Phylogeny of Six Genera of the Subclass Haptoria (Ciliophora,Litostomatea) Inferred from Sequences of the Gene Coding for Small Subunit Ribosomal RNA

ABSTRACT. The small subunit ribosomal RNA genes of nine species belonging to six genera of litostome ciliates, namely Amphileptus aeschtae, Chaenea teres, Chaenea vorax, Lacrymaria marina, Litonotus paracygnus, Loxophyllum sp.‐GD‐070419, Loxophyllum jini, Loxophyllum rostratum, and Phialina salinarum, were sequenced for the first time. Phylogenetic trees were constructed using different methods to assess the inter‐ and intra‐generic relationships of haptorians, of which Chaenea, Lacrymaria, Litonotus, and Phialina were analyzed for the first time based on molecular data. Monophyly of the order Pleurostomatida was strongly confirmed, and the two existing families of pleurostomatids, created on the basis of morphology, were confirmed by molecular evidence. Within the Pleurostomatida, Siroloxophyllum utriculariae occupied a well‐supported position basal to the Loxophyllum clade, supporting the separation of these genera from one another. Both the subclass Haptoria and the order Haptorida were partially unresolved, possibly paraphyletic assemblages of taxa in all analyses, creating doubts about the traditional placement of some haptorid taxa. The existing sequence of L. rostratum in GenBank (DQ411864) was conspicuously different from that of the isolate from Qingdao, China sequenced in the present work, indicating that they are different species. The isolate from Qingdao was verified as L. rostratum by morphological analysis, and the published morphology of existing GenBank record of L. rostratum is different from it. Based on both morphological and molecular evidence, the latter may be congeneric with an undescribed species of Loxophyllum from Guangdong Province, China.     

How Quaternary geologic and climatic events in the southeastern margin of the Tibetan Plateau influence the genetic structure of small mammals: inferences from phylogeography of two rodents, <Emphasis Type="Italic">Neodon irene</Emphasis> and <Emphasis Type="Italic">Apodemus latronum</Emphasis>

Phylogeographical studies that focus on the southeastern margin of the Tibetan Plateau are limited. The complex terrain and unique geological history make it a particularly unusual region of the Tibetan Plateau. We carried out a phylogeographical study of two rodent species Neodon irene and Apodemus latronum using the mitochondrial cytochrome b gene sequences. High genetic diversities and deep phylogenetic splits were detected in both rodents. Some haplotypes from one sampling region fell into different evolutionary clades, but most haplotypes from the same sampling regions were clustered together with each other. The results of isolation by distance analysis further substantiated that their genetic diversities were structured along geography. Thus, there were high levels of geographical structure for both rodents. Demographic analyses implied a relatively constant population size for all samples of N. irene and A. latronum in history. However, clade B of N. irene and clade 3 of A. latronum experienced population expansions at 105–32 and 156–47 Kya, respectively. Through comparison with previous studies, we suggest the high mitochondrial DNA diversities in them are probably not a species-specific feature, but a common pattern for small mammals in this unique area. Details of the historical demography of these rodents revealed in this study could provide new insights into how rodents and possibly other small mammals in this region responded to the geological and climatic events.     

Revision of the Helicellinae of Crete (Gastropoda: Hygromiidae)

This paper presents a comprehensive revision of the Helicellinae of Crete (Gastropoda: Hygromiidae) based on characters of the shell and the genitalia. The native fauna includes eleven Xerocrassa species, two Pseudoxerophila species, and perhaps one Xeromunda species. One additional Xerocrassa species and eight species of the genera Trochoidea, Xeropicta, Xerolenta, Xerotricha, Microxeromagna, and Cernuella were probably introduced by man to Crete. The distribution patterns of the endemic Xerocrassa and Pseudoxerophila species do not provide evidence for the hypothesis that these radiations were caused by the fragmentation of the region of present‐day Crete into several palaeoislands in the late Miocene and Pliocene.     

