Morphology and morphogenesis of Apoholosticha sinica n. g., n. sp. (Ciliophora,Hypotrichia), with consideration of its systematic position among urostylids

This paper investigates the morphology, morphogenesis and SSU rRNA gene-based phylogeny of Apoholosticha sinica n. g., n. sp., isolated from mangrove wetland in Shenzhen, southern China. The new genus Apoholosticha is characterized by its bipartite adoral zone, clearly differentiated frontal cirri arranged in a bicorona, midventral complex composed of midventral pairs only, one marginal cirral row on each side, presence of frontoterminal and transverse cirri, and the lack of a buccal cirrus and caudal cirri. The type species, Apoholosticha sinica n. sp. is diagnosed by the elongated body shape and two kinds of cortical granules. Its main morphogenetic features are similar to that of Pseudokeronopsis except for (1) no buccal cirrus is formed and (2) its macronuclear nodules fuse into a single mass during cell division. Phylogenetic analyses for the new taxon indicate that Apoholosticha n. g. is most closely related to Nothoholosticha and Heterokeronopsis, and falls into the family Pseudokeronopsidae within the core Urostylida clade. In addition, a species that had been misidentified in previous literature is here recognized and assigned to the new genus as Apoholosticha sepetibensis (Wanick and Silva-Neto, 2004) n. comb. (basionym: Pseudokeronopsis sepetibensis Wanick and Silva-Neto, 2004).     

A Molecular Phylogenetic Investigation of Bakuella,Anteholosticha, and Caudiholosticha (Protista,Ciliophora, Hypotrichia) Based on Three‐Gene Sequences

Traditionally classifications of the Urostyloida have been mainly based on morphology and morphogenesis. Recent molecular phylogenetic analyses have been largely based on single‐gene data for a limited number of taxa. Consequently, incongruence has arisen between the morphological/morphogenetic and the molecular data. In this study, the three phylogenetic markers (SSU rDNA, ITS1‐5.8S‐ITS2 region, and LSU‐rDNA) of three urostyloid genera represented by four species (Bakuella granulifera, Anteholosticha monilata, Caudiholosticha sylvatica, and C. tetracirra) were sequenced to investigate their phylogeny. The results show that: (1) all three genera should be regarded as the members of the order Urostyloida within the subclass Hypotrichia, as indicated by morphological characters; (2) phylogenetic analyses and sequence similarities both indicate that neither Anteholosticha nor Caudiholosticha are monophyletic and the systematic assignment of both genera awaits further evaluation; and (3) Bakuella has a closer relationship with Urostyla than with bakuellids (e.g. Apobakuella and Metaurostylopsis), suggesting Bakuella may belong to the family Urostylidae rather than the family Bakuellidae.     

Morphology and taxonomy of Pseudokeronopsis rubra (Ehrenberg, 1836) and Pseudokeronopsis parasongi sp. nov. (Ciliophora,Hypotrichia, Urostylida) from the Yellow Sea

Pseudokeronopsis rubra (Ehrenberg, 1836), type species of Pseudokeronopsis, is a widely reported but taxonomically confused species with most available sequences either misidentified or dubious. Based on the Yellow Sea population, of which the molecular data have been analyzed in a previous work, we redescribe P. rubra and try to clarify the identifications of the populations designated as P. rubra or those allocated to this species. Nonetheless, there is still a need of integrated investigation of the species combing both morphological and molecular data based on specimens from its type locality. Recent molecular analyses indicate a Yellow Sea population of Pseudokeronopsis (marked as P. cf. songi) likely represents a new species. Here we describe it as P. parasongi sp. nov. based on the specimens from the same population. The new species possesses a yellow-brownish body with both pigmented cortical granules and colorless blood cell-shaped structures underneath the body surface, invariably one contractile vacuole at 66% of body length and occasionally a second one at 25%, and the midventral complex terminating 2–23 μm ahead of 2–4 transverse cirri. It differs from closely related species by its yellow-brownish body color and separates from them in the ITS1-5.8S-ITS2 and COI genetic distances.     

