Effects of Vertically Heterogeneous Soil Salinity on Genetic Polymorphism and Productivity of the Widespread Halophyte Bassia prostrata

Salinity is one of the environmental factors that affects both productivity and genetic diversity in plant species. Within the soil profile, salinity is a dynamic indicator and significantly changes with depth. The present study examined the effects of the vertical heterogeneity of soil salinity chemistry on the plant height, fresh and dry biomass accumulation, water content, level of genetic polymorphism, and observed and expected heterozygosity in seven populations of halophyte Bassia prostrata in natural habitats. Soil salinity ranged from slight (Ssalts = 0.11–0.25%) to extreme (Ssalts = 1.35–2.57%). The main contributors to salinity were Na+, Ca2+, and Mg2+. Multivariate analysis revealed that biomass accumulation is positively affected by moderate/high salinity in 20–60 cm soil layers, which may be associated with the salt required for the optimal growth of the halophyte B. prostrata. The formation of seed genetic diversity is negatively affected by slight/moderate salinity in the 0–40 cm layers. An increase in divalent ion content can reduce genetic diversity and increase the local adaptation of B. prostrata to magnesium–calcium sulfate salinity. The effect of the in-depth distribution of soil salinity on productivity and genetic diversity may be related to seasonal variables during biomass accumulation (summer) and seed formation (autumn).


Introduction
Salinity is a significant environmental problem that limits plant productivity, especially in arid and semiarid regions that cover approximately 40% of the globe. Semiarid regions are projected to become drier and more saline due to rising global temperatures [1][2][3]. Vegetation survival and productivity are primarily regulated by the water balance in soil, which affects the water balance and photosynthetic rate in plants [4]. Soils in drylands are usually heterogeneous in space and time due to the presence of biotic and abiotic elements. Spatiotemporal variations in soil salinity and water content are well documented [5]. Salinity amplifies the effects of soil drought on plants by creating additional osmotic pressure. Soil is considered saline when the salt content exceeds 3-5 g salt/L in the soil solution, when electrical conductivity (EC) exceeds 2-4 dS/m, or when the sum of salts exceeds 0.15-0.2%, creating osmotic pressure above 0.2 MPa, which significantly reduces the yield of the most crops [2,6]. Salinity reduces plant growth and prematurely ages mature leaves, which leads to a decrease in the functional leaf area. A decrease in plant biomass is also influenced by Na + and Cl − toxicity and the accompanying oxidative stress [2,7]. Halophytes are highly salt-tolerant plants but underutilized resources that occupy naturally saline soil environments in coastal estuaries and inland salt flats in arid and semiarid zones [8]. Nowadays, climate-smart agriculture (CSA) practices increasingly use wild The present study aims to investigate the effects of the level and chemistry of salinity within different soil layers (including horizontal and vertical variations in the soil characteristics) on the productivity and genetic diversity of the halophyte B. prostrata to clarify the adaptive mechanism it uses to withstand fluctuations of salt accumulation along soil depth profiles.

Study Area
Studies were carried out in the northwestern Caspian Lowland (Russia) ( Figure 1A). The region is a flat marine accumulative plain, which is characterized by the almost complete absence of surface and subsurface runoff. According to climate parameters ( Figure 1B), the region is arid, with an average annual temperature of 8.7 °С and precipitation of 291 mm. In Caspian Lowland plain landscapes, solonetzic complexes are widespread; depressions and other negative relief elements (microdepressions, depressions, estuaries) are characterized by dark-colored chernozem-like or meadow-chestnut soils [29]. Seven typical habitats of B. prostrata were selected for the study based on differences in the soil salinity levels (No. 1-7 in Figure 1A,). Five of them were located near the salt lakes Bulukhta and Elton at different distances from the coastline. The other two habitats were in the plain part between these lakes ( Figure 1A). B. prostrata habitats were characterized mainly by solonetzic and/or light chestnut solonetzic soils and desert steppe vegetation.

