Sensitivity of <Emphasis Type="Italic">Eisenia fetida</Emphasis> in comparison to <Emphasis Type="Italic">Aporrectodea caliginosa</Emphasis> and <Emphasis Type="Italic">Lumbricus terrestris</Emphasis> after imidacloprid exposure. Body mass change and histopathology

Purpose  

The use of only one or a few species—representing an entire taxon—in ecotoxicological standard tests poses risk of underestimating the impact of toxicants on the environment. In earthworm ecotoxicity tests, the species Eisenia fetida or Eisenia andrei are commonly used, and there is evidence that these species respond relatively insensitive towards environmental pollution. With the present study, we wanted to evaluate the risk of underestimating effects of the insecticide imidacloprid in soil organisms by comparing E. fetida with two other earthworm species (Aporrectodea caliginosa and Lumbricus terrestris) regarding their sensitivities towards soil contaminated with this widely used insecticide.     

Dehydration does not affect the radial pressures produced by the earthworm <Emphasis Type="Italic">Aporrectodea caliginosa</Emphasis>

As a soil dries, the earthworms in that soil dehydrate and become less active. Moisture stress may weaken an earthworm, lowering the radial pressure that the animal can produce. This possibility was investigated for the earthworm Aporrectodea caliginosa (Savigny). Pressures were compared for saturated earthworms (worms taken from saturated soil) and stressed earthworms (worms that had been partially dehydrated by leaving them in dry soil). A load cell was used to record the forces that earthworms produced as they moved through artificial burrows (holes that had been drilled through blocks of aluminium or Perspex). The radial pressure was calculated using the forces exerted and the dimensions of the artificial burrows. There was a negative correlation between burrow diameter and radial pressure, although radial pressure was independent of the length of the block through which the earthworms had burrowed. The highest radial pressures were produced by the anterior segments of the animal. Partial dehydration caused the earthworms to become quiescent, but did not decrease the radial pressure that the earthworms produced. It is suggested that coelomic fluid is retained in the anterior segments while the rest of the animal dehydrates. Dehydrated earthworms became lethargic, and we suggest that lethargy is due to the loss of coelomic fluid from the posterior segments. Coelomic fluid is known to be lost through dorsal pores. In burrowing species of earthworm such as Aporrectodea caliginosa, these pores are only present on the posterior segments.     

Transformation of nitrogen compounds and dynamics of microbial biomass in fresh casts of <Emphasis Type="Italic">Aporrectodea caliginosa</Emphasis>

A comparative assessment of the contents of total nitrogen, ammonium, nitrates, and fungal and bacterial biomasses and of the activity of nitrogen transformation in fresh casts of Aporrectodea caliginosa and in a soddy-podzolic soil was performed. The total content of nitrogen in the casts was similar to or slightly higher than that in the soil; the content of inorganic forms of nitrogen in the casts was significantly higher than in the soil. The intensities of ammonification, nitrification, and denitrification and the pool of microbial biomass with a predominance of fungi during the 1.5-week-long incubation were also significantly higher in the casts. The activity of nitrogen fixation in the casts was lower than that in the soil. In the course of the incubation, the values of these parameters in the casts became closer to those in the soil. The inhibition of fungi in the casts with cycloheximide resulted in an increasing content of inorganic nitrogen and a higher activity of denitrification. In our opinion, this phenomenon is related to the fact that limitation of the fungal growth decreases the intensity of immobilization of nitrates and the fungal competition with denitrifying bacteria for available carbon. It was supposed that the activation of the fungal growth via application of plant substrates into the soil with a high population density of earthworms could suppress the emission of gaseous nitrogen compounds. The soil processing by earthworms had a positive effect on the soil properties.     

