Pollen Flavonoid/Phenolic Acid Composition of Four Species of Cactaceae and its Taxonomic Significance

The pollen flavonoid/phenolic acid composition of 14 individual of Stenocactus multicostatus subsp. zacatecasensis (Britton et Rose) U. Guzmán et Vázquez-Benitez, comb. et stat. nov., 11 of Echinocereus enneacanthus Engelmann, 8 of Echinocereus pectinatus (Scheidweiler) Engelmann and 12 of Mammillaria heyderi sensu lato (all belonging to Cactaceae) was analyzed by HPLC/DAD with the aim of determining the role of pollen profiles of flavonoid/phenolic acid as valuable markers in those taxa. The flavonoid/phenolic acid composition of pollen was evaluated within and among the taxa analyzed. The results suggest that (a) the flavonoid/phenolic acid profiles of these species are among the most complex reported, (b) that quercetin, kaempferol and herbacetin glycoside derivatives are the major phenols found in the pollen of these species of cactus, (c) that some intrapopulation variability is present in all four species and (d) that these pollen flavonoid/phenolic acid profiles tend to be species-specific, so that they can represent important taxonomic markers in Cactaceae.


INTRODUCTION
Cactaceae is a native family of American Continent; its natural distribution is from southwestern Canada to Chile. However, Mexico is where this family reaches its highest level of genera and species diversity [1] . The family Cactaceae has between 1500 and 1800 species in approximately 100 genera [2] . It is estimated that in Mexico around 707 species and 58 genera are found [3] , with around 14 endemic genera and 400 endemic species [4] .
The delimitation of taxa of this family and the understanding of their taxonomic relationships becomes a hard task because of many species and genera pertaining to Cactaceae, have been partially described or the description has been based solely on one individual, has not been typified or has been made without denoting the origin [5,6] . That is the case of Stenocactus multicostatus subsp. zacatecasensis [5,6] , Echinocereus enneacanthus, Echinocereus pectinatus and Mammillaria heyderi sensu lato [5] . The great morphological variability and the high hybridization capacity are two additional factors making more difficult the taxonomy of these groups.
Stenocactus (= Echinofossulocactus), Mammillaria and Echinocereus belong to subfamily Cactoideae [7] . Stenocactus and Mammillaria share, according to morphological features, a closer taxonomic relationship, since both belong to tribe Cacteae and to subtribe Cactinae, than between either of them and Echinocereus, which belongs to tribe Echinocereae [5] .
Chemical features, like flavonoid profiles, have been considered as significant taxonomic markers in the delimitation of species of different plant families [8][9][10][11][12] . Almost every species of plant synthesizes some kinds of flavonoids. Pollen is a very important site of synthesis and accumulation of flavonoids and phenolic acids [13,14] . Recently, reports of the taxonomic significance of pollen flavonoid/phenolic acid profiles have appeared [14,15] , this is relevant especially for chemotaxonomic studies of Cactaceae, in which many elements are fixed in some dangerous status, since the determination of those profiles represent a non plant destructive analysis.
The present study was conducted to investigate the intra-and interspecific variations in pollen flavonoid/phenolic acid compositions among one species of Mammillaria, one species of Stenocactus and two species of Echinocereus and establish their taxonomic significance in these taxa of Cactaceae.

MATERIALS AND METHODS
Sampling: Pollen of one species of Stenocactus, two of Echinocereus and one de Mammillaria were collected in different locations of Durango, Mexico (Table 1). Information about environmental conditions where each taxon develops was compiled. The flavonoid/phenolic acid composition was interpreted considering the previous identification of material based on morphological markers. A voucher of each population collected was deposited at the Herbarium CIIDIR.  HPLC/DAD analysis: Ten milligrams of dried pollen of each sample were sonicated in an ethanol-water solution (1mL, 50% v/v) for 60 min. The resultant mixtures were centrifuged at 15269 g for 10 min and the supernatants were used for HPLC/DAD analysis as previously described [14] . Extracts (20 µL) were analyzed on a Gilson 170 and Waters Spherisorb ODS2 (5µm) column (4.6×250 mm) by an acidified acetonitrile-water gradient. Standard chromatograms were plotted at λ max 260 and 340 nm. Spectral data for all peaks were accumulated in the range 220-400 nm using DAD (Gilson 170). The phenolic profile of each sample was made up of all compounds resolved in its respective chromatogram. Each compound was treated as a single chemical character. The structural information was made by direct comparison of retention time and absortion spectra with standards and according to the information compiled by Campos and Markham [16] .

