phylogeny of Pseudodiophrys nigricans n. g., n. sp., and Paradiophrys zhangi n. sp., isolated from a sandy beach near Qingdao, China, were investigated. Pseudodiophrys is characterized by the Diophrys‐like ciliature pattern, but having only a single, reduced undulating membrane. Pseudodiophrys nigricans, the type species by monotypy, is described from live and silver‐impregnated specimens. Paradiophrys zhangi is similar to the type species Paradiophrys irmgard but can be recognized by its border body and the number (7 vs. 8–10) and arrangement (in rows vs. sparsely distributed) of the frontoventral cirri. Small subunit (SSU) rRNA gene sequence data support the validity of both species. Phylogenetic analyses based on the SSU rRNA gene sequence data currently available for uronychiids and other related taxa indicate that P. nigricans is most closely related to Diophrys scutum and Diophrys apoligothrix, while P. zhangi clusters most closely with Apodiophrys ovalis within a clade that also includes two other Paradiophrys species.     

Selenidium fauchaldi sp. n. and S. telepsavi (Stuart) comb. nov. (Protozoa,Apicomplexa), Gregarines from Polychaetes*

Gregarine Selenidium fauchaldi sp. n. is described from the intestinal lumen of the polychaete Phragmatopoma californica (Fewkes, 1889) in the intertidal zone of the Pacific Ocean on Santa Catalina Island, California. In addition, the new combination Selenidium telepsavi (Stuart, 1871) comb. nov. is introduced for the gregarine originally described as Monocystis telepsavi from the polychaete Telepsavus castarum in the Black Sea.     

Molecular Phylogenetic Positions and Ultrastructure of Marine Gregarines (Apicomplexa) Cuspisella ishikariensis n. gen., n. sp. and Loxomorpha cf. harmothoe from Western Pacific scaleworms (Polynoidae)

Marine gregarines are unicellular parasites of invertebrates commonly found infecting the intestine and coelomic spaces of their hosts. Situated at the base of the apicomplexan tree, marine gregarines offer an opportunity to explore the earliest stages of apicomplexan evolution. Classification of marine gregarines is often based on the morphological traits of the conspicuous feeding stages (trophozoites) in combination with host affiliation and molecular phylogenetic data. Morphological characters of other life stages such as the spore are also used to inform taxonomy when such stages can be found. The reconstruction of gregarine evolutionary history is challenging, due to high levels of intraspecific variation of morphological characters combined with relatively few traits that are taxonomically unambiguous. The current study combined morphological data with a phylogenetic analysis of small subunit rDNA sequences to describe and establish a new genus and species (Cuspisella ishikariensis n. gen., n. sp.) of marine gregarine isolated from the intestine of a polynoid host (Lepidonotus helotypus) collected from Hokkaido, Japan. This new species possesses a set of unusual morphological traits including a spiked attachment apparatus and sits on a long branch on the molecular phylogeny. Furthermore, this study establishes a molecular phylogenetic position for Loxomorpha cf. harmothoe, a previously described marine gregarine, and reveals a new group of gregarines that infect polynoid hosts.     

Molecular Phylogeny and Surface Morphology of Thiriotia hyperdolphinae n. sp. and Cephaloidophora oradareae n. sp. (Gregarinasina,Apicomplexa) Isolated from a Deep Sea Oradarea sp. (Amphipoda) in the West Pacific

In an effort to broaden our understanding of the biodiversity and distribution of gregarines infecting crustaceans, this study describes two new species of gregarines, Thiriotia hyperdolphinae n. sp. and Cephaloidophora oradareae n. sp., parasitizing a deep sea amphipod (Oradarea sp.). Amphipods were collected using the ROV Hyper‐Dolphin at a depth of 855 m while on a cruise in Sagami Bay, Japan. Gregarine trophozoites and gamonts were isolated from the gut of the amphipod and studied with light and scanning electron microscopy, and phylogenetic analysis of 18S rDNA. Thiriotia hyperdolphinae n. sp. was distinguished from existing species based on morphology, phylogenetic position, as well as host niche and geographic locality. Cephaloidophora oradareae n. sp. distinguished itself from existing Cephaloidophora, based on a difference in host (Oradarea sp.), geographic location, and to a certain extent morphology. We established this latter new species with the understanding that a more comprehensive examination of diversity at the molecular level is necessary within Cephaloidophora. Results from the 18S rDNA molecular phylogeny showed that T. hyperdolphinae n. sp. was positioned within a clade consisting of Thiriotia spp., while C. oradareae n. sp. grouped within the Cephaloidophoridae. Still, supplemental genetic information from gregarines infecting crustaceans will be needed to better understand relationships within this group of apicomplexans.     

