Playing hide-and-seek with the tiny dragonfly: DNA barcoding discriminates multiple lineages of <Emphasis Type="Italic">Nannophya pygmaea</Emphasis> in Asia

We examined the utility of DNA barcode data for assessing genetic diversity of the tiny dragonfly Nannophya pygmaea Rambur in Asia. Data analyses inferred from the barcode region of cytochrome oxidase subunit I (COI) were performed with Malaysian N. pygmaea, along with the existing COI haplotypes distributed in Asia. We applied four species delimitation analyses [automatic barcode gap discovery (ABGD), generalised mixed yule coalescent (GMYC), poisson tree processes maximum likelihood (PTP_ML) and poisson tree processes simple heuristic solutions (PTP_sh)] to investigate potential lineages in this geographically widespread species. Based on our dataset, we provisionally recognize four distinct lineages or operational taxonomic units of N. pygmaea, which were represented by the taxa from Japan/Korea, China/Laos/Taiwan, Malaysia and Vietnam, respectively. Phylogenetic analyses showed two well-supported assemblages of N. pygmaea: one restricted to the taxa from Malaysia and Vietnam; and the other covering all populations further north (i.e., China, Japan, Korea, Laos and Taiwan). An extraordinarily high degree of genetic distance (up to >12 %) was detected between these two assemblages—suggesting they represent two separate species.     

Phylogenetic Analysis of Cryptosporidium Parasites Based on the Small-Subunit rRNA Gene Locus

Biological data support the hypothesis that there are multiple species in the genus Cryptosporidium, but a recent analysis of the available genetic data suggested that there is insufficient evidence for species differentiation. In order to resolve the controversy in the taxonomy of this parasite genus, we characterized the small-subunit rRNA genes of Cryptosporidium parvum, Cryptosporidium baileyi, Cryptosporidium muris, and Cryptosporidium serpentis and performed a phylogenetic analysis of the genus Cryptosporidium. Our study revealed that the genus Cryptosporidium contains the phylogenetically distinct species C. parvum, C. muris, C. baileyi, and C. serpentis, which is consistent with the biological characteristics and host specificity data. The Cryptosporidium species formed two clades, with C. parvum and C. baileyi belonging to one clade and C. muris and C. serpentis belonging to the other clade. Within C. parvum, human genotype isolates and guinea pig isolates (known as Cryptosporidium wrairi) each differed from bovine genotype isolates by the nucleotide sequence in four regions. A C. muris isolate from cattle was also different from parasites isolated from a rock hyrax and a Bactrian camel. Minor differences were also detected between C. serpentis isolates from snakes and lizards. Based on the genetic information, a species- and strain-specific PCR-restriction fragment length polymorphism diagnostic tool was developed.     

One or three species in Megadenia (Brassicaceae): insight from molecular studies

Megadenia Maxim. is a small genus of the Brassicaceae endemic to East Asia with three disjunct areas of distribution: the eastern edge of the Qinghai–Tibetan Plateau, the Eastern Sayan Mountains in southern Siberia, and Chandalaz Ridge in the southern Sikhote-Alin Mountains. Although distinct species (M. pygmaea Maxim., M. bardunovii Popov, and M. speluncarum Vorob., Vorosch. and Gorovoj) have been described from each area, they have lately been reduced to synonymy with M. pygmaea due to high morphological similarity. Here, we present the first molecular study of Megadenia. Using the sequences of 11 noncoding regions from the cytoplasmic (chloroplast and mitochondrial) and nuclear genomes, we assessed divergence within the genus and explored the relationships between Megadenia and Biscutella L. Although M. bardunovii, M. speluncarum, and M. pygmaea were found to be indiscernible with regard to the nuclear and mitochondrial markers studied, our data on the plastid genome revealed their distinctness and a clear subdivision of the genus into three lineages matching the three described species. All of the phylogenetic analyses of the chloroplast DNA sequences provide strong support for the inclusion of Megadenia and Biscutella in the tribe Biscutelleae. A dating analysis shows that the genus Megadenia is of Miocene origin and diversification within the genus, which has led to the three extant lineages, most likely occurred during the Early–Middle Pleistocene, in agreement with the vicariance pattern. Given the present-day distribution, differences in habitat preferences and in some anatomical traits, and lack of a direct genealogical relationship, M. pygmaea, M. bardunovii, and M. speluncarum should be treated as distinct species or at least subspecies.     

