Trade-off strategy of leaf functional traits of desert halophyte Lycium ruthenicum in the lower reaches of Heihe River, Northwest China: response to soil moisture and salinity

Abstract Background: Understanding salinity resistance and water utilization on shrub species is a challenge to the management and conservation of desert halophytes. Lycium ruthenicum Murr.with a significant soil and water conservation capacity, is one of the dominant shrubs and halophytes in the lower reaches of the Heihe River, Northwest China. In this paper, the effects of two depths (0-40 and 40-80 cm) of soil salinities and water contents on the leaf functional traits of eight L. ruthenicum communities in different distances from the main channel were studied. Fourteen leaf water physiological and ecological stoichiometric traits were investigated, linking with soil factors to explain desert plant trade-off strategies. Results: Specific leaf volume (SLV), specific leaf area (SLA), leaf thickness (LT), nitrogen (N), C:N, C:P could serve as good indicators of drought and saline resistance. Low N, specific leaf area (SLA) indicated that the plant was located at the slow investment-return axis of the species resource utilization. Low C:N, C:P showed that L. ruthenicum had a defensive life history strategy at drought and salinity areas. The RDA results showed that 0-40 and 40-80 cm soil properties respectively explained 93.45% and 99.96% leaf traits variation. Soil water contents, HCO3- had extremely positive correlation (P<0.01) with leaf functional traits. Shallow soil water contents significantly affects P, and deeper soil water contents significantly responds C and N; shallow soil salinity significantly affected LT, C and N contents, whereas deeper soil salinity significantly affected N and SLV. Conclusions: L. ruthenicum had a foliar resource acquisition and resource conservation trade-off with a defensive life history strategy in the area of drought and salinity. This finding provides baseline information to facilitate the management and restoration of arid-saline desert ecosystem. This study found that the patterns of ruthenicum in arid and saline environments have a tendency of low leaf SLA , LDMC , C , N and N:P levels, but high LT , Suc , P content and C:N performance, and leaf average N:P <14. Our findings are as follows: (1) The ratio of leaf N:P was more stable than C:N and C:P adaptation to drought-salt stress. There was no significant difference in average SLA trait values between eight different habitats, indicating that intra-specific variation in SLA at a finer ecological scale was minimal or non-existent. (2) Resource acquisition and resource conservation trade-off; (3) Shallow soil moisture significantly affects leaf P , and deep soil moisture significantly responds leaf C and N ; Shallow soil salinity significantly affected LT , C and N contents, whereas deep soil salinity significantly affected leaf N and SLV . HCO 3-and soil water contents have significant effects on leaf functional traits. (4) Given that these remarkable features we recommend L. ruthenicum had a defensive life history strategy at drought and salinity areas. Although this paper proved to some extent the effect of soil water and salt on functional traits, however, other larger scale studies are needed to determine the drivers of functional characteristics.

