Phylogenetic confirmation of the genus Robbea (Nematoda: Desmodoridae,Stilbonematinae) with the description of three new species

ABSTRACT

The Stilbonematinae are a monophyletic group of marine nematodes that are characterized by a coat of thiotrophic bacterial symbionts. Among the ten known genera of the Stilbonematinae, the genus Robbea Gerlach 1956 had a problematic taxonomic history of synonymizations and indications of polyphyletic origin. Here we describe three new species of the genus, R. hypermnestra sp. nov., R. ruetzleri sp. nov. and R. agricola sp. nov., using conventional light microscopy, interference contrast microscopy and SEM. We provide 18S rRNA gene sequences of all three species, together with new sequences for the genera Catanema and Leptonemella. Both our morphological analyses as well as our phylogenetic reconstructions corroborate the genus Robbea. In our phylogenetic analysis the three species of the genus Robbea form a distinct clade in the Stilbonematinae radiation and are clearly separated from the clade of the genus Catanema, which has previously been synonymized with Robbea. Surprisingly, in R. hypermnestra sp. nov. all females are intersexes exhibiting male sexual characters. Our extended dataset of Stilbonematinae 18S rRNA genes for the first time allows the identification of the different genera, e.g. in a barcoding approach.

http://zoobank.org/urn:lsid:zoobank.org:pub:D37C3F5A-CF2B-40E6-8B09-3C72EEED60B0     

A widespread cyanobacterium supported by polyphasic approach: proposition of Koinonema pervagatum gen. & sp. nov. (Oscillatoriales)1

Several new genera originally classified as the genus Phormidium, a polyphyletic and taxonomically complex genus within the Oscillatoriales, were recently described. The simple morphology of Phormidium does not reflect its genetic diversity and the delimitation of a natural group is not possible with traditional classification systems based on morphology alone. Therefore, this study used morphological, ecological, and molecular approaches to evaluate four populations morphologically similar to Ammassolinea, Kamptonema, and Ancylothrix (simple, curved, and gradually attenuated at the ends trichome), found in subtropical and tropical Brazilian regions. 16S rRNA gene sequences grouped all the strains in a highly supported clade with other two European strains isolated from thermal springs surrounding areas. The 16S‐23S ITS secondary structure corroborated the phylogenetic analysis with all the strains having similar structures. Consequently, a genetically well‐defined and cryptic new genus, Koinonema gen. nov., is proposed containing the aquatic, mesophilic, and morphologically homogeneous new species, Koinonema pervagatum sp. nov.     

MOLECULAR AND MORPHOLOGICAL CHARACTERIZATION OF TEN POLAR AND NEAR‐POLAR STRAINS WITHIN THE OSCILLATORIALES (CYANOBACTERIA)1

An approximately 1400‐bp region of the 16S rRNA gene was sequenced for 10 polar or near‐polar strains putatively placed in the Oscillatorialean genera Oscillatoria, Phormidium, and Lyngbya obtained from the University of Toronto Culture Collection to assess phylogenetic relationships. The strains were also examined for thylakoid structure and cell division type with TEM as well as traditional morphology with LM. Phylogenetic trees constructed using parsimony, distance, and maximum likelihood methods were similar in topology. If the original epithets applied to the sequenced strains (both polar and those from GenBank) were used, it was clear that taxa were not monophyletic. However, using the revised taxonomic system of Anagnostidis and Komárek, we were able to reassign these strains to their current correct taxa (species, genus, and family). When these assignments were made, it was determined that the molecular sequence data analyses were congruent with morphology and ultrastructure. Nine of the polar strains were found to be new species, and eight were described as such: Arthronema gygaxiana Casamatta et Johansen sp. nov., Pseudanabaena tremula Johansen et Casamatta sp. nov., Leptolyngbya angustata Casamatta et Johansen sp. nov., Phormidium lumbricale Johansen et Casamatta sp. nov., Microcoleus glaciei Johansen et Casamatta sp. nov., Microcoleus rushforthii Johansen et Casamatta sp. nov., Microcoleus antarcticus Casamatta et Johansen sp. nov., Microcoleus acremannii Casamatta et Johansen sp. nov. Some genera (Leptolyngbya and Microcoleus) were clearly not monophyletic and require future revision.     