A new peronosporomycete, <Emphasis Type="Italic">Halioticida noduliformans</Emphasis> gen. et sp. nov., isolated from white nodules in the abalone <Emphasis Type="Italic">Haliotis</Emphasis> spp. from Japan

Four strains belonging to the Peronosporomycetes (formerly Oomycetes) were isolated from white nodules found on the mantle of three species of abalone. In artificial seawater, the four isolates formed fragments such as in the genus Haliphthoros, but the protoplasm constriction was weaker, and fragments were longer, with smaller spaces between them, than those of Haliphthoros. The four strains form one or more discharge tubes from each zoosporangium. The four strains were similar, but not identical, to the genus Haliphthoros based on morphological characteristics. As a result, the four isolates were classified in a new genus and species, Halioticida noduliformans gen. et sp. nov. Phylogenetic analysis of the D1/D2 region of the large subunit ribosomal RNA gene (LSU rDNA) was performed, and the four isolates showed 100%–99.8% concordance. In the phylogenetic tree, the four isolates were not classified in the subclass Peronosporomycetidae, Saprolegniomycetidae, or Rhipidiomycetidae. However, the four isolates formed a new clade with genera Haliphthoros and Halocrusticida in Peronosporomycetes. Within this new clade, the four isolates, Haliphthoros spp. and Halocrusticida spp., were grouped in their respective independent subclades. These results showed that these were the new genus and species from the morphological characteristics.     

Scanning electron microscopy of Heterocapsa pygmaea sp. nov., and evidence for polyploidy as a speciation mechanism in dinoflagellates

The thecal morphology of three isolates of the marine dinoflagellateHeterocapsa pygmaea sp. nov. are here examined by manning electronand light microscopy. The thecal tabulation of these isolatesis p.p., c.p., 5', 3a, 7', 6c, a.s., r.s., l.a.s., l.p.s.,[?a.a.s. and p.a.s.], 5' ' and 2' ' and is identical to thatof H. niei and H. illdefina. The assignment of thecal platesto various series is based on interpretation of plate homologiesamong peridinioid genera. The above formula represents the basicpattern for Heterocapsa. The cell dimensions of four Heterocapsaspecies are determined; Heterocapsa pygmaea is the smallestspecies. Heterocapsa pygmaea differs from the next largest species,H. niei, in having approximately half the number of chromosomesand as such can be interpreted as a case of polyploidy. If so,this is the first evidence of polyploidy as a speciation mechanismin the Pyrrhophyta. ¹Present address: Department of Botany, Duke University, DurhamNorth Carolina 27706 ²Present address: The Biological Laboratories, Harvard University,Cambridge, Massachusetts 02138     

A mycological survey of the South Adriatic Sea

A total of 498 fungal isolates belonging to 33 genera and 90 species were identified in the course of a 2-yr survey of the southern part of the territoreal waters of the Yugoslav Adriatic Sea. The best represented genus was Penicillium (32.12% of the total number of isolates), the P. chrysogenumnotatum series being the most abundant, followed by P. verrucosum var. cyclopium, and P. brevicompactum The genus Aspergillus represented 22.28% of the total. A. versicolor being the most ubiquitous. The Dematiaceae as a group, with 148 isolates included in 14 genera and 26 species, accounted for 29.71% of the total; the dominant genera were Cladosporium, Alternaria, and Ulcladium, with 14 25, 5.42 and 4.21%, respectively of the total number of isolates. Hyphomycetes with hyaloconidia, with 46 isolates included in 9 genera and 20 species, constituted about 8.83%, the best represented genus in this group being Fusarium, with 10 identified species. The Sphaeropsidales accounted for 5.42% of the total number of isolates, the genus Phoma being the most abundant.The number of isolates obtained from surface waters accounted for 85% of the total. The decreasing number of isolates in the water column was particularly evident in the case of the most ubiquitous species, a fact which may be explained by the influence of the contact of water masses with the atmosphere and the lower salinity of the surface waters. The distance from the shore as well as the human factor is reflected in the number of isolates and species. The ecological factors influenced not only the fungal population densities, but also their quality.     

