Twenty-three flavonoids were isolated from the leaves of two species of Sophora (Fabaceae) from the Juan Fernández Islands (S. fernandeziana and S. masafuerana), two related species from continental Chile (S. macrocarpa and S. microphylla) and one sample from New Zeland (S. microphylla). A phenetic analysis was used to evaluate chemical similarities. Flavonoid profiles suggest that Chilean and New Zealand populations of S. microphylla are most closely related to each other. Sophora masafuerana appears most closely related to S. microphylla and this pair of species is also fairly closely related to S. fernandeziana. S. macrocarpa is more distantly related to the cluster of other species. The results suggest that the two Juan Fernandez endemic species are derived from two separate introductions. The exact origin of S. masafuerana remains unclear, as the evidence does not enable us to distinguish between or clarify its possible relationships with New Zealand or South American populations.

KEY WORDS: Chemotaxonomy, Juan Fernandez, Sophora, flavonoids.

Se aislaron veintitrés flavonoides de hojas de dos especies de Sophora (Fabaceae) endémicas de Juan Fernández: S. fernandeziana y S. masafuerana, dos especies de Chile continental, S. microphylla y S. macrocarpa y una población de S. microphylla proveniente de Nueva Zelanda. Se realizó un análisis fenético para evaluar la similitud química entre las especies en estudio. Las dos poblaciones de S. microphylla están más relacionadas entre sí. S. masafuerana está más relacionada a S. microphylla, formando un grupo que se relaciona con S. fernandeziana. S. macrocarpa se ubica algo distante en el fenograma. Los resultados sugieren que las dos especies de Juan Fernández derivaron de dos introducciones. No existe suficiente evidencia para relacionar el origen de S. masafuerana con representantes de Oceanía o de Sudamérica.

PALABRAS CLAVES: Quimiotaxonomía, Juan Fernández, Sophora, Flavonoides.

The Juan Fernandez Archipelago is located 667 km west off continental Chile at 33° S latitude. This archipelago comprises two major islands: Masatierra (or Robinson Crusoe Island) and Masafuera (or Alejandro Selkirk Island). Off the western end of Masatierra is another small island, Santa Clara. Masatierra, the nearest of the three islands to the South American continen, has an area of 48 km² and is 4 million years old¹⁾. Masafuera, with an area of 50 km², is located 150 km further west and is 1-2 million years old¹⁾. Several genera of plants are represented by different species on the two islands, providing interesting opportunities for evolutionary studies.

The genus Sophora L. (Fabaceae) consists of about 50 species. Two of the four Chilean species of this genus occur in the Juan Fernandez archipelago. S. fernandeziana (Phil.) Skottsb. is endemic to Masatierra and S. masafuerana (Phil.) Skottsb. is found only in Masafuera. Two other species of Sophora occur in continental Chile. S. macrocarpa Sm. in central and south-central Chile, and S. microphylla Aiton in south-central and southern Chile²⁾. On the basis of pollen micromorphology, Peña et al.³⁾ hypothesized separate origins for the two species of Sophora on the Juan Fernandez archipelago. Their results suggested that S. masafuerana was most closely-related to South American populations of S. microphylla, whereas other South American species may be the closest relatives of S. fernandeziana. Cladistic analysis⁴⁾, based on morphology and alkaloid contents, gave a cladogram with very low ralationships for all Chilean species. When morphological characters were treated separately, S. masafuerana showed a much closer relationship with S. microphylla than with S. fernandeziana. S. macrocarpa appears rather distant from other Chilean species, but within the same branch of the genus. Hoeneisen et al.⁵⁾ isolated 12 alkaloids from six populations of Sophora: two populations of S. microphylla (from Chile and New Zealand) and one population from each of S. macrocarpa, S. masafuerana, S. fernandeziana and S. linearifolia Griseb, (the latter species from Argentina). Alkaloid distribution profiles were very similar for S. macrocarpa and S. linearifolia. The two populations of S. microphylla also showed strong similarities with each other. Alkaloid distribution profiles of S. masafuerana and S. fernandeziana were both fairly well differentiated from those of the other species, although these two species did not show strong similarities with each other.

To test the hypothesis about the origin of two species of Juan Fernandez Islands, leaf flavonoids from the endemic species were examined, and compared to flavonoids in S. macrocarpa and S. microphylla from continental Chile, as well as those from New Zealand populations of S. microphylla. The objectives of this research were (1) to examine flavonoids in these four species of Sophora; (2) to evaluate the relationships among these species based on flavonoid chemistry; (3) to compare these relationships with those derived from morphology and alkaloids⁴⁾ or alkaliids⁵⁾; (4) to help clarify the origins of the two endemic Juan Fernandez species, with particular emphasis on the question of whether they are originated from one or two introductions.

Material of S. microphylla and S. macrocarpa was obtained from regions V to X of continental Chile (32°59'S - 42°38'S). Leaves of S. fernandeziana and S. masafuerana were collected in both major islands of the Juan Fernandez archipelago, Masatierra and Masafuera. Plant material from New Zealand was sent by Dr. P.J. Garnock-Jones of the Department of Scientific and Industrial Research, New Zealand. Collected samples are deposited in the Herbarium of University of Concepción (CONC) (Tablel I).

TABLE I. Plant material collected.

