Redescription of Atopospira galeata (Kahl, 1927) nov. comb. and A. violacea (Kahl, 1926) nov. comb. with redefinition of Atopospira Jankowski, 1964 nov. stat. and Brachonella Jankowski, 1964 (Ciliophora,Armophorida)

The taxonomy of the Metopidae (Ciliophora, Armophorida) remains poorly understood since most of its members have not been studied by modern morphologic and molecular methods. Recent molecular investigations have indicated that the two most species-rich genera, Metopus and Brachonella, are likely nonmonophyletic with at least one well-supported 18S rDNA clade comprised of a species from each of these genera (Brachonella galeata and Metopus violaceus). We investigated these two species with silver impregnation and scanning electron microscopy. Both taxa share important morphologic characteristics not described in other species of Metopus or Brachonella. These synapomorphies include: (1) a diplostichomonad paroral membrane, (2) a bipartite adoral zone with a short buccal part composed of ordinary membranelles and a longer distal part composed of much smaller membranelles bearing a single cilium or none and extending the same length as the perzonal ciliary stripe. We transfer Brachonella galeata (Kahl, 1927) Jankowski, 1964 and Metopus violaceus Kahl, 1926 to genus Atopospira Jankowski, 1964 nov. stat. Pending detailed morphologic and molecular characterization, Brachonella campanula, B. cydonia and B. pyriforma, B. intercedens, and B. lemani remain in Brachonella Jankowski 1964.     

Two Anaerobic Ciliates (Ciliophora,Armophorea) from China: Morphology and SSU rDNA Sequence,with Report of a New Species,Metopus paravestitus nov. spec

The morphology and phylogeny of two metopid ciliates, collected from anaerobic habitats in China, were investigated using live observation, protargol staining method, and SSU rDNA sequencing. The new species Metopus paravestitus nov. spec. can be distinguished by a combination of the following features: oblong cell with densely arranged ectobiotic prokaryotes perpendicular to cell surface, filiform intracytoplasmic structures packed in the anterior portion of the cell. Our work also demonstrates the wide geographical distribution of Metopus es (Müller, 1776) Lauterborn, 1916. The order Metopida is consistently depicted as a paraphylum in SSU rDNA phylogeny. Metopus paravestitus nov. spec. is closely related to its marine congeners than to freshwater forms. The present study confirms once again the non-monophyly of the genus Metopus and genus Metopidae.     

Morphology and Phylogeny of the Soil Ciliate Metopus yantaiensis n. sp. (Ciliophora,Metopida), with Identification of the Intracellular Bacteria

The morphology and infraciliature of a new ciliate, Metopus yantaiensis n. sp., discovered in coastal soil of northern China, were investigated. It is distinguished from its congeners by a combination of the following features: nuclear apparatus situated in the preoral dome; 18–21 somatic ciliary rows, of which three extend onto the preoral dome (dome kineties); three to five distinctly elongated caudal cilia, and 21–29 adoral polykinetids. The 18S rRNA genes of this new species and two congeners, Metopus contortus and Metopus hasei, were sequenced and phylogenetically analyzed. The new species is more closely related to M. hasei and the clevelandellids than to other congeners; both the genus Metopus and the order Metopida are not monophyletic. In addition, the digestion‐resistant bacteria in the cytoplasm of M. yantaiensis were identified, using a 16S rRNA gene clone library, sequencing, and fluorescence in situ hybridization. The detected intracellular bacteria are affiliated with Sphingomonadales, Rhizobiales, Rickettsiales (Alphaproteobacteria), Pseudomonas (Gammaproteobacteria), Rhodocyclales (Betaproteobacteria), Clostridiales (Firmicutes), and Flavobacteriales (Bacteroidetes).     

