Annesorhiza calcicola (Apiaceae), a new limestone endemic species from the Western Cape Province of South Africa

The new species, Annesorhiza calcicola Magee and J.C. Manning, is described. It is known only from a few limestone outcrops around Jacobsbaai on the Saldanha Peninsula on the West Coast of South Africa. The species differs from all others in the genus in its small stature (inflorescence less than 0.3 m and leaves less than 150 mm long), the hysteranthous, pilose leaves which are completely absent at flowering (in the other species dead or dying at flowering and usually glabrous), the greyish-black, pilose inflorescence (yellowish-brown and glabrous in the other species) and the smaller homomericarpic fruit less than 5 mm long.     

Physiological,morphological and anatomical trait variations between winter and summer leaves of Cistus species

Morphological, anatomical and physiological summer and winter leaf traits of Cistus incanus subsp. incanus, C. salvifolius and C. monspeliensis growing at the Botanical garden of Rome were analyzed. With regard to differences between summer and winter leaves of the considered species, leaf thickness (L) was 21% higher in summer than in winter leaves (mean of the considered species) and this increase was mostly the result of the increased palisade parenchyma thickness over the spongy parenchyma one (24 and 16% higher in summer than in winter leaves, respectively). Leaf mass area (LMA) and leaf tissue density (LTD) were 38% and 17% higher in summer than in winter leaves, respectively (mean of the considered species). The photosynthetic rate (P_N), stomatal conductance (g_s) and chlorophyll content (Chl) of summer leaves were 54%, 17% and 14% lower, respectively, than in winter leaves. C. monspeliensis summer leaves had the highest LMA, LTD, adaxial cuticle thickness (14.6 ± 1.8 mg cm⁻², 1091 ± 94 mg cm⁻³, and 5.8 ± 1.7 μm, respectively) and the lowest mesophyll intercellular spaces (f_ias 38 ± 3%). Moreover, C. monspeliensis had the highest P_N in summer (2.6 ± 0.1 μmol m⁻² s⁻¹) and C. incanus the highest P_N and WUE (84% and 59% higher than the other species) in the favorable period, associated to a higher f_ias (42 ± 2%). C. salvifolius had the highest P_N (54% higher than the other species) in winter. The plasticity index could allow a better interpretation of the habitat preference of the considered species. The physiological plasticity (PI_p = 0.39, mean value of the considered species) was higher than the morphological (PI_m = 0.22, mean value) and anatomical (PI_a = 0.13, mean value) plasticity. Moreover, among the considered species, C. salvifolius and C. incanus are characterized by a larger PI_a (0.14, mean value) which seems to be correlated with their wider ecological distribution and the more favorable conditions of the environments where they naturally occur. The highest PI_m (0.29) of C. monspeliensis indicates that it can play a high adaptive role in highly stressed environments, like fire degraded Mediterranean areas in which it occurs.     

Kneallhazia carolinensae sp. nov., a microsporidian pathogen of the thief ant, Solenopsis carolinensis

A new species of microsporidia is described from adults of the thief ant, Solenopsis carolinensis, collected in Florida, USA. Morphological and genetic characterization of this new species showed that it is most closely related to the genus Kneallhazia and is therefore formally designated, Kneallhazia carolinensae sp. nov. Masses of ovoid, binucleate spores were localized to fat body of adult workers and measured 6.2 ± 0.1 × 3.1 ± 0.1 μm (fresh) and 6.0 ± 0.1 × 3.4 ± 0.1 μm (fixed). These spores were in direct contact with the cell cytoplasm and contained an isofilar polar filament with 12-15 coils. Blastn analysis revealed that the K. carolinensae 16S rDNA sequence exhibited 91% identity with the 16S rDNA gene of K. solenopsae. The morphological and sequence data support the conclusion that K. carolinensae is a novel microsporidian species distinct from K. solenopsae.     

