Molecular phylogeny of selected Old World Astragalus (Fabaceae): incongruence among chloroplast trnL-F, ndhF and nuclear ribosomal DNA ITS sequences

This study reports maximum parsimony and Bayesian phylogenetic analyses of selected Old World Astragalus using two chloroplast fragments including trnL-F and ndhF and the nuclear ribosomal internal transcribed spacer (nrDNA ITS). A total of 52 taxa including 34 euploid Old World and New World Astragalus , one aneuploid species from the Neo-Astragalus clade as a representative and 14 other Astragalean taxa, plus Cheseneya astragalina and two species of Caragana as outgroups were analyzed for both trnL-F and nrDNA ITS regions. ndhF was analyzed in 30 taxa and the same number for the combination of these three datasets were examined. In general, the trnL-F dataset and the ndhF and nrDNA ITS datasets generated more or less the same clades within Astragalus . However, in the trnL-F and ndhF phylogenies, Astragalus species are not gathered in a single clade, the so-called Astragalus s.s., as indicated by the nrDNA ITS tree. Visual inspection of these three phylogenies revealed that they were inconsistent regarding the position and relationships of Astragalus hemsleyi , A. ophiocarpus , A. annularis–A. epiglottis / Astragalus pelecinus, A. echinatus and A. arizonicus . Incongruence length difference test suggested that the trnL-F , ndhF and nrDNA ITS datasets were incongruent. In spite of this, phylogenetic analyses of the combined datasets as one unit or as three partitions generated trees that were topologically similar as a mix of the cpDNA and the nrDNA ITS trees. However, the combined dataset provided more resolved and statistically supported clades. The recently described A. memoriosus appeared closely related to A. stocksii (both from sect. Caraganella ) based on both trnL-F and nrDNA ITS sequences.     

The evolution of the diatoms (Bacillariophyta). I. Origin of the group and assessment of the monophyly of its major divisions

The diatoms are one of the best characterised algal groups. Despite this, little is known of the evolution of the group from the earliest cell to the myriad of taxa known today. Relationships among taxa at the family or generic level have been recognised in some diatoms. However, relationships at higher taxonomic levels are poorly understood and have often been strongly influenced by the first appearances of key taxa in the fossil record. An independent assessment of relationships among the diatoms at these higher taxonomic levels has been made using rRNA sequence data to infer phylogenetic relationships. In this paper we present an analysis of 18S rRNA data from several chosen centric, araphid and raphid pennate taxa. The phylogenetic inferences from these 18S rRNA sequences are supported by evidence from the fossil record and evidence from ontogenetic data. Ribosomal RNA data indicate that both the centric and araphid pennate lineages may not be monophyletic.     

Preliminary comparison of quantification efficiency between DNA-derived dataset and cell-derived dataset of mixed diatom sample based on rDNA-ITS sequence analysis

Quantification of eukaryotes, such as diatom within a water community is extremely difficult due to the different copy number of the measured gene. ITS sequence was considered to be useful in defining intra-specific or population-level differences for their fast evolving. To assess the possibility of exploring rDNA-ITS region in quantification analysis of environment samples, the fragment of 5.8S + ITS-2 was amplified from extracted DNA and sedimental cell samples derived from the same natural-simulated water community, respectively. Two clone libraries were built from the above DNA-derived (DD) and cell-derived (CD) amplifications. The operational taxonomic units (OTUs) of the libraries were calculated. In most cases, number of OTUs for CD dataset was smaller than DD dataset. For the DD and CD datasets, 112 sequences yielded 6 OTUs-96 and 4 OTUs-97 and were assigned to six and four species, respectively. Intraspecific variations existed in these cultivations. The sequences percentage in datasets did not match with the cell portions in mixed sample, which revealed that 5.8S + ITS-2 copies varied between different diatom species, and it was not suitable for quantitative analysis of diatom community structures in mixed sample. More researches are necessary to seek for proper gene to quantify diatoms in the environmental samples.     

