How do abiotic stresses influence the seed germination and vigor of tree species from the Pampa biome?

Abstract

The Pampa biome is increasingly threatened by anthropogenic pressure and climate change, which intensify thermal extremes, water scarcity, and soil salinity, negatively affecting seed production and dispersal. This study evaluated the effects of thermal, water, and salinity stresses on the germination and vigor of Neltuma affinis and Vachellia caven, two ecologically and economically important Fabaceae species native to the Pampa parklands. The seeds were subjected to different temperatures (15, 25, 30, and 35 °C), polyethylene glycol (PEG) concentrations (0.0, -0.41, -0.58, and -0.79 MPa), and NaCl concentrations (0.0, -0.46, -0.92, and -1.37 MPa). Germination of N. affinis was not observed at 15 or 35 °C, whereas V. caven failed to germinate only at 15 °C. Under water stress, both species showed reduced germination and germination speed index (GSI). While V. caven demonstrated tolerance to salinity, N. affinis germination and GSI were inversely proportional to salinity. Overall, V. caven showed tolerance to thermal and saline stress, whereas N. affinis was significantly more sensitive to abiotic stresses. Given the limited distribution of N. affinis in Brazil, together with ongoing anthropogenic pressure and increasing climatic extremes, this species may be at risk of extinction due to reduced germination capacity and seed vigor.

Key words
Neltuma affinis; Vachellia caven; water stress; salt stress; thermal stress; forest restoration

INTRODUCTION

The Pampa biome spans Brazil, Argentina, Uruguay, and Paraguay, covering approximately 1 million km² and consisting predominantly of grassland ecosystems (Altmann & Filho 2020). In addition to grassland physiognomies, the Pampa includes forested areas, shrublands, wetlands, and rocky outcrops (Overbeck et al. 2022, Andrade et al. 2023). Its native vegetation provides essential ecosystem services, including carbon storage, soil protection, and habitat provision (Silva et al. 2025). However, the biome is increasingly threatened by intense anthropogenic pressure and climate change (Baeza et al. 2022, Brendel et al. 2025). Despite its ecological importance, the Pampa remains underrepresented in public policy agendas and insufficiently studied scientifically (Baeza et al. 2022; Overbeck et al. 2022).

The expansion of monocultures and pastures composed of exotic species is rapidly degrading the natural landscapes of the Pampa biome (Altmann & Filho 2020, Caumo et al. 2021, Baeza et al. 2022). Vegetation monitoring data indicate a loss of approximately 2.4 million hectares of native grassland between 2001 and 2018, primarily due to the conversion of native vegetation into agricultural land (Baeza et al. 2022). Intensified agricultural practices, including mechanized farming, irrigation, and soil preparation techniques, have been major drivers of land-use change, disrupting soil structure and reducing organic matter content (Foucher et al. 2023, Song et al. 2021). In livestock-dominated areas, overgrazing further contributes to the degradation of native grasslands and biodiversity loss (Fedrigo et al. 2018). Additionally, inadequate legislation can weaken the protection of native grasslands and facilitate their conversion to exotic pastures in the Brazilian Pampa (Brancalion et al. 2016). Without land-use planning, these changes result in biodiversity loss through the decline of species with forage, food, ornamental, and medicinal value, as well as the degradation of ecosystem services (Caumo et al. 2021, Pometti et al. 2021, Milagre et al. 2025). In this context, maintaining Legal Reserve and Permanent Preservation Areas (Tomas et al. 2024) and expanding conservation units are essential, as these areas serve as refuges for native species within the biome (Silva et al. 2021, Ribeiro et al. 2021b).

In addition to the conversion of native vegetation, studies indicate that the Pampa biome is becoming increasingly vulnerable to climate change (Brendel et al. 2025, Pinho et al. 2020, Ribeiro et al. 2021a). Climate projections suggest a 5.0–10.0% increase in precipitation and a rise of up to 1.0 °C in mean annual temperature by 2040 (PBMC 2020). In this scenario, higher air temperatures, altered precipitation patterns, and increased droughts frequencies (Chou et al. 2014, MCTI/Brasil 2020, Rocha et al. 2021) may impair the adaptive capacity of native species. Species responses to climate change include developing tolerance mechanisms, adapting to adverse conditions, or migrating to more favorable environments, strategies that may reduce extinction risk (Moritz & Agudo 2013). However, extreme climatic events and rising temperatures can also intensify soil weathering and promote the accumulation of saline elements at the soil surface, thereby compromising the establishment of species sensitive to salinity (Eswar et al. 2021). In addition, the excessive use of mineral fertilizers in agricultural systems contributes to increased soil salinity (Figueiredo 2005). The Pampa also has highly degraded areas with sandy soils, known as “areais”, which pose a challenge to environmental restoration and the survival of native species (Suertegaray 2012, Mendes et al. 2024).

Seeds subjected to water deficit exhibit alterations in germination potential, including reduced germination percentage and germination speed (Li et al. 2022, Luo et al. 2022). When combined with elevated temperatures, water deficit also affects intracellular solute concentrations (Dambros et al. 2024). Temperature affects the rate of water imbibition and the enzymatic and biochemical reactions involved in the germination process (Flores et al. 2014). Identifying the optimal temperature is essential in seed analysis, as it maximizes germination in the shortest time. In contrast, germination is significantly reduced at minimum and maximum temperatures (Nascimento et al. 2021, Luo et al. 2022). This data reflects the regenerative capacity of species when extreme weather conditions were rare, in contrast to what is observed today. High salinity levels can induce enzymatic changes, interfere with cell signaling, and affect the use of available reserves during seed germination (Zhu 2016). In seedlings, the accumulation of Na+ and Cl- ions can reduce photosynthetic efficiency, growth, and promote premature fall of young leaves (Skirycz et al. 2009, Nicotra et al. 2010, Zhu 2016).

