Ecology

The keystone species concept is an important aspect of population ecology, community ecology, and conservation biology1,2, and its application is likely to be critical with ongoing climate change3. Keystone species can be identified because they have a larger effect on communities and ecosystems than would be predicted based on their abundance or dominance. Loss of keystone species within communities and ecosystems is likely to result in secondary extinction events, and in extreme cases these events can lead to community and ecosystem collapse4. The critical importance of keystone species is derived from the wide range of biotic interactions they engage in with other community members (predation, competition, herbivory, mutualism, facilitation, etc.) and their influence on abiotic environmental conditions2. Keystone species have been described in a range of ecosystems (e.g., marine, fresh water, terrestrial, etc.) and have included a variety of taxa (e.g., fungi, animals, and plants)1,3,5.
Plant communities consisting of isolated or scattered trees occur across the globe, and such trees have been described as keystone species, or “keystone structures”6. This certainly applies to trees and shrubs that are members of plant communities in arid and semi-arid habitat7. Many members of Acacia s.l. (Fabaceae: Mimosoideae8), which are broadly distributed around the world, are considered keystone species within the communities they reside. For example, they are considered keystone species in parts of Australia9, Pakistan10, the Kalahari Desert, Botswana11, Tunisia12,13,14, the Sinai Desert, Egypt15,16, and south-eastern Egypt16,17. As pointed out by Abdallah et al.12, isolated trees in arid habitats, including Vachellia species., have several characteristics that contribute to their keystone status: (1) shade from their canopies prevents extreme temperature fluctuations, increases soil moisture levels, and provides shelter for wildlife, (2) they improve soil conditions through biological nitrogen fixation and litter fall by increasing soil nitrogen content, organic carbon, and water-holding capacity, (3) they increase plant and animal biodiversity as a consequence of characteristics one and two, (4) they provide a source of food for wildlife, and (5) they provide a source of fuel, fodder, and medicines for local people and their domesticated animals. Because of their critical importance, a full characterization of keystone species and the roles they play within communities and ecosystems is urgently needed; especially as they are adversely impacted by various human activities.
The Gebel Elba mountain range is an extension of the Afromontane “biodiversity hotspot” and is at the northern limit of the Eritreo-Arabian province and the Sahel regional transition zone18. The relatively high abundance of moisture of this mountain range leads to higher plant biodiversity than reported elsewhere in Egypt, it consists of 458 species, which constitutes approximately 21% of the Egyptian flora19,20. According to the plant checklist provided by Boulos21, the flora of Egypt consists of 2100 taxa belonging to 755 genera and 129 families; including 45 genera and 228 taxa in the Fabaceae. Gebel Elba is one of the seven main phytogeographical regions in Egypt21. Additionally, the region’s tree and shrub species diversity is higher than in any other regions in Egypt19, with some Sahelian woody elements restricted to the Gebel Elba region and not reported elsewhere in Egypt. Of the 10 Vachellia (synonym: Acacia8) species reported in Egypt, seven are known to occur in the Gebel Elba region, with Vachellia asak (synonym: Acacia asak) and Vachellia oerfota subsp. oerfota (synonym: Acacia oerfota subsp. oerfota) restricted to this region.
An analysis of the plant communities of wadi Yahmib and three of its tributaries, on the north-western slopes of Gebel Elba, revealed the presence of seven plant communities, with these communities being arrayed across an elevational (environmental) gradient17. The Vachellia tortilis subsp. tortilis (synonym: Acacia tortilis subsp. tortilis) community was the main vegetation type on Gebel Elba. This community type occurred commonly in the water channels of wadis and gravel terraces from low to mid elevations (130–383 m), and the species was a member of all of the other six communities in the study area17. In addition, Vachellia tortilis subsp. raddiana (synonym: Acacia tortilis subsp. raddiana) was an overstory co-dominant species in another community on Gebel Elba. Finally, a third acacia species, Vachellia etbaica (synonym: Acacia etbaica), was also detected in this study.
Within arid and semi-arid ecosystems across north Africa and the Arabian Peninsula, plant ecologists have focused their attention on describing the vegetation of wadis that drain to the Red Sea, with these studies focusing on keystone Vachellia species12,13,14,15,16,17,22,23. The present study aimed to contribute to this body of knowledge by determining the distribution, abundance, and describing the growth characteristics of three Vachellia tree taxa in wadi Khoda and wadi Rahaba, in Gebel Elba National Park, south-eastern Egypt. These data will allow us to provide detailed descriptions of the characteristics of these three taxa. This study is essential at this moment because these tree taxa are keystone species within these ecosystems, and their presence and conservation are likely to be threatened by human activities and ongoing climate change. More