Data analysis:
The diversity of the profile of Flavonoid/phenolic acid for each taxon was derived using the Shannon index (H). A presence-absence matrix formed by all individual samples vs. all resolved compounds (45 individuals vs. 46 compounds) was analyzed using the UPGMA method (clustering program NTSYSpc 1.8 [17] .

RESULTS AND DISCUSSION
HPLC/DAD analysis: A total of 46 compounds were resolved by HPLC/DAD (Table 2). Flavonoids and phenolic acids were the only two classes of phenolics found in the pollen of all species of Cactaceae analyzed; 3-O-flavonolglycosides were the major structures among the flavonoids. The analysis revealed 16 kaempferol derivatives, 7 quercetin derivatives, 5 herbacetin derivatives, 4 unidentified flavonoids, 3 unidentified phenols and 11 phenolic acid derivatives.
Flavonoids are present in pollen of many species of Angiosperm and Gymnosperm and in spores of moss and ferns [18] . Particularly kaempferol and quercetin are two required flavonols to pollen tube germination and growth in tobacco [18] , petunia and maize [19] . However, these two flavonols have not been shown to carry a similar function in Arabidopsis [20] . The abundance of kaempferol and quercetin derivatives in pollen of Stenocactus multicostatus subsp. zacatecasensis, Echinocereus pectinatus, E. enneacanthus and Mammillaria heyderi sensu lato could suggest that those flavonols could serve an essential function during reproduction of these species of Cactaceae.
Phenolic acids derivatives were more abundant in the pollen of Stenocactus multicostatus subsp. zacatecasensis (8 structures, Table 3) than in the pollen of Mammillaria heyderi sensu lato (2 structure,  Table 4) and Echinocereus pectinatus (one structure, Table  5), while in the pollen of Echinocereus enneacanthus no phenolic acid was found ( Table 6).
The pollen phenolic profiles of four all species of Cactaceae analyzed were relatively complex (22 phenolic compounds in Stenocactus multicostatus subsp. zacatecasensis, 17 in Mammillaria heyderi sensu lato, 17 in E. pectinatus and 9 in Echinocereus enneacanthus). The phenols richness found in this study represents a contrast with other reports on pollen phenols obtained by the same method; for example those of Zea mays with six compounds, Bidens odorata with three [15] ; Eucalyptus globulus with seven and Erica australis with two [14] . A similar complex composition has been found in the perianth parts of Echinocereus triglochidiatus var. gurneyi (Cactaceae), but in contrast to pollen composition, dihydroflavonols and dihydroflavonol 7-O-gycosides, besides flavonol glycosides were present [21] . Other      Species of Cactaceae investigated for their composition of phenolics in the tepals, as a part of perianth, are Astrophytum ornatum Web., Notocactus apricus A. Berg.,