Knowledge of the tolerance of some forest species to stress conditions is still incipient (Jagadish et al. 2021). This is also true for species of the Fabaceae family, such as Neltuma affinis (Spreng.) C.E. Hughes & G.P. Lewis (commonly known as inhanduvá), formerly classified as Prosopis affinis Spreng. (Hughes et al. 2022), and Vachellia caven (Molina) Seigler & Ebinger (espinilho). This information is relevant for species conservation, as it supports the implementation of restoration projects in degraded areas in the Pampa (Savacinski et al. 2023) and the management of conservation units. N. affinis and V. caven are adapted to sedimentary, stony, dry, and saline soils (Pometti et al. 2021), which may help them cope with increasing drought frequency and soil salinity under climate change. Both species occur naturally in Argentina, Paraguay, Uruguay, Bolivia, and Brazil, with V. caven also native to Chile (Velasco et al. 2023, SpeciesLink 2024). Given the high density and durability of N. affinis wood, it was intensively exploited in the 19th century to produce fence posts (Marchiori & Alves 2014). Consequently, the species is categorized as “vulnerable to extinction in Brazil” (Rio Grande do Sul 2014). In contrast, V. caven is used in silvopastoral systems, and is suitable for beekeeping and wood production for energy purposes (Pometti et al. 2021).

The need to provide information for the conservation and use of these species, as well as for the restoration of altered areas, demands research on the silviculture of native species. Such research should aim to conserve and sustainably develop the Pampa. Thus, this study aimed to evaluate the effects of thermal, hydric, and saline stress on the germination and vigor of N. affinis and V. caven seeds. Seeds of both species were subjected to thermal (15, 25, 30, and 35 °C), water (PEG 0.0, -0.41, -0.58, and -0.79 MPa), and salinity (NaCl 0.0, -0.46, -0.92, and -1.37 MPa) stress conditions to assess their responses. We hypothesize that the species exhibit distinct responses to abiotic stress (thermal, water, and salinity) due to species-inherent ecological adaptations associated with the prevailing edaphoclimatic conditions of their natural distribution ranges.

Abbreviations

ANOVA - analysis of variance

GSI - germination speed index

PEG - polyethylene glycol

RS - Rio Grande do Sul

T₅₀ - time to 50% germination

MATERIALS AND METHODS

Seed material

Seeds were collected in the Espinilho State Park, a protected area located in the Barra do Quaraí municipality, Rio Grande do Sul (RS), southern Brazil (30°12’26“S, 57°33’17” W; 53 m altitude) (Figure 1). The Park is a state conservation unit situated on a sedimentary plain. Its soils are composed of Chernosols, Vertisols, and carbonate Gleysols, associated with volcanic rocks of the Serra Geral Formation and sandstones of the Botucatu Formation (Cunha et al. 2001, Streck et al. 2018). The soils of Espinilho State Park present high salinity, with average values ranging from 141 to 268 mg dm−3 of Na+ depending on the park environment (Fockink et al. 2026). The vegetation is classified as Steppe Savannah Park type (IBGE 2012), characterized by a continuous lower stratum dominated by grasses and herbaceous species, interspersed with sparse woody species. The tree layer is dominated by Fabaceae species, including N. affinis, V. caven, and Neltuma nigra (Griseb.) C.E. Hughes & G.P. Lewis. Another common tree species is Aspidosperma quebracho-blanco Schltdl., from the Apocynaceae family (Hasenack et al. 2019, Baeza et al. 2022). Within the 10 km buffer zone of Espinilho State Park, land use is predominantly anthropogenic: 52.7% of the area is occupied by rice fields, agricultural mosaics, and degraded forest, while only 21.3% retains natural vegetation, and 26% consists of abandoned fields (Ribeiro et al. 2021b). According to the Köppen classification, the region has a humid subtropical climate (Cfa), with a mean annual temperature of 18.5 °C and an annual precipitation of 1,468 mm (Alvares et al. 2013).

Figure 1
Table SI.

Fruits were collected from 10 mother trees per species during January-February 2021 (V. caven) and March-April 2021 (N. affinis). The climatic data for the year of seed collection was obtained from the meteorological station located in the municipality of Uruguaiana, approximately 67 km from the headquarters of Espinilho State Park (Figure 2).

Figure 2
Location of the collection area for Neltuma affinis and Vachellia caven seeds (a and b), with details of the anthropogenic pressure condition (c) of the Espinilho State Park (delimited in red), Barra do Quaraí municipality, state of Rio Grande do Sul, southern Brazil.

Fruits were transported to the Silviculture and Forest Nursery Laboratory of the Federal University of Santa Maria, located in Santa Maria, RS. Seeds were manually extracted using pruning shears. After processing, seeds were placed in glass jars and stored at 3.4 °C and 46.5% relative humidity under refrigeration. The initial seed batches’ characterization was performed according to Brasil (2009). Briefly, it considers seed moisture content, determined by oven drying at 105 ± 3 °C for 24 h, with four replicates of 20 seeds (Equation 1) for each species. Thousand-seed weight was determined using eight replicates of 100 seeds, and the number of seeds per kilogram was subsequently calculated (Equation 2).

$M \%=\frac{100(W-w)}{W-t}$ (1)

where M% represents the seed moisture content; W is the initial weight, corresponding to the combined weight of the container with its lid and the wet seeds; w is the final weight, corresponding to the combined weight of the container with its lid and the dried seeds; and t is the tare weight of the container with its lid.

$N_{kg}=\frac{1000\mathrm{ }\mathrm{g}}{W_{1000}} \mathrm{x}\mathrm{ }1000$ (2)

where N_kg is the number of seeds per kilogram (seeds/kg) and W₁₀₀₀ represents the weight of one thousand seeds (g).

Germination under thermal, water, and saline stress

The seeds of each species were evaluated separately, and experiments were conducted to assess three stress conditions. Accordingly, three experiments were performed for each species (N. affinis and V. caven). Thermal stress was evaluated at four temperatures (15, 25, 30, and 35 °C). Water stress was induced using four polyethylene glycol (PEG 6000) concentrations (0, -0.41, -0.58, and -0.79 MPa). PEG was selected because it is an inert, non-toxic compound that does not penetrate seed cells (Marcos-Filho 2015). Saline stress was assessed using four NaCl concentrations (0, -0.46, -0.92, and -1.37 MPa). The PEG 6000 and NaCl solutions were prepared according to the temperature used in the germination test (25 °C), following the method described by Sun (2002).

Physical dormancy in N. affinis seeds was overcome by mechanical scarification using a nail clipper. Scarification was performed at the lateral region of the upper third of the seeds, opposite the micropyle. For V. caven, mechanical scarification was performed with No. 60 sandpaper on the opposite side to the micropyle. Subsequently, seeds were subjected to asepsis by immersion in a 2.5% sodium hypochlorite solution for 10 minutes, followed by immersion in 70% ethanol for two minutes. After each sterilization step, seeds were rinsed three times with deionized water.

