Short- and long-term responses to fescue toxicosis at different ambient temperatures

Intake of endophyte-infected tall fescue by cattle results in fescue toxicosis, which is characterized by increased hyperthermia during heat stress and concomitant reductions in feed intake and growth. Rats were monitored at 21 or 31 °C for short- or long-term periods to determine temporal changes associated with the intake of endophyte-infected (E+) or uninfected (E−) fescue seed diets. Core temperature only changed in rats fed E+ diet at 31 °C. Intake of E+ diet reduced feed intake, daily gain, and serum prolactin. There were temporal and thermal differences in the response to endophytic toxins, with short-term changes diminishing over time at 21 °C, but increasing for certain parameters at 31 °C.     

Physiological and morphological short-term responses to light and temperature in two Nothofagus species of Patagonia, South America

The study of plant responses to environmental stress factors is essential for management of plant systems and for anticipating their response to climate change. The main goal of this study was to determine morphological and physiological responses of Nothofagus obliqua and N. nervosa seedlings to light and temperature, two of the main stress factors acting in their current natural distribution in NW Patagonia. Responses to light were evaluated analyzing growth and survival, as well as morphological and physiological traits related to them, in seedlings subjected to three contrasting light conditions (full-sun conditions, 50% of sunlight and 20% of sunlight) during one growth season. Temperature photosynthetic responses were evaluated in seedlings subjected to temperature treatments between ?5 and 40°C for 2 and 4 h. Growth rate and biomass partition were similar between light treatments in both species. High apical meristem damage and decreased photosynthetic capacity of preformed leaves were observed under full-sun conditions, suggesting that high light levels have a deleterious effect on plant yield. Both species produced neoformed leaves during the growing season with better photosynthetic capacity than preformed leaves under full sun conditions, contributing to plant acclimation. Almost no plasticity was observed in morphological traits in response to shade. Both species differed in optimum temperature for photosynthesis, with a wider temperature range at which high photosynthesis is maintained in N. obliqua. In both species the higher values of net photosynthetic rate were found at higher temperatures than the mean annual temperature of its current natural distribution range. Under no water-stress conditions, future higher temperatures could increase carbon fixation of these species, with a little advantage of N. obliqua if temperature variance is high. Synergy effect of various environmental stress factors, particularly considering cultivation of these species outside their current natural distribution sites require further studies.     

Water relations of ponderosa pines in Patagonia Argentina: implications for local water resources and individual growth

Replacement of grasslands by forests may result in increased water consumption, and the magnitude of this will depend on stand density. To test this hypotheses and evaluate the impact of pine plantations on hydric resources in Patagonia Argentina, we measured over two seasons (1999–2000 and 2000–2001) and at two densities of ponderosa pine plantations (350 and 500 trees ha^?1) the following variables: soil water content, leaf water potential (ψ), individual tree growth, individual sap flow, and response of sap flow density (u) to vapor pressure deficit (VPD). Stand transpiration (T) and whole-plant liquid-phase hydraulic conductance (L) were also estimated. Pre-dawn ψ varied from about –0.5 to –1.0 MPa. No differences were found in midday maximum u (1100–1800 hours) on clear days between the 2 measurement years, throughout each season, or between different densities of plantation. Sapflow density was also not correlated with soil water storage up to 1.4 m soil depth. Sapflow increased until VPD of about 2.3 kPa, and decreased at VPD >4 kPa, describing a hysteresis in the afternoon. Values of L in Patagonian trees were similar to those recalculated from published data for pines of the same height in the USA. Average stand transpiration increased with increased density (2.07 and 3.08 mm day^-1 for 350 and 500 pines ha^-1, respectively) and size of the trees. We conclude that ponderosa pines in Patagonia Argentina use more water, in a magnitude depending on the density of the trees, than native grasslands. Accordingly, ponderosa pines use deep water to maintain high water potential and transpiration rates even during the dry season.     

