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Seedling emergence and survival of invasive pines in Argentinian mountain grasslands

Natural regeneration is a critical step for the dispersal of alien plants and the establishment of new invasive foci. The adequate timing and location of germination, as well as the survival of the seedlings, have direct effects on the recruitment of the population, and are expected to depend on habitat characteristics. Understanding the dynamics of these processes, and their variations according to the landscape at different scales, can be particularly important to design and implement control strategies. In this article, we evaluate differences in seedling emergence and establishment of Pinus halepensis in highland grasslands of the Argentine pampas, according to altitude, orientation of the slope and vegetation cover. Our results showed spatial variation on the rate of seedling emergence, with topography as a determining factor, possibly due to a facilitating effect on the slopes with higher incident solar radiation (NE at the study site). Slope exposition, on the contrary, seemed to have the opposite effect on the survival of seedlings. Once established, seedlings also survived better in sites with less solar exposure, probably due to protection from high evapotranspiration during the summer. These results could explain variations in the invasive performance of the species and guide management actions aimed at preventing its further expansion in the study area and in other habitats with similar characteristics.


Introduction
Invasive alien species are considered one of the main global threats to biodiversity (Lövei and Lewinsohn 2012;Simberloff et al. 2013;Alexander et al. 2014). Woody plant invasions, in particular, have the ability to dominate the landscape and dramatically change the dynamics of grassland ecosystems (Williams and Wardle 2005). Several species of the Pinaceae family (hereafter Pines), almost exclusively native to the northern hemisphere, have been introduced and planted in different regions of the southern hemisphere for commercial exploitation, landscaping and control of soil erosion, and many of them have become invasive (Richardson and Higgins 2000; Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10530-020-02410-6) contains supplementary material, which is available to authorized users. et al. 2010). Pines can change the dominant life forms, increase biomass, thus modifying the frequency and intensity of the fires (Richardson 1998;Taylor et al. 2017), alter hydrological regimes (Gorgens and Van Wilgen 2004;Jobbágy et al. 2013) and change soil properties, as well as the availability and flow of nutrients in the ecosystem (Jobbágy and Jackson 2003;Amiotti et al. 2007;Dickie et al. 2014).
Natural regeneration is a key process in the population dynamics of plant species (Manso et al. 2013), and thus, a critical step in the dispersal and establishment of invasive plants. Seed germination represents a risky transition from a stage that is more tolerant to environmental conditions to the weakest and most vulnerable stage in plant development, the seedling (Harper 1977;Baskin and Baskin 2014). As it is also an irreversible process, the adequate timing and location of germination has a direct impact on the recruitment of the population (Baskin and Baskin 2014). Seeds are dispersed to a variety of microhabitats with a range of different biotic and abiotic conditions that affect seed germination, emergence, survival and growth of seedlings. Recruitment may be limited by different reasons, including the efficiency of seed dispersal, seed predation, abiotic stresses such as drought and light limitations, and availability of suitable microsites for seed germination and establishment of seedlings (Retana et al. 1999). In plant species with wide geographical distributions, the different populations are usually subject to different environmental conditions, and therefore to environments with diverse ecological constraints (Castro et al. 2004), which a priori could result in a greater diversity of capacities and colonization strategies and, consequently, in a greater aptitude to become established and to invade new environments.
Pine recruitment could be strongly influenced by the interaction between the seedlings and the resident vegetation (Richardson and Bond 1991;Langdon et al. 2019). The quality of the habitat at the local level can be a determining factor for both germination and establishment (Castro et al. 2005;Del Campo et al. 2007). Simberloff et al. (2010) highlight the importance of propagule pressure to define the fate of the colonization process in conifers. The discussion about the availability of seeds versus that of appropriate microsites as determinants for plant establishment is not new, and the combined limitation of both factors could be particularly significant for the regulation of recruitment in plant populations (Eriksson and Ehrlén 1992;Turnbull et al. 2000). Pines usually produce large amounts of seed (Richardson and Rundel 1998), and therefore the availability of propagules is usually not a limiting factor. Thus, understanding the effects of the characteristics of the seed arrival sites on their germination and on seedling survival would be particularly important for the establishment of new foci of invasion.
