Short rDNA Barcodes for Species Identification in Foraminifera

ABSTRACT. Ribosomal DNA (rDNA) sequences have been shown to be very useful for identification of microbial eukaryotes. Usually, complete or long partial sequences of the rDNA genes are analysed. However, the development of new massive sequencing technologies producing a large amount of relatively short sequences raises the question about the minimum length of rDNA fragments necessary for species distinction in environmental sampling. To answer this question, we compared six variable regions of the small subunit (SSU) rDNA of foraminifera, known to have rapidly evolving ribosomal genes. For each region, we analysed (1) the sequence divergence between and within foraminiferal morphospecies, (2) the intraspecific polymorphism, and (3) the ability of each region to recognize the phylotypes inferred from analysis of a longer fragment. Our results show that although the variable regions differ considerably between taxonomic groups, most of them perform very well as species identifiers. Taking into account different analyses, the expansion segment of Helix 37 appears to be the best candidate for barcoding foraminifera. We propose that this relatively short region, averaging 50–60 nt in length, could be an ideal barcode for identification of foraminifera in environmental samples using massive sequencing approach.     

Discovery of a group I intron in the SSU rDNA of Ploeotia costata (Euglenozoa)

The gene coding for the small ribosomal subunit RNA of Ploeotia costata contains an actively splicing group I intron (Pco.S516) which is unique among euglenozoans. Secondary structure predictions indicate that paired segments P1-P10 as well as several conserved elements typical of group I introns and of subclass IC1 in particular are present. Phylogenetic analyses of SSU rDNA sequences demonstrate a well-supported placement of Ploeotia costata within the Euglenozoa; whereas, analyses of intron data sets uncover a close phylogenetic relation of Pco.S516 to S-516 introns from Acanthamoeba, Aureoumbra lagunensis (Stramenopila) and red algae of the order Bangiales. Discrepancies between SSU rDNA and intron phylogenies suggest horizontal spread of the group I intron. Monophyly of IC1 516 introns from Ploeotia costata, A. lagunensis and rhodophytes is supported by a unique secondary structure element: helix P5b possesses an insertion of 19 nt length with a highly conserved tetraloop which is supposed to take part in tertiary interactions. Neither functional nor degenerated ORFs coding for homing endonucleases can be identified in Pco.S516. Nevertheless, degenerated ORFs with His-Cys box motifs in closely related intron sequences indicate that homing may have occurred during evolution of the investigated intron group.     

Origin and Evolution of the Lichenized Ascomycete Order Lichinales: Monophyly and Systematic Relationships Inferred from Ascus, Fruiting Body and SSU rDNA Evolution

Abstract: The Lichinales are a group of lichenized ascomycetes that almost exclusively possess cyanobacteria as their primary photobiont and are hitherto separated from the Lecanorales, the major group of lichenized ascomycetes, by thallus structure, ascoma ontogeny, ascus structure and ascus function. The relationship of the two families Peltulaceae and Lichinaceae, both placed within the Lichinales, with the Heppiaceae, placed within the Lecanorales, was investigated, as well as a possible sister group relationship of the Lichinales to the Lecanorales. Phylogenetic analyses included non-molecular data as well as 18S rDNA sequence data. The monophyly of the Lichinales including the family Heppiaceae and a sister group relationship of Lichinales and Lecanorales, based on the shared presence of lecanoralean asci, are proposed in a morphological hypothesis. Parsimony and distance analyses of 18S rDNA sequence data strongly support the monophyly of the Lichinales, including all three families. Therefore, the presence of rostrate, lecanoralean asci in Peltula and part of the Lichinaceae suggests that this ascus type is an autapomorphy of the monophyletic Lichinales. Furthermore, the occurrence of prototunicate asci in the Heppiaceae and most of the Lichinaceae is autapomorphic and was gained independently by reduction of the rostrate ascus. The 18S rDNA analysis did not reject the non-molecular hypothesis of a sister group relationship of the Lichinales and the Lecanorales as based on ascus characters. The alternative placement of the Lichinales as the sister group of all inoperculate euascomycetes excluding the Sordariomycetes and most of the Leotiales in the gene tree received unsufficient bootstrap support and no support from any non-molecular data and consequently was rejected.     

