Molecular evolution of Cinetochilum and Sathrophilus (Protozoa,Ciliophora, Oligohymenophorea), two genera of ciliates with morphological affinities to scuticociliates

Zhang, Q., Miao, M., Strüder‐Kypke, M. C., Al‐Rasheid, K. A. S., Al‐Farraj, S. A. & Song, W. (2011). Molecular evolution of Cinetochilum and Sathrophilus (Protozoa, Ciliophora, Oligohymenophorea), two genera of ciliates with morphological affinities to scuticociliates. —Zoologica Scripta, 40, 317–325. The ciliate order Loxocephalida sensu Li et al. (2006) has been considered to be systematically uncertain within the subclass Scuticociliatia. Loxocephalids display mixed morphological features and morphogenetic patterns that are found in two different oligohymenophorean subclasses: scuticociliates and hymenostomes. To reveal their phylogenetic positions, molecular information on this group is urgently needed but still inadequate. In the present study, we have sequenced the small subunit rRNA gene of two newly described loxocephalids, Cinetochilum ovale Gong & Song 2008; and Sathrophilus planus Fan et al. 2010; which have never been discussed based on molecular analysis. Results show: (i) all phylogenetic trees are nearly identical in placing Cinetochilum closest to the subclass Apostomatia and form a monophyletic group divergent from the typical scuticociliates, (ii) the genus Sathrophilus, together with Anoplophrya, a poorly known Astomatia, forms a peripheral branch separated from the scuticociliatian assemblage and (iii) the affiliation of the loxocephalid genera sensu Li et al. (2006) is not confirmed due to a dispersion in four deeply diverged clades. In addition, the polyphyly of the genus Cyclidium, shown in previous studies, is confirmed by our phylogenetic analyses and supported by the approximately unbiased test based on the new database in this work.     

Phylogenetic position of the marine ciliate, Cardiostomatella vermiforme (Kahl, 1928) Corliss, 1960 inferred from the complete SSrRNA gene sequence, with establishment of a new order Loxocephalida n. ord. (Ciliophora, Oligohymenophorea)

The small subunit rRNA (SSrRNA) gene was sequenced for Cardiostomatella vermiforme, a large marine ciliate the systematic position of which is uncertain but which has been regarded as a scuticociliate for about forty years. The present work indicates that this organism, together with a closely related species, Dexiotrichides pangi, always form a separate assemblage as a sister group to the scuticociliates sensu stricto. The fact that the clade comprising Cardiostomatella and Dexiotrichides branches between the typical scuticociliates and Hymenostomatia, and shares a series of morphological and morphogenetical characters with both, supports the conclusion that it belongs to an intermediate group between the two. We suggest that this group represents a new order, Loxocephalida n. ord. within the subclass Scuticociliatia, which possibly contains all taxa within the families Loxocephalidae and Cinetochilidae and with Loxocephalidae as the type family.     

Starvation Induces Tomitogenesis in Miamiensis avidus Strain Ma/21

Miamiensis avidus is a marine polymorphic scuticociliate that undergoes microstome to tomite transformation when nutrients are depleted. This transformation takes place as a result of two consecutive divisions and does not involve a cyst. Tomitogenesis was induced by nutrient deprivation. When returned to nutrient medium tomites transformed into microstomes. Changes in population were noted at one-hour intervals. Tomites of Miamiensis avidus Ma/2 are compared to dispersal stages in other scuticociliates and hymenostomes.     