Unsuitability of Charidotis pygmaea for biocontrol of Lantana camara in Africa

The host range of the tortoise beetle,Charidotis pygmaea Klug (Coleoptera:Chrysomelidae), was studied under quarantinelaboratory conditions to evaluate the insect'ssuitability for release as a biological controlagent for the noxious weed, Lantanacamara L. (Verbenaceae) in South Africa.Culturing on the target plant, L. camara,proved problematic with high larvalmortalities. Host-specificity studies showedthat four species in the genus Lantana,and two species in the genus Lippiawere acceptable as host plants. Duringlarval development trials, the insect performedbetter on the indigenous Lantana rugosaThunb. (Verbenaceae) and the introduced,commercially used L. montevidensis(Spreng.) Briq. (Verbenaceae), than on any ofthe weedy South African L. camaravarieties tested. Adult multi-choice trialsindicated that the beetle preferred to ovipositon L. rugosa and L. montevidensis.It is therefore recommended that C.pygmaea not be released against L.camara in Africa.     

Queens defense by workers in the highly polygynous ant Crematogaster pygmaea (Hymenoptera: Myrmicinae)

Some aspects of the biology of Crematogaster pygmaea, a highly polydomous and polygynous ant, are more commonly found in monogynous species. One such characteristic is the high attractiveness of its queens. In this study, this attractiveness was assessed under varying experimental conditions to investigate the factors responsible for its expression and variation, and to identify the nature of queen attractiveness. It was shown (1) that C. pygmaea queens are highly attractive to the workers that cluster on and around them (retinue), (2) that the attractiveness of C. pygmaea queens is context-dependent, i.e., it increases with increasing degree of potential danger to the queen, (3) that the attractiveness signal of C. pygmaea queens is chemically based, and (4) that this signal is persistent and apparently not colony-specific. The proposed hypothesis is that the C. pygmaea queens constantly release an attractiveness signal that is “read” by the workers, in a dependent way linked to the context, and that the main function of this attractiveness is to protect queens. This protection would have a high adaptive value in the context of the social structure and the reproductive strategies in C. pygmaea.     

Influence of light intensity,temperature and humidity on photosynthesis and transpiration ofSasa nipponica andArundinaria pygmaea

Photosynthesis and transpiration were simultaneously measured under different light intensity, temperature and humidity conditions inSasa nipponica andArundinaria pygmaea grown in exposed and shaded habitats. Both species showed a saturated light curve for photosynthetic rate. The saturation point was lower in shaded plants. The apparent quantum yields were larger inS. nipponica and in shaded plants, while the maximum photosynthesis was higher inA. pygmaea and exposed plants. The temperature response of photosynthesis showed an optimum curve in both species. The optinum temperatures were 20 C inS. nipponica and 25 C inA. pygmaea. The influence of humidity on photosynthesis was insignificant for both species. The responses of transpiration to light intensity and relative humidity showed a saturated curve and an optimal one, respectively. There was a significant relationship between transpiration and stomatal frequency, both of which were higher inS. nipponica, while water use efficiency was higher inA. pygmaea. These results suggest thatS. nipponica adapts itself better to shaded, low temperature and less water stress habitats as compared withA. pygmaea.     

