The quantification of genetic variability and the description of how the distribution of genotypes among and within natural populations are fundamental for the development of strategies of conservation and use of natural resources in pre-improvement programs (Cruz, Ferreira & Pessoni, 2011; Delialioğlu et al., 2022; Akan, 2022). However, the drastic reduction of natural populations of shrubby plants in Brazil neotropical savanna has led them to the loss of genetic variability by drift (Sebbenn & Ettori, 2001). This loss of variability results in the reduction of the species’ adaptation capacity to environmental factors, as well as in the loss of genotypic and phenotypic characteristics (Ritland, 1996; Campus & Maia, 2019). Additionally, the loss of genetic diversity is also associated with the reduction of the species evolutionary potential and the reduction of the reproduction rate (Allendorf, Hohenlohe & Luikart, 2010).

The Brazilian neotropical savanna presents among 39 (priorities) and 76 fruit species with economic exploration potential. Knowing the physico-chemical characteristics, the nutritional and functional value of the Cerrado fruits are means of encouraging consumption and the creation of new products (Vieira et al., 2016). The physical characterization, of macros and micronutrients and the existing compounds in these fruits enables a better indication regarding their use and better use in the food industry (Soares et al., 2019).

Among the species with high sustainable exploration potential found in the Cerrado biome, the Anacardium othonianum Rizz, can be highlighted (Agostini-Costa et al., 2016), popularly known as shrubby cashew from Cerrado or small cashew from Cerrado, considered the main cashew plant in the mid-west region, and presents reddish yellow fruits, with acid flavor and high succulence (Silva, Silva & Oliveira, 2007; Bessa et al., 2013). This fruit stands out for presenting economic potential for use in cooking and in natura consumption (Allendorf, Hohenlohe & Luikart, 2010), besides presenting potential for use in pharmaceutical industry (Padilha et al., 2020). Cashew is a non-climacteric fruit that presents high vitamin C content; it is a source of antioxidants and bioactive components (Silva et al., 2021). The true fruit also constitutes an important source of food and can be used as an oil source or consumed when toasted (Sousa et al., 2017; Silva et al., 2017).

The shrubby cashew from Cerrado can reach three to four meters high, for three to four meters in canopy diameter; it is native to the Cerrado area “stricto sensu”, big Cerrado and dirty fields (Silva et al., 2001; Agostini-Costa et al., 2010; Agostini-Costa et al., 2016). They occur mainly in acidic latosol areas with low nutrient availability. Its flowering and fruiting begin during the dry season, between May and June, and end in October. The fruit production per plant concentrates between 200 and 600 fruits, with total weight between 5 and 12 grams (Agostini-Costa et al., 2010). The species under study presents potential for genotypes selection with desired behaviors for use in genetic improvement programs (Belo et al., 2019).

The present study aimed to characterize fruits and pseudofruits 15 natural subpopulations of A. othonianum species, in order to contribute to the elaboration of efficient strategies for its use and genetic conservation.

2.1 Study area and collection

The material collection for this study was performed in 15 subpopulations distributed in the state of Goiás (Fig. 1). The term “subpopulation” was assigned to the group of sampled plants, considering a minimum distance of approximately 20 km, as indicated by Silva, Chaves e Naves (2001). In each area, matrix trees were identified through field surveys in their respective occurrence sites (Cerradão, Campo Sujo, and pasture), followed by the pre-selection of individuals with good phytosanitary conditions and fruits suitable for collection.

Ripe fruits were collected directly from the matrix tree or from the ground, provided they showed no signs of deterioration. Subpopulations were considered suitable for data collection if they had at least 15 adult cashew trees with sufficient production (12 fruits). The matrices were randomly sampled within each subpopulation. If more than one phytophysiognomy was present at the collection site, efforts were made to sample the different phytophysiognomies where the species occurs. The fruiting of shrubby cashew occurs, normally, in the months of June to October (Agostini-Costa et al., 2016).

The collection was carried out in areas of Latossolo, a predominant soil class in the Cerrado biome, according to the classification of the Brazilian Soil Classification System (EMBRAPA, 1999). Within this class, variations such as Red-Yellow Latosol and Red Latosol stand out, which are widely representative of the Cerrado.

The specific environmental conditions of the study area in the Cerrado are predominantly characterized by an Aw climate type, according to the Köppen-Geiger classification, which describes a climate with a rainy summer and dry winter (Köppen & Geiger, 1961; Silva, Assad & Evangelista, 2008). In the collection area, located in the Central-West region of the Cerrado, the annual precipitation is approximately 600 mm.