Genetic diversity and phylogenetic position of the subclass Astomatia (Ciliophora) based on a sampling of six genera from West African oligochaetes (Glossoscolecidae, Megascolecidae), including description of the new genus Paraclausilocola n. gen

To more confidently assess phylogenetic relationships among astome ciliates, we obtained small subunit (SSU) rRNA sequences from nine species distributed in six genera and three families: Almophrya bivacuolata, Eudrilophrya complanata, Metaracoelophrya sp. 1, Metaracoelophrya sp. 2, Metaracoelophrya intermedia, Metaradiophrya sp., Njinella prolifera, Paraclausilocola constricta n. gen., n. sp., and Paraclausilocola elongata n. sp. The two new species in the proposed new clausilocolid genus Paraclausilocola n. gen. are astomes with no attachment apparatus, two files of contractile vacuoles, and an arc-like anterior suture that has differentiations of thigmotactic ciliature on the anterior ends of the left kineties of the upper surface. Phylogenetic analyses were undertaken using neighbor-joining, Bayesian inference, maximum likelihood, and maximum parsimony. The nine species of astomes formed a strongly supported clade, showing the subclass Astomatia to be monophyletic and a weakly supported sister clade to the scuticociliates. There were two strongly supported clades within the astomes. However, genera assigned to the same family were found in different clades, and genera assigned to the same order were found in both clades. Thus, astome taxa appear to be paraphyletic when morphology is used to assign species to genera.     

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

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

PHYLOGENY OF THE EUGLENALES BASED UPON COMBINED SSU AND LSU RDNA SEQUENCE COMPARISONS AND DESCRIPTION OF DISCOPLASTIS GEN. NOV. (EUGLENOPHYTA)1

A Bayesian analysis, utilizing a combined data set developed from the small subunit (SSU) and large subunit (LSU) rDNA gene sequences, was used to resolve relationships and clarify generic boundaries among 84 strains of plastid‐containing euglenophytes representing 11 genera. The analysis produced a tree with three major clades: a Phacus and Lepocinlis clade, a Discoplastis clade, and a Euglena, Colacium, Trachelomonas, Strombomonas, Monomorphina, and Cryptoglena clade. The majority of the species in the genus Euglena formed a well‐supported clade, but two species formed a separate clade near the base of the tree. A new genus, Discoplastis, was erected to accommodate these taxa, thus making the genus Euglena monophyletic. The analysis also supported the monophyly of Colacium, Trachelomonas, Strombomonas, Monomorphina, and Cryptoglena, which formed two subclades sister to the Euglena clade. Colacium, Trachelomonas, and Strombomonas, all of which produce copious amounts of mucilage to form loricas or mucilaginous stalks, formed a well‐supported lineage. Our analysis supported retaining Strombomonas and Trachelomonas as separate genera. Monomorphina and Cryptoglena formed two well‐supported clades that were sister to the Colacium, Trachelomonas, and Strombomonas clade. Phacus and Lepocinclis, both of which have numerous small discoid chloroplasts without pyrenoids and lack peristaltic euglenoid movement (metaboly), formed a well‐supported monophyletic lineage that was sister to the larger Euglena through Cryptoglena containing clade. This study demonstrated that increased taxon sampling, multiple genes, and combined data sets provided increased support for internal nodes on the euglenoid phylogenetic tree and resolved relationships among the major genera in the photosynthetic euglenoid lineage.     

Morphology and phylogeny of three trachelocercids (Protozoa,Ciliophora, Karyorelictea), with description of two new species and insight into the evolution of the family Trachelocercidae

Although trachelocercid ciliates are common in marine sandy intertidal zones, methodological difficulties mean that their biodiversity and evolutionary relationships have not been well documented. This paper investigates the morphology and infraciliature of two novel Trachelolophos and one rarely known form, Tracheloraphis similis Raikov and Kovaleva, 1968, collected from the coastal waters of southern and eastern China. The small subunit (SSU) rRNA gene sequences of two of the species are presented, allowing the phylogenetic position of the genus Trachelolophos to be revealed for the first time. Phylogenetic analyses based on SSU rRNA gene sequences indicate that Trachelolophos branches with Kovalevaia and forms a sister clade with the group including Prototrachelocerca, Trachelocerca and Tracheloraphis. The monophyly of Trachelocerca is not rejected by the approximately unbiased (AU) test (P = 0.209, > 0.05) but that of Tracheloraphis is rejected (P = 3e‐033, < 0.05). The newly sequenced genus Trachelolophos, and recent studies on the morphology and phylogeny of the family Trachelocercidae, suggest two new hypotheses about the evolution of the seven genera within Trachelocercidae, based on either infraciliature or molecular evidence. Both hypotheses suppose the compound circumoral kineties in the oral apparatus is a plesiomorphic feature while the single circumoral kinety is synapomorphic. More evidence is still needed, however, as to whether the closed circumoral kinety with no brosse feature in Trachelocerca is ancestral or secondarily reduced. © 2015 The Linnean Society of London     