Diurnal and seasonal responses of stomatal conductance for cowpea plants subjected to different levels of environmental drought

Summary Previously we reported that leaf conductance of cowpea (Vigna unguiculata) decreased with small changes in soil water status without associated changes in leaf water status. In these studies a larger range of soil water deficits was imposed in a rain-free environment by prolonged soil drying, and by weekly irrigation with different amounts of water. With progressive soil water deficits, leaf conductance and xylem pressure potential both declined, but in a manner which indicated that they were not related. Diurnal courses of leaf conductance usually indicated that stomatal opening occurred in the morning, and partial or complete stomatal closure occurred during midday and afternoon. This stomatal closure was associated with increases in air vapor pressure deficit. Day-to-day increases in leaf conductance, at times when radiation was not limiting stomatal opening, were associated with decreases in air vapor pressure deficits. However, maximum leaf conductances and their responses to vapor pressure deficit were generally smaller for plants subjected to greater depletion of soil water.     

Water flux and canopy conductance of natural versus planted forests in Patagonia, South America

In NW Patagonia, South America, natural shrublands and mixed forests of short Nothofagus antarctica (G. Forst.) Oerst. trees are currently being replaced by plantations with Pseudotsuga menziesii (Mirb) Franco. This land use change is controversial because the region is prone to drought, and replacement of native vegetation by planted forests may increase vegetation water use. The goal of this study was to examine the physiological differences, especially the response of water flux and canopy conductance to microclimate, that lead to greater water use by exotic trees compared to native trees. Meteorological variables and sapflow density of P. menziesii and four native woody species were measured in the growing season 2005–2006. Canopy conductance (gc) was estimated for both the exotic (monoculture) and native (multi-species) systems, including the individual contributions of each species of the native forest. Sapflow density, stand-level transpiration and gc were related to leaf-to-air vapor pressure difference (VPD). All native species had different magnitudes and diurnal patterns of sapflow density compared to P. menziesii, which could be explained by the different gc responses to VPD. Stomatal sensitivity to VPD suggested that all native species have a stronger stomatal control of leaf water potential and transpiration due to hydraulic limitations compared to P. menziesii. In conclusion, differences in water use between a P. menziesii plantation and a contiguous native mixed forest of similar basal area could be explained by different gc responses to VPD between species (higher sensitivity in the native species), in addition to particular characteristics of the native forest structure.     

Diverse functional responses to drought in a Mediterranean-type shrubland in South Africa

? Mediterranean-type ecosystems contain 20% of all vascular plant diversity on Earth and have been identified as being particularly threatened by future increases in drought. Of particular concern is the Cape Floral Region of South Africa, a global biodiversity hotspot, yet there are limited experimental data to validate predicted impacts on the flora. In a field rainout experiment, we tested whether rooting depth and degree of isohydry or anisohydry could aid in the functional classification of drought responses across diverse growth forms. ? We imposed a 6-month summer drought, for 2?yr, in a mountain fynbos shrubland. We monitored a suite of parameters, from physiological traits to morphological outcomes, in seven species comprising the three dominant growth forms (deep-rooted proteoid shrubs, shallow-rooted ericoid shrubs and graminoid restioids). ? There was considerable variation in drought response both between and within the growth forms. The shallow-rooted, anisohydric ericoid shrubs all suffered considerable reductions in growth and flowering and increased mortality. By contrast, the shallow-rooted, isohydric restioids and deep-rooted, isohydric proteoid shrubs were largely unaffected by the drought. ? Rooting depth and degree of iso/anisohydry allow a first-order functional classification of drought response pathways in this flora. Consideration of additional traits would further refine this approach.     

Evolutionary responses of Drosophila melanogaster to selection at different larval densities: changes in genetic variation,specialization and phenotypic plasticity

Abstract We studied the evolutionary response to novel environments by applying artificial selection for total progeny biomass in populations of Drosophila melanogaster maintained at three different larval population densities. We found the relative amount of genetic variability for characters related with biomass to be lower and the correlation between them more negative at the intermediate density, and that selection resulted in changes in phenotypic plasticity and in patterns of resource allocation between traits. We found some evidence for tradeoffs between densities, which suggests that populations living at heterogeneous densities might be subject to disruptive selection. Our results show that adaptation to new environments may be a complex process, involving not only changes in trait means, but also in correlations between traits and between environments.     