A Molecular Study of Euglenoid Phylogeny using Small Subunit rDNA

The euglenoids are an ancient and extremely diverse lineage of eukaryotic flagellates with unclear relationships among taxa. Synapomorphies for the euglenoids include a surface pellicle and a closed mitosis with a series of separate sub-spindles. The taxonomy currently in use is inconsistent with the available data and needs revision. Most euglenoid phylogenies are largely intuitive reconstructions based on a limited number of morphological characters. Therefore, we have added molecular characters from the Small Subunit (SSU) rDNA to generate an overall phylogenetic framework for the euglenoids. SSU rDNA sequences from photosynthetic, osmotrophic, and phagotrophic euglenoids were aligned based on secondary structure. Phylogenetic analysis using the conserved areas of the sequence was performed using parsimony, maximum likelihood, and distance methods. Trees derived using different criteria are in agreement. The euglenoids form a distinct monophyletic clade with phagotrophic members diverging prior to the phototrophic and osmotrophic members. Among photosynthetic members, the biflagellate form diverged prior to the uniflagellate form. Additionally, the genus Euglena appears to be paraphyletic, with osmotrophic taxa, such as Astasia and Khawkinea, diverging independently within the clade containing the photosynthetic genus Euglena.     

A New Freshwater Amoeba: Cochliopodium pentatrifurcatum n. sp. (Amoebozoa,Amorphea)

Cochliopodium pentatrifurcatum n. sp. (ATCC^© 30935^TM) is described based on light microscopic morphology, fine structure, and molecular genetic evidence. Cochliopodium pentatrifurcatum n. sp. (length ~ 25 μm) is characterized by surface microscales (0.3 μm tall) containing a circular porous base (~ 0.6 μm diam.) with a thin peripheral rim. Five radially arranged feet, emanating from the base, support a short central column terminating apically as a funnel‐shaped collar (~ 0.5 μm diam.) composed of five radial, trifurcate rays extending from the center toward a thin peripheral rim. The central spine is 0.5–0.6 μm long. The comparative morphologies and combined molecular genetic evidence, SSU‐rDNA and COI, indicate that the new species falls in a clade sufficiently different from other species to suggest that it is a valid new species.     

Species boundaries in gregarine apicomplexan parasites: a case study-comparison of morphometric and molecular variability in Lecudina cf. tuzetae (Eugregarinorida, Lecudinidae)

Trophozoites of gregarine apicomplexans are large feeding cells with diverse morphologies that have played a prominent role in gregarine systematics. The range of variability in trophozoite shapes and sizes can be very high even within a single species depending on developmental stages and host environmental conditions; this makes the delimitation of different species of gregarines based on morphological criteria alone very difficult. Accordingly, comparisons of morphological variability and molecular variability in gregarines are necessary to provide a pragmatic framework for establishing species boundaries within this diverse and poorly understood group of parasites. We investigated the morphological and molecular variability present in the gregarine Lecudina cf. tuzetae from the intestines of Nereis vexillosa (Polychaeta) collected in two different locations in Canada. Three distinct morphotypes of trophozoites were identified and the small subunit (SSU) rDNA was sequenced either from multicell isolates of the same morphotype or from single cells. The aim of this investigation was to determine whether the different morphotypes and localities reflected phylogenetic relatedness as inferred from the SSU rDNA sequence data. Phylogenetic analyses of the SSU rDNA demonstrated that the new sequences did not cluster according to morphotype or locality and instead were intermingled within a strongly supported clade. A comparison of 1,657 bp from 45 new sequences demonstrated divergences between 0% and 3.9%. These data suggest that it is necessary to acquire both morphological and molecular data in order to effectively delimit the "clouds" of variation associated with each gregarine species and to unambiguously reidentify these species in the future.     

A Phylogenetic Study of the Anopheles punctulatus Group of Malaria Vectors Comparing rDNA Sequence Alignments Derived from the Mitochondrial and Nuclear Small Ribosomal Subunits

A phylogenetic study of the members of the Anopheles punctulatus group was performed using structural and similarity-based DNA sequence alignments of the small ribosomal subunit (SSU) from both the nuclear and the mitochondrial genomes. The mitochondrial SSU gene (12S, 650 bp) proved to be highly restricted by its secondary structure and displayed little informative sequence variation. Consequently, it was considered unsuitable for a phylogenetic study of these closely related mosquito species. A structural alignment of the nuclear ribosomal DNA SSU (18S, 2000 bp) proved to be more informative than similarity-based alignments. Analyses showed the A. punctulatus group to be monophyletic with two major clades; a Farauti clade containing members displaying an all-black-scaled proboscis (A. farauti 1–3 and 5–7) and the Punctulatus clade containing members displaying extensive white scaling on the apical half of the proboscis (A. farauti 4, A. punctulatus, and An. sp. near punctulatus). Anopheles koliensis was positioned basal to the Farauti clade.     

