Screening for Cold Tolerance during Germination within Sweet and Fiber Sorghums [ Sorghum bicolor (L.) Moench] for Energy Biomass

: Within the project “BIOSEA” by the Italian Agriculture and Forestry, a preliminary laboratory test was conducted to assess the variability for cold tolerance during germination in 30 cultivars of biomass sorghum, among ﬁber and sweet types. Seed germination (%) and mean germination time (MGT) were examined at seven constant temperatures (from 8 ◦ C to 35 ◦ C) and base temperature ( T b) and thermal time ( θ T ) for 50% germination were calculated. A wide genetic diversity in the germination response of sorghum was ascertained at 8 ◦ C (CV 45%) and 10 ◦ C (CV 25.4%). At 8 ◦ C, in cultivars of ‘Padana 4’, ‘PR811F’, ‘PSE24213’, ‘PR849’ and ‘Zerberus’, seed germination exceeded 80%. Seeds of ‘Zerberus’ were also the fastest, requiring less than 13 days for ﬁnal germination at this low temperature. Great differences were found in T b and θ T among cultivars. T b varied between 7.44 ◦ C (‘PR811F’) and 13.48 ◦ C (‘Nectar’). Thermal time ( θ T ) was, on average, 24.09 ◦ Cd − 1 , and ranged between 16.62 (‘Nectar’) and 33.42 ◦ Cd − 1 (‘PSE24213’). The best combination of the two germination parameters (i.e., low T b and θ T ) corresponded to ‘Zerberus’, ‘Sucrosorgo 506’, ‘Jumbo’ and ‘PR811F’. Accordingly, these cultivars are more tolerant to cold stress during germination and, thus, more adapt to early spring sowings in Mediterranean areas (March-April). Cultivars ‘PR811F’ (ﬁber type) and ‘Sucrosorgo 506’ (sweet type) also combine high cold tolerance with good productivity in terms of ﬁnal dry biomass, as assessed in open-ﬁeld conditions (late spring sowing). The genetic variation in the germination response to a low temperature is useful for the identiﬁcation of genotypes of sorghum suitable to early sowings in semi-arid areas. Selection within existing cultivars for cold tolerance during germination may also contribute to the expansion of biomass sorghum into cooler cultivation areas, such as those of Northern Europe, which are less suitable to this warm season crop. curation, V.C. Funding C.P. C.P. and A.S. Methodology, C.P. and S.L.C. Software, C.P. and S.L.C. Validation, C.P. Writing—original draft, C.P. and V.C. Writing—review


Introduction
Sweet and fiber sorghums [Sorghum bicolor (L.) Moench], as fast growing and highyielding species under a wide range of soil and environmental conditions [1,2], are considered promising industrial crops for the European Community, for the bioethanol (sweet types) and the combustion (fiber types) chains [3]. Moreover, both types of sorghums produce lignocellulose that could serve as feedstock for second generation biofuel [4].
Sorghum is a C4 plant, native to tropical areas, that can be adapted to most of the temperate and sub-tropical climates as annual crop, but it grows well on marginal and non-irrigated lands under Mediterranean-like climates [5]. Field experiments conducted in different areas of Europe confirmed the high yield potential of this crop under no water limitations [4,6].

Plant Material
The experiment was conducted in the laboratory on the seeds of 30 cultivars of biomass sorghum (Sorghum bicolor L. Moench) among fiber and sweet biomass types. The list of cultivars and their provenance are reported in Table 1. The cv. 'Keller' of sweet sorghum was adopted as a control, as it has been proven to be the most adaptable and best producing energy biomass in the Mediterranean environment [6,16]. When the experiment was conducted, the seeds were less than 12 months old, and kept at room temperature (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) • C) before being tested.

Germination Tests
The seeds of the 30 cultivars of sorghum were germinated at seven constant temperatures (T): 8,10,15,20,25 and 30 • C, with 25 • C considered as the optimum for seed germination of sorghum [17]. The germination tests were made in a thermostatically controlled (±1 • C) incubator. Samples of 150 seeds (three replicates of 50 seeds each) were placed in Petri dishes containing a single sheet of paper tissue, moistened with 7 mL of distilled water. Petri dishes were hermetically sealed with Parafilm to prevent evaporation. Thus, they were randomised within each temperature and incubated in the dark. Seeds germinated (those whose radicle reached at least 2 mm of length) were recorded and removed daily. The seed germination test was stopped when no additional germination occurred for 72 h. The germination tests were conducted in 2010, in a time span of approximately 6 months, starting in the spring.