The Pampas biome is one of the most intensely affected by human activities in South America. Due to its mild climate and the aptitude of the soils, it has been historically used for agriculture and livestock husbandry (Bilenca and Miñarro 2004;Fonseca et al. 2013;Pretelli et al. 2015). Many of the valuable remaining grassland in the region have been invaded by pine trees (Pinus halepensis, P. radiata, P. pinaster and P. elliottii, among others) dispersed from nearby cultivated areas (Zalba and Villamil 2002;Yezzi et al. 2018). The Ventania mountains, at the southern tip of the South American Pampas, constitute a particularly valuable relict, where the rocky outcrops prevented the advance of the agricultural frontier. The area, however, undergoes an intense process of colonization by invasive woody plants, especially Aleppo pine (Pinus halepensis, Zalba and Villamil 2002). Aleppo pines, a native species of the Mediterranean basin, are capable of growing in the full height range and respond positively to the two main structuring forces of grassland plant communities, fire and herbivory, thus representing the main threat to the conservation of this refuge for the regional biodiversity (Zalba et al. 2008;de Villalobos and Zalba 2010;de Villalobos et al. 2011).
Knowing the specific characteristics of favorable sites for pine recruitment can help in the development of more effective management actions by mapping particularly suitable areas for the establishment of pine trees, and thus identifying sites where to intensify the search for new sources of invasion and targeting associated seed sources for elimination, according to the direction of the prevailing winds (Brancatelli and Zalba 2018).
One of the main forces regulating the growth of Aleppo pines (Pinus halepensis) in their native range is the availability of water (Rathgeber et al. 2005;Olivar et al. 2012Olivar et al. , 2014Bueis et al. 2017). The correlation between precipitation and growth of this species is usually significant and positive (Condés and García-Robredo 2012). Therefore, topography at the local level is relevant since it partially regulates water flows as well as soil characteristics that affect its availability by plants (Vennetier et al. 2018). In Jordan, Omary (2011) found differential growth of Aleppo pines according to the orientation of the slopes where they were growing that could be attributed to local differences in rainfall. Del Río et al. (2008), in Spain, indicated higher recruitment on sunny slopes than in shady areas. Nevertheless, high temperatures cause greater evapotranspiration resulting in water stress on seedlings (Condés and García-Robredo 2012). Consistently, summer time, associated with high temperatures and marked drought, constitutes the phase of greatest risk for newly emerged seedlings in the region where the species is native (Rathgeber et al. 2005;Del Río et al. 2008;Papadopoulos et al. 2009). The first summer is estimated to be the period of greatest mortality for the species . Another important factor that affects establishment in P. halepensis is the interaction with the surrounding vegetation (Richardson and Bond 1991). Competition with abundant herbaceous cover can result in a high recruitment failure rate (Rubira et al. 1996;Puértolas et al. 2003). However, bare soil seems not to be beneficial either (Gasque and García-Fayos 2004). Climate, topography and the regime of disturbances particularly affect the recruitment of pines and their capacity to invade natural ecosystems in mountain ranges (Mast et al. 1997;Ayari et al. 2011), by affecting their development, fecundity and dispersal (Vennetier et al. 2018). A previous study in our area showed differences in growth rate, as well as in the age at maturity of Aleppo pines growing in peaks, compared to those growing on hillsides and valleys (Brancatelli et al. 2020), while the effect of environmental variables on the germination and establishment of this invasive species was unknown until this publication.
The objective of this study is to evaluate differences in seedling emergence and establishment of Pinus halepensis in highland grasslands of the Argentine pampas, according to altitude, orientation of the slope and vegetation cover. We expect to find a better performance both in seedling emergence, survival and growth on the NE side of the mountains, where a higher average annual precipitation coincides with more sunny hillsides (Gentili and Gil 2013). At high altitudes (900-1200 m a.s.l.), a decrease in emergency and establishment can be expected due to environmental stress caused by low temperatures and exposure to stronger winds Frangi 1995a, 1996). At the same time, recruitment is expected to be more successful in sites with intermediate plant cover, since both extremes: bare soil (due to excess radiation, higher predation, etc.) and consolidated grassland vegetation (due to competition), limit survival and growth of seedlings (Zalba et al. 2008;de Villalobos et al. 2011).