Arenavirus Coinfections Are Common in Snakes with Boid Inclusion Body Disease

Recently, novel arenaviruses were found in snakes with boid inclusion body disease (BIBD); these form the new genus Reptarenavirus within the family Arenaviridae. We used next-generation sequencing and de novo sequence assembly to investigate reptarenavirus isolates from our previous study. Four of the six isolates and all of the samples from snakes with BIBD contained at least two reptarenavirus species. The viruses sequenced comprise four novel reptarenavirus species and a representative of a new arenavirus genus.     

Description of the two novel species of the genus Helicobacter: Helicobacter anatolicus sp. nov., and Helicobacter kayseriensis sp. nov., isolated from feces of urban wild birds

A total of 26 Gram-negative, motile, gently curved, and rod-shaped isolates were recovered, during a study to determine the faeco-prevalence of Helicobacter spp. in urban wild birds. Pairwise comparisons of the 16S rRNA gene sequences indicated that these isolates belonged to the genus Helicobacter and phylogenetic analysis based on the 16S rRNA gene sequences showed that the isolates were separated into two divergent groups. The first group consisted of 20 urease-positive isolates sharing the highest 16S rRNA gene sequence identity levels of 98.5–98.6% to H. mustelae ATCC 43772^T, while the second group contained six urease-negative isolates with the sequence identity level of 98.5% to the type strain of H. pametensis ATCC 51478^T. Five isolates were chosen and subjected to comparative whole-genome analysis. The phylogenetic analysis of the 16S rRNA, gyrA and atpA gene sequences showed that Helicobacter isolates formed two separate phylogenetic clades, differentiating the isolates from the other Helicobacter species. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses between strains faydin-H8^T, faydin-H23^T and their close neighbors H. anseris MIT 04-9362^T and H. pametensis ATCC 51478^T, respectively, confirmed that both strains represent novel species in the genus Helicobacter. The DNA G+C contents of the strains faydin-H8^T and faydin-H23^T are 32.0% and 37.6%, respectively. The results obtained for the characterization of the wild bird isolates indicate that they represent two novel species, for which the names Helicobacter anatolicus sp. nov., and Helicobacter kayseriensis sp. nov., are proposed, with faydin-H8^T(=LMG 32237^T = DSM 112312^T) and faydin-H23^T(=LMG 32236^T = CECT 30508^T) as respective type strains.     

Riboprinting and 16S rRNA Gene Sequencing for Identification of Brewery Pediococcus Isolates

A total of 46 brewery and 15 ATCC Pediococcus isolates were ribotyped using a Qualicon RiboPrinter. Of these, 41 isolates were identified as Pediococcus damnosus using EcoRI digestion. Three ATCC reference strains had patterns similar to each other and matched 17 of the brewery isolates. Six other brewing isolates were similar to ATCC 25249. The other 18 P. damnosus brewery isolates had unique patterns. Of the remaining brewing isolates, one was identified as P. parvulus, two were identified as P. acidilactici, and two were identified as unique Pediococcus species. The use of alternate restriction endonucleases indicated that PstI and PvuII could further differentiate some strains having identical EcoRI profiles. An acid-resistant P. damnosus isolate could be distinguished from non-acid-resistant varieties of the same species using PstI instead of EcoRI. 16S rRNA gene sequence analysis was compared to riboprinting for identifying pediococci. The complete 16S rRNA gene was PCR amplified and sequenced from seven brewery isolates and three ATCC references with distinctive riboprint patterns. The 16S rRNA gene sequences from six different brewery P. damnosus isolates were homologous with a high degree of similarity to the GenBank reference strain but were identical to each other and one ATCC strain with the exception of 1 bp in one strain. A slime-producing, beer spoilage isolate had 16S rRNA gene sequence homology to the P. acidilactici reference strain, in agreement with the riboprint data. Although 16S rRNA gene sequencing correctly identified the genus and species of the test Pediococcus isolates, riboprinting proved to be a better method for subspecies differentiation.     