Cyanomargarita gen. nov. (Nostocales,Cyanobacteria): convergent evolution resulting in a cryptic genus

Two populations of Rivularia‐like cyanobacteria were isolated from ecologically distinct and biogeographically distant sites. One population was from an unpolluted stream in the Kola Peninsula of Russia, whereas the other was from a wet wall in the Grand Staircase‐Escalante National Monument, a desert park‐land in Utah. Though both were virtually indistinguishable from Rivularia in field and cultured material, they were both phylogenetically distant from Rivularia and the Rivulariaceae based on both 16S rRNA and rbcLX phylogenies. We here name the new cryptic genus Cyanomargarita gen. nov., with type species C. melechinii sp. nov., and additional species C. calcarea sp. nov. We also name a new family for these taxa, the Cyanomargaritaceae.     

Species diversity of Onchidiidae (Eupulmonata: Heterobranchia) on the mainland of China based on molecular data

The identification of members of the Onchidiidae is based on morphological characters; this is often time-consuming and can be inconclusive. In order to explore the species diversity of onchidiids in China, we provide a phylogeny constructed using partial sequences of two mitochondrial genes (16S rRNA and COI) and one nuclear ribosomal RNA gene (28S rRNA) from 32 samples comprising five genera. The topology, using both Bayesian and Maximum Likelihood inference methods, showed that the taxa clustered in two main groups of six species, one of which included Platevindex mortoni, Platevindex sp. and Onchidium ‘struma’; the other included Paraoncidium reevesii, Onchidella sp. and Peronia verruculata. It is clear that COI will be useful in discriminating onchidiid species-group taxa.     

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.     

New evidence of cryptic speciation in the family Longidoridae (Nematoda: Dorylaimida)

Longidorid nematodes comprise more than 500 species, and Longidorus and Xiphinema are the most diversified, prevalent, and cosmopolitan genera within plant-parasitic nematodes. This increases the risk of species misidentification. We conducted an integrative morphometric and genetic study on two longidorid species to elucidate the existence of new cases of cryptic speciation within the genera Longidorus and Xiphinema. Detailed morphological, morphometrical, multivariate, and genetic studies were carried out, and mitochondrial and nuclear haploweb analyses were used to differentiate species within the L. iliturgiensis and X. hispanum complexes. Species delimitation using multivariate and haplonet tools of L. iliturgiensis species complex clearly separated L. tabernensis sp. nov. from L. iliturgiensis and L. indalus, and X. subbaetense sp. nov. from X. hispanum and X. adenohystherum. D2-D3, partial 18S, and partial coxI regions were used for inferring their phylogenetic relationships with other species in each genus. The present study provides new insights into the diversity of Longidorus and Xiphinema species detected in southern Spain, and new evidence of cryptic speciation in both genera. These results support our hypothesis that the biodiversity of Longidoridae in southern Europe is higher than previously supposed and is still not fully clarified.     

Etheliaceae fam. nov. (Gigartinales,Rhodophyta), with a clarification of the generitype of Ethelia and the addition of six novel species from warm waters

Based upon COI‐5P, LSU rDNA, and rbcL sequence data and morphological characteristics, six new members of the noncalcified crustose genus of red algae Ethelia are described in a new family, Etheliaceae (Gigartinales), sister to the recently described Ptilocladiopsidaceae. The novel species are described from subtropical to tropical Atlantic and Indo‐Pacific Ocean basins; E. mucronata sp. nov. and E. denizotii sp. nov. from southern and northern Western Australia respectively, E. wilcei sp. nov. from the Cocos (Keeling) Islands of Australia, E. suluensis sp. nov. from the Philippines, E. umbricola sp. nov. from Bermuda and E. kraftii sp. nov. from Lord Howe Island, Australia. The generitype, Ethelia biradiata, originally reported from the Seychelles, Indian Ocean, is added to the Western Australian flora.     

The Extremophile Endolithella mcmurdoensis gen. et sp. nov. (Trebouxiophyceae,Chlorellaceae), A New Chlorella-like Endolithic Alga From Antarctica

The McMurdo Dry Valleys constitute the largest ice-free region of Antarctica and one of the most extreme deserts on Earth. Despite the low temperatures, dry and poor soils and katabatic winds, some microbes are able to take advantage of endolithic microenvironments, inhabiting the pore spaces of soil and constituting photosynthesis-based communities. We isolated a green microalga, Endolithella mcmurdoensis gen. et sp. nov, from an endolithic sandstone sample collected in the McMurdo Dry Valleys (Victoria Land, East Antarctica) during the K020 expedition, in January 2013. The single non-axenic isolate (E. mcmurdoensis LEGE Z-009) exhibits cup-shaped chloroplasts, electron-dense bodies, and polyphosphate granules but our analysis did not reveal any diagnostic morphological characters. On the basis of phylogenetic analysis of the 18S rRNA (SSU) gene, the isolate was found to represent a new genus within the family Chlorellaceae.     