Echinopsis huotii
Lab., Aylostera pseudodeminuta Backbg. and Neochilenia napina Backberg.; in them no dihydroflavonol was detected [22] .    Among the four species analyzed, Stenocactus multicostatus subs. zacatecasensis had the pollen phenolic profile most complex (Table 3), with  22 structures: 1 unidentified flavonoid, 8 phenolic acid  derivatives and 13 flavonol glycosides, from which 2  are  gycosilherbacetin  derivatives,  2  are  glycosilquercetin  derivatives  and  9 are glycosilkaempferol derivatives. The most simple pollen phenolic profile was that of Echinocereus enneacanthus (Table 6), with 9 structures, 2 glycosilherbacetin derivatives, 2 glycosilquercetin derivatives and 5 glicosilkaempferol derivatives. These figures suggest that the pollen of every these four species of Cactaceae synthesizes more than the fifty per cent of its phenolics in a very conservative way.
Mammillaria heyderi sensu lato is a complicated taxonomic group due to high polymorphism and hybridizing capacity [5] . This taxon and Echinocereus pectinatus both are distributed through a very broad zone, practically in a continuous way in State of Durango, Mexico. They are found either in arid zones or in template Pinus-Quercus forest. Their distributions contrast with that of Stenocactus multicostatus subsp. zacatecasensis, which is found in scarce and disjunctive populations [25] . These three species can be found as sympatric populations in zones of Quercus forest with few grasses and rocky substratum, like it is the growing habitats of Stenocactus multicostatus subsp. zacatecasensis. Mammillaria heyderi sensu lato and Echinocereus pectinatus both with a broad distribution, showed a relative high flavonoid/phenolic acid variability in pollen (H = 2.254 and H = 1.494, respectively). These results were expected, since according to Sosa et al. [26] the increment in the morphological and genetic variability is associated to the increment in the distribution area. However, the high variability in the phenolic composition of pollen of Stenocactus multicostatus subsp. zacatecasensis (H = 2.107), which is similar to that of Mammillaria heyderi sensu lato, can not be associated to a broad distribution, but to a high intrapopulation genetic variability. The high chemical variability found in the pollen phenol composition of Stenocactus multicostatus subs. zacatecasensis is according to the Bravo-Hollis and Sánchez-Mejorada [5] 'statement about this taxon is in a present speciation process; in this taxon both chemical and morphological variability may be maintained by a heterogeneous environmental pressure of selection.
The fenetic analysis of pollen phenol profiles groups together Mammillaria heyderi sensu lato (Tribe Cacteae) and the two species of Echinocereus (Tribe Echinocereae) and place Stenocactus multicostatus subsp. zacatecasensis in a separated group, (Fig. 1). However, on a morphological basis, Stenocactus multicostatus subsp. zacatecasensis shares along to Mammillaria heyderi sensu lato the same tribe Cacteae [5] . Nevertheless, in spite of the suggestion given by these results it is necessary to investigate the distribution of pollen flavonoid/phenolic acid profiles and the correlations between chemical and morphological features, among a higher number of populations, species and genera of Cactaceae before considering a closer taxonomic relationship between Mammillaria and Echinocereus than between Mammillaria and Stenocactus.
Pollen phenolic profiles confirmed taxonomic separation of the four species made on morphological basis. The fenetic analysis (Fig. 1) clearly distinguishes four groups that correspond to the four morphologically-based taxa, Stenocactus multicostatus subsp. zacatecasensis, Echinocereus enneacanthus, Echinocereus pectinatus and Mammillaria heyderi sensu lato. This indicates that each of four species can be distinguished from the others by a unique flavonoid/phenolic acid profile. Pollen phenolic profiles are species-specific and each species express a variable number of unique flavonoids or phenolic acid.
In spite of the intrapopulation variability, the pollen flavonoid/phenolic acid profiles of Stenocactus multicostatus subsp. zacatecasensis, Echinocereus enneacanthus, Echinocereus pectinatus and Mammillaria heyderi sensu lato are so stable for each species that type profiles by species can be recognized. These profiles can be considered as valuable chemical markers at the specific level. The refereed profiles, rich in quercetin, kaempferol and herbacetin glycosil derivatives, are among the most complex reported. Although more population studies on the phenolic composition of pollen of Cactaceae are needed, over all in species with a broad distribution, these results suggest that flavonoid/phenolic acid profiles of pollen could be specific taxonomic markers in this family of plants.