Austrocedrus chilensis growth decline in relation to drought events in northern Patagonia, Argentina

The significant mortality of the Austrocedrus chilensis (D. Don) Pic. Serm. et Bizarri forests, locally known as “Mal del Ciprés”, has been reported since 1945 for most sites across its distribution in Argentina. However, the cause of this decline is still a topic of discussion. In this study, radial growth patterns from symptomatic and asymptomatic A. chilensis trees were analyzed to determine the influence of drought events on tree growth. Fifty pairs of symptomatic and asymptomatic trees with similar DBH, competition, and microsite conditions were cored at five pure A. chilensis stands near El Bolsón, Río Negro, Argentina. A reference chronology from nonaffected trees was used to cross-date all cores and to determine the relationship between A. chilensis radial growth and climate. The growth of A. chilensis is favored by above average precipitation in late spring–early summer (November and December). A strong relationship was also observed between radial growth patterns and the Palmer drought severity index, a measure of the regional water deficit. Significant differences in growth patterns were recorded between symptomatic and asymptomatic trees. Following extreme drought events, the growth of symptomatic trees is consistently lower than in asymptomatic trees. Based on the larger number of droughts recorded during the past decades and on future climatic predictions suggesting increasing trends in the frequency and intensity of drought events in northern Patagonia, a gradual increase in the number of trees affected by “Mal del Ciprés” along the twenty-first century is likely expected.     

Photosynthetic functions of cembran pines and dwarf pines during winter at timberline as regulated by different temperatures,snowcover and light

Trees at timberline in the high Alps are exposed to a variety of climatic conditions. Most climatic stresses occur during winter and spring, when frost, occasionally low snow cover, and high irradiation interact. In this study, we follow reactions of photosynthesis from high winter to spring in two dominating tree species of the alpine timberline, which may indicate the status of stress response to a changing environment. The results indicate a level of physiological stability in trees, which are important for stabilising natural high mountain ecosystems. Trees of Pinus cembra and of Pinus mugo were selected at altitudes between 1850 m a.s.l. and 1950 m a.s.l. near innsbruck, Austria. At six sampling times from January to May, fast chlorophyll fluorescence was measured in the field and twigs were collected for further investigation in the laboratory. The following measurements were taken: photosynthetic oxygen formation, needle chlorophyll and carotenoid determination, and kinetic studies of the xanthophyll cycle. In general, both tree species showed similar results in most parameters studied. P. mugo seems to have some advantages if winter precipitation is high, when, because of its growth habitus, most needles will be snow covered. Primary photochemistry (trapping per reaction centre) in PS II does not change with sampling dates despite the fact that temperature and light are changing. However, first events in electron transport and whole needle photosynthesis are strongly affected by light and temperature conditions during the days before sampling. The kinetics of the xanthophyll cycle indicate not only light, but also strong temperature effects. P. mugo photosynthesis seems to have a higher stability under changing weather. Both tree species are well prepared to start with photosynthesis in winter, if favourable conditions, like foehn events, occur.     

Short- and long-term skin graft survival in cattle clones with different mitochondrial haplotypes

In contrast to nuclear DNA, cytoplasmic genes may differ among cloned animals due to the presence of polymorphic mitochondrial DNA haplotypes in the host oocytes, raising doubts about histocompatibility among clones. Three bovine clones were generated by nuclear transfer; dermal fibroblasts from a fetus were used as donor cells, whereas oocytes from abbatoir-derived ovaries were used as recipient cells. The mitochondrial DNA (sequencing of coding and non-coding regions) and nuclear DNA (13 microsatellite markers) of cloned and control animals were characterized to identify potential polymorphisms. Skin auto- and allografts were transplanted on the adult clones and a non-related animal as a measure of immunological reactivity. Nuclear DNA of cloned animals was genetically identical but differed in all microsatellites of the non-related control. Amounts of donor cell mitochondrial DNA in the skin ranged from 1 to 2.6% among clones. Few differences in heteroplasmy were observed between skin and WBC of the clones, indicating limited mitochondrial DNA segregation in tissues during pre- and post-natal development to adulthood. Sequencing of the remaining oocyte-derived mitochondrial DNA haplotype identified polymorphisms in coding and non-coding regions, confirming their origin from unrelated maternal lineages. Nonetheless, skin transplants between clones were accepted for the 92 d study period, whereas third-party grafts were rejected. In conclusion, the nuclear transfer-generated adult bovine clones used in this study were immunologically compatible with one another despite differences in their mitochondrial DNA haplotypes.     