Behavioural versus physiological photoprotection in epipelic and epipsammic benthic diatoms

Benthic diatoms are dominant primary producers in intertidal marine sediments, which are characterized by widely fluctuating and often extreme light conditions. To cope with sudden increases in light intensity, benthic diatoms display both behavioural and physiological photoprotection mechanisms. Behavioural photoprotection is restricted to raphid pennate diatoms, which possess a raphe system that enables motility and hence positioning in sediment light gradients (e.g. via vertical migration into the sediment). The main physiological photoprotection mechanism is to dissipate excess light energy as heat, measured as Non-Photochemical Quenching (NPQ) of chlorophyll fluorescence. A trade-off between vertical migration and physiological photoprotection (NPQ) in benthic diatoms has been hypothesized before, but this has never been formally tested. We exposed five epipelic diatom species (which move in between sediment particles) and four epipsammic diatom species (which live in close association with individual sand grains) to high light conditions, and characterized both NPQ and the relative magnitude of the migratory response to high light. Our results reveal the absence of a significant downward migratory response in an araphid diatom, but also in several raphid epipsammic diatoms, while all epipelic species showed a significant migratory response upon high light exposure. In all epipsammic species the upregulation of NPQ was rapid and pronounced; NPQ relaxation in low light conditions, however, occurred faster in the araphid diatom, compared with the raphid epipsammic species. In contrast, all epipelic species lacked a strong and flexible NPQ response and showed higher susceptibility to photodamage when not able to migrate. While overall our results support the vertical migration-NPQ trade-off, the lack of strong relationships between the capacity for vertical migration and NPQ within the epipsammic and epipelic groups suggests that other factors as well, such as cell size, substrate type and photoacclimation, may influence photoprotective strategies.     

A polyphasic approach to the delimitation of diatom species: a case study for the genus Pinnularia (Bacillariophyta)

Diatoms are one of the most abundant and arguably the most species‐rich group of protists. Diatom species delimitation has often been based exclusively on the recognition of morphological discontinuities without investigation of other lines of evidence. Even though DNA sequences and reproductive experiments have revealed several examples of (pseudo)cryptic diversity, our understanding of diatom species boundaries and diversity remains limited. The cosmopolitan pennate raphid diatom genus Pinnularia represents one of the most taxon‐rich diatom genera. In this study, we focused on the delimitation of species in one of the major clades of the genus, the Pinnularia subgibba group, based on 105 strains from a worldwide origin. We compared genetic distances between the sequences of seven molecular markers and selected the most variable pair, the mitochondrial cox1 and nuclear encoded LSU rDNA, to formulate a primary hypothesis on the species limits using three single‐locus automated species delimitation methods. We compared the DNA‐based primary hypotheses with morphology and with other available lines of evidence. The results indicate that our data set comprised 15 species of the P. subgibba group. The vast majority of these taxa have an uncertain taxonomic identity, suggesting that several may be unknown to science and/or members of (pseudo)cryptic species complexes within the P. subgibba group.     

Origin and evolution of the canal raphe system in diatoms

One lineage of pennate diatoms has a slit through the siliceous cell wall, called a "raphe," that functions in motility. Raphid pennate diatoms number in the perhaps tens of thousands of species, with the diversity of raphe forms potentially matching this number. Three lineages-the Bacillariales, Rhopalodiales, and Surirellales-possess a complex and presumably highly derived raphe that is physically separated from the cell interior, most often by a set of siliceous braces. Because the relationship among these three lineages is unclear, the number of origins of the canal raphe system and the homology of it and its constitutive parts among these lineages, is equally unclear. We reconstructed the phylogeny of raphid pennate diatoms and included, for the first time, members of all three canal raphid diatom lineages, and used the phylogeny to test specific hypotheses about the origin of the canal raphe. The canal raphe appears to have evolved twice, once in the common ancestor of Bacillariales and once in the common ancestor of Rhopalodiales and Surirellales, which form a monophyletic group in our analyses. These results recommend careful follow-up morphogenesis studies of the canal raphe in these two lineages to determine the underlying developmental basis for this remarkable case of parallel evolution.     