PHYLOGENY OF PHAGOTROPHIC EUGLENIDS (EUGLENOZOA): A MOLECULAR APPROACH BASED ON CULTURE MATERIAL AND ENVIRONMENTAL SAMPLES1

Molecular studies based on small subunit (SSU) rDNA sequences addressing euglenid phylogeny hitherto suffered from the lack of available data about phagotrophic species. To extend the taxon sampling, SSU rRNA genes from species of seven genera of phagotrophic euglenids were investigated. Sequence analyses revealed an increasing genetic diversity among euglenid SSU rDNA sequences compared with other well‐known eukaryotic groups, reflecting an equally broad diversity of morphological characters among euglenid phagotrophs. Phylogenetic inference using standard parsimony and likelihood approaches as well as Bayesian inference and spectral analyses revealed no clear support for euglenid monophyly. Among phagotrophs, monophyly of Petalomonas cantuscygni and Notosolenus ostium, both comprising simple ingestion apparatuses, is strongly supported. A moderately supported clade comprises phototrophic euglenids and primary osmotrophic euglenids together with phagotrophs, exhibiting a primarily flexible pellicle composed of numerous helically arranged strips and a complex ingestion apparatus with two supporting rods and four curved vanes. Comparison of molecular and morphological data is used to demonstrate the difficulties to formulate a hypothesis about how the ingestion apparatus evolved in this group.     

MULTIGENE ANALYSES OF PHOTOSYNTHETIC EUGLENOIDS AND NEW FAMILY,PHACACEAE (EUGLENALES)1

Bayesian and maximum‐likelihood (ML) analyses of the combined multigene data (nuclear SSU rDNA, and plastid SSU and LSU rDNA) were conducted to evaluate the phylogeny of photosynthetic euglenoids. The combined data set consisted of 108 strains of photosynthetic euglenoids including a colorless sister taxon. Bayesian and ML analyses recovered trees of almost identical topology. The results indicated that photosynthetic euglenoids were divided into two major clades, the Euglenaceae clade (Euglena, Euglenaria, Trachelomonas, Strombomonas, Monomorphina, Cryptoglena, Colacium) and the Phacaceae clade (Phacus, Lepocinclis, Discoplastis). The Euglenaceae clade was monophyletic with high support and subdivided into four main clades: the Colacium, the Strombomonas and Trachelomonas, the Cryptoglena and Monomorphina, and the Euglena and Euglenaria clades. The genus Colacium was positioned at the base of the Euglenaceae and was well supported as a monophyletic lineage. The loricate genera (Strombomonas and Trachelomonas) were located at the middle of the Euglenaceae clade and formed a robust monophyletic lineage. The genera Cryptoglena and Monomorphina also formed a well‐supported monophyletic clade. Euglena and the recently erected genus Euglenaria emerged as sister groups. However, Euglena proxima branched off at the base of the Euglenaceae. The Phacaceae clade was also a monophyletic group with high support values and subdivided into three clades, the Discoplastis, Phacus, and Lepocinclis clades. The genus Discoplastis branched first, and then Phacus and Lepocinclis emerged as sister groups. These genera shared a common characteristic, numerous small discoid chloroplasts without pyrenoids. These results clearly separated the Phacaceae clade from the Euglenaceae clade. Therefore, we propose to limit the family Euglenaceae to the members of the Euglena clade and erect a new family, the Phacaceae, to house the genera Phacus, Lepocinclis, and Discoplastis.     

