Assessing the potential of poplar short rotation plantations to contribute to a low-carbon bioeconomy under water-limited conditions.

Poplar short rotation coppice (SRC) systems are important for biomass production and for short-to medium-term carbon (C) sequestration, contributing to a low-carbon bioeconomy and thus helping to mitigate global warming. The productivity and profitability of these plantations are, however, challenged under restrictive irrigation associated with climate change. This study compares the above- and below-ground C sequestration potential and economic viability of a 12-year plantation cycle (4 rotations of 3 years each) under Mediterranean conditions with optimum irrigation (T1) and 50% irrigation reduction (T2), analysing other promising biomass uses in the form of bioproducts. A total of 138 trees of the highly productive hybrid ('AF2') in a SRC-trial were sampled monthly (first rotation). Additionally, data from an extensive poplar plantation network (30 sites) was used to complete data for the plantation cycle. The average C content for above- and below-ground biomass was 17.04 Mg C ha-1 yr-1 (T1), falling by 24% in T2. The net present value (NPV) in T1 (6461 € ha-1) was 52% lower under T2 conditions. Extra payments for C sequestration increased the NPV to 8023 for T1 and 4331 € ha-1 for T2. Roots represent an important C storage in the soil, accumulating 29.9 Mg C ha-1 (T1) and 22.8 Mg C ha-1 (T2) by the end of the cycle in our study. The mitigation potential is strongly fortified when the share of bioproducts in biomass end-use increases. Assuming a distribution of 50% bioenergy and 50% bioproducts, emission were reduced between -114 Mg CO2eq ha-1 (T1) and -88 Mg CO2eq ha-1 (T2) compared to BAU until end of the century. This scenario plays a crucial sink-effect role by storing C contained in biomass, which is not immediately released into the atmosphere.

Responses of photosynthesis and component processes to drought and temperature stress: are Mediterranean trees fit for climate change?

Global warming is raising concerns about the acclimatory capacity of trees and forests, especially in Mediterranean-type ecosystems. The sensitivity of photosynthesis to temperature is a key uncertainty for projecting the magnitude of terrestrial feedbacks on future climate change. While boreal, temperate and tropical species have been comparatively well investigated, our study provides the first comprehensive overview of the seasonal acclimatory responses of photosynthesis and its component processes to temperature in four Mediterranean climax species under natural conditions. We quantified seasonal changes in the responses of net photosynthesis (Anet), stomatal conductance (gs), mesophyllic conductance (gm) and electron-transport rate (Jcf), and investigated their sensitivity to drought and temperature stress in sunlit and shaded leaves of four Mediterranean tree species (Quercus ilex L., Pinus halepensis Mill., Arbutus unedo L. and Quercus pubescens Willd.). Sunlit leaves, but not shaded leaves, showed a pronounced seasonality in the temperature responses of Anet, gs, gm and Jcf. All four species and variables showed a remarkably dynamic and consistent acclimation of the thermal optimum (Topt), reaching peaks in summer ~29-32 °C. Changes in the shape of the response curves were, however, highly species-specific. Under severe drought, Topt of all variables were on average 22-29% lower. This was accompanied by narrower response curves above all in P. halepensis, reducing the optimal range for photosynthesis to the cooler morning or evening periods. Wider temperature-response curves and less strict stomatal control under severe drought were accompanied by wilting and drought-induced leaf shedding in Q. ilex and Q. pubescens and by additional branch dieback in A. unedo. Mild winter conditions led to a high Topt (~19.1-22.2 °C), benefitting the evergreen species, especially P. halepensis. Seasonal acclimation of Anet was explained better by gs and gm being less pronounced in Jcf. Drought was thus a key factor, in addition to growth temperature, to explain seasonal acclimation of photosynthesis. Severe drought periods may exceed more frequently the high acclimatory capacity of Mediterranean trees to high ambient temperatures, which could lead to reduced growth, increased leaf shedding and, for some species such as A. unedo, increased mortality risk.

Balance between carbon gain and loss under long-term drought: impacts on foliar respiration and photosynthesis in Quercus ilex L.