Plant Sampling
Bassia prostrata (L.) A.J. Scott (Kochia prostrata (L.) Schrad.) (Chenopodiaceae) is a typical perennial C 4 halophyte native to arid and semiarid rangelands in Central Eurasia and the Western United States. B. prostrata naturally occurs in all kinds of soils, such as saline, sandy, rocky, and poor soils [24,30,31]. B. prostrata has a thick, woody root system that can penetrate 3-6.5 m depths and lateral roots stretching 130-160 cm that mine for moisture in the upper (up to 60 cm) soil layers [30,31]. This is the reason for studying  Figure 1A). The aboveground parts of five plants were harvested in each habitat in the middle of September for biomass analysis. More than 100 seeds from 10 to 15 mother plants from each habitat (population) were collected at the beginning of November and combined to generate a seed pool for population genetic analysis.

Soil Sampling and Analysis
Seven habitats of B. prostrata soil pits (Nos. 14,11,15,10,18,7, and 6, corresponding to habitats Nos. 1 to 7 in Figure 1A) were excavated. Profiles were examined to depths of 0 to 60 cm. Three soil samples (n = 3) were used for the analysis of each soil layer of each habitat. Chemical and physicochemical analyses were performed at the Analytical Laboratory of the V.V. Dokuchaev Soil Science Institute using standard methods [32]. Calcium and magnesium concentrations in water extracts (1:5) were determined with the complexometric titration method; sodium and potassium concentrations were determined with the flame photometry method; the total alkalinity was determined using titration with sulfuric acid (with methyl orange indicator); the concentration of chlorine ions was determined with argentometry (according to Mohr); and the concentration of sulfate ions was determined using titration with BaCl 2 . The content of ions Na + , K + , Ca 2+ , Mg 2+ , Cl − , SO 4 2− , and HCO 3 − are presented in cmol(eq)/kg. The sum of salts (S salts ) represents the sum of the mass fraction of ions from the solid soil residue (%) [6].

Plant Biomass and Water Content
Plant height, fresh (FW) and dry (DW) biomass, and water content (W) were assessed for aboveground parts of B. prostrata plants (n = 5) from seven habitats. Biomass was estimated for fresh and dry shoots. Plant samples were dried at 80 • C for two days until reaching a constant mass to quantitatively measure the dry shoot matter. The water content in the shoots was calculated according to the following formula: (1)

Population Genetic Analysis
Genetic diversity can be studied using neutral markers (based on differences in DNA sequences) and partially selective markers (isozymes), which can reflect changes in environmental conditions [33,34]. In this study, we used isozymes (alternative forms of the enzymes encoded by different alleles of the same gene) to assess the genetic diversity of the populations.
For each population of B. prostrata, 50 seeds from the seed pool (more than 100 seeds from 10 to 15 mother plants) were germinated, and all good germinated seeds (n = 25-35 per population) were analyzed for genetic polymorphism. Population genetic analysis was performed on embryos using starch gel electrophoresis of the following enzymatic systems: glutamate oxaloacetate transaminase (GOT (AAT), E.C. 2.6.1. The seeds were cleaned of their wings and soaked in water for 12 h and homogenized in 80 µL of Tris-HCl buffer with KCl, MgCl 2 , EDTA, Triton X-100, and PVP. Enzymes were separated in 10% starch gel using two buffer systems. In system 1, the electrode buffer was 160 mM Tris-50 mM citric acid, pH 8.0; the gel buffer was prepared by diluting 10 mL of the electrode buffer with 90 mL H 2 O. In system 2, the electrode buffer was 300 mM boric acid-60 mM NaOH, pH 8.2; the gel buffer was 80 mM Tris-9 mM citric acid, pH 8.7. Electrophoresis was performed at 90 V, 40-50 mA in buffer system 1 or at 210 V, and 70-80 mA in buffer system 2 for 4-6 h at 5 • C. Staining of particular enzymes and genetic interpretation of the results followed standard techniques according to Soltis and Soltis [35] and Spooner et al. [33]. The level of genetic polymorphism was estimated

Statistical Analysis
Principal component analysis (PCA) was carried out using R software (version 3.6.1). Table 1 and Figure 2 show the means of the obtained values and their standard errors (n = 3 for soil samples and n = 5 for plant samples).