Novel diversity within Roseofilum (Desertifilaceae,Cyanobacteria) from marine benthic mats with description of four new species

Benthic cyanobacterial mats (BCMs) are natural phenomena in marine environments. Reports of BCMs occurring across coastal marine environments have increased, partly driven by nutrient loading and climate change; thus, there is a need to understand the diversity involved in the proliferations and potential toxicity of the BCMs. Furthermore, marine cyanobacterial mats are observed growing on and affecting the health of corals with one specific cyanobacterial genus, Roseofilum, dominating the microbial mats associated with black band disease (BBD), a destructive polymicrobial disease that affects corals. To explore the diversity of Roseofilum, cyanobacterial mats from various marine habitats were sampled, and individual isolates were identified based on morphology, 16S rRNA gene phylogenies, 16S–23S ITS rRNA region sequence dissimilarities, and phylogenomics. Four novel species of Roseofilum were isolated from benthic marine mats, three from the coasts of Florida, United States (R. capinflatum sp. nov., R. casamattae sp. nov., and R. acuticapitatum sp. nov.) and one from the coast of France (R. halophilum sp. nov.). Our analyses revealed that Roseofilum associated with coral BBD and those not associated with corals but rather from coastal benthic mats are systematically distinct based on both phylogenetic and phylogenomic analyses. Enzyme-linked immunosorbent assay (ELISA) and LC–MS data indicated that microcystin production was found in one of the four species.     

Crusticorallina gen. nov., a nongeniculate genus in the subfamily Corallinoideae (Corallinales,Rhodophyta)

Molecular phylogenetic analyses of 18S rDNA (SSU) gene sequences confirm the placement of Crusticorallina gen. nov. in Corallinoideae, the first nongeniculate genus in an otherwise geniculate subfamily. Crusticorallina is distinguished from all other coralline genera by the following suite of morpho‐anatomical characters: (i) sunken, uniporate gametangial and bi/tetrasporangial conceptacles, (ii) cells linked by cell fusions, not secondary pit connections, (iii) an epithallus of 1 or 2 cell layers, (iv) a hypothallus that occupies 50% or more of the total thallus thickness, (v) elongate meristematic cells, and (vi) trichocytes absent. Four species are recognized based on rbcL, psbA and COI‐5P sequences, C. painei sp. nov., the generitype, C. adhaerens sp. nov., C. nootkana sp. nov. and C. muricata comb. nov., previously known as Pseudolithophyllum muricatum. Type material of Lithophyllum muricatum, basionym of C. muricata, in TRH comprises at least two taxa, and therefore we accept the previously designated lectotype specimen in UC that we sequenced to confirm its identity. Crusticorallina species are very difficult to distinguish using morpho‐anatomical and/or habitat characters, although at specific sites, some species may be distinguished by a combination of morpho‐anatomy, habitat and biogeography. The Northeast Pacific now boasts six coralline endemic genera, far more than any other region of the world.     

Franciscideres gen. nov. – a new,highly aberrant kinorhynch genus from Brazil,with an analysis of its phylogenetic position

A new genus and species of Kinorhyncha, Franciscideres kalenesos gen. et sp. nov., is described from tidal and subtidal sandy habitats in Brazil. The new genus and species is characterized by an extremely flexible trunk without pachycycli that appears perfectly circular in cross-section, segments 1, 2 and 11 consisting of closed rings and 3 to 10 of single, bent plates with midventral articulations, a neck without placids that resembles an additional segment, densely packed scale-like, cuticular hairs, and a terminal segment with a middorsal spine and two sets of lateral terminal spines, but no midterminal spine. Phylogenetic analyses of 18S rRNA of Franciscideres kalenesos gen. et sp. nov. and 47 other kinorhynch ingroup taxa suggest that the new genus is a basal homalorhagid, whereas comparison of morphological characters indicates affinities between the new genus and the peculiar cyclorhagid Cateria.

