Light Microscopic Observations, Ultrastructure, and Molecular Phylogeny of Hicanonectes teleskopos n. g., n. sp., a Deep-Branching Relative of Diplomonads

ABSTRACT. We describe Hicanonectes teleskopos n. g., n. sp., a heterotrophic flagellate isolated from low-oxygen marine sediment. Hicanonectes teleskopos has a ventral groove and two unequal flagella, and rapidly rotates during swimming. At the ultrastructural level H. teleskopos is a "typical excavate": it displays flagellar vanes, a split right microtubular root, "I,""B," and "C" fibres, a singlet microtubular root, and a possible composite fibre. Small subunit rRNA (SSU rRNA) gene phylogenies and an "arched" B fibre demonstrate that H. teleskopos belongs to Fornicata (i.e. diplomonads, retortamonads, and relatives). It forms a clade with the deep-branching fornicate Carpediemonas , with moderate-to-strong bootstrap support, although their SSU rRNA gene sequences are quite dissimilar. Hicanonectes differs from Carpediemonas in cell shape, swimming behaviour, number of basal bodies (i.e. 4 vs. 3), number of flagellar vanes (i.e. 2 vs. 3), anterior root organization, and by having a cytopharynx. Like Carpediemonas and Dysnectes, Hicanonectes has conspicuous mitochondrion-like organelles that lack cristae and superficially resemble the hydrogenosomes of parabasalids, rather than the mitosomes of their closer relatives the diplomonads (e.g. Giardia ).     

Andalucia (n. gen.)--the deepest branch within jakobids (Jakobida; Excavata), based on morphological and molecular study of a new flagellate from soil

A new heterotrophic flagellate (Andalucia godoyi n. gen. n. sp.) is described from soil. Earlier preliminary 18S rRNA analyses had indicated a relationship with the phylogenetically difficult-to-place jakobid Jakoba incarcerata. Andalucia godoyi is a small (3-5 mum) biflagellated cell with a ventral feeding groove. It has tubular mitochondrial cristae. There are two major microtubular roots (R1, R2) and a singlet root associated with basal body 1 (posterior). The microtubular root R1 is associated with non-microtubular fibres "I,"B," and "A," and divides in two parts, while R2 is associated with a "C" fibre. These structures support the anterior portion of the groove. Several features of A. godoyi are characteristic of jakobids: (i) there is a single dorsal vane on flagellum 2; (ii) the C fibre has the jakobid multilaminate substructure; (iii) the dorsal fan of microtubules originates in very close association with basal body 2; and (iv) there is no "R4" microtubular root associated with basal body 2. Morphological analyses incorporating the A. godoyi data strongly support the monophyly of all jakobids. Our 18S rRNA phylogenies place A. godoyi and J. incarcerata as a strong clade, which falls separately from other jakobids. Statistical tests do not reject jakobid monophyly, but a specific relationship between Jakoba libera and J. incarcerata and/or A. godoyi is rejected. Therefore, we have established a new genus Andalucia n. gen. with the type species Andalucia godoyi n. sp., and transfer Jakoba incarcerata to Andalucia as Andalucia incarcerata n. comb.     

Ultrastructure and ribosomal RNA phylogeny of the free-living heterotrophic flagellate Dysnectes brevis n. gen., n. sp., a new member of the Fornicata

Dysnectes brevis n. gen., n. sp., a free-living heterotrophic flagellate that grows under microaerophilic conditions possesses two flagella. The posterior one lies in a ventral feeding groove, suggesting that this flagellate is an excavate. Our detailed electron microscopic observations revealed that D. brevis possesses all the key ultrastructural characters considered typical of Excavata. Among the 10 excavate groups previously recognized, D. brevis displays an evolutionary affinity to members of the Fornicata (i.e. Carpediemonas, retortamonads, and diplomonads). Firstly, a strong D. brevis-Fornicata affinity was recovered in the phylogenetic analyses of small subunit ribosomal RNA (SSU rRNA) sequences, albeit the internal branching pattern of the D. brevis+Fornicata clade was not resolved with confidence. Corresponding to the SSU rRNA phylogeny, D. brevis and the Fornicata shared the following components of the flagellar apparatus: the arched B fiber bridging the right root; a posterior basal body; and a left root. Combining both morphological and molecular phylogenetic analyses, D. brevis is classified as a new free-living excavate in the Fornicata incertae sedis.     

