Towards a molecular phylogeny of the fungus gnat genus Boletina (Diptera: Mycetophilidae)

Martinsson, S., Kjærandsen, J. & Sundberg, P. (2011). Towards a molecular phylogeny of the fungus gnat genus Boletina (Diptera: Mycetophilidae). —Zoologica Scripta, 40, 272–281. Boletina is a species rich genus of fungus gnats (Diptera: Mycetophilidae) with a mainly Holarctic distribution. The systematics within the genus has gained little attention and this is a first attempt to shed some light over the systematics of Boletina and to test the segregation of the genera Saigusaia and Aglaomyia from Boletina. The nuclear marker 28S and mitochondrial 16S, COI and CytB were amplified and sequenced for 23 taxa that were analysed separately and together with a broad sample of outgroup taxa obtained from GenBank, where also 18S sequences were added. Phylogenies were estimated using maximum likelihood, Bayesian inference and parsimony. We strengthen the hypothesized sister‐group relationship between Docosia and Boletina, but the genus Boletina as currently delimited appears to be paraphyletic and nested in a clade together with Aglaomyia, Coelosia and Gnoriste. The genus Saigusaia, on the other hand, seems to be well separated from Boletina. The Boletina erythropyga species group is consistently found as a distinct basal clade within Boletina s.l. The results obtained are otherwise ambiguous both for the taxa in focus and in some analyses globally with a statistically supported total breakdown of the traditional higher classification into tribes, subfamilies and even families. Interestingly, this breakdown almost disappeared when additional 18S sequences were added.     

Towards a species level tree of the globally diverse genus Chenopodium (Chenopodiaceae)

Chenopodium is a large and morphologically variable genus of annual and perennial herbs with an almost global distribution. All subgenera and most sections of Chenopodium were sampled along with other genera of Chenopodieae, Atripliceae and Axyrideae across the subfamily Chenopodioideae (Chenopodiaceae), totalling to 140 taxa. Using Maximum parsimony and Bayesian analyses of the non-coding trnL-F (cpDNA) and nuclear ITS regions, we provide a comprehensive picture of relationships of Chenopodium sensu lato. The genus as broadly classified is highly paraphyletic within Chenopodioideae, consisting of five major clades. Compared to previous studies, the tribe Dysphanieae with three genera Dysphania, Teloxys and Suckleya (comprising the aromatic species of Chenopodium s.l.) is now shown to form one of the early branches in the tree of Chenopodioideae. We further recognize the tribe Spinacieae to include Spinacia, several species of Chenopodium, and the genera Monolepis and Scleroblitum. The Chenopodium rubrum and the Ch. murale-clades were newly discovered as distinct major lineages but their relationships within Chenopodioideae will need further evaluation. Based on our results, we suggest the delimitation of Chenopodium to include Einadia and Rhagodia because these are part of the crown group composed of species of subg. Chenopodium that appear sister to the Atripliceae. The tetraploid crops such as Ch. berlandieri subsp. nuttalliae and Ch. quinoa also belong to Chenopodium sensu stricto. Trees derived from trnL-F and ITS were incongruent within this shallow crown group clade. Possible biological causes are discussed, including allopolyploidization.     

Towards a molecular phylogeny of colonial spumellarian radiolaria

Gene sequence data from the small-subunit ribosomal RNA coding region were used to explore evolutionary relationships among the colonial spumellarian radiolaria (Polycystinea). Representatives from the two spumellarian families known to form colonies were considered including the following taxa: Sphaerozoidae: Collozoum pelagicum; Collozoum serpentinum; Rhaphidozoum acuferum; Sphaerozoum punctatum; and Collosphaeridae: Collosphaera globularis–huxleyi; Acrosphaera (circumtexta?); and Siphonosphaera cyathina. Our molecular analyses support the monophyly of the Collosphaeridae in all analyses used, but only distance analyses support the monophyly of the Sphaerozoidae. Within the Sphaerozoidae, two species of Collozoum (C. serpentinum and C. pelagicum) failed to branch together, indicating a more distant relationship than first suggested, a conclusion further supported by recent ultrastructural studies (see adjoining paper). Branching patterns within the Collosphaeridae indicate that Siphonosphaera diverged prior to the split between Collosphaera and Acrosphaera, a result which challenges evidence based on data from the radiolarian fossil record.     