Putative Role of <Emphasis Type="Italic">Flavobacterium</Emphasis>, <Emphasis Type="Italic">Dokdonella</Emphasis> and <Emphasis Type="Italic">Methylophilus</Emphasis> Strains in Paracetamol Biodegradation

Paracetamol, the most widely and globally used analgesic and antipyretic, is easily accumulated in aquatic environments. In the present study, the biodegradation of paracetamol in different media (one for general growth, one specific for sulfate reducing bacteria, a mineral salts medium and municipal wastewater) inoculated with two types of sludge (from anaerobic lagoon and from oxidation ditch) under different oxygenic conditions (anoxic; moderate oxygenation in open flasks and high oxygenation by aeration) was investigated. In addition, bacteria with relative abundances increasing simultaneously with paracetamol degradation, when this drug was the only carbon source, thus with a putative role in its degradation, were identified using 16S rRNA gene sequences. The results show that aerobic microorganisms had a major role in the degradation of paracetamol, with 50 mg/L totally removed from municipal wastewater after 2 days incubation with aeration, and that the metabolites 4-aminophenol and hydroquinone plus one compound not identified in this work were produced in the process. The identification of bacteria with a role in the degradation of paracetamol revealed a strain from genus Pseudomonas with the highest final relative abundance of 21.2%, confirming previous works reporting strains of this genus as paracetamol decomposers. Besides, genera Flavobacterium, Dokdonella and Methylophilus were also in evidence, with initial relative abundances of 1.66%, 1.48 and 0.00% (not detected) in the inoculum and 6.91%, 3.80 and 3.83% after incubation, respectively. Therefore, a putative role of these genera in paracetamol biodegradation is suggested for the first time.

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Graphical Abstract ?

     

Production and cytogenetics of trigeneric hybrid involving <Emphasis Type="Italic">Triticum</Emphasis>, <Emphasis Type="Italic">Psathyrostachys</Emphasis> and <Emphasis Type="Italic">Secale</Emphasis>

Trigeneric hybrids may help establish evolutionary relationships among different genomes present in the same cellular-genetic background, and also offers the possibility to transfer different alien characters into cultivated wheat. In this study, a new trigeneric hybrid involving species from the Triticum, Psathyrostachys and Secale was synthesized by crossing wheat-P. huashanica amphiploid (PHW-SA) with wheat-S. cereale amphiploid (Zhongsi 828). The crossability of F₁ hybrid was high with 35.13%, and the fertility was 41.95%. The morphological characteristics of F₁ plants resembled the parent Zhongsi 828. The trigeneric hybrids pollen mother cells (PMCs) regularly revealed averagely 19.88 univalents, 9.63 ring bivalents, 3.97 rod bivalents, 0.60 trivalents and 0.03 tetravalents per cell. Multivalents consisted of trivalents and tetravalents can be observed in 52.7% of cells. A variation of abnormal lagging chromosome, micronuclei and chromosome bridge were formed at anaphase I and telophase II. The mean chromosomes number of F₂ progenies was 2n = 46.13, and the distribution range was 42–53. GISH results revealed that most F₂ plants had 6–12 S. cereale chromosomes, and only 0–2 P. huashanica chromosomes were detected. The results indicated that S. cereale chromosomes can be preferentially transmitted in the F₂ progenies of trigeneric hybrid than P. huashanica chromosomes. A survey of disease resistances revealed that the stripe rust resistance from the PHW-SA were completely expressed in the F₁ and some F₂ plants. The trigeneric hybrid could be a useful bridge for the transference of P. huashanica and S. cereale chromatins to common wheat.     

Metal soil pollution differentially affects both the behaviour and exposure of <Emphasis Type="Italic">A. caliginosa</Emphasis> and <Emphasis Type="Italic">L. terrestris</Emphasis>: a mesocosm study

The effects on two earthworm species of a gradient of metal contamination in soil collected close to a 50-year-old lead recycling factory were investigated in mesocosms filled with soil sampled at three distances from the factory (10, 30 and 60 m). After 5 weeks of exposure, earthworm litter consumption and weight change were measured. Burrow systems were analysed using X-ray tomography, and water infiltration was measured. No significant differences in earthworm weight or activity were observed between mesocosms filled with soil from 30 and 60 m. In contrast, both earthworm species significantly lost weight and burrowed less in the soil sampled at 10 m. In the cores filled with the soil collected at 10-m distance, Aporrectodea caliginosa avoided the highly contaminated first layer (0–5 cm) and burrowed deeper whereas Lumbricus terrestris burrowed relatively more in this layer. We assume that these different reactions are associated with their ecological types. Epi-anecic earthworms forage litter at the soil surface, whereas endogeic earthworms are geophagous and thus are able to forage deeper. This was further corroborated by the bioaccumulation factors measured for each species: for L. terrestris, BAF values for Pb and Cd only decreased slightly in the 10-m soil correlating with their overall reduced activity. However, BAF values for A. caliginosa were 20-fold lower compared to those observed in soil from 30 and 60 m. These modifications in burrowing behaviour in the 10-m mesocosms resulted in a significant and marked decrease in water infiltration rates but only for L. terrestris.     