A wide diversity of previously undetected free‐living relatives of diplomonads isolated from marine/saline habitats

Over the last 15 years classical culturing and environmental PCR techniques have revealed a modest number of genuinely new major lineages of protists; however, some new groups have greatly influenced our understanding of eukaryote evolution. We used culturing techniques to examine the diversity of free‐living protists that are relatives of diplomonads and retortamonads, a group of evolutionary and parasitological importance. Until recently, a single organism, Carpediemonas membranifera, was the only representative of this region of the tree. We report 18 new isolates of Carpediemonas‐like organisms (CLOs) from anoxic marine sediments. Only one is a previously cultured species. Eleven isolates are conspecific and were classified within a new genus, Kipferlia n. gen. The remaining isolates include representatives of three other lineages that likely represent additional undescribed genera (at least). Small‐subunit ribosomal RNA gene phylogenies show that CLOs form a cloud of six major clades basal to the diplomonad‐retortamonad grouping (i.e. each of the six CLO clades is potentially as phylogenetically distinct as diplomonads and retortamonads). CLOs will be valuable for tracing the evolution of diplomonad cellular features, for example, their extremely reduced mitochondrial organelles. It is striking that the majority of CLO diversity was undetected by previous light microscopy surveys and environmental PCR studies, even though they inhabit a commonly sampled environment. There is no reason to assume this is a unique situation – it is likely that undersampling at the level of major lineages is still widespread for protists.     

On Core Jakobids and Excavate Taxa: The Ultrastructure of Jakoba incarcerata

The cellular organisation of the 'excavate' flagellate Jakoba incarcerata Bernard, Simpson and Patterson 2000 is described. Cells have one nucleus and dictyosome. The putative mitochondria lack cristae. Two flagella (anterior and posterior) insert anterior to the feeding groove. The posterior flagellum bears a dorsal vane. An 'anterior' microtubular root arises against the anterior basal body. Two main microtubular roots, left and right, and a singlet 'root' arise around the posterior basal body and support the groove. Non-microtubular fibres termed 'A', 'B', 'I', and 'composite' associate with the right root. A multilaminar 'C' fibre associates with the left root. The cytoskeleton of J. incarcerata indicates a common ancestry with other excavate taxa (i.e. diplomonads, retortamonads, heteroloboseids, 'core jakobids', Malawimonas, Carpediemonas, and Trimastix). Overall, J. incarcerata is most similar to (other) core jakobids, namely Jakoba libera, Reclinomonas, and Histiona. We regard J. incarcerata as a core jakobid and identify the group by the synapomorphy 'vanes restricted to dorsal side of the posterior flagellum'. The anterior root and position of the B fibre (and presence of dense inclusions in the cartwheels and a conscpicuous singlet root-associated fibre) in J. incarcerata are novel for core jakobids and argue for close relationships with Trimastix and/or Heterolobosea. The C fibre is similar in substructure to the costal fibre of parabasalids and it is possible that the structures are homologous.     

Multigene phylogenies of diverse Carpediemonas-like organisms identify the closest relatives of 'amitochondriate' diplomonads and retortamonads

Diplomonads, retortamonads, and "Carpediemonas-like" organisms (CLOs) are a monophyletic group of protists that are microaerophilic/anaerobic and lack typical mitochondria. Most diplomonads and retortamonads are parasites, and the pathogen Giardia intestinalis is known to possess reduced mitochondrion-related organelles (mitosomes) that do not synthesize ATP. By contrast, free-living CLOs have larger organelles that superficially resemble some hydrogenosomes, organelles that in other protists are known to synthesize ATP anaerobically. This group represents an excellent system for studying the evolution of parasitism and anaerobic, mitochondrion-related organelles. Understanding these evolutionary transitions requires a well-resolved phylogeny of diplomonads, retortamonads and CLOs. Unfortunately, until now the deep relationships amongst these taxa were unresolved due to limited data for almost all of the CLO lineages. To address this, we assembled a dataset of up to six protein-coding genes that includes representatives from all six CLO lineages, and complements existing rRNA datasets. Multigene phylogenetic analyses place CLOs as well as the retortamonad Chilomastix as a paraphyletic basal assemblage to the lineage comprising diplomonads and the retortamonad Retortamonas. In particular, the CLO Dysnectes was shown to be the closest relative of the diplomonads + Retortamonas clade, with strong support. This phylogeny is consistent with a drastic degeneration of mitochondrion-related organelles during the evolution from a free-living organism resembling extant CLOs to a probable parasite/commensal common ancestor of diplomonads and Retortamonas.     