The Klingon batbugs: Morphological adaptations in the primitive bat bugs,Bucimex chilensis and Primicimex cavernis,including updated phylogeny of Cimicidae

The Cimicidae is a family of blood‐dependent ectoparasites in which dispersion capacity is greatly associated with host movements. Bats are the ancestral and most prevalent hosts for cimicids. Cimicids have a worldwide distribution matching that of their hosts, but the global classification is incomplete, especially for species outside the most common Cimicidae taxa. In this study, we place a little‐studied cimicid species, Bucimex chilensis, within a comprehensive molecular phylogeny of Cimicidae by sequencing the genomic regions of this and other closely related species. For this study, we collected B. chilensis females from Myotis chiloensis in Tierra del Fuego, 1,300 km further south than previously known southernmost distribution boundary. We also sequenced COI regions from Primicimex cavernis, a species which together with B. chilensis comprise the entire subfamily Primiciminae. Using Bayesian posterior probability and maximum‐likelihood approaches, we found that B. chilensis and P. cavernis clustered close to each other in the molecular analyses, receiving support from similar morphological features, agreeing with the morphology‐based taxonomic placement of the two species within the subfamily Primiciminae. We also describe a previously unrecognized morphological adaptation of the tarsal structure, which allows the austral bat ectoparasite, B. chilensis, to cling on to the pelage of its known host, the Chilean myotis (Myotis chiloensis). Through a morphological study and behavioral observation, we elucidate how this tarsal structure operates, and we hypothesize that by clinging in the host pelage, B. chilensis is able to disperse effectively to new areas despite low host density. This is a unique feature shared by P. cavernis, the only other species in Primiciminae.     

Morphological and molecular analysis of centropagids from the high Andean plateau (Copepoda: Calanoidea)

In small metazoan invertebrates classical taxonomic analyses can be ambiguous due to the limited number of morphological characters available. This difficulty can yield incorrect estimates of species richness or taxa distribution. The Boeckella genus has been described as the dominant taxon of zooplankton assemblages in the Andean biogeographical region. In this genus, taxonomic classification and delimitation of boundaries between species has long been problematic and controversial. Among South American centropagids Boeckella gracilipes has been regarded as one of the most broadly distributed species, its presence having been reported from Ecuador to Tierra del Fuego. However, in the high Andean plateau some centropagid populations identified as Boeckella gracilipes have also been considered as B. gracilipes titicacae or even identified as a different species, namely Boeckella titicacae. In an attempt to resolve the taxonomic status of the Centropagidae family from the high Andean plateau, we combined traditional and multivariate morphological analyses (integral approach) with the molecular phylogenetic approach. The results obtained allow us to conclude that centropagids collected from the high Andean plateau actually represent a different species, B. titicacae, not B. gracilipes. The phylogenetic reconstruction of the South American Centropagidae family indicated that B. gracilipes represents a sister taxon to B. titicacae. The present study stresses the usefulness of integrating alfa-taxonomy with morphometric and molecular approaches in order to resolve species boundaries, to determine geographical distributions and to investigate evolutionary processes.     

Phylogenetic Relationships Among Hypotrichous Ciliates Determined with the Macronuclear Gene Encoding the Large, Catalytic Subunit of DNA Polymerase α

The complete macronuclear DNA polymerase α gene, previously sequenced in Oxytricha nova, has been cloned from a genomic macronuclear library and sequenced for the hypotrich O. trifallax. Macronuclear DNA clones of DNA polymerase α encoding ∼1000 amino acids, or approximately two-thirds of the open reading frame, have been obtained by PCR and sequenced for Halteria grandinella, Holosticha species, Paraurostyla viridis, Pleurotricha lanceolata, Stylonychia lemnae Teller, Sty. mytilus, Uroleptus gallina, and Urostyla grandis. Phylogenetic relationships inferred from DNA polymerase α amino acid sequences have been used to clarify taxonomic relationships previously determined by morphology of the cell cortex. Hypotrich phylogenies based on DNA polymerase α amino acid sequences are incongruent with morphological and other molecular phylogenies. Based upon these data, we assert that, contrary to morphological data, O. nova and O. trifallax are different species, and we propose that the oligotrich Halteria grandinella be reclassified as a hypotrich. This work also extends the available data base of eukaryotic DNA polymerase α sequences, and suggests new amino acid sequence targets for mutagenesis experiments to continue the functional dissection of DNA pol α biochemistry at the molecular level. Received: 7 January 1997 / Accepted: 7 April 1997     

