Under Stress: Searching for Genes Involved in the Response of Abies pinsapo Boiss to Climate Change.

Currently, Mediterranean forests are experiencing the deleterious effects of global warming, which mainly include increased temperatures and decreased precipitation in the region. Relict Abies pinsapo fir forests, endemic in the southern Iberian Peninsula, are especially sensitive to these recent environmental disturbances, and identifying the genes involved in the response of this endangered tree species to climate-driven stresses is of paramount importance for mitigating their effects. Genomic resources for A. pinsapo allow for the analysis of candidate genes reacting to warming and aridity in their natural habitats. Several members of the complex gene families encoding late embryogenesis abundant proteins (LEAs) and heat shock proteins (HSPs) have been found to exhibit differential expression patterns between wet and dry seasons when samples from distinct geographical locations and dissimilar exposures to the effects of climate change were analyzed. The observed changes were more perceptible in the roots of trees, particularly in declining forests distributed at lower altitudes in the more vulnerable mountains. These findings align with previous studies and lay the groundwork for further research on the molecular level. Molecular and genomic approaches offer valuable insights for mitigating climate stress and safeguarding this endangered conifer.

Faecal nutrient deposition of domestic and wild herbivores in an alpine grassland.

The contribution of herbivores to ecosystem nutrient fluxes through dung deposition has the potential to, directly and indirectly, influence ecosystem functioning. This process can be particularly important in nutrient-limited ecosystems such as alpine systems. However, herbivore dung content (carbon, C; nitrogen, N; phosphorus, P; potassium, K) and stoichiometry (C/N) may differ among species due to differences in diet, seasonality, body type, feeding strategy, and/or digestive system with consequences for soil biogeochemistry. Here we explore how species, body size, and seasonality may result in differences in dung stoichiometry for four alpine herbivores (chamois, sheep, horse, and cattle). We found that herbivore dung nutrient content often varies among species as well as with body size, with the dung of small herbivores having larger C, N, and P faecal content. Seasonality also showed marked effects on faecal nutrient content, with a general pattern of decreasing levels of faecal P, N and an increase of C/N as the summer progresses following the loss of nutrient value of the vegetation. Moreover, we showed how herbivores play an important role as natural fertilizers of C, N, and P in our study area, especially cattle. Our study highlights the importance of considering the relative contribution of different herbivores to ecosystem nutrient fluxes in management practices, especially with ongoing changes in wild and domestic herbivore populations in alpine ecosystems.

Transcriptome Analysis and Intraspecific Variation in Spanish Fir (Abies pinsapo Boiss.).

Spanish fir (Abies pinsapo Boiss.) is an endemic, endangered tree that has been scarcely investigated at the molecular level. In this work, the transcriptome of Spanish fir was assembled, providing a large catalog of expressed genes (22,769), within which a high proportion were full-length transcripts (12,545). This resource is valuable for functional genomics studies and genome annotation in this relict conifer species. Two intraspecific variations of A. pinsapo can be found within its largest population at the Sierra de las Nieves National Park: one with standard green needles and another with bluish-green needles. To elucidate the causes of both phenotypes, we studied different physiological and molecular markers and transcriptome profiles in the needles. "Green" trees showed higher electron transport efficiency and enhanced levels of chlorophyll, protein, and total nitrogen in the needles. In contrast, needles from "bluish" trees exhibited higher contents of carotenoids and cellulose. These results agreed with the differential transcriptomic profiles, suggesting an imbalance in the nitrogen status of "bluish" trees. Additionally, gene expression analyses suggested that these differences could be associated with different epigenomic profiles. Taken together, the reported data provide new transcriptome resources and a better understanding of the natural variation in this tree species, which can help improve guidelines for its conservation and the implementation of adaptive management strategies under climatic change.

Unexpected resilience in relict Abies pinsapo Boiss forests to dieback and mortality induced by climate change.