Terrestrial carbon exchange is a key process of the global carbon cycle consisting of a delicate balance between photosynthetic carbon uptake and respiratory release. We have, however, a limited understanding how long-term decreases in precipitation induced by climate change affect the boundaries and mechanisms of photosynthesis and respiration. We examined the seasonality of photosynthetic and respiratory traits and evaluated the adaptive mechanism of the foliar carbon balance of Quercus ilex L. experiencing a long-term rainfall-exclusion experiment. Day respiration (Rd) but not night respiration (Rn) was generally higher in the drought treatment leading to an increased Rd/Rn ratio. The limitation of mesophyll conductance (gm) on photosynthesis was generally stronger than stomatal limitation (gs) in the drought treatment, reflected in a lower gm/gs ratio. The peak photosynthetic activity in the drought treatment occurred in an atypical favourable summer in parallel with lower Rd/Rn and higher gm/gs ratios. The plant carbon balance was thus strongly improved through: (i) higher photosynthetic rates induced by gm; and (ii) decreased carbon losses mediated by Rd. Interestingly, photosynthetic potentials (Vc,max, Jmax, and TPU) were not affected by the drought treatment, suggesting a dampening effect on the biochemical level in the long term. In summary, the trees experiencing a 14-year-long drought treatment adapted through higher plasticity in photosynthetic and respiratory traits, so that eventually the atypical favourable growth period was exploited more efficiently.

Seasonal variability of foliar photosynthetic and morphological traits and drought impacts in a Mediterranean mixed forest.

The Mediterranean region is a hot spot of climate change vulnerable to increased droughts and heat waves. Scaling carbon fluxes from leaf to landscape levels is particularly challenging under drought conditions. We aimed to improve the mechanistic understanding of the seasonal acclimation of photosynthesis and morphology in sunlit and shaded leaves of four Mediterranean trees (Quercus ilex L., Pinus halepensis Mill., Arbutus unedo L. and Quercus pubescens Willd.) under natural conditions. Vc,max and Jmax were not constant, and mesophyll conductance was not infinite, as assumed in most terrestrial biosphere models, but varied significantly between seasons, tree species and leaf position. Favourable conditions in winter led to photosynthetic recovery and growth in the evergreens. Under moderate drought, adjustments in the photo/biochemistry and stomatal/mesophyllic diffusion behaviour effectively protected the photosynthetic machineries. Severe drought, however, induced early leaf senescence mostly in A. unedo and Q. pubescens, and significantly increased leaf mass per area in Q. ilex and P. halepensis. Shaded leaves had lower photosynthetic potentials but cushioned negative effects during stress periods. Species-specificity, seasonal variations and leaf position are key factors to explain vegetation responses to abiotic stress and hold great potential to reduce uncertainties in terrestrial biosphere models especially under drought conditions.

The actin loci in the genus Drosophila: establishment of chromosomal homologies among five palearctic species of the Drosophila obscura group by in situ hybridization.

Chromosomal homologies among the four palearctic Drosophila obscura group species D. ambigua, D. tristis, D. obscura, and D. subsilvestris and the "trans-palearctic" species D. bifasciata were established by in situ hybridization using the 5C actin gene of D. melanogaster as a probe. In all species two labeling sites were detected in each of chromosomal elements C and E and one in each of chromosomal elements A and D. In addition one labeling site was detected on element B for the species D. subsilvestris and D. bifasciata. The conservative distribution pattern of the genes of the actin multigene family, the similarities of the locations of the actin genes in the chromosomes of the five species studied, together with the concordant evidence of synteny of visible and other genetic markers as well as the similarities in banding patterns, all agree with the conclusion that the chromosomal elements have retained their essential identity throughout the evolution of these species. Using in situ hybridization detailed information of some homologous regions of chromosomes can also be established.

A species specific satellite DNA family of Drosophila subsilvestris appearing predominantly in B chromosomes.

This paper describes a species specific satellite DNA family (pSsP216) of Drosophila subsilvestris, a palearctic species of the D. obscura group. The pSsP216 family consists of tandemly arranged 216 bp repetitive units that are predominantly localized on B chromosomes. These chromosomes appear in variable numbers in the karyotype of this species. Some pSsP216 repeats can also be detected in the centromeric heterochromatin of the acrocentric A chromosomes. Two strains, one with and the other without B chromosomes, were investigated for sequence variability and for the location of this satellite DNA on the chromosomes. Among 16 clones of the 216 bp basic repeat unit an overall similarity of about 93% and no strain specific differences were found, indicating that the B chromosomes may have derived from the A chromosomes (probably the dots) by spontaneous amplification of the pSsP216 satellite DNA family.