were constrained by variation among different functional groups and scaled N with respect to C content in foliage [11,13]. In addition, the ratio of C:N and C:P indicates the ability of plants to assimilate C while simultaneously absorbing N and P. Comparatively, the ratio of N:P can reflect a dynamic balance between soil nutrients and plant nutrition demands [10,15]. Over the past decade, distribution patterns of C, N, and P in plant leaves at global or regional scales as well as environmental factor relationship research has received widespread attention [12,13,16]. Recent studies tend to explain the temporal and spatial variability of plant functional traits under adverse (salinity, drought and frost stress) conditions [17][18][19][20][21].
Among many soil characteristics, salinity and moisture are important conditions affecting plant growth [22]. In arid environments, drought exerts a strongly selective pressure on morphological-chemical traits and plant life histories [1,4,23]. Salinity is one of the major environmental factors limiting plant growth, development, productivity and distribution pattern [24][25][26]. Excessive accumulation of salt in the soil imposes physiological limitations on plants, including osmotic stress, ion imbalance, oxidative stress and photosynthesis, thereby affecting plant growth [27][28][29]. This situation is exacerbated by the impact of human over-exploitation on land and the initial lack of water in the desertoasis eco-interlaced zone in arid and semi-arid regions [30]. Severe water and salinity stress decreases a plant growth rate, leaf area, biomass accumulation [31]. However previous studies have suggested that appropriate saline conditions can enhance biological C fixation of halophytes [32]. Another stoichiometrical research in an oasis-desert also indicated that soil conductivity was highly positively correlated with leaf C, N contents [20]. But there was a significant negative correlation between leaf P content and soil salt, conversely, a positive correlation was found between the ratios of leaf C:P, N:P and soil salt [33]. The regression analyses for three functional groups along salinity gradients indicated that the salinity decreases leaf C:N , and increases N:P, but salinity was not the driver of leaf C:N:P stoichiometry in halophytes [24]. In summary, plants responses to stress have attracted much attention possibly due to the ecosystem degradation over the past decades. However, the adaptive strategy and tolerance of L.ruthenicum to drought and salinity stresses is not well understood.
Many studies have shown that L. ruthenicum is an important medicinal desert halophyte in the arid and saline land [34]. In addition to their nutritive values, L. ruthenicum can adapt to high salinity and drought through morphology adjustments in both carbon assimilation and metabolism, and can be a colony species with defensive functions on desert salinealkali land [35]. It can prevent soil desertification and reduce the salinity and alkalinity through the special physiological characteristics of stress environment [36]. Therefore, it is of great significance to study the functional traits of L. ruthenicum in the desert salinealkali region where plant species diversity is lacking. In this paper, we measured the leaf water physiological and ecological stoichiometry traits of L. ruthenicum and different depths of soil salinity and water contents, selected an approximately 17 km long northsouth transect of eight L. ruthenicum communities living under different salinity and moisture regimes from the lower reaches of the Heihe River of China. The objective of the present study was to explore: (1) Trade-off strategies between leaf functional traits under salinity and drought stress habitats; (2) What are the relationships between leaf functional traits and soil factors? ; And (3) Find major environmental factors that affect plant traits.

Study site
The Heihe River is an inland river located in an extremely arid and fragile ecological environment in northwestern China. The desert ecosystem extends from the upstream to the downstream, with unique ecological structures and plant communities, dominated by shrubs [37]. The Ejina desert area is located in the lower reaches of the Heihe River Basin.
According to the data of Ejinaqi Weather Station from 1957 to 2011, the annual average temperature is 8.77°C, the relative humidity is 33.9%, the annual precipitation is 37.40 mm, and the annual evaporation is 3390. 26 mm. In the environment with rare precipitation, the water supply in the Ejina desert area mainly comes from the Heihe River Basin, and the riverside vegetation is mainly assembled by shrubs and grasses. Shrubs are mainly Tamarix chinensis followed by Lycium ruthenicum, Nitraria tangutorum and Alhagi sparsifolia [38]. In Ejina desert, the plant species are poor, and the vegetation types are mainly dry and salt-tolerant desert plants, mainly distributed in the Heihe River and the lake plains of Ejina Banner. The soil types of the entire Heihe River series include brown calcium, desert calcium, meadows, salt and sand [37].

Sampling protocol and community characteristics
This study was conducted in early August 2017 within a 17 km long north-south transect in the lower reaches of the Heihe River Basin. Study area was flat and far from the village.
We Selected 8 different communities of moisture and salinity conditions from the near to the far from the main river channel. Main distribution areas of plots and different plant habitat types were shown in Table 1. Three quadrats (5×5 m) were established within each selected community and their geographic information (latitude, longitude), desert types and plant community structure were investigated by GPS eXplorist 510 (Magellan, USA). Sunny side and fully expanded mature leaves (n>30) were collected from 15 individuals of each L. ruthenicum communities, and all foliage sampled from 3 squares were combined into a mixed sample.

