Local Adaptation of Frankia to Different Discaria (Rhamnaceae) Host Species Growing in Patagonia

Frankia BCU110601 (Da) and Frankia BCU110345 (Dc) were isolated from root nodules of Discaria articulata and Discaria chacaye, respectively; Frankia BCU110501 (Dt) was previously isolated from Discaria trinervis. The strains were identical at the 16S sequence and after analysis of RFLP of 16S and 23S rDNA intergenic region. Diversity was revealed at the molecular level after fingerprint analysis by BOX–polymerase chain reaction. The strains were infective and effective on the original host plants. A cross-inoculation assay intra Discaria genus, including D. trinervis, D. articulata, and D. chacaye, with each of these isolated Frankia strains caused effective symbioses with a similar dry weight in each plant species regardless of the inoculated strain. Nevertheless, a differential degree of recognition was revealed: Homologous symbiotic pairs in the case of D. chacaye–Frankia BCU110345 (Dc), D. articulata–Frankia BCU110601 (Da), and D. trinervis–Frankia BCU110501 (Dt) had faster nodulation rates than heterologous pairs. The differences in nodulation rate would suggest the existence of a subspecific level of recognition within a certain cross-inoculation group, pointing to subspecific adaptation occurring in this actinorhizal symbiosis.     

Variation of 15N natural abundance in leaves and nodules of actinorhizal shrubs in Northwest Patagonia

This study was performed to assess the N₂-fixing capability of the native actinorhizal species Ochetophila trinervis (sin. Discaria trinervis) and Discaria chacaye (Rhamnaceae) in Northwest Patagonia. We measured the N concentration and ¹⁵N natural abundance in leaves and nodules of O. trinervis and D. chacaye, in leaves of associated non-actinorhizal vegetation, and in the soils under each sampled plant. O. trinervis and D. chacaye had foliar N concentrations that were about twice that of non-actinorhizal shrubs growing at the same sites, even though soils varied four-fold in total N across the sites. Leaves of both actinorhizal plants had a similar δ¹⁵N at any site and were close to atmospheric values. The foliar δ¹⁵N of non-actinorhizal plants and soil δ¹⁵N were strongly correlated across the sites. Nodules were depleted in δ¹⁵N relative to the foliage of the respective actinorhizal species. In conjunction with the uniformly high foliage N concentration of these actinorhizal plants and the universal presence of vesicles observed in root nodules, these data strongly suggest that O. trinervis and D. chacaye obtain a significant amount of their N from N₂ fixation. To calculate the proportion of N derived from atmosphere, theoretical B-values were estimated. In all cases where the δ¹⁵N of fixing and reference foliage were significantly different, O. trinervis and D. chacaye obtained almost all of their N from N₂ fixation. These results are the first to demonstrate N₂ fixation by O. trinervis and D. chacaye in the field and therefore suggest an important role for these actinorhizal plants in the N economy of ecosystems in northwest Patagonia as well as their potential use for restoration of degraded lands in this region.     

Effective nodulation of rhamnaceous actinorhizal plants induced by air dry soils

Air dry soil samples stored at room temperature for more than one and a half years, were used as inocula for actinorhizal plants. Seedlings of Colletia paradoxa, Discaria americana, D. articulata and D. trinervis (Rhamnaceae) cultivated in nitrogen-free nutrient solution, were inoculated by adding dry soil to the solution. All the soil samples tested were able to induce nodulation, showing the presence of infective propagules of Frankia. Healthy growth of nodulated plants suggested the occurrence of nitrogen fixation.     