Gradual evolution of a specific satellite DNA family in Drosophila ambigua, D. tristis, and D. obscura.

The highly repetitive satellite DNA family "ATOC180" is specific for the three closely related species Drosophila obscura, D. ambigua, and D. tristis but does not occur in their closest relatives D. subsilvestris and D. bifasciata. Approximately 10,000 copies/haploid genome of approximately 180-bp repetition units are tandemly arranged in the centromeric heterochromatin of all chromosomes of all three species. Molecular analysis of 29 cloned repeats shows much intra- and interspecific sequence homogeneity. Single nucleotide changes are the main source of variability and distinguish the sequence-, subfamily- and species-specific ATOC180 repeats from each other. Based on these nucleotide differences, phylogenetic dendrograms were constructed and compared with published trees for other traits. The data indicate that the sequences of the ATOC180 satellite DNA family probably arose in a phylogenetically "short period" during the anagenetic evolution of the common ancestor of D. obscura, D. tristis, and D. ambigua, as a consequence of a process of genome reorganization, followed by a "long period" of entirely gradual sequence evolution. For the latter period, an evolutionary rate of 3 x 10(-8) substitutions/site/year was calculated.

Conservation and change of the developmentally crucial fushi tarazu gene in Drosophila.

We have studied the evolutionary changes occurring in the noncoding regions around the developmentally important fushi tarazu (ftz) gene in a total of 11 species in the genus Drosophila. Previous molecular developmental studies have identified DNA elements both 3' and 5' to the coding region which are important in proper regulation of expression of the Drosophila melanogaster ftz gene. We show here that these same elements are the most evolutionarily conserved regions in the vicinity of the gene homologs. Parts of some control elements are more conserved than exonic sequences. Not only is there sequence conservation, but the relative position, orientation, and distances among the control elements remain conserved. One quite significant difference does exist between the two major subgenera studied, Sophophora and Drosophila: namely, an inversion of the ftz unit with respect to other genes in the Antennapedia complex, ANT-C. As a comparison, we applied similar analysis to a "housekeeping" gene-rosy (ry), or Xdh. In contrast, DNA sequences 5' to the ry coding region revealed little evolutionary conservation. These studies bear out the proposition that functionally important DNA sequences remain more conserved through evolutionary time than do less functionally important sequences. This proposition could be tested in the present case because we could predict a priori from the developmental studies which DNA regions should be most conserved.

Stable heteroplasmy for a large-scale deletion in the coding region of Drosophila subobscura mitochondrial DNA.

Due to the extremely economic organization of the animal mitochondrial genome, large-scale deletions are rarely found in animal mtDNA. We report the occurrence of a massive deletion in the coding region of mtDNA in Drosophila subobscura. Restriction mapping and nucleotide sequence analysis revealed that the deletion encompasses six protein genes and four tRNAs. All individuals of an isofemale strain proved to be heteroplasmic for normal and deficient mtDNA molecules. This type of heteroplasmy resembles one observed in patients with mitochondrial myopathies but differs in that the fitness of heteroplasmic flies is not significantly reduced even though the mutant mtDNA constitutes 50-80% of total mtDNA in most of the individuals studied. The heteroplasmic strain is genetically stable: despite extensive screening not a single homoplasmic fly was observed since the foundation of the line.

Cloning and characterization of KM190, a specific satellite DNA family of Drosophila kitumensis and D. microlabis.

The nucleotide sequences of nine clones, pKA191/1-4 from Drosophila kitumensis and pMR190/1-5 from D. microlabis, were determined. They represent a tandemly arranged and highly repetitive satellite DNA family, KM190, which is specific for the two species.

Nuclear and mitochondrial ribosomal RNA variability in the obscura group of Drosophila.

Parts of 28S (nuclear) and 12S (mitochondrial) ribosomal RNA of Palearctic, Nearctic and African species of the obscura group have been sequenced by the direct method of sequencing. Rates of nucleotide substitutions in both molecules were compared. The nucleotide divergence is higher in the mitochondrial rRNA. Average distances of species taken in pairwise were compared to results obtained with the melanogaster subgroup: the divergence of nuclear rRNA appears lower, that of the mtDNA higher whereas genetic distances (allozymes) and sncDNA distances are similar. Noticeable variability of evolutionary rates can be observed even in low taxonomical levels. Phylogenetic trees for the obscura group are in general agreement with those obtained with other characters.