At least 12 undamaged fruits of ten matrices were collected from each of the 15 sampled subpopulations. This is a sampling considered suitable for studies of this nature, according to simulations made by Goudet and Buchi (Goudet & Buchi, 2006). The fruit collection was performed between August and October, respecting its physiological maturation point. Once collected, the fruits were packaged in plastic bags, stored in a thermal box with ice and transported to the Microbiology Laboratory of the Goianésia Evangelical Faculty (FACEG), in Goianésia, GO, Brazil. The fruits were kept in freezer (-18 °C) until the quantitative data were collected, without deterioration.

Figure 1 – Location of the collection areas of the 15 Anacardium othonianum Rizz. subpopulations sampled in the Cerrado of the state of Goiás, Brazil

Source: Gustavo Henrique Mendes Brito, 2021 (QGIS)

2.2 Characterization of fruits and pseudofruits

Ten fruits per matrix were evaluated and 10 matrices for each of the 15 subpopulations, except for 6 (Ceres) and 12 (Anápolis) (Fig. 1), where 9 and 8 matrices were evaluated, respectively. The total number of fruits and pseudofruits of A. othonianum characterized was 1470. In the fruit evaluation, performed between the months of August and October 2020, the following morphological descriptors were used: Total Mass (MT), Pseudofruit Mass (PFM) and Fruit Mass (FM) - with the aid of an analytical scale, expressed in grams; Total Length (TL), Pseudofruit length (PFL), Pseudofruit Larger Width (PFLW), Pseudofruit Smaller Width (PFSW), Pseudofruit Thickness (PFTh), Fruit Length (FL), Fruit Larger Width (FLW), Fruit Smaller Width (FSW) and Fruit Thickness (FTh) - data taken with the aid of a digital caliper, expressed in millimeters. The coloration was determined through a color scale and the pseudofruit format was classified in apple, pyriform and pyramidal (Fig. 2). These biometric characteristics were selected because they are fundamental in the selection of plants for the genetic improvement of cultivated cashews. They directly influence the productivity and quality of the fruits, which are crucial aspects for both the market and the conservation of the species (Aliyn & Awopetu, 2011; Rossetti, Vidal-Neto & Barros, 2019).

The biometric characteristics were measured using standardized methods to ensure accuracy and consistency in the results. The mass of the pseudofruits and fruits was determined using a high-precision analytical balance, equipped with a glass enclosure to protect against air currents and other interferences. The balance was calibrated before each weighing session to ensure measurement accuracy. Before weighing, the pseudofruits and fruits were carefully dried with absorbent paper to remove any surface moisture, minimizing potential weight variations. Additionally, size measurements of the fruits and pseudofruits were taken with a high-precision digital caliper. The caliper’s display was zeroed (calibrated) before each new measurement to guarantee accuracy and repeatability. All measurements were conducted under controlled environmental conditions, such as temperature (20 °C) and humidity (60%), to avoid fluctuations that could affect the results.

Figure 2 – Patterns for the classification of cashew peduncles. I. Color scale (1- red, 2- orange red, 3- orange, 4- yellow, 5- orange yellow, 6- light yellow) and II. Formats: A- apple, B- piriform and C- pyramidal. Adapted from Silva (2001)

Source: Design made by the plastic artist Leonardo Santana Mendes Moreira (2021)

2.3 Statistical analyses

The data were submitted to descriptive statistics and, subsequently, to the variance analysis based on a hierarchical model that considers the effect of subpopulations, matrices within subpopulations and fruits within matrices. The adapted model (Mota et al., 2020) was:

(1)

in which:,: phenotypic fruit value (or pseudofruit) k of the matrix j of the subpopulation i; : general average of observations;: random effect of the subpopulation i, i = 1, 2, ..., S; : random effect of the matrix j within the subpopulation i, j = 1, 2, ..., mi; : random effect of the fruit/pseudofruit k within the matrix j of the subpopulation i, k =1, 2, ..., fj.

In the present study, the number of matrices per subpopulation (mi = 10 - exception for Ceres and Anápolis) and fruits per matrix (fj = 10) were constant. According to the adopted statistical model, the variance components were estimated among subpopulation, among matrices within subpopulations and residual (fruits/pseudofruits within matrices) according to the expected average squares. Subsequently, the proportions of the total phenotypic variation were estimated, which is due to the difference among subpopulations (PS), difference among matrices within subpopulations (PM/S) and difference among fruits/pseudofruits within matrices within subpopulations (PF/M) (Mota et al., 2020). Through the variance components we estimated the PST parameter, which measures the quantitative phenotypic divergence among subpopulations, using the following formulas:

(2);

(3);

(4);

(5).