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Virus and cells
RVFV strain 35/74 was originally isolated from the liver of a sheep that died during a RVFV outbreak in the Free State province of South Africa in 197421. The strain was previously passaged four times in suckling mouse brain and three times in BHK cells. The virus used for IV inoculation of sheep was prepared by a further amplification in BHK-21 cells (ATCC CCL-10) cultured in CO2-independent medium (CIM, Invitrogen), supplemented with 5% FBS (Bodinco) and 1% Pen/Strep (Invitrogen).
To prepare a virus-spiked blood meal for membrane feeding of mosquitoes, the virus was amplified in Aedes albopictus C6/36 cells (ATCC CRL-1660). To this end, C6/36 cells were inoculated with a multiplicity of infection of 0.005 and cultured at 28 °C in absence of CO2 in L-15 medium (Sigma) supplemented with 10% fetal bovine serum (FBS), 2% Tryptose Phosphate Broth (TPB) and 1% MEM nonessential amino acids solution (MEMneaa). At 4 days post infection, culture medium was harvested, cleared by slow-speed centrifugation and titrated using Vero-E6 cells (ATCC CRL-1586), grown in DMEM supplemented with GlutaMAX, 3% FBS, 1% Pen/Strep and 1% Fungizone (DMEM +) at 37 °C and 5% CO2. Titers were determined using the Spearman-Kärber algorithm22,23.
Mosquito rearing and feeding on lambs
Rockefeller strain Ae. aegypti mosquitoes (Bayer AG, Monheim, Germany) were maintained at Wageningen University, Wageningen, the Netherlands, as described24. Briefly, mosquitoes were kept in Bugdorm-1 rearing cages at a temperature of 27 °C with a 12:12 light:dark cycle and a relative humidity of 70% with a 6% glucose solution provided ad libitum. Mosquitoes were subsequently transported to biosafety level three (BSL-3) facilities of Wageningen Bioveterinary Research (Lelystad, the Netherlands), where the mosquitoes were maintained with sugar water (6% sucrose in H2O), provided via soaked cotton pads covered with a lid to prevent evaporation in an insect incubator (KBWF 240, Binder) at 28 °C at a humidity of 70% and a 16:8 light:dark cycle.
Mosquito feeding on lambs was preceded by sedating the lambs with IV administration of medetomidine (Sedator). When fully sedated, cardboard boxes containing 40–50 female mosquitoes were placed on the shaved inner thigh of each hind leg (Fig. 1b,c). After 20 min of feeding, cardboard boxes were removed and atipamezol (Atipam) was administered via intramuscular (IM) route to wake up the animals. Fully engorged mosquitoes were collected using an automated insect aspirator and maintained with sugar water (6% sucrose in H2O), provided via soaked cotton pads covered with a lid to prevent evaporation, in an insect incubator (KBWF 240, Binder) at 28 °C at a humidity of 70% and a 16:8 light:dark cycle.
Feeding of mosquitoes using a Hemotek system
Blood meals to be used for Hemotek membrane feeding were prepared essentially as described before25. Briefly, erythrocytes were harvested from freshly collected bovine EDTA blood by slow-speed centrifugation (650 xg), followed by three wash steps with PBS. Washed erythrocytes were resuspended in L15 complete medium (L15 + 10% FBS, 2% TPB, 1% MEMneaa) to a concentration that is four times higher than found in blood. To prepare a blood meal, one part of the erythrocyte suspension was mixed with two parts of culture medium containing RVFV resulting in a final titer of 107.5 TCID50/ml as determined on Vero-E6 cells.
Mosquitoes were allowed to take a RVFV-spiked blood meal through a Parafilm M membrane using the Hemotek PS5 feeding system (Discovery Workshops, Lancashire, United Kingdom). Feeding was performed in plastic buckets (1 l) covered with mosquito netting. After blood feeding for approximately 1.5–2 h, fully engorged mosquitoes were collected using an automated insect aspirator and maintained with sugar water (6% sucrose in H2O), provided via soaked cotton pads covered with a lid to prevent evaporation in an insect incubator (KBWF 240, Binder) at 28 °C at a humidity of 70% and a 16:8 light:dark cycle.
