GRACILARIOPSIS MCLACHLANII SP. NOV. AND GRACILARIOPSIS PERSICA SP. NOV. OF THE GRACILARIACEAE (GRACILARIALES,RHODOPHYCEAE) FROM THE INDIAN OCEAN1

Two new species of Gracilariopsis from the Indian Ocean are proposed—Gracilariopsis (Gp.) mclachlanii Buriyo, Bellorin et M. C. Oliveira sp. nov. from Tanzania and Gracilariopsis persica Bellorin, Sohrabipour et E. C. Oliveira sp. nov. from Iran—based on morphology and DNA sequence data (rbcL gene and SSU rDNA). Both species fit the typical features of Gracilariopsis: axes cylindrical throughout, freely and loosely ramified up to four orders, with an abrupt transition in cell size from medulla to cortex, cystocarps lacking tubular nutritive cells and superficial spermatangia. Nucleotide sequence comparisons of rbcL and SSU rDNA placed both species into the Gracilariopsis clade as distinct species from all the accepted species for this genus, forming a deeply divergent lineage together with some species from the Pacific. The new species are very difficult to distinguish on morphological grounds from other species of Gracilariopsis, stressing the importance of homologous molecular marker comparisons for the species recognition in this character‐poor genus.     

Phylogeny and Genetic/Morphological Variation of Strombidinopsis minima‐like Species (Ciliophora: Choreotrichia)

Six isolates of mineral‐enveloped Strombidinopsis minima‐like species were collected from the coastal waters across several regions in Korea. Morphological observations and molecular analyses were performed. The ribosomal DNA sequences (including small subunit ribosomal DNA, internal transcriber spacer 1‐5.8S ribosomal DNA‐internal transcriber spacer 2; and part of large subunit ribosomal DNA) of these six isolates were compared. Their morphological characteristics were also compared with those of S. minima populations reported. The marked genetic differences (with a similarity range of 96.85–98.48%) in SSU rDNA among these S. minima‐like entities suggest the existence of multiple species. This finding is also supported by morphological variations detected in this study and reported in the literature (e.g. 15–32 collar membranelles in different populations). In addition, S. minima‐like species are clustered with S. batos and S. sinicum, and therefore, our SSU rDNA results support previous results suggesting that the genus Strombidinopsis is not monophyletic in origin. Further collection of morphological and molecular data may facilitate the determination of a new genus carrying mineral‐enveloped Strombidinopsis species.     

New considerations on the phylogeny of cyrtophorian ciliates (Protozoa,Ciliophora): expanded sampling to understand their evolutionary relationships

To rationalize the confusing relationships among the cyrtophorian ciliates, we expanded the taxon sampling by sequencing the small subunit ribosomal RNA (SSU rRNA) gene of representatives of 12 genera (20 species, 23 new sequences). The SSU rRNA sequences of Spirodysteria, Agnathodysteria, Brooklynella and Odontochlamys are reported for the first time. Phylogenetic trees were constructed, and secondary structures of variable region 4 (V4) of all genera for which SSU rRNA gene sequence data are available were predicted. The results indicate that (i) Brooklynella is likely an intermediate taxon between Dysteriidae and Hartmannulidae; (ii) the genus Dysteria is paraphyletic with Spirodysteria and Mirodysteria nested within it; (iii) the genus Agnathodysteria is well separated from Dysteria based on both molecular and morphological data; and (iv) Trithigmostoma is a basal genus of Chilodonellidae, based on both the morphological and molecular data.     

Cryptic Speciation in the Genus Cryptoglena (Euglenaceae) Revealed by Nuclear and Plastid SSU and LSU rRNA Gene