Plant Growth and Water Content
B. prostrata plants varied significantly in growth parameters between populations ( Figure 2). The greatest plant heights were in populations Nos. 2, 4, and 7 (Figure 2A mined with the flame photometry method; the total alkalinity was determined using ti-tration with sulfuric acid (with methyl orange indicator); the concentration of chlorine ions was determined with argentometry (according to Mohr); and the concentration of sulfate ions was determined using titration with BaCl2. The content of ions Na + , K + , Ca 2+ , Mg 2+ , Cl − , SO4 2− , and HCO3 − are presented in cmol(eq)/kg. The sum of salts (Ssalts) represents the sum of the mass fraction of ions from the solid soil residue (%) [6].

Plant Biomass and Water Content
Plant height, fresh (FW) and dry (DW) biomass, and water content (W) were assessed for aboveground parts of B. prostrata plants (n = 5) from seven habitats. Biomass was estimated for fresh and dry shoots. Plant samples were dried at 80 °C for two days until reaching a constant mass to quantitatively measure the dry shoot matter. The water content in the shoots was calculated according to the following formula:

Population Genetic Diversity
An analysis of eight enzyme systems in seven B. prostrata populations revealed ten loci; one of them (Sod) was monomorphic in all populations. The other nine loci were polymorphic: G6pd in all populations; Me in six populations; Gdh in four populations; Got, 6pgd, and Mdh-A in 3 populations; and Dia-A, Dia-B, and Mdh-B in one population. Values of observed heterozygosity (H o ) varied from 5 to 47% among polymorphic loci and populations ( Figure 3A), whereas expected heterozygosity (H e ) varied from 5% to 59% ( Figure 3B). The average (for all loci) observed heterozygosity varied from 5.5% to 11.1%, and expected heterozygosity varied from 6.2% to 15.9% in populations of B. prostrata ( Figure 3C). The polymorphic loci proportion (P) among the populations was 20-70% ( Figure 3C). On average, populations Nos. 2, 3, 5, and 6 were more polymorphic than populations Nos. 1, 4, and 7 ( Figure 3).

Plant-Soil Interaction
Principal component analysis (PCA) did not reveal significant correlations between B. prostrata fresh and dry biomass and soil properties in 0-20 cm soil layers ( Figure 4A). There were significant positive correlations between B. prostrata fresh and dry biomass Life 2023, 13, 56 7 of 12 and the sum of salts and the sum of the contents of anions Ca 2+ , Mg 2+ , and SO 4 2− in the 20-40 cm and, to a lesser degree, 40-60 cm soil layers ( Figure 4B), as well as with K + content in 40-60 cm layers ( Figure 4C).
PCA revealed the negative dependencies of genetic polymorphism parameters (P, H e , and H o ) on K + , Ca 2+ , and sulfate ions contents and, to a lesser degree, on the sum of salts and the sum of anions in the 0-20 cm soil layers ( Figure 4D). In addition, a negative correlation was observed between P, H e , and H o from one side and Mg 2+ , K + , Ca 2+ , and SO 4 2− contents in 20-40 cm soil layers from the other side ( Figure 4E). There were no correlations between genetic polymorphism parameters and soil properties in the 40-60 cm layers ( Figure 4F).