Phylogenomic analysis of pseudocryptic diversity reveals the new genus Deltalsia (Rhodomelaceae,Rhodophyta)

Molecular analyses, in combination with morphological studies, provide invaluable tools for delineating red algal taxa. However, molecular datasets are incomplete and taxonomic revisions are often required once additional species or populations are sequenced. The small red alga Conferva parasitica was described from the British Isles in 1762 and then reported from other parts of Europe. Conferva parasitica was traditionally included in the genus Pterosiphonia (type species P. cloiophylla in Schmitz and Falkenberg 1897), based on its morphological characters, and later transferred to Symphyocladia and finally to Symphyocladiella using molecular data from an Iberian specimen. However, although morphological differences have been observed between specimens of Symphyocladiella parasitica from northern and southern Europe they have yet to be investigated in a phylogenetic context. In this study, we collected specimens from both regions, studied their morphology and analyzed rbcL and cox1 DNA sequences. We determined the phylogenetic position of a British specimen using a phylogenomic approach based on mitochondrial and plastid genomes. Northern and southern European populations attributed to S. parasitica represent different species. Symphyocladiella arecina sp. nov. is proposed for specimens from southern Europe, but British specimens were resolved as a distant sister lineage to the morphologically distinctive Amplisiphonia, so we propose the new genus Deltalsia for this species. Our study highlights the relevance of using materials collected close to the type localities for taxonomic reassessments, and showcases the utility of genome-based phylogenies for resolving classification issues in the red algae.     

Morphological vs. molecular delineation of taxa across montane regions in Europe: the case study of Gammarus balcanicus Schäferna, (Crustacea: Amphipoda)

Mountainous areas are characterized by substantial biodiversity and endemicity due to their complex geological history and habitat fragmentation. Hence, it can be assumed that particularly high species richness can be found in organisms with limited dispersal capabilities that inhabit mountain streams. A number of scientific papers focus on molecular phylogeography or traditional taxonomy of species or species groups inhabiting such habitats. However, there is a lack of studies that integrate morphological and molecular data to identify and delineate cryptic species. For practical reasons, uncovering cryptic diversity is crucial in taxa used in biomonitoring. Distinct species, hard to separate based on morphology only, may have different tolerance ranges towards a variety of factors. Thus, our goal is to combine the two approaches to reveal potential patterns of diversification within a species widely distributed across European mountains: the amphipod crustacean Gammarus balcanicus. The data were obtained from 13 populations spread across the range of the species. Individuals were initially ascribed to G. balcanicus based on conventional fauna key morpho‐anatomical diagnostic features and were further analysed for 23 additional features to explore any putative diversification. Morphometric data were analysed with use of the multiple correspondence analysis and anova . Molecular distances were calculated for 551‐bp‐long COI sequences. Test for isolation by distance was performed for both morphological and molecular data. The morphometric studies showed that some of the analysed features differed significantly between populations, although there was only a weak correlation between the morphological divergence and the between‐population geographical distances. Moreover, high morphological diversity was present within sites. A set of 42 COI haplotypes was identified among the 135 individuals sequenced. No haplotype was shared among populations. The molecular p‐distances within the nine localities presenting more than one haplotype were either almost null (ca. <0.003 for 7 localities) or relatively low (ca. 0.01–0.02 for 2 localities). In opposite, the molecular p‐distances between localities were mostly at a high level (94% of pairwise comparisons being >0.14), similar as between other well‐defined species of the genus Gammarus. Surprisingly, G. balcanicus appears to be polyphyletic based on topology of the neighbour‐joining tree. The level of genetic distance between localities was not correlated with their geographical proximity. Globally, combining spatial patterns of morphological versus molecular divergence indicates a high level of cryptic diversity within a species conventionally defined based upon fauna key morphological features. In this context, the name G. balcanicus should be applied only to the population from locus typicus, while the other populations represent a number of putative distinct species. We may expect that such phenomenon would apply also to other animal taxa with conserved morphology, which are widespread over different mountain ranges in Europe.     