The photosynthetic euglenoid genus Cryptoglena is differentiated from other euglenoid genera by having a longitudinal sulcus, one chloroplast, two large trough‐shaped paramylon plates positioned between the chloroplast and pellicle, and lack of metaboly. The genus contains only two species. To understand genetic diversity and taxonomy of Cryptoglena species, we analyzed molecular and morphological data from 25 strains. A combined data set of nuclear SSU and LSU and plastid SSU and LSU rRNA genes was analyzed using Bayesian, maximum likelihood, maximum parsimony, and distance (neighbor joining) methods. Although morphological data of all strains showed no significant species‐specific pattern, molecular data segregated the taxa into five clades, two of which represented previously known species: C. skujae and C. pigra, and three of which were designated as the new species, C. soropigra, C. similis, and C. longisulca. Each species had unique molecular signatures that could be found in the plastid SSU rRNA Helix P23_1 and LSU rRNA H2 domain. The genetic similarity of intraspecies based on nr SSU rDNA ranged from 97.8% to 100% and interspecies ranged from 95.3% to 98.9%. Therefore, we propose three new species based on specific molecular signatures and gene divergence of the nr SSU rDNA sequences.     

Morphology and Phylogeny of Two Species of Loxodes (Ciliophora,Karyorelictea), with Description of a New Subspecies,Loxodes striatus orientalis subsp. n.

The morphology and phylogeny of Loxodes vorax and L. striatus orientalis subsp. n. were investigated based on infraciliature and small subunit (SSU) rRNA gene sequence data. Loxodes striatus orientalis subsp. n. was separated from L. striatus striatus stat. n. by having fewer dikinetids in the intrabuccal kinety (35–55 vs. 50–70) and a variable number of macronuclei (2–4 vs. 2). In addition, the SSU rRNA gene sequence of the new subspecies differs in 13 and 11 nucleotides from that of two populations of the nominotypic subspecies. We also summarized the morphological differences between Loxodes and Remanella based on the data available. Phylogenetic analyses revealed that the genus Loxodes was monophyletic and nested within Remanella species. This study might, therefore, support the hypothesis that the freshwater genus Loxodes evolved from the marine genus Remanella.     

Phylogeny and Establishment of a New Ciliate Family,Wilbertomorphidae fam. nov. (Ciliophora,Karyorelictea), a Highly Specialized Taxon Represented by Wilbertomorpha colpoda gen. nov., spec. nov.

The morphology and molecular phylogeny of a new marine interstitial karyorelictean ciliate, Wilbertomorpha colpoda gen. nov., spec. nov., isolated from a sandy beach at Qingdao, China, are investigated. The ciliature is mainly on the right surface with the left surface barren except for a single marginal kinety, which is typical of the order Loxodida. Nevertheless, other morphological features, such as the highly reduced buccal structure, the absence of a buccal cavity and the presence of extremely specialized cortical granules, prevent its assignation to any known family. Furthermore, the secondary structure of the variable region 2 of the SSU rRNA gene is unique among loxodids. Thus, we establish a new family, Wilbertomorphidae fam. nov. for the genus Wilbertomorpha. Phylogenetic analyses based on SSU rRNA gene sequence data suggest that this taxon is a sister group to the family Geleiidae which belongs to the order Protoheterotrichida. However, morphologically Wilbertomorpha bears a much stronger resemblance to Loxodidae which belongs to the order Loxodida. Based on these findings, we tentatively place Wilbertomorphidae nov. fam. incertae sedis within Karyorelictea.     

Ulva (Chlorophyta,Ulvales) Biodiversity in the North Adriatic Sea (Mediterranean,Italy): Cryptic Species and New Introductions

Ulva Linnaeus (Ulvophyceae, Ulvales) is a genus of green algae widespread in different aquatic environments. Members of this genus show a very simple morphology and a certain degree of phenotypic plasticity, heavily influenced by environmental conditions, making difficult the delineation of species by morphological features alone. Most studies dealing with Ulva biodiversity in Mediterranean waters have been based only on morphological characters and a modern taxonomic revision of this genus in the Mediterranean is not available. We report here the results of an investigation on the diversity of Ulva in the North Adriatic Sea based on molecular analyses. Collections from three areas, two of which subject to intense shipping traffic, were examined, as well as historical collections of Ulva stored in the Herbarium Patavinum of the University of Padova, Italy. Molecular analyses based on partial sequences of the rbcL and tufA genes revealed the presence of six different species, often with overlapping morphologies: U. californica Wille, U. flexuosa Wulfen, U. rigida C. Agardh, U. compressa Linnaeus, U. pertusa Kjellman, and one probable new taxon. U. californica is a new record for the Mediterranean and U. pertusa is a new record for the Adriatic. Partial sequences obtained from historical collections show that most of the old specimens are referable to U. rigida. No specimens referable to the two alien species were found among the old herbarium specimens. The results indicate that the number of introduced seaweed species and their impact on Mediterranean communities have been underestimated, due to the difficulties in species identification of morphologically simple taxa as Ulva.     