A molecular phylogeny of the Sylvia cantillans complex: cryptic species within the Mediterranean basin

The subalpine warbler Sylvia cantillans is formally considered a polytypic species, with four subspecies, European S. c. cantillans, albistriata, moltonii (recently resumed name: subalpina) and North African S. c. inornata. They are very similar in external morphology but clearly differ in their vocalizations. We evaluated their uncertain taxonomic status reconstructing the phylogenetic and phylogeographic relationships among populations sampled across major biogeographical areas in the European species’ range, using nucleotide sequences of the mitochondrial cytochrome b gene (mtDNA cyt b). A variety of phylogenetic analyses concordantly led to identify four major groups, only partially corresponding to the three European nominal subspecies. Phylogenetic trees showed a monophyletic group including all moltonii individuals, well diverged from all other taxa. Populations taxonomically assigned to cantillans were polyphyletic being split into two distinct clades (western and southern cantillans), with monophyletic albistriata closely related to southern cantillans. Individuals of moltonii and southern cantillans sampled in sites of sympatry in central Italy were assigned to their respective groups, with perfect concordance between phenotypic and genetic identifications. All findings indicate that moltonii should be ranked as a distinct species. Former subspecies cantillans is polyphyletic, but additional data are needed to define the taxonomic status of its two clades. Albistriata is phylogenetically related to southern cantillans and should be provisionally kept as a subspecies of S. cantillans. The cantillans complex thus provides an interesting case-study illustrating geographical structuring across small geographical ranges, and it exemplifies speciation through differentiation in allopatry leading to reproductive isolation after a secondary contact.     

Towards a phylogeny of the genus Vibrio based on 16S rRNA sequences.

The inter- and intrageneric relationships of the genus Vibrio were investigated by performing a comparative analysis of the 16S rRNAs of 10 species, including four pathogenic representatives. The results of immunological and 5S rRNA studies were confirmed in that the genus is a neighboring taxon of the family Enterobacteriaceae. With regard to the intrageneric structure, Vibrio alginolyticus, Vibrio campbellii, Vibrio natriegens, Vibrio harveyi, Vibrio proteolyticus, Vibrio parahaemolyticus, and Vibrio vulnificus form the core of the genus, while Vibrio (Listonella) anguillarum, Vibrio diazotrophicus, and Vibrio hollisae are placed on the outskirts of the genus. Variable regions around positions 80, 180, and 450 could be used as target sites for genus- and species-specific oligonucleotide probes and polymerase chain reaction primers to be used in molecular identification.     

Northern species of earth tongue genus Thuemenidium revisited, considering morphology, ecology and molecular phylogeny

Thuemenidium is a small earth tongue genus with three recognized white-spored species. Within Thuemenidium, T. atropurpureum and T. arenarium have been reported only from the northern hemisphere while T. berteroi is known solely in the southern hemisphere. We reviewed the ecology, examined the morphology and inferred the systematic positions of northern species of Thuemenidium from LSU-rDNA gene phylogeny of 48 taxa in Pezizomycotina including recent collections. Our results suggest that Thuemenidium in its current sense is polyphyletic and that T. atropurpureum, closely related to Microglossum and Leotia species, is a member of Leotiaceae (Helotiales, Leotiomycetes). Our phylogeny placed the other northern species, T. arenarium, in Geoglossaceae (Geoglossales, Geoglossomycetes), retaining genus Thuemenidium, pending further investigation.     

Molecular definition and the ubiquity of species in the genus Naegleria

To investigate the variability within species of the genus Naegleria, the ITS1,5.8S and ITS2 rDNA were sequenced of several strains of N. lovaniensis and its Western Australian variants, N. australiensis, N. fowleri, N. andersoni, N. jamiesoni, N. tihangensis, N. pringsheimi, N. pagei, N. gruberi sensu lato and a Naegleria lineage that lost a group I intron from the SSUrDNA twintron. As a result, it is possible to define a molecular species within the Naegleria genus. In addition, one strain of each different allozyme cluster was sequenced to investigate whether they belong to described species or should be treated as distinct new species. This leads to the proposal of eleven new species. The sequencing results from those Naegleria spp. of which several strains are available indicate that these species are ubiquitous. The only exception might be the species represented by the WA variants. However, there are still many Naegleria spp. for which only one strain has been isolated, hence, it is important that the search for more isolates should be continued worldwide.     

