Profiling of Phenolic Compounds of Somatic and Reproductive Tissues of Agave Durangensis Gentry (Agavaceae)

Problem statement: In Durango, Mexico, mescal is elaborated from wild p ants of Agave durangensis. This species shows a high morphological variabili ty within and among populations, what makes its taxonomic delimitation a hard task. Approach: In this study the pollen and foliar phenolic compositions of Agave durangensis were analyzed by HPLC/DAD with the aim of determin ing the significance of phenol profiles to delimit this tax on. Results: The foliar phenol compositions were evaluated within and among two populations and betw e n juvenile and adult plants. Agave asperrima Jacobi, Dasylirion sp. and juvenile samples of A. shrevei Gentry subsp. shrevei, A. shrevei Gentry subsp. matapensis Gentry and A. wocomahi Gentry, were also analyzed to stand comparisons wi th. The results from this study indicated that pollen and f oliar tissues of Agave durangneisis were rich in kaempferol glycoside derivatives (13 and 23 differe nt compounds can be present, respectively). Principal coordinates analysis (PCO), based on foli ar profiles of adults, indicated the presence of several chemotypes within the Type locality of Agave durangensis and revealed chemical differences between the both analyzed populations. Conclusion/Recommendations: Chemical and morphological differences and biogeographical evidence suggest th recognition of two different taxonomic entities i n this morphological variable group.


INTRODUCTION
Agave is the biggest genus of the family Agavaceae, with around 166 species, from which 125 grow in Mexico [1,2] . Relevant ethnobotanic relationships have been established between the elements of this genus and the ancient and present cultures of Mexico [3,4] . Several authors have described the use of Agave as source of fibers, food and beverages [5] . In addition, Agave is used as natural fences to avoid the soil erosion and as cattle food [6] .
At the present, the relevance of Agave has increased meanly because of the increased demand of alcoholic beverages like tequila and mescal [7] and the research on potential sources of prebiotics [8] . In all the cases, the authentification of the species of Agave to be used is an important requirement in the quality control of the manufacturing processes. In Mexico, with exception of Agave tequilana Weber var. azul, species like Agave durangensis, which support local industries of alcoholic beverages and which with just begin an industrialization process, techniques are needed to guarantee the botanical origin of plants, according to the respective origin denomination statement.
Agave durangensis Gentry is one of the 24 species of genus Agave occurring in Durango, Mexico [9] . This species belongs to group Ditepalae of subfamily Agavoideae, proposed by Gentry [3] and can be found from Southern Durango to Northern Zacatecas [3,10] .
In two localities of Durango ("Sierra of Registro", the type locality and "Mezquital"), populations of Agave, traditionally called "agave mezcalero" or "agave cenizo", presumably A. durangensis, maintains a thriving mescal industry. Actually, studies have not been done to confirm that in all the cases the raw material to elaborate mescal is composed of that species [10] . In Durango, the manufacturing of mescal is mainly based on the gathering of agaves from their natural populations [7] . Recently, producers have been interested in the establishment of plantations of A. durangensis and then, typification methods are needed to guarantee the botanical origin of plants, since genus Agave is taxonomically difficult, owing to the high degree of phenotypic plasticity, the occurrence of several ploidy levels and the high capacity of hybridization [3,4] . Previous reports [3] and our own field observations in the Type locality ("Sierra of Registro", Durango, Mexico) indicate that A. durangensis is highly variable in size, color of leaf and size and form of teeth, in such a way that it has been suggested as a complex of species more than a single species by some authors [7] . Chemical characterization is an important technique, which with plant typification and identification can be made in a relative easy and fast manner, as it has been reported for many groups of plants [11,12] .
In the literature there are many reports describing the significance of phenol profiles to discriminate among different related taxa [13][14][15] . The species-specific tendency of pollen [12,16,17] and somatic tissues phenol profiles [18,19] has been reported for many species of plants. In spite of the economic and ecologic importance and the taxonomic controversies prevailing in the delimitation of the different species of the genus Agave and in the other hand, in spite of the taxonomic relevance of phenol composition, the efforts focused in determining the taxonomic significance of the phenol profiles in this genus has been limited.
Agave is rich in saponins and it has been better analyzed by its saponin composition [20][21][22][23] than any other secondary metabolite. One of the few species of Agave analyzed by its phenol composition is Agave americana, in which complex flavonoids has been found in somatic tissues, like 5,7-dihydroxy-6,5'-dimethoxy-3',4'methylenedioxyflavanona [24] and kaempferol-3glycoside derivatives were detected in flowers [25] . In this study the phenol characterization of somatic and reproductive tissues of Agave durangensis was performed, using high pressure liquid chromatography/diode array detector (HPLC/DAD) profiles in order to establish the specific taxonomic significance of these markers and to detect intra and interpopulation variability. Moreover, to our knowledge, the phenolic composition of Agave durangensis and A. asperrima is reported for the first time.  Table 1.