Species boundaries in tintinnid ciliates: a case study--morphometric variability, molecular characterization, and temporal distribution of Helicostomella species (Ciliophora, Tintinnina)

Tintinnids are a large group of planktonic ciliates with diverse morphologies. The range of variability in lorica shapes and sizes can be very high even within a single species depending on life stages and environmental conditions, which makes the delimitation of different species based on morphological criteria alone very difficult. Accordingly, comparisons of morphological and molecular variability in tintinnids are necessary to provide a pragmatic approach for establishing species boundaries within this diverse and poorly understood group. We investigated the temporal distribution of species of the hyaline tintinnid Helicostomella (Ciliophora, Tintinnina), which were collected daily from September 2008 to August 2009 in Jangmok Bay of Geoje Island on the south coast of Korea. Two forms - a long form and a short form, were discovered. The long form was found in cold waters in February and March whereas the short form occurred in warm waters from June to October. Thus, these two forms were seasonally isolated. However, all the morphological characteristics for these two forms overlap to some degree. A comparison of the small subunit (SSU) rDNA, ITS1-5.8S-ITS2, and ITS2 sequences from two forms revealed 0.5%, 3.8%, and 5.6% divergences, respectively. Morever, one compensatory base change (CBC) and three hemi-CBCs were identified from ITS2 secondary structures of these two forms. All these data suggest that these two forms represent two distinct species despite their highly similar lorica morphology. The phylogenetic position of the genus Helicostomella was also examined using SSU rDNA sequences.     

A Light and Scanning Electron Microscopic Study of the Closing Apparatus in Tintinnid Ciliates (Ciliophora,Spirotricha, Tintinnina): A Forgotten Synapomorphy

ABSTRACT. A membranous closing apparatus shuts the lorica opening in disturbed tintinnids of six genera belonging to four families. The homology of the apparatuses is investigated, using data from the literature and Mediterranean tintinnids studied in vivo and by scanning electron microscopy. Morphological and functional similarities indicate that the foldable closing apparatus is not only a synapomorphy of the genera Codonella (Codonellidae) and Dictyocysta (Dictyocystidae), as suggested 80 years ago, but also of Codonaria (Codonellidae) and Codonellopsis (Codonellopsidae). In Codonaria, Codonella, and Dictyocysta, the apparatuses merge posteriorly into membranous lorica sacs, which probably represent homologous structures. The diagnoses of these genera are improved according to the new findings. The close relationship of Codonella, Codonellopsis, and Dictyocysta is also inferred from small subunit rRNA phylogenies and the ultrastructure of the capsules. It contradicts the current lorica‐based classification of the tintinnids. The assumption that the diaphragm‐like apparatus in the genera Salpingacantha and Salpingella is not homologous to the foldable ones in the genera mentioned above is supported by molecular and cytological features.     

Evaluation of Morphological Variation in the Lichen Diploschistes ocellatus (Ascomycota, Ostropales): Evidence from Nuclear rDNA ITS Sequence Data

Abstract: A morphologically distinct population (var. almeriensis ) of the lichen species Diploschistes ocellatus from southern Spain is examined using morphological and molecular methods. While the morphological examination revealed some unique features of var. almeriensis, the ITS sequence data did not support a distinction from D. ocellatus s. str. The Kishino-Hasegawa test did not rule out trees distinguishing between the two morphotypes, although spectral analytical data support an inclusion of var. almeriensis in D. ocellatus. The influence of potentially erronously aligned ambiguous sites is evaluated by analysis of different alignments. However, all alignments agree in placing all specimens of D. ocellatus together and, consequently, in the consensus tree D. ocellatus s. lat. forms a monophyletic clade with high bootstrap support.     