Distribution and molecular phylogeny of the dinoflagellate genus Ostreopsis in Thailand

The toxic benthic dinoflagellate genus Ostreopsis has been connected to the production of palytoxin and its analogs in many tropical and temperate areas. Although the type species, O. siamensis, was originally described from the Gulf of Thailand in 1901, little is known about the species composition and distribution of the genus Ostreopsis in Thailand. In this study, a total of 64 Ostreopsis strains isolated from the Andaman Sea as well as the Gulf of Thailand were investigated by analyzing the nucleotide sequences of the LSU rDNA D1/D2, D8/D10 and ITS-5.8S rDNA regions. Phylogenetic analyses (BI and ML) resulted in some of the strains being assigned to previously described clades, O. cf. ovata and Ostreopsis sp. 6, and revealed the existence of a novel clade named Ostreopsis sp. 7, which exhibited large genetic distances from the other clades. Among O. cf. ovata, several strains from Thailand were formed into a new subclade, the Thailand subclade, whereas a few strains belonged to the South China Sea subclade. Morphometric characteristics such as the cell sizes of the two O. cf. ovata subclades and those of Ostreopsis sp. 7 were not significantly different from each other (p > 0.05). Their characteristics were similar but slightly different from those of O. ovata and were significantly different from those of Ostreopsis sp. 6 (p < 0.05). Toxicities of Ostreopsis from Thailand were evaluated using mouse bioassay. Strains of Ostreopsis sp. 6 and Ostreopsis sp. 7 tested were highly toxic, while the two subclades of O. cf. ovata strains seemed to be nontoxic. This study suggests that toxic Ostreopsis sp. 7 is distributed in the Andaman Sea, whereas the two subclades of O. cf. ovata and toxic Ostreopsis sp. 6 are distributed in the Gulf of Thailand.     

DNA barcoding and phylogeny in neotropical species of the genus Spondias

The genus Spondias belongs to the Anacardiaceae family, with about 18 species, having significant economic and social importance and with some species used in the agricultural industry, however, problems are encountered when trying to identify phylogenetic relationships among the species. The use of DNA barcoding is of importance to this group, allowing species identification at the molecular level and in determining the phylogenetic relationships within the group. The objective of this study is to obtain DNA barcoding and to determine the phylogenetic relationships among the species. For this, DNA from six species of the genus was extracted and amplified by PCR using sequences from the rbcL and matK genes and the trnH-psbA spacer gene, followed by sequencing using the Sanger method. The results show that the matK and rbcL genes cannot be used for DNA barcoding, because their discriminatory level between species is low. On the other hand, trnH-psbA shows a high level of discrimination, allowing most of the species to be identified. However it is not possible to separate Spondias venulosa and Spondias tuberosa. Phylogenetic analysis shows that Spondias mombim and S. tuberosa are distinct “umbucajá” clades, suggesting a non-hybrid origin for “umbucajá”.     

A molecular phylogeny for the large African orchid genus Disa

Phylogenetic relationships were inferred for the African subtribe Disinae (Orchidoideae, Orchidaceae), which include the large genus Disa and the small genus Schizodium. One nuclear (ITS) gene region and two plastid (trnLF and matK) gene regions were sequenced for 136 ingroup, representing 70% of all known Disinae species, as well as for 7 outgroup taxa. The combined data matrix contained 4094 characters and was analysed using parsimony and Bayesian inference. Our results show that the generic status of Schizodium can no longer be supported, as it is deeply embedded within the genus Disa. Furthermore, the currently recognised subgenera do not reflect the phylogenetic relationships and should be rejected. Several of the currently recognised sections are monophyletic, others contain misplaced elements, while some are polyphyletic. Morphological divergence, rather than convergence, has hampered previous attempts at a phylogenetic classification of the Disinae. On the basis of our molecular phylogenetic hypothesis, we propose a monotypic subtribe Disinae and a subdivision of the genus Disa into 18 sections.     