Germination tests were performed in transparent Gerbox-type boxes with lids, lined with two sheets of blotting paper. The substrate was moistened with deionized water, PEG 6000, or NaCl solutions, depending on the treatment, at a volume equivalent to 2.5 times the weight of the paper (Brasil 2009). The blotting paper, water, and solutions were replaced every seven days to maintain the osmotic potential. The boxes were placed in a Mangelsdorf germination chamber under a 24h photoperiod. Temperatures were adjusted according to the thermal stress treatments (15, 25, 30, and 35 °C), whereas water and salinity stress treatments were maintained at 25 °C. The experiment followed a completely randomized design, with four replicates of 25 seeds per treatment.

Germination was recorded at 7-day intervals for up to 30 days after the initiation of the experiments under thermal, water, and salinity stress conditions. Germination was assessed using the technological criterion of normal seedling formation, defined by the development of all essential structures, including the primary root, hypocotyl, and cotyledons (Brasil 2009). Based on the germination data, the germination speed index (GSI) (Equation 3) was calculated according to Maguire (1962), and the time required to reach 50% germination (T₅₀) (Equation 4) was calculated following Farooq et al. (2005), as follows:

$\mathrm{G}\mathrm{S}\mathrm{I}={\sum }_{i=1}^n \frac{G_i}{T_i}$ (3)

where GSI is the germination speed index; G_i is the number of seeds germinated on day i; and T_i is the number of days since sowing

$T_{50}= t_i+\frac{ \left(\raisebox{1ex}{$N$}\!\left/ \!\raisebox{-1ex}{$2$}\right.-n_i\right)\left(t_j-t_i\right)}{(n_j-n_i)}$ (4)

where T₅₀ represents the time required to reach 50% germination (days); N is the final number of germinated seeds; t_i is the last day on which cumulative germination was below 50%; t_j is the first day on which cumulative germination exceeded 50%; n_i is the cumulative number of germinated seeds on day t_i; and n_j is the cumulative number of germinated seeds on day t_j.

As germination was recorded at 7-day intervals, T₅₀ was estimated by linear interpolation between consecutive sampling points where cumulative germination crossed 50%. Therefore, T₅₀ values should be interpreted as estimates with limited temporal resolution due to the sampling interval.

Statistical analysis

Data were tested for normality and homogeneity of variances using the Shapiro–Wilk and Bartlett tests, respectively. When these assumptions were not met, the arcsine transformation (√x/100) (Santana & Ranal 2004) was applied, and the data were retested. Once the assumptions of normality and homoscedasticity were met, an analysis of variance (ANOVA) and a Tukey test (p<0.05) were performed. All analyses were performed with RStudio software version 4.2.1 (R Core Team 2023).

RESULTS

Seed characterization

The seeds of N. affinis and V. caven had moisture contents of 5.84 ± 0.15% and 4.55 ± 0.27%, respectively (mean ± standard deviation). The weight of a thousand seeds was 48.76 ± 2.74 g for N. affinis and 58.70 ± 2.75 g for V. caven. The number of seeds per kilogram was 20,567 ± 1.155 and 17,068 ± 793 for N. affinis and V. caven, respectively.

Thermal stress

Seeds of N. affinis germinated only at 25 °C and 30 °C (Figure 3a), exhibiting significantly higher germination at these temperatures than at 15 °C and 35 °C, but similar rates between these two temperatures (Supplementary Material - Table SI). In contrast, V. caven seeds did not germinate at 15 °C, whereas no significant differences were observed among 25 °C, 30 °C, and 35 °C (Figure 3b). Accordingly, germination percentage, germination speed index (GSI), and time to 50% germination (T₅₀) were null at 15 °C and 35 °C for N. affinis, and at 15 °C for V. caven (Figures 3c–3f). No differences were observed for GSI and T₅₀ were observed among the remaining temperatures for either species, although a trend toward higher values was noted at 25 °C and 30 °C.

Figure 3
Minimum and maximum temperature (°C) monthly averages and accumulated precipitation (mm) in 2021 obtained from the meteorological station located in Uruguaiana municipality, Rio Grande do Sul state, southern Brazil. The bars represent precipitation, and the symbols represent temperature. Source: SEAPDR/DDPA (2021).

Water stress

Under water-restricted conditions during germination, both species exhibited reduced germination percentage and germination speed index (GSI) (Table SI). Compared with the control (0 MPa), the highest water stress level (-0.79 MPa) reduced germination by more than 50% in N. affinis and V. caven (Figures 4a and 4b). A GSI reduction was also observed under increasing water stress (Figures 4c and 4d), especially in V. caven seeds subjected to -0.79 MPa. The T₅₀ of N. affinis seeds did not differ significantly among water potential treatments (p>0.05) (Figure 4e). In contrast, the T₅₀ of V. caven seeds increased progressively with increasing polyethylene glycol concentration (Figure 4f). Given the lower water potential of V. caven, the T₅₀ occurred 22 days after the start of the experiment, twice the time required under control conditions.

Figure 4
Germination (%) (a and b), germination speed index (GSI) (c and d), and time to 50% germination (T₅₀) in days (d) (e and f) of Neltuma affinis and Vachellia caven species at different temperatures (15, 25, 30, and 35 °C). Values ​​are presented as mean ± standard error. Different letters indicate statistically significant differences (p<0.05) between temperatures assessed by Tukey’s test. ‘’ns’’ indicates not significant (p>0.05).

Saline stress

When saline stress conditions (-1.37 MPa), N. affinis seeds exhibited a 64% reduction in germination percentage and a 68% reduction in GSI compared with the control (Table SI, Figures 5a and 5c). On the other hand, V. caven showed tolerance to the tested saline concentrations, maintaining high germination in all water potentials (Figure 5b). Furthermore, V. caven showed no significant differences in GSI (p=0.204) or T₅₀ (p=0.059) across osmotic potentials (Figures 5d and 5f). Both species showed T₅₀ close to 12 days (Figures 5e and 5f).

Figure 5
Germination (%) (a and b), germination speed index (GSI) (c and d), and time to 50% germination (T₅₀) in days (d) (e and f) of Neltuma affinis and Vachellia caven species at different concentrations of PEG 6000 (0, -0.41, -0.58, and -0.79 MPa). Values are presented as mean ± standard error. Different letters indicate statistically significant differences (p<0.05) between the water potentials evaluated by Tukey’s test. ‘’ns’’ indicates not significant (p>0.05).