Phylogenetic positions and taxonomic assignments of the systematically controversial genera,Spirotrachelostyla, Uroleptopsis and Tunicothrix (Protozoa,Ciliophora, Stichotrichia) based on small subunit rRNA gene sequences

The phylogenetic positions of three systematically controversial genera of ciliates, Spirotrachelostyla, Uroleptopsis and Tunicothrix, have never been established by molecular data. The small subunit rRNA genes of three species, S. tani, U. citrina and T. wilberti, were sequenced and added to existing sequences of stichotrichs and other ciliates to construct phylogenetic trees. Results indicate the following: (1) Uroleptopsis is most closely related to species of Pseudokeronopsis, supporting its assignment to the family Pseudokeronopsidae; (2) one sampled Tunicothrix branches sister to the two sampled Parabirojimia, and this supports the placement of Tunicothrix in the Parabirojimidae; (3) Spirotrachelostyla clusters consistently with Trachelostyla to form a distinct, divergent clade that associates with Amphisiella at the base of the entire sporadotrich-urostylid clade, confirming the hypothesis that Spirotrachelostyla should be placed in the family Trachelostylidae.     

Morphology,Ontogeny, and Phylogeny of Two Brackish Urostylid Ciliates (Protist,Ciliophora, Hypotricha)

The diversity of hypotrichous ciliates has encouraged numerous researchers to use a combination of morphological, morphogenetic, and phylogenetic data to provide a better understanding of the evolutionary relationships within this complex group. In this study, we investigate the morphology and morphogenesis of Pseudourostyla subtropica sp. nov., isolated from mangrove wetland. The new species can be distinguished from its congeners by the huge body size, many more adoral membranelles and marginal cirral rows, and numerous macronuclear nodules. In addition, we provide a morphological characterization of a population of Pseudourostyla nova Wiackowski 1988 from an estuarine habitat. The main events during binary fission of P. subtropica sp. nov. and the Chinese population of P. nova are also revealed to be conservative. The morphological, ontogenetic, and phylogenetic analyses based on the SSU rDNA sequences corroborate the monophyly of Pseudourostyla Borror, 1972, which corresponds well with previous research. The phylogenetic analyses also show that Pseudourostyla and Hemicycliostyla Stokes, 1886, both of which are assigned to the family Pseudourostylidae based on morphological and morphogenetic data, in fact fall into separated clades. The approximately unbiased tests, however, do not reject the possibility that the family Pseudourostylidae is a monophyletic lineage.     

Molecular phylogeny of Nothoholosticha (Protozoa,Ciliophora, Urostylida) and systematic relationships of the Holosticha-complex

The marine ciliate Nothoholosticha is characterized by having a Holosticha-like ciliature pattern but without migratory frontoterminal cirri. However, its systematic position and its relationship to other members of the Holosticha-complex have not yet been resolved. In order to gain deeper insights into these relationships, the small subunit rRNA (SSrRNA) gene and the rRNA internal transcribed spacer and 5.8SrRNA (ITS1–5.8S-ITS2) region of two marine Anteholosticha species, as well as the ITS1–5.8S-ITS2 region of N. fasciola, were sequenced, and molecular trees (BI, ML, NJ and MP trees) were constructed. Although our analyses failed to conclusively resolve the phylogeny of this assemblage, certain conclusions could be drawn. Firstly, Nothoholosticha is a valid genus that is more closely related to Pseudokeronopsis than to other Holosticha-complex genera. Secondly, sequence analyses and phylogenetic trees of several Anteholosticha species revealed a high molecular diversity, which does not support the monophyly of this genus. Thirdly, the current assignment of certain well-known genera, e.g. Holosticha, Anteholosticha, Apokeronopsis, Parabirojimia, Psammomitra, Diaxonella, Metaurostylopsis and Thigmokeronopsis, to the families Bakuellidae (sensu Berger 2006), Urostylidae (sensu Berger 2006) or Holostichidae (sensu Berger 2006) is challenged by the molecular data presented here. And fourthly, the families Holostichidae and Pseudokeronopsidae (sensu Lynn 2008) are probably paraphyletic, and their systematic assignments await further evaluation.     