Virus isolation
Virus isolation from plasma samples was performed using BHK-21 cells, seeded at a density of 20,000 cells/well in 96-wells plates. Serial dilutions of samples were incubated with the cells for 1.5 h before medium replacement. Cytopathic effect was evaluated after 5–7 days post infection. Virus titers (TCID50/ml) were determined using the Spearman-Kärber algorithm22,23.
To check for positive saliva, mosquitoes were sedated on a semi-permeable CO2-pad connected to 100% CO2 and wings and legs were removed. Saliva was collected by forced salivation using 20 µl filter tips containing 7 µl of a 1:1 mixture of FBS and 50% sucrose (capillary tube method). After 1–1.5 h, saliva samples were collected and used to inoculate Vero-E6 cell monolayers. Cytopathic effect (CPE) was scored 5–7 days later.
Serology
Weekly collected serum samples were used to detect RVFV-specific antibodies using the ID Screen Rift Valley Fever Competition Multi-species ELISA (ID-VET). This ELISA measures percentage competition between antibodies present in test sera and a monoclonal antibody. Neutralizing antibodies were detected using the RVFV-4 s-based virus neutralization test as described26.
RT-qPCR
Viral RNA was isolated with the NucliSENS easyMAG system according the manufacturer’s instructions (bioMerieux, France) from 0.5 ml plasma samples. Briefly, 5 µl RNA was used in a RVFV RT-qPCR using the LightCycler one-tube RNA Amplification Kit HybProbe (Roche, Almere, The Netherlands) in combination with a LightCycler 480 real-time PCR system (Roche) and the RVS forward primers (AAAGGAACAATGGACTCTGGTCA), the RVAs (CACTTCTTACTACCATGTCCTCCAAT) reverse primer and a FAM-labelled probe RVP (AAAGCTTTGATATCTCTCAGTGCCCCAA). Primers and probes were earlier described by Drosten et al.27. Virus isolations were performed on RT-qPCR positive samples with a threshold above 105 RNA copies/ml as this was previously shown to be a cut-off point below which no live virus can be isolated.
Pathology and (immuno)histopathology
Liver samples were placed on ice during the necropsies and subsequently stored at − 80 °C until virus isolations and RT-qPCR Tissue samples for histology and IHC were collected, placed in 10% neutral buffered formalin, embedded into paraffin and prepared for H&E staining or IHC staining for RVFV antigen using the RVFV Gn-specific 4-D4 mAb as described5.
Statistics
For statistical analysis, mosquito feeding and mosquito saliva positive rates per group were compared by fitting logistic regression mixed models where lamb or membrane were introduced as random effects. To compare viremia (based on virus isolation results) the area under the curve (AUC) representing the overall viremia during the infected period was calculated for each infected sheep. This AUC and peak of viremia was used for comparison between groups, which was done by fitting linear regression models.
Additionally we also assessed the variability observed between groups on the above mentioned variables (feeding and saliva positive rates, AUC and peak viremia). For these comparisons, data were first assessed for normality using the Shapiro–Wilk test. If data from all groups were normally distributed, the Bartlett’s test of homogeneity of variance was used. If the data did not have a normal distribution, the Fligner-Killeen test was applied.
Survival of infected lambs (time to death) was compared between experiment groups using Kaplan–Meier survival analysis and the mortality rates were compared fitting a logistic regression model.
For all comparisons, the threshold for significance was p  More

