Core and symbiotic genes reveal nine Mesorhizobium genospecies and three symbiotic lineages among the rhizobia nodulating Cicer canariense in its natural habitat (La Palma,Canary Islands)

Cicer canariense is a threatened perennial wild chickpea endemic to the Canary Islands. In this study, rhizobia that nodulate this species in its natural habitats on La Palma (Canary Islands) were characterised. The genetic diversity and phylogeny were estimated by RAPD profiles, 16S-RFLP analysis and sequencing of the rrs, recA, glnII and nodC genes. 16S-RFLP grouped the isolates within the Mesorhizobium genus and distinguished nine different ribotypes. Four branches included minority ribotypes (3–5 isolates), whereas another five contained the predominant ribotypes that clustered with reference strains of M. tianshanense/M. gobiense/M. metallidurans, M. caraganae, M. opportunistum, M. ciceri and M. tamadayense. The sequences confirmed the RFLP groupings but resolved additional internal divergence within the M. caraganae group and outlined several potential novel species. The RAPD profiles showed a high diversity at the infraspecific level, except in the M. ciceri group. The nodC phylogeny resolved three symbiotic lineages. A small group of isolates had sequences identical to those of symbiovar ciceri and were only detected in M. ciceri isolates. Another group of sequences represented a novel symbiotic lineage that was associated with two particular chromosomal backgrounds. However, nodC sequences closely related to symbiovar loti predominated in most isolates, and they were detected in several chromosomal backgrounds corresponding to up to nine Mesorhizobium lineages. The results indicated that C. canariense is a promiscuous legume that can be nodulated by several rhizobial species and symbiotypes, which means it will be important to determine the combination of core and symbiotic genes that produce the most effective symbiosis.     

Xenopsylla spp. (Siphonaptera: Pulicidae) in murid rodents from the Canary Islands: An update

The geographical and host distributions of Xenopsylla fleas parasitizing murid rodents on the Canary Islands have been reported. Three Xenopsylla species, X. cheopis, X. brasiliensis and X. guancha, have been detected on two rodents species, Mus musculus and Rattus rattus. X. guancha has been the most prevalent species detected, specifically on M. musculus, the most abundant rodent, but it has been detected only on three eastern islands, where the species is endemic. X. cheopis has been shown to be the most widely distributed species throughout the archipelago and the species most frequently found on R. rattus. X. brasiliensis has been shown to be the least prevalent Xenopsylla species, with the lowest geographical distribution on the Canary Islands and focused only on R. rattus. The detection of both X. cheopis and X. brasiliensis on the island of Lanzarote, and of X. guancha on the island of Fuerteventura and the islet of La Graciosa represents the first report of these species on those particular Canary Islands.     

Molecular and phenotypic characterization of strains nodulating Anthyllis vulneraria in mine tailings, and proposal of Aminobacter anthyllidis sp. nov., the first definition of Aminobacter as legume-nodulating bacteria

Bacterial strains from Zn-Pb mine tailings were isolated by trapping with Anthyllis vulneraria, a legume-host suitable for mine substratum phytostabilisation. Sequence analysis of the 16S rRNA gene and three housekeeping genes (atpD, dnaK and recA) showed that they were related to those of the genus Aminobacter. DNA-DNA relatedness of representative isolates supported the placement of novel strains in Aminobacter as a new species. Phenotypic data emphasize their differentiation from the other related species of Aminobacter and Mesorhizobium. Aminobacter isolates exhibited nodA sequences tightly related with M. loti as the closest nodA relative. By contrast, their nodA sequences were highly divergent from those of M. metallidurans, another species associated with A. vulneraria that carries two complete copies of nodA. Therefore, the novel bacterial strains efficient on A. vulneraria represented the first occurrence of legume symbionts in the genus Aminobacter. They represent a new species for which the name Aminobacter anthyllidis sp. nov. is proposed (type strain STM4645(T)=LMG26462(T)=CFBP7437(T)).     