Morphology,molecules and the phylogeny of Characidae (Teleostei,Characiformes)

This is the most comprehensive phylogenetic analysis of the Characidae to date and the first large-scale hypothesis of the family, combining myriad morphological data with molecular information. A total of 520 morphological characters were analysed herein, of which 98 are newly defined. Among the analysed taxa, 259 species were coded by examining specimens, three fossil species were coded from the literature, one species was coded almost completely from published figures, 122 were partially coded from the literature, and 88 were analysed exclusively from molecular data. The total number of species in the analysed dataset is 473. Analyses were made by parsimony under equal and extended implied weighting with a broad range of parameters. The final hypothesis was selected using a stability criterion that chooses among the most parsimonious trees of all searches. It was found by weighting molecular characters with the average homoplasy of entire partitions (markers). The resulting hypothesis is congruent with previous molecular-based phylogenies of the family. The Characidae are monophyletic, with four main clades: the Spintherobolinae new subfamily; an expanded Stethaprioninae including the Grundulini, Gymnocharacini, Rhoadsiini and Stethaprionini; the Stevardiinae; and a clade composed of the Aphyocharacinae, Characinae, Cheirodontinae, Exodontinae and Tetragonopterinae. Also, a stem Characidae was found, as formed by the Eocene–Oligocene genera †Bryconetes and †Paleotetra as successive sister groups of extant members of the family. A subfamilial classification is proposed, but deep changes in the systematics that are beyond the scope of this study are still needed to classify the Characidae into monophyletic genera.     

Phylogeny of some systematically uncertain urostyloids--Apokeronopsis, Metaurostylopsis, Thigmokeronopsis (Ciliophora, Stichotrichia) estimated with small subunit rRNA gene sequence information: discrepancies and agreements with morphological data

The small subunit rRNA (SSrRNA) genes of seven species of urostyloids representing four genera were sequenced. These were: Apokeronopsis crassa, A. bergeri, Anteholosticha sp-QD-1, Metaurostylopsis sp-QD-1, M. sp-QD-2, M. sp-QD-3 and Thigmokeronopsis sp-QD-1. Gene trees were constructed in order to investigate their phylogenetic relationships. The results indicate that: (1) Apokeronopsis, Thigmokeronopsis and Metaurostylopsis form a well-supported, clearly isolated, monophyletic group; (2) Metaurostylopsis species analysed consistently group together indicating that it is a well-outlined genus; (3) the validity of the genus Apokeronopsis is supported; (4) the separation of Holosticha and Anteholosticha is supported although Anteholosticha species exhibit a high molecular diversity; (5) Pseudokeronopsis and Thigmokeronopsis, which have been considered closely related, may not share a recent common ancestor, casting doubt on the monophyly of the family Pseudokeronopsidae.     

Molecular phylogeny,analysis of character evolution,and submersible collections enable a new classification of a diverse group of gobies (Teleostei: Gobiidae: Nes subgroup), including nine new species and four new genera

The Nes subgroup of the Gobiosomatini (Teleostei: Gobiiformes: Gobiidae) is an ecologically diverse clade of fishes endemic to the tropical western Atlantic and eastern Pacific oceans. It has been suggested that morphological characters in gobies tend to evolve via reduction and loss associated with miniaturization, and this, coupled with the parallel evolution of adaptations to similar microhabitats, may lead to homoplasy and ultimately obscure our ability to discern phylogenetic relationships using morphological characters alone. This may be particularly true for the Nes subgroup of gobies, where several genera that are diagnosed by ‘reductive characters’ have been shown to be polyphyletic. Here we present the most comprehensive phylogeny to date of the Nes subgroup using mitochondrial and nuclear sequence data. We then evaluate the congruence between the distribution of morphological characters and our molecular tree using maximum‐likelihood ancestral state reconstruction, and test for phylogenetic signal in characters using Pagel's λ tree transformations (Nature, 401 , 1999 and 877). Our results indicate that all of the characters previously used to diagnose genera of the Nes subgroup display some degree of homoplasy with respect to our molecular tree; however, many characters display considerable phylogenetic signal and thus may be useful in diagnosing genera when used in combination with other characters. We present a new classification for the group in which all genera are monophyletic and in most cases diagnosed by combinations of morphological characters. The new classification includes four new genera and nine new species described here, many of which were collected from rarely sampled deep Caribbean reefs using manned submersibles. The group now contains 38 species in the genera Carrigobius gen. nov., Chriolepis, Eleotrica, Gobulus, Gymneleotris, Nes, Paedovaricus gen. nov., Pinnichthys gen. nov., Psilotris, and Varicus. Lastly, we provide a key to all named species of the Nes subgroup along with photographs and illustrations to aid in identification.     