Comparing Morphological and Molecular Estimates of Species Diversity in the Freshwater Genus Synura (Stramenopiles): A Model for Understanding Diversity of Eukaryotic Microorganisms

We performed a comparison of molecular and morphological diversity in a freshwater colonial genus Synura (Chrysophyceae, Stramenopiles), using the island of Newfoundland (Canada) as a case study. We examined the morphological species diversity in collections from 79 localities, and compared these findings to diversity based on molecular characters for 150 strains isolated from the same sites. Of 27 species or species-level lineages identified, only one third was recorded by both molecular and morphological techniques, showing both approaches are complementary in estimating species diversity within this genus. Eight taxa, each representing young evolutionary lineages, were recovered only by sequencing of isolated colonies, whereas ten species were recovered only microscopically. Our complex investigation, involving both morphological and molecular examinations, indicates that our knowledge of Synura diversity is still poor, limited only to a few well-studied areas. We revealed considerable cryptic diversity within the core S. petersenii and S. leptorrhabda lineages. We further resolved the phylogenetic position of two previously described taxa, S. kristiansenii and S. petersenii f. praefracta, propose species-level status for S. petersenii f. praefracta, and describe three new species, S. vinlandica, S. fluviatilis, and S. cornuta. Our findings add to the growing body of literature detailing distribution patterns observed in the genus, ranging from cosmopolitan species, to highly restricted taxa, to species such as S. hibernica found along coastal regions on multiple continents. Finally, our study illustrates the usefulness of combining detailed morphological information with gene sequence data to examine species diversity within chrysophyte algae.     

Assessment of variation in Aceria cajani using analysis of rDNA ITS regions and scanning electron microscopy: implications for the variability observed in host plant resistance to pigeonpea sterility mosaic disease

Aceria cajani on pigeonpea (Cajanus cajan) is the vector of the agent of pigeonpea sterility mosaic disease (PSMD), a very damaging virus‐like disease in the Indian subcontinent. PCR was used to amplify A. cajani nuclear ribosomal DNA (rDNA) internal transcribed spacers (ITS) and associated rDNA genes. They were assessed for variation in this genome region by nucleotide sequencing and RFLP. A. cajani‐specific rDNA primers are described. Several A. cajani populations were collected from pigeonpea plants from various PSMD endemic locations in India, Nepal and Myanmar. No significant variation was identified in rDNA regions, or in morphological features. These results suggest strongly that A. cajani on pigeonpea across the Indian subcontinent constitutes one species and that no other Aceria species and probably no A. cajani biotypes that differ in vectoring ability are involved in the transmission of the agent of PSMD. The implications of these findings for the variability observed in PSMD‐resistant pigeonpea genotypes across various locations in India are discussed.     

A 'living fossil' eel (Anguilliformes: Protanguillidae, fam. nov.) from an undersea cave in Palau

We report the discovery of an enigmatic, small eel-like fish from a 35 m-deep fringing-reef cave in the western Pacific Ocean Republic of Palau that exhibits an unusual suite of morphological characters. Many of these uniquely characterize the Recent members of the 19 families comprising the elopomorph order Anguilliformes, the true eels. Others are found among anguilliforms only in the Cretaceous fossils, and still others are primitive with respect to both Recent and fossil eels. Thus, morphological evidence explicitly places it as the most basal lineage (i.e. the sister group of extant anguilliforms). Phylogenetic analysis and divergence time estimation based on whole mitogenome sequences from various actinopterygians, including representatives of all eel families, demonstrate that this fish represents one of the most basal, independent lineages of the true eels, with a long evolutionary history comparable to that of the entire Anguilliformes (approx. 200 Myr). Such a long, independent evolutionary history dating back to the early Mesozoic and a retention of primitive morphological features (e.g. the presence of a premaxilla, metapterygoid, free symplectic, gill rakers, pseudobranch and distinct caudal fin rays) warrant recognition of this species as a 'living fossil' of the true eels, herein described as Protanguilla palau genus et species nov. in the new family Protanguillidae.     

A comparative study in birds: call-type-independent species and individual recognition using four machine-learning methods and two acoustic features

Species- and individual-specific animal calls can be used in identification as verified in playback experiments and analyses of features extracted from these signals. The use of machine-learning methods and acoustic features borrowed from human speech recognition to identify animals at the species and individual level has increased recently. To date there have been few studies comparing the performances of these methods and features used for call-type-independent species and individual identification. We compared the performance of four machine-learning classifiers in the identification of ten passerine species, and individual identification for three passerines using two acoustic features. The methods did not require us to pre-categorize the component syllables in call-type-independent species and individual identification systems. The results of our experiment indicated that support vector machines (SVM) performed best generally, regardless of which acoustic feature was used, linear predictive coefficients (LPCs) increased the recognition accuracies of hidden Markov models (HMM) greatly, and the most appropriate classifiers for LPCs and Mel-frequency cepstral coefficients (MFCCs) were HMM and SVM respectively. This study will assist researchers in selecting classifiers and features to use in future species and individual recognition studies.     