<Emphasis Type="Italic">Rhizobium</Emphasis>,<Emphasis Type="Italic"> Bradyrhizobium</Emphasis> and<Emphasis Type="Italic"> Agrobacterium</Emphasis> strains isolated from cultivated legumes

The present study was conducted to isolate and characterize rhizobial strains from root nodules of cultivated legumes, i.e. chickpea, mungbean, pea and siratro. Preliminary characterization of these isolates was done on the basis of plant infectivity test, acetylene reduction assay, C-source utilization, phosphate solubilization, phytohormones and polysaccharide production. The plant infectivity test and acetylene reduction assay showed effective root nodule formation by all the isolates on their respective hosts, except for chickpea isolate Ca-18 that failed to infect its original host. All strains showed homology to a typical Rhizobium strain on the basis of growth pattern, C-source utilization and polysaccharide production. The strain Ca-18 was characterized by its phosphate solubilization and indole acetic acid (IAA) production. The genetic relationship of the six rhizobial strains was carried out by random amplified polymorphic DNA (RAPD) including a reference strain of Bradyrhizobium japonicum TAL-102. Analysis conducted with 60 primers discriminated between the strains of Rhizobium and Bradyrhizobium in two different clusters. One of the primers, OPB-5, yielded a unique RAPD pattern for the six strains and well discriminated the non-nodulating chickpea isolate Ca-18 from all the other nodulating rhizobial strains. Isolate Ca-18 showed the least homology of 15% and 18% with Rhizobium and Bradyrhizobium, respectively, and was probably not a (Brady)rhizobium strain. Partial 16S rRNA gene sequence analysis for MN-S, TAL-102 and Ca-18 strains showed 97% homology between MN-S and TAL-102 strains, supporting the view that they were strains of B. japonicum species. The non-infective isolate Ca-18 was 67% different from the other two strains and probably was an Agrobacterium strain.     

Morphological variation in <Emphasis Type="Italic">Laelia anceps</Emphasis> subsp. <Emphasis Type="Italic">dawsonii</Emphasis> f. <Emphasis Type="Italic">chilapensis</Emphasis> Soto-Arenas Orchidaceae in traditional home gardens of Chilapa,Guerrero, Mexico

Calaverita an endangered Mexican orchid is one of the most elegant and showy orchids of the genus Laelia and essential symbol within the local traditional celebrations of the Day of the Dead, in the region of Chilapa-Guerrero. We investigated morphological variation in flowers of la calaverita (Laelia anceps Lind. subsp. dawsonii (J. Anderson) Rolfe f. chilapensis Soto-Arenas, Orchidaceae) using the labellum of the specimens cultivated in traditional home gardens in the region of Chilapa, Guerrero, Mexico. The use of morphometric traits of the labellum is useful in appraising infra-subspecific morphological variation within the chilapensis form. Floral morphological variation exists among the specimens examined. These specimens, found exclusively in traditional home gardens, were grouped into four morphotypes: NAT, Topiltepec, Xulchuchío, and Terrero. The study of variation of la calaverita revealed the existence of four local morphotypes that originated from a long and complex process of selection of traits which responds to traditional preferences and knowledge about specific biotic and agroecological conditions. The cultural and aesthetic influence of traditional homegarden owners in the Chilapa region acts directly on selection, use and conservation of the morphological traits of la calaverita.     