Evolution of the Drosophila melanogaster-sigma virus system in a natural population from Tübingen.

In natural populations of D. melanogaster, usually, a minority of individuals are infected by a Rhabdovirus called sigma. This virus is not contagious but is vertically transmitted through the gametes. In D. melanogaster, a polymorphism for two alleles (O, permissive and P, restrictive) of a gene responsible for resistance to the virus is regularly observed in the wild. On the virus side two types are found, which differ in their sensitivity to the P allele: Type I is very sensitive, and Type II more resistant. Previous findings had led to the hypothesis that an invasion of Type II clones, starting from central France, might be spreading over European populations. This replacement of viral Type I by viral Type II in natural populations could be observed in Languedoc (southern France), where it led to a dramatic increase in the frequency of infected flies. The invasion hypothesis is confirmed by the data from samples collected at Tübingen, where the frequency of Type II clones increased from 0.27 to 0.93 over a 6-year period (1985-1991). However, over the same period, no increase in the frequency of infected flies was observed. The evolution of other viral characteristics is discussed.

Mitochondrial DNA evolution in the obscura species subgroup of Drosophila.

Mitochondrial DNA (mtDNA) restriction site maps for nine species of the Drosophila obscura subgroup and for Drosophila melanogaster were established. Taking into account all restriction enzymes (12) and strains (45) analyzed, a total of 105 different sites were detected, which corresponds to a sample of 3.49% of the mtDNA genome. Based on nucleotide divergences, two phylogenetic trees were constructed assuming either constant or variable rates of evolution. Both methods led to the same relationships. Five differentiated clusters were found for the obscura subgroup species, one Nearctic, represented by Drosophila pseudoobscura, and four Palearctic, two grouping the related triads of species Drosophila subobscura, Drosophila madeirensis, Drosophila obscura, Drosophila subsilvestris, and two more represented by one species each, Drosophila bifasciata, and Drosophila tristis. The different Palearctic clusters are as distant between themselves as with the Nearctic one. For the related species D. subobscura, D. madeirensis, and D. guanche, the pair D. subobscura-D. madeirensis is the closest one. The relationships found by nucleotide divergence were confirmed by differences in mitochondrial genome size, with related species sharing similar genome lengths and differing from the distant ones. The total mtDNA size range for the obscura subgroup species was from 15.5 kb for D. pseudoobscura to 17.1 for D. tristis.

Mitochondrial DNA variation and genetic structure in Old-World populations of Drosophila subobscura.

To discover the relation between mitochondrial DNA (mtDNA) polymorphism and the geographic population structure of Drosophila subobscura previously established for other genetic traits, a wide Paleartic survey was carried out. A total of 24 nucleomorphs was observed among 261 isofemale lines assayed by 11 restriction endonucleases with 38 different sites in the mtDNA cleavage map. The differentiation of the Canary Islands populations (delta = 0.0119) compared with the mean among all the other continental and insular populations (delta = 0.0002) is striking. Both the great divergence among Canary Islands nucleomorphs (delta = 0.021) compared with the maximum nucleomorph distance in all other populations (delta = 0.017) and the abundance of endemic nucleomorphs (11) on the Canary Islands (50% of the total number of different nucleomorphs found in the entire distribution area) suggest that this molecular differentiation most probably results from the very old age of the Canary Islands populations rather than from drift and founder effects.

Evolution of a telomere associated satellite DNA sequence in the genome of Drosophila tristis and related species.

A highly repetitive satellite DNA sequence from the genome of Drosophila tristis with a length of 181 bp has been cloned in the pUC plasmid. The sequence hybridizes to the telomeres of all chromosomes but the Y of D. tristis and produces a ladderlike hybridization pattern with filterbound genomic DNA of D. tristis digested with Eco RI or Pst I with the hybridization bands at fragment lengths in multiples of 181 bp. A similar pattern is found when the genomic DNA comes from D. ambigua or, though less clear, from D. microlabis. Additional bands appear in the zones of high fragment lengths, too. In D. obscura and D. kitumensis, however, the 181 bp sequence is found in fragments with a length of a few kb only. The 181 bp sequence is tandemly arranged in the genome of D. tristis and has a copy number of about 82,000 per haploid genome (i.e. 10 per cent of the total DNA). A sequence comparison among four independently cloned copies of the family from D. tristis and another homologous sequence from D. obscura, found by chance, shows a one to six per cent variation in basepair composition. However, low divergence (only one per cent) between two copies of D. tristis and between the one of D. obscura and one of D. tristis was observed, and high divergence (six per cent) between these two pairs. This is discussed and explained as the evolutionary consequence of an existing homogenization process by unequal crossing over.

