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.     

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 ).     

Analysis of Kozhowia Vasiljeva & Smirnov, 1969 (Chydoridae,Anomopoda, Branchiopoda), with a description of Parakozhowia n. gen.

The five endemic species of Kozhowia Vasiljeva & Smirnov, 1969 (Branchiopoda: Anomopoda: Chydoridae) are redescribed based on material from Lake Baikal, including types of all species. Two well-defined groups were found. A new genus Parakozhowia n.gen., with one species, P. baicalensis Vasiljeva & Smirnov, 1969 n. comb., is erected. An extended diagnosis for the genus Kozhowia is presented. Parakozhowia n. gen. differs from Kozhowia in (1) antennal formula; (2) proportions of postabdomen; (3) number of setae on exopodite IV; (4) presence of soft setae of endopodite IV; (5) presence of a limb VI, and (6) presence of an inflated rostrum in the adult male of Kozhowia. An improved key for the discrimination of the species is given. Thoracic limbs of all species were given special attention. There are six setae on exopodite IV in Parakozhowia n.gen., as normal for Aloninae, while only four setae and a rudimentary hillock were detected on exopodite IV in the species of Kozhowia. Kozhowia is a true member of the Aloninae, and the closest relative of Parakozhowia and Camptocercus, meaning that the diagnosis of the Aloninae must be amended: the number of setae on exopodite IV in Aloninae may vary from 4 to 6.     

Phylogenetic position of Sphaerospora testicularis and Latyspora scomberomori n. gen. n. sp. (Myxozoa) within the marine urinary clade

An amendment of the family Sinuolineidae (Myxosporea) is proposed in order to include a newly described genus Latyspora n. gen. The type species Latyspora scomberomori n. gen. n. sp. is a coelozoic parasite in the kidney tubules of Scomberomorus guttatus. In addition to the morphological and molecular characterization of L. scomberomori n. gen. n. sp., we also present novel SSU rDNA data on Sphaerospora testicularis, a serious parasite of Dicentrarchus labrax. Performed phylogenetic analyses revealed that both species cluster within the marine urinary clade encompassing the representatives with a shared insertion within their V4 SSU rRNA region and grouping according to the shape of their spores' sutural line and their similar tissue tropism in the host. Sphaerospora testicularis is the closest relative to Parvicapsula minibicornis within the Parvicapsula subclade and L. scomberomori n. gen. n. sp. is the basal species of the Zschokkella subclade. The phylogenetic position of S. testicularis, outwith the basal Sphaerospora sensu stricto clade, and its morphology suggest it being a non-typical Sphaerospora. The sequence data provided on S. testicularis can help in future revisions of the strongly polyphyletic genus Sphaerospora. We recommend re-sequencing of several sphaerosporids as an essential step before such taxonomic changes are accomplished.     

Spinalona anophtalma, n. gen. n. sp. (Anomopoda, Chydoridae) a blind epigean cladoceran from the Neovolcanic Province of Mexico

Spinalona anophtalma n. gen. n. sp. is describedon parthenogenetic and ephippial females andmales from material collected in a temporary lagoonlocated in the Neovolcanic Province from Mexico at analtitude of 2507 m above sea level. It ischaracterized by a strong armature of the antenna,postabdomen and postabdominal claw, no compound eyeor ocellus, the exopod of thoracic limb IIIwith only four setae and that of P5 with only three setae.This new taxon has no relationwith blind Alona from hypogean habitats.     

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.     

DNA sequence and secondary structure of the mitochondrial small subunit ribosomal RNA coding region including a group-IC2 intron from the cultivated basidiomycete Agrocybe aegerita

Due to their structural complexity and their evolutionary dimension, rRNAs are the most investigated nucleic acids in prokaryotes, eukaryotes and organelles. However, no complete sequence of a mitochondrial small subunit (SSU) rRNA was available in the basidiomycotina subdivision. The mitochondrial gene encoding the SSU rRNA of the cultivated basidiomycete Agrocybe aegerita was cloned and its complete nucleotide sequence achieved; the 5′- and 3′-ends were localized by nuclease S1 mapping, leading to a size of 3277 nt. The secondary structure of the SSU rRNA (1906 nt in size) possessed all the helices and loops of the prokaryotic model; a unique modification was found in a conserved nucleotide predicted by the model: the nt 487 was A instead of C. The same modification, has been found in all the partial basidiomycete mitochondrial sequences available in databases. The Agrocybe aegerita SSU rRNA was characterized by large and unusual extensions leading to additional helices in the variable domains V4, V6 and V9, which were the longest of the known prokaryotic or mitochondrial SSU rRNAs. Nucleotide sequence analysis indicated a 1371-bp intron, belonging to subgroup-IC2, located in a conserved loop in the 3′-part of the SSU rRNA. This intron, which is the second example reported in a fungal mitochondrial SSU rDNA, encoded a putative protein (407 aa) sharing homologies with endonucleases involved in group-I intron mobility. This report constitutes the first complete mitochondrial SSU rRNA sequence and secondary structure of any member of the basidiomycotina subdivision.     