<Emphasis Type="Italic">Trichoderma</Emphasis> improves the growth of <Emphasis Type="Italic">Leymus chinensis</Emphasis>

Bio-fertilizer application has been proposed as a strategy for enhancing soil fertility, regulating soil microflora composition, and improving crop yields, and it has been widely applied in the agricultural yields. However, the application of bio-fertilizer in grassland has been poorly studied. We conducted in situ and pot experiments to investigate the practical effects of different fertilization regimes on Leymus chinensis growth, with a focus on the potential microecological mechanisms underlying the responses of soil microbial composition. L. chinensis biomass was significantly (P?<?0.05) increased by treatment with 6000 kg ha^?1 of Trichoderma bio-fertilizer compared with other treatments. We found a positive (R² =?0.6274, P <?0.001) correlation between bacterial alpha diversity and L. chinensis biomass. Hierarchical cluster analysis and nonmetric multidimensional scaling (NMDS) revealed that soil bacterial and fungal community compositions were all separated according to the fertilization regime used. The relative abundance of the most beneficial genera in bio-fertilizer (BOF) (6000 kg ha^?1Trichoderma bio-fertilizer) was significantly higher than in organic fertilizer (OF) (6000 kg ha^?1 organic fertilizer) or in CK (non-amend fertilizer), there the potential pathogenic genera were reduced. There were significant negative (P?<?0.05) correlations between L. chinensis biomass and the relative abundance of several potential pathogenic genera. However, the relative abundance of most beneficial genera were significantly (P?<?0.05) positively correlated with L. chinensis biomass. Soil properties had different effects on these beneficial and on these pathogenic genera, further influencing L. chinensis biomass.     

A new mycorrhizal helper bacterium, <Emphasis Type="Italic">Ralstonia</Emphasis> species,in the ectomycorrhizal symbiosis between <Emphasis Type="Italic">Pinus thunbergii</Emphasis> and <Emphasis Type="Italic">Suillus granulatus</Emphasis>

In a Robinia-pseudoacacia-dominated coastal forest in Tottori prefecture Japan, the growth and survival of Pinus thunbergii seedlings and the natural regeneration of P. thunbergii was disturbed by R. pseudoacacia. In order to improve the growth of P. thunbergii seedling in the Tottori sand dune, we tried to find a mycorrhiza helper bacteria (MHB) from P. thunbergii mycorrhizosphere in a Tottori sand dune. Two MHB, Ralstonia sp. and Bacillus subtilis, were selected from the nine bacterial species isolated from the mycorrhizosphere of P. thunbergii. The bacterial effect on the ectomycorrhizal fungus Suillus granulatus was investigated by confrontation assay and a microcosm experiment. The confrontation assay showed that Ralstonia sp. promoted the hyphal growth of S. granulatus. Moreover, the S. granulatus–P. thunbergii symbiosis was significantly stimulated by Ralstonia sp. and B. subtilis. Ralstonia sp. and B. subtilis were regarded as MHB associated with P. thunbergii. This is the first report of Ralstonia sp. as an MHB.     

Exploitation of <Emphasis Type="Italic">Malus</Emphasis> EST-SSRs and the utility in evaluation of genetic diversity in <Emphasis Type="Italic">Malus</Emphasis> and <Emphasis Type="Italic">Pyrus</Emphasis>

A total of 8117 suitable SSR-contaning ESTs were acquired by screening from a Malus EST database, among which dinudeotide SSRs were the most abundant repeat motif, within which, CT/TC followed by AG/GA were predominant. Based on the suitable sequences, we developed 147 SSR primer pairs, of which 94 pairs gave amplifications within the expected size range while 65 pairs were found to be polymorphic after a preliminary test. Eighteen primer pairs selected randomly were further used to assess genetic relationship among 20 Malus species or cultivars. As a result, these primers displayed high level of polymorphism with a mean of 6.94 alleles per locus and UPGMA cluster analysis grouped twenty Malus accessions into five groups at the similarity level of 0.6800 that were largely congruent to the traditional taxonomy. Subsequently, all of the 94 primer pairs were tested on four accessions of Pyrus to evaluate the transferability of the markers, and 40 of 72 functional SSRs produced polymorphic amplicons from which 8 SSR loci selected randomly were employed to analyze genetic diversity and relationship among a collection of Pyrus. The 8 primer pairs produced expected bands with the similar size in apples with an average of 7.375 alleles per locus. The observed heterozygosity of different loci ranged from 0.29 (MES₉₆) to 0.83 (MES₁₃₈), with a mean of 0.55 which is lower than 0.63 reported in genome-derived SSR marker analysis in Pyrus. The UPGMA dendrogram was similar to the previous results obtained by using RAPD and AFLP markers. Our results showed that these EST-SSR markers displayed reliable amplification and considerable polymorphism in both Malus and Pyrus, and will contribute to the knowledge of genetic study of Malus and genetically closed genera.     