http://zoobank.org/urn:lsid:zoobank.org:pub:64E29D97-DE1D-4511-8683-C969DD2EED43     

Morphological and molecular taxonomy of a new Daptonema (Nematoda,Xyalidae) with comments on the systematics of some related taxa

A new species of Daptonema is described based upon morphological characters and 18S rRNA sequence. Daptonema matrona sp. nov. was collected in Pina Basin (north‐eastern Brazil). It differs from all other species of the genus by the presence of reduced cephalic setae and straight spicules. These features require an adaptation of the generic diagnosis. Moreover, the females are characterized by intra‐uterine development of the offspring, considered herein as their major autapomorphic feature. Molecular systematic analyses supported Daptonema matrona sp. nov. as a distinct genetic and evolutionary lineage. The data also indicate hypotheses of taxonomic synonymies amongst some related taxa from Xyalidae as well as the paraphyly of Daptonema. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 158 , 1–15.     

Application of multigene phylogenetics and site‐stripping to resolve intraordinal relationships in the Rhodymeniales (Rhodophyta)

Previous molecular assessments of the red algal order Rhodymeniales have confirmed its monophyly and distinguished the six currently recognized families (viz. Champiaceae, Faucheaceae, Fryeellaceae, Hymenocladiaceae, Lomentariaceae, and Rhodymeniaceae); however, relationships among most of these families have remained unresolved possibly as a result of substitution saturation at deeper phylogenetic nodes. The objective of the current study was to improve rhodymenialean systematics by increasing taxonomic representation and using a more robust multigene dataset of mitochondrial (COB, COI/COI‐5P), nuclear (LSU, EF2) and plastid markers (psbA, rbcL). Additionally, we aimed to prevent phylogenetic inference problems associated with substitution saturation (particularly at the interfamilial nodes) by removing fast‐evolving sites and analyzing a series of progressively more conservative alignments. The Rhodymeniales was resolved as two major lineages: (i) the Fryeellaceae as sister to the Faucheaceae and Lomentariaceae; and (ii) the Rhodymeniaceae allied to the Champiaceae and Hymenocladiaceae. Support at the interfamilial nodes was highest when 20% of variable sites were removed. Inclusion of Binghamiopsis, Chamaebotrys, and Minium, which were absent in previous phylogenetic investigations, established their phylogenetic affinities while assessment of two genera consistently polyphyletic in phylogenetic analyses, Erythrymenia and Lomentaria, resulted in the proposition of the novel genera Perbella and Fushitsunagia. The taxonomic position of Drouetia was reinvestigated with re‐examination of holotype material of D. coalescens to clarify tetrasporangial development in this genus. In addition, we added three novel Australian species to Drouetia as a result of ongoing DNA barcoding assessments—D. aggregata sp. nov., D. scutellata sp. nov., and D. viridescens sp. nov.     

PCR fishing for red endophytes in British Columbia Kallymeniaceae (Gigartinales,Florideophyceae) uncovers the species‐rich Kallymenicola gen. nov. (Palmariales,Nemaliophycidae)

Unexpected contaminants uncovered during routine COI‐5P DNA barcoding of British Columbia Kallymeniaceae indicated the presence of a novel lineage allied to the family Meiodiscaceae, Palmariales. Available rbcL data for species of this family were used to design specific primers to screen for the presence of the meiodiscacean species in 534 kallymeniacean specimens primarily from British Columbia, Canada. Ultimately, 43 positive PCR products representing six diverse genetic groups from nine host species were uncovered; three are described here in the new genus Kallymenicola gen. nov., viz., K. invisiblis sp. nov., K penetrans sp. nov., and K superficialis sp. nov. Although genetic groups loosely displayed evidence of host specificity and cospeciation, examples of host switching with interesting biogeographical patterns were also documented.     

Albertania and Egbenema gen. nov. from Nigeria and the United States,expanding biodiversity in the Oculatellaceae (cyanobacteria)