Characterization of Selenaion koniopes n. gen., n. sp., an Amoeba that Represents a New Major Lineage within Heterolobosea,Isolated from the Wieliczka Salt Mine

A new heterolobosean amoeba, Selenaion koniopes n. gen., n. sp., was isolated from 73‰ saline water in the Wieliczka salt mine, Poland. The amoeba had eruptive pseudopodia, a prominent uroid, and a nucleus without central nucleolus. Cysts had multiple crater‐like pore plugs. No flagellates were observed. Transmission electron microscopy revealed several typical heterolobosean features: flattened mitochondrial cristae, mitochondria associated with endoplasmic reticulum, and an absence of obvious Golgi dictyosomes. Two types of larger and smaller granules were sometimes abundant in the cytoplasm—these may be involved in cyst formation. Mature cysts had a fibrous endocyst that could be thick, plus an ectocyst that was covered with small granules. Pore plugs had a flattened dome shape, were bipartite, and penetrated only the endocyst. Phylogenies based on the 18S rRNA gene and the presence of 18S rRNA helix 17_1 strongly confirmed assignment to Heterolobosea. The organism was not closely related to any described genus, and instead formed the deepest branch within the Heterolobosea clade after Pharyngomonas, with support for this deep‐branching position being moderate (i.e. maximum likelihood bootstrap support—67%; posterior probability—0.98). Cells grew at 15–150‰ salinity. Thus, S. koniopes is a halotolerant, probably moderately halophilic heterolobosean, with a potentially pivotal evolutionary position within this large eukaryote group.     

Polkepsilonema mombasae gen. et sp.n. andPternepsilonema servaesae gen. et sp.n. (Nematoda,Epsilonematidae) from East African coasts

Two new genera and species from Kenyan tidal areas,Polkepsilonema mombasae gen. et sp.n. andPternepsilonema servaesae gen. et sp.n., are described. Both are characterized by the presence of at least fourteen subcephalic setae and by thick thorns on the ventral body region of males. In the first genus, eight to ten subcephalic setae are situated anterior to the amphid, and the ambulatory setae are bisinuous. In the second, the subcephalic setae are situated at the posterior edge of the rostrum, and the ambulatory setae are straight.A key to the 13 genera of the Epsilonematidae is presented.Abbreviations a body length divided by maximum body diameter - abd body diameter at level of anus - amph % diameter of amphid as a percent of head diameter - Asl length of anteriormost ambulatory seta of external subventral row - b body length divided by pharyngeal length - c body length divided by tail length - cs length of cephalic setae - dcs distance from rostrum edge to cephalic setae - gub length of gubernaculum - L body length - lct length of copulatory thorns - lpt length of precloacal thorns - mbd maximum body diameter of posterior body region - (mdb) minimum body diameter - mbd/(mbd) maximum body diameter divided by minimum body diameter - mbd ph body diameter at level of pharyngeal bulb - N number of body rings - ph length of pharynx - spic length of spicule measured along the arc - SSph length of subdorsal somatic setae in pharyngeal region - t tail length - tmr length of nonannulated tail region - V position of vulva as a percentage of total body length from anterior     

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.     

Parobia husbandi gen. n., sp. n. (Acari: Podapolipidae): subelytral parasites of paropsine beetles (Coleoptera: Chrysomelidae)

Abstract Parobia husbandi gen. n., sp. n. is described from material collected from the subelytra of the southern eucalyptus leaf beetle, Chrysophtharta agricola (Chapuis), in Tasmania, Australia. Parobia gen. n. is distinguished from other podapolipid genera adult males and females with four pairs of legs and no setae on genua I−IV and femora III−IV, adult females with setae v1 and sc2 < 20 µm, and at least two pairs of coxal setae peg-like, adult males with dorso-terminal genitalia, dorsal shields C-D-E fused, and physogastric larval females with plates C and D entire. A series of morphological plesiomorphies and biological apomorphies link Parobia gen. n. with Chrysomelobia Regenfuss, the most basal genus of Podapolipidae.     