Six new species of Pythiogeton in Taiwan,with an account of the molecular phylogeny of this genus

Pythiogeton is a little-studied genus of pythialean Oomycete. The genus is characterized by producing its zoospores outside of the sporangium within an apparently naked protoplasmic mass, which formed from a discharge tube-vesicle complex. A total of nine morphologically distinct Pythiogeton species were identified, of which six were new species (Pythiogeton abundans, Pythiogeton microzoosporum, Pythiogeton oblongilobum, Pythiogeton paucisporum, Pythiogeton proliferatum, and Pythiogeton puliensis). A phylogenetic analysis based on internal transcribed spacer sequences revealed that all isolates of Pythiogeton formed a highly supported clade, nested within the wider clade of Pythium species. Each newly recognized Pythiogeton species that was established on the basis of morphological characters was found to occur in a well-supported subgroup within the Pythiogeton clade, confirming their assignment to new species. Pythiogeton shares a common ancestor with the monophyletic group of Pythium species that have predominantly filamentous sporangia rather than with the separate clade of Pythium species that have predominantly globose or ovoid sporangia. This study confirms that Pythium is an extremely heterogenous and polyphyletic genus containing a number of distinct clades of species, including Pythiogeton, which possess morphologically distinguishable characters. A synoptic key to all the described Pythiogeton species is provided.     

Distribution of a novel mycolic acid in species of the genus Mycobacterium

We found that Mycobacterium porcinum ATCC 33776T (T = type strain) contains a new kind of mycolic acid with a methoxy group at the omega-1 position. This mycolic acid was identified by comparing it with the previously described methoxymycolic acids. The patterns of mycolic acid methyl esters from 418 strains belonging to 44 species of mycobacteria were studied by using thin-layer chromatography. In addition to M. procinum ATCC 33776T, representative strains of M. porcinum, Mycobacterium fortuitum, "Mycobacterium peregrinum," Mycobacterium senegalense, and a recently isolated Mycobacterium sp. contained appreciable amounts of the newly described mycolic acid.     

Systematic revision of the genus Psithyristria Stål (Hemiptera: Cicadidae) with seven new species and a molecular phylogeny of the genus and higher taxa

The cicada genus Psithyristria Stål (Hemiptera: Cicadidae), endemic to Luzon, the Philippines, is reviewed, and its higher taxonomic affiliations are determined using morphology and molecular phylogenetics. Seven new Psithyristria species, P. grandis, sp.n. , P. albiterminalis, sp.n. , P. incredibilis, sp.n. , P. paraspecularis, sp.n. , P. peculiaris, sp.n. , P. moderabilis, sp.n. and P. genesis, sp.n. , are described. A key to the 12 known species is provided. The molecular phylogenetic analyses of Psithyristria and putatively related taxa showed some unexpected generic relationships, leading to changes in higher taxonomic placement for a number of Asian cicada genera. Psithyristria falls within the tribe Cicadini Latreille, 1802, and the tribe Psithyristriini Distant, 1905 syn.n. is therefore synonymized with Cicadini. The subtribe Psithyristriina Distant, 1905 stat.n. is recognized within Cicadini and defined for the genera Psithyristria, transferred from Psithyristriini, and Basa Distant, Pomponia Stål and Semia Matsumura, transferred from Cicadina, synonymizing Pomponiina Kato, 1932 syn.n. with Psithyristriina. The Cicadini subtribe Leptopsaltriina Moulton, 1923 is redefined to include the genera Terpnosia Distant, Euterpnosia Matsumura, Leptosemia Matsumura, Neocicada Kato, Puranoides Moulton, Leptopsaltria Stål, Tanna Distant, Purana Distant, Formosemia Matsumura, Maua Distant, Nabalua Moulton, Taiwanosemia Matsumura, Gudaba Distant and Calcagninus Distant, synonymizing Terpnosiina Kato, 1932 syn.n. The tribe Cicadatrini Distant, 1905 is recognized and redefined to include the genera Cicadatra Kolenati, Psalmocharias Kirkaldy, Mogannia Amyot & Audinet‐Serville, Nipponosemia Kato and Emathia Stål, synonymizing Moganniini Distant, 1905 syn.n. The nine Psithyristria species included in the molecular analyses have an uncorrected mitochondrial genetic distance of 2–9%, and most species appear to be related through a single radiation event that occurred approximately 5–10 Ma. No phylogenetic structure was found in two unusually divergent characters, the forewing venation and the shape of the uncal lobes. Psithyristria is found in the northern Luzon mountains and is closely related to Semia of Taiwan and eastern China, Pomponia of east Asia and the Indonesian/Malaysian islands, and probably most closely to Basa of India. Psithyristria may be a cicadid example of a small number of Philippine organisms that trace their ancestry directly back to the temperate Asian mainland, rather than through the island chains to the south of the country.     