Cytological localization and organization of dispersed middle repetitive DNA sequences of Drosophila subobscura.

To study the middle repetitive fraction of the Drosophila subobscura genome, 26 phage clones containing repetitive sequences were examined by Southern DNA blot analysis and by in situ hybridization to polytene chromosomes. These results led to a classification of the clones according to five different types of hybridization patterns. Two types, each containing seven clones, are characterized by hybridization at 100 to 300 sites dispersed over the euchromatic parts of the chromosome band. One of these two classes also showed strong labelling of the chromocentre. The remaining types of hybridization pattern lacked a prominent band but showed hybridization either to the euchromatic regions or to the chromocentre or both. Chromosome A (= X) was the preferred location of prominently labelled bands and it also showed an excess of labelling by some clones. Some of the cloned dispersed sequences were localized cytologically on chromosomes of larvae from crosses between different strains of D. subobscura and between two closely related species, in order to detect heterozygosity at hybridization sites. Comparisons of the chromosomal distribution of labelling sites showed differences in number and location, indicating the possibility of transposition events.

Conditioning the behavior of Drosophila melanogaster by means of electric shocks.

Eighty male Drosophila melanogaster were observed individually for extended periods (1-2 hr) as they moved about in a small chamber consisting of a narrow, clear plastic ring whose vertical walls were thin epoxy plates with electric grids by means of which an 80-V shock could be delivered to the fly. Males (whether initially subjected to 0, 1, 2, or 3 electric shocks) that were observed for 10 consecutive 5-min sessions, given a supplemental electric shock (followed by a 2-min rest), and then observed for 5 additional 5-min sessions spent only 88.9% as much time on the grids after the supplemental shock as they had been spending earlier. This avoidance of the potentially electrified grids corresponds to that observed in tests involving the exposure of flies to 0, 1, 2, or 3 electric shocks preceding the 10 5-min sessions. The results reveal (i) an extreme variation in the normal behavior among unshocked (control) males and (ii) an exponential decline in the length of time spent on the grids immediately after receiving 1, 2, or 3 shocks. These data confirm earlier reports on the conditioning of behavior in Drosophila by means of electric shocks; in addition, they remove an ambiguity inherent in the earlier studies, which relied on a presumed exposure of each of 150 or more flies to each shock as it was delivered.

Cytogenetic mapping of marker genes on the chromosome elements C and E of Drosophila pseudoobscura and D. subobscura.

Enzyme loci, visible marker genes and lambda-cloned DNA-sequences from a D. miranda library were mapped cytologically on the chromosome elements C and E of D. pseudoobscura and D. subobscura. New data are incorporated into the linkage maps of the two species. Homologous segments can now be localized in the polytene chromosomes with these markers. A comparison of the chromosome elements E of D. melanogaster and D. subobscura shows 12 conserved subsections which have been rearranged by paracentric inversions in the evolution of the two lineages.

Cytogenetic Mapping of Enzyme Loci on Chromosomes J and U of DROSOPHILA SUBOBSCURA.

Enzyme loci located on chromosome J and U were mapped cytologically by means of a Y translocation technique. A linkage map of the two chromosomes was established in a parallel experiment and the recombination frequency in different regions of the chromosomes determined. A comparison of the cytogenetic localization of the enzyme genes in D. subobscura and D. melanogaster indicates that many paracentric inversions must have taken place in the course of divergent evolution. However, no displacements of genes from one element to another due to pericentric inversions, reciprocal translocations or transposing elements can be observed. In spite of the large number of structural rearrangements that have occurred in the phylogeny of the genus Drosophila, gross similarities of banding pattern in homologous regions of the chromosomes of the two species become apparent.

A Y-translocation method for localizing enzyme genes on Drosophila polytene chromosomes.

X-Ray induced translocations between autosomes and the Y-chromosome giving balanced and aneuploid (partially trisomic) male offspring proved useful for a rather precise localization of enzyme loci in the subsections of the polytene chromosomes of Drosophila subobscura.