Effects of calcium in the nitrogen-fixing symbiosis between actinorhizal <Emphasis Type="Italic">Discaria trinervis</Emphasis> (Rhamnaceae) and <Emphasis Type="Italic">Frankia</Emphasis>

We have analysed the growth and symbiotic performance of actinorhizal Discaria trinervis at various Ca supply regimes. We aimed at discriminating between specific, if any, effects on nodulation and general growth stimulation by Ca. The hypothesis that a high Ca supply would interfere with nodulation by Frankia was also tested. Results showed that plant growth increased with Ca supply. Nodulation was stimulated by moderate levels of Ca, but inhibited by Ca higher than 0.77 mM. Growth of nodules was less affected by Ca than shoot and root growth. Ca concentration of symbiotic plants increased with Ca supply, but nitrogen concentration was independent of Ca at concentrations which did not impair plant growth. All together, these results show that Ca has a positive effect on the establishment and functioning of the symbiosis between Discaria trinervis and Frankia. However, the positive influence of Ca was more likely due to a promotion of plant growth rather than a direct effect on nodule growth and nitrogen fixation itself. At high levels of Ca supply nodulation was impaired. Given the intercellular infection pathway in Discaria trinervis, we suggest that the increment of Ca availability would strengthen its root cell walls, thus decreasing Frankia penetration of the root.     

Genetic relationships of Nuphar in central to western Japan as revealed by allozyme analysis

The genetic relationships of Nuphar japonica, N. oguraensis, N. subintegerrima and two morphologically intermediate plant groups (J-O and J-S) found in central to western Japan were investigated. Allozyme analyses (analysis of allele frequencies and principal coordinate analysis, based on shared allele distance among the 162 multi-locus genotypes) showed the three Nuphar species to be distinguished, and indicated that the intermediate plant groups were genetically intermediate either between two or among three of these species. Natural hybridization and introgression were found to have occurred among these three species in central to western Japan, producing genetically varied intermediate plant groups. Many of the intermediate plants (74.4% of the total numbers of genotypes of the intermediate plants) did not show additive combinations of parental allozymes, which were expected in F₁ hybrids. The absence of characteristic parental bands suggested that sexual reproduction had continued among the hybrids beyond the F₁.     

Diversity of Frankia Strains in Root Nodules of Plants from the Families Elaeagnaceae and Rhamnaceae

Partial 16S ribosomal DNAs (rDNAs) were PCR amplified and sequenced from Frankia strains living in root nodules of plants belonging to the families Elaeagnaceae and Rhamnaceae, including Colletia hystrix, Elaeagnus angustifolia, an unidentified Elaeagnus sp., Talguenea quinquenervia, and Trevoa trinervis. Nearly full-length 16S rDNAs were sequenced from strains of Frankia living in nodules of Ceanothus americanus, C. hystrix, Coriaria arborea, and Trevoa trinervis. Partial sequences also were obtained from Frankia strains isolated and cultured from the nodules of C. hystrix, Discaria serratifolia, D. trinervis, Retanilla ephedra, T. quinquenervia, and T. trinervis (Rhamnaceae). Comparison of these sequences and other published sequences of Frankia 16S rDNA reveals that the microsymbionts and isolated strains from the two plant families form a distinct phylogenetic clade, except for those from C. americanus. All sequences in the clade have a common 2-base deletion compared with other Frankia strains. Sequences from C. americanus nodules lack the deletion and cluster with Frankia strains infecting plants of the family Rosaceae. Published plant phylogenies (based on chloroplast rbcL sequences) group the members of the families Elaeagnaceae and Rhamnaceae together in the same clade. Thus, with the exception of C. americanus, actinorhizal plants of these families and their Frankia microsymbionts share a common symbiotic origin.     

Nodule distribution on the roots of actinorhizal Discaria trinervis (Rhamnaceae) growing in pots

Roots of actinorhizal plants can develop nitrogen-fixing nodules with actinomycetic bacteria of the genus Frankia. We aimed to know if unrestricted growth of roots in pots could influence the pattern of nodule development that we had previously observed for Discaria trinervis growing in pouches. Growth pouches, although being a space saving device convenient for the analysis of nodule development, do restrict root growth. Thus, the pattern of root nodule development was analysed in actinorhizal D. trinervis growing in pots with inert substrates. Inoculation of axenic seedlings growing in perlite resulted in clustering of nodules in a defined region of the taproot and upper lateral roots. When surface sterilized seeds were sown in pots containing vermiculite that had been previously inoculated with Frankia cells, nodules were again concentrated in defined portions of the main and lateral roots. Potted plants developed comparable numbers of nodules with respect to plants grown in pouches. However, a significant proportion of nodules appeared in lateral roots. As it was first inferred from field grown plants, these results confirm that D. trinervis plants growing in pots display the same autoregulatory mechanism for nodule formation that was previously observed in growth pouches.     