<Emphasis Type="Italic">Achromobacter insolitus</Emphasis> and <Emphasis Type="Italic">Zoogloea ramigera</Emphasis> associated with wheat plants (<Emphasis Type="Italic">Triticum aestivum</Emphasis>)

This study reports for the first time the presence of diazotrophic bacteria belonging to the genera Achromobacter and Zoogloea associated with wheat plants. These bacterial strains were identified by the analysis of 16S rDNA sequences. The bacterium IAC-AT-8 was identified as Azospirillum brasiliense, whereas isolates IAC-HT-11 and IAC-HT-12 were identified as Achromobacter insolitus and Zoogloea ramigera, respectively. A greenhouse experiment involving a non-sterilized soil was carried out with the aim to study the endophytic feature of these strains. After 40 days from inoculation, all the strains were in the inner of roots, but they were not detected in soil. In order to assess the location inside wheat plants, an experiment was conducted under axenic conditions. Fifteen days after inoculation, preparations of inoculated plants were observed by the scanning electron microscope, using the cryofracture technique, and by the transmission electron microscope. It was observed that all isolates were present on the external part of the roots and in the inner part at the elongation region, in cortex cells, but not in the endodermis or in the vascular bundle region. No colonizing bacterial cells were observed in wheat leaves.     

Karyotypic variation in <Emphasis Type="Italic">Nicotiana</Emphasis> section Suaveolentes

Nicotiana section Suaveolentes (Solanaceae) currently includes 28 species and subspecies that are endemic to Australasia and the South Pacific and one African species, N. africana. The section is monophyletic and of allotetraploid origin, but relationships among the species in it and its diploid progenitors are poorly understood. Here we report chromosome numbers for 20 of the 29 taxa from the Suaveolentes, including a count for one recently proposed species for which no number has previously been available. Many of the published chromosome numbers for the Suaveolentes are confirmed in this study. However, six counts were different from the published numbers including n = 15 for N. maritima and N. suaveolens, which is a new chromosome number for the genus. Nicotiana goodspeedii and N. rotundifolia were n = 16, and the same number was found in the suggested species N. sp. ‘Corunna’. Nicotiana suaveolens contains polyploid races of n = 32 and here we report the probable existence of an n = 31 race as well. Karyotypic variation within species and within the section is apparently much greater than previously thought and further investigation is warranted.     

Salt tolerance in <Emphasis Type="Italic">Zea mays</Emphasis> (L). following inoculation with <Emphasis Type="Italic">Rhizobium</Emphasis> and <Emphasis Type="Italic">Pseudomonas</Emphasis>

This study aimed to investigate the effect of inoculation with plant growth-promoting Rhizobium and Pseudomonas species on NaCl-affected maize. Two cultivars of maize (cv. Agaiti 2002 and cv. Av 4001) selected on the basis of their yield potential were grown in pots outdoors under natural conditions during July. Microorganisms were applied at seedling stage and salt stress was induced 21 days after sowing and maintained up to 50% flowering after 120 days of stress. The salt treatment caused a detrimental effect on growth and development of plants. Co-inoculation resulted in some positive adaptative responses of maize plants under salinity. The salt tolerance from inoculation was generally mediated by decreases in electrolyte leakage and in osmotic potential, an increase in osmoregulant (proline) production, maintenance of relative water content of leaves, and selective uptake of K ions. Generally, the microbial strain acted synergistically. However, under unstressed conditions, Rhizobium was more effective than Pseudomonas but under salt stress the favorable effect was observed even if some exceptions were also observed. The maize cv. Agaiti 2002 appeared to be more responsive to inoculation and was relatively less tolerant to salt compared to that of cv. Av 4001.     