Malawimonas jakobiformis n. gen., n. sp. (Malawimonadidae n. fam.): A Jakoba-like Heterotrophic Nanoflagellate with Discoidal Mitochondrial Cristae

Malawimonas jakobiformis n. gen., n. sp., is established for a bacterivorous heterotrophic nanoflagellate isolated from the Malawi shore of Lake Nyasa (eastern Africa). Trophic stages observed were anteriorly biflagellate and naked. The posterior flagellum of a trophic cell resided in a conspicuous groove on the ventral surface, and bore a prominent vane. A Golgi stack and a mitochondrion with discoidal cristae were present anterior to the nucleus. The kinetid consisted of two short, slightly separated basal bodies, four microtubular roots, and associated fibers and bands. The three microtubular roots associated with the posterior basal body were associated with the ventral groove, while the single root associated with the anterior basal body gave rise to secondary cytoskeletal microtubules. Dividing cells became rounded, with persistent flagella. Cysts were uninucleate, and had thin organic walls without clearly differentiated apertures or ornamentation but with conspicuous attachment pads. Kinetid elements were present within cysts. On the basis of microscopical features, especially those of the kinetid, the nearest relatives of M. jakobiformis are the mitochondriate “jakobid” protists (families Histionidae and Jakobidae) and the amitochondriate retortamonads. Malawimonadidae n. fam. is established to accommodate this species.     

Pelagostrobilidium liui n. sp. (Ciliophora,Choreotrichida) from the Coastal Waters of Northeastern Taiwan and an Improved Description of Pelagostrobilidium minutum Liu et al., 2012

The number of somatic kineties in Pelagostrobilidium ranges from 4 to 6 according to the present state of knowledge. This study investigates Pelagostrobilidium liui n. sp. using live observation, protargol stain, and small subunit rDNA data sequencing. Pelagostrobilidium liui n. sp. is characterized by having a spherical‐shaped body, four somatic kineties, with kinety 2 spiraled around the left side of body, about six elongated external membranelles, and invariably no buccal membranelle. It differs from its most similar congener, Pelagostrobilidium minutum Liu et al., 2012 , in (i) cell shape; (ii) macronucleus width; (iii) oral apparatus; (iv) anterior orientation of kinety 2; (v) location where kinety 2 commences; (vi) arrangement of kinety 1; (vii) distance between the anterior cell end and the locations where kineties commence; and (viii) the presence of 12 different bases (including two deletions) in the small subunit rDNA sequences. The diagnosis of P. minutum Liu et al., 2012 is also improved to include the following new characteristics: invariably four somatic kineties; kineties 2 and 4 alone commence at the same level; kinety 2 originates from right anterior cell half on ventral side, extends sinistrally posteriorly, over kinety 1, around left posterior region, terminates near posterior cell end on dorsal side; kinety 1 commences below anterior third of kinety 2.     

Morphology,Morphogenesis, and Molecular Phylogeny of Paraparentocirrus sibillinensis n. gen., n. sp., a “Stylonychine Oxytrichidae” (Ciliophora,Hypotrichida) Without Transverse Cirri

A terrestrial oxytrichid ciliate Paraparentocirrus sibillinensis n. gen., n. sp., which was found in soil samples of a beech forest stand within the National Park of Sibillini Mountains, Italy, was investigated using live observation and protargol impregnation. The morphology of interphase, morphogenesis, and molecular phylogeny inferred from SSU rDNA sequences of this ciliate were studied. Paraparentocirrus n. gen., is mainly characterized by a semirigid body, an undulating membrane in the Oxytricha pattern, six fronto‐ventral (FV) rows, the absence of transverse cirri, one right and one left row of marginal cirri, four dorsal kineties, two dorsomarginal rows, and caudal cirri at the end of dorsal kinety 4. During morphogenesis, oral primordia develop through the proliferation of basal bodies from some cirri of FV rows 4 and 5, and FV row 6 takes part in the anlagen formation of the proter. The dorsal morphogenesis was typical of oxytrichids, with simple fragmentation of dorsal kinety 3, and the dorsomarginal rows developed from the right marginal row. Phylogenetic analyses based on the SSU rDNA sequences support the classification of this new genus in the stylonychines.     

Macrobothridium rhynchobati n. g., n. sp. from the elasmobranch Rhynchobatus granulatus,representing a new family of diphyllidean cestodes,the Macrobothridiidae

Macrobothridium rhynchobati n. g., n. sp. from the guitarfish Rhynchobatus granulatus in Kuwaiti waters in the Arabian Gulf is described and figured. The new genus differs from Echinobothrium in having a short unarmed peduncle and a posterior sucker-like structure in the terminal proglottid. It differs from Ditrachybothridium in having a powerful rostellum with dorsal and ventral groups of hooks and in having unarmed bothridia. A new family, the Macrobothridiidae, assigned to the order Diphyllidea is erected. Larval forms are described and figured.     