MATERIALS AND METHODS
Additionally, information about the morphological features was recorder for each sample.
Phenol extraction: Each sample was individually treated. Five grams of foliar dried grinded tissue were firstly extracted by maceration for 24 h, in 100 mL 60% methanol (v/v), in darkness and at room temperature. The extracts were centrifuged (5000 rpm), for 10 min, at room temperature. The supernatants were separated. The pellets were reextracted in 100 mL 60% methanol (v/v) by maceration for 3 h. The extracts were centrifuged at the same conditions. The similar supernatants were brought together and formed the total extracts. Each total extract was concentrated under vacuum to dryness and then resolved in 5 mL methanol; aliquots were taken to be used in the HPLC/DAD analysis. Samples of pollen collected directly from anthers were individually extracted according to Campos [11] , with ethanol-water (50% v/v) and sonicated for 60 min. The resultant mixtures were centrifuged for 10 min and the supernatants used for HPLC/DAD analysis.

HPLC/DAD analysis:
To determine the HPLC/DAD phenolic profiles, extracts (20 µL) were analyzed as previously described [16] , on a Perkin Elmer HPLC system and Perkin Elmer Brownlee Analytical C18 column (4.6×250 mm, 5 µm), by an acidified acetonitrile-water gradient. Standard chromatograms were plotted at 260 and 340 nm. Spectral data for all peaks were accumulated in the range 220-400 nm using diode-array detection (Perkin Elmer Series 200). The structural identifications were made by direct comparison of retention time and UV spectra of standards and according to [26,27] .

Data analysis:
The phenol profile of each sample was made up of all compounds resolved in their respective HPLC/DAD chromatograms. Each compound was treated as a single chemical character. A binary matrix of presence-absence formed by all individual samples vs. all resolved compounds, for each adult and juvenile samples, were analyzed by principal coordinates analysis (PCO) from NTSyS 1.8 [28] .

Pollen phenol composition:
The relation of phenolic compounds found in pollen of Agave durangensis is shown in Table 2. Retetion times and the chemical identification are included. Individual variability can be observed.

Foliar phenol composition:
The resolved flavonoid compounds for all the 23 individuals of Agave durangensis from Sierra of Registro are presented in Table 3. The individual foliar phenolic profiles of each sample from the population of "Temoaya" are indicated in Table 4; while those corresponding to A. asperrima are shown in Table 5. In all these cases, the retention times and the chemical identification are included. As in pollen, variability among the individual profiles can be observed.
The foliar phenol profiles of each of the four analyzed individuals of Dasylirion sp. are showed in Table 6. Contrary to that observed for samples of Agave, individual variability was not observed.