Acute and early symptoms of forest dieback linked to climate warming and drought episodes have been reported for relict Abies pinsapo Boiss. fir forests from Southern Spain, particularly at their lower ecotone. Satellite, orthoimages, and field data were used to assess forest decline, tree mortality, and gap formation and recolonization in the lower half of the altitudinal range of A. pinsapo forests (850-1550 m) for the last 36 years (1985-2020). Field surveys were carried out in 2003 and in 2020 to characterize changes in stand canopy structure and mortality rates across the altitudinal range. Time series of the Normalized Difference Vegetation Index (NDVI) at the end of the dry season (derived from Landsat 5 and 7 imagery) were used for a Dynamic Factor Analysis to detect common trends across altitudinal bands and topographic solar incidence gradients (SI). Historical canopy cover changes were analyzed through aerial orthoimages classification. Here we show that extensive decline and mortality contrast to the almost steady alive basal area for 17 years, as well as the rising photosynthetic activity derived from NDVI since the mid-2000s and an increase in the forest canopy cover in the late years at mid and high altitudes. We hypothesized that these results suggest an unexpected resilience in A. pinsapo forests to climate change-induced dieback, that might be promoted by compensation mechanisms such as (i) recruitment of new A. pinsapo individuals; (ii) facilitative effects on such recruitment mediated by revegetation with other species; and (iii) a 'release effect' in which surviving trees can thrive with fewer resource competition. Future research is needed to understand these compensation mechanisms and their scope in future climate change scenarios.

Drought, axe and goats. More variable and synchronized growth forecasts worsening dieback in Moroccan Atlas cedar forests.

Understanding how climate warming and land-use changes determine the vulnerability of forests to drought is critical. However, we still lack: (i) robust quantifications of long-term growth changes during aridification processes, (ii) links between growth decline, changes in forest cover, stand structure and soil conditions, and (iii) forecasts of growth variability to projected climate warming. We investigated tree-ring records over the past 400-700 years, quantified changes in grazing area and forest cover during the 20th century, sampled current stand structure, and analyzed soil organic carbon δ13C and total nitrogen δ15N of Atlas cedar (Cedrus atlantica (Endl.) Manetti ex Carrière) Moroccan forests to characterize their dieback. Atlas cedar forests experienced massive dieback after the 1970s, particularly in the xeric High Atlas region. Forest cover increased in the less xeric regions (Middle Atlas and Rif) by almost 20%, while it decreased about 18% in the High Atlas, where soil δ13C and δ15N showed evidences of grazing. Growth declined and became more variable in response to recent droughts. The relative growth reduction (54%) was higher in the Middle Atlas than elsewhere (Rif, 32%; High Atlas, 36%). Growth synchrony between forests located within the Middle and High Atlas regions increased after the 1970s. Simulations based on a worst-case emission scenario and rapid warming forecast a stronger limitation of growth by low soil moisture in all regions, but particularly in the Middle Atlas and after the mid-21st century. Climate warming is expected to strengthen growth synchronization preceding dieback of conifer forests in xeric regions. The likelihood of similar dieback episodes is further exacerbated by historical degradation of these forests.

Morphostatic Speciation within the Dagger Nematode Xiphinema hispanum-Complex Species (Nematoda: Longidoridae).

Dagger nematodes of the genus Xiphinema include a remarkable group of invertebrates of the phylum Nematoda comprising ectoparasitic animals of many wild and cultivated plants. Damage is caused by direct feeding on root cells and by vectoring nepoviruses that cause diseases on several crops. Precise identification of Xiphinema species is critical for launching appropriate control measures. We deciphered the cryptic diversity of the Xiphinema hispanum-species complex applying integrative taxonomical approaches that allowed us to verify a paradigmatic example of the morphostatic speciation and the description of a new species, Xiphinema malaka sp. nov. Detailed morphological, morphometrical, multivariate and genetic studies were carried out, and mitochondrial and nuclear haploweb analyses were used for species delimitation of this group. The new species belongs to morphospecies Group 5 from the Xiphinema nonamericanum-group species. D2-D3, ITS1, partial 18S, and partial coxI regions were used for inferring the phylogenetic relationships of X. malaka sp. nov. with other species within the genus Xiphinema. Molecular analyses showed a clear species differentiation not paralleled in morphology and morphometry, reflecting a clear morphostatic speciation. These results support the hypothesis that the biodiversity of dagger nematodes in southern Europe is greater than previously assumed.

Using ForeStereo and LIDAR data to assess fire and canopy structure-related risks in relict Abies pinsapo Boiss. forests.