Tintinnophagus acutus n. g., n. sp. (Phylum Dinoflagellata), an Ectoparasite of the Ciliate Tintinnopsis cylindrica Daday 1887, and Its Relationship to Duboscquodinium collini Grassé 1952

ABSTRACT. The dinoflagellate Tintinnophagus acutus n. g., n. sp., an ectoparasite of the ciliate Tintinnopsis cylindrica Daday, superficially resembles Duboscquodinium collini Grassé, a parasite of Eutintinnus fraknoii Daday. Dinospores of T. acutus are small transparent cells having a sharply pointed episome, conspicuous eyespot, posteriorly positioned nucleus with condensed chromosomes, and rigid form that may be supported by delicate thecal plates. Dinospores attach to the host via a feeding tube, losing their flagella, sulcus, and girdle to become spherical or ovoid cells. The trophont of T. acutus feeds on the host for several days, increasing dramatically in size before undergoing sporogenesis. Successive generations of daughter sporocytes are encompassed in an outer membrane or cyst wall, a feature not evident in trophonts. Tintinnophagus acutus differs from D. collini in host species, absence of a second membrane surrounding pre‐sporogenic stages, and failure to differentiate into a gonocyte and a trophocyte at the first sporogenic division. Phylogenetic analyses based on small subunit (SSU) ribosomal DNA (rDNA) sequences placed T. acutus and D. collini in the class Dinophyceae, with T. acutus aligned loosely with Pfiesteria piscicida and related species, including Amyloodinium ocellatum, a parasite of fish, and Paulsenella vonstoschii, a parasite of diatoms. Dubosquodinium collini nested in a clade composed of several Scrippsiella species and Peridinium polonicum. Tree construction using longer rDNA sequences (i.e. SSU through partial large subunit) strengthened the placement of T. acutus and D. collini within the Dinophyceae.     

Morphology and Molecular Phylogeny of Trimyema koreanum n. sp., a Ciliate from the Hypersaline Water of a Solar Saltern

ABSTRACT. A new ciliate, Trimyema koreanum n. sp., isolated from hypersaline water (salinity of 293‰) from a solar saltern in Korea, was investigated using live observation, protargol impregnation, and gene sequencing. Trimyema koreanum is about 30 × 13 μm in vivo, has usually 23 longitudinal ciliary rows forming two distinct ciliary girdles visible both in vivo and in protargol impregnation. A third indistinct ciliary girdle as well as a girdle of mucocysts is distinguishable only in impregnated cells. We suggest T. koreanum as a new species, differing from the most similar species, T. marinum, by the presence of two distinct ciliary girdles (T. marinum usually has six ciliary girdles clearly visible in living cells and three anterior spirals that encircle the cell completely). Although the number of known 18S rRNA sequences in the genus Trimyema was limited, the Trimyema group including T. koreanum forms a strong clade. The phylogenetic position confirms that the isolate belongs to the genus Trimyema and is different from previously sequenced species. Trimyema koreanum is able to consume both prokaryotes and small eukaryotes (specifically, the alga Dunaliella sp.).     

Tylenchocriconema alleni n. g., n. sp. from Guatemala (Tylenchocriconematidae n. fam.; Tylenchocriconematidae n. superfam.; Nematoda)

Tylenchocriconema alleni n.g.n.sp, is described from soil about roots of a bromeliad from Guatemala City, Guatemala. It has characteristics of both the Tylenchoidea and Criconematoidea. Tylenchocriconematidae, n. fam., and Tylenchocriconematoidea, n. superfam., are proposed to express the relationship of this species to other taxa. Tylenchulidae and Paratylenchidae are considered closely related, and Tylenchulidoidea, n. rank, is proposed to indicate their relationship.     