Two new deer truffle species,Elaphomyces marmoratus and Elaphomyces fuscus spp. nov., from a secondary forest in Japan

We describe two new species of deer truffle, Elaphomyces marmoratus and E. fuscus spp. nov., collected from a secondary forest dominated by Quercus serrata, in Tokyo, Japan. Both species have morphological similarities to E. muricatus and E. granulatus; however, the former has a gleba without a purplish color and the latter has reticulated ascospores more closely resembling E. asperulus. Molecular phylogenetic analyses also support the assignment of these specimens as new species. ITS rDNA homologies with known species were low (<91% and <97%). In addition, phylogenetic trees using neighbor-joining and maximum likelihood methods showed that the sequences of the two new species each formed a monophyletic group within section Elaphomyces with bootstrap support of 99%. Analyses of ectomycorrhizal roots collected concurrently with the ascomata revealed that E. marmoratus is associated with at least Pinus densiflora, while E. fuscus is associated with at least Q. serrata.     

Identification of photobionts from the lichen family Physciaceae using algal-specific ITS rDNA sequencing

Abstract:The identity of photobionts from 20 species of the Physciaceae from different habitats and geographical regions has been determined by ITS rDNA sequence comparisons in order to estimate the diversity of photobionts within that lichen group, to detect patterns of specificity of mycobionts towards their photobionts and as a part of an ongoing study to investigate possible parallel cladogenesis of both symbionts. Algal-specific PCR primers have been used to determine the ITS rDNA sequences from DNA extractions of dried lichens that were up to 5 years old. Direct comparisons and phylogenetic analyses allowed the assignment of Physciaceae photobionts to four distinct clades in the photobiont ITS rDNA phylogeny. The results indicate a diversity within the genus Trebouxia Puymaly and Physciaceae photobionts that is higher than expected on the basis of morphology alone. Physciaceae photobionts belonged to 12 different ITS lineages of which nine could unambiguously be assigned to six morphospecies of Trebouxia. The identity of the remaining three sequences was not clarified; they may represent new species. Specificity at the generic level was low as a whole range of photobiont species were found within a genus of Physciaceae and different ranges were detected. The photobionts ofPhyscia (Schreb.) Michaux were closely related and represented one morphospecies of Trebouxia, whereas the algal partners of Buellia De Not and Rinodina (Ach.) S. Gray were in distant lineages of the ITS phylogeny and from several Trebouxia morphospecies. Photobiont variation within a genus of Physciaceae may be due to phylogeny, geographical distance or because photobionts from neighbouring lichens were taken (‘algal sharing’). At the species level Physciaceae mycobionts seem to be rather selective and contained photobionts that were very closely related within one morphospecies of Trebouxia.     

Systematic and morphological diversity of endosymbiotic methanogens in anaerobic ciliates

The identities and taxonomic diversity of the endosymbiotic methanogens from the anaerobic protozoaMetopus contortus, Metopus striatus, Metopus palaeformis, Trimyema sp. andPelomyxa palustris were determined by comparative analysis of their 16S ribosomal RNA sequences. Fluorescent oligonucleotide probes were designed to bind to the symbiont rRNA sequences and to provide direct visual evidence of their origins from methanogenic archaea contained within the host cells. Confocal microscopy was used to analyze the morphology of the endosymbionts in whole cells ofMetopus palaeformis, Metopus contortus, Trimyema sp. andCyclidium porcatum. The endosymbionts are taxonomically diverse and are drawn from three different genera;Methanobacterium, Methanocorpusculum andMethanoplanus. In every case the symbionts are closely related to, but different from, free-living methanogens for which sequences are available. It is thus apparent that symbioses have been formed repeatedly and independently. Ciliates which are unrelated to each other (Trimyema sp. andMetopus contortus) may contain symbionts which are closely related, and congeneric ciliates (Metopus palaeformis andM. contortus) may contain symbionts which are distantly related to each other. This suggests that some of the symbiotic associations must be relatively recent. For example, at least one of the symbioses inMetopus must postdate the speciation ofM. palaeformis andM. contortus. Despite this,Metopus contortus, Trimyema sp., Cyclidium porcatum and their respective endosymbionts show sophisticated morphological interactions which probably facilitate the exchange of materials between the partners.     