Gibbsiella greigii sp. nov., a novel species associated with oak decline in the USA

In 2010, cream-coloured, Gram-negative staining, facultatively anaerobic enterobacteria were isolated from a single black oak tree (Quercus kelloggii) exhibiting decline symptoms in southern California, USA. These 12 isolates were tentatively identified as Gibbsiella quercinecans based on partial gyrB sequencing. Closer examination of the strains using multilocus sequence analysis, based on partial sequences of gyrB, rpoB, infB and atpD genes, and almost complete 16S rRNA gene sequencing suggested that the isolates belong to a novel taxon within the genus Gibbsiella with G. quercinecans as their closest phylogenetic relative. DNA–DNA relatedness studies confirmed that the strains belong to a single taxon in Gibbsiella, which can be differentiated from other members of the genus by several phenotypic traits. Therefore, the name Gibbsiella greigii sp. nov. is proposed for this novel species isolated from symptomatic Q. kelloggii in the USA with FRB 224^T (=LMG 27716^T = NCPPB 4583^T) as the type strain.     

Description of Brenneria roseae sp. nov. and two subspecies, Brenneria roseae subspecies roseae ssp. nov and Brenneria roseae subspecies americana ssp. nov. isolated from symptomatic oak

Gram-negative, facultatively anaerobic bacteria were isolated from symptomatic oak tissue in the UK and USA. Partial gyrB sequencing placed ten strains in the genus Brenneria, with B. goodwinii as the closest phylogenetic relative. The strains were investigated further using a polyphasic approach including MLSA (based on partial gyrB, rpoB, infB and atpD gene sequences), 16S rRNA gene sequencing, DNA–DNA relatedness studies and both phenotypic and chemotaxonomic assays. The MLSA and 16S rRNA gene analyses separated the strains into two groups based on origin, suggesting that they belong to Brenneria as two novel species. However, the DNA–DNA relatedness values revealed a closer relationship between the groups and indicated that they should belong to the same species. As the two groups of strains from the UK and USA can be differentiated from each other phenotypically and by ERIC PCR fingerprints, it is proposed to classify them as novel subspecies of a novel Brenneria species. The name Brenneria roseae sp. nov. (FRB 222^T = LMG 27714^T = NCPPB 4581^T) is proposed, with Brenneria roseae subsp. roseae ssp. nov. (FRB 222^T = LMG 27714^T = NCPPB 4581^T) for the strains from the UK and Brenneria roseae subsp. americana ssp. nov. (FRB 223^T = LMG 27715^T = NCPPB 4582^T) for the strains from the USA.     

Marichromatium litoris sp. nov. and Marichromatium chrysaorae sp. nov. isolated from beach sand and from a jelly fish (Chrysaora colorata)

Three strains (JA349^T, JA553^T, JA439) of phototrophic sulphur bacteria were isolated from marine habitats of India. 16S rRNA gene sequence of the three strains clustered phylogenetically with members of the genus Marichromatium of the family Chromatiaceae belonging to the class Gammaproteobacteria. All the strains shared highest sequence similarity with the type strains of Marichromatium spp. (96-99% sequence similarity) and the new strains were characterized based on polyphasic taxonomy. Strains JA349^T and JA553^T can be distinguished from closest relative species of the genus Marichromatium with respect to distinct differences in cellular polar lipids, fatty acids and carbon/nitrogen sources utilization. Both strains were distinctly related (<50% based on DNA-DNA hybridization) with the type strains of the genus Marichromatium. Multilocus Sequence Analysis (MLSA) of the concatenated five protein coding genes (fusA, pufM, dnaK, recA, soxB) along with internal transcribed spacer (ITS; 16S-23S rRNA) had sequence similarity of less than 92% with the type strains of Marichromatium spp. Distinct phenotypic, chemotaxonomic and molecular differences allow the separation of strains JA349^T and JA553^T into new species of the genus Marichromatium for which, we propose the names Marichromatium litoris sp. nov. and Marichromatium chrysaorae sp. nov., respectively.     

Characterization of six microsatellite loci in Myrica faya (Myricaceae) and cross amplification in the endangered endemic M. rivas-martinezii in Canary Islands, Spain

Six novel polymorphic microsatellite markers were isolated from enriched libraries in Myrica faya Ait., recently renamed Morella faya, (fayatree, firetree, or firebush) in order to examine the genetic diversity in natural populations. Also, test cross-specific amplification and genetic diversity in Myrica rivas-martinezii, which is endemic on the Canary islands. Microsatellite loci were screened in 225 individuals of both species from different islands of the Canarian archipelago. All markers were successfully amplified from both Myrica species, with an average number of 6.5 and 9.3 alleles per locus in M. rivas-martinezii and M. faya, respectively. There was no evidence for linkage disequilibrium between loci, and the probability of null alleles ranged from 0.01 to 0.17.     

Presence of Hypolagus Dice, 1917 (Mammalia, Lagomorpha) in the Neogene of the Balearic Islands (Western Mediterranean): Description of Hypolagus balearicus nov. sp.

