CHEMICAL INDEXES CALCULATED FOR 8 , 11 , 13-TRIEN-ABIETANE DITERPENOIDS ISOLATED FROM SWARTZIA SPECIES

The disparity found in the molecular structures of compounds isolated from nine plants of the Swartzia genus indicates that the Swartzia species that furnished cassane diterpenoids and triterpenoidal saponins are more recent, since these metabolites have adopted the mevalonic acid route of formation, abandoning the shikimic acid/acetate route that produces the isoflavonoids found in the remaining species. Chemical indexes calculated from the molecular structure diversities of sixteen 8,11,13-trien-abietane diterpenoids isolated from Swartzia langsdorffii and S. arborescens indicate that S. arborescens is more recent than S. langsdorffii. The results suggest a more evolved position in Swartzia species of the section Possira.

Nevertheless, studies conducted after this review showed, directly or indirectly, the need of a revision of the infrageneric classifi cation, due the artifi ciality of some series [3][4][5][6][7][8][9][10]. In fact, some of the Swartzia species are included in two distinct subsections or series and varieties of the same species have been placed in different series 11].Cowan attempted to order the species in his monograph along putative phylogenetic lines, but his infrageneric groups were not intended to refl ect common ancestry.Indeed, a systematic study of Swartzia, based on morphological and molecular characters, could provide an evolutionary basis to evaluate the infrageneric taxonomy of Cowan [2].
Recently, it was pointed out [11] that Swartzia should be retained without further division, despite its large number of species and considerable morphological diversity, because it was otherwise resolved as monophyletic, albeit with limited support.The present resolution of subclades of Swartzia should be used as a basis for revising the highly artifi cial infrageneric classifi cation of Cowan [2].
Additionally, an updated monograph of the entire genus has taxonomic and phylogenetic signifi cance because of the position of Swartzieae in the Papilionoideae classifi cation.Molecular data strongly suggest that Swartzieae sens.strict.should be redefi ned to include only the genera Swartzia, Bobgunnia, Bocoa, Candolleodendron, Trischidium, Cyathostegia, and Ateleia [1,12,13].
Cladistic analyses showed the group of Myrospermum, Myroxylon and Myrocarpus of Sophoreae to be associated with the Aldinoid clade of the Swartzieae and closely to Swartzieae sens.strict.[12].The reclassifi cation of Swartzieae sens.strict., and realignment of the remaining swartzioid genera in other tribes, needs to be corroborated by further evidence [1,13].
On the other hand, evolutionary changes in plants can also be evaluated through the chemical structures of the special metabolites found in them, based on the disparity (related to the biogenetic route) and the diversity of their molecular structures [14].The methodology is very attractive because it does not imply knowledge of the complete biogenetic route of metabolites and has already indicated evolutionary tendencies for the Swartzia species that produce isofl avonoids [15].In this paper, the methodology is used to suggest evolutionary directions for two Swartzia species that, instead of isofl avonoids [16][17][18][19][20], furnish terpenoids.