Comparison between Colony Morphology and Molecular Phylogeny in the Caribbean Scleractinian Coral Genus Madracis

A major challenge in coral biology is to find the most adequate and phylogenetically informative characters that allow for distinction of closely related coral species. Therefore, data on corallite morphology and genetic data are often combined to increase phylogenetic resolution. In this study, we address the question to which degree genetic data and quantitative information on overall coral colony morphologies identify similar groupings within closely related morphospecies of the Caribbean coral genus Madracis. Such comparison of phylogenies based on colony morphology and genetic data will also provide insight into the degree to which genotype and phenotype overlap. We have measured morphological features of three closely related Caribbean coral species of the genus Madracis (M. formosa, M. decactis and M. carmabi). Morphological differences were then compared with phylogenies of the same species based on two nuclear DNA markers, i.e. ATPSα and SRP54. Our analysis showed that phylogenetic trees based on (macroscopical) morphological properties and phylogenetic trees based on DNA markers ATPSα and SRP54 are partially similar indicating that morphological characteristics at the colony level provide another axis, in addition to commonly used features such as corallite morphology and ecological information, to delineate genetically different coral species. We discuss this new method that allows systematic quantitative comparison between morphological characteristics of entire colonies and genetic data.     

Morphology and Morphogenesis of Metopus hasei Sondheim, 1929 and M. inversus (Jankowski, 1964) nov. comb. (Ciliophora, Metopida)

ABSTRACT The morphology and morphogenesis of Metopus hasei Sondheim, 1929 and M. inversus (Jankowski, 1964) n. comb, were investigated using live observation, silver impregnation, and scanning electron microscopy. Metopus has a spiral body organization and the ventral margin of the preoral dome bears five specialized ciliary rows, that form the so-called perizonal stripe. Division is homothetogenic, occurs in freely motile (i. e. non-encysted) condition, and includes a partial reorganization of the parental oral apparatus. During division, the complicated cell shape becomes ellipsoidal and all ciliary rows arrange meridionally. Stomatogenesis is entirely somatic (≅ pleurotelokinetal) and commences with the formation of kinetofragments in some dorsolateral kineties. The fragments become the opisthe's adoral membranelles, while the paroral membrane is generated by the left two perizonal ciliary rows, which proliferate kinetids intrakinetally. The perizonal stripe of the opisthe is generated by the three right parental perizonal kineties, which divide, and by two dorsolateral ciliary rows, which are added. The morphogenetic processes, especially the unique mode of formation of the paroral membrane, are used to define the order Metopida Jankowski, 1980 n. stat. more properly. The ontogenetic, ultrastructural, and sequence data available give no clear indication about metopid phylogeny, but definitely exclude metopids from the classical heterotrichs, with which they were classified for more than 100 years. Accordingly, we place the Metopida as incertae sedis in the subphylum Intramac-ronucleata Lynn, 1996.     

Speciation slowing down in widespread and long-living tree taxa: insights from the tropical timber tree genus Milicia (Moraceae)

The long generation time and large effective size of widespread forest tree species can result in slow evolutionary rate and incomplete lineage sorting, complicating species delimitation. We addressed this issue with the African timber tree genus Milicia that comprises two morphologically similar and often confounded species: M. excelsa, widespread from West to East Africa, and M. regia, endemic to West Africa. We combined information from nuclear microsatellites (nSSRs), nuclear and plastid DNA sequences, and morphological systematics to identify significant evolutionary units and infer their evolutionary and biogeographical history. We detected five geographically coherent genetic clusters using nSSRs and three levels of genetic differentiation. First, one West African cluster matched perfectly with the morphospecies M. regia that formed a monophyletic clade at both DNA sequences. Second, a West African M. excelsa cluster formed a monophyletic group at plastid DNA and was more related to M. regia than to Central African M. excelsa, but shared many haplotypes with the latter at nuclear DNA. Third, three Central African clusters appeared little differentiated and shared most of their haplotypes. Although gene tree paraphyly could suggest a single species in Milicia following the phylogenetic species concept, the existence of mutual haplotypic exclusivity and nonadmixed genetic clusters in the contact area of the two taxa indicate strong reproductive isolation and, thus, two species following the biological species concept. Molecular dating of the first divergence events showed that speciation in Milicia is ancient (Tertiary), indicating that long-living tree taxa exhibiting genetic speciation may remain similar morphologically.     