Principal coordinates analysis:
The discrimination of taxa based on the foliar phenol composition of adults, according to the PCO analysis, is showed in Fig. 1. The clear segregation of Dasylirion sp., A. asperrima and A. durangensis from "Temoaya" can be observed. However, three subgroups in the mayor group of A. durangensis from Sierra of Registro can be distinguished. The PCO analysis, based on the foliar phenol composition of juvenils, is showed in Fig. 2. The segregation of samples of A. shrevei subsp. shrevei, A. shrevei subsp. matapensis and A. wocomahi and the inclution of the sample of A. durangensis from the Botanic Garden, UNAM, in the mayor group formed by the most of the juvenile samples of A. durangensis from Sierra of Registro can be observed.    (Table 2). Flavonoids were the only class of phenolics found in the pollen of Agave durangensis. The analysis revealed 12 kaempferol glycosides and one myricetin glycoside. Derivatives of both quercetin and kaempferol are the most abundant phenols in pollen [11,12,17,29] . They along with myricetin are involved in such as important functions as the pollen tube germination and growth in several species of plants [30,31] ; however, in spite of quercetin glycoside derivatives are almost ubiquitous, it was significantly absent from Agave durangensis pollen. The role of the abundant kaempferol glycosides in pollen of Agave durangensis is left for determining.
The pollen phenol profile of the two individuals of Agave durangensis coming from the population of "Mezquital" were identical between them (six flavonols and the presence of one myricetin glycoside) but some different of those of the two individuals from "Temoaya", which were very similar between them but a little variability was detected (Table 2). These two localities are separated one from each other by 15 Km and similar morphological traits between both populations were observed.
Variability in pollen phenol profiles has been showed for other plants, including plants sharing the habitat of A. durangensis in the semiarid zones of Durango, like some species of Cactaceae [12] .
The importance of pollen phenol profiles as specific taxonomic markers has been reported [16,17] . These results suggest that, although only two individuals from each population were analyzed (inflorescences of Agave durangensis are difficult to find due to it is a monocarpic species and due to when plants are ready to flower, the inflorescences stems are cut to prepare the plants for mescal manufacturing), this kind of profiles could be even used to discriminate among populations. This would be relevant because of the taxonomic controversy in the specific delimitation of A. durangensis, which is, in turn, a consequence of the high morphological variability found in this taxon [3,10] and of the little effort dedicated to study this group. However, to propose these profiles as a specific and infraspecific marker, it is necessary to carry out more population studies, with a higher number of individuals, throughout all the geographical distribution.