Pseudonotohymena antarctica n. g., n. sp. (Ciliophora,Hypotricha), a New Species from Antarctic Soil

A new soil ciliate, Pseudonotohymena antarctica n. g., n. sp., from King George Island, Antarctica, is described based on live observation, protargol impregnation, and its 18S rRNA gene. The new genus Pseudonotohymena is morphologically similar to the genus Notohymena Blatterer and Foissner 1988 in the following characteristics: 18 fronto‐ventral‐transverse cirri, a flexible body, undulating membranes, dorsomarginal kineties, and the number of cirri in the marginal rows. However, Pseudonotohymena differs from Notohymena particularly in the dorsal ciliature, that is, in possessing a nonfragmented dorsal kinety (vs. fragmented). In addition, the molecular phylogenetic relationship of the new species differs from that of Notohymena species. On the basis of the morphological features, the genetic data, and morphogenesis, we establish P. antarctica n. g., n. sp. In addition, the cyst morphology of this species is described.     

Gibbsia archiuli (Apicomplexa, Eucoccidiorida) n. g., n. sp. from the Millipede Archiulus moreleti

ABSTRACT. Gibbsia archiuli n. g., n. sp. (Eucoccidiorida, Adeleidae) is named for the apicomplexan protozoon described by Gibbs (1952) from the blood cells of the South African garden millipede Archiulus moreleti. Merogony, gamogony, and sporogony all take place in the host's blood cells. The oocysts contain four sporocysts, each with one sporozoite. The microgametes are not flagellated.     

Morphology and Phylogeny of Two Novel Ciliates,Arcanisutura chongmingensis n. gen., n. sp. and Naxella paralucida n. sp. from Shanghai,China

The morphologies of two novel ciliates, Arcanisutura chongmingensis n. gen., n. sp. and Naxella paralucida n. sp., collected from Shanghai, China, have been investigated using live observation and silver staining methods. Arcanisutura n. gen. can be easily distinguished from related genera by its inconspicuous, oblique anterior suture. Arcanisutura chongmingensis n. sp. is mainly recognized by its elongated body with a tail‐like posterior end, 25–33 somatic kineties, and 4–11 excretory pores. Naxella paralucida n. sp. can be distinguished from its congeners based on its two short nassulid organelles, fusiform trichocysts, 37–49 somatic kineties, and 16 nematodesmal rods. The small‐subunit (SSU) rRNA gene sequences of these two species are presented, revealing the phylogenetic positions of Arcanisutura and Naxella. Phylogenetic analyses show that Arcanisutura forms a sister clade to other synhymeniid genera, namely, Chilodontopsis, Orthodonella, and Zosterodasys; Naxella is most closely related to Nassula spp. and is located within the monophyletic clade of the family Nassulidae.     

Myosporidium merluccius n. g., n. sp. infecting muscle of commercial hake (Merluccius sp.) from fisheries near Namibia

A new species of Microsporidia classified to a new genus was observed in the trunk muscle of commercial hake (Merluccius capensis/paradoxus complex) from Namibian fisheries. Macroscopic examination revealed thin and dark filaments inserted among muscle fibers. Inside the filaments were many sporophorous vesicles with about 30-50 spores per vesicle. The shape of the spore was pyriform and the extruded polar filament was of moderate length (up to 4.29 microm, n=12). This new species of Microsporidia is described using macrophotography, microphotography, staining, and transmission electron microscopy (TEM), as well as molecular methods. Its 16S rRNA was found to be similar to that of Microsporidium prosopium Kent et al., 1999, while both sequences were quite different from 16S rRNA sequences known for other Microsporidia. Nevertheless, this new species is separated morphologically from M. prosopium by the presence of 11-12 anisofilar coils and the formation of the xenoma at the site of infection. Type species.     

