2.1 Study Region
The town of Alcañiz is located in the southern part of the central Ebro basin (also sometimes referred to as the Ebro depression) in the Teruel Province, northeast Spain. The Ebro River, located ~ 22 km to the north of Alcañiz, bisects the basin as it flows from the northwest towards the Mediterranean Sea in the southeast. The river receives much of its discharge from snowmelt and rain-fed tributaries originating in the Pyrenees to the north and maintains perennial flow but the central basin has one of the highest annual water deficits in Europe (Herrero and Snyder, 1997), with a mean air temperature of ~ 15°C, an annual precipitation of 300–350 mm, and a mean annual potential evapotranspiration exceeding 1000 mm (e.g. Valero-Garcés et al., 2000a; Gutiérrez et al., 2005). Despite this widespread aridity, areas both south (Bajo Aragón region, including Alcañiz) and north (Monegros region) of the Ebro River host numerous saladas ranging in sizes up to ~ 200 ha (2 km2) (e.g. Sánchez et al., 1998; Gutiérrez et al., 2005; Castañeda et al., 2005; Domínguez-Beisiegel et al., 2013b). Many previous investigations have focused on the saladas in the Monegros region, particularly with respect to their geomorphological development and Quaternary history, and their present-day hydrology, geochemistry and ecology (e.g. Vizcayno et al., 1995; Samper-Calvete and García-Vera, 1998; Castañeda et al. 2005, 2013; Castañeda and García-Vera, 2008; González-Sampériz et al., 2008; Conesa et al., 2011; Mees et al., 2011; Domínguez-Beisiegel et al. 2013a; Gutiérrez et al., 2013; Casamayor et al., 2013). By contrast, and with a few exceptions (e.g. Ibáñez, 1973, 1976; Macklin et al., 1994; Gutiérrez et al., 2005), the saladas near Alcañiz have been subject to fewer investigations. To the best of our knowledge, our study is is the first to quantify soil C stocks and determine CO2 efflux for any of the saladas in the central Ebro basin, and the first to describe in detail the microbial communities of the saladas near Alcañiz.
Across the central Ebro basin, the saladas are commonly found in depressions formed by physical weathering, dissolution, and aeolian deflation of fine-grained sedimentary rocks, principally siltstones, mudstones, gypsiferous units, and limestones (Sánchez et al., 1998; Castañeda et al., 2005; Gutiérrez et al., 2005). Consequently, many saladas are partially bordered by a bedrock escarpment or elevated ground and contain a central flat area that is periodically inundated for varying durations (Domínguez-Beisiegel et al., 2013a). Approximately 50% of the water input into the saladas derives directly from rainfall, with 40% from groundwater and the remainder from surface runoff (Castañeda and García-Vera, 2008; Castañeda et al., 2013). Local geological structure and lithology can exert strong controls on water supply and quality; for example, in the Monegros region, numerous northwest-southeast trending lineaments and faults (Arlegui and Soriano, 1998) contribute to a complex hydrology and hydrochemistry that can vary markedly between adjacent saladas (Castañeda et al., 2013). Nevertheless, during the northern hemisphere autumn and winter (October through late February), the salada surfaces are typically saturated and/or inundated. The water in the saladas becomes increasingly saline as it evaporates during the spring and summer. Water tables are shallow and saline (> 100 g L− 1), with the high evaporation rates leading to a concentration of evaporites within the soils on the soil surfaces (Vizcayno et al., 1995; Samper-Calvete and García-Vera, 1998). In the Monegros region, for example, the evaporites take several forms, and include gypsum and halitic-mirabillitic efflorescences on soil surfaces, mirabillite crystallisation in small depressions, and saline crusts formed by precipitation of bloedite and halite (Vizcayno et al., 1995).
Across the central Ebro basin, salada soils are fine grained (silt and clay dominated), and classified as Typic Haplogypsids, Typic Haplocalcids, and Gypsic Aquisalids (Soil Survey Staff, 2010, cited in Castañeda et al., 2013). Soils are typically saline, gypsiferous and enriched in organic matter compared to the surrounding soils, owing to the remains of plants, insects, algae, extremophile microbes (Domínguez-Beisiegel et al., 2013a) and crustaceans, including in some saladas the brine shrimp Artemia salina (Valero-Garcés et al., 2000a, b). Most salada surfaces are devoid of vascular plants, but halophytes can be found around the margins where moisture and salinity conditions are more favourable (Castañeda et al., 2013). In the Monegros region, many saladas have been degraded or lost due to ploughing or levelling, with numbers declining from 136 in 1927 to 96 in 2006 (Domínguez‐Beisiegel et al., 2013b). Since the 1960s, large parts of the central Ebro basin have been irrigated, leading to water seepage, leaching, salt redistribution, and local water table uplift within and around some saladas (Vizcayno et al., 1995). These changes have led to the disappearance of some protected halophyte species such as Chenopodioideae (Conesa et al., 2011; Domínguez‐Beisiegel et al., 2013b).