Population Genetic Diversity
An analysis of eight enzyme systems in seven B. prostrata populations reveale loci; one of them (Sod) was monomorphic in all populations. The other nine loci polymorphic: G6pd in all populations; Me in six populations; Gdh in four popula Got, 6pgd, and Mdh-A in 3 populations; and Dia-A, Dia-B, and Mdh-B in one popul Values of observed heterozygosity (Ho) varied from 5 to 47% among polymorphi and populations ( Figure 3A), whereas expected heterozygosity (He) varied from 5 59% ( Figure 3B). The average (for all loci) observed heterozygosity varied from 5. 11.1%, and expected heterozygosity varied from 6.2% to 15.9% in populations of B trata ( Figure 3C). The polymorphic loci proportion (P) among the populations wa 70% ( Figure 3C). On average, populations Nos. 2, 3, 5, and 6 were more polymo than populations Nos. 1, 4, and 7 ( Figure 3).  He, and Ho) on K + , Ca 2+ , and sulfate ions contents and, to a lesser degree, on the sum of salts and the sum of anions in the 0-20 cm soil layers ( Figure 4D). In addition, a negative correlation was observed between P, He, and Ho from one side and Mg 2+ , K + , Ca 2+ , and SO4 2− contents in 20-40 cm soil layers from the other side ( Figure 4E). There were no correlations between genetic polymorphism parameters and soil properties in the 40-60 cm layers ( Figure 4F).