Open Field Experiment
In the same year of germination tests in the laboratory (2010), a field experiment was conducted in a flat site of the Eastern coast of Sicily (South Italy, 10 m a.s.l., 37 • 25 N Lat, 15 • 30 E Long), on a Vertic Xerochrepts soil. The same 30 cultivars of sorghum tested in the laboratory assay were evaluated for biomass yield in the field, in a complete randomized blocks' experimental design with three replicates. Sowing was hand made on 3 June, in single plots of 6 m 2 (2 m long × 3 m wide). A 0.50 m row distance and a 12 plants m −2 Agronomy 2021, 11, 620 4 of 16 final density were adopted. Before sowing, 100 kg ha −1 of N (as ammonium sulfate) and 100 kg ha −1 of P 2 O 5 (as mineral perphosphate) were distributed. Approximately a month after seedling emergence, a further 70 kg ha −1 N (as ammonium nitrate) were distributed as top dressing. During the growing season, the main meteorological variables (maximum and minimum air temperature, rainfall, reference evapotranspiration-ET 0 ) were recorded, using a weather station connected to a data logger (CR10, Campbell Scientific, Inc., Logan, UT, USA).
Irrigation was applied to meet crop requirements (100% evapotranspiration restoration) along the growing season. Total 3450 m 3 ha −1 were distributed to the crop. Final harvest was made on 11 November, on plants of central rows, after removing the border plants. At harvest, the total above-ground dry biomass was estimated. To this end, total biomass samples were oven-dried at 65 • C until constant weight for a dry matter measurement.

Calculations and Data Analysis
At the end of germination tests, final germination (FG, %) and Mean Germination Time (MGT, days) were calculated.
The time course of cumulative values of seed germination was described by a nonlinear iterative regression method (SIGMAPLOT ® 9.0 software, Systat Software Inc., San Jose, CA, USA) using the following sigmoidal model with three parameters.
where a is the maximal value of y (i.e., maximum germination), x is the time (days) after seed imbibition, x 0 is the time (days) to reach 50% of maximal germination, and b is a fitting parameter of the curve. The x value on the curve corresponding to 50% germination (y value of the curve) was assumed as theoretical time to 50% germination or t 50 (days) [18,19]. Data set of germination rates of 50% germination fraction (1/t 50 or GR 50 ) of seed population, resulting from the germination time course (see Figure 1), was plotted against T, separately for the cultivars. The linear regression of GR 50 vs. T allowed us to calculate the theoretical minimum or base temperature (Tb) at which seed germination of each cultivar is reduced to 50%. The slope b of the regression line is the germination rate with a decreasing temperature (the higher the b, the faster the germination with the temperature increase). The abscissa intercept is an estimate of the theoretical minimum temperature of germination (Tb) [19,20]. Thermal time (θ T ) to achieve 50% germination (θ T(50) ) at each temperature was calculated for each cultivar using the equation below.
where θ T(50) = thermal time needed for 50% germination ( • Cd), T = actual germination temperature ( • C, constant in controlled environment), Tb = base germination temperature, t 50 = the time to 50% germination (median response time) [19,20]. In order to compare the two methods of θ T(50) calculation, thermal time was also calculated as the inverse of the slope b of the regression line used for Tb estimation.
Data of the final percentage germination, previously arcsine transformed, and those of MGT, were statistically analysed by a completely randomised two-way (temperature × cultivar) analysis of variance (ANOVA) using COSTAT version 6.003 (CoHort Software, Monterey, CA, USA). When 'F' ratios were significant, means were separated by the Least Significant Difference (LSD) test at p ≤ 0.05. A one-way ANOVA was also performed on data of final dry biomass yield, using 'cultivar' as a source of variation, and means were separated by the LSD test at p ≤ 0.05.  In order to compare the two methods of θT(50) calculation, thermal time was also calculated as the inverse of the slope b of the regression line used for Tb estimation.
Data of the final percentage germination, previously arcsine transformed, and those of MGT, were statistically analysed by a completely randomised two-way (temperature × cultivar) analysis of variance (ANOVA) using COSTAT version 6.003 (CoHort Software, Monterey, CA, USA). When 'F' ratios were significant, means were separated by the Least Significant Difference (LSD) test at p ≤ 0.05. A one-way ANOVA was also performed on data of final dry biomass yield, using 'cultivar' as a source of variation, and means were separated by the LSD test at p ≤ 0.05.