Morphology,Morphogenesis, and Molecular Phylogeny of Neobakuella aenigmatica n. sp. (Ciliophora,Spirotrichea, Bakuellidae)

The morphology and morphogenesis of a new ciliate species, Neobakuella aenigmatica n. sp., which was discovered in an estuary in Korea, were investigated, using live observation, protargol impregnation, and scanning electron microscopy. This new species is characterized by a large (185–300 × 55–105 μm in vivo), elongate‐ellipsoidal, flexible but not contractile body. It has ellipsoidal, yellowish cortical granules, 1.3 × 1.0 μm in size. The species has invariably 3 frontal and 2 frontoterminal cirri, about 5–10 buccal and 1–6 parabuccal cirri, 7 midventral rows, and 1 right and 2–4 left marginal rows. The outer left marginal row(s) consists of 1–7 short rows of cirri. The nuclear apparatus comprises 130 macronuclear nodules and 2 spherical micronuclei on average. The dorsal ciliature consists of 3 dorsal kineties. The leftmost left marginal row(s) likely develops from anlagen originating from both the rightmost and leftmost left marginal row(s). The molecular phylogenetic tree based on SSU rDNA suggests the nonmonophyly of the genus Neobakuella.     

Ultrastructure and Molecular Phylogeny of Mesodinium coatsi sp. nov., a Benthic Marine Ciliate

Mesodinium is a globally distributed ciliate genus forming frequent and recurrent blooms in diverse marine habitats. Here, we describe a new marine species, Mesodinium coatsi n. sp., originally isolated from interstitial water of surface sand samples collected at Mohang Beach, Korea. The species was maintained under a mixotrophic growth condition for longer than 1 yr by providing a cryptomonad, Chroomonas sp., as the sole prey. Cell morphology and subcellular structure were examined by light microscopy, scanning, and transmission electron microscopy, and molecular phylogeny was inferred from nuclear‐encoded 18S rDNA sequence data. Like other Mesodinium species, M. coatsi consisted of two hemispheres separated by two types of kinetids, and had tentacles located at the oral end of the cell. Several food vacuoles were observed in the cytoplasm, and partially digested prey cells sometimes existed in food vacuoles. Kinetids and the associated accessory structures were quite similar to those previously reported, but M. coatsi was differentiated from other marine Mesodinium species by ultrastructural characters of the dikinetids, polykinetids, and tentacles. We also provided a detailed illustration of infraciliature. Molecular phylogeny revealed that M. coatsi and Mesodinium chamaeleon were closely related to each other.     

Molecular Characterization of Gregarines from Sand Flies (Diptera: Psychodidae) and Description of Psychodiella n. g. (Apicomplexa: Gregarinida)

ABSTRACT. Sand fly and mosquito gregarines have been lumped for a long time in the single genus Ascogregarina and on the basis of their morphological characters and the lack of merogony been placed into the eugregarine family Lecudinidae. Phylogenetic analyses performed in this study clearly demonstrated paraphyly of the current genus Ascogregarina and revealed disparate phylogenetic positions of gregarines parasitizing mosquitoes and gregarines retrieved from sand flies. Therefore, we reclassified the genus Ascogregarina and created a new genus Psychodiella to accommodate gregarines from sand flies. The genus Psychodiella is distinguished from all other related gregarine genera by the characteristic localization of oocysts in accessory glands of female hosts, distinctive nucleotide sequences of the small subunit rDNA, and host specificity to flies belonging to the subfamily Phlebotominae. The genus comprises three described species: the type species for the new genus— Psychodiella chagasi ( Adler and Mayrink 1961 ) n. comb., Psychodiella mackiei ( Shortt and Swaminath 1927 ) n. comb., and Psychodiella saraviae ( Ostrovska, Warburg, and Montoya-Lerma 1990 ) n. comb. Its creation is additionally supported by sequencing data from other gregarine species originating from the sand fly Phlebotomus sergenti . In the evolutionary context, both genera of gregarines from mosquitoes ( Ascogregarina ) and sand flies ( Psychodiella ) have a close relationship to neogregarines; the genera represent clades distinct from the other previously sequenced gregarines.