Evidence for extensive cryptic speciation in trematodes of butterflyfishes (Chaetodontidae) of the tropical Indo-West Pacific

Molecular data from the cytochrome c oxidase subunit I (cox1) mitochondrial DNA gene and the second internal transcribed spacer (ITS2) nuclear rDNA region were used to test the current morphologically-based taxonomic hypothesis regarding species of Monorchiidae (Hurleytrematoides) from chaetodontid and tetraodontid fishes from six sites in the tropical Indo-West Pacific (TIWP): Heron and Lizard Islands off the Great Barrier Reef (GBR, Australia), Moorea (French Polynesia), New Caledonia, Ningaloo Reef (Australia) and Palau. The 16 morphospecies analysed differed from each other by a minimum of 55 bp (9.1%) over the mitochondrial cox1 and 8 bp (1.6%) over the ITS2 DNA regions. For two species, Hurleytrematoides loi and Hurleytrematoides sasali, specimens from the same host species in sympatry differed at levels comparable to those between pairs of distinct morphospecies for both cox1 and ITS2 sequences. We take this as evidence of the presence of combinations of cryptic species; however, we do not propose new species for these taxa because we lack identified morphological voucher specimens. For seven species, Hurleytrematoides coronatum, Hurleytrematoides deblocki, Hurleytrematoides faliexae, H. loi, Hurleytrematoides morandi, H. sasali and Hurleytrematoides sp. A, samples from some combinations of localities had base pair differences that were equal to or greater than differences between some pairs of distinct morphospecies for one or both cox1 and ITS2 sequences. For three species, H. coronatum, H. loi and H. morandi, one haplotype differed from every other haplotype by more than the morphospecies benchmark. In these cases morphological specimens could not be distinguished by morphology. These data suggest extensive cryptic richness in this genus. For the present we refrain from dividing any of the morphospecies. This is because there is a continuum of levels of intra- and interspecific genetic variation in this system, so that distinguishing the two would be largely arbitrary.     

Morphology,morphogenesis, and molecular phylogeny of a novel saline soil ciliate,Heterourosomoida sinica n. sp. (Ciliophora,Hypotrichia)

The living morphology, infraciliature, morphogenesis and phylogenetic position of a new hypotrichous ciliate, Heterourosomoida sinica n. sp., discovered in saline soil in Northwest of China, were investigated. Heterourosomoida sinica n. sp. is characterized as follows: body size about 70–95 × 20–30 μm in vivo, ellipsoid or elongate; cortical granules dark green, 0.5 μm across, arranged around dorsal cilia and in short irregular rows on ventral side; consistently 18 frontoventral-transverse cirri; left and right marginal rows composed of 17–24 and 13–21 cirri, respectively; two macronuclear nodules and one or two micronuclei. The detailed morphogenesis of a species of Heterourosomoida is reported for the first time. The ontogenesis on dorsal side is basically similar to that in Urosomoida-species and the process on ventral side is characterized by: 18 frontoventral-transverse cirri develop from six anlagen with the anlagen V and VI formed in the primary mode. Phylogenetic analyses based on SSU rDNA sequence data reveal that Heterourosomoida sinica n. sp. clusters with Kleinstyla dorsicirrata and forms a clade with the type species H. lanceolata.     

Atlantic moonfishes: independent pathways of karyotypic and morphological differentiation

Fish of the genus Selene, known as lookdowns or moonfish, are one of the most morphologically derived groups of the family Carangidae, whose phylogenetic relationships are still largely unknown. In this study, we discuss karyoevolutionary aspects of three representatives of this genus from the Western Atlantic: Selene brownii (2n = 48; FN = 48), Selene setapinnis (2n = 46; FN = 48), and Selene vomer (2n = 48; FN = 50). Their body patterns were also investigated and compared to one another and in relation to two other species of different genera. Two mechanisms of karyotypic evolution seem to have acted in the diversification of this genus, namely pericentric inversions and centric fusions. Mapping of rDNA sequences showed that chromosome pairs bearing 5S rDNA sites are similar, whereas those bearing 18 rDNA sites are morphologically distinct while apparently also exhibiting interspecies synteny. Although the nucleolar organizer-bearing chromosomes are extremely efficient cytotaxonomic markers among Selene species, others cytogenetic patterns of these species are relatively conserved. Hybridization with telomeric probes (TTAGGG)_n did not exhibit interstitial telomeric sites (ITS), especially in S. setapinnis, where, along with a reduction in diploid number, a large metacentric pair derived from centric fusion is present. Data obtained by geometric morphometrics enable a clear morphological distinction among the three species, as well as in relation to two other species of the genus Caranx and Oligoplites. Data obtained suggest that morphologic evolution in Selene species was primarily dissociated from visible changes that occurred at the chromosomal level.     