Study area
The study area was the Amazon basin (Fig. 6)42. The basin was sub-divided in four regions with markedly different forest dynamics, geography and substrate origin, adapted from the classification of Feldpausch et al.33: West (parts of Brazil, Colombia, Ecuador and Peru), Southeast (Bolivia and Brazil), Central-East (Brazil) and North (Brazil, Guyana, French Guiana and Venezuela). The natural vegetation mainly corresponds to broadleaf moist forests and tropical seasonal forests, with both terra firme and seasonally flooded forests. Across the Amazon, there is a wide range of average monthly rainfall (100–300 mm) and dry season length (DSL) (0–8 months)43.
Figure 6

The Amazon in South America with colored regions, defined in Feldpausch et al.33, indicating faster (West and Southeast) and slower forest dynamics (Central-East and North). Small black lines represent single-date airborne lidar data acquisitions from the EBA project (n = 610 flight lines). Red triangles illustrate multi-temporal lidar data acquisition over five sites (BON, DUC, FN1, TAL and TAP). Circles indicate the location of field inventory plots (n = 181). R v4.0.2 was used to plot this figure32.

Full size image

The five sites selected for the multi-temporal assessment of the static and dynamic gaps relationship (red triangles in Fig. 6) were: Adolpho Ducke forest (DUC), Tapajós National Forest (TAP), Feliz Natal (FN1), Bonal (BON) and Talismã (TAL). These areas were chosen to represent distinct forest types, vegetation structure and biomass stocks. The predominant vegetation types consisted of dense rain forests (DUC and TAP), seasonal forests (FN1), and open rain forests (TAL and BON). DUC and FN1 are mostly undisturbed forests, while TAP underwent fire and/or selective logging in the past. TAL and BON were affected by a known fire occurrence in 2010. The sites cover a gradient of aboveground biomass (AGB) that increase, in average, from TAL (185 Mg ha−1), FN1 (235 Mg ha−1), BON (251 Mg ha−1), and DUC (327 Mg ha−1) to TAP (364 Mg ha−1)44.
Data acquisition and pre-processing
Airborne lidar data
Multi-temporal lidar data were obtained by an airplane at each of the five sites (red triangles in Fig. 6), as part of the Sustainable Landscapes Brazil project. The time-interval window was close to 5 years and was sufficient to measure the long-term aggregated dynamics of tree mortality. The area covered by lidar in the 2012–2018 period was ~ 43 km2, ranging from 480 ha at TAL site to 1200 ha at DUC site (Supplementary Table S5).
In addition to the multi-temporal datasets, 610 single-date airborne discrete-return lidar data strips (approx. 300 m wide by 12.5 km long; ~ 3.75 km2 each) were acquired during 2016 (acquisition dates in Supplementary Figure S6A) using the Trimble HARRIER 68i system at an airplane. The average flight height was 600 m above ground and the scan angle was 45° (dataset from the EBA project31).
For both lidar datasets, multiple lidar returns were recorded with a minimum point density of 4 points m−2. Horizontal and vertical accuracy ranging from 0.035 to 0.185 m and from 0.07 to 0.33 m, respectively.
Following the procedures described by Dalagnol et al.19, the lidar point clouds were processed into canopy height models (CHM) of 1-m spatial resolution. The steps of CHM processing included the: (a) classification of the points between ground and vegetation using the lasground, lasheight, and lasclassify functions from the LAStools 3.1.145; (b) creation of a Digital Terrain Model (DTM) using the TINSurfaceCreate function from FUSION/LDV 3.646; (c) normalization of the point cloud height to height above ground using the DTM; and (d) CHM generation by extracting the highest height of vegetation using the CanopyModel function from FUSION.
Environmental and climate data
To analyze the environmental and climatic drivers of gap dynamics, we used a spatialized set of variables and products for the whole Amazon, including: (a) HAND product at 30 × 30 m47; (b) slope calculated from the Shuttle Radar Topography Mission (SRTM) at 30 × 30 m48; (c) soil fertility proxied by SCC at 11 × 11 km37; (d) floodplain cover map at 30 × 30 m49; (e) forest degradation proxied by a non-forest distance map derived from the 30-m global forest change dataset v1.4 (2000–2016)50; (f) monthly mean rainfall (mm), climate water deficit (mm) and wind speed (m s−1), obtained from the TerraClimate dataset at 5 × 5 km (1958–2015)43; and (g) DSL at 28 × 28 km51. All variables and products, except HAND and slope, were resampled to the predominant spatial resolution of most datasets (5 km × 5 km), especially the climate data. We used the SRTM instead of the lidar DTM because the very narrow lidar DTMs (300–500 m) would not permit to determining the lowest point in the terrain to accurately calculate the HAND for every pixel.
Long-term field inventory data
We used data from 181 long-term field inventory plots from the RAINFOR network (Fig. 6)5. The data were collected at closed canopy mixed forests with vegetation structure preserved from fire and logging. All trees with diameter at breast height (DBH) ≥ 10 cm were measured at least twice5. These plots had 852 censuses from 1975 to 2013 with median plot size of 1 ha. The mean re-census interval was 3 years. Tree stem mortality rates (m; % year−1) were calculated as the coefficient of exponential mortality for each census interval and each plot52 (Eq. 1). The m estimates were then averaged by plot and were weighted by the censuses interval length, in years1.