Study area
This study was carried out at the Ernesto Tornquist Provincial Park (ETPP) that covers an area of ca. 6700 hectares in the Ventania mountains (38°10 0 S, 62°8 0 W), Argentina, and includes natural grassland in a range that extends from basal valleys at 450 m a.s.l. to some of the highest peaks in the pampas region, which barely exceed 1200 m above sea level (Kristensen and Frangi 1995b).
The climate is temperate, with a mean annual temperature of 14.6°C. Average annual rainfall ranges from 600 to 800 mm, especially concentrated in spring and part of summer (Frangi and Botinno 1995). The mountain topography determines local variations of the climate based on altitude, exposure and slope (Kristensen and Frangi 1995b). Thus, for example, habitats at higher altitudes are characterized by a high evaporation, strong winds and shallow soils (Kristensen and Frangi 1996). The orientation of the slope determines the amount of sunshine it receives, being the south-west facing areas more shady, with lower evaporation and higher humidity; while the slopes to the northeast are sunny and warmer, and hence with higher evaporation. These conditions determine differences in the composition and the structure of vegetation (Kristensen and Frangi 1995b).The area belongs to the Southern District of the Pampas Phytogeographic Region, where grass steppe is the dominant vegetation (Cabrera 1976;Frangi and Botinno 1995;Long et al. 2004). Although the region was almost deprived of trees before European settlement (Parodi 1942), it is currently under an invasion process that quickly replaces the grassland ecosystem with monospecific pine stands (Pinus halepensis and P. radiata), among other invasive species (Zalba and Villamil 2002;Amodeo et al. 2017).

Collection of seeds
During the months of January and February 2018, twenty individuals growing at altitudes between 450 and 500 m a.s.l. were selected. Five full-grown but still closed cones were collected from each of them, all located at a height that facilitated extraction (less than 2.5 m).Cones were placed near a heat source to promote their opening and the released seeds were kept in paper envelopes until the time of planting.

Laboratory germination experiment
Pinus halepensis seeds do not require stratification since they have an endogenous rhythm that regulates germination under the right conditions (Skordilis and Thanos 1995). To estimate the germination potential of the collected seeds, a test was carried out in a germination chamber using five Petri dishes with 20 seeds each (100 seeds in total), at constant humidity with a photoperiod of 13 h of light at 20°C and 11 h of darkness at 15°C (optimal germination conditions for Aleppo pine, Skordilis and Thanos 1997). Germination was registered when a radicle of 5-10 mm long emerged.

Irrigated experiment
A control site was placed outdoors in the Pillahuincó Botanic Garden, located in a valley (460 m a.s.l.) of the ETPP. This site included five plots (of approximately 1 m 2 each) with 40 seeds each, planted manually in small excavations of 1 cm depth covered by the substrate. Control plots received periodic irrigation (biweekly), in order to free them from water stress. The volume of water was calculated so that the sum of rainfall and irrigation reaches at least the monthly average rainfall according to the historical records in the area for the period 2000-2016 (Table S1 supplementary material, data provided by Ernesto Tornquist Provincial Park staff).

Field experiment
Twelve sampling sites were distributed on the two main orientations of the mountains: northeast and southwest; and at three different altitudes: low (450-500 m a.s.l.), mid (600-650 m a.s.l.) and high (900-950 m a.s.l.). This resulted in a crossed design between the two factors with two replicates for each combination. At each experimental site five plots of 1 9 1 m were located randomly. In April 2018, 40 seeds per plot were planted in the same way as was done for those under control conditions. Seeds did not receive any previous treatment and individual planting sites were marked by labeled stakes. A total of 2600 seeds were sown and monitored every 2 weeks for a total period of 2 years, recording seedling emergence and mortality, and measuring their total longitude (from ground to the highest leaf).