Gut passage and insect grazer selectivity of lotic diatoms

SUMMARY 1. Grazing experiments were conducted in a small Kentucky stream to determine if epilithic diatom taxa differed in susceptibility to grazing by the larval caddisfly, Neophylax autumnus , and whether ingested taxa varied in digestibility. Live/dead ratios of diatom cells from N. autumnus faecal material were compared with live/dead ratios of cells taken from adjacent epilithic habitats to determine diatom digestibility. Selectivity was studied by comparing relative abundances of diatom taxa on substrata with the relative abundances of these taxa in grazer faecal material.
2. 73% of the diatoms collected from epilithic habitats contained intact chloroplasts and were characterized as live cells. Only 42% of diatoms eliminated in caddisfly faeces were living. The five dominant diatom taxa did not differ in digestibility. Elimination of viable diatoms in grazer faecal material may contribute to diatom drift in streams, and thus supply individuals for downstream recolonization. Failure to document live/dead ratios of diatoms from grazed substrata can lead to overestimation of the digestibility of taxa and misinterpretation of results.
3. Grazers in this investigation were more successful at ingesting large, high-profile diatom taxa (i.e. Cymbella, Meridion and Gomphonema ) and less able to remove small, adnate forms ( Achnanthes minutissima ). Such 'selectivity'may be one mechanism by which A. minutissima maintains dominance in this system.     

Growth form defines physiological photoprotective capacity in intertidal benthic diatoms

In intertidal marine sediments, characterized by rapidly fluctuating and often extreme light conditions, primary production is frequently dominated by diatoms. We performed a comparative analysis of photophysiological traits in 15 marine benthic diatom species belonging to the four major morphological growth forms (epipelon (EPL), motile epipsammon (EPM-M) and non-motile epipsammon (EPM-NM) and tychoplankton (TYCHO)) found in these sediments. Our analyses revealed a clear relationship between growth form and photoprotective capacity, and identified fast regulatory physiological photoprotective traits (that is, non-photochemical quenching (NPQ) and the xanthophyll cycle (XC)) as key traits defining the functional light response of these diatoms. EPM-NM and motile EPL showed the highest and lowest NPQ, respectively, with EPM-M showing intermediate values. Like EPL, TYCHO had low NPQ, irrespective of whether they were grown in benthic or planktonic conditions, reflecting an adaptation to a low light environment. Our results thus provide the first experimental evidence for the existence of a trade-off between behavioural (motility) and physiological photoprotective mechanisms (NPQ and the XC) in the four major intertidal benthic diatoms growth forms using unialgal cultures. Remarkably, although motility is restricted to the raphid pennate diatom clade, raphid pennate species, which have adopted a non-motile epipsammic or a tychoplanktonic life style, display the physiological photoprotective response typical of these growth forms. This observation underscores the importance of growth form and not phylogenetic relatedness as the prime determinant shaping the physiological photoprotective capacity of benthic diatoms.     

A review of long-branch attraction

The history of long‐branch attraction, and in particular methods suggested to detect and avoid the artifact to date, is reviewed. Methods suggested to avoid LBA‐artifacts include excluding long‐branch taxa, excluding faster evolving third codon positions, using inference methods less sensitive to LBA such as likelihood, the Aguinaldo et al. approach, sampling more taxa to break up long branches and sampling more characters especially of another kind, and the pros and cons of these are discussed. Methods suggested to detect LBA are numerous and include methodological disconcordance, RASA, separate partition analyses, parametric simulation, random outgroup sequences, long‐branch extraction, split decomposition and spectral analysis. Less than 10 years ago it was doubted if LBA occurred in real datasets. Today, examples are numerous in the literature and it is argued that the development of methods to deal with the problem is warranted. A 16 kbp dataset of placental mammals and a morphological and molecular combined dataset of gall waSPS are used to illustrate the particularly common problem of LBA of problematic ingroup taxa to outgroups. The preferred methods of separate partition analysis, methodological disconcordance, and long branch extraction are used to demonstrate detection methods. It is argued that since outgroup taxa almost always represent long branches and are as such a hazard towards misplacing long branched ingroup taxa, phylogenetic analyses should always be run with and without the outgroups included. This will detect whether only the outgroup roots the ingroup or if it simultaneously alters the ingroup topology, in which case previous studies have shown that the latter is most often the worse. Apart from that LBA to outgroups is the major and most common problem; scanning the literature also detected the ill advised comfort of high support values from thousands of characters, but very few taxa, in the age of genomics. Taxon sampling is crucial for an accurate phylogenetic estimate and trust cannot be put on whole mitochondrial or chloroplast genome studies with only a few taxa, despite their high support values. The placental mammal example demonstrates that parsimony analysis will be prone to LBA by the attraction of the tenrec to the distant marsupial outgroups. In addition, the murid rodents, creating the classic “the guinea‐pig is not a rodent” hypothesis in 1996, are also shown to be attracted to the outgroup by nuclear genes, although including the morphological evidence for rodents and Glires overcomes the artifact. The gall wasp example illustrates that Bayesian analyses with a partition‐specific GTR + Γ + I model give a conflicting resolution of clades, with a posterior probability of 1.0 when comparing ingroup alone versus outgroup rooted topologies, and this is due to long‐branch attraction to the outgroup. © The Willi Hennig Society 2005.     