$$m = left[ {lnleft( {N0} right) – lnleft( {Nt} right)} right]/t$$
(1)

where N0 and Nt are the initial and final number of trees, and t is the censuses interval.
Data analysis
Gap definition and static–dynamic gaps relationship
Dynamic gaps were detected using multi-date lidar data at the five study sites: DUC, TAP, FN1, BON, and TAL. We define here dynamic gaps as those opened between two periods of observation associated with canopy turnover events, including tree mortality. For this purpose, we calculated a delta height difference of 10 m between the two acquisitions (~ 5 years apart) and filtered for detections with area greater than 10 m2. This height difference was strongly correlated with tree loss at the canopy level in previous studies19, 20. Because standing dead trees do not necessarily generate gaps, we assume that the dynamic gaps are mostly related to the felled canopy trees associated with broken and uprooted mode of death.
Static gaps were delineated using the CHM from the second lidar acquisition at the five sites (Supplementary Material S1). We applied and compared two types of gap delineation: a traditional method based on a fixed height cutoff (H = 2, 5 or 10 m), and an alternative method based on the relative height (RH = 33, 50, and 66% maximum tree height) around a neighborhood (W = 5–45 m). Since the relative height method did not depend on absolute height values, it should better account for local canopy variability and lower stature vegetation, as opposed to the fixed height method. For both methods, we tested a variety of parameters in the search of an optimal calibration amongst the sites. We filtered gaps for a minimum area of 10 m2, which corresponded to an approximation of the mean canopy area of trees greater than 5-cm DBH in tropical forests21. We also filtered them for a maximum area of 1 ha to automatically exclude open areas that very likely did not correspond to small-scale disturbance from treefall gaps21.
The spatial match between each static and dynamic gap event was assessed by intersecting the detections and calculating metrics of precision (p), recall (r) and F1-score (F) (Eqs. 2–4) (more information at Supplementary Material S1). p represents the percentage of total correct detections, r represents the percentage of reference data correctly mapped, and F represents the harmonic mean between p and r, that is, a balance between commission and omission errors. Methods and parameters were compared to determine the optimal method for static gap delineation, i.e. higher F means greater agreement between static and dynamic gaps.

$$Precisionleft( p right) = true , positives/number , of , gap , polygons$$
(2)

$$Recallleft( r right) = true , positives/number , of , mortality , polygons$$
(3)

$$F1 – scoreleft( F right) = left( {2 times p times r} right)/left( {p + r} right)$$
(4)