Molecular and ultrastructural characterization of Andreanna caspii n. gen., n. sp. (Microsporida: Amblyosporidae), a parasite of Ochlerotatus caspius (Diptera: Culicidae)

A new genus and species of microsporidia, Andreanna caspii n. gen., n. sp. is described from the mosquito, Ochlerotatus caspius (Pallas) based on ultrastructural morphology, developmental characteristics, and comparative sequence analyses of the small subunit (SSU) ribosomal DNA (rDNA). Parasite development is confined to fat body tissue and infected larvae appear swollen with dull white masses within the thorax and abdomen. Meronts have diplokaryotic nuclei and are delineated by a simple plasmalemma contiguous with the host cell cytoplasm. Merogony occurs by synchronous binary division followed by cytokinesis. Diplokaryotic sporonts undergo meiosis and synchronous nuclear division forming sporogonial plasmodia with two, four and eight nuclei enclosed within a persistent sporophorous vesicle. Cytokinesis of sporogonial plasmodia results in the formation of eight uninucleate spores. The episporontal space of early sporonts is filled with a homogeneous accumulation of electron dense granular inclusions and ovoid vesicles of various dimensions, transforming into an interwoven matrix during the initial phase of sporogenesis. Spores are oval, uninucleate and measure 4.8 ± 0.3 × 3.1 ± 0.4 μm (fixed). The spore wall is 260 μm thick with an irregular exospore consisting of two layers (150-170 μm) and a thinner endospore (90-100 μm). The anchoring disk is well developed and is contiguous with a lamellar polaroplast that occupies the anterior third of the spore and possess more narrow lamellae on the posterior end. The polar filament is gradually tapered and arranged in a single row consisting of six coils ranging from 180 to 150 μm in diameter. The posterior vacuole (posterosome) is moderately sized and filled with a matrix of moderate electron density. Phylogenetic analysis of SSU rDNA from A. caspii and 30 other species of microsporidia including 11 genera parasitic in mosquitoes using maximum parsimony, neighbor joining and maximum likelihood methods showed A. caspii to be a sister group to the clade containing all of the Amblyospora species, including Culicospora, Edhazardia and Intrapredatorus, as well as Culicosporella and Hyalinocysta thus providing strong support for establishment of Andreanna as a separate genus.     

Biometry of Upper Cretaceous (Cenomanian–Maastrichtian) coccoliths – a record of long-term stability and interspecies size shifts

Biometric measurements of Mesozoic coccoliths (coccolith length and width) have been used in short-term biostratigraphic, taxonomic and palaeoecologic studies, but until now, not over longer time scales. Here, we present a long time-series study (∼ 30 million years) for the Upper Cretaceous, which aims to identify broad trends in coccolith size and to understand the factors governing coccolith size change over long time scales. We have generated biometric data for the dominant Upper Cretaceous coccolith groups, Broinsonia/Arkhangelskiella, Prediscosphaera, Retecapsa and Watznaueria, from 36 Cenomanian–Maastrichtian (100.5–66 Ma) samples from Goban Spur in the northeast Atlantic (DSDP Site 549). These data show that the coccolith sizes within Prediscosphaera, Retecapsa and Watznaueria were relatively stable through the Late Cretaceous, with mean size variation less than 0.7 μm. Within the Broinsonia/Arkhangelskiella group there was more pronounced variation, with a mean size increase from ∼ 6 μm in the Cenomanian to ∼ 10 μm in the Campanian. This significant change in mean size was largely driven by evolutionary turnover (species origination and extinctions), and, in particular, the appearance of larger species/subspecies (Broinsonia parca parca, Broinsonia parca constricta, Arkhangelskiella cymbiformis) in the early Campanian, replacing smaller species, such as Broinsonia signata and Broinsonia enormis. Shorter-term size fluctuations within Broinsonia/Arkhangelskiella, observed across the Late Cenomanian–Turonian and Late Campanian–Maastrichtian intervals, may, however, reflect changing palaeoenvironmental conditions, such as sea surface temperature and nutrient availability.     