Estimation of the Germination Date in an Open Field
In order to reproduce potential scenarios, the date of occurrence of 50% seed germination in the field was estimated for each cultivar of sorghum, considering three hypo-

Estimation of the Germination Date in an Open Field
In order to reproduce potential scenarios, the date of occurrence of 50% seed germination in the field was estimated for each cultivar of sorghum, considering three hypothetical early sowing dates: March 1, March 15, and April 1. For the prediction of the germination date in the field, individual thermal time and Tb values of each cultivar, and mean soil temperatures from March to April, as measured over a 10-year period (2005-2014) by a weather station connected to a data logger (CR10, Campbell Scientific, Inc., Logan, UT, USA), located in the same site of the 2010 field experiment, were considered.

Cumulative Germination Time Course
The cumulative seed germination time course of 30 sorghums during imbibition at different constant temperatures is illustrated in Figure 1. The course is well described Agronomy 2021, 11, 620 6 of 16 (R 2 > 0.90) by a three-parameter sigmoidal function whose trend reveals an initial phase of low germination, which is followed by a step rise up to a maximum. After that, germination stopped (at warmer temperatures) or slowly proceeded (at lower temperatures). The length of the initial phase of slow germination, minor or null at temperatures ≥ 25 • C, progressively increased as temperature decreases.
The lowering of the temperature from that optimal progressively inhibited and delayed germination to a different extent, depending on the cultivar. Some sorghums (e.g., 'Padana 4', 'PR811F', 'PSE24213', 'PR849', 'Zerberus') only postponed the start of the exponential increase of germination at lower temperatures, while exhibiting an overall similar cumulative germination trend and final germination percentage at all temperatures. In other cultivars ('Jumbo', 'PSE98456', 'Sugargraze', 'PSE22053', 'Sucrosorgo 506', 'ABF26', 'Keller'), the thermal lowering slowed down and depressed seed germination (i.e., final percentage of seeds germinated) only at the lowest temperature (8 • C). In some others (e.g., 'Padana 1', 'Hay Day', 'Nectar'), the depressive effects of thermal lowering on the germination time course were progressive, since a clear response was observed at 15 • C. Overall, the higher the temperature, the faster the germination process. However, at 30 • C, the final germination percentage in some cases was lower than that at 25 • C.

Mean Germination Time in Response to Temperatures
Mean Germination Time (MGT) varied with temperature and cultivar. Germination was faster at the highest temperatures and, at 30 • C, all cultivars were germinated in less than two days (Table 4). The lowering of temperature from that optimal level led to a progressive increase in germination time to a different extent depending on the cultivar (C × T significant, p ≤ 0.001) ( Table 5). At 10 • C, the genetic variability for the germination rate was the greatest (28.49%), since cultivars responded quite differently from this cold temperature in terms of germination speed. 'Nectar' was the slowest (17.45 days MGT) under this temperature as well, while 'PR811F' was the fastest, reaching its final germination in a 5.45-day MGT.
At 8 • C, germination further slowed down and seeds took, on average, 18 days (MGT) to germinate. At this temperature, seeds of 'Zerberus' germinated the fastest, requiring less than 13 days to achieve final germination.

Final Germination vs. MGT
The relationship of final germination vs. mean germination time (MGT) at 8 • C was studied ( Figure 2). An exponential decay model best fitted the data (R 2 = 0.50), whose trend reveals that cold-tolerant cultivars, i.e., those best performing as the total number of seeds germinated at this temperature, tend to germinate faster than those susceptible (i.e., little germinating) to a low temperature. At 10 °C, the genetic variability for the germination rate was the greatest (28.49%), since cultivars responded quite differently from this cold temperature in terms of germination speed. 'Nectar' was the slowest (17.45 days MGT) under this temperature as well, while 'PR811F' was the fastest, reaching its final germination in a 5.45-day MGT.
At 8 °C, germination further slowed down and seeds took, on average, 18 days (MGT) to germinate. At this temperature, seeds of 'Zerberus' germinated the fastest, requiring less than 13 days to achieve final germination.