Morphological and molecular identification of helminths of the greater bulldog bat Noctilio leporinus (Quiroptera: Noctilionidae) from Campeche,Mexico

Surveys on parasites of bats from the Americas have been conducted, but information on helminths is still scarce, especially in the Neotropical region. In Mexico, there are species of bats that lack of a record for helminth species, such as members of the family Noctilionidae. The present study describes for the first time the helminths of Noctilio leporinus in Campeche, Mexico. In 2017, six specimens of N. leporinus were studied for helminths. The species identification of helminths was based on morphological studies and molecular analysis of fragments of the 28S rDNA. All bat specimens were infected for at least one helminth species. Three helminth taxa were identified: the trematode Pygidiopsis macrostomum, and the nematodes Tricholeiperia cf. proencai, and Heligmonellidae gen. sp. The morphological identification of P. macrostomum was confirmed by sequence analysis of 28S rDNA gene. The phylogeny of P. macrostomum grouped our sequence with other sequences of the same species collected in Brazil. The phylogenetic tree of Heligmonellidae gen. sp. indicated that the helminth belongs to clade formed by the species Odilia bainae, Nippostrongylus magnus and Nippostrongylus brasiliensis of the family Heligmonellidae. The phylogenetic analysis of the 28S sequences of T. cf. proencai did not show any similarity or close affinity with nematodes from which that gene has been sequenced to date. The findings of the present study increase the number of helminth species parasitizing bats in Mexico.     

Phylogeny of Cucurbitaceae species in Korea based on 5S rDNA non-transcribed spacer

The 5S rDNA coding region and its spacer have been successfully utilized in phylogenetic studies of plants. However, it has not been utilized in the phylogenetic analysis of Cucurbitaceae. Here, we obtained the 5S rDNA sequences of 12 Cucurbitaceae species by direct PCR or cloning. The 5S rDNA sequences ranged from 275 to 359 bp, and the coding regions of all species were 120 bp long, except for that of Cucurbita, which was 119 bp. Some genus-specific SNPs were observed in the coding regions of Cucurbita, Lagenaria, Melothria, and Tricosanthes. The GC content of the coding regions was generally higher than that of the NTS regions, and the difference in GC content between the coding and NTS regions varied among species, with Gynostemma pentaphyllum having the greatest difference of 20.3. The phylogenetic trees generated using maximum parsimony and maximum likelihood were congruent and well supported by the recently published classification of Cucurbitaceae. These results demonstrated the utility of the 5S rDNA sequence in inferring phylogenetic relationships among 12 Cucurbitaceae species, and its utility could be extended by using a greater number of species in future studies.     

Morphology,ontogeny and molecular phylogeny of a new brackish water ciliate Bakuella subtropica sp. n. (Ciliophora,Hypotricha) from southern China

This paper investigates the morphology, ontogenesis and small subunit (SSU) rRNA gene-based phylogeny of a new urostylid ciliate, Bakuella subtropica sp. n., discovered from the estuary of the Pearl River in Guangzhou, southern China. The new species is diagnosed by its elongate body, one buccal and one parabuccal cirrus, midventral complex comprised of 9–23 midventral pairs and one or two midventral rows extending to four fifths of body length, yellow-brown to yellow-greenish cortical granules and an estuary habitat. Its main ontogenetic features are: (1) in the proter, the parental adoral zone of membranelles is completely renewed by new structures and old midventral pairs join the formation of frontal-midventral-transverse cirral anlagen (FVT-anlagen); (2) in the opisthe, the oral primordium originates apokinetally, FVT-anlagen are formed besides and some old midventral cirri join the formation; (3) the anlagen for marginal rows and dorsal kineties develop intrakinetally; and (4) the numerous macronuclear nodules fuse into a single mass before dividing. Based on the SSU rDNA sequences, phylogenetic analyses show a close relationship between Bakuella subtropica sp. n., Apobakuella and Neobakuella, forming a clade separated from the other genera in the family Bakuellidae. Available morphological and ontogenetic data challenge the monophyly of Bakuellidae.     