Salinity increases the triterpenoid content of a salt secretor and a non-salt secretor mangrove

The present study describes the effect of salinity on the triterpenoid content of the salt secretor mangrove Avicennia marina and the non-secretor Rhizophora stylosa. Mangrove seedlings were grown for eight months in 0%, 0.5%, 1.5%, 2.0% and 3.0% salt concentration. The growth of both species was increased by salt with maximal stimulation at 1.5%, and this elevation appeared to be attenuated by increasing the salt concentration above 1.5%. The triterpenoid compositions of three types of chemical structures, lupane (lupeol, lupenone), oleanane (β-amyrin, taraxerol, germanicol), and ursane (α-amyrin), were studied. In addition, the phytosterol components campesterol, stigmasterol and β-sitosterol were analyzed. The total triterpenoid contents in the roots and leaves of A. marina for the 0% group were 87.0 and 66.2 μg g⁻¹, respectively, and increased significantly to 173.1 and 142.6 μg g⁻¹ with 3% salinity. The higher salinity also significantly increased the total concentration of phytosterols in the leaves and roots of this species. A similar increase in the concentration of both triterpenoids and phytosterols was observed in the roots and leaves of R. stylosa with increasing salt concentration. Thus, the triterpenoid concentration was increased by salinity in the roots and leaves of both A. marina and R. stylosa irrespective of their differences in salt management by salt excretion or by a non-excretion mechanism. Comparison of the triterpenoid concentration in four species of growing mangrove seedlings revealed a correlation between the total triterpenoid content and the salt tolerance based on the habitat zonation on Iriomote Island. A. marina thrives closest to sea and had the highest content of triterpenoids (173.1 μg g⁻¹ in 3% salt group). Therefore, it is likely that the triterpenoid content play an important role in mangrove plants for protection from salinity in both salt-secretors and non-secretors.     

Identification of zoonotic Giardia genotypes in fish

Apart from a single record in a shark, there have been no published studies conducted on Giardia genotypes in fish. The present study investigated the prevalence of Giardia in cultured fingerlings (n = 227), wild freshwater (n = 227) and wild marine/estuarine species (n = 255) of fish in Western Australia by PCR amplification at the 18S rRNA, glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi) and beta-giardin (bg) loci. Results revealed a low prevalence of Giardia, 3.8% (27/709), in fish hosts. The zoonotic Giardia species, Giardia duodenalis assemblages A, B as well as G. duodenalis assemblage E and Giardia microti were detected. The identification of zoonotic species of Giardia highlights the public health importance of investigating parasites within fish host species.     

Diversity of Bacteria and Archaea from a landfill in Chandigarh,India as revealed by culture-dependent and culture-independent molecular approaches

The bacterial community structure of a municipal landfill in Chandigarh, India was analysed by culture-dependent as well as culture-independent molecular approaches, and archaeal structure by the latter method. Samples were collected in two phases from the surface and a depth of 0.91 m in June, 2004 and from 0.91 m, 1.52 m and 1.68 m in May, 2005. After serial dilutions, samples were plated onto tryptic soy agar (TSA), plate count agar (PCA), tryptic soy broth agar (TSBA) and TSBA100 (TSBA diluted 100 times and solidified with agarose), and incubated aerobically at 30 °C. The number of bacteria (CFU) on different media ranged between 9.4 × 10⁵ g⁻¹ (on PCA) and 1.9 × 10⁷ g⁻¹ (on TSA) (wet weight). The numbers of bacteria enumerated from plates incubated anaerobically (anaerobic agar and reinforced clostridial agar) were 2.1 × 10⁷ and 1.7 × 10⁶ g⁻¹, respectively. Of the 468 isolated and purified bacteria (183 in the first phase and 285 in the second phase), 135 were characterised using phenotypic characteristics as well as 16S rRNA gene sequence analysis. It was found that members of the phylum Firmicutes were overwhelmingly predominant (86.6%) in the landfill, followed by Actinobacteria (9.6%) and Proteobacteria (3.7%). Among the Firmicutes, at least 17 species from the single genus Bacillus were the most abundant inhabitants of the landfill. Detailed polyphasic characterisation of many of these isolates led to the discovery of a novel genus Paenisporosarcina (and the species P. quisquiliarum), a novel species of Microbacterium, M. immunditiarum, and reclassification of Sporosarcina macmurdoensis, Pelagibacillus goriensis, Bacillus silvestris, Bacillus insolitus, Bacillus psychrotolerans and Bacillus psychrodurans. Culture-independent analysis of two 16S rRNA gene libraries also revealed that the phylum Firmicutes was the predominant group in this community. The diversity of Archaea was found to be limited mainly to members of two orders: Methanosarcinales and Methanomicrobiales of the phylum Euryarchaeota. When these results were compared to those reported earlier on similar studies, it was found that irrespective of differences in composition of municipal solid waste (especially compostable organic matter and paper) and climate, the members of bacterial and archaeal communities in landfills of many countries remained broadly similar.     