The phenotypic correlation coefficients were also estimated among the evaluated characters. The analyses were performed based on genetic and statistical procedure of the R software version 3.4.3 (R DEVELOPMENT CORE TEAM, 2017).

The largest phenotypic variations occurred among the characters related to the sample mass and pseudofruit coloration. The total mass (TM), pseudofruit mass (PFM) and fruit mass (FM) presented the following general averages: 11.977 g, 9.260 g and 2.718 g, respectively, as well as the highest coefficient of variation (CVs%) among the evaluated characteristics, higher than 31% (Table 1).

The highest estimated averages for the characters related to the mass were observed in subpopulations 6 (Ceres), 9 (Uruaçu) and 10 (Souzalândia). While the lowest average for TM and PFM were observed in subpopulations 4 (Jaraguá) and 11 (Pirenópolis). It is noted that, in the case of subpopulation 4, the average value found for TM was approximately 49% lower than the value found for the same characteristic in the subpopulation 6. As for the FM, the lowest averages were observed by subpopulations 1 (Santa Familia River) and 3 (Vila Propício) (Table 1).

Table 1 – Averages per subpopulation, general average (per plant), maximum and minimum values and coefficient of variation of quantitative fruit and pseudofruits of Anacardium othonianum Rizz. from 15 native subpopulations from the Cerrado in the state of Goiás, Brazil

TM: total mass (g); PFM: pseudofruit mass; FM: fruit mass (g); TL: total length (mm); PFL: pseudofruit length (mm); FL: fruit length; PFLW: pseudofruit larger width (mm); PFSW: pseudofruit smaller width (mm); PFTh: pseudofruit thickness (mm); FLW: fruit larger width (mm); FSW: fruit smaller width (mm); FTh: fruit thickness (mm). Source: the authors (2021)

Regarding the characters based on the sample length, the pseudofruit length (PFL) presented the highest coefficient of variation (CV%= 26), corroborated by its variation amplitude of 10.7 mm to 49.7 mm. The total length (TL) and fruit length (FL) manifested similar CVs, close to 16%. The subpopulations 6 (Ceres) and 9 (Uruaçu) presented the highest values for TL and PFL. Most of the matrices of the two subpopulations mentioned above were sampled in pasture environment, probably with soils that are more fertile and less shade, factors that may have influenced the size of the TL and PFL. For the FL, the highest averages were observed by the subpopulations 15 (Faina), 9 and 8 (Barro Alto) (Table 1).

The characters pseudofruit larger (PFLW), small (PFSW) width and thickness (PFT) presented CVs considered average for Cerrado fruits and general averages of 22.57 mm, 16.95 mm and 21.43 mm, respectively. The subpopulations 6 (Ceres), 10 (Souzalândia) and 9 (Uruaçu) presented the highest averages for PFLW and PFTh. For the characteristic PFSW, the subpopulations that presented the highest average values were 10, 9 and 3 (Vila Propício) (Table 1).

The characteristics related to the width (FLW and FSW) and fruit thickness (FTh) presented the lowest variations among the quantitative variables evaluated, based on their CVs and variation, minimum and maximum amplitudes. It was noted that the subpopulations 9 (Uruaçu), 2 (Goianésia) and 15 (Faina) obtained higher averages for FLW, and the subpopulations 9, 2 and 3 (Vila Propício) manifested higher averages for FSW. For the variable FTh, the highest averages were presented by the subpopulations 15, 13 (Silvânia) and 12 (Anápolis) (Table 1).

The fruits presented a predominance of 47% for the pyriform format, followed by 34% for the apple format and 19% for the pyramidal form. Using the patterns for the classification of cashew peduncles by the color variable, it was verified that all six colors are present in the sampled subpopulations and twelve of the fifteen subpopulations presented predominance of color 2 (orange red) (Table 1). The subpopulations that presented pseudofruit coloration different from the general average were 7 (Placa) - red color, 13 (Silvânia) - orange color and 15 (Faina) - yellow color.