DISCUSSION

In the present study, N. affinis was found to be sensitive to all extreme conditions evaluated, whereas V. caven exhibited tolerance to thermal and saline stress. Temperature is a key environmental factor influencing seed germination (Kim & Han 2018). Within the optimal temperature range, seeds reach maximum germination in a shorter time, whereas germination may be inhibited or entirely suppressed at temperatures above or below this range (Mayer & Poljakoff-Mayber 2014). Germination of N. affinis and V. caven was maximized at 25 °C and 30 °C, respectively. These optimal temperatures differ from those reported for other South American Neltuma species. For instance, Neltuma chilensis (Molina) C.E. Hughes & G.P. Lewis germinates between 25 and 40 °C, whereas Neltuma flexuosa (DC.) C.E. Hughes & G.P. Lewis show optimal germination between 20 and 25 °C (Villagra et al. 2010). In contrast, the species Neltuma argentina (Burkart) C.E. Hughes & G.P. Lewis and Neltuma alpataco (Phil.) C.E. Hughes & G.P. Lewis exhibit higher optimal germination temperatures around 35 °C (Villagra 1995). Such interspecific variation in thermal requirements suggests a considerable degree of ecological plasticity within the genus, likely reflecting adaptations to distinct environmental conditions across its distribution range. This differentiation may confer varying levels of resilience to climate variability, with implications for species selection.

Differences in germination across the evaluated temperatures indicate that neither N. affinis nor V. caven germinates at low temperatures (15 °C) (Figure 3a). In N. affinis, germination was completely inhibited at the highest temperature tested (35 °C). In contrast, V. caven tolerated 35 °C, suggesting that this species may be less affected by future temperature changes in the Pampa region (Rocha et al. 2021) than N. affinis. The observed trend toward reduced germination and seed vigor at 35 °C suggests that the establishment of both species could be compromised under extreme heat conditions, with more pronounced effects expected for N. affinis. Elevated temperatures can negatively impact seed metabolic activity by disrupting enzymatic processes essential for germination and early seedling development, potentially reducing recruitment success under future climate scenarios (Vicente & Garzón 2024, Zhou et al. 2023).

One factor to consider is that the ideal germination temperature varies according with the species’ natural distribution, encompassing both intra- and interspecific variation (Dürr et al. 2015, Carvalho et al. 2021). V. caven has a broader natural distribution than N. affinis, largely due to its adaptation to a wider temperature range and a greater tolerance to salinity (Pometti et al. 2021, SpeciesLink 2024, Velasco et al. 2023). Moreover, V. caven exhibits high phenotypic plasticity (Pometti et al. 2021). The species is widely distributed in rural areas of RS and other regions of South America, where daily temperature fluctuations are more pronounced (Marchiori & Alves 2012, Velasco et al. 2023). In Espinilho State Park, where the seeds for this study were collected, the distribution of N. affinis and V. caven regenerants is associated with land-use history (e.g., presence or absence of livestock) and physical soil characteristics such as bulk density and penetration resistance (Redin et al. 2017).

Water stress can negatively affect seedling establishment and growth in tree species (Tiebel et al. 2023). Under control conditions, normal seedlings developed rapidly, reaching 50% germination (T₅₀) in 11 days, allowing efficient water use for the development of essential structures when water availability is not limited. In contrast, germination and GSI of N. affinis and V. caven were markedly reduced under water-restricted conditions (Figure 4). Although the reduction in germination at the lowest water potential (-0.79 MPa) was similar for both species, reduced water availability had a greater impact on V. caven vigor, as evidenced by lower GSI values and higher T₅₀ than in the control. Germination is generally impaired at water potential below -1.4 MPa under both salinity and water stress conditions. Species adapted to arid environments often exhibit some degree of tolerance to water deficit, depending on soil characteristics (Villagra et al. 2010). Species within the genus Prosopis, to which N. affinis was formerly assigned (Hughes et al. 2022), have evolved several morphophysiological mechanisms that confer tolerance to water deficit. Such mechanisms include pronounced xeromorphism, reduced leaf area, thick leaf cuticles and bark, petiolar glands, leaf movements (nasties), reduced CO₂ assimilation, and tight stomatal regulation, among others (Villagra et al. 2010). It is important to note that tolerance to abiotic stresses may involve ecological trade-offs, in which adaptation to high salinity conditions may result in reduced performance under water deficit, or vice versa, depending on the origin of each species. With increasing drought frequency or the intensification of soil salinization, these differences may influence germination and species distribution, favoring those with a greater capacity to tolerate the predominant stress in each environment (Contreras-Negrete et al. 2021).

N. affinis seeds were sensitive to moderate salinity, while V. caven seeds were tolerant. This sensitivity in N. affinis is likely related to reduced water uptake under high salinity, which can inhibit or delay germination (Ibrahim 2016). Some plant species are more adapted to saline environments and can tolerate elevated salt concentrations (halophytic species), while others are salt-sensitive (Polash et al. 2019). Salinity stress restricts plant growth primarily through osmotic effects, leading to water deficit and the accumulation of toxic ions such as Na⁺ and Cl⁻ (Santos et al. 2022). For instance, a marked reduction in germination percentage was reported for N. flexuosa at NaCl concentrations above 0.2 M (-0.92 MPa at 25 °C) (Catalán et al. 1994). On the other hand, Neltuma juliflora (Sw.) Raf., Sylva Tellur. demonstrates higher salinity tolerance (El-Keblawy & Al-Rawai 2005, Nasr et al. 2012), exhibiting increased germination and seedling biomass production at 0.1 M NaCl (-0.46 MPa at 25 °C) (Nasr et al. 2012). Notably, both N. flexuosa and N. juliflora were previously classified within the genus Prosopis (Hughes et al. 2022). Together, these contrasting responses among Neltuma species highlight substantial ecological variability within the genus. This intra-generic variation should be explicitly considered in management or restoration programs, as it directly influences species selection and may determine establishment success under stress-prone conditions.