Amounts of nuclear DNA in marine halophytes

The amount of nuclear DNA, expressed as the C-value, was estimated for 13 marine halophytic plant species from six families. Plant material was collected in the nature reserve of the Strunjan saltpan in the Northern Adriatic and comprised all halophytic species inside the investigated area. Reproductive region of the shoot or root tips of halophytes were dissected, nuclei were Feulgen stained and 2C-values were measured by DNA image cytometry as follows: Crithmum maritimum (4.38 pg DNA), Artemisia caerulescens (6.43 pg), Aster tripolium (21.43 pg), Inula crithmoides (3.63 pg), Atriplex portulacoides (1.83 pg), A. prostrata (1.51 pg), Salicornia europaea (2.75 pg), Salsola soda (2.62 pg), Sarcocornia fruticosa (5.91 pg), Suaeda maritima (2.11 pg), Limonium angustifolium (5.06 pg), Puccinellia palustris (8.15 pg) and Ruppia cirrhosa (4.65 pg). With the exception of the C-value estimate for A. caerulescens, which has been listed in the Plant DNA C-values Database, the C-values represent the first estimates for all the examined species. In addition, the C-value for R. cirrhosa is also the first report for the family Ruppiaceae. The investigated halophytes had a smaller genome size compared to other known C-values for species within a particular family and also when compared to the mean values of dicots and monocots. The study also showed that halophylic annuals have a smaller genome size (2.49 pg) than perennial ones (7.45 pg DNA).     

New microsporidia parasitizing bark lice (Insecta: Psocoptera)

Two species of bark lice, Xanthocaecilius sommermanae Mockford and Polypsocus corruptus Hagen, collected in a canopy Malaise trap placed in Great Smoky Mountains National Park as part of a survey of the park’s fauna, were found to be infected with microsporidia. Diagnosis was originally based on light microscopy, and was confirmed by PCR amplification and electron microscopy. This is the first record of microsporidia infection in the insect order Psocoptera. Four morphological spore types corresponded to four original SSUrDNA sequences (Genbank accession no. FJ865221-24), suggesting infection with four microsporidia species. Two of those species were examined by electron microscopy. We describe here one new genus and two new species based on morphological and sequence data: Antonospora psocopterae sp. n. with elongated diplokaryotic spores, 4.4 ± 0.05 × 1.9 ± 0.03 μm and Mockfordia xanthocaeciliae gen. n. sp. n. with ovocylindrical monokaryotic spores, 2.5 ± 0.10 × 1.4 ± 0.02 μm. A. psocopterae displayed high sequence (95%) and structural similarity with Antonospora scoticae, fell within a well supported dichotomy with A. scoticae inside the Antonospora-Paranosema clade in phylogenetic analyses by NJ, PS and ML. M. xanthocaeciliae did not exhibit much sequence or structural similarity with any of known microsporidia species, except Encephalitozoon spp. M. xanthocaeciliae fell within one clade with Encephalitozoon spp. in phylogenies and shared with encephalitozoons structural resemblance and about 80% of SSUrDNA sequence identity. The other two species were not described and provisionally were placed to the collective genus Microsporidium as Microsporidium sp. 1 and Microsporidium sp. 4 from bark lice because of insufficient morphological data. The finding that samples fixed and stored for months in propylene glycol (“antifreeze”) are good enough for DNA sequence analysis and can be used for morphological analyses (if no better fixation alternatives are available), is promising for future surveys for microsporidia.     

