FA profiles were shown to be taxonomically useful at distinct hierarchic levels in Pimentinae: a) at the generic level, by allowing a pronounced distinction between Psidium and Campomanesia; b) at the species level, due to distinct combinations of saturated and unsaturated FA, as well as the presence/absence of palmitoleic and azelaic acids (Fig. 1). Azelaic acid is an uncommon C9 dicarboxylic acid, also present in barley, rye and wheat grains. It has antibacterial and tyrosinase activity, being used in formulations for skin treatment (Nazzaro-Porro, 1987). The utility of seed FA profiles as taxonomic markers has been often observed, as in Pinaceae (Wolff et al., 2001) and Malvaceae (Fernandes et al., 2021). Mayworm and Salatino (2002) observed that the distribution of seed FA characterizes species of Qualea and Vochysia (Vochysiaceae), irrespective of the geographic location of samples of the same species.
A visual inspection of Fig. 1 denotes that FA profiles are useful for characterizing species of Campomanesia. FA composition was shown to be a conservative character among different populations, with only slight deviations being noted comparing profiles of individuals of the same species, irrespective of geographic origins. This is visible also in the biplot of Fig. 2, which denotes chemical affinities between species by the proximity between individuals of some pairs of species, such as C. eugenioides-C. phaea, C. pubescens-C. xanthocarpa and C. guaviroba-C. guazumifolia. The biplot evidences also the intraspecific stability of the FA profiles, the spots corresponding to individuals of the same species distributed in high proximity (Fig. 2). This aspect strengthens the taxonomic utility of the seed FA as species markers. In this regard, it is worth observing that areas delimited by individuals of one species never merge with areas corresponding to individuals of another species (Fig. 2), a reflection of the fact that, in Campomanesia, interspecific distances are larger than intraspecific.
A deeper evaluation of the usefulness of FA profiles in the systematics of Campomanesia may be reached by comparing chemical and molecular data. Figure 3 depicts chemical and phylogenetic affinities among the same set of species. A comparison of the cladogram (left side) with the phenogram (right side) reveals more points of agreement than incongruence. Thus, chemical affinities between C. pubescens-C. xanthocarpa and C. guaviroba-C. neriiflora are reflected in identical and strongly supported phylogenetic affinities, derived from ITS region and maximum likelihood (Fig. 3). Points of disagreement between the two dendrograms are restricted to links among C. guazumifolia, C. eugenoides, and C. phaea, although both sources of evidence agree that the three species are closely related (Fig. 3). Reports about the convergence of affinities based on analysis of FA profiles and molecular phylogeny are common in scientific literature. They have been shown in studies involving Coffea (Dussert et al., 2008) and Arecaceae (Guerin et al., 2020).
The results of the phylogenetic analysis are congruent with the phylogeny obtained by Oliveira et al. (2021), with C. phaea, which was not included in the previous analysis, emerging as a sister to the clade C. eugenioides-C guazumifolia (Fig. 3). Another congruent aspect is the failure to recover the “C. xanthocarpa complex” (Landrum, 1986). The profile of C. eugenioides (a member of “C. xanthocarpa complex”) exhibits a distinct FA profile in comparison with other species of the complex, such as C. pubescens and C. xanthocarpa. Instead, C. eugenioides is chemically close to C. phaea, with which it holds phylogenetic links (Fig. 3). The present results also support proposals by Oliveira et al. (2021), recognizing a “C. eugenioides group” and a “C. xanthocarpa group”, the latter including C. pubescens.
Excepting C. guazumifolia, whose seed oil contains approximately 75% of unsaturated FA (with predominance, however, of oleic, rather than linoleic acid), the seed oils of all other species of Campomanesia have proportions of not much different of saturated and unsaturated FA (Figs. 1 and 4B). High contents of unsaturated FA are not exclusive of Psidium among genera of Pimentinae. Andrade et al. (2012) detected 92% of unsaturated FA (chiefly linoleic acid) in the seed oil of Feijoa sellowiana. Further work is needed to widen the knowledge about seed oil FA affinities among genera and subtribes of Myrteae.
Results obtained in the present work indicate that the evolution of FA profiles in Campomanesia followed different paths, all of them leading to decreases in the proportion of linoleic acid and increase of oleic (a mono-unsaturated FA), stearic and palmitic acids (both saturated FA) (Fig. 4A).