Since it is estimated that 97% of the seeds produced by an adult individual are concentrated at less than 20 m from the tree (Nathan et al. 2000), each plot was located more than 30 m away from any adult pine in order to minimize the probability of arrival of exogenous seeds. Only some of the sites in the SW hillside had a few adult specimens of Aleppo pine about 50-150 m away. Most of the plots were located in sites with no pines in the surrounding areas (more than 200 m).
Vegetation and rainfall were recorded at sampling sites every 2 months. Percentage cover of bare soil and rocks (BSR), grasses and herbs (GHR), and shrubs and subshrubs (SHR) was assessed in each plot. We also recorded the average height of the vegetation at five random points within each plot. A plastic rain gauge, with a wide mouth and 140 mm of total capacity, was placed at each site to assess local rainfall.

Statistical analyses
We analyzed seedling emergence and survival separately by means of time-to-event analyses (McNair et al. 2012). Since only two seedlings emerged during the second year, we only included data of the first year in the emergence analysis. Time since sowing and time since emergence were used as time scales for the analysis of emergence and survival, respectively. Seeds that did not germinate during the experiment were treated as censored data and they were given a censoring time (302 days) that was defined as the last day of the germination period, after which germination did not occur. The same was applied for seedling survival (699 days assigned for seeds that did not die during the whole experimental time). We compared seedling emergence and survival across the different slope orientations and altitudes using proportional hazards regressions (or Cox regressions). The Cox model is based on a hazard function defined by the product of a baseline hazard function that depends only on time, and a modifier function that depends on the covariates (McNair et al. 2012). No parametric assumptions are made about the baseline hazard function, while the covariate portion does depend on parameters. The hazard ratio obtained from this analysis represents the ratio of the intensity of emergence (or mortality) in two comparing groups. To account for random factors that arise from our experimental design (sites and plots), we incorporated a frailty term to the Cox regressions. Frailty terms are random effects allowing the emergence/survival rate to vary among these categories. The proportional hazards model assumes that the hazard ratio is constant with respect to time. We checked this assumption using graphical methods following McNair et al. (2012). Potential multicollinearity of the covariates was also assessed using variance inflation factor analysis (VIF). Neither of these evaluations showed clear violations of the model assumptions: no decisive crossings of the functions in the proportionality diagnostic plot were detected, and the analysis of VIF, showed no evidence of collinearity. For graphical representation we used Kaplan-Meier curves for showing the survival data and ''1 minus Kaplan-Meier'' curves for emergence data to show the proportion of seed that had germinated on any given day of the experiment (Andersen et al. 2016).
The length of the seedlings was compared among altitudes and orientations for measurements at their median age (140 days) using GLM with Gamma distribution and inverse link function, and Tukey post hoc comparisons. The distribution was chosen based on QQplots.
Temporal variation in microhabitat conditions across all sampling dates for each site was assessed using coefficients of variation at each site. Average values were compared between sites at different altitudes and orientations using GLM with binomial distribution for the proportion of cover of soil and rocks, grasses and herbs and shrubs and subshrubs. A GLM with Gamma distribution was used for comparing vegetation mean height. Local variation of precipitation was evaluated using Pearson correlation coefficients. The correlation of monthly precipitations across experimental sites was analyzed by Pearson correlation pairwised between all the sites.

Irrigated experiment
Twenty-four seedlings emerged in the control site during the irrigation experiment, from a total of 200 seeds that were sown. Emergence occurred in three of the five plots between 51 (late May) and 95 days (early July) after sowing, leading to a general final emergence percentage of 12% (SE = 7.39). The plots received an average irrigation of 34.02 mm, with a maximum value of 80.12 and a minimum of 0 (Table S1 supplementary material). All emerged seedlings died during the experiment with a median survival time of 49 days. Along the whole time of emergence in the control site, the environment was characterized by a 100% of soil cover of grasses and herbs with an average height of 6.96 cm (SE = 0.76).