Finally, considering the optimal gap delineation method, we modeled the relationship between static-dynamic gaps at the landscape scale using a linear regression. For this purpose, annualized dynamic gap fraction and static gap fraction (i.e., the area occupied by gaps in relation to the total area of the flight line) were calculated at the 5-ha scale. Following the strategy by Wagner et al.53, we defined this value after several simulation tests between variable estimates, change rates and plot area (Supplementary Figure S7). Data and residuals were inspected for normality, and variables were transformed to the logarithmic scale prior to the linear model fitting. To assess the model, we calculated the coefficient of determination (R2), absolute Root Mean Square Error (RMSE) and relative RMSE (%) (ratio of RMSE and the mean of observations). To obtain more reliable and unbiased estimates of the model predictive performance, we calculated the RMSE considering out-of-sample values with a leave-one-site-out cross-validation (CV) strategy. Thus, we fitted the model with four sites and calculated the RMSE with predicted and observed values for the site not used in the modeling. We repeated this process for all five sites. A 95% prediction interval described the variability of tree mortality estimates from the gap fraction.
Spatial variability of static gaps across the Brazilian Amazon
We delineated static gaps on the single-date airborne lidar datasets (n = 610 flight lines) using the optimal gap delineation method and parameters assessed in the previous section. To characterize the gaps variability across the region, we calculated the gap fraction and mean gap size for each site.
Assessment of landscape- and regional-scale drivers of static canopy gaps
To quantify the relationship between static gaps and landscape- and regional-scale predictors, we employed correlation matrices and generalized linear models (GLM). Binomial GLM and Gaussian GLM were applied for landscape and regional models, respectively (detailed information at Supplementary Material S2). Models were assessed using a tenfold CV approach with 30 repetitions. The gap data used in this analysis were those obtained from the 610 single-date lidar data. We defined landscape-scale drivers as those showing great heterogeneity intra-site such as the topography (HAND and slope variables). We defined regional-scale drivers as those having great variability across sites such as the rainfall (Mean_pr and SD_pr), wind speed (Mean_vs and SD_vs), climate water deficit (Mean_def and SD_def), DSL, SCC, floodplains, and non-forest distance.
Through the modeling we evaluated if gap occurrence (presence or absence) and gap size increased at valleys and steep terrains of the Amazon, represented by low HAND and high slope, respectively. As previously demonstrated with tree mortality ground observations, we also tested if gap fraction would increase with: (1) higher water stress, represented by low Mean_pr, and high SD_pr, Mean_def, SD_def, and DSL; (2) higher soil fertility, expressed by high SCC; (3) higher wind speed, proxied by high Mean_vs and SD_vs; (4) higher forest degradation/fragmentation, represented by low non-forest distance; and (5) areas of seasonally flooded forests, expressed by high floodplains cover. Model residuals were inspected in comparison to fitted values using also variogram and Moran’s I analyses to assess for potential biases and spatial correlation (detailed information in Supplementary Material S2). Static gap fraction and Nonforest_dist were transformed to log-scale due to non-normality data.
Amazon-wide dynamic gaps mapping and relationship with tree mortality
To obtain a map of dynamic gap estimates over the Amazon, we first applied the GLM model based on environmental and climate drivers to estimate static gap fractions for the whole region. We then applied the static–dynamic gaps relationship to estimate annualized dynamic gap fraction (% year−1). To explore the opportunities for tree mortality estimates based on gap dynamics, we compared the spatialized dynamic gap estimates with time-averaged mortality rates from long-term field inventory data using a linear model. The model was assessed using a tenfold CV approach with 30 repetitions and the RMSE calculated out-of-sample. We acknowledge that the comparison between field tree mortality and lidar gap estimates is not trivial. However, it is the best source available of independent mortality data to compare the results. Field plot-estimates located within the same 5-km cell of the lidar gap estimates were averaged, resulting in 88 pairs of lidar- and field-estimates samples for validation. The mean annualized dynamic gap fraction per Amazonian region (Fig. 6) was extracted and compared using one-way ANOVA and post-hoc Tukey–Kramer tests. More

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Biodegradability of crude oil by two enrichment cultures
The enrichment culture H7S showed no obvious proliferation in the first five days because sample 7S was a sulphide rock, while H11S showed visible proliferation after the fourth day. After 14 days of cultivation, gravimetric analysis demonstrated that the enrichment cultures H7S and H11S exhibited similar oil-degrading abilities and degraded 54% and 56% of the crude oil, respectively (Fig. 1).
Figure 1

The oil degradation efficiency of the two enrichment cultures H7S and H11S.

Full size image

The biodegradation percentages for total n-alkanes (C10–C34) and polycyclic aromatic hydrocarbons (PAHs) were calculated by comparing the two enrichment cultures with the negative controls (Fig. 2). Based on evaluation with C17/pristane and C18/phytane, the degradation efficiencies of the two enrichment cultures were significantly better than those of the negative controls (P  More

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