Katatopygia gen. n., a monophyletic branch segregated from Boletina (Diptera, Mycetophilidae)

The genus Katatopygia gen. n. is proposed for the Boletina erythropyga/punctus-group that was first introduced by Garrett (1924, 1925) and currently comprises eight described species. Molecular studies have strongly indicated that this group forms a monophyletic sister-group to a clade consisting of all other Boletina, Coelosia and Gnoriste, and its monophyly is supported by morphological data as well. The new genus includes the following species: Katatopygia antoma (Garrett, 1924), comb. n., Katatopygia antica (Garrett, 1924), comb. n., Katatopygia erythropyga (Holmgren, 1883), comb. n.,Katatopygia hissarica (Zaitzev & Polevoi, 2002), comb. n., Katatopygia magna (Garrett, 1925), comb. n., Katatopygia laticauda (Saigusa, 1968), comb. n., Katatopygia neoerythropyga (Zaitzev & Polevoi, 2002), comb. n. andKatatopygia sahlbergi (Lundström, 1906), comb. n., all transferred from Boletina. Katatopygia sahlbergi is found to be a senior synonym of Boletina punctus Garrett, 1925, syn. n. A phylogeny based on morphological data and using parsimony analysis yielded four most parsimonious trees where the new genus is retrieved as monophyletic with high support. Katatopygia neoerythropyga is found to be the sister-taxon to all other species that form two clades, one with Katatopygia sahlbergi-like species and one with Katatopygia erythropyga-like species. A key to males of Katatopygia is provided.     

The colonial rock-forming microfossils of the Bohemian Upper Proterozoic (Czechoslovakia), “Bohemipora Pragensis’ n.g., n.sp.

Large amounts of well preserved microfossils have been reported from the cherts of the Upper Proterozoic of the Bohemian Massif (Middle Europe). They resemble those described by Cayeux (1894) from the Upper Proterozoic (Brioverian) of Bretagne (France). It is shown, unlike the views of Cayeux and his followers (Deflandre, 1955, and Graindor 1957), that the observed structures did not belong to individuals but to colonies of filamentous prokaryotic organisms, most probably blue-green algae (Cyanophyta). These produced specific crystal-like mineral aggregation round each filament. Scanning microscope examination has revealed that the individual facets of these mineral crystals were perforated by the openings through which the thread-like bodies of these primitive organisms protruded. It is shown that these microorganisms were attached to the cells of other, bigger microorganisms and enveloped them. Some of these substrate organisms might have been eukaryotic algae. The thecae gradually accumulated around the cells of these carrier organisms and after death the colonies disintegrated to constitute the main component of the sediment. The microfossils described are just a major component of a complicated fossil assemblage comprising coccoid and filamentous blue-green algae and bacteria. There are indications that several eukaryotic species might also have been present.The following new taxa are described:Thecophytales, new order,Cayeuxidae (Graindor) family emend.,Bohemipora n. gen.,B. pragensis, n. sp.     

Corollonema thylogale n. g., n. sp. (Nematoda: Strongyloidea), parasitic in the pademelons Thylogale thetis and T. stigmatica (Marsupialia: Macropodidae) from Queensland, Australia

Corollonema thylogale n. g., n. sp. is described from the stomachs of Thylogale thetis and T. stigmatica from south-eastern Queensland. The new genus is characterised by four branches to the dorsal ray, a J-shaped ovejector, a cylindrical buccal capsule reduced to a posterior, thickened annulus and a leaf-crown of eight elements originating from the base of the buccal capsule. On the basis of these features, the new genus is placed in the subfamily Cloacininae Stossich, 1899 and the tribe Coronostrongylinea Beveridge, 1986. The presence of an internal leaf crown is unique within the tribe and the new genus is erected on this basis.     

Kiitricha minuta n. sp., a peculiar hypotrichous ciliate (Ciliophora, Spirotrichea) from the Yellow Sea