Bryoerythrophyllum duellii Blockeel (Bryophyta: Pottiaceae), a new moss species from Greece and Cyprus,and its molecular affinities

A moss first collected on Crete by Prof. Ruprecht Düll in 1976 but never satisfactorily identified is described as a new species, Bryoerythrophyllum duellii. It is known from five localities in Greece and Cyprus. It belongs to the B. recurvirostrum group, and its status as a distinct taxon is supported by molecular data. The molecular relationships of the genus Bryoerythrophyllum are discussed, and they suggest that the genus is monophyletic only if Erythrophyllopsis, Mironia and Saitobryum are included. The B. recurvirostrum and B. wallichii groups apparently form a young complex of taxa that are not very diverse molecularly, but sometimes have a distinctive morphology. Bryoerythrophyllum duellii is one of several lineages within this complex that may have experienced a longer period of reproductive isolation leading to the fixation of distinctive morphological and molecular features.     

Four novel yeasts from decaying organic matter: Blastobotrys robertii sp. nov., Candida cretensis sp. nov., Candida scorzettiae sp. nov. and Candida vadensis sp. nov

Four novel yeast species are described, two from decaying mushrooms, viz. Candida cretensis and Candida vadensis, and two from rotten wood, viz. Blastobotrys robertii and Candida scorzettiae. Accession numbers for the CBS and ARS Culture Collections, and GenBank accession numbers for the D1/D2 domains of the large subunit of ribosomal DNA are: B. robertii CBS 10106^T, NRRL Y-27775, DQ839395; C. cretensis CBS 9453^T, NRRL Y-27777, AY4998861 and DQ839393; C. scorzettiae CBS 10107^T, NRRL Y-27665, DQ839394; C. vadensis CBS 9454^T, NRRL Y-27778, AY498863 and DQ839396. The GenBank accession number for the ITS region of C. cretensis is AY498862 and that for C. vadensis is AY498864. C. cretensis was the only species of the four that displayed fermentative activity. All four type strains grew on n-hexadecane. C. scorzettiae is the only one of the new species that assimilates some phenolic compounds, viz. 3-hydroxy derivatives of benzoic, phenylacetic and cinnamic acids, but not the corresponding 4-hydroxy acids. This is indicative of an operative gentisate pathway.     

Morphological and Molecular Identification of the New Species,Trichodina pseudoheterodentata sp. n. (Ciliophora,Mobilida, Trichodinidae) from the Channel Catfish,Ictalurus punctatus,in Chongqing China

It is difficult to differentiate similar trichodinids solely based on morphological examination, thus other identification methods, such as molecular identification, are necessary for identification. One mobilid ciliate named Trichodina pseudoheterodentata sp. n. was isolated from the gills of channel catfish, Ictalurus punctatus, in Chongqing, China. In the present study, its SSU rDNA was sequenced for the first time. Based on the results from both morphological identification and SSU rDNA sequencing, the new species was identified and compared with similar species. The morphological analysis revealed that T. pseudoheterodentata is a large Trichodina species (cell diameter 73.0–82.5 μm) and possesses robust denticles with broad blades and well‐developed blade connections. Characterization of its primary and secondary SSU rDNA structures indicated that T. pseudoheterodentata was distinctly different from congeneric species in H12, H15, E10_1, and V4 regions. Phylogenetic analysis revealed that the genetic distances among the new species and similar species reached interspecific levels, furthermore, the phylogenetic study also validated the identification of T. pseudoheterodentata and its placement in the genus Trichodina.     