Knowledge of the tropical terrestrial cyanobacterial flora from the African continent is still limited. Of 31 strains isolated from soil and subaerial samples collected in Lagos State, Nigeria, three were found to be in the Oculatellaceae, including two species in a new genus. Subsequently, isolates from microbial mats in White Sands National Park in New Mexico, United States, and from a rock near the ocean in Puerto Rico, United States, were found to belong to the new genus as well. Cyanobacterial isolates were characterized microscopically, sequenced for the 16S rRNA gene and associated ITS region, and phylogenetically analyzed. Egbenema gen. nov., with three new species, as well as two new species of Albertania were differentiated from all other Oculatellaceae. Both genera belong to a supported clade within the Oculatellaceae that includes Trichotorquatus and Komarkovaea. The two new species of Albertania, A. egbensis and A. latericola, were from the same sample, but were evolutionarily separate based on 16S rRNA gene phylogenies, percent identity below the 98.7% threshold, and ITS rRNA percent dissimilarity >7.0%. Egbenema aeruginosum gen. et sp. nov. was phylogenetically separated from Trichotorquatus and Albertania but was in a clade with other strains belonging to Egbenema. The two Egbenema strains from the United States are here named Egbenema epilithicum sp. nov. and Egbenema gypsiphilum sp. nov. Our results support the hypothesis that further species discoveries of novel cyanobacteria will likely be made in soils and subaerial habitats, as these habitats continue to be studied, both in tropical and temperate biomes.     

Closely coupled evolutionary history of ecto‐ and endosymbionts from two distantly related animal phyla

The level of integration between associated partners can range from ectosymbioses to extracellular and intracellular endosymbioses, and this range has been assumed to reflect a continuum from less intimate to evolutionarily highly stable associations. In this study, we examined the specificity and evolutionary history of marine symbioses in a group of closely related sulphur‐oxidizing bacteria, called Candidatus Thiosymbion, that have established ecto‐ and endosymbioses with two distantly related animal phyla, Nematoda and Annelida. Intriguingly, in the ectosymbiotic associations of stilbonematine nematodes, we observed a high degree of congruence between symbiont and host phylogenies, based on their ribosomal RNA (rRNA) genes. In contrast, for the endosymbioses of gutless phallodriline annelids (oligochaetes), we found only a weak congruence between symbiont and host phylogenies, based on analyses of symbiont 16S rRNA genes and six host genetic markers. The much higher degree of congruence between nematodes and their ectosymbionts compared to those of annelids and their endosymbionts was confirmed by cophylogenetic analyses. These revealed 15 significant codivergence events between stilbonematine nematodes and their ectosymbionts, but only one event between gutless phallodrilines and their endosymbionts. Phylogenetic analyses of 16S rRNA gene sequences from 50 Cand. Thiosymbion species revealed seven well‐supported clades that contained both stilbonematine ectosymbionts and phallodriline endosymbionts. This closely coupled evolutionary history of marine ecto‐ and endosymbionts suggests that switches between symbiotic lifestyles and between the two host phyla occurred multiple times during the evolution of the Cand. Thiosymbion clade, and highlights the remarkable flexibility of these symbiotic bacteria.     

Gymnoxanthella radiolariae gen. et sp. nov. (Dinophyceae), a dinoflagellate symbiont from solitary polycystine radiolarians

The symbiotic dinoflagellate Gymnoxanthella radiolariae T. Yuasa et T. Horiguchi gen. et sp. nov. isolated from polycystine radiolarians is described herein based on light, scanning and transmission electron microscopy as well as molecular phylogenetic analyses of SSU and LSU rDNA sequences. Motile cells of G. radiolariae were obtained in culture, and appeared to be unarmored. The cells were 9.1–11.4 μm long and 5.7–9.4 μm wide, and oval to elongate oval in the ventral view. They possessed an counterclockwise horseshoe‐shaped apical groove, a nuclear envelope with vesicular chambers, cingulum displacement with one cingulum width, and the nuclear fibrous connective; all of these are characteristics of Gymnodinium sensu stricto (Gymnodinium s.s.). Molecular phylogenetic analyses also indicated that G. radiolariae belongs to the clade of Gymnodinium s.s. However, in our molecular phylogenetic trees, G. radiolariae was distantly related to Gymnodinium fuscum, the type species of Gymnodinium. Based on the consistent morphological, genetic, and ecological divergence of our species with the other genera and species of Gymnodinium s.s., we considered it justified to erect a new, separate genus and species G. radiolariae gen. et sp. nov. As for the peridinioid symbiont of radiolarians, Brandtodinium has been erected as a new genus instead of Zooxanthella, but the name Zooxanthella is still valid. Brandtodinium is a junior synonym of Zooxanthella. Our results suggest that at least two dinoflagellate symbiont species, peridinioid Zooxanthella nutricula and gymnodinioid G. radiolariae, exist in radiolarians, and that they may have been mixed and reported as “Z. nutricula” since the 19th century.