In this paper we describe a new species of Hypolagus coming from an early Pliocene karstic deposit near Caló den Rafelino (Manacor, Mallorca). It represents the westernmost European record for the genus. The faunal assemblage of this deposit represents an early phase of the second insular faunal episode of Mallorca, related with the Messinian regressive episode (latest Miocene). Although the postcranial are relatively robust, the size of all these elements is included in the range of continental leporids. It is unknown if they display significant allometric changes, as occurred in other insular leporids. p3 is small and displays a markedly trapezoidal outline and a deep hypoflexid (between 54 and 56% of the total tooth width). The presence of Hypolagus in the Neogene of the Balearic Islands is in agreement with the faunal scenario for the European continent during the late Miocene and the Pliocene, where this genus is abundant and widely distributed.     

Notes on the biology of Cancer bellianus (Brachyura, Cancridae) around the Canary Islands

Information on biometric and biological parameters of Cancer bellianus Johnson, 1861 (Crustacea, Decapoda, Brachyura, Cancridae) off the Canary Islands is given. Crabs examined were collected during experimental fishing surveys during 1974–1998. Carapace length, carapace width, total wet weight, sex and ovigerous condition were determined. This species was caught at depths from 153 to 750 m, the deepest ever recorded. Size frequency distributions were assembled and size-weight relationships were estimated by sex. Sex-ratio as a function of size and depth was determined. The size at first maturity was calculated by analysing the relative growth between the carapace length and the left chela width: 103.5 mm CL in males, 101.2 mm CL in females. Ovigerous females, egg size and fecundity estimates are reported apparently for the first time.     

Stygofauna of the Canary Islands, 6 A new Rhipidogammarus (Crustacea,Amphipoda) from Tenerife: first record of the genus outside the Mediterranean region and its biogeographic implications

The discovery of a new species ofRhipidogammarus, Rh. nivariae n. sp., in water supply shafts of Tenerife (Canary Islands), presents an interesting biogeographic problem. Up to now, members of the genus were known exclusively from stygohabitats in the peri-Mediterranean belt. Its sister-genus is found in very shallow waters of the Mediterranean, and on morphological grounds the genus seems to have got adapted only fairly recently to continental hypogean waters. The occurrence of a member ofRhipidogammarus on an oceanic island like Tenerife can be explained by one of the following two scenarios: (1) Tenerife is a fragment of the African plate, or (2) the island's volcanic outcrops arose from a very shallow submarine bank and drifted later into deeper waters.     

Colonisation and diversification of the blue tits (Parus caeruleus teneriffae-group) in the Canary Islands

The blue tit (Parus caeruleus teneriffae group) is proposed to have colonised the Canary Islands from North Africa according to an east-to-west stepping stone model, and today, the species group is divided into four subspecies, differing in morphological, acoustic, and ecological characters. This colonisation hypothesis was tested and the population structure between and within the islands studied using mitochondrial DNA sequences of the non-coding and relatively fast evolving control region. Our results suggest that one of the central islands, Tenerife, was colonised first and the other islands from there. Three of the presently recognised four subspecies are monophyletic, exception being the subspecies teneriffae, which consists of two monophyletic groups, the one including birds of Tenerife and La Gomera and the other birds of Gran Canaria. The Gran Canarian birds are well differentiated from birds of the other islands and should be given a subspecies status. In addition, the teneriffae subspecies group is clearly distinct from the European caeruleus group, and therefore the blue tit assemblage should be divided into two species.     

Data on the family Pandalidae around the Canary Islands, with first record of Plesionika antigai (Caridea)