The decrease of unsaturated FA (mainly linoleic acid) and concomitant increases of oleic acid and saturated FA in the evolution of Campomanesia (Fig. 4.A, B), may have consequences in aspects of seed physiology. High levels of unsaturated FA may increase the tolerance of cell membranes to low water potentials (Uemura and Steponkus 1994; Uemura et al., 1995).
Similar patterns of FA profiles, with high concentrations of saturated FA, are observed in species of Myrteae with recalcitrant (intolerant to desiccation) seeds, such as Myrcianthes pungens (Guardia et al., 2020) and Eugenia uniflora (Delgado and Barbedo, 2012), the former with 31% and the latter with 45% of saturated FA (Andrade et al., 2012). Instead, orthodox (tolerant to desiccation) seeds of Myrtus communis (Franchi et al., 2011) exhibit high concentrations (86%) of unsaturated FA (Wannes and Marzouk, 2016). Liu et al. (2006) also observed that orthodox seeds have higher concentrations of unsaturated FA (mainly linoleic) than recalcitrant seeds. Germination studies indicate an orthodox behaviour by seeds of Psidium (Silva et al., 2011; Masetto et al., 2014; Gentil et al., 2018; Fernandes et al., 2022), while seeds of Campomanesia are generally classified as recalcitrant or intermediate (Melchior et al., 2006; Dousseau et al., 2011; Dresch et al., 2014, 2015; Leão-Araújo et al., 2019, 2022; Vieira, 2021; Elias et al., 2023). Species of Campomanesia assumed as orthodox, have higher moisture content and lower desiccation tolerance than seeds of Psidium (Maluf and Pisciottano-Ereio, 2005; Santoro et al., 2020).
The evolutionary change in the proportion of un- and saturated FA may also impact germination time, as saturated FA provide a higher amount of energy per carbon unit and higher melting points, compared to unsaturated FA (Lehninger, 2014). It is expected that in environments with warmer temperatures, the germination time after sowing is shorter by seeds rich in saturated FA, compared with seeds rich in unsaturated FA (Linder, 2000). According to data yet to be published by the Secretariat of Green and Environment of São Paulo - Brazil (SVMA-PMSP), the average time for the first germination is 18 days after sowing for seeds of Campomanesia and 40 days for Psidium. In addition to the contribution of other seed morpho-physiological attributes, comparing seeds of the two genera, differences in the saturation degree of the seed FA observed in the present study may be relevant to account for the mentioned differences of germination time.
High proportions of unsaturated FA, and consequent lower melting points and seed orthodoxy, are expected in colder and drier climates, while high proportions of saturated FA, higher melting points and seed recalcitrance, are expected in warmer and more humid places (Linder, 2000; Liu et al., 2006; Zhang et al., 2015; Guerin et al., 2020). Zhang et al. (2015) investigated the seed FA composition of 747 species (95 plant families) across China, and noted a negative correlation between, on one side, the mean annual temperature and precipitation, and on the other side, the concentration of unsaturated FA. Linder (2000) and Guerin et al. (2020) found a positive correlation between latitude and unsaturated FA concentration. Despite the possible selective pressures exerted by temperature and precipitation on the evolution of FA profiles, genetic drift has stronger influences on the development of FA profiles than environment (Aitzetmüller, 1995; Velasco and Goffman, 1999; Zhang et al., 2015; Guerin et al., 2020). In addition, differences between FA saturation degree observed in this study (Figs. 1, 4A and B) are unlikely to be a response to latitudinal patterns, such as described by Linder (2000) and Guerin et al. (2020), since all specimens in the present study were collected in very similar latitudes (Table 1). On the other hand, the biogeographic history of Pimentinae may have influenced the patterns of saturation and unsaturation of Campomanesia FA. Data of the present work may suggest a shift in selective pressure, starting with the ancestor shared by Psidium and Campomanesia, from colder and/or drier climate, which later generated progenies that evolved to give rise to Campomanesia lineages, containing FA characteristic of plant taxa from warmer and/or wetter climate. This shift could also have influenced aspects of success in competition, as quicker germination may enhance reproductive success (Pelc and Linder, 2014).