Natural Hybridization between Rhododendron delavayi and R. cyanocarpum (Ericaceae), from Morphological, Molecular and Reproductive Evidence

The natural hybridization that occurs between two sympatric species of Rhododendron subgenus Hymenanthes in Yunnan, China, was investigated. In field observations, it was noted that the putative hybrids between R. delavayi Franch. and R. cyanocarpum (Franch.) Franch. ex W.W. Sm. had intermediate morphologies. On the basis of morphology, chloroplast DNA (trnL-rpl32) and nuclear DNA (waxy), hybrids and parental species were identified. Hybridization occurred in both directions, but was asymmetrical, with R. delavayi as the major maternal parent. Reciprocal hand pollination treatments showed that either species, as pollen donor or pollen receiver, could produce fruits. It was noted that fruit set varied among treatments. The same pollinators (bumblebees) were shared in both parental species. From these results, we conclude that individuals with intermediate morphologies are indeed of hybrid origin from natural hybridization between R. cyanocarpum and R. delavayi. Furthermore, we presume the hybridization at the study site could have been initiated by habitat disturbance in the 1950s, and we may hence witness the early stages of hybrid swarm formation.     

Studies of two Frankia strains isolated from Trevoa trinervis Miers

The Frankia strains TtI 11 and TtI 12 isolated from T. trinervis Miers were characterized regarding their carbon source utilization, intrinsic antibiotic resistance, infectivity, and effectivity on the original host. Both strains grew on BAP medium supplemented with glucose, maltose, and sucrose, but differed in their ability to use other carbon sources such as propionate, pyruvate, acetate, succinate, citrate, and mannitol. The isolates were sensitive to five of the twelve antibiotics tested at 1 μg mL⁻¹ concentration: chloramphenicol, tobramycin, eritromycin, streptomycin, and rifampicin. They exhibited a variable degree of resistance at 1 μg mL⁻¹ concentraction to penicillin G, 4-fluorouracil, oleandomycin, and lincomycin. Both isolates were able to infect and nodulate the original host plant, and thus represent the first reported infective and effective microsymbionts for T. trinervis Miers, a rhamnaceous actinorhizal host. R O D Dixon Section editor     

Discordant patterns of morphological and genetic divergence in the closely related species Schizanthus hookeri and S.?grahamii (Solanaceae)

In this study I have examined the patterns of morphological and genetic differentiation between two species of the Andean genus Schizanthus that differ in their pollination and mating systems. Schizanthus hookeri has a bee pollination syndrome and is strongly dependent on pollinators for seed set. In contrast, S.?grahamii has a hummingbird pollination syndrome and exhibits late autonomous selfing. Southern populations of the latter species have red flowers (reddish morph), while northern populations have yellow (yellowish morph) or pink flowers (pinkish morph). I used two noncoding chloroplast DNA (cpDNA) regions to investigate the genetic affinities between S.?hookeri and the three morphs of S.?grahamii. I also performed intra- and interspecific crosses to assess whether gene flow between species was possible. Phylogenetic analyses supported the existence of two differentiated clades that did not match currently accepted taxonomic classification. Accordingly, genetic distance did not correlate significantly with morphological distance. No fruits were produced from interspecific crosses, and there were no individuals with intermediate morphology that could indicate current and frequent hybridization events between species.?I propose that the discordance between cpDNA data and conventional taxonomy could be explained by parallel evolution, or alternatively, by a very sporadic hybridization.     