Carbon storage and fluxes in ponderosa pine forests at different developmental stages

We compared carbon storage and fluxes in young and old ponderosa pine stands in Oregon, including plant and soil storage, net primary productivity, respiration fluxes, eddy flux estimates of net ecosystem exchange (NEE), and Biome‐BGC simulations of fluxes. The young forest (Y site) was previously an old‐growth ponderosa pine forest that had been clearcut in 1978, and the old forest (O site), which has never been logged, consists of two primary age classes (50 and 250 years old). Total ecosystem carbon content (vegetation, detritus and soil) of the O forest was about twice that of the Y site (21 vs. 10 kg C m^?2 ground), and significantly more of the total is stored in living vegetation at the O site (61% vs. 15%). Ecosystem respiration (R_e) was higher at the O site (1014 vs. 835 g C m^?2 year^?1), and it was largely from soils at both sites (77% of R_e). The biological data show that above‐ground net primary productivity (ANPP), NPP and net ecosystem production (NEP) were greater at the O site than the Y site. Monte Carlo estimates of NEP show that the young site is a source of CO₂ to the atmosphere, and is significantly lower than NEP(O) by c. 100 g C m^?2 year^?1. Eddy covariance measurements also show that the O site was a stronger sink for CO₂ than the Y site. Across a 15‐km swath in the region, ANPP ranged from 76 g C m^?2 year^?1 at the Y site to 236 g C m^?2 year^?1 (overall mean 158 ± 14 g C m^?2 year^?1). The lowest ANPP values were for the youngest and oldest stands, but there was a large range of ANPP for mature stands. Carbon, water and nitrogen cycle simulations with the Biome‐BGC model suggest that disturbance type and frequency, time since disturbance, age‐dependent changes in below‐ground allocation, and increasing atmospheric concentration of CO₂ all exert significant control on the net ecosystem exchange of carbon at the two sites. Model estimates of major carbon flux components agree with budget‐based observations to within ± 20%, with larger differences for NEP and for several storage terms. Simulations showed the period of regrowth required to replace carbon lost during and after a stand‐replacing fire (O) or a clearcut (Y) to be between 50 and 100 years. In both cases, simulations showed a shift from net carbon source to net sink (on an annual basis) 10–20 years after disturbance. These results suggest that the net ecosystem production of young stands may be low because heterotrophic respiration, particularly from soils, is higher than the NPP of the regrowth. The amount of carbon stored in long‐term pools (biomass and soils) in addition to short‐term fluxes has important implications for management of forests in the Pacific North‐west for carbon sequestration.     

Correction to: New Specimens of Reigitherium bunodontum from the Late Cretaceous La Colonia Formation,Patagonia, Argentina and Meridiolestidan Diversity in South America

Journal of Mammalian Evolution -     

Genetic structure and outcrossing rates in Flourensia cernua (Asteraceae) growing at different densities in the South-western Chihuahuan Desert

BACKGROUNDS AND AIMS: Flourensia cernua is a partially self-incompatible, wind-pollinated shrub that grows in two scrub types of contrasting densities. It was anticipated that differences in plant density would affect the amount of genotype availability, and thus higher outcrossing rates and less genetic differentiation would be found at high-density sites. METHODS: At five high-density sites and at five low-density sites, 11 allozyme loci were analysed in adults. Outcrossing rates were estimated using five allozyme loci sampled from eight families from each scrub type. KEY RESULTS: High levels of genetic variation were found at all sites (ranging from P = 82-100 %, He = 0.33-0.45, and Ho = 0.4-0.59). Heterozygotes were found in excess (FIS = -0.15 +/- 0.06 s.d.), suggesting that natural selection favours heterozygosity, and there was little differentiation between sites (FST = 0.08 +/- 0.02 s.d.). Life history attributes, such as long-lived habit and wide geographic distribution, as well as the presence of a self-incompatibility system may explain these results. Outcrossing rates did not differ from 1.0 in both scrub types, and there was no genetic differentiation between scrub types (FST = -0.01 +/- 0.004 s.d.). CONCLUSIONS: The high rate of outcrossing favoured by partial incompatibility may generate unrestricted gene flow between scrub types and thus may explain the lack of differentiation between them. High heterozygosity could be expected in long-lived plants of arid zones as they confront a variable and stressing environment.     