The molecular phylogeny of the genus Rhizopus based on rDNA sequences

In order to establish the molecular phylogeny of the genus Rhizopus, three molecules of the ribosomal RNA-encoding DNA (rDNA), complete 18S, internal transcribed spacer (ITS)1-5.8S-ITS2, and 28S D1/D2 regions of all the species of the genus were sequenced. Phylogenetic trees showed three major clusters corresponding to the three groups in the current morphological taxonomy, microsporus-group, stolonifer-group, and R. oryzae. R. stolonifer var. lyococcos was clustered independently from the major clusters. R. schipperae clustered differently in all trees. Strains of R. sexualis had multiple ITS sequences. A. rouxii clustered with R. oryzae. These results indicate the possibility of molecular identification of species groups using rDNA sequencing. Reclassification of the genus might be appropriate.     

A molecular analysis of relationships and biogeography within a species complex of Holarctic fish (genus Osmerus)

Episodes of trans-Arctic faunal exchange and isolation between the north Pacific and Atlantic ocean basins have been implicated as important historic geological events contributing to extant patterns of genetic diversity and structure in Holarctic faunas. We made a further test of the significance of such biogeographic events by examining mitochondrial DNA (mtDNA) restriction fragment length and cytochrome b sequence polymorphism among north Pacific and Arctic, north-western Atlantic (north-eastern North American), and north-eastern Atlantic (European) regional forms of the boreal smelt, genus Osmerus. Our analyses also assessed whether the regional forms within this ‘species complex’: (i) represent a single widely distributed and polytypic species, or is composed of three geographically distinct species, and (ii) resulted from a single split from north Pacific ancestral Osmerus or two independent Pacific-Atlantic divergences. MtDNA sequence divergence estimates among forms ranged from 5.6–8.9% and from 6.1–8.5% based on restriction fragment and 300 base pairs of cytochrome b sequencing, respectively. Divergence within forms averaged less than 0.5% for fragment analysis and no differences were detected from sequence analysis. Provisional dating of lineage separations in Osmerus based on our sequence divergence estimates suggested a mid-Pliocene to early Pleistocene time frame for diversification among the forms. These estimated lineage separation dates support the idea that geological events in ‘Beringia’ and the surrounding trans-Arctic area (e.g. opening of the Bering Seaway, Pleistocene glacial advances), occurring over a similar time frame, have influenced radiation in Osmerus. Phenetic and parsimony analyses of the sequence divergence estimates and of sequence polymorphisms suggested that the north Pacific/Arctic form and the northwestern Atlantic form shared a common ancestor more recently than either has with the north-eastern Atlantic form, thus supporting the hypothesis that the species complex has arisen from two independent Pacific-Atlantic divergences probably beginning during the mid-Pliocene.     

Dating of divergences within the Rattus genus phylogeny using whole mitochondrial genomes

The timing and order of divergences within the genus Rattus have, to date, been quite speculative. In order to address these important issues we sequenced six new whole mitochondrial genomes from wild-caught specimens from four species, Rattus exulans, Rattus praetor, Rattus rattus and Rattus tanezumi. The only rat whole mitochondrial genomes available previously were all from Rattus norvegicus specimens. Our phylogenetic and dating analyses place the deepest divergence within Rattus at approximately 3.5 million years ago (Mya). This divergence separates the New Guinean endemic R. praetor lineage from the Asian lineages. Within the Asian/Island Southeast Asian clade R. norvegicus diverged earliest at approximately 2.9Mya. R. exulans and the ancestor of the sister species R. rattus and R. tanezumi subsequently diverged at approximately 2.2Mya, with R. rattus and R. tanezumi separating as recently as approximately 0.4Mya. Our results give both a better resolved species divergence order and diversification dates within Rattus than previous studies.     

Mycobacterium valentiae sp. nov., a new species of the genus Mycobacterium isolated from soil

One strain of rapidly growing scotochromogenic mycobacteria was isolated. This study comprises 101 biochemical, cultural and morphological characteristics and reports the behavior towards several concentrations of the most commonly used antituberculous drugs. The organism is considered to belong to a new species of the genus Mycobacterium and has been deposited in the American Type Culture Collection, Rockville, Maryland, USA (ATCC 29356) and in the Czechoslovak Collection of Microorganisms, Czechoslovak National Collection of Type Cultures, Institute of Hygiene and Epidemiology, Srobárova 48, 100 42 Prague 10 under the My 220/77.