Vertebrate faeces as sources of nodulating Frankia in Patagonia

Frankia strains nodulate the native actinorhizal plant Ochetophila trinervis (sin. Discaria trinervis), which grows in stream margins and nearby areas in northwest Patagonia (Argentina). Infective Frankia are found in soils with presence of host plants but also may be found in areas lacking them. This may be partly explained by water transport of Frankia propagules but there are other possible sources. The aim of this study was to discover whether the faeces of introduced mammalian herbivores, including cows (Bos taurus, adult and calf), horses (Equus caballus), sheep (Ovis aries), red and/or fallow deer (Cervus elaphus and Dama dama, respectively), wild boar (Sus scrofa), European hare (Lepus capensis), or the native upland goose (Chloephaga picta), could be a source of infective Frankia, and enhance its dispersal. Faecal material and soil samples were aseptically sampled in different plant communities, and tested via plant bioassays using O. trinervis. The faeces of all animals contained infective Frankia and led to an effective symbiosis with this plant. Faeces of large introduced herbivores gave rise to higher nodulation (number of nodulated plants with respect to the total number of inoculated plants) than faeces of hare and upland goose. Soils from the sites where the cow (two sites), sheep, wild boar and deer faeces were collected did not contain infective Frankia. This suggests that the animals may have ingested Frankia from plant material and that the Frankia propagules passed through the digestive tracts of the animals without losing its infectivity. We conclude that the faeces of large introduced herbivores contribute to the dispersal of infective Frankia in Northwest Patagonia.     

Two new genomes in the Oryza complex identified on the basis of molecular divergence analysis using total genomic DNA hybridization

The genus Oryza to which cultivated rice belongs has 24 species (2n?=?24 or 48), representing seven genomes (AA, BB, CC, EE, FF, BBCC and CCDD). The genomic constitution of five of these species is unknown. These five species have been grouped into two species complexes, the tetraploid ridleyi complex (O. ridleyi, O.?longiglumis) and the diploid meyeriana complex (O.?granulata, O. meyeriana, O. indandamanica). To evaluate the genomic structure of these species in terms of divergence at the molecular level vis-à-vis other known genomes of Oryza, we used the total genomic DNA hybridization approach. Total genomic DNA (after restriction digestion) of 79 accessions of 23 Oryza species, 6 related genera, 5 outgroup taxa (2 monocots, 3 dicots) and 6 F₁s and BC₁s derived from crosses of O.?sativa with wild species were hybridized individually with ³²P-labeled total genomic DNA from 12 Oryza species: O. ridleyi, O.?longiglumis, O. granulata, O.?meyeriana, O. brachyantha, O. punctata, O. officinalis, O. eichingeri, O. alta, O. latifolia, O. australiensis, and O.?sativa. The labeled genomic DNAs representing the ridleyi and meyeriana complexes cross-hybridized best to all the accessions of their respective species, less to those representing other genomes of Oryza and related genera, and least to outgroup taxa. In general, the hybridization differential measured in terms of signal intensities was >50-fold under conditions that permit detection of 70–75% homologous sequences, both in the presence and in the absence of O. sativa DNA as competitor. In contrast, when total DNAs representing other Oryza genomes were used as probes, species of the O.?ridleyi and O.?meyeriana complexes did not show any significant cross-hybridization (<5%). These results demonstrate that the genome(s) of both of these complexes are highly diverged and distinct from all other known genomes of Oryza. We, therefore, propose new genomic designations for these two species complexes: GG for the diploid O. meyeriana complex and HHJJ for the allotetraploid O. ridleyi complex. The results also suggest that the uniqueness of these genomes is not restricted to species-specific highly repetitive DNA sequences, but also applies to dispersed sequences present in single or low to moderate copy numbers. Furthermore these appear to share relatively more genome-specific repeat sequences between themselves than with other genomes of rice. The study also demonstrates the potential of total genomic DNA hybridization as a simple but powerful tool, complementary to existing approaches, for ascertaining the genomic makeup of an organism.     