2.2 Study saladas
Data were collected in May 2018 at Salada Grande, Salada Pequeña and Salada de la Jabonera de las Torrazas (hereafter shortened to Salada Jabonera), located ~ 5 km west of Alcañiz (Fig. 1a). The area covered by Salada Grande, Pequeña and Jabonera is ~ 1.24, 0.19 and 0.19 km2, respectively. The saladas have developed in an area of sub-horizontal Miocene siltstones, mudstones and ribbon or sheet sandstones and microconglomerates (sensu Friend et al., 1979). Preferential weathering of the less resistant argillaceous strata has tended to give rise to the depressions, leaving some of the more resistant calcareous sandstone and microconglomerate bodies in positive relief in the form of sinuous, inverted palaeochannels on the salada margins (Sánchez et al., 1998; Gutiérrez et al., 2005). All three saladas form part of a 7 km2 Natura 2000 site (Saladas de Alcañiz, site code ES2420114), which was designated on account of being one of the most important endorheic areas on the Iberian Peninsula, with plant communities to adapted to saline conditions and a rich fauna linked to seasonal inundation (European Commission, no date). There is a topographic gradient from the surface of Salada Pequeña at ~ 359 m above sea level (masl), through Salada Grande at ~ 351 masl, and to Salada Jabonera at ~ 340 masl. Although topographically the highest, Pequeña is the wettest of the three saladas (Fig. 1a-b). Shallow (< 0.5 m deep) but frequently prolonged standing water occurs over much of the salada surface, partly because of rainfall and runoff but also because of contributions from groundwater and a small stream that periodically inflows from the southwest (Fig. 1a). Salada Grande is drier and Salada Jabonera drier still, but both typically experience shallow standing water over at least part of their surfaces for some of the year as a result of rainfall and runoff, and possibly some limited groundwater contributions. In all three saladas, the ephemeral nature of the standing water, combined with the widespread dissolution of halite and gypsum and its subsequent evaporative concentration, leads to a high soil pH (Gutiérrez et al., 2005). Analysis of water extracts from saturated pastes of Salada Grande sediments indicates that soils are dispersive when wet but prone to pelletisation when desiccating, which promotes aeolian deflation during dry periods (Gutiérrez et al., 2005). The surfaces of Pequeña and Grande are mostly unvegetated, except at the fringes where soils are elevated above the salada surface by a few metres (Fig. 1a-b). Salada Jabonera, the driest of the saladas, has a patchy cover of vascular halophytic shrubs and grasses across the surface (Fig. 1a-b), including Halopeplis amplexicaulis (Vahl) Ung.-Sternb., Arthrocnemum macrostachyum (Moric.) K. Koch, Microcnemum coralloides (Loscos & Pardo) and Atriplex halimus L. (Amaranthaceae). Around all three saladas, there are also localised zones where overland flow has transported fine-grained soil and associated organic matter from the surrounding slopes short distances onto the salada surface, and wind erosion may also contribute soil and organic matter. The lower topographic setting of Salada Jabonera with its steeper northern and eastern margins (Fig. 1b) may result in an enhanced contribution of soil and organic matter from overland flow.
2.3 Surface water coverage
To quantify the areal extent and frequency/duration of water coverage on each salada over six years, data were extracted from a time series of 86 cloud-free Landsat 8 OLI (2013–2016) and Sentinel-2 L1C (2016, 2017 and 2018) satellite images. Scenes were downloaded via EarthExplorer (USGS) and converted to Top of Atmosphere Reflectance using the Semi-Automatic Classification Plugin (SCP) in QGIS 3.4. Red, green and blue bands were stacked to generate true-colour composite images, with near infrared and shortwave infrared bands also used to aid water detection. A Modified Normalised Difference Water Index (MNDWI) (Eq. 1) was used to enhance the identification of surface water (Xu, 2007; Du et al., 2016; Li et al., 2018, 2021).