Determination of leaf water physiological and stoichiometric traits
The caliper with an accuracy of 0.02 mm was used to measure the thickness of the upper, middle and lower sides of the leaf (LT, mm). The leaf area was determined by a combination of a scanner (EPSON DS-1610) and ImageJ software [39]. The specific leaf area (SLA, leaf area per unit dry mass), the specific leaf volume (SLV, leaf volume per unit dry mass) was determined by a drainage method using a 10 mL cylinder, leaf dry matter content (LDMC, leaf dry mass per unit fresh mass), relative water content (RWC, %). Total leaf water content (TWC, %), degree of the leaf succulence measured by saturation fresh weight minus dry weight divided by surface area (Suc, g·cm -2 ), leaf tissue density (the ratio of leaf dry weight to volume, LD, g·cm -3 ). Since the leaves dehydrate after leaving the branches, the LT, SLA, and SLV properties were all completed in the field, and then samples were taken back to the laboratory dried at 80 °C for 48h to constant weight as well as other traits. Dried leaves were ground to a 0.15 mm powder using a sample pulverizer to measure the carbon (C), nitrogen (N) and phosphorus (P) contents, and calculate the stoichiometric ratio. C content was determined using the K 2 Cr 2 O 7 -H 2 SO 4 external heating method in oil bath, N content was determined by the Semi-automatic Kjeldahl procedure, which involves digestion with concentrated H 2 SO 4 followed by measurement of NH 3 on an auto analyzer (Hanon K9840, Jinan, China), and leaf P was digested by H 2 SO 4 -H 2 O 2 and then measured by the molybdenum antimony method.

Measurement of soil moisture, salinity and ion contents
Soil was collected at 0-40 and 40-80 cm depths of 8 plant communities. We collected soil samples without rain within 7-10 days. The samples were first passed through a 2 mm screen to remove roots and other impurities, and then dried at 80 ℃ for moisture content analysis. Electrical conductivity (EC) measured using a DDS-307a portable conductivity meter (Leici Instrument, Shanghai, China). We have previously established a standard curve between the soil salinity and electrical conductivity of saline-alkaline soil in the study area as y = 217.73 x -22.723, R 2 = 0.994, and the unit of soil salinity is g·kg -1 . Soil samples were analyzed within 20 days of collection for carbonate (CO 3 2-), bicarbonate (HCO 3 -), chloride (Cl -), sulfate (SO 4 2-), sodium (Na + ), potassium (K + ), calcium (Ca 2+ ) and magnesium (Mg 2+ ) following methods described by the US Salinity Laboratory Staff [40].

Statistical analysis
All one-way analysis of variance (ANOVA) used by the SPSS 19

Characteristic of the leaf functional traits in different communities of L. ruthenicum
In this study, we measured 14 leaf functional traits ( In eight different moisture and salinity environment sites (Table 1), we compared the differences between L. ruthenicum functional traits and found that larger leaf thickness appeared in severe saline site Ⅷ, significantly different from moderately saline Gobi siteⅠand low saline site Ⅵ with lower leaf thickness (Table 2). In addition, the largest SLV, Suc, TWC and RWC were also observed to appear in severe saline sites (Site Ⅶ and Ⅷ).
Conversely, LDMC, LD, and N contents had the lowest values in heavy saline areas. Leaf N concentration was the least variable in different regions, but it still showed the effect of heavy saline on L. ruthenicum stress, and leaf C content was significantly lower. Different degrees of drought stress (eight different soil moisture sites) had significant effects on leaf P content ( Fig.2), statistical analysis showed that the ratio of N:P was more stable than C:N and C:P adaptation to drought-salt stress. Moreover, we found that there was no significant difference in the SLA trait values between eight different habitats, meaning that intra-specific variation in SLA at our finer ecological scale was minimal or nonexistent. RWC was highly negatively correlated with N:P and C:P (r=-0.56, r=-0.62, P<0.05).