Field experiment
The vegetation cover at the different sites did not show a marked temporal variation for all cover types (coefficients of variation across dates were low for most of the cases, third quartile = 72.5%, Table S2 supplementary material). Therefore, an average value was calculated to characterize each site. In general terms, vegetation cover was dominated by grasses and herbs and tended to be lower at the northeastern hillside, with a higher proportion of rocks and bare soil with respect to the southwestern side (GLM, Likelihood Ratio Tests, p \ 0.05, Table 1). Shrubs and subshrubs cover did not differ significantly between altitudes and orientations (GLM Likelihood Ratio Test, Deviance = 4.32, p = 0.12). The height of the vegetation showed a peak during the summer months in all the plots. Vegetation tended to be higher at low and mid altitudes of the southwestern hill-side with respect to the northeast side. Minimum values were recorded at the highest sites, regardless of orientation (Fig. 1).
Monthly precipitation across experimental sites ranged from 0 to 140 mm with an average of 39.2 mm (SE = 0.14, n = 246), during the first year of the experiment (April 2018-March 2019). This was the year in which the entire emergency occurred and therefore this is the precipitation value considered for the analysis. It has to be noted that rainfall during these months was below the average at the reserve for 2000-2016 (670.4 mm vs. 942.2 mm). The accumulated rainfall during the summer months (December, January, February) was 129.7 mm, a 43.7% of the historical average for those months (296.8 mm). The precipitation curve across the dates showed a high correlation among sites (all the pairwise correlation coefficients between sites were highly significant and ranged between 0.947 and 0.996, with an average of 0.976, SE = 0.0002, n = 66; Fig. 2).
Field experiment-emergence From a total of 2400 seeds sown in the field, 476 seedlings emerged during the first year (19.8% overall emergence) between 33 (early May) and 302 days (late January) after sowing. Only two seedlings emerged during the second season (0.08% overall emergence), both 363 days after sowing (late March). The results shown below correspond to the emergence during the first year. The final emergence percentage at each site ranged from 3.5% (SE = 1.2%) to 44.5% (SE = 2.4%) and varied significantly with site orientation and altitude (Tables 2, 3).
All Cox regressions were run with a frailty term for Site and Plot, showing significant effects on the parameter estimates. This is evidence of significant variation in seedling emergence rate with location. The effect of topographic variables on emergence was significant and strong as indicated by the hazard functions (Table 3). Emergence in sites located in southwest hillside was lower and took more time than in sites at the northeast hillsides, as indicated by the lower hazard function for emergence (single model, southwest orientation HR 0.318, CI [0.195-0.519], p \ 0.001). The effect of altitude showed a significant interaction with slope orientation. At the northeast hillside, sites at mid and high altitude showed a lower hazard function for emergence than those at low altitude (0.48 and 0.36 times, respectively). At the southwest slope, on the contrary, the mid and upper altitude sites were associated with higher hazard functions with respect to those at the lowest altitude (HR 6.488/0.081 = 80.1 and HR 8.419/0.081 = 103.9 times, respectively Table 3, Fig. 3).  (Table 4). Seedling survival showed very different patterns depending on orientation and altitude of the site, denoting an interaction between the effects of both habitat variables. All sites facing to the northeast showed null or very low (below 5%) seedling survival at the end of the experiment, whereas survival on the opposite side of the mountains reached higher values (34.4%, Table 2). Highest sites on the northeast hillside showed a steeper decay in survival (median survival at 106 days) compared to those at low and mid altitudes (median survival at 190 and 200 days, respectively, Table 2, Fig. 4). Survival curves decayed faster in sites on the southwest slope (median survival of 64, 106 and 141 days), with higher survival in mid altitudes at the end of the experiment (34.4%, Table 2, Fig. 4). Mid and upper altitude sites at the southwest hillside showed a lower hazard function than those at the lowest altitude (HR 0.156/ 3.361 = 0.046, and HR 0.214/3.361 = 0.064, respectively), leading to a much slower fall of the survival   curve and resulting in censored seedlings that remained alive at the end of the experiment (Table 4).