Final Germination vs. MGT
The relationship of final germination vs. mean germination time (MGT) at 8 °C was studied ( Figure 2). An exponential decay model best fitted the data (R 2 = 0.50), whose trend reveals that cold-tolerant cultivars, i.e., those best performing as the total number of seeds germinated at this temperature, tend to germinate faster than those susceptible (i.e., little germinating) to a low temperature.

Base Temperature and Thermal Time
A linear model was used to estimate the critical germination temperature, based on the germination rate GR 50 (i.e., 1/t 50 ). Minimum or base temperature allowing germination was 8.75 • C, on average of cultivars ( Table 6). The base temperature varied with the cultivar (CV 18.7%), from 7.44 • C ('PR811F') to 13.48 • C ('Nectar'). It is interesting to notice that, among the thirty sorghums, more than half exhibited a low Tb (<8 • C), including 'Keller' (7.95 • C). Conversely, a high thermal threshold for germination (Tb > 10 • C) was calculated, beside 'Nectar', in 'PR895', 'HayDay', 'Biomass H133' and 'Biomass 150', which indicates a great sensitivity to suboptimal temperatures during germination.  Cultivars with similar Tb had different germination rates (i.e., b slope of linear regression of GR 50 vs. T), thus, revealing a faster or slower response to increasing (or decreasing) temperature. In particular, while for most cultivars, a b ranging from 0.04 to 0.05 d −1 • C −1 was calculated, in 'Biomass 150', this value increased to 0.07 d −1 • C −1 , which indicates a great seed responsiveness to temperature shifting (above or below) from the optimum one. In this cultivar, seeds reached 100% germination at 25 • C, but at 8 • C, germination dropped to 18.7%. Differently, low b coefficients (0.032-0.033 d −1• C −1 ) calculated in 'PSE27677', 'PSE24213' and 'PSE22043', reveal a low sensitivity to increasing or decreasing temperature, little varying their final germination with a changing temperature.
Values of θ T as estimated from the model (Equation (2)), closely matched those calculated (from the inverse of the slope b of x-axis intercept of GR 50 vs. T). When calculated as 1/b, the average θ T was 23.50 • Cd −1 , with a 16.9% CV.
The observations of the graph divided in four quadrants allows us to point out the best combination, i.e., low Tb and low θ T (Figure 3, quadrant A). A good combination of the two parameters (Tb and θ T ) corresponded to 'Zerberus', 'Sucrosorgo 506', 'Jumbo' and 'PR811F' (all in quadrant A). Contrastingly, in the upper right side of the figure (quadrant C), the worst combination corresponded to cultivars 'HayDay' and 'PR895'. culated (from the inverse of the slope b of x-axis intercept of GR50 vs. T). When calcula as 1/b, the average θT was 23.50 °Cd −1 , with a 16.9% CV.
The observations of the graph divided in four quadrants allows us to point out best combination, i.e., low Tb and low θT (Figure 3, quadrant A). A good combination the two parameters (Tb and θT) corresponded to 'Zerberus,' 'Sucrosorgo 506,′ 'Jum and 'PR811F' (all in quadrant A). Contrastingly, in the upper right side of the figure (qu rant C), the worst combination corresponded to cultivars 'HayDay' and 'PR895.′

Biomass Yield in an Open Field
Final dry biomass measured in the thirty cultivars of sorghum under open field c ditions was, on average, 17.51 ± 5.51 t ha −1 (Figure 4). A wide variability (CV 31.5%) amo cultivars was calculated. Dry yield was maximized in 'PR811′ (31.32 t ha −1 ), which sig icantly differed from all cultivars of sorghum examined. Low biomasses (<11 t ha −1 ) w harvested in 'Maya' and 'ABF26′.

Biomass Yield in an Open Field
Final dry biomass measured in the thirty cultivars of sorghum under open field conditions was, on average, 17.51 ± 5.51 t ha −1 (Figure 4). A wide variability (CV 31.5%) among cultivars was calculated. Dry yield was maximized in 'PR811' (31.32 t ha −1 ), which significantly differed from all cultivars of sorghum examined. Low biomasses (<11 t ha −1 ) were harvested in 'Maya' and 'ABF26'.