New Specimens of Reigitherium bunodontum from the Late Cretaceous La Colonia Formation,Patagonia, Argentina and Meridiolestidan Diversity in South America

We describe the first maxillae and additional new specimens of Reigitherium bunodontum, a small meridiolestidan from the Late Cretaceous La Colonia Formation, Patagonia, Argentina. The new material supports a dental formula of I?, C1, P4, M3, resolves postcanine positional uncertainty and corrects previous interpretations. Our phylogeny recovers Reigitherium as a meridiolestidan allied to other bunodont Mesungulatoidea, as the sister group of the Paleocene Peligrotherium. Posterior premolars/molars of Reigitherium, and to a smaller degree Peligrotherium, are dominated by an incomplete transverse ridge running between the protoconid-metaconid in the lowers and the paracone-stylocone in the uppers, semi-symmetrical basins developing mesially and distally from these central ridges. The trigonid-derived single transverse crest results from a mesial shift of the robust metaconid, an enhancement of the basin crest stretching from the protoconid/metaconid, and a shallower trigonid basin. The mesungulatoid condition, with its complete absence of talonid, contrasts sharply with that of therians with lophs, or transverse ridges, which involved at least one talonid-derived loph resulting in two transverse crests per tooth. Mesungulatoid meridiolestidans achieved complex tooth-on-tooth occlusion with a predicted increase in herbivory/omnivory, departing from the traditional sharp-cusp insectivores plesiomorphic for meridiolestidans and Mesozoic mammals in general. Reigitherium’s dramatic remodeling of the primitive meridiolestidan molar morphology, extensive continuous occlusal surface, accessory cuspules, and highly textured crenulated enamel illustrates one of most distinctive adaptations to herbivory among Mesozoic mammals.

     

Seasonal fluctuation of different edaphic microarthropod population densities in relation to soil moisture and temperature in a pine,Pinus kesiya Royle plantation ecosystem

Seasonal fluctuations of soil and litter microarthropod populations in a pine,Pinus kesiya Royle plantation of North Eastern India were investigated between November 1976 and November 1977. Three major groups were recognized: (a) Collembola, (b) Acarina and (c) miscellaneous. Collembola was the most abundant group and was dominated byIsotoma trispinata (MacGillivray). The total microarthropod density ranged from 26,800 per m² to 145,200 per m². Collembola densities ranged from 10,000 to 121,200 per m², Acarina densities ranged from 8,800 to 41,600 per m², and the miscellaneous group ranged from 1,200 to 6,400 per m². Soil moisture was positively correlated with total arthropod, Collembola and Acarina densities. Soil temperature was positively correlated only with Acarina. Densities of Collembola and Acarina were negatively correlated.The work described in this paper was carried out while the author was at the Department of Zoology, North Eastern Hill University, Shillong, Mehgalaya (India).It was presented at the Ninth International Biometeorological Congress, 23rd Sptember–1st October, 1981, Osnabrück and Stuttgart-Hohenheim, FRG.     

Biting patterns and seasonal densities of Anopheles mosquitoes in the Cayo District, Belize, Central America with emphasis on Anopheles darlingi.

The present study utilized an experimental hut to conduct human-baited landing collections for characterizing the all-night biting patterns and seasonal densities of adult Anopheles darlingi in the centrally located Cayo District of Belize, Central America. A total of 25 all-night collections (i.e., sunset to sunrise) were conducted from January 2002 to May 2003, capturing a total of 18,878 An. darlingi females. Anopheles darlingi exhibited a bimodal nightly biting pattern with one predominate peak occurring three h after sunset and a smaller peak occurring one h prior to sunrise. Biting females were collected throughout the night in higher densities indoors (9,611) than outside (9,267) the experimental hut (O:I=1.00:1.04). Seasonal adult collections show An. darlingi densities were highest during the transitional months between the end of the wet and beginning of the dry season (January) and the end of the dry season and beginning of the wet season (May). A total of 2,010 An. darlingi females was captured in 31 two-h, human-baited landing collections performed from January to October 2002. Anopheles darlingi monthly population densities were found to have no significant associations with high or low temperatures, precipitation, or river level. However, qualitative data examination indicates an inverse relationship between river level and An. darlingi adult collections suggesting a disturbance of larval habitats. All-night biting and seasonal distribution patterns for other anopheline species are also described. None of the adult specimens collected throughout the entire study tested positive for Plasmodium spp. infection using the VecTest rapid diagnostic kit.     