Taxonomic
implications of foliar phenol composition: The flavonoids of the Agave family, the Agavaceae, have not been well investigated; just a few species have been analyzed [32] , so that, this survey, basically of two species of Agave (A. durangensis and A. asperrima), is useful in indicating the variation that may be encountered in these plants.
A total of 32 compounds were resolved in the foliar samples of adult plants by HPLC/DAD ( Table 3). The analysis revealed 23 flavonol glycosides present in the foliar profiles of Agave durangensis. Twenty of those were kaempferol glycoside derivatives. Three different quercetin glycoside derivatives were found in samples 203, 225 and 228. More phenol compounds could be present but at a very low concentration in such a way that their identification was not possible.
A high variability was found in the individual phenol profiles of the population from Sierra of Registro, which is the Type locality of Agave durangensis. Our own field observations revealed also a high morphological variability concerning the leaves form and length, adult plant height, number of teeth in 10 cm and terminal spine length, this variability agree to that found in the phenol composition. The profiles varied from three compounds in four individuals (200, 201,206 and 232) to nine in the individual 230 (Table 3).
The PCO analysis based on the foliar phenol composition of the individuals from Sierra of Registro revel the presence of three major groups; these could represent three chemotypes (Fig. 1). The morphological and chemical variations could reflect a present evolution process in this taxon, which has been put under an intense environmental pressure due to the overexploitation and deforestation of the area several decades ago.
As in the case of pollen, the dominance of kaempferol derivatives was clear in the foliar phenol composition of A. durangensis (and also of Agave asperrima, Table 5). It has been suggested that certain compounds may be induced by herbivore predators or microbial attackers [33] , this could explain the presence of quercetin derivatives in few individuals (three) of Agave durangensis from the population Sierra of Registro (Table 3).
The homogeneity in the foliar phenol profiles of individuals of Agave durangensis from the population of "Temoaya" represents a strong contrast with that found for the population Sierra of Registro. Assumed as Agave durangensis, the individuals from "Temoaya" showed a profile of only three to four kaempferol glycosides ( Table 4). The plants growing in "Temoaya" are in average higher (101.3±38.718 cm) than the plants from Sierra of Registro (78.25±23.686 cm) and have longer leaves (73.7±26.765 cm and 54.795±16.130 cm, respectively), with similar middle-leaf wide (19.2±4.134 cm and 18.3±4.401 cm, respectively) and similar spine length (4.5±1.08 and 4.2±1.10 cm, respectively). Both populations (Sierra of Registro and "Temoaya") are separated one from each other by around 50 Km, each with different environmental conditions (Table 1). This could explain the two classes of profiles found in one and another population, since it has been stated that the biosynthesis and accumulation of secondary metabolites depends on highly regulated processes, requiring, among others, environment-specific controls [34] . However, it has been reported that enzymes catalyzing modification reactions of simple flavonoids generally exhibit high substrate specificity, implying that many reactions proceed in a defined sequential order, which seems to be specific for each plant species [35] ; then, according to this and to the PCO analysis, the foliar phenol compositions of both populations are different in such a way that each could represent an independent taxon (Fig. 1).
González-Elizondo et al. [10] report Agave angustifolia, A. wocomahi, A. shrevei ssp. magna and A. maximiliana occurring also in Southern Durango. The samples of "Temoaya" could belong to some of those species; however their morphological attributes do not correspond to those given by [10] for each of mentioned species.        The foliar phenol profiles of all individuals identified, on a morphological basis, as Agave asperrima (samples 300-333) showed a very simple foliar phenol profile. The individual profiles were very homogeneous. The patterns were formed for only one or two among three different kaempferol glycosides ( Table 5). The PCO analysis clearly separates the samples of A. asperrima from all the other individuals (Fig. 1).
None kaempferol glycoside derivative was found as part of foliar phenol profile of Dasylirion sp., but one quercetin glycoside, one luteolin glycoside, probably one gossypetin glycoside and four unidentified flavones glycosides were detected (Table 6). The PCO analysis place Dasylirion sp., which belongs to family Nolinaceae [9] , clearly separated from Agave (Fig. 1). The foliar phenol profiles of juvenile samples of Agave durangensis were more complex and variable than those belonging to adults (Table 7). A total of 28 different kaempferol glycosides were found and from three to eleven could be present in any sample. Compounds J10, J14 and J22 (Table 7) are shared with adults. However, a considerable difference exists between juvenile and adult profiles. This difference suggests that, contrary to reported for others species of plants by Abou-Zaid and Nozzolillo [36] and Almaraz-Abarca et al. [15] , who did not find any age-related variability in foliar flavonoid composition, the qualitative expression of A. durangensis foliar flavonoids is not defined from the juvenile stage. The role of all these compounds in the growth and development processes of this species is left for determining.
The phenol foliar profile of juvenile sample of A. durangensis donated by the Botanic Garden, UNAM, was formed by four kaempferol glycoside derivatives,   (Table 7). That donated sample is situated among most of the A. durangensis samples collected from Sierra of Registro by the PCO analysis.
Other three groups are distinguished, each formed by one to three individuals; these groups could represent different chemotypes (Fig. 2). Agave wocomahi, which belongs to the same section Ditepalae than Agave durangensis [3] , showed profiles formed by four kaempferol derivatives (J8, J9, J13 and J15), all, except J13, are also present in juvenile A. durangensis samples ( Table 7). The PCO analysis places this species close to the juvenile sample 221b of A. durangensis.
Concerning to the foliar phenol profile of A. shrevei subsp. shrevei and A. shrevei subsp. matapensis (belonging also to the same section Ditepalae), their profiles were different to that of A. wocomahi and that of A. durangensis. They were formed by three kaempferol glycoside derivative, from which one (J9) was common to both ( Table 7). The PCO analysis of foliar profiles of juvenile samples groups together both subspecies of A. shrevei (Fig. 2).

CONCLUSION
The foliar and pollen phenol compositions of Agave durangensis are rich in glycosides derivatives of the flavonol kaempferol. Actually, this kind of compounds domain in all species of Agave studied. These taxa could be distinguished by the foliar phenol components and some species-specific patterns could be discerned. This reveals the flavonoid foliar profiles as valuable specific chemical markers in Agave.
In spite of the intrapopulation variability detected within the population of Agave durangensis from Sierra of Registro, the presence of specific flavonoid components in the leaves of each analyzed population (Sierra of Registro and "Temoaya") suggest the recognition of two independent taxonomic entities within this morphologically variable group.