Effects of long-term mineral fertilization on microbial biomass,microbial activity,and the presence of <Emphasis Type="Italic">r</Emphasis>- and <Emphasis Type="Italic">K</Emphasis>-strategists in soil

Long-term effects of mineral fertilization on microbial biomass C (MBC), basal respiration (R _B), substrate-induced respiration (R _S), β-glucosidase activity, and the r–K-growth strategy of soil microflora were investigated using a field trial on grassland established in 1969. The experimental plots were fertilized at three rates of mineral N (0, 80, and 160 kg ha⁻¹ year⁻¹) with 32 kg P ha⁻¹ year⁻¹ and 100 kg K ha⁻¹ year⁻¹. No fertilizer was applied on the control plots (C). The application of a mineral fertilizer led to lower values of the MBC and R _B, probably as a result of fast mineralization of available substrate after an input of the mineral fertilizer. The application of mineral N decreased the content of C extracted by 0.5 M K₂SO₄ (C _ex). A positive correlation was found between pH and the proportion of active microflora (R _S/MBC). The specific growth rate (μ) of soil heterotrophs was higher in the fertilized than in unfertilized soils, suggesting the stimulation of r-strategists, probably as the result of the presence of available P and rhizodepositions. The cessation of fertilization with 320 kg N ha⁻¹ year⁻¹ (NF) in 1989 also stimulated r-strategists compared to C soil, probably as the result of the higher content of available P in the NF soil than in the C soil.     

Genetic Characterization of Brazilian Annual <Emphasis Type="Italic">Arachis</Emphasis> Species from Sections <Emphasis Type="Italic">Arachis</Emphasis> and <Emphasis Type="Italic">Heteranthae</Emphasis> using RAPD Markers

The genus Arachis is divided into nine taxonomic sections. Section Arachis is composed of annual and perennial species, while section Heteranthae has only annual species. The objective of this study was to investigate the genetic relationships among 15 Brazilian annual accessions from Arachis and Heteranthae using RAPD markers. Twenty-seven primers were tested, of which nine produced unique fingerprintings for all the accessions studied. A total of 88 polymorphic fragments were scored and the number of fragments per primer varied from 6 to 17 with a mean of 9.8. Two specific markers were identified for species with 2n = 18 chromosomes. The phenogram derived from the RAPD data corroborated the morphological classification. The bootstrap analysis divided the genotypes into two significant clusters. The first cluster contained all the section Arachis species, and the accessions within it were grouped based upon the presence or absence of the ‘A’ pair and the number of chromosomes. The second cluster grouped all accessions belonging to section Heteranthae.     

Nitrification and denitrification activities of zinc-treated soils worked by the earthworm<Emphasis Type="Italic"> Pheretima</Emphasis> sp.

Little information is available on the roles of earthworms in N transformation and biological activities in heavy metal-contaminated soils. The purpose of the present paper is to examine the effects of earthworm on nitrifying and denitrifying enzyme activities of Zn-treated soils. ZnCl₂ solution was added to soils at rates ranging from 0 to 400 mg Zn kg^–1 and was allowed to fractionate and stabilize for 60 days. The Zn-tolerant earthworm Pheretima sp., which had been previously screened, was then introduced into Zn-treated soils and allowed to work for 40 days. Original soils without the addition of Zn and introduction of earthworms but incubated under the same conditions were used as controls. Zn-treated soils without the introduction of earthworms were also used as controls. Results showed that earthworm activity increased both nitrifying and denitrifying enzyme activities of the soils. These beneficial effects of earthworms were particularly evident in the treatments where Zn had an adverse effect. Zn added at a rate 200 mg Zn kg^–1 generally had a stimulating effect on the enzyme activities measured. However, Zn added at a rate >200 mg Zn kg^–1 had an adverse effect on nitrifying and denitrifying enzyme activities, which could be alleviated or even completely eliminated by earthworm activities.     