Evolution of the order Urostylida (Protozoa, Ciliophora): new hypotheses based on multi-gene information and identification of localized incongruence

Previous systematic arrangement on the ciliate order Urostylida was mainly based on morphological data and only about 20% taxa were analyzed using molecular phylogenetic analyses. In the present investigation, 22 newly sequenced species for which alpha-tubulin, SSU rRNA genes or ITS1-5.8S-ITS2 region were sampled, refer to all families within the order. Following conclusions could be drawn: (1) the order Urostylida is not monophyletic, but a core group is always present; (2) among the family Urostylidae, six of 10 sequenced genera are rejected belonging to this family; (3) the genus Epiclintes is confirmed belonging to its own taxon; (4) the family Pseudokeronopsidae undoubtedly belongs to the core portion of urostylids; however, some or most of its members should be transferred to the family Urostylidae; (5) Bergeriellidae is confirmed to be a valid family; (6) the distinction of the taxon Acaudalia is not supported; (7) the morphology-based genus Anteholosticha is extremely polyphyletic; (8) ITS2 secondary structures of Pseudoamphisiella and Psammomitra are rather different from other urostylids; (9) partition addition bootstrap alteration (PABA) result shows that bootstrap values usually tend to increase as more gene partitions are included.     

Reappraisal of Primula ranunculoides (Primulaceae), an endangered species endemic to China,based on morphological,molecular genetic and reproductive characters

The taxonomic status of Primula ranunculoides and other members of section Ranunuculoides is reappraised here based on data from morphological, reproductive and molecular characters. Multivariate analysis of morphological characters indicates that P. ranunculoides is a coherent species that can be distinguished from its sectional congeners P. cicutariifolia and P. merrilliana by the characters of simple kidney‐shaped outer leaves and the unique clonal reproductive ability by which apices of the scape differentiate into bulblets at the late phases of flowering. Recognition of P. ranunculoides at the specific level is also supported by palynological characters, breeding system, cross pollination results and molecular phylogenetic analysis of nrDNA internal transcribed spacer sequences. A taxonomic revision of section Ranunuculoides is presented and a possible mechanism of speciation discussed. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169 , 338–349.     

Phylogenetic relationships of the family Axinellidae (Porifera: Demospongiae) using morphological and molecular data