There were highly significant variations for all the evaluated characters, in all hierarchical levels, Eq. (01): among subpopulations (PS) – Eq. (2), among matrices within subpopulations (PM/S) – Eq. (3) and among fruits within matrices (PF/M) – Eq. (4). However, higher levels of variation were found among matrices within subpopulations, with the highest values presented by the TL and PFL. Subsequently, the variability was verified among fruits within matrices, except for the pseudofruit thickness variable, which presented the highest variation at the subpopulation level (Table 2). The variables that presented the highest values for the PST parameter (divergence among subpopulations – Eq. (5),) were pseudofruit thickness (PFTh) and pseudofruit smaller width (PFSW) (Table 2).

Table 2 – Analysis of variance and quantitative parameters estimates of 15 native subpopulations of Anacardium othonianum Rizz

***, Significant F test at 0.01% of probability : variance among subpopulations : variance among matrices; : variance among fruits ; PS% : proportion of the total variance that is due to the variation among fruits; PST: quantitative genetic divergence among subpopulation; TM: total mass; TL: total length; PFL: pseudofruit length; PFlW: pseudofruit larger width; PFSW: pseudofruit smaller width; PFTh: pseudofruit thickness; PFM: pseudofruit mass; FM: fruit mass; FL: fruit length; FLW: fruit larger width; FSW: fruit smaller width; FTh: fruit thickness. Source: the authors (2021)

High and significant correlation levels were observed among the mass and length measurements of the pseudofruit with the total mass and length measurements (Table 3), indicating that the higher the pulp mass (pseudofruit), the higher the total size, or vice versa. In addition to this, the PFLW presented a high and significant correlation with the PFT (0.906), and partially high correlation between the PFLW vs. PFSW (0.826) and PFLW vs. PFM (0.855). As observed in Table 1, the average length and weight of the pseudofruit represent 56.7% and 77.3% of the total length averages and total mass of the samples, explaining the high correlation levels found among the pseudofruit measurements and the total samples measurements for length and mass (Table 3).

The TM also presented moderate correlation levels with the TL variables (0.717) and PFT (0.784). It was also observed partially high correlation among TM vs. PFLW (0.847). The PFM presented average and significant correlation with the other peduncle dimension measurements: PFL (0.723) PFSW (0.702) and PFT (0.797) (Table 3).

Table 3 – Phenotypic correlation among physical variables of 15 native subpopulations of Anacardium othonianum Rizz

*,**, Significant at 0.5%, 0.01% of probability. NS Not significant. TM: total mass; TL: total length; PFL: pseudofruit length; PFLW: pseudofruit larger width; PFSW: pseudofruit smaller width; PFTh: pseudofruit thickness; PFM: pseudofruit mass; FM: fruit mass; FL: fruit length; FLW: fruit larger width; FSW: fruit smaller width; FTh: fruit thickness. Source: the authors (2021)

In two studies conducted with subpopulations of A. othonianum in the state of Goiás (Corrêa et al., 2008; Borges, 2012) and one in the city of Teresina – Piauí (Rocha et al., 2013), observed mean total mass values of 11.14 g, 8.99 g and 7.9 g, respectively, lower than the general average found in the present study. The researcher in the previously mentioned study (Borges, 2012) also observed lower averages than the present study for the FM variables (2.21 g) and PFM (8.93 g). The studies with Cerrado’s native fruit have demonstrated that the characteristics related to the fruit mass present phenotypic variation amplitudes higher than the other quantitative fruit variables (Moura, Chaves & Naves, 2013; Boaventura-Novaes, 2018; Mota et al., 2020).

As for the characters related to the cashew size, in a study (Borges, 2012) that evaluated 22 shrubby cashew subpopulations from the Cerrado in the state of Goiás, verified general averages for fruit length of 20.84 mm and pseudofruit of 25.40 mm, similar to the present study. A research conducted in the Brazilian Central Plateau (Rizzini, 1969) described fruits with TL lower than the ones reported in this research, between 15 mm and 25 mm.

The phenotypic variations observed for the characteristics related to fruit/pseudofruit mass and size are desirable for the potential commercial exploitation of the species and/or possible genetic improvement programs. The higher the fruit mass, the higher the capacity to generate products that will be used as raw material, besides, knowing the biometric characteristics of the fruits is of extreme importance for the equipment dimensioning used by the industry (Silva & Abud, 2017).

A study conducted with tree cashew from the Piauí Cerrado (Rocha et al., 2013) verified average value for the pseudofruit width of 21.8 mm, similar to the average (per fruit) found in this study for the PFLW. In another study conducted with fruit-bearing plants from the Cerrado (Corrêa et al., 2008), the authors observed fruits with average width of 15.39 mm, slightly higher than the FSW and lower than the FLW reported in the present study.