Abiotic factors can affect seed germination capacity either independently or in combination (Pearson et al. 2002, Thomas et al. 2010). In arid environments, for example, interactions between temperature and salinity may modulate salinity tolerance during germination in species of the genus Neltuma (El-Keblawy & Al-Rawai 2005). In the present study, the 25 °C temperature used in the saline stress assays may have contributed to the absence of significant effects of NaCl on germination percentage, GSI, and T₅₀ in V. caven (Figure 5). V. caven naturally occurs in areas with alkaline deposits and imperfect drainage in Uruguay (Inofuentes & Säumel 2022) and in parkland formations of RS (Marchiori et al. 2014), suggesting an inherent tolerance to saline conditions. Although salinity tolerance is species-specific, it was not verified for N. affinis, as both germination percentage and GSI were negatively affected by salinity even at 25 °C. The combined influence of multiple stressors has also been reported for Neltuma caldenia (Burkart) C.E. Hughes & G.P. Lewis, whose germination percentage was significantly reduced at a water potential of −1.0 MPa under a constant temperature regime of 35 °C (Villalobos & Peláez 2001). However, it is important to note that, in the present study, abiotic factors were evaluated independently, which may limit the detection of potential interactive effects. Future studies should investigate the combined influence of temperature, salinity, and water availability to better reflect natural environmental conditions.

Tolerance to abiotic stressors can be acquired even before seed formation or induced after exposure to a specific stress, in a process known as cross-tolerance (Shibata et al. 2021). Tolerance to water deficit and saline conditions during germination appears to be closely linked to soil characteristics. Species originating from environmentally harsh conditions are generally less affected by reduced soil moisture and elevated soil salinity than species from more favorable environments (Villagra et al. 2010). Accordingly, studies have shown that Neltuma alpataco (Phil.) C.E. Hughes & G.P. Lewis exhibit greater tolerance to NaCl, likely because it naturally occurs in sites with moderate to high salinity (Villagra 1997, Villagra et al. 2010). Neltuma argentina (Burkart) C.E. Hughes & G.P. Lewis is typically found in non-saline soils (Villagra 1997). Soil salinity is a prominent feature in the region where the seeds used in this study were collected, as well as in other areas where the species naturally occur. This condition is particularly pronounced in flat terrains that are prone to periodic flooding (Marchiori et al. 2014). In these environments, the accumulation of salts at the soil surface contributes to patchy thinning of plant cover, especially for N. affinis, leading to soil exposure during dry periods (Marchiori et al. 2014).

After seed dispersal, N. affinis and V. caven may exhibit limited or inhibited germination due to environmental constraints intensified by climate change which can compromise natural regeneration and increase conservation risks (Dias et al. 2024). Abiotic stress associated with environmental extremes - such as thermal, water, and saline stress - can negatively affect species establishment in the Pampa biome by reducing seed germination and vigor. N. affinis regenerants are more abundant in areas without grazing, while V. caven predominates in areas with livestock presence. Ungrazed areas typically exhibit higher density of grass and herbaceous plants, resulting in greater soil moisture and lower mechanical resistance to root penetration, which favors N. affinis germination (Redin et al. 2017). On the other hand, in addition to its greater phenotypic plasticity, the regeneration of V. caven in grazed areas may be associated with seed dispersal by cattle (Ferreras et al. 2018). These patterns help explain the observed germination of V. caven at 35 °C and the absence of germination in N. affinis at the same temperature, as herbaceous vegetation is the primary component responsible for buffering soil temperature extremes.

As heliophilous species, N. affinis and V. caven require light incidence for successful establishment (Redin et al. 2017). However, high temperatures and the associated reduction in soil moisture appear to be limiting factors for N. affinis germination. Thermal stress negatively affects plants by inhibiting photosynthesis and respiration and by reducing Rubisco efficiency, which is optimal within a temperature range of approximately 20-30 °C (Santos et al. 2022). The requirement of N. affinis for lower temperatures poses additional challenges for its conservation. In addition to grazing pressure, regenerants in the milder environments, such as those under the shade of dispersing trees, face increased competition and disturbance from livestock. Cattle frequently seek shade beneath the scattered trees to cool down, leading to trampling and further compromising the establishment of natural regeneration.

N. affinis and V. caven are considered “nurse” species, as they create favorable conditions for the establishment and growth of other plants under their canopies (Inofuentes & Säumel 2022). The biological capacity of V. caven for nitrogen fixation (Root-Bernstein et al. 2017), combined with its greater tolerance to thermal and saline stress, makes this species a strong candidate for restoring degraded ecosystems, particularly in areas affected by sandification. Moreover, V. caven can be integrated into silvopastoral systems, providing shade for livestock while supporting biodiversity in the Pampa biome. In addition to restoration actions in ecosystems where N. affinis and V. caven were suppressed, it is necessary to develop strategies to conserve the remaining populations of these species. This is especially critical for N. affinis, which has a restricted geographical distribution, lower tolerance to abiotic stress, and is currently classified as vulnerable to extinction.

CONCLUSIONS

Germination and vigor of N. affinis seeds were markedly reduced under extreme temperatures (15 and 35 °C), water deficit, and salinity. In contrast, V. caven showed tolerance to high temperatures (35 °C) and salinity but was sensitive to water stress during germination. For both species, the optimal temperature for seed germination was 25 °C. Thus, under extreme climatic conditions within their natural range, such as dry summers, harsh winters, and periods of excessive rainfall, the regenerative potential of N. affinis may be severely compromised. Therefore, future studies focusing on the regeneration dynamics of this species are strongly suggested. Our findings underscore the importance of developing targeted conservation strategies for N. affinis and V. caven, considering their ecological roles, adaptive traits and potential contributions to the sustainable management of the Pampa biome.

SUPPLEMENTARY MATERIAL

Table SI.

Table SI.

Acknowledgements

The authors would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Brasil – CAPES (Finance Code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) (Call ARD 10/2021 – Grant Agreement No. 22/2551-0000623-0), and the staff of Espinilho State Park.

• Data availability
  Data of this study are available from the corresponding author upon reasonable request.