Of the 20 pandalid shrimps species and subspecies reported for the Eastern Central Atlantic (26–36° N), 16 were found in one or more Macaronesian archipelagos (Azores, Madeira, Canary Islands and Cape Verde Islands) (14–40° N), and 11 of them were recorded to date in the Canary Island waters (27° 30–29° 30 N): Bitias stocki Fransen, 1990; Heterocarpus ensifer ensifer A. Milne-Edwards, 1881; Heterocarpus grimaldii A. Milne-Edwards & Bouvier, 1900; Heterocarpus laevigatus Bate, 1888; Plesionika edwardsii (Brandt, 1851); Plesionika ensis (A. Milne-Edwards, 1881); Plesionika holthuisi Crosnier & Forest, 1968; Plesionika martia martia (A. Milne-Edwards, 1883); Plesionika narval (J.C. Fabricius, 1787); Plesionika williamsi Forest, 1964; and Stylopandalus richardi Coutière, 1905. In the present work, Plesionika antigai Zariquiey Álvarez, 1955 is recorded for the first time from the Canary Islands. As a result of many fishing surveys around the Canary Islands at 27–1550 m depth between 1985 and 1998, information on bathymetric distribution, habitat, size and biology of the 12 Canarian pandalid species is given. The geomorphologic, geographic and oceanographic characteristics of the Canary Islands marine ecosystems could explain the great diversity in the biogeographic patterns of the pandalid species inhabiting this area. The distribution patterns found were: Macaronesian (1 spec.), Atlanto-Mediterranean (1 spec.), Eastern Atlantic warm-temperate (1 spec.), amphi-Atlantic warm (2 spec.), amphi-Atlantic warm-temperate (1 spec.), pantropical (5 spec.), and cosmopolitan (1 spec.).     

Hatching Behavior of Potato Cyst Nematodes from the Canary Islands

The present work investigated early hatching differences in naturally occuring field populations and newly reared populations of potato cyst nematodes from the Canary Islands. Hatching behavior of the two species appears to be distinct, with more juveniles hatched from G. pallida that hatch earlier and over a shorter time than G. rostochiensis. The hatching rate of 3-year-old PCN populations was more than double (mean 44.5% ñ 1) that shown by newly reared populations (mean 19.1% ñ 12.5), and those that could be classified as pathotype Pa 1 (Pa 1 and P 13) were found to hatch particularly poorly. Significant differences were also observed in the juveniles released in tap water between newly reared populations of both species, with mean hatch significantly higher for G. rostochiensis. The results are discussed in relation to the implication that these findings may have for competition between the two species of PCN in the field.     

A Mesorhizobium lipopolysaccharide (LPS) specific lectin (CRL) from the roots of nodulating host plant, Cicer arietinum

A 30 kDa rabbit erythrocyte agglutinating glycoprotein isolated and characterized from the roots of Cicer arietinum and designated as cicer root lectin (CRL). Hemagglutination activity of CRL is strongly inhibited by cell surface LPS of nodulating cicer specific Rhizobium. CRL agglutinates mesorhizobial cells and not Escherichia coli or yeast cells. It binds to immobilized LPS of cicer specific Rhizobium only. The primary structure of CRL as predicted by peptide mass fingerprinting by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) indicated ∼54% amino acid sequence homology with C. arietinum seedling lectin (Accession no. gi/3204123) and ∼26% with C. arietinum (Accession no. gi/110611256), and Pisum sativum (Accession nos. gi/230612, gi/6729956, gi/126148) lectins. These suggested CRL to be a member of vegetative tissue lectin. Circular dichroism analysis indicated that the secondary structure of CRL consists of 48% β-sheets, 26% random coils, and 11% α-helix. CRL has six isoforms of closely associated molecular mass with differential acidic pI of 5.30, 5.20, 5.15, 5.05, 5.00, 4.80. Identity of these isoforms was confirmed from their binding with cicer specific Rhizobium LPS. All the isoforms of CRL are differentially glycosylated as identified by deglycosylation and monosaccharide analysis using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). All these results suggest that unlike other plant lectins CRL is a LPS-binding lectin.     

The symbiont Capsaspora owczarzaki,nov. gen. nov. sp., isolated from three strains of the pulmonate snail Biomphalaria glabrata is related to members of the Mesomycetozoea

While investigating the resistance of some strains of Biomphalaria glabrata to infection with Schistosoma mansoni, a unicellular eukaryotic symbiont was noted in the snail haemolymph. It was similar in appearance to Nuclearia sp. reported from B. glabrata. Sequences comprising the 18S, ITS1, 5.8S, ITS2 and the beginning of the 28S rDNA gene regions were obtained from symbionts isolated from three strains of B. glabrata, and compared with the same sequences obtained from a culture of Nuclearia sp. 18S rDNA sequences were identical for all four isolates. 18S rDNA sequences were used in a phylogenetic analysis to produce minimum evolution, maximum parsimony, maximum likelihood and Bayesian trees. All four analyses indicated that the B. glabrata symbiont is not closely related to Nuclearia but instead to the Mesomycetozoea, a recently recognised clade of symbiotic eukaryotes. Based on phylogenetic analysis, life history and morphological differences, the symbiont is described as a new genus and species, Capsaspora owczarzaki. Distinguishing characters are the presence of life cycle stage(s) that occur within snail haemolymph; ability to kill and ingest digenetic trematode larvae; ability to undergo asexual fission to produce daughter cells; absence of flagella, a mucous sheath and membranes containing chitin, elastin, or collagen; and presence of long unbranching pseudopodia and a penetration process. Using both polymerase chain reaction (PCR) and culturing techniques, the S. mansoni-resistant Salvador and 13-16-R1 strains were found to be significantly more likely to harbour the symbiont than the susceptible M line strain. Small but consistent sequence differences were noted among symbiont isolates from different snail strains, raising the possibility that the symbiont has diverged in different snail lineages. This suggests further that the symbiont is not restricted to albino lab-reared snails. A role, if any, of the symbiont in resistance awaits further study.     