Prediction of Germination Date in an Open Field
The dates of occurrence of 50% seed germination in the field in relation to three hypothetical times of sowing, as predicted for each cultivar of sorghum by θT and Tb values, are reported in Table 7

Prediction of Germination Date in an Open Field
The dates of occurrence of 50% seed germination in the field in relation to three hypothetical times of sowing, as predicted for each cultivar of sorghum by θ T and Tb values, are reported in Table 7. To this end, the mean soil temperatures recorded in the thirty-year period of 2005-2014 in the experimental site of the 2010 open field, were considered ( Figure 5). Table 7. Date of occurrence of 50% seed germination in the field in relation to three hypothetical early sowing dates, as predicted by thermal time. With sowing in early March, predicted germination occurs over a window of almost two months (from March 9 to May 4). Cultivars that are better performing are 'Zerberus', 'Jumbo' and 'PR811F', that achieve 50% seed germination just after 6 ('Zerberus') and 8 days ('Jumbo' and 'PR811F'). Cultivars, as 'HayDay', 'Biomass H133', 'Biomass 150', take long to germinate (more than 1 month). 'Nectar' requires almost 2 months to 50% germination.
When the date of sowing is split to mid-March, the variability among cultivars decreases. Predicted germination occurs by the end of March in most cultivars, except 'Padana 1,′ 'HayDay,' 'Biomass H133,′ 'Biomass 150,′ whose germination is delayed to April, and 'Nectar' that completes germination in early May. With a sowing in early April, 50% seeds germinate in 4-5 days in most cultivars. 'HayDay,' 'Biomass H133,′ and 'Nectar' take much longer to germinate (20-30 days).

Discussion
Seeds have a critical temperature for germination, which means that those having a base temperature (Tb) above actual T, fail to germinate. This occurs in all plants, including sorghum. Base temperature indicates the level of thermal stress the seeds may suffer, and the lower the Tb, the greater the tolerance to stress due to a low temperature [14]. However, other factors, besides cold, which may affect the germination of seeds in soil, must be considered. In this regard, it has been observed that a delayed germination in cold soil, combined with a low seed tannin content, made the seeds of sorghum more susceptible to mould and other pathogens [21].