Field experiment-seedling growth The length of 140 days old seedlings varied with orientation and altitude showing no significant interaction between both factors (GLM, interaction term LRT, p = 0.28). The length of seedlings at mid altitudes tended to be higher than in the other altitudes at both sides of the mountain range, with significant differences only at the southwest slope (GLM, Tukey tests, Fig. 5).

Discussion
This work documents for the first time the effect of topography and local habitat variables on the emergence and establishment of Pinus halepensis in mountain grasslands of the Pampas biome. We found differences in both demographic parameters that could explain variations in the invasive performance of the species and guide management actions aimed at preventing its further expansion in the study area and in other habitats with similar characteristics. Recruitment capacity in the field has been traditionally identified as a key component of invasive behavior, since it determines colonization ability and population dynamics (Boulant et al. 2008;Steinitz et al. 2011). In spite of the extensive knowledge available regarding this components of the life history for Aleppo pine in its native region (Trabaud et al. 1985;Thanos et al. 1996;Nathan et al. 2000;Nathan and Ne'eman 2004;Omary 2011;Sagra et al. 2019), until this work almost nothing was known about the same components of its population dynamics in  Our results show an effect of the site on the seedling emergence rate, with topography as a determining factor. As we expected, germination was lower and slower in sites located on the southwest slope of the mountains compared to those on the northeast. During the germination season, NE orientation receives more solar radiation, which determines higher ground temperatures than at the other slopes (Kristensen and Frangi 1995b). Higher insolation and temperature could favor the emergence of pine trees on these slopes. It was found that long exposure to diurnal light always promotes the germination in P. halepensis Thanos and Daskalakou 2000). This relationship has been also recorded for other pine species, like Pinus sylvestris in the southeast of Spain, where seeds showed a higher germination rate in microhabitats directly exposed to the sun than in shady microhabitats (Castro et al. 2005).
On the contrary, the survival of seedlings once established is lower in sites facing the northeast in our study area. In Aleppo pine the correlation between growth and temperature is usually negative, since higher temperatures result in higher evapotranspiration and reduce water reserves (Condés and García-Robredo 2012). This could be the cause of the highest mortality observed in the NE sites at the study site. Del Campo et al. (2007) report that mild to cold temperature regimes would have improved survival in Aleppo pine plantation in eastern Spain. In regions where this species is native, duration and intensity of the summer drought periods are also the main factors limiting its survival (Broncano et al. 1998;Puértolas et al. 2003;Rathgeber et al. 2005).
Altitude is also recognized as a feature with significant effects on seedling emergence and survival. In the Ventania mountains, height seems to have contrasting effects on germination depending on the orientation of the slopes. Thus, while sites located northeast of the mountain range showed less germination at mid and high altitudes (650-950 m a.s.l.), germination reached its highest values at sites located in the same altitude ranges, but on the southwestern slope. Survival at high and mid altitudes seems to be higher than at low areas. This effect, in turn, is more intense on the southwest slope. These results contradict our expectations of finding lower emergency and establishment on the higher habitats, associated to environmental stressors like low temperatures and stronger winds Frangi 1995a, 1996). A possible explanation for this discordance could be related to the competition of the seedlings with local vegetation at each site. Basal habitats on the southwest side of the hills are characterized by having the maximum values of plant cover and plant height throughout the year (Table 1). In general terms, pine recruitment is strongly related to interaction between pine seedlings and the resident vegetation. Pines invasion is most prevalent where competition in the regeneration niche is limited, and it occurs more often and intensely in habitats where this trees contrast the most with the dominant vegetation, such as grasslands (Richardson and Bond 1991). Other authors also agree that competition from an abundant herbaceous cover may be responsible for temporal variation in pine recruitment (Rubira et al. 1996;Puértolas et al. 2003;Simberloff et al. 2010;Ayari and Khouja 2014 argue that herbivory may favor the establishment of pines. Absence or low herbivory intensity can lead to greater cover of grasses and herbs, and a greater resistance to invasion. The characteristics of the vegetation at basal sites on the southwest slope in our study could be similar to those. Conversely, a high grazing intensity causes a decrease in grass cover and greater vulnerability to the establishment of pine seedlings. This is the case at the lower