The origin and diversity of Drilus Olivier, 1790 (Elateridae: Agrypninae: Drilini) in Crete based on mitochondrial phylogeny

We investigated the phylogeography and speciation of Drilus in the Mediterranean, with focus on the Aegean and especially Crete. Altogether 12 species were sequenced for two fragments of the cytochrome c oxidase subunit I gene (cox1–3′ and cox1–5′ mtDNA) and provided 1381 nucleotides. Both fragments were analysed under the maximum likelihood criterion and Bayesian inference separately and concatenated as a single dataset. The Drilus species from the Peloponnese, the Ionian islands and Crete did not form a monophylum. Drilus sp. E from the Peloponnese and Zakynthos was sister to D. mauritanicus Lucas from Spain in most cases. The remaining Greek Drilus species formed a robustly supported clade in all analyses; however, the species from Crete do not seem to be monophyletic. Estimating species divergences using BEAST, we found out that the key dates in the west Aegean Drilus phylogeography appeared to be the Tortonian Crete–Peloponnese separation (12–9 Mya), the desiccation of the Mediterranean basin during the Messinian Salinity Crisis (5.96–5.33 Mya), and the repeated fragmentation of Crete during the Pliocene and Pleistocene. Within the Drilus lineages, we obtained a substitution rate estimate of 2.75% divergence per million years, which is in excellent agreement with previous studies. A Generalized Mixed Yule Coalescent (GMYC) analysis suggested the presence of six Drilus species in Crete (seven species in total, since the DNA sequences were not available for D. creticus Pic); however, we formally identified and (re)described only four which can be morphologically defined: D. creticus, D. longulus Kiesenwetter, D. horasfakionus sp. nov. and D. baenai sp. nov. These species are endemic to Crete and surrounding islets. Their diagnostic characters are illustrated and an identification key to males of these species is provided. The intraspecific variability, distribution and ecology of all species are discussed and suggestions for further research are given.

http://zoobank.org/urn:lsid:zoobank.org:pub:F8DDBCC1-C2D1-48F7-BE99-55D9ED4C2234     

Age estimates of Frullania (Frullaniaceae,Porellales) main lineages: another example of rapid and recent diversification in liverwort evolution

Frullania Raddi is an extant genus of liverworts (Bryophytes) widespread around the world. It belongs to the family Frullaniaceae Lorch., with a large number of species distributed into several subgenera, sections and subsections according with different morphological classifications. As shown in previous studies, most Frullania sub-generic classifications are supported by molecular data, indicating that morphological characters appear well suitable to discriminate species. However, deep among-clade relationships remain unclear, probably due to the rapid diversification of main clades, paralleling other plant lineages. In this study, we reconstruct Frullania phylogeny based on available molecular data used in previous studies, and we present time estimates for the origin of its main branches. Results supported the monophyly of most subgenera as demonstrated in previous studies and supported a rapid diversification of these main lineages. Time estimates under a relaxed molecular clock and with integrated fossil evidence and nucleotide mutation rates further suggested a Jurassic origin of the genus and a rapid diversification during Palaeogene and Neogene. This may have been influenced by geographical and climate changes during these periods as predicted for most leafy liverworts.     

Molecular and chemical characters to evaluate species status of two cuckoo bumblebees: Bombus barbutellus and Bombus maxillosus (Hymenoptera,Apidae, Bombini)

Many methods, based on morphological, molecular or chemical characters, have been used to address the question of species taxonomic status. Integrative taxonomy aims to define stronger supported taxonomic hypotheses by considering complementary datasets from different characters. By following an integrative approach, the present study includes molecular, chemical and morphological criteria to establish the taxonomic status of two rare and doubtful cuckoo bumblebee taxa: Bombus (Psithyrus) barbutellus and Bombus (Psithyrus) maxillosus. These two sympatric taxa are discriminated by few morphological criteria (mainly wing darkness and hair length). We used these morphological character diagnoses to establish an a priori status of our samples (23 specimens). We developed a combined molecular dataset from one nuclear gene, elongation factor 1α (EF‐1α), and one mitochondrial gene, cytochrome c oxidase subunit I (COI), spanning 1623 bp, and a chemical dataset of sexual marking pheromones (73 compounds). The molecular data were subjected to maximum‐likelihood and Bayesian phylogenetic inference under partitioned model and maximum parsimony. The chemical data were analysed by clustering and the two‐group k‐means method to test divergences between the two species. The resulting phylogenetic trees show no consistent divergence between the two taxa. Moreover, we found no divergence in the sexual marking pheromones in the clustering and two‐group k‐means analyses. These converging results support the conspecificity of both taxa. Nonetheless, our determinations using the traditional morphological criteria separated our samples into two taxa. We conclude that the morphological criteria seem to relate to intraspecific variations: B. maxillosus is regarded as a syn.n. of B. barbutellus.     