Methodology
Disparity Evaluation-The disparity of metabolites reported in Swartzia genera were evaluated by comparing their chemical structures since, according to Gottlieb's methodology [14], disparity in the molecular structure of special metabolites indicates an evolutionary move and occurs by the replacement of compounds generated from the shikimic acid/acetate biogenetic route by compounds generated from the acetated route (or mevalonic acid route).
Accordingly, the species were separated into two groups, those that furnished special metabolites originating from the shikimic acid/acetate route -the isofl avonoids, and those that furnished compounds formed by the mevalonic acid route -the terpenoids (Table 1).Diversity Evaluation-For contextualization and better visualization, the biodiversity observed for ten 8,11,13-trien-abietane diterpenoids isolated from S. langsdorffi , A, B, and 1-4 [21], including a pair of epimers, 5-8 [22] is explained through a series of methylation and oxidation reactions rationalized using a proposed biogenetic route (Fig. 2).The initial part of proposed route is very similar to that reported for the C-12 methoxy derivative of ferruginol [23] including a 1,2-methyl si migration.However, the shift now proposed occurs between C-13 and C-14, and not between C-13 and C-15, and is reinforced by the detection of C-14 methylated 8,11,13-trien-abietane compounds in the species Myrocarpus frondosus [24], a species of the Sophoreae tribe, which is closely related to Swartzieae sens.strict.. Some cyclization processes are proposed leading to the compounds that were isolated from Swartzia species and from the species Myrocarpus frondosus.The metabolites carbon skeletal difference is in ring D, as, in case of the metabolites furnished by M. frondosus , ring D would be formed by an oxidative cyclization reaction between C-16 and C-12, allowed by the absence of the protective C-12 methoxy group of swartziarboreols.In the case of swartziarboreols, however, oxidative steps would occur before a fi nal cyclization reaction between C-16 and C-17 that would give rise to the isocoumarin ring D.Then, the other compounds would be generated by a series of oxidative and methylation processes.
However, when Gottlieb's chemical index methodology is applied, a knowledge of such biogenetic pathways of special metabolites is not necessary since the evolutionary evaluation is made by means of two chemical indexes, the oxidative evolutive advance index, EA O , and the skeleton complexity evolutive advance index, EA E , of each plant species.
The indexes EA O and EA E are found by calculating the averages: EA O = Σ / N and EA E = Σ  / N, where the value N refers to the number of metabolite occurrences, and the O and E values are the chemical indexes that quantify the oxidative degree of the molecular structures (O) and the carbon skeleton specialization (E) of each metabolite.
The oxidative degree indexes (O) were calculated by the equation, O = (x-h)/ n, where the letters n, h and x refer, respectively, to the total number of carbon atoms (n), the number of C-H bonds (h) and the number of C-heteroatom bonds (x) of the terpenoid skeleton, while the carbon skeleton specialization indexes (E) were calculated by comparing the carbon skeleton of each diterpenoid chemical structure in focus with the carbon skeleton of a hypothetic common precursor (compound A; Fig. 1), by using the equation: E = (q + f + c +u) / n, where the letters n, q, f, c, and u, represent, respectively, the total number of carbon atoms (n), the number of C-C bonds broken (q), the number of C-C bonds formed (f), the number of rings formed with a heteroatom, (c), and the number of additional carbon atoms (u).Table 2 shows the O and E values found for ten compounds identifi ed from S. langsdorffi i.The same calculations were applied to the fi ve diterpenoids reported for S. arborescens [25] (Table 3).Finally, the oxidative advance index, EA O , and the skeleton specialization advance index, EA E, of each species are displayed in Table 4.
Since the carbon skeleton is the same for all the isolated swartziarboreols, the carbon skeleton specialization index (E) is zero for all of them, and the value of EA E = Σ E/N is also zero.The differences between metabolites are related only to the oxidation indexes (O) calculated for each metabolite (Tables 2 and 3).Finally, Table 4 shows the EA O and EA E values found for each species, and indicate that, according to the chemical index methodology, S. arborescens is a more recent evolutionarily species than S. langsdorffi i.