Alvarez, B., Crisp, M.D., Driver, F., Hooper, J.N.A. & Van Soest, R.W.M. (2000). Phylogenetic relationships of the family Axinellidae (Porifera: Demospongiae) using morphological and molecular data. —Zoologica Scripta, 29, 169–198. Twenty‐seven species of marine sponges belonging to Axinellidae and related groups (Halichondriidae, Dictyonellidae, Agelasida) were selected to test the monophyly of Axinellidae and investigate their phylogenetic relationships using parsimony and maximum likelihood methods. Partial 28S rDNA sequences, including the D3 domain, and traditional morphological characters (mainly skeletal ones) were used independently to construct phylogenetic trees. Sequences were aligned using the appropriate model of secondary structure of the RNA and compared to that produced by the multiple sequence alignment program, ClustalW. The alignment using secondary structure constraints produced a better estimate of the phylogeny and was demonstrated to be an effective and objective method. Results of the cladistic analyses of the molecular and morphological data sets were not fully congruent; the morphological data suggest that Axinellidae is monophyletic, however, the molecular data suggest that it is nonmonophyletic. The single most‐parsimonious tree derived from the molecular data showed that species of Axinella (except A. polypoides) are united in a clade that is more closely related to members of Agelasida than to other species of Axinellidae; the remaining members of Axinellidae form a monophyletic group that is closely related to the families Dictyonellidae and Halichondriidae. The consensus tree of 20 most‐parsimonious trees from the morphological analysis, on the other hand, showed that all the sampled species of Axinellidae belong to a monophyletic group which is closely related to the species of Dictyonellidae and Halichondriidae. Only two branches were identical in both cladograms, the one uniting the species of Ptilocaulis and Reniochalina and the one with the species of Dictyonellidae. The robustness of the molecular and morphological trees (or parts of the trees), was tested using bootstrap, jack‐knife, PTP and T‐PTP tests. The results of the PTP test were significant indicating significant cladistic structure in both data sets. The bootstrap and jack‐knife values indicate that the molecular tree is in general better supported than the morphological one. The lack of morphological characters and the homoplastic nature of some may explain the weak support of the morphological tree. A T‐PTP test of nonmonophyly showed that the nonmonophyly of Axinellidae, as indicated by the results of the molecular analysis, is not significant; however, a T‐PTP test of monophyly of Axinellidae, as indicated by the morphological tree, produced significant results. This indicates that the monophyly of Axinellidae based on morphological data cannot be rejected; the family however, cannot be defined in terms of a unique diagnostic character common to all members of the ingroup. Tests of heterogeneity (reciprocal T‐PTP and partition homogeneity test) indicated that the data partitions are heterogeneous, which could be due to sampling errors (in either data set) or differences in the underlying phylogenies; therefore data were not combined in a single analysis. Further, both data sets are unequally sized (95 informative molecular characters vs. 16 informative morphological characters), which means that the molecular signal could swamp the morphological signal if the data is combined. Nonmonophyly of Axinellidae is supported by chemical and genetic evidence available in the literature and DNA sequences data of axinellid species from New Zealand. However, this needs to be confirmed using independent evidence from different genes (or gene regions), biochemistry, histology or cell ultrastructure. Therefore, no changes to the taxonomic position of the family in the higher classification are proposed at this stage.     

An integrative approach for species delimitation in the spider genus Grammostola (Theraphosidae,Mygalomorphae)

The mygalomorph genus Grammostola (family Theraphosidae) is endemic to South America. The species Grammostola anthracina is one of the largest spiders in Uruguay and reputed to be the longest lived tarantula in the world. This nominal species has two distinct colour morphs comprising black and reddish‐brown forms with controversial taxonomic status. Here, we present a phylogenetic study based on molecular characters (cytochrome c oxidase subunit I) of haplotypes of G. anthracina and closely related species. Our analysis together with new morphological data and biogeographical information indicates that the two morphs of G. anthracina constitute different species that are not sister to each other. Consequently, a new species, Grammostola quirogai is described, diagnosed and illustrated to encompass the black morph. Phylogenetic relationships and new taxonomic characters for Grammostola species included in this study are discussed.     

Hidden biodiversity: total evidence phylogenetics and evolution of morphological traits in a highly diverse lineage of endogean ground beetles,Typhlocharis Dieck, 1869 (Carabidae,Trechinae, Anillini)

Typhlocharis is the most diverse eyeless endogean ground beetle genus known to date, with 62 species all endemic to the West Mediterranean region. The lineage is characterized by a conservative and singular body plan within Carabidae that contrasts with a high morphological diversity in many traits. We provide an exhaustive phylogeny of the lineage through the study of 92 morphological characters from all 62 described species and 45 potential new species from 70 additional populations, and the combination of morphological and available molecular data, in the first total evidence phylogenetic approach for a highly diverse endogean lineage. We tracked the evolution of morphological traits over the obtained phylogenies. Results suggest eight morphologically distinct clades, which do not correspond to the species groups proposed formerly. Ancestral state reconstructions and phylogenetic signal analyses of morphological traits revealed that some of the previously key characters to the classification of Typhlocharis, such as the umbilicate series or the apical denticles of elytra, are highly homoplasic, whereas other characters show stronger phylogenetic signal, including structures in the antennae, gula, pronotum and last abdominal ventrite. This evidence supports the split of Typhlocharis into three genera: Lusotyphlus gen. nov. ; Typhlocharis Dieck, 1869 and Microcharidius Coiffait, 1969 (revalidated), forming the subtribe Typhlocharina Jeanne, 1973.     