altitude sites on the northeast slope, where the proportion of rocks and bare soil are the highest and probably result in greater availability of microsites appropriate for pine recruitment and reduced competition at the regeneration niche. De Villalobos et al. (2011) report an average seedling survival at 1 year of 16.5% and 49.7%, in grazing exclusion areas and in areas under grazing of wild horses, respectively. In our study, the overall average survival at 1 year was lower (10.5%), which could be due to the variety of habitats considered in the experimental design of our work, including sites under more extreme environmental conditions. The highest survival rate in our trial occurred in the mid altitude sites on the southwest slope, which according to the characteristics of the vegetation could be similar to an intermediate grazing situation. The establishment of P. halepensis seedlings could also be affected by the seasonality of grazing in the study area (de Villalobos and Schwerdt 2020). They found that the defoliation of perennial grasses during autumn-winter generates loss of vigor in them, reducing competition at the time of emergence of pine seedlings. This process could facilitate the establishment of this and other invasive species. In our study, however, there are no seasonal differences in vegetation cover. So the emergence and differential survival found could not be explained by this process. Water availability is recognized as a determining factor for the germination and establishment of Aleppo pine in its native range (Rathgeber et al. 2005;Olivar et al. 2012Olivar et al. , 2014Bueis et al. 2017). Although there are some studies that found variations in rainfall at the local scale at our study area (Frangi and Botinno 1995;Gentili and Gil 2013), our experiments did not detect significant differences among experimental sites. The year in which the test was conducted was not typical in terms of rainfall. Precipitation records showed a water deficit in comparison to the historical records, especially during the summer. In our control site, however, where water stress was avoided through irrigation, both emergence and survival were among the lowest of all sites. This could be indicating the importance of other factors such as microhabitat conditions and the interaction with the surrounding vegetation at the local level. In this regard it is important to understand that the rain, by itself, is not enough to reflect the availability of water for the seedlings. Evapotranspiration, competition and physical parameters of the soil also play a role in this regard (Bueis et al. 2017;Helluy et al. 2020). Potential evapotranspiration, which depends on the net radiation and on temperature, reduces water reserves available to the vegetation (Rathgeber et al. 2005). The lower seedling survival we found at northeast hillside is in concordance with this idea and could be explained by solar exposure. The amount of water that is actually available to plant roots is also strongly related to the properties of the soil and its retention capacity (Bueis et al. 2017). Del Río et al. (2014) found that sites with clay soils presented higher growth rates of P. halepensis than sites with other soil types. Knowing the effects of the substrate on the performance of Aleppo pines would probably help to better explain the differences found in the recruitment of the species. Competition, on the other hand, also influences the availability of resources, and may cause differences in the individual growth of plants (Calama et al. 2019). As mentioned before, our results regarding vegetation cover and height are indicative that the competition with the surrounding vegetation can be crucial for recruitment.
Beyond improving knowledge about the relationship between the population dynamics of the species under study and the environmental variables of the areas it colonizes, our results allow adjusting and optimizing the efficiency of management actions aimed at reducing its current and potential effects on the grasslands affected by the invasion that are currently on place. According to our results, the sites at mid and high altitudes on the southwest side of the mountain range, would meet the most appropriate conditions for the establishment and survival of P. halepensis seedlings, and thus, are particularly prone for the start of new invasion foci in the study area. From this point of view, it is a priority to detect and eliminate trees that act as seed sources with the potential to reach these areas (those growing upwind and above this sites). These activities must be complemented by actions aimed at detecting young individuals growing in these environments before they reach their minimum reproductive age (5 years, Cuevas 2010).
Biological invasions are complex processes that challenge the management capacity of protected areas. Our work shows variations in germination and seedling survival in response to the characteristics of the different environments exposed to invasion. This information can be very useful to develop management strategies for the control of Pinus halepensis in order to optimize the available resources and to increase the chances for successful intervention to slow down the spread of the species.