Three novel species of Stemphylium from Sinkiang, China: their morphological and molecular characterization

Three new species of Stemphylium were isolated from diseased leaves of Luffa cylindrica, Lycium chinense and Cucumis melo growing in the Sinkiang province of Northwest China. Stemphylium luffae, S. lycii and S. cucumis are described by morphological and molecular phylogenetic analyses. The principal morphological characteristics of these three species are congruent with those of Stemphylium. They are distinguished from morphologically similar Stemphylium species based on characteristics of their conidia and conidiophores. In order to establish the molecular phylogenetic status of these species, DNA loci including the internal transcribed spacer (ITS) nuclear rDNA region and the glyceraldehyde-3-phosphate dehydrogenase (gpd) genes were amplified and sequenced. Phylogenetic analyses using Maximum-Parsimony (MP) and Neighbor-Joining (NJ) were performed on the combined DNA sequences of ITS and gpd gene regions. Both morphological observations and molecular analyses support S. luffae, S. lycii and S. cucumis as three new taxa.     

Phylogeny in Echinocereus (Cactaceae) based on combined morphological and molecular evidence: taxonomic implications

Echinocereus is a morphologically diverse genus that includes 64 species grouped into eight taxonomic sections based on morphological traits. In previous molecular phylogenetic analyses, the relationships amongst Echinocereus species were not entirely revealed and useful characters to recognize clades were not provided. The inclusion of several sources of evidence in a phylogenetic analysis is likely to produce more supported hypotheses. Therefore, we performed a combined phylogenetic analysis with a set of 44 morphological characters and six chloroplast DNA sequences. Topologies from parsimony and Bayesian analyses were mostly congruent. However, the relationships of E. poselgeri were not consistent between analyses. A second Bayesian analysis using a long-branch extraction test resulted in a topology with the morphological position of E. poselgeri congruent with that in parsimony analysis. Parsimony and Bayesian analyses corroborated the monophyly of Echinocereus, which included eight monophyletic groups. The combined phylogeny integrated into different clades those taxa that were not determined in previous analyses and changed the relationships of some recognized clades. The clades did not recover the recent infrageneric classification. In the present study, a new sectional classification for Echinocereus is proposed based on the eight recovered clades, which is supported by a combination of morphological and molecular characters. An identification key for sections in the genus is included.     

Legs of deception: disagreement between molecular markers and morphology of long‐legged flies (Diptera,Dolichopodidae)

Conflicting hypotheses in phylogenetics and systematics, generated by different data sets (e.g. morphological versus molecular), are common in biology. The clarification of such instances may allow understanding general mechanisms involved in the speciation process in an evolutionary light. Here, we present and discuss the case of the Dolichopus plumipes species group in the long‐legged flies, Dolichopodidae. A phylogenetic survey was performed based on both morphological and molecular data. The full data set comprises 31 morphological characters and 2252 molecular characters (mitochondrial – COI: 810; 12S: 343; 16S: 514; nuclear – ITS2: 585) of 49 different species, represented by 82 specimens. The molecular phylogenetic analysis revealed a clade (composed by the species D. plumipes, Dolichopus wahlbergi, Dolichopus polleti, Dolichopus simplex, and Dolichopus nigricornis) that disagrees with the traditional morphological view based on external characters. In particular, specimens of the species D. plumipes and D. simplex were indistinguishable with the molecular markers used here. Yet, we still consider D. plumipes and D. simplex as two distinct taxa and provide explanatory hypotheses on the evolutionary background. The conspicuous male secondary sexual characters (present in plumipes but not in simplex) are key factors in sexual selection and their presumably rapid reduction in D. simplex is thought to be of main importance for the explanation of the speciation process. The plumipes–simplex case may therefore be viewed as a paradigmatic illustration showing that a better integration of the molecular and morphological approaches is needed to understand and clarify the, in some cases, complex systematics and phylogeny of organisms.