Sinharaja gen. n., a new Uropodina mite genus from Sri Lanka with description of two new species (Acari: Mesostigmata: Uropodidae)

A new Uropodina genus (Sinharaja gen. n.) is described on the basis of two new species (S. ceylonensis sp. n. and S. sinhala sp. n.) from Sri Lanka. The new genus differs by the strongly sclerotised dorsal lines, pentagonal pygidial shield, the shape and length of dorsal setae and the shape of first and fourth hypstomal setae, internal malae and epistome from the other known Uropodina genera.     

Two New Diophrys‐Like Genera and Their Type Species,Apodiophrys ovalis n. g., n. sp. and Heterodiophrys zhui n. g., n. sp. (Ciliophora: Euplotida), with Notes on Their Molecular Phylogeny

ABSTRACT. Based on both morphological and molecular information, two new euplotid genera Apodiophrys n. g. and Heterodiophrys n. g. are described in the present paper. Apodiophrys n. g. is defined as sculptured Diophryinae with bipartite adoral zone; frontoventral cirri arranged in Diophrys‐pattern; marginal cirri located in two clearly separated groups. Heterodiophrys n. g. is recognizable by the combination of Diophrys‐like frontoventral cirri and the unique structure of several marginal cirri that are arranged in a long row. The type species for both new genera, Apodiophrys ovalis n. sp. and Heterodiophrys zhui n. sp., collected from southern China sea, are described. The small subunit rRNA (SSU rRNA) gene sequences for both new taxa are determined. Phylogenetic analyses based on these data indicate that Apodiophrys is most closely related to Paradiophrys, which then clusters with Uronychia species. Thus, Apodiophrys–Paradiophrys is separated from other typical Diophrys‐like genera in the SSU rRNA gene trees. The new genus Heterodiophrys is basal to the sister group of Diophrys–Diophryopsis, hence belongs to the “core”Diophrys‐complex.     

Leptojacobus dorci n. gen., n. sp. (Nematoda: Diplogastridae), an Associate of Dorcus Stag Beetles (Coleoptera: Lucanidae)

A new species of diplogastrid nematode, Leptojacobus dorci n. gen., n. sp., was isolated from adults of the stag beetle Dorcus ritsemae (Coleoptera: Lucanidae) that were purchased from a pet shop in Japan. Leptojacobus n. gen. is circumscribed by a very thin, delicate body and by a small stoma with minute armature. A combination of other stomatal characters, namely the division of the cheilostom into adradial plates, the symmetry of the subventral stegostomatal sectors, and the presence of a thin, conical dorsal tooth, further distinguishes Leptojacobus n. gen. from other genera of Diplogastridae. Phylogenetic analysis of nearly full-length SSU rRNA sequences support the new species, together with an isolate identified previously as Koerneria luziae, to be excluded from a clade including all other molecularly characterized diplogastrids with teeth and stomatal dimorphism. Therefore, the new species will be of importance for reconstruction of ancestral character histories in Diplogastridae, a family circumscribed by a suite of feeding-related novelties.     

Morphology and Molecular Phylogeny of Pseudocyrtohymena koreana n. g., n. sp. and Antarctic Neokeronopsis asiatica Foissner et al., 2010 (Ciliophora,Sporadotrichida), with a Brief Discussion of the Cyrtohymena Undulating Membranes Pattern

We discovered a new brackish water oxytrichid Pseudocyrtohymena koreana n. g., n. sp. in South Korea and investigated the new species on the basis of morphology, ontogenesis, and 18S rRNA gene sequences. The new genus has the 18 frontal‐ventral‐transverse cirri of typical oxytrichids with flexible body, cortical granules, Cyrtohymena undulating membranes (UM), and one left and one right marginal cirral row. Ontogenesis of the new species indicated that dorsal kinety anlage 3 stretches within the parental row without any fragmentations (Urosomoida pattern) and exclusively forms all caudal cirri. The new genus is morphologically similar to Cyrtohymena Foissner, 1989, but has the following distinctive features: (i) caudal cirri absent in dorsal kineties 1 and 2 (vs. present in Cyrtohymena); and (ii) dorsal kinety 3 nonfragmented (vs. fragmented in Cyrtohymena). Further, we collected an additional species Neokeronopsis asiatica Foissner et al. 2010, from King George Island, Antarctica, and the species shares the morphology of UM with Cyrtohymena. Herein, we describe the previously unidentified characteristics of N. asiatica (i.e., cortical granules, body flexibility, contractile vacuole, and 18S rRNA gene sequence). In addition, we obtained two 18S rRNA gene sequences from Cyrtohymena muscorum and Parasterkiella thompsoni to expand samples for phylogenetic analysis. Our 18S rRNA gene tree supports the hypothesis that the Cyrtohymena UM pattern might have evolved several times in hypotrichs (e.g., Neokeronopsidae, Oxytrichinae, and Stylonychinae).     