Equation 1.
For each image, surface water area was manually digitised using ArcGIS 10.5. Mapping is deemed accurate to +/- 1 pixel (20 m for Sentinel and 30 m for Landsat MNDWI) resulting in a maximum uncertainty of +/- 12%, although in reality, uncertainty is much lower owing to the distinct boundaries between the water surface and adjacent bare soil surface in the true-colour composites.
2.4 Soil moisture and temperature
The moisture content of salada soils to 5 cm depth was recorded in a grid pattern every 10 m across the surface of each salada using a DeltaT SM150 probe (n = 100, 120 and 180 on Saladas Pequeña, Grande and Jabonera, respectively). The grids corresponded approximately to the area of the sampling sites for CO2 efflux determination (Fig. 1a), although additional soil moisture readings were taken with the DeltaT probe adjacent to the chambers used to determine CO2 efflux (Sect. 2.6).
2.5 Soil physical and chemical properties
On each salada, four pits up to 0.5 m deep were excavated at equal distances within the area of the soil moisture sampling grid. From each pit, up to five soil samples were collected from a range of depths (see Supplementary Information), then air dried and bagged prior to analyses. H+ ion activity was determined in a 1:2.5 soil-water mixture using a pH probe. Approximately 5 g of each sample was dried at 105°C to a constant weight and heated at 430°C for 16 hours in a muffle furnace to determine organic matter by loss-on-ignition. Bulk density was determined after weighing the oven dry mass of a soil sample collected from the subsurface using a stainless steel tube with an internal volume of 99 cm3.
An elemental analyser (vario PYRO cube, Elementar UK Ltd.) was used to determine the total C, N and S content of 10–30 mg sub-samples in tin capsules (the mass was dependent on the amount of loss-on-ignition). C stable isotope measurements were made on the same sub-samples using a coupled mass spectrometer (visION, Elementar UK Ltd.). Organic C content and isotopic compositions were determined from separate sub-samples, after removal of inorganic C with 50 µL of 10% HCl in silver capsules (Brodie et al., 2011) and then overnight drying at 60°C. Stable isotope data are reported as δ¹³CVPDB values, the proportion per thousand (‰) variation from the ratio of 13C/12C in V-Pee Dee Belemnite (VPDB) (Thompson et al., 2006; Ibell et al., 2013) (Eq. 2):
δ ¹³CVPDB = (RsampleC-RVPDB)/RVPDB*1000 Eq. 2.
where RsampleC indicates the ratio of 13C/12C in the sample, and RVPDB is the ratio of 13C/12C in the VPDB. Isotope ratio measurements were calibrated to the VPDB scale using commercially available standard reference materials (B2205 EMA P2, B2153 low organic content soil, B2151 high organic content sediment and B2159 sorghum flour, all from Elemental Microanalysis, UK). Further elemental analysis on each sediment sample was performed with a Nito XL3t 950 GOLDD + portable X-ray fluorescence spectrometer (pXRF). Data in the results section of this article are reported as depth averages, with depth specific data reported in the Supplementary Information (S1).
2.6 Soil CO2 efflux
To quantify soil CO2 efflux, respiration chambers were positioned at 12 locations in a grid across the water-free surface of each salada (Fig. 1a). Maximum distances between chambers were c. 50 m on Salada Pequeña, c. 120 m on Salada Grande and c. 70 m on Salada Jabonera. Comprehensive details of the chamber design and method can be found in Thomas (2012) and Thomas et al. (2018) but, in summary, are made from white uPVC and comprise two parts: i) a lower chamber that when pushed 3 cm into the surface forms an air-tight seal; and ii) a screw-on lid that enables soil gases to accumulate inside the chamber and to be sampled during measurement cycles. The chamber lids contain a sampling port covered with a Suba seal for gas extraction and a two-way valve to ensure any pressure differences between the chamber and atmosphere are minimal and rapidly equilibrated. Chamber surface area is 83 cm2 and in this study chamber volume ranged from 0.48 to 0.52 litres depending on insertion depth. Heat sinks mounted through the chamber walls ensured the internal air temperatures were not elevated above ambient. Three of the 12 chambers were equipped with a sensor (USB502, Adept Science, UK) to record the air temperature and humidity inside the chamber at 2 minute intervals.