In a study with a population of early dwarf cashew trees clones (Moura et al., 2001), the authors reported that 44% of the evaluated cashews presented the pyriform format, also predominant in this study. The authors also classified this format as ideal for use in commercial packaging.

A study characterizing pseudofruits of different subpopulations of A. occidentale (local) species (Paiva et al., 1998), verified variations among the color patterns red, orange and orange red, and did not report results for the other cashew coloration patterns. The consumer market of the pseudofruit mainly in natural form prefers peduncles that present handling resistance, piriform format and orange and red colors (Silva et al., 2014).

Studies on intra and interspecific genetic variability in natural populations of tropical regions prove that the native species present wide intrapopulational variability (Boaventura-Novaes et al., 2018; Mota et al., 2020). In addition, the reproductive system of the species, effective population size, gene flow and geographical distribution (Paiva et al., 1998) also influence the distribution of this variability.

Regarding the genetic structure presented by the shrubby cashew from Cerrado, higher variation levels among matrices within subpopulations were also observed in studies with different fruit species from Cerrado: mangaba (Hancornia speciosa Gomes) (Almeida et al., 2019), jatobá-do-Cerrado (Hymenaea stigonocarpa Mart. ex Hayne) (Tiago et al., 2018), cagaita (Eugenia dysenterica DC.) (Boaventura-Novaes et al., 2018) and with baru (Dipteryx alata Vog.) (Mota et al., 2020). For pequi tree (Caryocar brasiliense Camb.), the studies (Vera et al., 2007; Moura et al., 2013) found a genetic structure in which the majority of the variability is among subpopulations or regions and subsequently among matrices within subpopulations.

The PST, Eq. (5), is a parameter that portrays the phenotypic differentiation among the subpopulations, and then, a measure that resembles the QST, which measures the quantitative genetic differentiation among the subpopulations (Brommer & Whither, 2011). The estimated PST, Eq. (5), values did not detect high phenotypic differentiation among the subpopulations, indicating that probably the sampled subpopulations still share genes among the individuals. When studying the genetic diversity in wild populations of the salt marshes ecotype of A. occidentale in the coast of Piaui, the authors (Santos et al., 2019) detected strong genetic heterogeneity among the wild populations studied, despite the probable gene flow among them. Therefore, studies with genetic markers that enable a better estimate of genetic divergence among the A. othonianum subpopulations, occurring in the mid-west region of Brazil are necessary.

In a study with A. othonianum, the author (Borges, 2012) also found positive and highly significant correlation (p<0.01) between TM vs. PFM (0.983) and TM vs. PFLW (0.815). When analyzing fruits and pseudofruits of early dwarf cashew clones, the authors (Pereira et al., 2005) observed high correlation (0.84) between fruit mass and pseudofruit. However, as expected in the present study, there was no correlation between the dimensional and mass measurements of fruits and pseudofruits, that is, pseudofruit length and mass do not influence the same parameters as the fruit, and there may be small and heavy fruits, and large and light pseudofruits. Similar results were obtained by a study with cashew tree (Borges, 2012), which also verified low correlation between the fruit and pseudofruit measurements. The disparity among the results obtained is explained by the fact that, as there was no selection of genotypes and phenotypic markers for the species, once the studies were performed in Cerrado native plants, it was not possible to standardize the results found among fruits and pseudofruit.

The biometric characteristics of fruits and pseudofruits of shrubby cashew species from Cerrado presented high phenotypic variation among all the evaluated hierarchical levels. As for the species genetic structure, most of the genetic variability is among matrices within subpopulations and among fruits within matrices. The subpopulations presented phenotypic differentiation based on the characteristics: pseudofruit larger width, pseudofruit smaller width and pseudofruit thickness, according to the PST parameter. The pseudofruit represents 78% of the total mass measure of the fruit, while the pseudofruit length constitutes 57% of the total fruit length. There is a predominance of the red-orange color of the pseudofruit of A. othonianum species in the sampled municipalities. The largest correlations were observed among the mass and length measurements of the pseudofruit and mass and total length. There is low correlation between the fruit and pseudofruits mass and size. Future studies should integrate genetic analyses and experiments under controlled cultivation conditions in order to disentangle genetic and environmental effects and to minimize phenotypic variation associated with contrasting environments, such as pasture areas.

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