CHROMOSOME COUNTS IN CHEILANTHES AND ASPIDOTIS WITH A CONSPECTUS OF THE CYTOLOGY OF THE SINOPTERIDACEAE

Confirmatory, corrective and new chromosome counts are listed for species in the genus Cheilanthes, and a new chromosome count is given for a member of the genus Aspidotis. An analysis of five collections of C. castanea revealed no significant morphological differences despite the different chromosome numbers. The ploidy level of all known species in genera of the Sinopteridaceae is summarized, revealing directions in which future research might proceed to complete our cytological knowledge of this family.     

MOLECULAR PHYLOGENY AND REVISION OF THE GENUS NETRIUM (ZYGNEMATOPHYCEAE,STREPTOPHYTA): NUCLEOTAENIUM GEN. NOV.1

Nuclear‐encoded SSU rDNA, chloroplast LSU rDNA, and rbcL genes were sequenced from 53 strains of conjugating green algae (Zygnematophyceae, Streptophyta) and used to analyze phylogenetic relationships in the traditional order Zygnematales. Analyses of a concatenated data set (5,220 nt) established 12 well‐supported clades in the order; seven of these constituted a superclade, termed “Zygnemataceae.” Together with genera (Zygnema, Mougeotia) traditionally placed in the family Zygnemataceae, the “Zygnemataceae” also included representatives of the genera Cylindrocystis and Mesotaenium, traditionally placed in the family Mesotaeniaceae. A synapomorphic amino acid replacement (codon 192, cysteine replaced by valine) in the LSU of RUBISCO characterized this superclade. The traditional genera Netrium, Cylindrocystis, and Mesotaenium were shown to be para‐ or polyphyletic, highlighting the inadequacy of phenotypic traits used to define these genera. Species of the traditional genus Netrium were resolved as three well‐supported clades each distinct in the number of chloroplasts per cell, their surface morphology (structure and arrangement of lamellae) and the position of the nucleus or nuclear behavior during cell division. Based on molecular phylogenetic analyses and synapomorphic phenotypic traits, the genus Netrium has been revised, and a new genus, Nucleotaenium gen. nov., was established. The genus Planotaenium, also formerly a part of Netrium, was identified as the sister group of the derived Roya/Desmidiales clade and thus occupies a key position in the evolutionary radiation leading to the most species‐rich group of streptophyte green algae.     

PHYLOGENY OF THE BASAL LINEAGES OF STREPTOPHYTA BASED ON RBCL AND ATPB GENE SEQUENCE DATA

The streptophytes comprise the Charophyceae sensu Mattox and Stewart (a morphologically diverse group of fresh‐water green algae) and the embryophytes (land plants). Several charophycean groups are currently recognized. These include the Charales, Coleochaetales, Chlorokybales, Klebsormidiales and Zygnemophyceae (Desmidiales and Zygnematales). Recently, SSU rRNA gene sequence data allied Mesostigma viride (Prasinophyceae) with the Streptophyta. Complete chloroplast sequence data, however, placed Mesostigma sister to all green algae, not with the streptophytes. Several morphological, ultrastructural and biochemical features unite these lineages into a monophyletic group including embryophytes, but evolutionary relationships among the basal streptophytes remain ambiguous. To date, numerous studies using SSU rRNA gene sequences have yielded differing phylogenies with varying degrees of support dependent upon taxon sampling and choice of phylogenetic method. Like SSU data, chloroplast DNA sequence data have been used to examine relationships within the Charales, Coleochaetales, Zygnemophyceae and embryophytes. Representatives of all basal streptophyte lineages have not been examined using chloroplast data in a single analysis. Phylogenetic analyses were performed using DNA sequences of rbcL (the genes encoding the large subunit of rubisco) and atpB (the beta‐subunit of ATPase) to examine relationships of basal streptophyte lineages. Preliminary analyses placed the branch leading to Mesostigma as the basal lineage in the Streptophyta with Chlorokybus, the sole representative of the Chlorokybales, branching next. Klebsormidiales and the enigmatic genus Entransia were sister taxa. Sister to these, the Charales, Coleochaetales, embryophytes and Zygnemophyceae formed a monophyletic group with Charales and Coleochaetales sister to each other and this clade sister to the embryophytes.     