References

• ALTMANN A & FILHO AGB. 2020. Certification and labeling for conservation of ecosystem services in the Pampa Biome: Case study of the Aliança do Pastizal scheme. Ecosyst Serv 46: 101209.
• ALVARES CA, STAPE JL, SENTELHAS PC, GONÇALVES JLDM & SPAROVEK G. 2013. Köppen’s climate classification map for Brazil. Meteorol Zeitschrift 22: 711-728.
• ANDRADE BO ET AL. 2023. 12,500+ and counting: biodiversity of the Brazilian Pampa. Front Biogeogr 15: e59288.
• BAEZA S ET AL. 2022. Two decades of land cover mapping in the Río de la Plata grassland region: The MapBiomas Pampa initiative. Remote Sens Appl Soc Environ 28: 100834.
• BRASIL. 2009. Regras para análise de sementes, Brasília: Ministério da Agricultura, Pecuária e Abastecimento-MAPA/ACS, 399 p.
• BRANCALION PHS, GARCIA LC, LOYOLA R, RODRIGUES RR, PILLAR VD & LEWINSOHN TM. 2016. A critical analysis of the Native Vegetation Protection Law of Brazil (2012): updates and ongoing initiatives. Natureza & Conservação 14: 1-15.
• BRENDEL AS, FERRELLI F & PICCOLO MC. 2025. Climate change scenarios and the increasing severity of thermal extremes in the pampas region. Environ Earth Sci 84: 238.
• CARVALHO ASR, ANDRADE LG & ANDRADE ACS. 2021. Germination of small tropical seeds has distinct light quality and temperature requirements, depending on microhabitat. Plant Biol 23: 981-991.
• CATALÁN L, BALZARINI M, TALEISNIK E, SERENO R & KARLIN U. 1994. Effects of salinity on germination and seedling growth of Prosopis flexuosa (D.C.). For Ecol Manage 63: 347-357.
• CAUMO M, FREITAS EM, SILVA VL, TOLDI M, ALVES LS, ORLANDI CR & FIOR CS. 2021. Grassland community structure in Permanent Preservation Areas associated with forestry and livestock in the Pampa biome, Southern Brazil. South African J Bot 139: 442-448.
• CHOU SC ET AL. 2014. Assessment of Climate Change over South America under RCP 4.5 and 8.5 Downscaling Scenarios. Am J Clim Chang 03: 512-527.
• CONTRERAS-NEGRETE G, PINEDA-GARCÍA F, NICASIO-ARZETA S, BARRERA DE LA E, & GONZÁLEZ-RODRÍGUEZ A. 2021. Differences in germination response to temperature, salinity, and water potential among Prosopis laevigata populations are guided by the tolerance-exploitation trade-off. Flora 285: 151963
• CUNHA NG, SILVEIRA RJC, MENDES RG & PEREIRA MR. 2001. Estudo de solos do município de Barra do Quaraí - RS, Circular t ed., Pelotas: Embrapa Clima Temperado, 84 p.
• DAMBROS VG, LOPES BC, CORRÊA BJS, SÁ ACS, SOARES CRB, FLORES AV & OLIVEIRA LM. 2024. Tolerância à dessecação de sementes de Butia eriospatha (Mart. Ex Drude) Becc. Ciência Florest 33: e67835.
• DIAS PB, KUNZ SH, PEZZOPANE JEM, XAVIER TMT, ZORZANELLI JPF, TOLEDO JV, GOMES LP & GORSANI RG. 2024. Water restriction alters seed bank traits and ecology in Atlantic Forest seasonal forests under climate change. Glob Chang Biol 30: e17494.
• DÜRR C, DICKIE JB, YANG X-Y & PRITCHARD HW. 2015. Ranges of critical temperature and water potential values for the germination of species worldwide: Contribution to a seed trait database. Agric For Meteorol 200: 222-232.
• EL-KEBLAWY A & AL-RAWAI A. 2005. Effects of salinity, temperature and light on germination of invasive Prosopis juliflora (Sw.) D.C. J Arid Environ 61: 555-565.
• ESWAR D, KARUPPUSAMY R & CHELLAMUTHU S. 2021. Drivers of soil salinity and their correlation with climate change. Curr Opin Environ Sustain 50: 310-318.
• FAROOQ M, BASRA SMA, AHMAD N & HAFEEZ K. 2005. Thermal Hardening: A New Seed Vigor Enhancement Tool in Rice. J Integr Plant Biol 47: 187-193.
• FEDRIGO JK, ATAIDE PF, FILHO JA, OLIVEIRA LV, JAURENA M, LACA EA, OVERBECK GE & NABINGER C. 2018. Temporary grazing exclusion promotes rapid recovery of species richness and productivity in a long-term overgrazed Campos grassland. Restor Ecol 26: 677-685.
• FERRERAS AE, MARCORA PI, VENIER MP & FUNES G. 2018. Different strategies for breaking physical seed dormancy in field conditions in two fruit morphs of Vachellia caven (Fabaceae). Seed Sci Res 28: 8-15.
• FIGUEIREDO AFR. 2005. Análise do risco de salinização dos solos da bacia hidrográfica do Rio Colônia - Sul da Bahia. Universidade Estadual de Santa Cruz, Ilhéus.
• FLORES AV, BORGES EEL, GUIMARÃES VM, GONÇALVES JFC, ATAÍDE GM & BARROS DP. 2014. Atividade enzimática durante a germinação de sementes de Melanoxylon brauna schott sob diferentes temperaturas. CERNE 20: 401-408.
• FOCKINK GD, BARICHELLO HA, COLLARES BB, GRANZOTTO F, SILVA PS, SCHENATO RB, ESSI L & ROVEDDER APM. 2026. Edaphic filters determine the floristic-structural composition of grassy-woody vegetation in park grassland in Southern Brazil. For Ecol Manage 601: 123374.
• FOUCHER A ET AL. 2023. Inexorable land degradation due to agriculture expansion in South American Pampa. Nat Sustain 6: 662-670.
• HASENACK H, WEBER EJ, VÉLEZ-MARTIN E, HOFMANN GS & DEWES H. 2019. Bioma Pampa: oportunidades e desafios de pesquisa para o desenvolvimento sustentável. In: Biomas e agricultura: oportunidades e desafios, p.304.
• HUGHES CE, RINGELBERG JJ, LEWIS GP & CATALANO SA. 2022. Disintegration of the genus Prosopis L. (Leguminosae, Caesalpinioideae, mimosoid clade). PhytoKeys 205: 147-189.