Assessing Health Risks from Pesticides in Recycled Water: A Case Study of Augmentation of Drinking Water Supplies in Perth,Western Australia

A characterization of pesticides in secondary treated effluent (STE) and a screening health risk assessment were conducted to evaluate potential health impacts of recycled water for potable reuse. Samples of STE were examined pre- and post-reverse osmosis (RO) treatment in two plants. Risk quotients (RQ) were estimated by expressing the maximum and median concentration as a function of the health value. Of 128 pesticides analyzed, 11 were detected in STE. Trifluralin was detected in 86.5% of the STE samples followed by metolachlor (67.6%), 2,4-D (50%), and propiconazole (42.5%). Metolachlor was the only pesticide detected post-RO in one sample (0.08 μg/L, limit of reporting = 0.05 μg/L). RQ(max) were all below health values in the STE with the exception of thiophanate methyl (RQ max = 2.4). RO was able to reduce the concentration of all pesticides below LOR. Median removal efficiency for RO ranged from 67.1% (atrazine) to 98.8% (2,4-D). The results indicate that, despite the detection of pesticides in STE, the impact on indirect potable reuse is negligible due to the low concentrations in STE and their subsequent removal during RO treatment. Implementation of risk management strategies to optimize safety and recommendations for a monitoring program are also discussed.     

Subsampling Herbarium Collections to Assess Geographic Diversity Gradients: A Case Study with Endemic Orchidaceae and Rubiaceae in Cameroon

We compiled herbarium specimen data to provide an improved characterization of geographic patterns of diversity using indices of species diversity and floristic similarity based on rarefaction principles. A dataset of 3650 georeferenced plant specimens belonging to Orchidaceae and Rubiaceae endemic to Atlantic Central Africa was assembled to assess species composition per half‐degree or one‐degree grid cells. Local diversity was measured by the expected number of species (S_k) per grid cell found in subsamples of increasing size and compared with raw species richness (S_R). A nearly unbiased estimator of the effective number of species per grid cell was also used, allowing quantification of ratios of ‘true diversity’ between grid cells. Species turnover was measured using a presence/absence‐based similarity index (Sørensen) and an abundance‐based index that corrects for sampling bias (NNESS). Our results confirm that the coastal region of Cameroon is more diverse in endemic species than those more inland. The southern part of this coastal forest is, however, as diverse as the more intensively inventoried northern part, and should also be recognized as an important center of endemism. A strong congruence between Sørensen and NNESS similarity matrices lead to similar delimitations of floristic units. Hence, heterogeneous sampling seems to confer more bias when measuring patterns of local diversity using raw species richness than species turnover using Sørensen index. Overall, we argue that subsampling methods represent a useful way to assess diversity gradients using herbarium specimens while correcting for heterogeneous sampling effort. Abstract in French is available in the online version of this article.     

Environmental Footprints and Scenario Analysis for Assessing the Impacts of the Agri‐Food Industry on a Regional Economy: A Case Study in Spain

The study of the environmental footprints of various sectors and industries is increasingly demanded by institutions and by society. In this context, the regional perspective is becoming particularly important, and even more so in countries such as Spain, where the autonomous communities have the primary responsibility for implementing measures to combat environmental degradation and promote sustainable development, in coordination with national strategies. Taking as a case study a Spanish region, Aragon, and significant economic sectors, including agriculture and the food industry, the aim of this work is twofold. First, we calculate the associated environmental footprints (of emissions and water) from the dual perspectives of production (local impacts) and consumption (final destination of the goods produced by the agri‐food industry). Second, through a scenarios analysis, based on a general equilibrium model designed and calibrated specifically for the region, we evaluate the environmental implications of changes in the agri‐food industry (changes in the export and import pattern, as well as in consumer behavior). This model provides a flexible approximation to the environmental impacts, controlling for a wider range of behavioral and economic interactions. Our results indicate that the agri‐food industry has a significant impact on the environment, especially on water resources, which must be responsibly managed in order to maintain the differential advantage that a regional economy can have, compared to other territories.