Molecular phylogeny confirms Conochilidae as ingroup of Flosculariidae (Rotifera,Gnesiotrocha)

We test the hypothesis that conochilid rotifers represent an independent family‐level taxon within Superorder Gnesiotrocha, by analysing their phylogenetic position based on the 18S rDNA sequence of a large number of representatives of this taxon and its putative relatives. Both Bayesian and maximum likelihood analysis confirm a monophyletic clade of all gnesiotrochans with strong branch supports. Within Gnesiotrocha, Conochilidae form a strongly supported clade with representatives of all but some genera of Flosculariidae. These results refute Conochilidae as separate family‐level taxon within Gnesiotrocha. This finding is also supported by a phylogenetic analysis using morphology, in particular new observations on trophi morphology. Conochilid rotifers are likely specialized Flosculariidae, which evolved to a planktonic lifestyle and reduction of coloniality within the group, in contrast to other Flosculariidae. Furthermore, our analysis reveals that two genera of Flosculariidae, Beachampia and Limnias, form a single, strongly supported clade in a sister‐group relation to a clade consisting of representatives of Order Collothecacea. The present results, both regarding position of the conochild rotifers and of two genera of Flosculariidae, highlight the need for a more extensive analysis of relationships within Gnesiotrocha.     

Observations on the Ultrastructure of the Buccal Apparatus in Thigmotrich Ciliates and their Bearing on Thigmotrich-Peritrich Affinities*

SYNOPSIS. In the first use of electron microscopy in a comparative investigation of ciliates belonging to the order Thigmotrichida, ultrastructural data were collected on ciliary organelles of the buccal area. Species studied included members of the genera Ancistrum and Boveria. Analysis of the findings suggest close affinities between thigmotrich and peritrich ciliates. Further comparison with studies on hymenostome (sensu lato) ciliates supportes the hypothesis of a common ancestral stock in the phylogenetic history of all 3 of these groups. Structures very likely homologous in species of hymenostomes, thigmotrichs, and peritrichs include: the undulating membrane, the polykineties, the oral ribs, the filamentous reticulum, and a small field of barren kinetosomes.     

Phylogenetic consideration of two scuticociliate genera, Philasterides and Boveria (Protozoa, Ciliophora) based on 18 S rRNA gene sequences

Many scuticociliates are facultative parasites of aquatic organisms and are among the most problematic ciliate taxa regarding their systematic relationships. The main reason is that most species, especially taxa in the order Thigmotrichida have similar morphology and have not been studied yet using molecular methods. In the present work, two scuticociliate genera, represented by two rare parasitic species, Philasterides armatalis (order Philasterida) and Boveria subcylindrica (order Thigmotrichida), were studied, and phylogenetic trees concerning these two genera were constructed based on their 18 S rRNA gene sequences. The results indicate that: 1) Philasterides forms a sister group with Philaster, supporting the classification that these two genera belong to the family Philasteridae; 2) it is confirmed that the nominal species, Philasterides dicentrarchi Dragesco et al., 1995 should be a junior synonym of Miamiensis avidus as revealed by both previous investigations and the data revealed in the present work; and 3) the poorly known form B. subcylindrica, the only member in the order Thigmotrichida, of which molecular data are available so far, always clusters with Cyclidium glaucoma, a highly specialized scuticociliate, indicating a sister relationship between the orders Thigmotrichida and Pleuronematida.     

Evolution of discocephalid ciliates: Molecular,morphological and ontogenetic data support a sister group of discocephalids and pseudoamphisiellids (Protozoa,Ciliophora) with establishment of a new suborder Pseudoamphisiellina subord. n.