Elongation factor-2: a useful gene for arthropod phylogenetics.

Robust resolution of controversial higher-level groupings within Arthropoda requires additional sources of characters. Toward this end, elongation factor-2 sequences (1899 nucleotides) were generated from 17 arthropod taxa (5 chelicerates, 6 crustaceans, 3 hexapods, 3 myriapods) plus an onychophoran and a tardigrade as outgroups. Likelihood and parsimony analyses of nucleotide and amino acid data sets consistently recovered Myriapoda and major chelicerate groups with high bootstrap support. Crustacea + Hexapoda (= Pancrustacea) was recovered with moderate support, whereas the conflicting group Myriapoda + Hexapoda (= Atelocerata) was never recovered and bootstrap values were always <5%. With additional nonarthropod sequences included, one indel supports monophyly of Tardigrada, Onychophora, and Arthropoda relative to molluscan, annelidan, and mammalian outgroups. New and previously published sequences from RNA polymerase II (1038 nucleotides) and elongation factor-1alpha (1092 nucleotides) were analyzed for the same taxa. A comparison of bootstrap values from the three genes analyzed separately revealed widely varying values for some clades, although there was never strong support for conflicting groups. In combined analyses, there was strong bootstrap support for the generally accepted clades Arachnida, Arthropoda, Euchelicerata, Hexapoda, and Pycnogonida, and for Chelicerata, Myriapoda, and Pancrustacea, whose monophyly is more controversial. Recovery of some additional groups was fairly robust to method of analysis but bootstrap values were not high; these included Pancrustacea + Chelicerata, Hexapoda + Cephalocarida + Remipedia, Cephalocarida + Remipedia, and Malaocostraca + Cirripedia. Atelocerata (= Myriapoda + Hexapoda) was never recovered. Elongation factor-2 is now the second protein-encoding, nuclear gene (in addition to RNA polymerase II) to support Pancrustacea over Atelocerata. Atelocerata is widely cited in morphology-based analyses, and the discrepancy between results derived from molecular and morphological data deserves greater attention.     

Complex repeat structures and novel features in the mitochondrial genomes of the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana

The mitochondrial genome of the raphid pennate diatom Phaeodactylum tricornutum has several novel features compared with the mitochondrial genomes of the centric diatom Thalassiosira pseudonana and the araphid pennate diatom Synedra acus. It is almost double the size (77,356 bp) due to a 35,454 bp sequence block consisting of an elaborate combination of direct repeats, making it the largest stramenopile (heterokont) mitochondrial genome known. In addition, the cox1 gene has a +1 translational frameshift involving Pro codons CCC and CCT, the first translational frameshift to be detected in an algal mitochondrial genome. The nad9 and rps14 genes are fused by the insertion of an in-frame sequence and cotranscribed. The nad11 gene is split into two parts corresponding to the FeS and molybdate-binding domains, but both parts are still on the mitochondrial genome, in contrast to the brown algae where the second domain appears to have been transferred to the nucleus. In contrast to P. tricornutum, the repeat region of T. pseudonana consists of a much smaller 4790 bp string of almost identical double-hairpin elements, evidence of slipped-strand mispairing and active gene conversion. The diatom mitochondrial genomes have undergone considerable gene rearrangement since the three lineages of diatoms diverged, but all three have kept their repeat regions segregated from their relatively compact coding regions.     

Seasonality of macroalgae and epilithic diatoms in spring-fed streams in Texas, USA