Morphological variability in rupicolous species of the Acianthera prolifera complex (Orchidaceae) occurring in southeastern Brazil

We carried out multivariate morphometric analysis of 23 floral characters in seven populations of a complex of four species of Acianthera (Orchidaceae) occurring in Brazilian campo rupestre (rocky field) vegetation (A.?hamosa, A.?limae, A.?modestissima, and A.?prolifera) that flower synchronously and are partially intercompatible, and one putative hybrid population between A.?limae and A.?prolifera. We also carried out cluster analysis involving these eight populations plus 21 populations of a previously published study belonging to another species complex of Acianthera occurring in campo rupestre, including 12 floral characters in the analysis. Allopatric species pollinated by the same group of Diptera showed higher floral similarity among themselves than to a sympatric species pollinated by another group of Diptera. Such patterns indicate the existence of floral convergence in allopatric species and/or radiation in sympatric species. The analysis also indicated that there is more floral similarity between species of different complexes but that share the same group of pollinators. Large overlap was observed between A.?limae and the putative sympatric hybrids, indicating the occurrence of later generations of hybrids and/or individuals of A.?limae with introgression. The results do not support A.?hamosa and A.?modestissima as distinct species. These taxa are geographically isolated, occurring in different environments, are recognized only by vegetative characters that show high phenotypic plasticity, and share the same pollinators, being interfertile.     

Confirmation of hybridization among sympatric insular populations of Orchis mascula and O. provincialis

The localized hybridization between Orchis mascula and O. provincialis in Sardinia is investigated. The two parental species are closely related and share the same pollinators but do not hybridize in any other areas of sympatry. Artificial crosses between the two species in areas of sympatry on the continent revealed that, in absence of an ethological isolation, a strong post-pollination barrier exists and preserves species integrity in the main part of their distribution. However, on Sardinia, morphological observations, karyological and molecular analyses of intermediate phenotypes confirmed the occurrence of hybridization between the insular O. mascula ssp. ichnusae and O. provincialis and the bi-directionality of pollen flow. The occurrence of hybrids on the island indicates a failure of otherwise strong post-pollination barriers between O. mascula and O. provincialis.     

Inter-specific hybridization underlies phenotypic variability in Daphnia populations

Summary In the glacial lakes of the Palaearctic three species of Cladocera commonly coexist: Daphnia hyalina, D. galeata, and D. cucullata. Frequently these populations contain not only animals which are morphologically typical for the species but also individuals of an intermediate phenotype. Electrophoretic investigations of allozyme-patterns in morphologically typical individuals reveal that each species is fixed for a different allele at the GOT locus. Morphologically intermediate animals are heterozygous for the alleles of the two species which they resemble. The allelic pattern at other loci is also consistent with the assumption that morphological intermediates are formed via interspecific hybridization. Very few backcrosses between galeata-hyalina hybrids and their parent species are found, and there is no indication of gene flow between D. cucullata and the other species.     

Presence of sporangia of Frankia in the rhizosphere of Discaria (Rhamnaceae)

Discaria trinervis and Discaria americana are native actinorhizal plants in Argentina. Discaria seedlings growing in N-free liquid culture, inoculated with dry soils, developed Frankia colonies in the rhizosphere. The occurrence of hyphae, vesicles and sporangia characteristic of Frankia are described in these colonies. The presence of sporangia of Frankia has previously neither been reported in the genus Discaria, nor in the other genera of the tribe Colletieae inside root nodules or outside roots. The infective capacity of the colonies has been demonstrated.     

Reproductive ecology of a perennial outcrosser with a naturally dissected distribution

We surveyed four populations of contrasting size (two isolated and two large stands) of a woody outcrossing species, Discaria trinervis (Rhamnaceae), which has a naturally dissected distribution. Our main goal was to investigate the floral biology, breeding system and pollination mechanism of the species, which in turn may help to explain the mechanisms underlying the survival of the isolated populations. Discaria trinervis is both wind- and insect-pollinated, showing interactions with a large fraction of the available flower visitors at both stand sizes. In the larger populations, stigmatic loads were larger, more pollen tubes reached the ovules, and fruit and seed set were higher; however, the reproductive output in the smaller populations (over 2000 seeds per plant per year) seemed adequate for population persistence. Human disturbance (e.g. destruction of trees for wood extraction) may put these smaller populations at risk through loss of incompatibility alleles. The results suggest that plant species with naturally dissected distributions may provide clues about which reproductive mechanisms ensure survival under fragmentation.     