Discocephalids and pseudoamphisiellids are possibly two of the most confused groups among hypotrichous/euplotid ciliates regarding their systematic position and phylogenetic relationships. The former were often regarded as related to euplotids while the latter, in the absence of molecular data, were mostly assigned to the urostylid-like hypotrichs. In the present work, the small subunit rRNA genes of several rarely observed discocephalid and pseudoamphisiellid genera were analyzed to obtain insights into the phylogenetic relationships of these highly ambiguous Spirotrichea. Four different tree reconstruction algorithms yielded nearly identical topologies, which indicated both groups belong to the same assemblage. This assemblage is clearly isolated as a deep-branching clade and invariably positioned between Euplotida and Hypotricha. The sister group relationship of the Pseudoamphisiellidae and Discocephalidae supports the previous suggestion that they might represent an ordinal taxon, the Discocephalida. Both morphological and morphogenetic features indicate that the pseudoamphisiellids should be placed in the order Discocephalida but as a sister group to other typical discocephalids. Thus we propose establishing a new suborder, Pseudoamphisiellina subord. n. The new taxon is diagnosed by the following characteristics: (i) two distantly separated midventral rows that are morphogenetically formed with an urostylid mode; (ii) absence of the “frontoterminal row”, which is formed from the posterior-most frontoventral-transverse cirral anlage in all other typical urostylids; (iii) numerous caudal cirri that derive from each of the dorsal kinety anlagen; (iv) right marginal row that has a unique de novo origin; and (v) inhabiting periphytic communities. The validity of the suborder Pseudoamphisiellina is firmly supported by molecular data.     

Two New Marine Ciliates,Caryotricha rariseta n. sp. and Discocephalus pararotatorius n. sp. (Ciliophora,Spirotrichea), with Phylogenetic Analyses Inferred from the Small Subunit rRNA Gene Sequences

The morphology, infraciliature, and molecular phylogeny of two novel spirotrich ciliates, Caryotricha rariseta n. sp. and Discocephalus pararotatorius n. sp., isolated from coastal waters of China, were investigated. Caryotricha rariseta n. sp. differs from its congeners mainly in possessing seven sparsely ciliated cirral rows that are conspicuously shortened posteriorly and three transverse cirri aligned in a row. Discocephalus pararotatorius n. sp. is characterized by the conspicuous spine‐like dorsal cilia, one extra (endoral?) membrane, and seven frontal, six ventral, and seven posterolateral marginal cirri. The small subunit rRNA gene was sequenced for both species. Complete SSU rRNA gene sequences of two Caryotricha spp. (including C. rariseta n. sp.) and two Discocephalus spp. (including D. pararotatorius n. sp.), along with those of 40 other ciliates, were used to determine their molecular phylogeny using maximum likelihood, neighbor joining and maximum parsimony analyses. The two Caryotricha species cluster with Kiitricha marina in the well‐supported Protohypotrichia clade that is basal to the main spirotrich assemblage. The two discocephalids form a clade that is sister to the Hypotrichia.     

Brandtia ciliaticola gen. et sp. nov. (Chlorellaceae,Trebouxiophyceae) a common symbiotic green coccoid of various ciliate species

Many freshwater protists harbor unicellular green algae within their cells and these host‐symbiont relationships slowly are becoming better understood. Recently, we reported that several ciliate species shared a single species of symbiotic algae. Nonetheless, the algae from different host ciliates were each distinguishable by their different genotypes, and these host‐algal genotype combinations remained unchanged throughout a 15‐month period of sampling from natural populations. The same algal species had been reported as the shared symbiont of several ciliates from a remote lake. Consequently, this alga appears to play a key role in ciliate‐algae symbioses. In the present study, we successfully isolated the algae from ciliate cells and established unialgal cultures. This species is herein named Brandtia ciliaticola gen. et sp. nov. and has typical ‘Chlorella‐like’ morphology, being a spherical autosporic coccoid with a single chloroplast containing a pyrenoid. The alga belongs to the Chlorella‐clade in Chlorellaceae (Trebouxiophyceae), but it is not strongly connected to any of the other genera in this group. In addition to this phylogenetic distinctiveness, a unique compensatory base change in the SSU rRNA gene is decisive in distinguishing this genus. Sequences of SSU‐ITS (internal transcribed spacer) rDNA for each isolate were compared to those obtained previously from the same host ciliate. Consistent algal genotypes were recovered from each host, which strongly suggests that B. ciliaticola has established a persistent symbiosis in each ciliate species.     