A seasonal study of two spring-fed stream systems in south-central Texas was undertaken over a 15-month period from June 1996 to September 1997. Relative abundance of the epilithic diatom flora, percent cover of macroalgae, and several physical and chemical conditions were monitored in one 20-m stretch in each of the streams at approximately two-month intervals. Six additional spring-fed stream segments were sampled in May 1996 for macroalgae and measured for the same conditions, in order to determine how representative the macroalgal floras of the study sites were of south-central and western Texas. Conditions in the two stream segments were relatively constant over the 15 months, except for maximum depth; this finding is similar those reported for other spring-fed systems worldwide. Sixty-eight diatom taxa and nine macroalgae taxa were identified from the San Marcos River, which compares to 46 diatom taxa and six macroalgae taxa from the Comal River. Several diatom and macroalgae species were significantly correlated to stream conditions (e.g. Dichotomosiphon tuberosus and Hildenbrandia angolensis with daylength, and Amphora pediculus with maximum depth). Twenty-one macroalgal taxa were identified from the six additional spring-fed steam segments. Conditions in these streams were very similar to those measured in the San Marcos River and Comal River, except that they had a higher average pH _x= 8.1 versus _x= 7.6). The epilithic diatom flora of the San Marcos River and the Comal River contains many of the diatom and some macroalgal taxa commonly reported from springs globally. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phylogeny of marattioid ferns (Marattiaceae): inferring a root in the absence of a closely related outgroup

Closely related outgroups are optimal for rooting phylogenetic trees; however, such ideal outgroups are not always available. A phylogeny of the marattioid ferns (Marattiaceae), an ancient lineage with no close relatives, was reconstructed using nucleotide sequences of multiple chloroplast regions (rps4 + rps4-trnS spacer, trnS-trnG spacer + trnG intron, rbcL, atpB), from 88 collections, selected to cover the broadest possible range of morphologies and geographic distributions within the extant taxa. Because marattioid ferns are phylogenetically isolated from other lineages, and internal branches are relatively short, rooting was problematic. Root placement was strongly affected by long-branch attraction under maximum parsimony and by model choice under maximum likelihood. A multifaceted approach to rooting was employed to isolate the sources of bias and produce a consensus root position. In a statistical comparison of all possible root positions with three different outgroups, most root positions were not significantly less optimal than the maximum likelihood root position, including the consensus root position. This phylogeny has several important taxonomic implications for marattioid ferns: Marattia in the broad sense is paraphyletic; the Hawaiian endemic Marattia douglasii is most closely related to tropical American taxa; and Angiopteris is monophyletic only if Archangiopteris and Macroglossum are included.     

Benthic diatoms as indicators of eutrophication in tropical streams

Diatoms are frequently used as indicators of eutrophication in temperate systems, but little is known about their application to impacted African tropical systems. Five streams located within Gombe Stream National Park and five streams supporting human settlements draining into Lake Tanganyika, East Africa, were investigated for species composition, richness and diversity of epilithic algae. In addition, a trophic diatom index (TDI) developed for monitoring European rivers was applied to these tropical systems. 54 specific and infraspecific diatom taxa representing 20 genera were identified for all sites with Achnanthes s.l., Gomphonema and Navicula s.l. being the most common genera. Species richness varied between 10 and 21 in disturbed streams and 13 and 19 in undisturbed streams. Nutrients were significantly enriched in streams draining the deforested watersheds but indices of diversity and evenness (Shannon H, J and Simpson–Yule D, E) did not show any significant differences between streams in forested and deforested watersheds. Significant differences were observed between pooled data for the TDI between forested and deforested watersheds. Analysis of percent pollution tolerant diatom taxa indicates that organic pollution of streams in deforested watersheds may be contributing to eutrophication. This study shows that African diatoms, cosmopolitan or resembling well-known North American and European taxa, allows for trophic indices tailored to the autecological preferences of species to be applied to new regions, although intensive studies on these African taxa will lead to more accurate results. Measures of species-richness and diversity, historically used to describe the state of an ecosystem, may not be suitable to evaluate streams which are not grossly polluted. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

3. Biodiversity, biogeography and conservation of diatoms

Recent morphometric and breeding studies of diatoms show that the present species classification is too coarse and hides significant diversity. Many species are subdivided into phenodemes, which often differ in cell size, shape, stria density and pattern, but may also have different ultrastructural features. In raphid diatoms these can include the form of the raphe endings, details of the pore occlusions, and the structure of the girdle, while chloroplast structure can also vary. The phenodemes can be sympatric or allopatric. In Sellaphora pupula and other species, sympatric phenodemes are reproductively isolated. It is recommended that such demes are recognized as separate species; the total number of diatom species worldwide may thus be at least 2 × 10⁵. Use of a fine-grained classification reveals that many diatom species may be endemics, some restricted to a single lake or catchment, others to wider areas. Environmental impact assessments and conservation strategies must begin to take account of endemism and rarity among microscopic algae and protists.     