Steinernema ichnusae sp. n. (Nematoda: Steinernematidae) a new entomopathogenic nematode from Sardinia Island (Italy)

A new Steinernema species was isolated from three different sandy soil samples along the Platamona Beach, in the north-west coast of Sardinia Island (Italy). This new species is characterized by the following morphological characters: infective third-stage juvenile with a body length of 866 ± 61 (767-969) μm, distance from head to excretory pore of 63 ± 2.7 (59-68) μm, tail length of 81 ± 3.2 (76-89) μm, ratio E (%) 77 ± 3.4 (68-83); male tail with a mucron only in the second generation, spicule length of 66 ± 1.4 (64-67) μm and gubernaculum length of 44 ± 1.4 (43-46) μm in the first generation male; female of first generation with a slight vulval protrusion and ratio D (%) of 53 ± 4.0 (47-63). The new species differs distinctly from the related species (S. feltiae, S. kraussei, S. litorale, S. oregonense and S. cholashanense) in some morphometric values such as percentage of hyaline portion, ratios of gubernaculum/spicule length, spicule head length/width. The DNA analyses of the internal transcribed spacers and D2D3 regions show that the studied nematode isolates are a new species. Cross hybridisation tests with S. feltiae, S. kraussei, S. litorale, S. weiseri and S. oregonense showed that these species were reproductively isolated.     

Clonal and spatial genetic structure in natural populations of Luohanguo (Siraitia grosvenorii), an economic species endemic to South China,as revealed by RAPD markers

Clonal growth is generally expected to have significant effects on the spatial genetic structure within populations. In this study, random amplified polymorphic DNA (RAPD) markers were used to reveal clonal and spatial genetic structure of four natural populations of Luohanguo (Siraitia grosvenorii), an economic vine species endemic to South China. A total of 351 ramets were assigned to 76 distinct multi-locus genotypes (i.e. genets), with the G/N varying from 0.121 to 0.350. No widespread genet was found across different populations. The clonal diversity (D) and evenness (E) ranged from 0.333 to 0.828 and from 0 to 0.741, respectively. While most genets consisted of fewer than five ramets, we observed some dominant genets that had much more (up to 69) ramets and spread over large areas. Spatial autocorrelation analyses revealed a spatial genetic structure (i.e. significant positive autocorrelation within 20 m and negative autocorrelation beyond 40 m) in one population, but not in other three populations with smaller population size. This study highlights the importance of clonal growth in shaping the spatial genetic structure in Luohanguo, which may have complex effects on the dynamics and evolution of its declining populations.     

Annesorhiza asparagoides (Apiaceae), a new species from the Cederberg Mountains, Western Cape Province, South Africa

A highly localised new species from the Cederberg Mountains near Wuppertal in the Western Cape Province is described. Annesorhiza asparagoides B.-E. Van Wyk, collected for the first time in 2009, differs from all other species of Annesorhiza (and the closely related Chamarea) in the unusual leaf structure, with crowded, subsessile, acicular leaf segments, resulting in dense, bottlebrush-like pinnae. The new species has a cluster of 10 or more slender roots, small (< 150 mm long), sparsely hairy leaves and small (± 8 mm long), oblong, conspicuously ribbed, homomericarpic fruits.     