Ciliates from eutrophized water in the northern Brazil and morphology of Cristigera hammeri Wilbert, 1986 (Ciliophora,Scuticociliatia)

Ciliates occur in all major aquatic and soil environments worldwide and are important links in the microbial food webs, which, along with other free-living protists, are generally overlooked in biodiversity conservation programs. In Brazil, the northern region comprises the Brazilian Amazonia, an area widely known for its huge biodiversity. However, the diversity of ciliates in that region is still almost unknown. As result of the present study, a total of 21 species of ciliates, distributed among 15 genera, were inventoried from samples of eutrophized water collected in the city of Belém, capital of the state of Pará, one of the states which comprise the Brazilian Amazonia. In addition, a local population of the rare scuticociliate Cristigera hammeri is described from optical and electron microscopy observations, and its synonymy with C. pleuronemoides is rejected based on new evidence.     

The Interrelationships of the Gastrotricha Using Nuclear Small rRNA Subunit Sequence Data, with an Interpretation Based on Morphology

Gastrotrichs are meiobenthic invertebrates of obscure origin and unclear phylogenetic alliances. Uncertainties also plague the intra-group relationship with major contrasts between the evolutionary scenarios inferred from morphology or molecules. In this study we analysed partial sequences of the 18S rDNA gene of 18 taxa (14 new and 4 published) to test morphological estimates of gastrotrich phylogeny and to verify whether controversial interrelationships from previous molecular data are due to poor sampling. Data were analysed using both maximum parsimony and maximum likelihood. MP topology was then forced to reflect published morphological estimates and the most parsimonious solutions from each constraint analysis was statistically compared against the unconstrained solution. MP analysis yielded a single tree with few nodes well supported by bootstrap resampling. These included the monophyly of the Chaetonotidae and the internal relationships of the members of this family, with Aspidiophorus appearing as the most basal member. The monophyly of the Turbanellidae was also well supported with some suggestion that its sister group might be Mesodasys. Lepidodasyidae was found to be an unnatural taxon with Lepidodasys forming a separated clade but unrelated also to the Thaumastodermatidae. With the exception of genera Lepidodasys and Neodasys, the Macrodasyida appeared to be resolved separately from the Chaetonotida, and Dactylopodola was resolved as the most basal macrodasyid. ML analysis yielded a tree not too dissimilar from MP, although Dactylopodola and Xenodasys were resolved as a clade. Statistics indicate that the output from our MP analysis is compatible with the classical view placing representatives of the two orders within two distinct evolutionary lines. Most of the constrained solutions, except the shortest, corroborate the monophyly of the two orders, whereas all five constrained solutions support also the notion that sees Neodasys as an early divergent clade along the Chaetonotida branch. Thus, results are generally compatible with the hypothesised evolutionary scenario based on morphological data, but are in contrast with previous findings from molecules. Future research should consider using the complete SSU rDNA gene sequence in their analysis and additional genes for deeper resolution.     

Complete small subunit rRNA gene sequence of the scuticociliate Miamiensis avidus pathogenic to olive flounder Paralichthys olivaceus

Eight isolates of Miamiensis avidus (scuticociliates) were collected from olive flounder Paralichthys olivaceus with symptoms of severe ulcers and haemorrhages at several culture farms in 1999 and 2003. Cloned strains were produced and the complete small subunit ribosomal RNA gene (SSU rRNA) of each strain was sequenced for classification and phylogenic study. The SSU rRNA is 1759 bp in length and the sequence was deposited in the GenBank under accession number AY550080. All 8 strains exhibited the same sequence, but this sequence did not match any previously deposited scuticociliate SSU rRNA sequence. Phylogenetic analysis placed Miamiensis avidus in a sister lineage to Cohnilembus verminus, Pseudocohnilembus hargisi and P. marinus.