Morphospecies and genospecies of Microcystis during blooms in eutrophic Lake Taihu (China) in autumn

Microcystis colonies collected in a hypertrophic lake (Lake Taihu, China) in October and November 2012 were divided into five subsamples according to colony size (<75, 75–150, 150–300, 300–500, and >500 μm). All the subsamples collected in November were dominated with Microcystis ichthyoblabe and the percentages of M. ichthyoblabe exceeded 83%. The percentages of Microcystis aeruginosa of the subsamples in >500 μm class collected in October was 93.5%. For the sample collected in October, the percentage of M. ichthyoblabe was more than 58% in <75, 75–150, 150–300 μm classes. The 16S rDNA as well as some polysaccharide biosynthesis-related genes were analyzed to understand the phylogeny of Microcystis species. There was no variant site presented in each Microcystis subsample but a single nucleotide polymorphism (SNP) was found in 16S rDNA alignment tested using MSR1 in subsamples between the two months in the current study. Our results also showed that samples collected in two months can by divided into two parts by the phylogenetic analysis using two polysaccharide biosynthesis-related genes (espL and TagH). All the results suggested that 16S rDNA was valuable to identify seasonal succession of Microcystis genospecies and the diversity of Microcystis morphospecies would be explained by these polysaccharide biosynthesis-related genes.     

Morphological and Molecular Characterization of a New Trichuris Species (Nematoda- Trichuridae), and Phylogenetic Relationships of Trichuris Species of Cricetid Rodents from Argentina

Populations of Trichuris spp. isolated from six species of sigmodontine rodents from Argentina were analyzed based on morphological characteristics and ITS2 (rDNA) region sequences. Molecular data provided an opportunity to discuss the phylogenetic relationships among the Trichuris spp. from Noth and South America (mainly from Argentina). Trichuris specimens were identified morphologically as Trichuris pardinasi, T. navonae, Trichuris sp. and Trichuris new species, described in this paper. Sequences analyzed by Maximum Parsimony, Maximum Likelihood and Bayesian inference methods showed four main clades corresponding with the four different species regardless of geographical origin and host species. These four species from sigmodontine rodents clustered together and separated from Trichuris species isolated from murine and arvicoline rodents (outgroup). Different genetic lineages observed among Trichuris species from sigmodontine rodents which supported the proposal of a new species. Moreover, host distribution showed correspondence with the different tribes within the subfamily Sigmodontinae.     

Biogeographically interesting planktonic Nostocales (Cyanobacteria) in the Czech Republic and their polyphasic evaluation resulting in taxonomic revisions of Anabaena bergii Ostenfeld 1908 (Chrysosporum gen. nov.) and A. tenericaulis Nygaard 1949 (Dolichospermum tenericaule comb. nova)

Questions of biogeography of freshwater cyanobacteria and their ability to colonize new areas have been recently discussed in connection with increasing occurrence of some formerly rare morphospecies in temperate zones. Nevertheless, the general knowledge about the distribution of cyanobacterial species is still fragmentary, and any new findings on cyanobacterial biogeography and spread are valuable. In this study, we provide updated information on the occurrence of Anabaena bergii, Raphidiopsis mediterranea, and Sphaerospermopsis aphanizomenoides in the Czech Republic. In addition, more nostocacean morphospecies are newly reported from the Czech Republic (A. fusca, A. tenericaulis, Dolichospermum curvum, D. mucosum, and S. reniformis). All of these morphospecies were characterized from a morphological point of view, and their phylogenetic affiliations were assessed on the basis of their 16S rRNA gene sequences. Based on these results, Anabaena bergii was reclassified into Chrysosporum gen. nov., and D. tenericaule comb. nova was established.