The effects of algae species and densities on the population growth of the marine rotifer, Colurella dicentra

Colurella dicentra clones isolated from bay water in the Mississippi Gulf Coast were cultured with artificial seawater. Experiments were conducted to determine the effects of six algae species (Nannochloropsis oculata, Tetraselmis chuii, Chaetoceros gracilis, Rhodomonas salina, Isochrysis galbana, and Prorocentrum micans), six C. gracilis densities, and six N. oculata densities (25,000, 50,000, 100,000, 250,000, 500,000, and 1,000,000 cells ml^− 1) on C. dicentra population growth. Algae type influenced rotifer production (p < 0.0001). C. gracilis treatment (9120 ± 3351SD) produced the highest number of rotifers followed by N. oculata (5760 ±2232SD). P. micans had the lowest number of rotifers, although not significantly different from numbers in T. chuii, R. salina, and I. galbana treatments (p > 0.05).The population growth rate (r) varied with algae species treatment. The highest values were recorded for C. gracilis treatment (0.22 to 0.26 d^− 1), followed by N. oculata (0.21 to 0.24 d^− 1), and the lowest for P. micans (− 0.19 to 0.14 d^− 1). C. gracilis and N. oculata densities had significant effects (p < 0.0001) on C. dicentra population growth. The highest rotifer production was recorded at a C. gracilis density of 100,000 cells ml^− 1, followed by 250,000 cells ml^− 1 and 50,000 cells ml^− 1. Algae densities of 500,000 cells ml^− 1 and above produced the lowest rotifer numbers. Population growth rate (r) varied with C. gracilis densities. The highest values were observed for C. gracilis concentrations of 100,000 cells ml^− 1 (0.17 to 0.19 d^− 1), and the lowest for concentrations of 500,000 cells ml^− 1 and above (− 0.19 to 0.09 d^− 1). The 100,000 cells ml^− 1N. oculata density gave the highest rotifer production followed by 50,000, 250,000, 25,000, and 500,000 cells ml^− 1. Algae densities of 1,000,000 cells ml^− 1 produced the lowest rotifer numbers. Population growth rate (r) varied with N. oculata densities, with the highest values obtained for algae densities of 100,000 cells ml^− 1 (0.35 to 0.40 d^− 1), and the lowest for concentrations of 1,000,000 cells ml^− 1 (0.05 to 0.012 d^− 1). This is the first report of C. dicentra in Mississippi Coastal waters, and perhaps the smallest marine rotifer species (93 by 49 μm) ever cultured successfully.     

Kinetic studies of oxygen atom transfer reactions from trans-dioxoruthenium(VI) porphyrins to sulfides

The kinetics of the reactions of three trans-dioxoruthenium(VI) porphyrin derivatives with organic sulfides were measured. The dioxo systems studied were 5,10,15,20-tetramesityl porphyrin-dioxoruthenium(VI) (2a), 5,10,15,20-tetraphenylporphyrin-dioxoruthenium(VI) (2b), and 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin-dioxoruthenium(VI) (2c). Species 2 were competent oxidants and reacted rapidly with thioanisoles to generate the corresponding sulfoxides. Typical second-order rate constants determined from pseudo-first-order kinetic studies for sulfoxidation reactions are 8-60 M^− 1 s^− 1, which are 3 orders of magnitude larger in comparison with those of well studied alkene epoxidations and activated C-H bond oxidations by the same dioxo species. For a given sulfide substrate, the reactivity order for the dioxoruthenium(VI) species was 2a < 2b < 2c, which is in agreement with expectation on the basis of the electron-withdrawing and steric effects of the porphyrin macrocycles. Various para-substituted thioanisoles react in a narrow kinetic range with the same dioxo species. The kinetic results obtained in this study indicate a concerted oxygen atom transfer and/or electron transfer followed by oxygen transfer mechanism from oxidant to sulfide. Competition kinetic reactions with a catalytic amount of porphyrin ruthenium(II) species and a terminal oxidant give relative rate constants for sulfoxidations of competing substrates that are somewhat smaller than the ratios of absolute rate constants, implying a multiple oxidant model for sulfoxidation reactions.     