In this study we combine information from aerial LIDAR and hemispherical images taken in the field with ForeStereo-a forest inventory device-to assess the vulnerability and to design conservation strategies for endangered Mediterranean fir forests based on the mapping of fire risk and canopy structure spatial variability. We focused on the largest continuous remnant population of the endangered tree species Abies pinsapo Boiss. spanning 252 ha in Sierra de las Nieves National Park (South Andalusia, Spain). We established 49 sampling plots over the study area. Stand structure variables were derived from ForeStereo device, a proximal sensing technology for tree diameter, height and crown dimensions and stand crown cover and basal area retrieval from stereoscopic hemispherical images photogrammetry. With this information, we developed regression models with airborne LIDAR data (spatial resolution of 0.5 points∙m-2). Thereafter, six fuel models were fitted to the plots according to the UCO40 classification criteria, and then the entire area was classified using the Nearest Neighbor algorithm on Sentinel imagery (overall accuracy of 0.56 and a KIA-Kappa Coefficient of 0.46). FlamMap software was used for fire simulation scenarios based on fuel models, stand structure, and terrain data. Besides the fire simulation, we analyzed canopy structure to assess the status and vulnerability of this fir population. The assessment shows a secondary growth forest that has an increasing presence of fuel models with the potential for high fire spread rate fire and burn probability. Our methodological approach has the potential to be integrated as a support tool for the adaptive management and conservation of A. pinsapo across its whole distribution area (<4,000 ha), as well as for other endangered circum-Mediterranean fir forests, as A. numidica de Lannoy and A. pinsapo marocana Trab. in North Africa.

Climate extremes and predicted warming threaten Mediterranean Holocene firs forests refugia.

Warmer and drier climatic conditions are projected for the 21st century; however, the role played by extreme climatic events on forest vulnerability is still little understood. For example, more severe droughts and heat waves could threaten quaternary relict tree refugia such as Circum-Mediterranean fir forests (CMFF). Using tree-ring data and a process-based model, we characterized the major climate constraints of recent (1950-2010) CMFF growth to project their vulnerability to 21st-century climate. Simulations predict a 30% growth reduction in some fir species with the 2050s business-as-usual emission scenario, whereas growth would increase in moist refugia due to a longer and warmer growing season. Fir populations currently subjected to warm and dry conditions will be the most vulnerable in the late 21st century when climatic conditions will be analogous to the most severe dry/heat spells causing dieback in the late 20th century. Quantification of growth trends based on climate scenarios could allow defining vulnerability thresholds in tree populations. The presented predictions call for conservation strategies to safeguard relict tree populations and anticipate how many refugia could be threatened by 21st-century dry spells.

Nutritional status of Abies pinsapo forests along a nitrogen deposition gradient: do C/N/P stoichiometric shifts modify photosynthetic nutrient use efficiency?

Chronic atmospheric N deposition has modified relative N availability, altering the biogeochemical cycles of forests and the stoichiometry of nutrients in trees, inducing P limitation, and modifying the N:P ratios of plant biomass. This study examines how the variation in the foliar stoichiometry of Abies pinsapo across an N deposition gradient affects foliar traits and photosynthetic rate. We measured the maximum net assimilation rates (Amax) and the foliar nitrogen (N) and phosphorus (P) concentrations in A. pinsapo needles of five age classes. The leaf mass per area and photosynthetic N and P use efficiencies (PNUE and PPUE, respectively) were also estimated. The results from the N-saturated stand (Sierra Bermeja, B) differed from the comparable N-limited stands under investigation (Yunquera, Y, and Sierra Real, SR). The trees from Y and SR exhibited a reduction in the N content in older needles, whereas the foliar N concentration at the B site increased with needle age. N and P were positively correlated at Y and SR, but not at B, suggesting that the overload of N in the trees at site B has exceeded the homeostatic regulation capacity of the N-saturated stand in terms of foliar stoichiometry. A max and PNUE were correlated positively with P and negatively with the N/P ratio at the three study sites. The foliar N concentration was positively correlated with A max at Y and SR. However, this relationship was negative for the B site. These findings suggest that the nutritional imbalance caused by increased chronic deposition of N and an insufficient supply of P counteracts the potential increase in net photosynthesis induced by the accumulation of foliar N.

Plant treatment, pollutant load, and soil type effects in rhizosphere ecology of trace element polluted soils.