• IBGE. 2012. Manual técnico da vegetação brasileira: sistema fitogeográfico: inventário das formações florestais e campestres: técnicas e manejo de coleções botânicas: procedimentos para mapeamentos, 2ª ed., Rio de Janeiro: IBGE, 272 p.
• IBRAHIM EA. 2016. Seed priming to alleviate salinity stress in germinating seeds. J Plant Physiol 192: 38-46.
• INOFUENTES PP & SÄUMEL I. 2022. Nurse species facilitate persistence of dry forests in agricultural landscapes in Uruguay. J Veg Sci 33.
• JAGADISH SVK, WAY DA & SHARKEY TD. 2021. Plant heat stress: Concepts directing future research. Plant Cell Environ 44: 1992-2005.
• KIM DH & HAN SH. 2018. Direct Effects on Seed Germination of 17 Tree Species under Elevated Temperature and CO₂ Conditions. Open Life Sci 13: 137-148.
• LI C, WAN Y, SHANG X & FANG S. 2022. Responses of Microstructure, Ultrastructure and Antioxidant Enzyme Activity to PEG-Induced Drought Stress in Cyclocarya paliurus Seedlings. Forests 13: 836.
• LUO Y, CHENG J, YAN X, ZHANG J & ZHANG J. 2022. Germination of Seeds Subjected to Temperature and Water Availability: Implications for Ecological Restoration. Forests 13: 1854.
• MAGUIRE JD. 1962. Speed of Germination-aid In selection and evaluation for seedling emergence and vigor. Crop Sci 2: 176-177.
• MARCHIORI JNC & ALVES FS. 2012. A região do Parque do Espinilho e a bacia hidrográfica do Rio Quaraí, segundo o inventário florestal contínuo do Rio Grande do Sul. Considerações finais. Balduinia 30-I: 01-08.
• MARCHIORI JNC & ALVES FDS. 2014. O inhanduvá (Prosopis affinis Spreng.) no Rio Grande do Sul. 8 - Aspectos fitogeográficos. Balduinia 2: 13-20.
• MARCHIORI JNC, ALVES FDS, DEBLE LP & DEBLE ASO. 2014. A vegetação do Parque Estadual do Espinilho. 2 - Origem do nome e considerações fitogeográficas. Balduinia 30-III: 01-06.
• MARCOS-FILHO J. 2015. Fisiologia de Sementes de Plantas Cultivadas, 2ª ed., Londrina: Abrates, 659 p.
• MAYER AM & POLJAKOFF-MAYBER A. 2014. The Germination of Seeds, 3ª ed., Oxford, 232 p.
• MCTI/BRASIL. 2020. Quarta Comunicação Nacional do Brasil à Convenção Quadro das Nações Unidas sobre Mudança do Clima, Brasília: Ministério da Ciência, Tecnologia e Inovações. Secretaria de Pesquisa e Formação Científica, 620 p.
• MENDES LJ, MILAGRE JC, DICK G, KULMANN MSS, ROSA KP, UGALDE E, ARAUJO EF & SCHUMACHER MV. 2024. Impact of different land uses on soil properties subject to sandyzation in the Brazilian Pampa biome. Can J For Res 55(23): 1-13.
• MILAGRE JC, MENDES LJ, SILVA PS, SANTOS WRT, FOCKINK GD, GRANZOTTO F, FONTANA AE, SCHENATO RB & ROVEDDER APM. 2025. Long-term effects of cattle rearing on vegetation and soil in riparian forests in southern Brazil. An Acad Bras Cienc 97: e20240333. https://doi.org/10.1590/0001-3765202520240333.
  » https://doi.org/10.1590/0001-3765202520240333
• MORITZ C & AGUDO R. 2013. The Future of Species Under Climate Change: Resilience or Decline? Science. 341: 504-508.
• NASCIMENTO JPB, DANTAS BF & MEIADO MV. 2021. Hydropriming changes temperature thresholds for seed germination of tree species from the Caatinga, a Brazilian tropical dry forest. J Seed Sci 43.
• NASR SMH, PARSAKHOO A, NAGHAVI H & KIANI SAVAD KOOHI S. 2012. Effect of salt stress on germination and seedling growth of Prosopis juliflora (Sw.). New For 43: 45-55.
• NICOTRA AB ET AL. 2010. Plant phenotypic plasticity in a changing climate. Trends Plant Sci 15: 684-692.
• OVERBECK GE ET AL. 2022. Placing Brazil’s grasslands and savannas on the map of science and conservation. Perspect Plant Ecol Evol Syst 56: 125687.
• PBMC. 2020. Mudanças ambientais de Curto e Longo Prazo: Projeções, Reversibilidade e Atribuição. In: Base científica das mudanças climáticas. Primeiro Relatório da Avaliação Nacional sobre Mudanças Climáticas - Atualizado 2020, Rio de Janeiro: COPPE - Universidade Federal do Rio de Janeiro, p.320-246.
• PEARSON TRH, BURSLEM DFRP, MULLINS CE & DALLING JW. 2002. Germination ecology of neotropical pioneers: interacting effects of environmental conditions and seed size. Ecology 83: 2798-2807.
• PINHO PF, ANJOS LJS, RODRIGUES-FILHO S, SANTOS DV & TOLEDO PM. 2020. Projections of Brazilian biomes resilience and socio-environmental risks to climate change. Sustentabilidade em Debate 11: 225-259.
• POLASH MAS, SAKIL MA & HOSSAIN MA. 2019. Plants responses and their physiological and biochemical defense mechanisms against salinity: A review. Trop Plant Res 6: 250-274.
• POMETTI C ET AL. 2021. Species Without Current Breeding Relevance But High Economic Value: Acacia caven, Acacia aroma, Acacia visco, Prosopis affinis, Prosopis caldenia and Gonopterodendron sarmientoi In: Low Intensity Breeding of Native Forest Trees in Argentina, Cham: Springer International Publishing, p. 295-318.
• R CORE TEAM. 2023. The R Project for Statistical Computing. Vienna: R Foundation for Statistical Computing. Available at: https://www.rproject.org/. Accessed: August 24, 2025.
  » https://www.rproject.org/.
• REDIN CG, LONGHI SJ, REICHERT JM, SOARES KP, RODRIGUES MF & WATZLAWICK LF. 2017. Grazing changes the soil-plant relationship in the tree-regeneration stratum in the Pampa of southern Brazil. CERNE 23: 193-200.
• RIBEIRO FL, GUEVARA M, VÁZQUEZ-LULE A, CUNHA AP, ZERI M & VARGAS R. 2021a. The impact of drought on soil moisture trends across Brazilian biomes. Nat Hazards Earth Syst Sci 21: 879-892.