Breoghania corrubedonensis gen. nov. sp. nov., a novel alphaproteobacterium isolated from a Galician beach (NW Spain) after the Prestige fuel oil spill,and emended description of the family Cohaesibacteraceae and the species Cohaesibacter gelatinilyticus

A Gram-negative bacterium designated UBF-P1^T was isolated from an enrichment culture established in nutrient supplemented artificial sea water with pyrene as a carbon source, and inoculated with a marine fuel oil-degrading consortium obtained from a sand sample collected from the beach of Corrubedo (A Coruña, Galicia, Spain) after the Prestige accidental oil spill. Phylogenetic analysis based on the almost complete 16S rRNA gene sequence affiliated strain UBF-P1^T with the family Cohaesibacteraceae, Cohaesibacter gelatinilyticus (DSM 18289^T) being the closest relative species with 92% sequence similarity. Cells were irregular rods, motile, strictly aerobic, catalase and oxidase positive. Ubiquinone 10 was the major respiratory lipoquinone. The major polar lipids comprised diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylmonomethylethanolamine (PME), and phosphatidylcholine (PC). The major fatty acids detected were C_18:1ω7c, C_19:0 cycloω8c, and C_16:0. The G + C content of strain UBF-P1^T was 63.9 mol%. The taxonomic comparison with the closest relative based on genotypic, phenotypic and chemotaxonomic characteristics supported that strain UBF-P1^T could be classified as a novel genus and species, for which the name Breoghania corrubedonensis gen. nov., sp. nov. is proposed. The type strain of this new taxon is UBF-P1^T (CECT 7622, LMG 25482, DSM 23382).     

Evolution and diversification of the forest and hypogean ground-beetle genus Trechus in the Canary Islands

The beetle genus Trechus (Carabidae) is represented in the Macaronesian Islands by 43 endemic species. The Canary Islands have 16 endemic species, with two adapted to hypogean life. Phylogenetic relationships among 177 individuals of 38 Canarian, Madeiran, Azorean and continental Trechus species were examined using mitochondrial DNA and nuclear internal transcribed spacer 2 (ITS2) sequence data. Results show two main lineages in the Canaries: one comprising two sister groups with species from the laurel forest of La Gomera and Tenerife, and the other containing the single species from Gran Canaria and a species complex in the four western islands including two troglobites. Calibrations were applied to a linearized tree using a relaxed molecular clock method to estimate the major evolutionary divergence times of the Canarian Trechus species. Although the species assemblage in this archipelago is relatively ancient (7-8 million years), much of the species diversity is recent. Transition to the hypogean environment is more consistent with the "adaptive shift" rather than with the competing "climatic relict" hypothesis.     

Ensifer mexicanus sp. nov. a new species nodulating Acacia angustissima (Mill.) Kuntze in Mexico

A new lineage of Ensifer nodulating the American legume Acacia angustissima in the tropical forest of Chiapas and Morelos, Mexico is described. Bacteria were identified as Ensifer with ssb or nolR specific primers. Phylogenetic analysis with partial sequences of the five chromosomal genes gyrA, nolR, recA, rpoB and rrs revealed that this new lineage is related to African Ensifer terangae. The results of total DNA-DNA hybridization and selected phenotypic tests among the A. angustissima strains and E. terangae indicated that they belong to different species. The phylogeny with the symbiotic nifH gene also separates this group as a different clade but with close affinities to bacteria belonging to the genus Ensifer isolated from American hosts. ITTG R7(T) (=CFN ER1001, HAMBI 2910, CIP 109033, ATCC BAA-1312, DSM18446) is the type strain of a new species for which the name Ensifer mexicanus sp. nov. is proposed.