Discussion
The habitats of Bassia prostrata in this study were characterized by significant diversity in the degree and chemistry of soil salinity; high salinity occurred at different soil depths (Table 1). B. prostrata has wide edaphic plasticity and can grow on various soil genesis, e.g., chestnut, light-chestnut alkaline soils, and solonetz, as well as on soil-forming rocks of different compositions, from light sandy to heavy loamy, stony, and gypsum [30,36].
Our results revealed differences in correlations between B. prostrata aboveground biomass accumulation and seed genetic polymorphism and the chemistry and degree of salinity of different soil layers. The genetic diversity level was affected by the salinity degree and the chemistry of the uppermost soil layers (0-20 cm, 20-40 cm), and biomass accumulation was mainly affected by the salinity of the 20-40 cm and 40-60 cm soil layers. Such differences may be associated with different seasons of aboveground biomass and seed pool formation. B. prostrata biomass accumulation (before flowering) occurs mainly in the summer, the hottest and driest season: 23-26 • C, 40-43% humidity, and 65.7 mm precipitation ( Figure 1B). In the summer, the drying of the uppermost soil layers can be observed, and plants receive water and dissolved salt ions from lower soil layers, affecting biomass formation. Our study showed a positive dependence of B. prostrata productivity on the degree of salinity in 20-40 cm soil layers ( Figure 4B). B. prostrata, as a halophyte, requires a certain amount of salt in the substrate for optimal growth [37] and has high productivity in soils with 20 dS/m (EC) salinity [31]. The content of the main plant nutrient K + in seven soil habitats decreased from the upper to lower layers, whereas the Na + concentration increased (means of K + /Na + were 0.45 and 0.01 in the 0-20 cm and 40-60 cm soil layers, respectively; Table 1). Despite the fact that plants growing in saline habitats have acquired mechanisms that allow for selective uptake of K + when Na + dominates in the substrate [37], in B. prostrata plants, K + content in tissues decreased when Na + exceeded 100-200 mM NaCl [38]. Thus, the selective absorption of K + from the 40-60 cm soil layer under conditions of increased competition with Na + affects B. prostrata biomass accumulation in natural habitats ( Figure 4C).
Ca 2+ and Mg 2+ ions are also essential mineral nutrients. Ca 2+ is a universal signal in all eukaryotic cells and participates in many other cellular processes, for example, in the maintenance of cell membrane integrity, cation-anion balance, and osmoregulation [39,40]. Mg 2+ is an activator of more than 300 enzymes, in particular, photosynthetic and respiratory ones, which are also needed for DNA and RNA synthesis [41,42]. It is well known that Ca 2+ plays a protective role in a plant's response to salinity. Much less is known about the role of Mg 2+ in the salt tolerance of plants [39]. However, it was shown that low concentrations of mixed salts with CaCl 2 , and MgSO 4 are necessary for the successful seed germination of B. prostrata [43]. Our study showed that Ca 2+ and Mg 2+ contents contributed significantly to soil salinity in B.prostrata habitats (Table 1). Positive correlations between biomass accumulation and Ca 2+ and Mg 2+ contents in the 20-40 cm soil layer ( Figure 4B) indicate their necessity for B. prostrata growth. The influence of magnesium in this soil layer can be associated with the optimal K + /Mg 2+ ratio. The K + /Mg 2+ ratio for soils and plant tissues is critical to maintaining optimal plant nutrition and, hence, plant productivity [42]. The K + /Mg 2+ ratio (0.09 ± 0.03) in the 20-40 cm soil layer in B. prostrata habitats was less than that of the 0-20 cm soil layer (0.56 ± 0.17) but higher than that of the 40-60 cm soil layer (0.02 ± 0.01).
B. prostrata seeds are formed in autumn, during a cooler and rainier period (1-16 • C, 49-81% humidity, 77.1 mm precipitation; Figure 1B) when the upper soil layers are moist and plants receive water and dissolved salt ions from them. At the same time, the need for water decreases due to lower air temperatures and higher humidity. Therefore, the formation of seed genetic diversity in B. prostrata, upon which the future stability of populations in changing environments depends [12,44,45], is affected by the salinity level and ionic composition of the 0-40 cm soil layers. In heterogeneous environments, the processes of gene flow, mutation, and sexual reproduction generate local genetic variation, providing material for local adaptation [45]. The influence of soil factors such as soil type, pH, moisture, and soil layer depth on population genetic diversity has been demonstrated in different plant species [15,[17][18][19]. A nine-year experiment on the influence of soil moisture and nitrogen, phosphorus, and potassium content in soil on allozyme frequency revealed an allele-habitat association in Festuca ovina [15]. It was found that in natural populations the Pgi-2-2 allele is significantly associated with soil moisture and is affected by nutrient/water treatments [15]. Negative correlations between B. prostrata genetic diversity with inorganic ion content (except for Na + and Cl − ) and the sum of salts in the 0-40 cm soil layers ( Figure 4D,E) indicate selection in favor of homozygotes. Since isozymes (allozymes) were also used in our study, a question arises regarding the functional significance of enzymes under selection. Loci G6pd and Me were the most polymorphic among the B. prostrata populations ( Figure 3A). They encode the enzymes glucose-6-phosphate dehydrogenase (G6PD) and malik-enzyme (NADP-Me), respectively, which are associated with the regulatory nodes of dark respiration and photosynthesis. G6PD is a key enzyme in the alternative apotomous oxidative pentose phosphate pathway (OPPP), whose role is enhanced under stress [46]. Malik-enzyme is involved in photosynthesis and is especially active in C 4 species, and it plays a vital role in the tolerance to salt stress [47]. The adaptive-compensatory reactions of plants under stress are always associated with additional energy costs, which leads to a change in the balance between photosynthesis and respiration [46]. Any shifts in this balance are reflected in the total plant productivity. Selection leads to local adaptation, and the strength of local adaptation depends on the strength of selection. Strong selection leads to strong local adaptation, which is significantly affected by landscape heterogeneity [48]. The negative influence of Ca 2+ , Mg 2+ , and SO 4 2− contents in the 0-40 cm soil layer on heterozygosity indicates the formation of the local adaptation of B. prostrata to magnesium-calcium sulfate soil salinity. The detected level of sodium chloride salinity did not negatively impact seed genetic polymorphism ( Figure 4D,E). This is probably due to the necessity of these ions in maintaining water balance in the aboveground organs of B. prostrata ( Figure 4C).

Conclusions
Our study demonstrates that in natural habitats the productivity and seed genetic polymorphism of halophytes may be affected by the salinity of different soil layers. These differential plant responses to vertically heterogeneous soil salinity could be attributed to seasonal variables during biomass accumulation (summer) and seed formation (autumn). An excess of some ions in the uppermost soil layers can lead to increased local adaptation to a certain type of salinity and the appearance of genotype-environment associations. Genotype-environment association analyses may allow us to develop adaptive measures for natural resource management, pasture improvement, and the phytoremediation and restoration of lands with different salinity chemistries.