Measurements were taken three times each day to capture a range of temperature and light conditions, giving a total of 36 measurements at each of the three saladas. Following previous sampling protocols to determine soil CO2 efflux (see for example Thomas, 2012; Thomas et al., 2018), the lid was placed on the chamber and 12 ml of gas was immediately extracted through the sample port using a syringe and hypodermic needle secured with a leur lock. After approximately five minutes, another syringe was gently pumped to mix the air within the chamber before a second sample was collected. Both sample CO2 concentrations were determined immediately after each of the three measurement cycles using an EGM-4 infrared gas analyser (PP Systems, Amesbury, USA). Mass CO2 flux in mg m− 2 hr− 1 was determined from the changes in CO2 concentration normalised to mean temperature and pressure during measurement (Kutzbach et al., 2007). To correct for the effect of any diffusion suppression owing to the accumulation of CO2 inside the chamber, a diffusion correction factor was applied (for details see Thomas, 2012). At the time of sampling, surface soil temperature and moisture adjacent to each respiration chamber were determined (n = 3) using an infra-red thermometer and a soil moisture probe (SM150, Delta-T Devices Ltd., Cambridge, UK).
2.7 DNA extraction and sequencing
At two locations on each salada, one gram of soil was collected from three depths (0–1 cm, 20–21 cm and 49–50 cm) for DNA extraction. Samples were stored in a 15 mL aseptic centrifuge tube containing 2 mL of Life Guard Soil Preservation solution (Qiagen, UK). The DNA was PCR amplified and sequenced for three primers, yielding 1, 863 and 925 valid sequences, although sequencing failed for six of the 54 analyses. Total DNA was extracted using a DNA extraction kit (Omega, USA), and DNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The quality of the extracted DNA was verified using 1% agarose gel electrophoresis, before amplification using a GeneAmp 9700 PCR System (ABI, USA). Prokaryotic bacterial 16S rRNA genes were amplified using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). Aarchaeal 16S rRNA genes were amplified using primers 3NDF (5'-GGCAAGTCTGGTGCCAG-3') and 4-euk-R2R (5'-ACGGTATCTRATCRTCTTCG-3'). Eukaryotic 18S rRNA genes were amplified using primers 524F10extF (5'-TGYCAGCCGCCGCGGTAA-3') and Arch958RmodR (5'-YCCGGCGTTGAVTCCAATT-3'). Each 20 µL of the PCR reaction system contained 0.2 µL bovine serum albumin (BSA), 4 µL of 5×FastPfu Buffer, 2 µL of 2.5 mM dNTPs, 0.4 µL of FastPfu polymerase, 1.6 µL of primers (0.8 µL and 5 µM each), and 10 ng of the template. The PCR amplification program comprised the following cycles: i) initial denaturation at 95°C for 3 min; ii) 29 (bacteria), 37 (archaea) and 37 (eukaryotes) cycles at 95°C for 30 s, annealing at 53°C for 30 s, and elongation at 72°C for 45 s; and iii) the final extension at 72°C for 10 min. The PCR products were extracted from a 2% agarose gel, and further purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, U.S.A.). The purified PCR products were then quantified using a QuantiFluor-ST quantitative system (Promega, USA) and sequenced on an Illumina MiSeq platform (Illumina, USA).
The raw sequencing data were quality filtered (< Q20) using a Trimmomatic trimmer, and merged using FLASH (Magoč and Salzberg, 2011). The chimera were eliminated using UCHIME, and the high-quality sequences were classified into different operational taxonomic units (OTUs) at a 97% similarity cut-off using UPARSE (Yuan et al., 2018). The taxonomic information from all the sequences was annotated by an RDP classifier using the Silva database (SSU132), with a confidence threshold set at 70%. Shannon index was analysed by Mothur (V1.30.1), with the details for this index calculation described at https://mothur.org/wiki/shannon/.
2.8 Statistical analyses
Statistical analyses of the soil chemical and CO2 efflux data were performed using SPSS (IBM v. 25). One-way analysis of variance (ANOVA) was used to test whether mean values of the dependent factors (moisture, temperature, total C, total N, S, pH, δ¹³C) were significantly different between saladas. For CO2 efflux, where multiple readings were taken at each site, repeated measure ANOVA was used. The Levene’s F statistic was used to test equality of variance. Although ANOVA can tolerate inhomogeneous variance, where these conditions were not met the more robust Welch and Brown Forsythe tests of significance were used. Tukey’s HSD post-hoc test was undertaken to determine whether saladas were significantly different with a probability of p < 0.05.