Detection and Identification of Bacterial Endosymbionts in Arbuscular Mycorrhizal Fungi Belonging to the Family Gigasporaceae

Intracellular bacteria have been found previously in one isolate of the arbuscular mycorrhizal (AM) fungus Gigaspora margarita BEG 34. In this study, we extended our investigation to 11 fungal isolates obtained from different geographic areas and belonging to six different species of the family Gigasporaceae. With the exception of Gigaspora rosea, isolates of all of the AM species harbored bacteria, and their DNA could be PCR amplified with universal bacterial primers. Primers specific for the endosymbiotic bacteria of BEG 34 could also amplify spore DNA from four species. These specific primers were successfully used as probes for in situ hybridization of endobacteria in G. margarita spores. Neighbor-joining analysis of the 16S ribosomal DNA sequences obtained from isolates of Scutellospora persica, Scutellospora castanea, and G. margarita revealed a single, strongly supported branch nested in the genus Burkholderia.     

Evaluation of using a short region of the recA gene for rapid and sensitive speciation of dominant bifidobacteria in the human large intestine

The feasibility of intragenerically characterizing bifidobacteria by a comparison of a short region within the recA gene was tested. An ∼300 bp fragment of the recA gene was PCR-amplified from six species from the genus Bifidobacterium using primers directed to two universally conserved regions of the recA gene. A phylogenetic analysis of the sequenced recA products compared favorably to classification based on the 16S rRNA sequences of the species tested. To apply this rapid methodology to unknown human intestinal bifidobacteria, 46 isolates were randomly chosen from the feces of four subjects and initially characterized by RFLP analysis of a PCR-amplified region of their 16S RNA genes. From a representative of the dominant RFLP family in each of the subjects, the recA segment was PCR-amplified, sequenced and phylogenetically analyzed. All four isolates were found to be related to one another and to B. longum and B. infantis. These results illustrate that the recA gene may be useful for intrageneric phylogenetic analysis as well as for the identification of unknown fecal bifidobacteria.     

SYSTEMATIC REVISION OF PATELLOIDA PYGMAEA (DUNKER, 1860) (GASTROPODA: LOTTIIDAE), WITH A DESCRIPTION OF A NEW SPECIES

Patelloida pygmaea (Dunker) and its closely allied species,P. heroldi (Dunker) and P. conulus (Dunker) have caused nomenclaturalconfusion because of their variable shell morphology and distinctivehabitats. According to current nomenclature, these species ofPatelloida have been synonymized and treated as one specieswith two ecological forms. Patelloida pygmaea lives on the shellof Crassostrea gigas (Ostreidae), P. pygmaea form heroldi occurson intertidal rocks on sheltered shores and P. pygmaea formconulus is found on the shell of Batillaria multiformis (Batillariidae).Their taxonomic relationships and possible species status are,however, unclear. Using two mitochondrial genes (fragments ofCOI and 16S ribosomal RNA; total 1192 sites), we analysed 88specimens of P. pygmaea, P. pygmaea form heroldi and P. pygmaeaform conulus from 37 localities in East Asia. In the resultingmolecular phylogenetic trees, the specimens of Patelloida fallinto four clades with high bootstrap probabilities; these cladescorrespond taxonomically to P. pygmaea, P. conulus, P. heroldiand a fourth previously unrecognized taxon, Patelloida ryukyuensisn. sp., described here. A minimum-spanning network for 29 uniquemitochondrial COI haplotypes obtained from 45 specimens in thesame bay in central Japan form three distinct clusters, consistingof P. pygmaea, P. conulus and P. heroldi, respectively. Thissuggests that reproductive isolation has been established betweeneach group. A detailed examination of radular and shell morphologiesof the four taxa clarifies the morphological distinction betweenthese species. The four species form a rather young clade inthe genus Patelloida that diverged during the Pliocene. Theyprovide an example of habitat segregation in a restricted marineenvironment. (Received 21 December 2004; accepted 11 March 2005)     

QTL mapping reveals a two-step model for the evolutionary reduction of inner microsporangia within the asteracean genus <Emphasis Type="Italic">Microseris</Emphasis>