Transferring the freshwater dinoflagellate Peridinium wisconsinense (Dinophyceae) to the family Thoracosphaeraceae,with the description of Fusiperidinium gen. nov.

Distinctive spindle‐shaped thecae first described by Samuel Eddy in 1930 and assigned to the genus Peridinium Ehrenberg are commonly reported from freshwater environments in eastern North America. We demonstrate that thecae incubated from cysts of Peridinium wisconsinense Eddy have six cingular plates and a protuberant apical pore complex characteristic of the family Thoracosphaeraceae Schiller 1930 emend. Tangen in Tangen et al . 1982. Small subunit ribosomal DNA (SSU rDNA) and internal transcribed spacer (ITS) sequences confirm the close genetic similarity with Chimonodinium lomnickii (Wo?oszyńska) Craveiro, Calado, Daugbjerg, Gert Hansen & Moestrup and with species recently reassigned to the genus Apocalathium Craveiro, Daugbjerg, Moestrup & Calado that was inferred from previously published LSU rDNA analysis of cysts of P. wisconsinense . Despite sharing identical tabulation with the thoracosphaeracean genera Chimonodinium Craveiro, Calado, Daugbjerg, Gert Hansen & Moestrup and Apocalathium , substantial morphological differences in the morphology of both the thecate and cyst stages of P. wisconsinense led us to reassign this species to the genus Fusiperidinium gen. nov. The phylogenetic position of Fusiperidinium wisconsinense comb. nov., inferred from concatenated data of SSU and LSU sequences, suggests that it evolved from the brackish Scrippsiella lineage, independently of the transition that produced the family Peridiniaceae. Cysts described as Geiselodinium tyonekensis Engelhardt from nonmarine strata from Alaska are apparently identical to the resistant cysts produced by F. wisconsinense . The palynologically‐constrained late Middle Miocene age for the Tyonek Formation provides a minimum age of 11.6 Ma for the evolution of this lineage, coinciding with a rapid glacioeustatic decline in sea level. Our findings also call into question the inclusion of the family Thoracosphaeraceae within the order Peridiniales Haeckel.     

Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. infecting invasive Chinese mitten crabs (Eriocheir sinensis) in Europe

We describe a microsporidian parasite infecting non-native Chinese mitten crabs (Eriochier sinensis) from Europe. Electron microscopy revealed merogonic and sporogonic life stages bound within a plasmalemma. The crab parasite develops polar tube precursors at the sporont stage but does not complete formation of the intact spore extrusion apparatus at the stage of the sporogonial plasmodium like Enterocytozoon bienuesi and other representatives of the Enterocytozoonidae. Its presence within an aquatic crustacean host, and a distinct molecular phylogeny based on partial small subunit ribosomal RNA (SSU rRNA) gene sequences also place it relatively close, though distinct to, existing genera within the Enterocytozoonidae. Consideration of morphological and phylogenetic characteristics of other hepatopancreas-infecting microsporidia from crustaceans suggests that certain ones (e.g. Enterospora canceri) are retained within the clade corresponding to the existing family Enterocytozoonidae, while others, including the parasite described here, may eventually be grouped in a sister taxon potentially of family rank. Based upon morphological and host similarity, it is likely that the parasite described here is the same as Endoreticulatus eriocheir (Wang and Chen, 2007), previously described from Chinese mitten crabs in Asia. However, using a combined taxonomic approach based upon morphological and phylogenetic data, we propose the formation of a new genus (Hepatospora) to replace the previous generic classification of the Asian parasite as Endoreticulatus. The microsporidian from the hepatopancreas of E. sinensis is named Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. It is assumed that the parasite was introduced during initial invasions of this crab to Europe during the early 20th Century.     