Characterization of fertile amphidiploid between <Emphasis Type="Italic">Raphanus sativus</Emphasis> and <Emphasis Type="Italic">Brassica alboglabra</Emphasis> and the crossability with <Emphasis Type="Italic">Brassica</Emphasis> species

A fertile amphidiploid × Brassicoraphanus (RRCC, 2n = 36) between Raphanus sativus cv. HQ-04 (2n = 18, RR) and Brassica alboglabra Bailey (2n = 18, CC) was synthesized and successive selections for seed fertility were made from F₄ to F₁₀. F₁₀ plants exhibited good fertility with 14.9 seeds per siliqua and 32.3 g seeds per plant. Cytological observation revealed that frequent secondary pairing occurred among 3 chromosome pairs in pollen mother cells of plants (F₄) with lower fertility, but not of plants with high fertility (F₁₀). GISH analysis indicated that these F₁₀ plants included the expected 18 chromosomes from R. sativus and B. alboglabra, respectively, but they lost approximately 27.6% R. sativus and 35.6% B. alboglabra AFLP (amplified fragment length polymorphism) bands. The crossability of the Raphanobrassica with R. sativus and 5 Brassica species (13 cultivars) were investigated. Seeds or F₁ seedlings were easy to be produced from crosses × Brassicoraphanus × R. sativus, and B. napus, B. juncea and B. carinata × Brassicoraphanus. Fewer seeds or seedlings were obtained from crosses × Brassicoraphanus × B. napus, B. juncea and B. carinata. However, few seeds were harvested in the reciprocals of × Brassicoraphanus with B. rapa and B. oleracea. The possible cause of fertility improvements and the potential of the present × Brassicoraphanus for breeding were discussed.     

Genetic and bioclimatic variation in <Emphasis Type="Italic">Solanum pimpinellifolium</Emphasis>

Solanum pimpinellifolium, due to its close relationship to S. lycopersicum, has been a genetic source for many commercially important tomato traits. It is a wild species found in the coastal areas of Peru and Ecuador. In this study, the genetic variation of S. pimpinellifolium was studied using the diversity found in 10 microsatellites in 248 plants spread throughout its entire distribution area, including Ecuador, which has been underrepresented in previous studies. Peruvian and Ecuadorian accessions are genetically quite differentiated. A possible cause of these differences could be the non-uniform nature of the coastal Ecuadorian and Peruvian climates, seeing as an important correlation between genetic differentiation and climate has been found. In addition, Ecuadorian and south Peruvian accessions have a lower genetic diversity and a higher homozygosity due to their higher autogamy, lower population size, and possible colonization bottlenecks. The Galápagos Islands population is an extreme case, with no diversity, likely caused by a recent colonization from the northern continental Ecuadorian region where genetically identical plants have been found. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Elena Zuriaga and José M. Blanca contributed equally to this work.     

Phylogenetic relationships between <Emphasis Type="Italic">Ensete</Emphasis> and <Emphasis Type="Italic">Musa</Emphasis> species as revealed by the <Emphasis Type="Italic">trnT trnF</Emphasis> region of cpDNA

Complete sequences of transcribed spacers and introns from the trnT trnF region of chloroplast DNA (cp DNA) were generated from Musaceae species to establish the phylogenetic relationships among 3 species of Ensete including the economically important Ensete ventricosum (Welw.) Cheesman and 13 species of Musa. Parsimony analysis and pair wise distance data produced a single tree, with Ensete and Musa as clearly distinguished clades. Six Musa and three Ensete clades were generated. The topology of these clades did not change when the data were split into spacers and introns, although the split resulted in poor bootstrap support. Removing a hotspot from the entire data set improved clade support. The clades produced are discussed with reference to existing taxonomic and phylogenetic treatments. In contrast to previous suggestions, most of the Rhodochlamys species that we investigated clustered together with strong support establishing their distinctiveness from the Musa species studied. Ensete glaucum (Roxb.) Cheesman and Musa beccartii Simmonds appear to represent ancestral forms of Ensete and Musa, respectively for the presently studied species, and both genera have a common ancestor that is yet to be established. Our data also show that E. ventricosum cannot be reduced to E. glaucum, nor can E. gilleti (De Wild.) Cheesman be reduced to E. ventricosum, as some authorities have suggested. Ensete gilleti or a species very close to it appears to be the ancestral species of E. ventricosum.