Using a starch-rich mutant of Arabidopsis thaliana as feedstock for fermentative hydrogen production

A mutant plant (Arabidopsis thaliana), sex1-1 (starch excess 1-1), accumulating high starch content in leaves was created to serve as better biomass feedstock for a H₂-producing strain Clostridium butyricum CGS2, which efficiently utilizes starch for H₂ production but cannot assimilate cellulosic materials. The starch content of the mutant plant increased to 10.67 mg/fresh weight, which is four times higher than that of wild type plant. Using sex1-1 mutant plant as feedstock, C. butyricum CGS2 could produce 490.4 ml/l of H₂ with a H₂ production rate of 32.9 ml/h/l. The H₂ production performance appeared to increase with the increase in the concentration of mutant plant from 2.5 to 10 g/l. The highest H₂ to plant biomass yield was nearly 49 ml/g for the mutant plant. This study successfully demonstrated the feasibility of using a starch-rich mutant plant for more effective bioH₂ production with C. butyricum CGS2.     

Coevolution of Cryptosporidium tyzzeri and the house mouse (Mus musculus)

Two house mouse subspecies occur in Europe, eastern and northern Mus musculus musculus (Mmm) and western and southern Mus musculus domesticus (Mmd). A secondary hybrid zone occurs where their ranges meet, running from Scandinavia to the Black Sea. In this paper, we tested a hypothesis that the apicomplexan protozoan species Cryptosporidium tyzzeri has coevolved with the house mouse. More specifically, we assessed to what extent the evolution of this parasite mirrors divergence of the two subspecies. In order to test this hypothesis, we analysed sequence variation at five genes (ssrRNA, Cryptosporidium oocyst wall protein (COWP), thrombospondin-related adhesive protein of Cryptosporidium 1 (TRAP-C1), actin and gp60) in C. tyzzeri isolates from Mmd and Mmm sampled along a transect across the hybrid zone from the Czech Republic to Germany. Mmd samples were supplemented with mice from New Zealand. We found two distinct isolates of C. tyzzeri, each occurring exclusively in one of the mouse subspecies (C. tyzzeri-Mmm and C. tyzzeri-Mmd). In addition to genetic differentiation, oocysts of the C. tyzzeri-Mmd subtype (mean: 4.24 × 3.69 μm) were significantly smaller than oocysts of C. tyzzeri-Mmm (mean: 4.49 × 3.90 μm). Mmm and Mmd were susceptible to experimental infection with both C. tyzzeri subtypes; however, the subtypes were not infective for the rodent species Meriones unguiculatus, Mastomys coucha, Apodemus flavicollis or Cavia porcellus. Overall, our results support the hypothesis that C. tyzzeri is coevolving with Mmm and Mmd.     

Flavonoid glycosides of the black locust tree, Robinia pseudoacacia (Leguminosae)

Four flavone glycosides isolated from extracts of the leaves of Robinia pseudoacacia (Leguminosae) were characterised by spectroscopic and chemical methods as the 7-O-β-d-glucuronopyranosyl-(1 → 2)[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranosides of acacetin (5,7-dihydroxy-4′-methoxyflavone), apigenin (5,7,4′-trihydroxyflavone), diosmetin (5,7,3′-trihydroxy-4′-methoxyflavone) and luteolin (5,7,3′,4′-tetrahydroxyflavone). Assignment of glycosidic ¹H and ¹³C resonances in their NMR spectra was facilitated by ²J_HC correlations detected using the H2BC (heteronuclear two-bond correlation) pulse sequence. Spectroscopic analysis of two known triglycosides, acacetin 7-O-β-d-glucopyranosyl-(1 → 2)[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside (previously unrecorded from this species) and acacetin 7-O-β-d-xylopyranosyl-(1 → 2)[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside (‘acacetin trioside’), enabled inconsistencies in the literature relating to these structures to be resolved. Comparison of the flavonoid chemistry of leaves and flowers of R. pseudoacacia using LC-UV and LC-MS showed that flavone 7-O-glycosides, particularly of acacetin, predominated in the former, whereas the latter comprised mainly flavonol 3,7-di-O-glycosides, including several examples new to this species. Tissue dependent differences in flavonoid chemistry were also evident from the glycosylation patterns of the compounds.