• RIBEIRO S, MOREIRA LFB, OVERBECK GE & MALTCHIK L. 2021b. Protected Areas of the Pampa biome presented land use incompatible with conservation purposes. J Land Use Sci 16: 260-272.
• RIO GRANDE DO SUL. 2014. Decreto n° 52.109, de 19 de dezembro de 2014. Declara as espécies da flora nativa ameaçadas de extinção no Estado do Rio Grande do Sul. 51.
• ROCHA NS, VEETTIL BK, CARVALHO CM, KÄFER PS, DIAZ LR, ROLIM SBA & CRUZ RC. 2021. Potential impacts of air temperature rise in the hydric balance of Brazilian Pampa biome. Acta Geophys 69: 1427-1445.
• ROOT-BERNSTEIN M, VALENZUELA R, HUERTA M, ARMESTO J & JAKSIC F. 2017. Acacia caven nurses endemic sclerophyllous trees along a successional pathway from silvopastoral savanna to forest. Ecosphere 8.
• SANTANA DG & RANAL MA. 2004. Análise da germinação - um enfoque estatístico, Brasília: Editora Universidade de Brasília, 248 p.
• SANTOS TB, FELICIO MS & DOMINGUES AD. 2022. A fisiologia dos estresses abióticos I: estresse hídrico, salino e altas temperaturas. In: Borém F-N (Ed), Melhoramento de Plantas para Estresses Abióticos, Viçosa: UFV, p. 312.
• SAVACINSKI S, LOUZADA P, HAIDUKI L, ROSA LMG, MÜLLER C, CANSIAN RL & SAUSEN TL. 2023. Assessing the role of light in flooding tolerance for tree species recommendation in the restoration of riparian subtropical forests. Trees 37: 403-415.
• SEAPDR/DDPA. 2021. Comunicado Agrometeorológico. Secr da Agric Pecuária e Desenvolv Rural (SEAPDR); Dep Diagnóstico e Pesqui Agropecuária.
• SHIBATA M, COELHO CMM, GARIGHAN JA, SANTOS HP, ARALDI CG & MARASCHIN M. 2021. Seed development of Araucaria angustifolia: plant hormones and germinability in 2 years of seeds production. New For 52: 759-775.
• SILVA CFM, CAMPOS MCC, SILVA COSTA DF, SCHNECK F & HEPP LU. 2025. Mapping and Identification of Ecosystem Services Hotspots in the Brazilian Pampa Biome. Environ Manage 75: 538-550.
• SILVA JMC, PINTO LP & SCARANO FR. 2021. Toward integrating private conservation lands into national protected area systems: Lessons from a megadiversity country. Conserv Sci Pract 3.
• SKIRYCZ A ET AL. 2009. Developmental Stage Specificity and the Role of Mitochondrial Metabolism in the Response of Arabidopsis Leaves to Prolonged Mild Osmotic Stress. Plant Physiol 152: 226-244.
• SONG X-P ET AL. 2021. Massive soybean expansion in South America since 2000 and implications for conservation. Nat Sustain 4: 784-792.
• SPECIESLINK. 2024. Search for Vacchelia caven and Neltuma affinis registers map.
• STRECK EV, KAMPF N, DALMOLIN RSD, KLAMT E, NASCIMENTO PC, ELVIOGIASSON & PINTO LFS. 2018. Solos do Rio Grande do Sul, 3ª ed., Porto Alegre: Emater/RS-Ascar, 254 p.
• SUERTEGARAY DMA. 2012. Erosão nos campos sulinos: arenização no sudoeste do Rio Grande do Sul. Rev Bras Geomorfol 12: 61-74.
• SUN W. 2002. Methods for the study of water relations under desiccation stress. In: Desiccation and survival in plants: Drying without dying, p. 47-91.
• THOMAS PB, MORRIS EC, AULD TD & HAIGH AM. 2010. The interaction of temperature, water availability and fire cues regulates seed germination in a fire-prone landscape. Oecologia 162: 293-302.
• TIEBEL K, KARGE A & WAGNER S. 2023. Does shading and ground cover of moss and litter improve germination and establishment of Betula pendula Roth, Salix caprea L. and Populus tremula L. seedlings during drought stress in climate change? - A greenhouse study. For Ecol Manage 544: 121212.
• TOMAS WM ET AL. 2024. Challenges in the conservation and management of legal reserve areas in Brazilian grassland and savanna ecosystems in the face of global climate change. Pesqui Agropecu Bras 59: e03491.
• VELASCO N, BUSTAMANTE R & SMIT C. 2023. Dispersal syndromes of Vachellia caven: Dismantling introduction hypotheses and the role of man as a conceptual support for an archaeophyte in South America. Heliyon 9: e17171.
• VICENTE E & GARZÓN MB. 2024. Tree Germination Sensitivity to Increasing Temperatures: A Global Meta-Analysis Across Biomes, Species and Populations. Global Ecology and Biogeography 33: 101036849.
• VILLAGRA PE. 1995. Temperature effects on germination of Prosopis argentina and P. alpataco (Fabaceae, Mimosoideae). Seed Sci Technol 23: 639-646.
• VILLAGRA PE. 1997. Germination of Prosopis argentina and P. alpataco seeds under saline conditions. J Arid Environ 37: 261-267.
• VILLAGRA PE, VILELA A, GIORDANO C & ALVAREZ JA. 2010. Ecophysiology of Prosopis Species From the Arid Lands of Argentina: What Do We Know About Adaptation to Stressful Environments? In: Desert Plants, Berlin, Heidelberg: Springer Berlin Heidelberg, p. 321-340.
• VILLALOBOS AE & PELÁEZ DV. 2001. Influences of temperature and water stress on germination and establishment of Prosopis caldenia Burk. J Arid Environ 49: 321-328.
• ZHOU C, WU S, LI C, QUAN W & WANG A. 2023. Response Mechanisms of Woody Plants to High-Temperature Stress. Plants 12: 3643.
• ZHU J-K. 2016. Abiotic Stress Signaling and Responses in Plants. Cell 167: 313-324.

Edited by

• Handling editor
  Yraima Cordeiro

Data availability

Data of this study are available from the corresponding author upon reasonable request.

Publication Dates

• Publication in this collection
  28 Aug 2026
• Date of issue
  2026

History

• Received
  15 Jan 2025
• Accepted
  17 May 2026