A further phylogenetic study of the heterotrophic dinoflagellate genus, Protoperidinium (Dinophyceae) based on small and large subunit ribosomal RNA gene sequences

The heterotrophic marine dinoflagellate genus Protoperidinium is the largest genus in the Dinophyceae. Previously, we reported on the intrageneric and intergeneric phylogenetic relationships of 10 species of Protoperidinium, from four sections, based on small subunit (SSU) rDNA sequences. The present paper reports on the impact of data from an additional 5 species and, therefore, an additional two sections, using the SSU rDNA data, but now also incorporating sequence data from the large subunit (LSU) rDNA. These sequences, in isolation and in combination, were used to reconstruct the evolutionary history of the genus. The LSU rDNA trees support a monophyletic genus, but the phylogenetic position within the Dinophyceae remains ambiguous. The SSU, LSU and SSU + LSU rDNA phylogenies support monophyly in the sections Avellana, Divergentia, Oceanica and Protoperidinium, but the section Conica is paraphyletic. Therefore, the concept of discrete taxonomic sections based on the shape of 1′ plate and 2a plate is upheld by molecular phylogeny. Furthermore, the section Oceanica is indicated as having an early divergence from other groups within the genus. The sections Avellana and Excentrica and a clade combining the sections Divergentia/Protoperidinium derived from Conica‐type dinoflagellates independently. Analysis of the LSU rDNA data resulted in the same phylogeny as that obtained using SSU rDNA data and, with increased taxon sampling, including members of new sections, a clearer idea of the evolution of morphological features within the genus Protoperidinium was obtained. Intraspecific variation was found in Protoperidinium conicum (Gran) Balech, Protoperidinium excentricum (Paulsen) Balech and Protoperidinium pellucidum Bergh based on SSU rDNA data and also in Protoperidinium claudicans (Paulsen) Balech, P. conicum and Protoperidinium denticulatum (Gran et Braarud) Balech based on LSU rDNA sequences. The common occurrence of base pair substitutions in P. conicum is indicative of the presence of cryptic species.     

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.     

Transfer of the Thecate Amoeba Lecythium mutabilis to a Novel Genus Omnivora (Fiscullidae,Thecofilosea, Cercozoa)

Thecofilosea is a class in Cercozoa (Rhizaria) comprising mainly freshwater‐inhabiting algivores. Recently, numerous isolates of thecofilosean amoebae have been cultured and were characterized by an integrated morphological and molecular approach. As attempts to establish a culture of Lecythium mutabilis repeatedly failed, it was not yet investigated by molecular means. We isolated single cells of L. mutabilis directly from their habitat and successfully sequenced the V4 region of their SSU rDNA. Phylogenetic analyses showed that L. mutabilis is not directly related to the genus Lecythium and instead branches within the Fiscullidae (Tectofilosida, Thecofilosea). Accordingly, we transfer the species L. mutabilis to a novel genus Omnivora gen. nov.     

Trichopodiella faurei n. sp. (Ciliophora,Phyllopharyngea, Cyrtophoria): Morphological Description and Phylogenetic Analyses Based on SSU rRNA and Group I Intron Sequences

ABSTRACT. A new marine cyrtophorian ciliate Trichopodiella faurei n. sp., which belongs to the order Dysteriida, family Hartmannulidae, was investigated at the morphological and molecular levels. A combination of morphological features of the organism including the oval body shape, 2–3 contractile vacuoles, 22–28 nematodesmal rods in the cytopharyngeal basket, and 31–39 somatic kineties, distinguishes it from all other known congeners. In reconstructed small subunit (SSU) rRNA phylogenies, T. faurei groups with Isochona, a representative genus of the subclass Chonotrichia. The similarity of the infraciliature between hartmannulids and several chonotrichian examples also suggests that these taxa should be closely related. A new S943 intron belonging to group IC1 was identified in the SSU rRNA gene of this species. This intron is phylogenetically related to the S891 introns previously found in the suctorians Acineta sp. and Tokophrya lemnarum, and their internal guide sequences share four nucleotides, indicating that these introns were vertically inherited from a common phyllopharyngean ancestor and that reverse splicing might have been involved in the transposition.