RDA restriction ordering of functional traits in soil water and salinity
In view of the significant effect of salinity on plant growth, we were not only interested in the effects of total soil salinity on leaf functional traits, but also on the exploration of  Table 3).
This suggested that models using these environmental factors can explain well the functional trait variation. In general, in the 0-40 cm soil layer, soil water contents significantly affected leaf N:P, C:P , P contents and RWC (Table 3, (Table 3), soil water contents significantly reduced leaf C and N contents, but highly positively with C:N (Fig.2b), while soil salinity only significantly reduced the leaf N contents and highly positively with C:N, leaf morphological trait SLV .
The spatial distribution of the eight community types may be driven by variation in soil chemical characteristics. Communities I, II and VII are close to each other due to their similar soil chemistry, as are VI and III, IV communities being close to each other.
However, the V and VIII communities are located away from other communities, so their soil properties may differ from other locations. The Eigen values and total cumulative variance of RDA axes 1 and 2 are also high, indicating that species data are strongly correlated with measured environmental variables (Table 3). in a new environment [42]. This study showed that the desert halophyte L. ruthenicum was characterized by low leaf SLA, LDMC, C, N and N:P levels, high LT, Suc, P content and C:N performance. SLA is one of the key leaf traits of plant carbon uptake strategy [43], it can reflect the distribution of plants and their adaptation to habitats [44]. LDMC mainly reflects the ability of plants to retain nutrients [45]. In addition, SLA and LDMC are the best variables for classifying plant species on the plant resource utilization classification axis [6]. This paper showed that L. ruthenicum was a resource reservation species for its low SLA, N content yet high C:N, this also indicated that L. ruthenicum is in "slow-return" end of the spectrum: Plants that invest in high LMA have a slower photosynthetic rate, but the leaf life is longer, so their slower income (carbon absorption) rate can be compensated by a longer income stream [6,46]. Furthermore, SLA and LDMC are two important soilfertility predictors as well as leaf N, P nutrient contents and N:P [15,[47][48][49]. The combination of these predictors indicates that soil fertility is lacking in the Ejina desert area in the lower reaches of Heihe River and the growth of L. ruthenicum is mainly restricted by N. Prior studies that have noted the importance of C:N and C:P ratios can effectively reflect the balance between competitive and defensive strategies [33]. When N and P contents were high, C:N and C:P ratios were comparatively low. Plants will apply competitive strategies at high photosynthetic rates. Conversely, when C content was high,  [62]. In general, salt stress causes the normal growth of plants to be inhibited to varying degrees, mainly because salt stress inhibits plant photosynthesis by reducing soil water potential and reducing leaf stomatal conductance [63][64]. Therefore, the fixed C ability of the blade will also be reduced significant, this was consistent with the low leaf C phenomenon in this study. Many studies have confirmed that under salt stress, especially the higher Clcontent in the soil will inhibit the plant's absorption of NO 3 -, so the NO 3 content in the leaves of the plants will decrease [65][66], The RDA results showed that SWC, HCO 3 -, Cl -1 and CO 3 2-can explain the variation of functional traits well. Surprisingly, Na + was not in this rank. Because the importance of Cland Na + has been mentioned in much salinity stress studies [67][68][69]. It was found that Na + is more effective than K + and Clin cell swelling, leaf succulence and shoot

Conclusion
This study found that the patterns of leaf functional traits in arid desert halophyte L.
ruthenicum in arid and saline environments have a tendency of low leaf SLA, LDMC , C, N and N:P levels, but high LT, Suc, P content and C:N performance, and leaf average N:P<14.
Our findings are as follows: (1) The ratio of leaf N:P was more stable than C:N and C:P adaptation to drought-salt stress. There was no significant difference in average SLA trait values between eight different habitats, indicating that intra-specific variation in SLA at a finer ecological scale was minimal or non-existent.   Multiple comparisons of traits between different communities using the tukey-HSD method, Different letters represent significant differences(P<0.05), ns means no difference. LT: leaf thickness (mm), SLA: specific leaf area (cm 2 ▪ mg -1 ), SLV: specific of leaf volume(cm 3 ▪ g -1 ), LDMC: leaf dry matter content (mg▪g -1 ); Suc: leaaf Sucuulent (g▪cm -2 ), LD: leaf tissue density (g▪cm -3 ), TWC: Total water content(%), RWC: relative water content(%), C: leaf carbon content(mg▪g -1 ), N: leaf nitrogen content(mg▪g -1 ), P: leaf phosphrous content(mg▪g -1 ), N:P, C:N, C:P: the ratios of C, N and P   Additional file 1.xlsx