Conclusions
When considering chemical index methodology for taxonomic problem elucidation, the idea is to show a relationship between the plant evolutionary order found by the use of chemical indexes and the plant evolutionary order already described in the literature based on plant morphology and other data.
The use of the chemical index methodology demonstrates that molecular structure differences observed for the metabolites isolated from the two species that produce 8,11,13-trien-abietane diterpenoids, the native Brazilian tree Swartzia langsdorffi Raddii, included in the series Recurvae of Swartzia sect.Swartzia, and S. arborescens (Aubl.)Pittier (Swartzia sect.Possira), are related to oxidative processes and indicated an evolutionary direction.
Based on morphological data of S. langsdorffi i, such as pattern venation, slender, axillary and shorter than leaf raceme, calyx shape, and androecium characteristics, and given the presence of terpenoids and terpenoidal saponins, we observe that this species is related to S. arborescens and to S. simplex, both pertaining to the section Possira.
Then, even though the aromatic diterpenoids found in S. langsdorffi i were very similar to those isolated from S. arborescens, the chemical index values obtained for the two species allow suggesting that S. arborescens is evolutionarily more recent than S. langsdorffi i.Those results corroborate the Cowan [2] evolutive line.
The Torke & Schaal's molecular phylogeny of the neotropical species of Swartzia [12] identifi es 11 nonoverlapping clades, few of which correspond exactly to previously published taxa [2].The grouping of the clades orthostyloid and benthamioid is a sister of the remaining Swartzia and, as mentioned above, is characterized by isofl avonoids.Thus, the presence of terpenoids in the clades possiroid and acutifolioid could be taken as an evolutionary novelty.
A weighted cluster analysis based on wood anatomical characters, which shows seven distinct groups within the Brazilian species of Swartzia [29], add data to this recent cladistic analysis and can be correlated with our results.The authors pointed out some similarity of their wood groups with Cowan's [2] classifi cation.Although the species, which were studied phytochemically, are not enough, the results can be useful to indicate phyllogenetic and taxonomic relationships in Swartzia.The correlation of chemical data and data reported by Angyalossy-Alfonso & Miller [29] suggest an evolutionary tendency from group 4 (where are the species of orthostyloid and benthamioid clades) to group 5 (where S. arborescens is included).In this sense, for example, the multiseriate rays in Swartzia can be interpreted as derived from the exclusively uniseriate rays.
Considering the presence of metabolites as one discriminating character for Swartzia sect.Swartzia, it seems that there are two subgroups: the orthostyloid clade characterized by isofl avonoids not containing a tetracyclic ring system and benthamioid, recurvoid and tournateoid clades characterized by the occurrence of pterocarpenoids.
Finally, our analysis compares favourably to some clades within cladistic analysis using molecular data of Torke & Schaal [12].The two main clades within Swartzia that were recovered by Torke & Schaal, the most interior node including most species and nine of the 11 supported subclades of Swartzia and other with orthostyloid and benthamioid clades, also fi nds support from the phytochemical data shown in this paper.

Results and Discussion
Disparity Evaluation-Comparing the chemical structures of metabolites reported for Swartzia species (Table 1) it is possible to observe two groups characterized by the presence of isofl avonoids or terpenoids.Phytochemically studied Swartzia species that furnished diterpenoids and triterpenoidal saponins (S. arborescens [25], S. simplex [26], S. schomburgkii [27] and S. langsdorffi i [21,22,28]) are more recent concerning the evolutionary aspects, since their metabolites were generated by the mevalonic acid route while the remaining species produced isofl avonoids that are formed by the shikimic acid/ acetate route.
Considering the clades obtained by Torke & Schaal [12], the presence of these substances could be used, in addition to other data, to defi ne the Swartzia clades: possiroid and acutifolioid clades characterized by terpenoids, and orthostyloid and benthamioid clades characterized by fl avonoids (Table 1).In this sense, the systematic position of S. schomburgkii is doubtful, appearing with 58% maximum parsimony bootstrap support in a combined chloroplast and nuclear sequences tree [12, Fig. 3].In the trees obtained in separate searches of combined chloroplast sequence data and ITS sequences, S. schomburgkii and S. polyphylla remain in distinct clades [12, Fig. 1 and 2].If the absence of diterpenoids in S. simplex would be confi rmed, this would point out the applicability of subcategories in Possira classifi cation.
Diversity Evaluation-The second evaluation was made by analysis of the diversity of the chemical structures of fi fteen 8,11,13 -trien--abietane diterpenoids identifi ed from the species S. arborescens and S. langsdorffi .
Different from isofl avonoid chemical index calculations, the chemical indexes for terpenoids, as mentioned above, is evaluated by the oxidative degree (O) and by the carbon skeleton specialization (E) of their molecular structures.Table 2 shows the values of the chemical indexes obtained for S. langsdorffi i metabolites.The same rationalization and kind of calculations led to the values displayed in Table 3 for the metabolites of S. arborescens.

Figure 1 .
Figure 1.Hypothetic common precursor which has been used to calculate skeleton complexity evolutive advance (EA c ) values for all swartziarboreols identifi ed.

Table 2 .
Oxidative evolutive index values (O), calculated for swartziarboreols isolated and identifi ed in the extracts of S. langsdorffi i (Magalhães et al. 2005; Santos et al. 2007).*O = x-h/ n, where the letters refer, respectively, to the total carbon atoms (n), the C-H bonds (h) and the C-heteroatom bonds (x) of the diterpenoid skeleton.** E = Carbon skeleton specialization chemical index.

Table 4 .
Values for oxidative evolutive advance and skeleton complexity evolutive advance, EA o and EA E, calculated using the chemical index values found for the metabolites identifi ed in the species S. langsdorffi i and S. arborescens.