Morphological characters add support for some members of the basal grade of Asteraceae

Recent molecular studies in Asteraceae have divided tribe Mutisieae (sensu Cabrera) into 13 tribes and eight subfamilies. Each of the major clades is well supported but the relationships among them are not always clear. Some of the new taxa are easily characterized by morphological data but others are not, chief among the latter being three subfamilies (Stifftioideae, Wunderlichioideae and Gochnatioideae) and the tribe Hyalideae. To understand evolution in the family it is critical to investigate potential morphological characters that can help to evaluate the basal lineages of the Asteraceae. The data for this study were taken from 52 species in 24 genera representing the basal groups in the family. Many characters were examined but most of the useful ones were from reproductive structures. Several apomorphies supported a few of the clades. For instance, members of subfamily Wunderlichioideae (Hyalideae and Wunderlichieae) share predominantly ten‐ribbed achenes and members of Wunderlichioideae + Stifftioideae share two synapomorphies: 100–150 (200) pappus elements, arranged in (three) four or five series. These apomorphies can be viewed as an indication of a sister‐group relationship between the two subfamilies as the placement of Stifftieae was not well resolved by the molecular data. Members of Wunderlichieae are characterized by having a paleaceous receptacle, style branches that are strongly papillose above and below the bifurcation, and a pappus of scales. Hyalis and Ianthopappus (Hyalideae) share venation type and an apiculate anther appendage but these are also found in Gochnatieae. Other clades have fewer supporting characters. These characters are just a beginning. Cladograms with morphology characters plotted, illustrations and a key to the basal grade of Asteraceae are provided. © 2013 The Linnean Society of London     

Molecular and morphological delimitation and generic classification of the family Oocystaceae (Trebouxiophyceae,Chlorophyta)

The family Oocystaceae (Chlorophyta) is a group of morphologically and ultrastructurally distinct green algae that constitute a well‐supported clade in the class Trebouxiophyceae. Despite the family's clear delimitation, which is based on specific cell wall features, only a few members of the Oocystaceae have been examined using data other than morphological. In previous studies of Trebouxiophyceae, after the establishment of molecular phylogeny, the taxonomic status of the family was called into question. The genus Oocystis proved to be paraphyletic and some species were excluded from Oocystaceae, while a few other species were newly redefined as members of this family. We investigated 54 strains assigned to the Oocystaceae using morphological, ultrastructural and molecular data (SSU rRNA and rbcL genes) to clarify the monophyly of and diversity within Oocystaceae. Oonephris obesa and Nephrocytium agardhianum clustered within the Chlorophyceae and thus are no longer members of the Oocystaceae. On the other hand, we transferred the coenobial Willea vilhelmii to the Oocystaceae. Our findings combined with those of previous studies resulted in the most robust definition of the family to date. The division of the family into three subfamilies and five morphological clades was suggested. Taxonomical adjustments in the genera Neglectella, Oocystidium, Oocystis, and Ooplanctella were established based on congruent molecular and morphological data. We expect further taxonomical changes in the genera Crucigeniella, Eremosphaera, Franceia, Lagerheimia, Oocystis, and Willea in the future.     

Morphology and morphogenesis of a new marine ciliate, Apokeronopsis bergeri nov. spec. (Ciliophora, Hypotrichida), from the Yellow Sea, China

The morphology and morphogenesis of a new marine hypotrich ciliate, Apokeronopsis bergeri nov. spec., collected from mussel-farming waters near Qingdao, China, are described from living and protargol-impregnated specimens. This ciliate has characteristics that place it in the family Pseudokeronopsidae, namely, two long rows of frontal cirri (bicorona), which are continuous with the long midventral rows, and a single row of marginal cirri on each side of the body. It shares with its only congener, Apokeronopsis crassa, the long rows of buccal and transverse cirri and the wide spacing between the midventral rows of cirri. These characters separate the genus Apokeronopsis from Pseudokeronopsis, which has a single buccal cirrus, fewer transverse cirri and midventral rows of cirri arranged in a typical zig-zag pattern. A. bergeri differs from A. crassa in its shape, colour and in the numbers of membranelles and transverse cirri. Although morphogenesis in A. bergeri is similar to that of A. crassa in most respects, the mode of formation of the buccal cirri is slightly different. The close relationship of A. bergeri with A. crassa, and the more distant relationship with three Pseudokeronopsis species, is supported by a comparison of the sequences of their ITS1-5.8S-ITS2 rDNA regions.