A preadipocyte clonal line from mouse brown adipose tissue. Short- and long-term responses to insulin and beta-adrenergics

A clonal cell line has been established from the interscapular brown adipose tissue (BAT) of the C57 BL/6J +/+ mouse. The line, designated BFC-1, is aneuploid and exhibits both morphological and biochemical properties characteristics of mature adipocytes. Adipose conversion begins after confluence and is accompanied by an early emergence of lipoprotein lipase; a later emergence of glycerol-3-phosphate dehydrogenase and acid: CoA ligase; an increase in the average triglyceride content. Adipose conversion, estimated by activities of enzyme markers, is enhanced at any given time by the continuous presence in the culture medium of insulin and triiodothyronine, both within their physiological range of concentrations. In addition to both hormones, chronic exposure of confluent cells to beta-adrenergics brings similar long-term effects on adipose conversion. The uptake of labelled 2-deoxyglucose by differentiated BFC-1 cells is stimulated by insulin; the half-maximum effect is observed at 1 nM insulin. Differentiated BFC-1 cells, in which endogenous triglycerides have been prelabelled on the fatty acid moiety, do respond to beta-adrenergics by releasing radioactive fatty acids. The agonist potency order and the EC50 value for each agonist are BRL 37344 (0.5 nM) greater than isoproterenol (1.5 nM) greater than norepinephrine (3 nM) greater than epinephrine (7 nM) greater than salbutamol (15 nM). The half-maximally and maximally effective concentrations of corticotropin to stimulate lipolysis are found to be 4 and 100 nM, respectively. The lipolytic response to isoproterenol is counteracted by prior addition of insulin or simultaneous addition of propranolol. Parallel studies performed on Ob17 cells, a clonal line established from mouse white adipose tissue (Négrel et al., Proc natl acad sci US 75 (1978) 6054), show that the agonist potency order and the EC50 value for each agonist are BRL 37344 (3 nM) greater than isoproterenol (10 nM) greater than norepinephrine (20 nM) greater than epinephrine (40 nM). Thus both BFC-1 cells and Ob17 cells show an atypical beta-adrenoreceptor similar to that described in rat adipocytes (Arch et al., Nature 309 (1984) 163), but the sensitivity of BFC-1 cells toward beta-agonists is found to be 6-fold higher than that of Ob17 cells. Thus the BFC-1 line represents a useful model for the study of short- and long-term responses to beta-adrenergics.     

Different responses to drought and freeze stress of Quercus ilex L. growing along a latitudinal gradient

The vulnerability to drought and freeze stress was measured in young plants of Quercus ilex L. growing in the field in two natural sites within the Italian distribution area of this species, i.e. Sicily (Southern Mediterranean Basin) and Venezia Giulia (Northeastern Italy), respectively. In particular, the resistance strategies adopted by Q. ilex to withstand the two stresses were estimated in terms of seasonal and/or diurnal changes in leaf conductance to water vapour (g_L), water potential (_L) and relative water content (RWC) as well as of xylem embolism in the stem and root hydraulic conductance (K_RL). Sicilian (SI) plants showed to reduce water loss by stomatal closure (g_L decreased) in summer, thus maintaining average RWCs at 88–90%. Moreover, SI plants showed considerable resistance to xylem cavitation in the stem (the loss of hydraulic conductance, PLC, was less than 12% throughout the year) and to maintain the hydraulic conductance of their roots (K_RL), constantly high even in summer. Plants growing in Venezia Giulia (VG plants), on the contrary, underwent leaf dehydration in the winter due to freeze stress so that RWC measured in April was still 78% on a diurnal basis. This was apparently due to consistent xylem embolism in the stem. In fact, PLC was as high as 40% between November and March. Only in the summer was PLC similar to that recorded in SI plants. Moreover, K_RL of VG plants decreased in November from about 1.5 to 0.8×10^–4 kg s^–1 m^–2 MPa^–1, i.e. about 50%, and in February K_RL dropped further to 0.4×10^–4 kg s^–1 m^–2 MPa^–1. On the basis of the above, we conclude that: (a) Q. ilex was more sensitive to freeze than to drought stress so that freeze stress can be considered as a factor limiting the distribution area of this species; (b) drought and freeze stress were faced by Q. ilex adopting two different resistance strategies, i.e. drought avoidance based on water saving in Sicily and freeze tolerance in Venezia Giulia.