A multi criterion approach for the selection of optimal outgroups in phylogeny: recovering some support for Mandibulata over Myriochelata using mitogenomics

The choice of an appropriate outgroup is a fundamental prerequisite when the difference between two conflicting phylogenetic hypotheses depends on the position of the root. This is the case for the myriapods that may group either with Pancrustacea forming a clade called Mandibulata, in accordance with morphological characters, or with chelicerates to form Myriochelata (also called Paradoxopoda) as has recently been proposed by mitochondrial and ribosomal RNA gene phylogenies. In order to understand the impact that outgroup choice may have on phylogenetic reconstruction, we have investigated compositional heterogeneity and genetic distance in mtDNA sequences of several different outgroups to the arthropods, selected from deuterostomes, lophotrochozoans and ecdysozoans, and have used them to root a phylogenetically balanced and compositionarily homogeneous arthropod dataset. Results indicate that some outgroups, in particular from lophotrochozoans, nematodes and an onychophoran have G+C content and strand specific biases which are very different from those of arthropods, suggesting that the use of such outgroups may interfere with the stationarity of the model to create a random outgroup effect. We suggest a multi criterion approach for the selection of optimal outgroup species on the basis of (1) low substitution rate, (2) ingroup-like G+C composition, (3) a new strand bias estimator called the skew index, (4) the ability of the outgroup to avoid a "random branching effect" and (5) phylogenetic proximity to arthropods. Inference of phylogeny using various outgroups shows that use of phylogenetically distant and compositionally distinct lophotrochozoans as outgroups strongly supports Myriochelata and use of more closely related, but fast evolving nematodes supports Mandibulata. A dataset comprising multiple ecdysozoan outgroups also supports Mandibulata, unless the compositionally distant Onychophora are included. A group of the best outgroups selected according to our multi criteria selection, and including the most closely related, least genetically distant and most compositionally similar outgroup, a priapulid worm, supports Mandibulata. We conclude that support for the Myriochelata hypothesis from mitochondrial sequences may depend on the nature of the outgroup sequences rather than a true phylogenetic signal. Finally, we advocate a careful analysis and an objective choice of outgroup when dealing with derived sequences, such as mitochondrial genomes.     

Assessing the short-term response of stream diatoms to acidity using inter-basin transplantations and chemical diffusing substrates

1. Acid‐base status has major effects on diatoms, but there is little information on their short‐term response to changing acidity. This is despite the use of diatoms as bioindicators in streams where acid episodes are important during rainstorms (hours to days) or snowmelt (days to weeks). In the Llyn Brianne experimental catchments (Wales, UK), we attempted to mimic the effects of short‐term acidification by (i) reciprocally transplanting diatoms between two streams of contrasting acidity and (ii) using acid‐diffusing substrates. 2. Diatom diversity decreased rapidly on substrata transplanted from the circumneutral into the acidic stream, and increased in the reciprocal transplantation. Changes in dominant taxa occurred within three days in the acidic stream because of the rapid growth of Eunotia exigua, and by nine days in the circumneutral stream because of the proliferation of Achnanthidium minutissimum. Transplants were near indistinguishable from ambient assemblages by day 12. 3. There were no effects of enclosures on assemblage composition, but diatoms responded more rapidly to altered chemistry in enclosures with coarse mesh (26 × 50 mm) than finer mesh (320 μm). 4. Chemical diffusing substrates comprised terracotta tiles attached to dosing reservoirs that created locally acid (using H₂SO₄) or metal‐rich conditions (using MnSO₄) in the circumneutral stream over 26 days. Diatom responses were compared with reference substrates dosed with deionised or circumneutral stream water, and we also assessed whether effects were moderated by macroinvertebrate grazers. 5. Surface pH was lower by 1–2 pH units on acid‐dosed substrates than on reference tiles or in surrounding streamwater. Grazed assemblages on acid‐dosed substrates differed significantly from ungrazed reference assemblages, acquiring significantly greater relative abundance of Eunotia spp. However, the magnitude of response was less than in the between‐stream transplantations either because (i) metal exposure and base cation concentrations differed between the transplants and dosing substrates or (ii) diatom response to reduced pH on the diffusing substrates was restricted by the scarcity of acidobiontic diatoms in the circumneutral stream. Similar filter, founder or dominance effects might also affect diatom responses to real acid episodes. 6. These data show that diatom assemblages can respond rapidly and directly to changes in acid‐base status, but short‐term acidification might affect diatoms more rapidly than subsequent recovery. Because the experimental methods used were imperfect representations of episodic effects, diatom response to real acid events requires further field evaluation.     