Discussion
Seeds have a critical temperature for germination, which means that those having a base temperature (Tb) above actual T, fail to germinate. This occurs in all plants, including sorghum. Base temperature indicates the level of thermal stress the seeds may suffer, and the lower the Tb, the greater the tolerance to stress due to a low temperature [14]. However, other factors, besides cold, which may affect the germination of seeds in soil, must be considered. In this regard, it has been observed that a delayed germination in cold soil, combined with a low seed tannin content, made the seeds of sorghum more susceptible to mould and other pathogens [21].
Results revealed that the seeds of sorghums studied responded differently when exposed to low temperature, both in terms of germination speed and extent. Significant variation of cold tolerance was also reported in literature in commercial sorghum hybrids under controlled low temperature in the laboratory [11]. In a previous research conducted on 242 accessions of sorghum at International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) for seed germination and seedling vigor at 12 • C as a measure of cold tolerance, only one marker locus (Locus 7-2) was found to be significantly associated with low-temperature germination [22]. The use of this locus in molecular breeding of sorghum for low-temperature tolerance during germination has been suggested [22]. Harris et al. [23], working with nine genotypes of sorghum at a range of temperatures between 8 and 48 • C, found that, although final germination varied very little with temperature, Tb widely differed among genotypes, between 8.5 and 11.5 • C. In our experiment, 18 of the 30 sorghums studied exhibited a Tb lower than 8 • C.
The occurrence of a thermal dormancy in some cultivars could be suggested, at least in fractions of seed population, when seeds are incubated at low temperatures, which does not occur at optimal or supra-optimal temperatures. This form of dormancy may be considered as an adaptation strategy of seeds to survive in stressful conditions and guarantee favorable conditions for seedling growth [24].
However, cultivars with a final germination < 50% at 8 • C or even at 10 • C, have no agronomic value since they are not suitable to those regions, such as South Italy, when early sowings in sorghum are recommended. In other cultivars ('Padana 4', PR811F', 'Zerberus', 'PR849'), low temperatures only delayed the start of germination with minor effects on the final germination percentage (>90% at 8 • C). In these seeds, once the germination at low T started, it proceeded regularly. It has been observed how temperatures lower than optimal slow the rate of water absorption and the metabolic activation in seeds of sorghum [18,20].
In the thermal time, the model describes the pattern of seed germination in response to T, using only two parameters to predict germination: Tb and θ T . Once parameters are known, the germination time course at any T can be predicted by varying the value of T in Equation (2). Since θ T is constant, the larger the difference between actual T and Tb is, the faster the germination process (t) is and vice versa.
Low Tb allows seed germination at temperatures that, conversely, would inhibit germination in seeds of sorghums with higher Tb. However, a prolonged germination time, even in cultivars with low Tb, may result in altered seed performance in the field. Extended germination in the field longer exposes seeds to soil seedbed injuries and may result in poor crop uniformity and unsuccessful seedling establishment in the field [25].
Tiryaki and Andrews [8] found that the germination rate in cold conditions gives good separation among genotypes of sorghum. The authors also reported a highly significant correlation (R = 0.66) between cold germination measurements in the growth chamber and the rate of emergence in a field experiment. Similarly, Salas et al. [26] observed that twelve of the top fifteen accessions of sorghum exhibiting cold tolerance during germination in a 7day test at 10 • C, were also ranked within the top 15 under field conditions. Accordingly, the authors suggested the breeders to perform a preliminary screening of sorghum germplasm for cold tolerant alleles adopting a 7-day cold test at 10 • C.
In our experiment, cultivars having similar Tb exhibited different θ T requirements to germinate. In fact, no significant relationship was found between Tb and θ T (R 2 = 0.14, data not shown). As an example, cultivars 'Bulldozer' and 'PSE23431' did not differ for Tb (7.98 and 7.97 • C, respectively), but the 'Bulldozer' needed to cumulate less θ T (21.39 • Cd) than 'PSE23431' (29.26 • Cd) since this last one took longer to germinate.
Thermal time calculated from the inverse of the slope b of the x-axis intercept of GR 50 vs. T, matched relatively well what was estimated by the model. In most cases (77% of cultivars), thermal time for germination was slightly overestimated by the model. These results highlight the validity of using both methods for thermal time estimation.
The wide genetic variability observed in thermal time requirements and Tb values was confirmed in the predicted time required by the different cultivars to reach 50% seed germination in the field, when three hypothetical dates of sowings (March 1, March 15, and April 1) are considered.
According to the results, it is possible to suggest the adoption of early sowings with seeds of 'Zerberus', 'Sucrosorgo 506', 'Jumbo' and 'PR811F', that may provide good plant stands at suboptimal soil temperature conditions (9-11 • C), as those occurring in late winter-early spring in a semi-arid environment.
Among all cultivars, only 'PR811F' (fiber type) and 'Sucrosorgo 506' (sweet type) combine good adaption to early sowings with high productivity in terms of final dry biomass. Cultivars such as 'Biomass H133' and 'Nectar' are high-yielding. However, they are not suitable to early sowings due to high cold sensitivity during germination.

Conclusions
The results of this study indicate that a decreasing temperature from the optimum level reduces seed germination of sorghum, to a greater extent, when the temperature is lower than 15 • C. However, wide differences for cold tolerance existed among sweet and fiber sorghums assessed under controlled temperatures in the laboratory. This genetic variation in germination response to a low temperature suggest the possibility of screening among cultivars for those with high thermal stress tolerance during germination, which are suitable to early sowings in semi-arid areas. In particular, criteria for selection are a low base temperature and low thermal time requirements (as observed in cultivars 'Jumbo', 'PR811F', 'Zerberus', all fiber types, and 'Sucrosorgo 506', sweet type) that, if coupled, ensure adequate seedling establishment standards when late winter-early spring sowings (March-April) are adopted in the Mediterranean environment. Among these cultivars, 'PR811F' and 'Sucrosorgo 506' combine high cold tolerance during germination with a good biomass yield potential.
The identification of cultivars cold tolerant during germination may also contribute to the expansion of biomass sorghum into cooler cultivation areas, such as those of Northern Europe, which are less suitable to this warm season crop.
Author Contributions: Conceptualization, C.P. and S.L.C. Data curation, C.P., V.C. and A.S. Formal analysis, C.P. and V.C. Funding acquisition, C.P. Investigation, C.P. and A.S. Methodology, C.P. and S.L.C. Software, C.P. and S.L.C. Validation, C.P. Writing-original draft, C.P. and V.C. Writing-review & editing, S.L.C. All authors have read and agreed to the published version of the manuscript.