The reduction of inner (adaxial) pollen sacs (microsporangia, MS) as a diagnostic character for the three asteracean species, Microseris bigelovii, Microseris elegans and Microseris pygmaea, was analysed in an interspecific cross between Microseris douglasii and Microseris bigelovii with 4 MS and 2 MS, respectively, using the average number of MS per plant as a quantitative character. A previous QTL (Quantitative Trait Locus) analysis had revealed one major QTL (3B) and three modifier QTLs (3A, 4A, 7A) with epistatic effects only on the homozygous recessive 2 MS genotype of QTL 3B. Here we performed a bulked segregant analysis on four 2 MS and four 4 MS DNA-bulks with 407 EcoRI/MseI AFLP-primer combinations each. In this way additional AFLP markers were mapped close to QTL 3B and QTL 3A. Three of them were converted to SCAR (Sequence Characterized Amplified region) markers. All markers were tested in natural populations of the disporangiate (2 MS) species M. bigelovii, M. elegans and M. pygmaea, and in different populations of tetrasporangiate (4 MS) M. douglasii. The marker distribution suggests that locus 3B mutated in a progenitor of the disporangiate species. QTL 3A has evolved in the 2 MS background of the major gene in the disporangiate species. Since M. pygmaea and M. bigelovii are the sister group to M. elegans, the 4 MS genotype for (markers of) QTL 3A in M. pygmaea populations is most likely due to a back mutation to the 4 MS state and could explain the slight instability of the 2 MS phenotype in this species.Communicated by O. Savolainen     

The large mimosoid genus Inga Mill. (tribe Ingeae,Caesalpinioideae) is nodulated by diverse Bradyrhizobium strains in its main centers of diversity in Brazil

Inga (Caesalpinioideae) is the type genus of the Ingeae tribe in the mimosoid clade. It comprises about 300 species, all trees or treelets, and has an exclusively neotropical distribution, with Brazil as its main center of diversity. In this study, we analyzed the diversity of 40 strains of rhizobia isolated from root nodules collected from ten species of Inga belonging to different types of vegetation in Brazil. Sequences of their housekeeping genes (dnaK, recA, rpoB, gyrB and glnII), 16S rRNA genes, internal transcribed spacer (ITS) regions, as well as their symbiosis-essential genes (nodC and nifH) were used to characterize them genetically. The ability of the rhizobia to form nodules on Inga spp., and on the promiscuous legume siratro (Macroptilium atropurpureum) was also evaluated. A multilocus sequence analysis (MLSA) combined with an analysis of the ITS region showed that the isolates were distributed into four main groups (A-D) within the large genus Bradyrhizobium. Analysis of the nodC and nifH genes showed that the isolates formed a separate branch from all described species of Bradyrhizobium, except for B. ingae. Most of the tested isolates formed nodules on siratro and all isolates tested nodulated Inga spp. Our results suggest a unique co-evolutionary history of Bradyrhizobium and Inga and demonstrate the existence of potential new species of microsymbionts nodulating this important and representative genus of leguminous tree from the Caesalpinioideae mimosoid clade.     

Meiothermus rufus sp. nov., a new slightly thermophilic red-pigmented species and emended description of the genus Meiothermus

Four red-pigmented isolates, with optimum growth temperatures of approximately 55–60 °C and an optimum pH for growth between 7.5 and 8.5, were recovered from hot springs in Central France. Phylogenetic analysis of the 16S rRNA gene sequences showed that these organisms represented a new species of the genus Meiothermus. The new isolates could be distinguished from other strains of the species of the genus Meiothermus primarily by the glycolipid profile and fatty acid composition because these organisms lacked the hydroxy fatty acids and the glycolipid variant GL-1a found in all other isolates of the species of Meiothermus examined. On the basis of the results presented here we propose the name Meiothermus rufus for the new species, which is represented by strains CAL-4^T (=DSM 22234^T=LMG 24878^T) and CAL-12 (=DSM 22235=LMG 24879). We also propose emending the genus Meiothermus to include strains that have only one glycolipid instead of two glycolipid variants.