Ammonium assimilation in root nodules of actinorhizal Discaria trinervis. Regulation of enzyme activities and protein levels by the availability of macronutrients (N,P and C)

Asparagine was found to be the main N compound exported from Discaria trinervis nodules. Aspartate (Asp), glutamate (Glu), alanine (Ala) and serine (Ser) were also detected in root xylem sap, but at lower concentrations. A comparable picture is found in nodulated alfalfa. We hypothesized that a similar set of enzymes for Asn synthesis was present in D. trinervis nodules. We demonstrate the expression of most of the enzymes involved in the synthesis of Asn from NH⁺ ₄ and oxoacids, in nodules – but not in roots – of fully symbiotic D. trinervis. By complementation of enzyme assays (^A) and immunodetection (^I) we detected glutamane-synthetase (GS^{A, I}), Asp-aminotransferase (AAT^A), malate-dehydrogenase (MDH^{A, I}, at least two isoforms), Glu-dehydrogenase (GDH^A), Glu-synthase (GOGAT^I) and Asn-synthetase (AS^I). PEP-carboxylase (PEPC) activity was not detected. We previously shown that N acts as a negative regulator of nodulation and nodule growth, while P is a strong stimulator for nodule growth. We present data on the regulation of nodule N metabolism by altering, during 4 weeks, the availability of N, P and light in symbiotic D. trinervis. NH₄NO₃ (2 mM) induced inactivation and degradation of nodule GS, MDH and AS, but activation of GDH and AAT; the amount of nitrogenase components was not affected. A 10-fold increase in P supply did not greatly affect activity and amount of enzymes, suggesting that N metabolism is not P-limited in nodules. On the other hand, suppression of P supply induced an important reduction of nodule GS, GOGAT, MDH and AS protein levels, although nitrogenase was not affected. GDH was the only measured activity that was stimulated by limiting P supply. Shading plants did result in complete degradation of nitrogenase and partial degradation of GS, AS and nodule-specific MDH isoform, but GDH and AAT were activated. These results are discussed in connection with the regulation of nodulation and nodule growth in D. trinervis.     

Floral scent in natural hybrids of Ipomopsis (Polemoniaceae) and their parental species

Background and Aims

Floral traits, such as floral volatiles, can contribute to pre-zygotic reproductive isolation by promoting species-specific pollinator foraging. When hybrid zones form, floral traits could also influence post-zygotic isolation. This study examined floral volatiles in parental species and natural hybrids in order to explore potential scent mediation of pre-zygotic and post-zygotic isolation.

Methods

Floral bouquets were analysed for the sister species Ipomopsis aggregata and I. tenuituba and their natural hybrids at two contact sites differing in both hybridization rate and temporal foraging pattern of hawkmoth pollinators. Floral volatiles were quantified in diurnal and nocturnal scent samples using gas chromatography–mass spectrometry.

Key Results

The bouquets of parental species and hybrids showed qualitative overlap. All flowers emitted similar sets of monoterpenoid, sesquiterpenoid, aliphatic and benzenoid compounds, but separated into groups defined by multivariate analysis of quantitative emissions. The parental species differed most strikingly in the nitrogenous compound indole, which was found almost exclusively in nocturnal bouquets of I. tenuituba. Natural hybrid bouquets were highly variable, and showed emission rates of several compounds that appeared transgressive. However, indole emission rates were intermediate in the hybrids compared with rates in the parents. Volatile bouquets at the contact site with lower hybridization did not show greater species specificity in overall scent emission, but I. tenuituba presented a stronger indole signal during peak hawkmoth activity at that site.

Conclusions

The two species of Ipomopsis differed in patterns of floral bouquets, with indole emitted in nocturnal I. tenuituba, but not in I. aggregata. Natural hybrid bouquets were not consistently intermediate between the parents, although hybrids were intermediate in indole emission. The indole signal could potentially serve as a hawkmoth attractant that mediates reproductive isolation both before and after hybrid formation.