Scratching the surface of the rare biosphere with ribosomal sequence tag primers

Increasingly large datasets of 16S rRNA gene sequences reveal new information about the extent of microbial diversity and the surprising extent of the rare biosphere. Currently, many of the largest datasets are represented by short and variable ribosomal sequence tags (RSTs) that are limited in their ability to accurately assign sequences to broad-scale phylogenetic trees. In this study, we selected 30 rare RSTs from existing sequence datasets and designed primers to amplify c. 1400 bases of the 16S rRNA gene to determine whether these sequences were represented by existing databases or if they might reveal new lineages within the Bacteria. Approximately one-third of the RST primers successfully amplified longer portions of these low-abundance 16S rRNA genes in a specific manner. Subsequent phylogenetic analysis demonstrated that most of these sequences were (1) distantly related to existing cultivated microorganisms and (2) closely related to uncultivated clone sequences that were recently deposited in GenBank. The presence of so many recently collected 16S rRNA gene reference sequences in existing databases suggests that progress is being made quickly towards a microbial census, one which has begun scratching the surface of the 'rare biosphere'.     

Morphological and genetic diversity of Beaufort Sea diatoms with high contributions from the Chaetoceros neogracilis species complex

Seventy‐five diatom strains isolated from the Beaufort Sea (Canadian Arctic) in the summer of 2009 were characterized by light and electron microscopy (SEM and TEM), as well as 18S and 28S rRNA gene sequencing. These strains group into 20 genotypes and 17 morphotypes and are affiliated with the genera Arcocellulus, Attheya, Chaetoceros, Cylindrotheca, Eucampia, Nitzschia, Porosira, Pseudo‐nitzschia, Shionodiscus, Thalassiosira, and Synedropsis. Most of the species have a distribution confined to the northern/polar area. Chaetoceros neogracilis and Chaetoceros gelidus were the most represented taxa. Strains of C. neogracilis were morphologically similar and shared identical 18S rRNA gene sequences, but belonged to four distinct genetic clades based on 28S rRNA, ITS‐1 and ITS‐2 phylogenies. Secondary structure prediction revealed that these four clades differ in hemi‐compensatory base changes (HCBCs) in paired positions of the ITS‐2, suggesting their inability to interbreed. Reproductively isolated C. neogracilis genotypes can thus co‐occur in summer phytoplankton communities in the Beaufort Sea. C. neogracilis generally occurred as single cells but also formed short colonies. It is phylogenetically distinct from an Antarctic species, erroneously identified in some previous studies as C. neogracilis, but named here as Chaetoceros sp. This work provides taxonomically validated sequences for 20 Arctic diatom taxa, which will facilitate future metabarcoding studies on phytoplankton in this region.     

A Mitogenomic Re-Evaluation of the Bdelloid Phylogeny and Relationships among the Syndermata

Molecular and morphological data regarding the relationships among the three classes of Rotifera (Bdelloidea, Seisonidea, and Monogononta) and the phylum Acanthocephala are inconclusive. In particular, Bdelloidea lacks molecular-based phylogenetic appraisal. I obtained coding sequences from the mitochondrial genomes of twelve bdelloids and two monogononts to explore the molecular phylogeny of Bdelloidea and provide insight into the relationships among lineages of Syndermata (Rotifera + Acanthocephala). With additional sequences taken from previously published mitochondrial genomes, the total dataset included nine species of bdelloids, three species of monogononts, and two species of acanthocephalans. A supermatrix of these 10-12 mitochondrial proteins consistently recovered a bdelloid phylogeny that questions the validity of a generally accepted classification scheme despite different methods of inference and various parameter adjustments. Specifically, results showed that neither the family Philodinidae nor the order Philodinida are monophyletic as currently defined. The application of a similar analytical strategy to assess syndermate relationships recovered either a tree with Bdelloidea and Monogononta as sister taxa (Eurotatoria) or Bdelloidea and Acanthocephala as sister taxa (Lemniscea). Both outgroup choice and method of inference affected the topological outcome emphasizing the need for sequences from more closely related outgroups and more sophisticated methods of analysis that can account for the complexity of the data.