Sarsicopia polaris gen. et sp.n., the first platycopioida (Copepoda: Crustacea) from the Arctic Ocean,and its phylogenetic significance

A new genus of Platycopioida is described from a boxcore sample taken at a depth of 534 m in the ArcticBarents Sea. This is the deepest record ofPlatycopioida so far. Sarsicopia gen. n. is thesistergroup of a taxon comprising Platycopia and Nanocopia; the sistergroup ofthese is Antrisocopia. Sarsicopia gen. n.is the only platycopioid to retain 2 inner setae onthe second endopod segment P2–P4, and 8 setae in thethird endopod segment of P2. The male antennnule isremarkable in having a geniculation located betweenancestral segments XX and XXI. It is suggested thatthis flexure zone was already present in thegroundpattern of Copepoda. Platycopia and Nanocopia have secondarily lost thisgeniculation.     

Dumontia oregonensis n. fam., n. gen., n. sp., a cladoceran representing a new family of `Water-fleas' (Crustacea,Anomopoda) from U.S.A., with notes on the classification of the Order Anomopoda

Dumontia oregonensis, a cladoceran representing a new family in the Order Anomopoda is described from rain pools in the Agate Desert, Oregon, U.S.A. The proposed family, Dumontiidae, is the newest family within Anomopoda that is not just a reshuffling of already-known species. The general appearance of this novel cladoceran is similar to that of members of the family Macrothricidae. However, a detailed examination of the trunk limbs, particularly of the second pair of limbs, showed that the new species lacks the scraper-setae typically observed in all members of the recently erected suborder Radopoda, to which macrothricids belong. Instead, limb morphology suggests a closer relation of Dumontiidae to the family Daphniidae. Dumontiidae appears to be a `missing' link between the suborder Radopoda and the `non-radopodid' anomopods. The families Daphniidae, Ilyocryptidae, Bosminidae, Moinidae and the new Dumontiidae are similar in that they lack of typical radopodid setae on the second pair of trunk limbs. Further studies on the limb morphology of non-radopodid cladocerans are required to solve the phylogenetic relationships among the members of the order Anomopoda.     

Secondary structure prediction for complete rDNA sequences (18S, 5.8S,and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.     

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.     

Analysis of the Primary Sequence and Secondary Structure of the Unusually Long SSU rRNA of the Soil Bug, Armadillidium vulgare

The complete nucleotide sequence of the SSU rRNA gene from the soil bug, Armadillidium vulgare (Crustacea, Isopoda), was determined. It is 3214 bp long, with a GC content of 56.3%. It is not only the longest SSU rRNA gene among Crustacea but also longer than any other SSU rRNA gene except that of the strepsipteran insect, Xenos vesparum (3316 bp). The unusually long sequence of this species is explained by the long sequences of variable regions V4 and V7, which make up more than half of the total length. RT-PCR analysis of these two regions showed that the long sequences also exist in the mature rRNA and sequence simplicity analysis revealed the presence of slippage motifs in these two regions. The putative secondary structure of the rRNA is typical for eukaryotes except for the length and shape variations of the V2, V4, V7, and V9 regions. Each of the V2, V4, and V7 regions was elongated, while the V9 region was shortened. In V2, two bulges, located between helix 8 and helix 9 and between helix 9 and helix 10, were elongated. In V4, stem E23-3 was dramatically expanded, with several small branched stems. In V7, stem 43 was branched and expanded. Comparisons with the unusually long SSU rRNAs of other organisms imply that the increase in total length of SSU rRNA is due mainly to expansion in the V4 and V7 regions. Received: 2 March 1999 / Accepted: 22 July 1999