Flavonoid analysis

Leaf material was extracted with methanol: water (80:20) which was partitioned with dichloromethane and ethyl acetate. Flavonoids were isolated from the purified ethyl acetate fraction by column chromatography on polyamide. The compounds were further purified by descending paper chromatography on Whatman N 3 MM paper with the following solvents: 15%, 25% and 40% acetic acid and TBA (tertiary butyl alcohol: acetic acid water; 3:1:1). Upon isolation, the flavonoids were characterized by ultraviolet spectroscopy⁶⁾ and gas-mass chromatography for aglycone and sugar fractions of each flavonoid. Compounds were identified by a combination of the color reactions when fumed with NH₃, R_r values on paper with two solvent systems and ultraviolet spectral properties. Similar R_r values in TBA, and 15%, 25% and 40% acetic acid were used to judge whether flavonoids from different collections represented the same compounds. Hydrolysis was done with hydrochloric acid 5N at 50° for 2 h, using 10 mg of compound. The aglycone was separated from sugars by partitioning with water/ethyl acetate. The acetylation was done dissolving 5-10 mg of compound in 1 mL of pyridine and acetic anhydride for 24 h. The precipitate was filtered and washed with distilled water and recovered with ethyl acetate.

Phenetic analysis

Flavonoid data were phenetically analyzed using Jaccard's association index and phenograms were constructed to reflect similarity among the flavonoids from the populatons of the four species. Correlation coefficients were used as a measure of chemical similarity. Cluster analysis was done using UPGMA with computer program NTSYS⁷⁾ to produce a phenogram.

The 23 flavonoids isolated from 13 populations of Sophora represent four different classes: flavonols (12), flavones (4), dihydroflavonols (4) and flavonones (3) (Table II). No new flavonoids were found in this study. No qualitative intraspecific flavonoid variation was detected except in S. microphylla material, sent from New Zealand, which had fewer flavonoids than S. microphylla from continental Chile.

The results in Table III show the distribution patterns of flavonoids in Sophora species. Only two species possess flavonones, S. macrocarpa and S. fernandeziana. Both species from Juan Fernandez Islands show balanced profiles of approximately equal numbers of two kind of flavonoids isolated, flavones and flavonols.

TABLE II. Flavonoids isolated from studied populations.

TABLE III. Distribution of flavonoids in the studied species of Sophora.

X = presence of flavonoid; NZ = New Zealand population, CH = Chilean population

Table IV shows the cophenetic values matrix, with Jaccard coefficient. The values of the phenetic relationship are higher between Juan Fernandez species and the New Zealand population than with the Chilean population of S. microphylla.

TABLE IV. Similarity matrix using association index (Jaccard)

The results of othe phenetic analysis of flavonoids are presented in Figure 1. The most closely interrelated populations are the two populations of S. microphylla. Both species from Juan Fernandez Islands are related to S. microphylla, but at different levels of similarity. S. masafuerana is more or less phenetically equidistant between S. fernandeziana and S. microphylla. On the other hand, S. macrocarpa forms a very different branch.

FIG. 1. Phenogram of relationship among species of Sophora based on flavonoid data. Cophenetic correlation index = 0.9.

The result in Table III show trends based on general classes of compounds. S. fernandeziana and S. macrocarpa possess flavanones but they are absent in S. microphylla and S. masafuerana.

The phenetic results in Figure 1 show that the two populations of S. microphylla are closely interrelated, and that they are also related to both the endemic species of Juan Fernandez. S. mcrocarpa is more distantly related to the other species. However, the two endemic island species, S. fernandeziana and S. masafuerana, do not form a cohesive unit, the latter being the more closely-related of the two to S. microphylla.

From a taxonomic perspective, Peña and Cassels⁴⁾ showed very close relationships among S. microphylla, S. masafuerana and S. macnabiana (collected in continental Chile). However, only two species are currently recognized in continental Chile. Sophora macrocarpa and S. microphylla⁸⁾. Sophora macnabiana (Grah.) Skottsb. ex G. Taylor is considered as a synonym of S. microphylla⁹⁾.

The results based on the distribution of flavonoids in the Sophora species suggest that the island species arose from two separate colonizations of the archipelago. S. masafuerana is closely related to S. microphylla, probably from continental Chile, and S. fernandeziana shows links with both S. microphylla and S. masafuerana. However, Skottsberg¹⁰⁾ suggested that S. fernandeziana and S. masafuerana are very similar to S. tetraptera J.F. Mill., from Chile and not identical with S. tetraptera from New Zealand. Although the evidence does not permit us to rule out a possible origin of S. masafuerana from New Zealand populations of S. microphylla, the most likely scenario is that both island species have their ancestors in continental Chile. There are strong parallels with the species of Myrceugenia (Myrtaceae) that are endemic to the Juan Fernandez archipelago. This genus consists of 39 species: 25 occur in Brazil, 12 in continental Chile, 1 in Masatierra (M. fernandeziana) and 1 in Masafuera (M. schulzei)¹¹⁾. Flavonoid profiles showed close relationships between insular endemic species and continental Chilean relatives, but suggested separate origins for the two island species¹²⁾.

Is our pleasue to acknowledge: FONDECYT, project 1960822, 7960015, Dirección de Investigación, