New contributions to the phylogeny of the ciliate class Heterotrichea (Protista,Ciliophora): analyses at family-genus level and new evolutionary hypotheses

Heterotrichous ciliates play an important role in aquatic ecosystem energy flow processes and many are model organisms for research in cytology, regenerative biology, and toxicology. In the present study, we combine both morphological and molecular data to infer phylogenetic relationships at family-genus level and propose new evolutionary hypotheses for the class Heterotrichea. The main results include:(1) 96 new ribosomal DNA sequences from 36 populations, representing eight families and 13 genera, including three poorly annotated genera, Folliculinopsis, Ampullofolliculina and Linostomella;(2) the earliest-branching families are Spirostomidae in single-gene trees and Peritromidae in the concatenated tree, but the family Peritromidae probably represents the basal lineage based on its possession of many "primitive" morphological characters;(3) some findings in molecular trees are not supported by morphological evidence, such as the family Blepharismidae is one of the most recent branches and the relationship between Fabreidae and Folliculinidae is very close;(4) the systematic positions of Condylostomatidae, Climacostomidae, and Gruberiidae remain uncertain based either on morphological or molecular data; and(5)the monophyly of each genus included in the present study is supported by the molecular phylogenetic trees, except for Blepharisma in the SSU r DNA tree and Folliculina in the ITS1-5.8 S-ITS2 tree.     

Molecular phylogeny of Stentor (Ciliophora: Heterotrichea) based on small subunit ribosomal RNA sequences

To determine the phylogenetic position of Stentor within the Class Heterotrichea, the complete small subunit rRNA genes of three Stentor species, namely Stentor polymorphus, Stentor coeruleus, and Stentor roeseli, were sequenced and used to construct phylogenetic trees using the maximum parsimony, neighbor joining, and Bayesian analysis. With all phylogenetic methods, the genus Stentor was monophyletic, with S. roeseli branching basally.     

Phylogeny and biogeography of the "Australian" trichostomes (Ciliophora: Litostomata)

The phylogenetic relationships of members of the ciliate class Litostomatea were determined by a molecular phylogeny using the small subunit of the ribosomal RNA (ssu-rRNA) gene and a morphological phylogeny based on ultrastructural analyses of the group. Molecular analyses consistently supported the monophyly of Trichostomatia, Entodiniomorphida and the "Australian" trichostomes but provided limited support for a monophyletic Vestibuliferida and Haptoria. The results of the morphological analyses depended on the way in which the dataset was treated: "unordered" and "ordered" recovered a monophyletic Trichostomatia, Haptoria and the "Australian" trichostomes but challenged the monophyly of Entodinimorphida and Vestibuliferida; "dollo" recovered a monophyletic Trichostomatia and Entodiniomorphida but at the cost of a greatly longer tree than either "unordered" or "ordered" datasets. The monophyly of each "Australian" trichostome family was supported in all analyses and by both approaches. These results suggest that the trichostome ciliates may have become associated with mammals in Gondwana with the "Australian" trichostome ciliates entering Australia with primitive herbivorous marsupials. Subsequent diversification of the "Australian" families was probably a result of dietary specialization and oral and cortical synapomorphies define each family. We decline at this time to erect a formal taxon name for the "Australian" trichostomes due to the instability of other superfamilial taxa within the Litosomatea and concerns about the stability of tree topology until a better taxon sample of litostome ciliates is available.     

Large-scale phylogenetic analysis provides insights into the diversification and evolution of sessilid peritrich ciliates (Protista: Ciliophora)

The Peritrichia is a speciose and morphologically distinctive assemblage of ciliated protists that was first observed by Antonie van Leeuwenhoek over 340 years ago. In the last two decades, the phylogenetic relationships of this group have been increasingly debated as morphological and molecular analyses have generated contrasting conclusions, mainly owing to limited sampling. In the present study, we performed expanded phylogenetic analyses of 152 sessilid peritrichs collected from 14 different provinces of China and 141 SSU rDNA peritrich sequences from GenBank. The results of the analyses revealed new divergent relationships between and within major clades that challenge the morphological classification of this group including, (1) the recovery of four major phylogenetically divergent clades in the monophyletic order Sessilida, (2) aboral structures such as the stalk and spasmoneme were evolutionary labile, (3) the stalk or/and spasmoneme was lost in each divergent clade indicating that parallel evolution occurred in sessilid peritrichs and (4) the life cycle and habit drive the diversity of aboral structures as well as diversification and evolution in peritrichs.     

Reconsideration of the Phylogenetic Positions of Five Peritrich Genera,Vorticella, Pseudovorticella,Zoothamnopsis, Zoothamnium,and Epicarchesium (Ciliophora,Peritrichia, Sessilida), Based on Small Subunit rRNA Gene Sequences

ABSTRACT. In order to re‐evaluate the systematics of sessilid peritrich ciliates, small subunit (SSU) rRNA gene sequences were determined for 12 species belonging to five genera: Vorticella, Pseudovorticella, Epicarchesium, Zoothamnium, and Zoothamnopsis. Phylogenetic trees were deduced using Bayesian inference, maximum parsimony, and maximum likelihood methods. The phylogenetic analyses suggest that (1) sessilids which have stalks with continuous myonemes that contract in a zig‐zag fashion form a separate clade from those which have stalks that contract independently and in a spiral fashion, supporting the separation of the family Zoothamniidae from the family Vorticellidae and (2) Epicarchesium and Pseudovorticella, both of which have reticulate silverline systems, are more closely related to each other than to other vorticellids, suggesting that differences in the silverline system (i.e. transverse vs. reticulate) may be the result of genuine evolutionary divergence among sessilid peritrichs. However, the newly sequenced Zoothamnopsis sinica, which has a reticulate silverline pattern, nests within the unresolved Zoothamnium species that have transverse silverline patterns. Thus, there were at least two evolutions of the reticulate silverline pattern character state from a plesiomorphic transverse state in the peritrichid ciliates. The molecular work demonstrates the genus Zoothamnium to be paraphyletic in relation to morphological studies, and suggests that Astylozoon, Opisthonecta, and Vorticella microstoma possibly share a SSU rRNA secondary structure in the helix E10‐1 region.     

Morphology and Molecular Phylogeny of Two New Brackish Water Ciliates of Bakuella (Ciliophora: Urostylida: Bakuellidae) from South Korea

Two new species of Bakuella, B. (B.) incheonensis n. sp. and B. (Pseudobakuella) litoralis n. sp., were discovered in brackish water near Aamdo Shore Park, Incheon, South Korea. We conducted a morphological study based on live observations and protargol‐impregnated specimens, and a molecular analysis using nuclear SSU rRNA gene sequences. Bakuella (B.) incheonensis is diagnosed by: body size 70–105 × 20–40 μm in vivo, 21–25 adoral membranelles, three or four frontoterminal cirri, midventral complex composed of 7–10 midventral pairs with one or two rows and terminating at about 62% of body length, 20–28 left and 25–32 right marginal cirri, 58–87 macronuclear nodules, and yellowish cortical granules. Bakuella (Pseudobakuella) litoralis is diagnosed by: body size 90–125 × 30–40 μm in vivo, 25–33 adoral membranelles, 3–5 buccal cirri, midventral complex composed 10–15 midventral pairs with one or two rows and terminated at 70% of body length, one or two pretransverse cirri, 3–6 transverse cirri, 26–39 left and 29–47 right marginal cirri, 49–84 macronuclear nodules, and two types of cortical granules. Molecular phylogeny using SSU rRNA gene sequences shows a nonmonophyletic relationship among Bakuella species and emphasizes the need for further morphogenetic studies of this genus and other related hypotrichs.     

Morphological and Molecular Identification of a New Ciliate,Zoothamnium palmphlatum nov. spec. (Ciliophora,Peritrichia) from North China

A new peritrichous ciliate, Zoothamnium palmphlatum nov. spec., was collected from an estuary in Yantai, China. It was investigated, using both live observation and silver staining. The new species can be identified by a palm‐shaped colony consisting of highly developed and alternately arranged secondary branches, a double‐layered peristomial lip, and an infundibular polykinety 3 composed of two parallel kinetosomal rows. Phylogenetic analyses of the small subunit (SSU) rRNA gene sequence show that Z. palmphlatum clusters with other members of the family Zoothamniidae. Furthermore, the comparison of primary and secondary SSU rDNA structures indicates that Z. palmphlatum is distinctly different from its morphologically similar species (93.2–97.0% in sequence similarity) in combination of H10 and H31 regions. Although Z. palmphlatum shares highest sequence similarity with Zoothamnium mucedo (98.9%), the new species has distinctly different structures in the H11, H12, H18, and H31 regions compared to Z. mucedo, which indicates that sequence similarity may not determine the similarity of the secondary structure.     

Morphological and Molecular Description of Three New Species of the Cyrtophorid Genus Chlamydodon (Ciliophora,Cyrtophoria)

This study investigates the morphology and molecular characteristics of three new cyrtophorid ciliates isolated from China seas: Chlamydodon salinus n. sp., Chlamydodon caudatus n. sp., and Chlamydodon paramnemosyne n. sp. Of these, C. salinus n. sp. differs from its congeners through a combination of body size, a cross‐striated band that is not continuous, the presence of 30–34 somatic kineties, 11–15 nematodesmal rods, and 13 contractile vacuoles. Chlamydodon caudatus n. sp., meanwhile, is characterized by having a conspicuous tail, a continuous cross‐striated band, 34–40 somatic kineties, about 15 contractile vacuoles, and 20–24 nematodesmal rods. Compared with other Chlamydodon species, the third new one, C. paramnemosyne n. sp., could be identified by its continuous cross‐striated band, 16–18 somatic kineties, 5 contractile vacuoles, and 9–12 nematodesmal rods. Based on the sequence of the small subunit (SSU) rRNA gene, the phylogeny of these three new species was analyzed, indicating that they all clustered with other congeners to form a monophyletic assemblage. Based on previous studies and the present work, a brief revision of the genus Chlamydodon is supplied, and a key to aid the identification of Chlamydodon species is given.     

A huge,undescribed soil ciliate (Protozoa: Ciliophora) diversity in natural forest stands of Central Europe

We investigated 12 natural forest stands in eastern Austria for soil ciliate diversity, viz., eight beech forests and two lowland and Pinus nigra forests each. The stands span a wide range of climatic (e.g., 543–1759 mm precipitation, 160–1035 m above sea-level) and abiotic (e.g., pH 4–7.4) factors. Samples were taken twice in autumn and late spring and analysed with the non-flooded Petri dish method. Species were identified in vivo, in silver preparations, and in the scanning electron microscope. A total of 233 species were found, of which 30 were undescribed, a surprising number showing our ignorance of soil ciliate diversity, even in Central Europe. Species number varied highly from 45 (acidic beech on silicate) to 120 (floodplain forest) and was strongly correlated with pH and overall habitat quality, as measured by climate, the C/P quotient (ratio of r-selected colpodean and k-selected polyhymenophorean ciliates), and the proportion of mycophagous ciliate species; multivariate analysis showed further important variables, viz., the general nutrient status (glucose, nitrogen, C/ N ratio) and microbial (urease) activity. The highest species number occurred in one of the two floodplain soils, supporting the intermediate disturbance hypothesis. The three main forest types could be clearly distinguished by their ciliate communities, using similarity indices and multidimensional scaling. Individual numbers varied highly from 135^–1 (lowland forest) to 10,925 ml^–1 (beech on silicate) soil percolate and showed, interestingly, a weak correlation with soil protozoan phospholipid fatty acids. Eight of the 30 new species found and a forgotten species, Arcuospathidium coemeterii (Kahl 1943) nov. comb., are described in detail, as examples of how species were recognized and soil protozoan diversity should be analyzed: Latispathidium truncatum bimicronucleatum, Protospathidium fusioplites, Erimophrya sylvatica, E. quadrinucleata, Paragonostomum simplex, Periholosticha paucicirrata, P. sylvatica, and Australocirrus zechmeisterae.     

Diversity of the cyrtophorid genus Chlamydodon (Protista,Ciliophora): its systematics and geographic distribution,with taxonomic descriptions of three species

The ciliate genus Chlamydodon is characterized by a unique cross-striated band (CSB) along the cell perimeter. To date, more than 15 nominal species have been assigned to this genus, all of which are exclusively from marine or brackish water. In the present work, we have revised the genus according to the available data and suggest an illustrated key to aid species diagnosis. In addition, the systematic relationships of chlamydodontid congeners were analysed based on SSU rRNA gene sequences, indicating that all congeners belong to a well-defined clade. Furthermore, we investigated three species from coastal areas of China, including two new species, Chlamydodon wilberti sp. nov. and C. bourlandi sp. nov., using morphological and phylogenetic criteria. Chlamydodon wilberti sp. nov. is characterized by a cell size of 65–105 × 35–60 µm, a complete CSB, and 38–49 somatic kineties. Chlamydodon bourlandi sp. nov. has a cell size of 150–250 × 65–150 µm, a complete CSB, a reddish to violet cell colour, 83–97 somatic kineties, and 40–68 contractile vacuoles. As a further contribution, a well-studied species, C. triquetrus (Müller, 1786) Kahl, 1931, is also re-described.

www.zoobank.org/urn:lsid:zoobank.org:pub:B83A5466-9D2B-4502-9A23-F16A61D48172.     

Morphology and molecular phylogeny of Holostichides terrae nov. spec. (Ciliophora: Spirotrichea) with discussion on the possible non-monophyly of Holostichides

In a study to investigate ciliate diversity, we discovered a new soil ciliate. Holostichides terrae nov. spec. was examined and identified based on observations of living cells and stained specimens. In addition, the nuclear SSU rRNA gene along with morphology was analyzed to infer its phylogenetic position. The new species closely resembles H. dumonti, but can be distinguished by the morphology of the pharynx (with rod-shaped structure vs. lacking) and the number of frontoterminal cirri (invariably two vs. usually more than two). Molecular analyses indicate that the genus Holostichides is not monophyletic, and H. terrae is closely related with the genera Birojimia and Hemicycliostyla, both of which have a pharynx with rod-shaped structures, as also seen in H. terrae.     

A Description of a New "Amoebozoan" Isolated from the American Lobster, Homarus americanus

ABSTRACT. Our knowledge of the diversity of amoeboid protists is rapidly expanding as new and old habitats are more fully explored. In 2003, while investigating the cause of an amoeboid disease afflicting lobsters on the East Coast, samples were examined for the presence of amoebae from the carapace washings of the American lobster, Homarus americanus . During this survey a unique community of gymnamoebae was discovered. Among the new taxa discovered was a small Thecamoeba -like organism with a single posteriorly directed pseudopodium. Although resembling Parvamoeba rugata , this amoeba displayed distinctive morphology from that isolate or any other amoebozoan. Phylogenetic analysis shows this amoeba is distantly related to the Thecamoebidae. In this paper we describe the unique morphology of a second species of Parvamoeba and discuss its phylogenetic position with respect to the "Amoebozoa."     

A unique euplotid ciliate, Gastrocirrhus (Protozoa, Ciliophora): assessment of its phylogenetic position inferred from the small subunit rRNA gene sequence

ABSTRACT. The morphologically unique genus Gastrocirrhus has been considered a distinct but systematically uncertain euplotid due to the absence of both morphogenetic and molecular information. Based on the small subunit rRNA gene sequence, the phylogenetic position of Gastrocirrhus monilifer Ozaki & Yagui, 1942 was re-addressed using multiple algorithms (neighbor-joining, maximum parsimony, least-squares, and Bayesian inference methods). Results indicate that: (1) all phylogenetic trees using different methods are nearly identical in topology, placing G. monilifer closest to Euplotidium arenarium ; (2) Gastrocirrhus and Euplotidium form a monophyletic group, namely the family Gastrocirrhidae, and appear to be intermediate taxa bridging the evolution of the Diophrys-Uronychia and Euplotes- complexes (i.e. Euplotes, Certesia , and Aspidisca ); (3) the order Euplotida is a paraphyletic group composed of three deeply diverged clades ( Euplotes–Certesia–Aspidisca – Gastrocirrhus–Euplotidium ; Uronychia – Diophrys ; and Prodiscocephalus ); (4) together with Prodiscocephalus , the Diophrys-Uronychia complex forms a group at the suborder level and is placed at the root of the order Euplotida, and (5) results from molecular analyses conspicuously challenge the conclusions deduced from morphological as well as morphogenetical investigations—the characteristics traditionally used to define the euplotid taxa at the generic level and/or above may not be uniformly reliable.     

Vannella epipetala n. sp. isolated from the leaf surface of Spondias mombin (Anacardiaceae) growing in the dry forest of Costa Rica

As part of a Microbial Observatory of Caterpillars located in the Area de Conservacíon Guanacaste (ACG) in northwestern Costa Rica, we isolated a novel species of the genus Vannella associated with the food of the caterpillars of the saturniid moth Rothschildia lebeau, namely the leaves of the dry forest deciduous tree Spondias mombin (Anacardiaceae). The new species can be distinguished from other described species of the genus by the presence of a plasmalemma coated with a thickened, osmiophilic lamina containing glycostyles, and by its unusual habitat, the leaf surfaces or phylosphere of S. mombin. We further established the novelty of our isolate by sequencing its nuclear small-subunit (SSU) rRNA gene and inferring its phylogenetic position among all other currently sequenced members of the genera Vannella and Platyamoeba. Our results reveal that our isolate shares most recent common ancestry with three strains of Platyamoeba placida, the type species of the genus Platyamoeba. Despite this placement, the isolate clearly possesses glycostyles that are the hallmark of the genus Vannella. In addition to the cultured isolate, we also present a closely related sequence from a SSU rRNA gene clone library constructed from a DNA extract of leaf-wash of S. mombin with sterile water.     

Taxonomic contributions to Hapalidiales (Corallinophycidae,Rhodophyta): Boreolithothamnion gen. nov., Lithothamnion redefined and with three new species and Roseolithon with new combinations

Phylogenetic analyses of rbcL gene sequences and of concatenated rbcL, psbA, and nuclear SSU rRNA gene sequences resolved the generitype of Lithothamnion, L. muelleri, in a clade with three other southern Australian species, L. kraftii sp. nov., L. saundersii sp. nov., and L. woelkerlingii sp. nov. Cold water boreal species currently classified in Lithothamnion and whose type specimens have been sequenced are transferred to Boreolithothamnion gen. nov., with B. glaciale comb. nov. as the generitype. The other species are B. giganteum comb. nov., B. phymatodeum comb. nov., and B. sonderi comb. nov., whose type specimens are newly sequenced, and B. lemoineae comb. nov., B. soriferum comb. nov., and B. tophiforme comb. nov., whose type specimens were already sequenced. Based on rbcL sequences from the type specimens of Lithothamnion crispatum, L. indicum, and L. superpositum, each is recognized as a distinct species and transferred to the recently described Roseolithon as R. crispatum comb. nov., R. indicum comb. nov., and R. superpositum com. nov., respectively. To correctly assign species to these three genera based only on morpho-anatomy, specimens must have multiporate conceptacles and some epithallial cells with flared walls. The discussion provides examples demonstrating that only with phylogenetic analyses of DNA sequences can the evolution of morpho-anatomical characters of non-geniculate corallines be understood and applied at the correct taxonomic rank. Finally, phylogenetic analyses of DNA sequences support recognition of the Hapalidiales as a distinct order characterized by having multiporate tetra/bisporangial conceptacles, and not as a suborder of Corallinales whose tetra/bisporangial conceptacles are uniporate.     

Species boundaries in tintinnid ciliates: a case study--morphometric variability, molecular characterization, and temporal distribution of Helicostomella species (Ciliophora, Tintinnina)

Tintinnids are a large group of planktonic ciliates with diverse morphologies. The range of variability in lorica shapes and sizes can be very high even within a single species depending on life stages and environmental conditions, which makes the delimitation of different species based on morphological criteria alone very difficult. Accordingly, comparisons of morphological and molecular variability in tintinnids are necessary to provide a pragmatic approach for establishing species boundaries within this diverse and poorly understood group. We investigated the temporal distribution of species of the hyaline tintinnid Helicostomella (Ciliophora, Tintinnina), which were collected daily from September 2008 to August 2009 in Jangmok Bay of Geoje Island on the south coast of Korea. Two forms - a long form and a short form, were discovered. The long form was found in cold waters in February and March whereas the short form occurred in warm waters from June to October. Thus, these two forms were seasonally isolated. However, all the morphological characteristics for these two forms overlap to some degree. A comparison of the small subunit (SSU) rDNA, ITS1-5.8S-ITS2, and ITS2 sequences from two forms revealed 0.5%, 3.8%, and 5.6% divergences, respectively. Morever, one compensatory base change (CBC) and three hemi-CBCs were identified from ITS2 secondary structures of these two forms. All these data suggest that these two forms represent two distinct species despite their highly similar lorica morphology. The phylogenetic position of the genus Helicostomella was also examined using SSU rDNA sequences.