Re-vegetation of trace element contaminated soils can alter the pH and chelating capacity in the rhizosphere, increasing the mobility of pollutants, which, in turn, may impact on rhizosphere ecology. In this study a short-term pot experiment was carried out in order to investigate the multi-factorial effects of: buffering capacity (sandy-loam and loam soils); pollutant load (0%, 1.3%, and 4% of pyrite sludge), and the presence/absence of plant (Lolium perenne L. and Medicago sativa L.) on the mobility of trace elements, soil biochemical functionality (hydrolase activities), and biological diversity (bacterial and nematode communities). The experiment was carried out with representative soils from the Guadiamar basin (SW Spain), an area where the Aznalcóllar mining spill affected over 4000ha. Results indicated that the development of rhizospheres in polluted soils (coarse-textured) increases the mobilization of trace elements. In general the presence of roots has stimulatory effects on soil quality indicators such as hydrolase activities and both bacterial and nematode communities. However, the presence of high amount of metals interferes with these beneficial effects. This study provided evidence about the complexity of the impact of growing plants on trace element polluted soils. Trace element mobilization, hydrolase activities and bacterial and nematode communities in the rhizosphere are dependent on plant species, soil type, and pollution dose.

Stable isotope views on ecosystem function: challenging or challenged?

Stable isotopes and their potential for detecting various and complex ecosystem processes are attracting an increasing number of scientists. Progress is challenging, particularly under global change scenarios, but some established views have been challenged. The IX meeting of the Spanish Association of Terrestrial Ecology (AAET, Ubeda, 18-22 October 2009) hosted a symposium on the ecology of stable isotopes where the linear mixing model approach of partitioning sinks and sources of carbon and water fluxes within an ecosystem was challenged, and new applications of stable isotopes for the study of plant interactions were evaluated. Discussion was also centred on the need for networks that monitor ecological processes using stable isotopes and key ideas for fostering future research with isotopes.

Shrub encroachment can reverse desertification in semi-arid Mediterranean grasslands.

The worldwide phenomenon of shrub encroachment in grass-dominated dryland ecosystems is commonly associated with desertification. Studies of the purported desertification effects associated with shrub encroachment are often restricted to relatively few study areas, and document a narrow range of possible impacts upon biota and ecosystem processes. We conducted a study in degraded Mediterranean grasslands dominated by Stipa tenacissima to simultaneously evaluate the effects of shrub encroachment on the structure and composition of multiple biotic community components, and on various indicators of ecosystem function. Shrub encroachment enhanced vascular plant richness, biomass of fungi, actinomycetes and other bacteria, and was linked with greater soil fertility and N mineralization rates. While shrub encroachment may be a widespread phenomenon in drylands, an interpretation that this is an expression of desertification is not universal. Our results suggest that shrub establishment may be an important step in the reversal of desertification processes in the Mediterranean region.

Competition and drought limit the response of water-use efficiency to rising atmospheric carbon dioxide in the Mediterranean fir Abies pinsapo.

The gas-exchange and radial growth responses of conifer forests to climatic warming and increasing atmospheric CO2 have been widely studied. However, the modulating effects of variables related to stand structure (e.g., tree-to-tree competition) on those responses are poorly explored. The basal-area increment (BAI) and C isotope discrimination (C stable isotope ratio; delta13C) in the Mediterranean fir Abies pinsapo were investigated to elucidate the influences of stand competition, atmospheric CO2 concentrations and climate on intrinsic water-use efficiency (WUEi). We assessed the variation in delta13C of tree-rings from dominant or co-dominant trees subjected to different degrees of competition. A high- (H) and a low-elevation (L) population with contrasting climatic constraints were studied in southern Spain. Both populations showed an increase in long-term WUEi. However, this increase occurred more slowly at the L site, where a decline of BAI was also observed. Local warming and severe droughts have occurred in the study area over the past 30 years, which have reduced water availability more at lower elevations. Contrastingly, trees from the H site were able to maintain high BAI values at a lower cost in terms of water consumption. In each population, trees subjected to a higher degree of competition by neighboring trees showed lower BAI and WUEi than those subjected to less competition, although the slopes of the temporal trends in WUEi were independent of the competitive micro-environment experienced by the trees. The results are consistent with an increasing drought-induced limitation of BAI and a decreasing rate of WUEi improvement in low-elevation A. pinsapo forests. This relict species might not be able to mitigate the negative effects of a decrease in water availability through a reduction in stomatal conductance, thus leading to a growth decline in the more xeric sites. An intense and poorly asymmetric competitive environment at the stand level may also act as an important constraint on the adaptive capacity of these drought-sensitive forests to climatic warming.