The morphology and infraciliature of a new ciliate, Kiitricha minuta n. sp., isolated from the Yellow Sea, were investigated using live observation and protargol impregnation. Kiitricha minuta represents a third member of the rarely known order Kiitrichida. It is unique in the subclass Hypotrichia in having many rows of small uniform cirri along the right side of the body and the dorsal kineties composed of dikinetids, most of which bear two cilia. Kiitricha minuta n. sp. is ovoid and measures about 60 x 45 microm in vivo. It has a huge buccal cavity occupying about 80% of the body length, numerous body extrusomes, one macronucleus and two micronuclei, 27-27 adoral membranelles, 9-12 frontoventral cirral rows, a submarginal row of 7-9 cirri, 6 or 7 transverse cirri, and roughly 7-9 dorsal kineties. This new species differs distinctly from its only congener Kiitricha marina by its smaller size (60 microm vs. 80-150 microm), the presence of body extrusomes (vs. absent), the different macronuclear pattern (one vs. two nodules), and the lower number of frontoventral cirral rows (9-12 vs. 21-26), which terminate at the anterior two-thirds of body (vs. extend to the posterior). The new term "submarginal cirral row" is introduced to distinguish from the marginal cirral row in typical hypotrichs sensu lato. Based on our new observations and the literature, an improved diagnosis for the genus Kiitricha is provided and its phylogenetic importance is discussed.     

Pararotylenchus n. gen. (Pararotylenchinae n. subfam., Hoplolaimidae) with Six New Species and Two New Combinations

A new subfamily, Pararotylenchinae, (Hoplolaimidae) is described. It includes a single genus, Pararotylenchus n. gen., six new species, attd two new combinations, Pararotylenchus (syn. Tylenchorhynchus) brevicaudatus (Hopper, 1959) n. comb. and Pararotylenchus (syn. Rotylenchus) pint (Mamiya, 1968) n. comb. Pararotylenchinae is similar to certain other Hoplolaimidae, such as Rotylenchinae, with respect to most characters including the short tail, the position of the phasmids near the anus, and the relatively great distance of the dorsal gland orifice from the base of the stylet knobs. The lip region, as observed with the scanning electron microscope, conforms to the basic pattern for Hoplolaimidae. The labial disc is round with slit-like amphidial openings at the lateral sides of the periphery of the disc. The distinctive anterior-most lip annule is segmented into six sectors. Unlike other Hoplolaimidae, however, the esophageal glands of Pararotylenchinae form a basal bulb similar to that of Tylenchorhynchidae; other characters do not resemble Tylencborhynchidae. Specimens of Pararotylenchus have been recovered only from cool regions at high elevations and Pacific coastal areas in the Western United States, Japan, and Korea.     

Coryphogonimus aglaos n. g., n. sp. (Digenea: Prosthogonimidae) from the Australian bush rat,Rattus fuscipes (Rodentia: Muridae), with notes on other prosthogonimids from Australian mammals

Coryphogonimus aglaos n. g., n. sp. (Digenea: Prosthogonimidae) is described from the liver of the Australian bush rat, Rattus fuscipes (Rodentia: Muridae). The new genus is distinguished from other prosthogonimids by the possession of the following combination of characters: oral and ventral suckers well developed; caeca terminating in front of testes; genital pore opening dorsal to oral sucker; vitelline follicles restricted to caecal region; uterus extending posterior to testes; and uterus without an egg reservoir. A single specimen of an as yet undetermined Coryphogonimus species is recorded from the Australian water rat, Hydromys chrysogaster (Rodentia: Muridae). A new host, Sminthopsis macroura (Marsupialia: Dasyuridae), is recorded for Coelomotrema antechinomes. Attention is drawn to the apparent rarity of the six known mammal prosthogonimids.     

Morphology and phylogeny of Sainouron acronematica sp. n. and the ultrastructural unity of Cercozoa

Sainouron are soil zooflagellates of obscure taxonomy. We studied the ultrastructure of S. acronematica sp. n. and sequenced its extremely divergent 18S rDNA and that of Cholamonas cyrtodiopsidis (here grouped as new family Sainouridae) to clarify their phylogeny. Ultrastructurally similar, they weakly group together, deeply within Monadofilosa. Sainouron has three cytoplasmic microtubules; all organelles specifically link to them or the nucleus. Mature centrioles have fibrous rhizoplasts. The posterior centriole bearing the motile cilium (with cortical filaments) has a transitional hub-lattice; a dense spiral fibre links its thicker rhizoplast and triplets; its ciliary root has two microtubules: mt1, underlying the plasma membrane, initiates at the spiral fibre; mt2, laterally attached to mt1 and nucleus, initiates in the amorphous centrosomal region. The anterior younger cilium, an immotile stub with submembrane skeleton as in Cholamonas, lacks axoneme, microtubular root, rhizoplasts and spiral fibre, but becomes the posterior one every cell cycle. The nuclear envelope donates coated vesicles directly to the Golgi, which makes kinetocyst-type extrusomes, concentrated at the cell anterior for extrusion into phagosomes. Ciliary transition region proximal hub-lattices (postulated to contain centrin) and distal nonagonal fibres are cercozoan synapomorphies, found with slight structural variation in all flagellate Cercozoa, but not in outgroups.     

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.