Effects of pyrite sludge pollution on soil enzyme activities: ecological dose-response model.

A laboratory study was conducted to evaluate the response of soil enzyme activities (acid and alkaline phosphatase, beta-glucosidase, arylsulfatase, urease and dehydrogenase) to different levels of trace elements pollution in soils representative of the area affected by the pyrite sludge mining spill of Aznalcóllar (Guadiamar basin, SW Spain). Three uncontaminated soils from the study area were mixed with different loads of pyrite sludge to resemble field conditions and criteria applied for reclamation practices following the pollution incident: 0% ("reference" or background level), 1.3% ("attention level", further monitoring required), 4% ("intervention level", further cleaning and liming required) and 13% (ten times the "attention level"). Enzyme activities were analysed 4, 7, 14, 21, 34 and 92 days after pollutant addition and those measured after 92 days were used to calculate the ecological dose value (ED50). Soil enzyme activities and pH decreased after the pyrite sludge addition with respect to the "reference level" (0% pyrite sludge), whereas soil bioavailable (DTPA-extractable) trace elements concentration increased. Arylsulfatase, beta-glucosidase and phosphatase activities were reduced by more than 50% at 1.3% pyrite sludge dose. Arylsulfasate was the most sensitive soil enzyme (in average, ED50=0.99), whereas urease activity showed the lowest inhibition (in average, ED50=7.87) after pyrite sludge addition. Our results showed that the ecological dose concept, applied to enzyme activities, was satisfactory to quantify the effect of a multi-metalic pollutant (pyrite sludge) on soil functionality, and would provide manageable data to establish permissible limits of trace elements in polluted soils. Additionally, we evaluate the recovery of enzyme activities after addition of sugar-beet lime (calcium carbonate) to each experimentally polluted soil. The amount of lime added to each soil was enough to raise the pH to the original value (equal to control soil), resembling field remediation practices. After lime amendment, soil recovery was still incomplete in terms of bioavailable trace elements. However, the recovery of soil enzyme activities varied widely, ranging from 0 to 100% depending on soil type and the specific enzyme.

Microbial response to heavy metal-polluted soils: community analysis from phospholipid-linked fatty acids and ester-linked fatty acids extracts.

Heavy metal pollution of soil is of concern for human health and ecosystem function. The soil microbial community should be a sensitive indicator of metal contamination effects on bioavailability and biogeochemical processes. Simple methods are needed to determine the degree of in situ pollution and effectiveness of remediating metal-contaminated soils. Currently, phospholipid-linked fatty acids (PLFAs) are preferred for microbial profiling but this method is time consuming, whereas direct soil extraction and transesterification of total ester-linked fatty acids (ELFAs) is attractive because of its simplicity. The 1998 mining acid-metal spill of >4000 ha in the Guadiamar watershed (southwestern Spain) provided a unique opportunity to study these two microbial lipid profiling methods. Replicated treatments were set up as nonpolluted, heavy metal polluted and reclaimed, and polluted soils. Inferences from whole community-diversity analysis and correlations of individual fatty acids with metals suggested Cu, Cd, and Zn were the most important in affecting microbial community structure, along with pH. The microbial stress marker, monounsaturated fatty acids, was significantly lower for reclaimed and polluted soil over nonpolluted soils for both PLFA and ELFA extraction. Another stress marker, the monounsaturated to saturated fatty acids ratio, only showed this for the PLFA. The general fungal marker (18:2omega6c), the arbuscule mycorrhizae marker (16:1omega5c), and iso- and anteiso-branched PLFAs (gram positive bacteria) were suppressed with increasing pollution whereas 17:0cy (gram negative bacteria) increased with metal pollution. For both extraction methods, richness and diversity were greater in nonpolluted soils and lowest in polluted soils. The ELFA method was sensitive for reflecting metal pollution on microbial communities and could be suitable for routine use in ecological monitoring and risk assessment programs because of its simplicity and reproducibility.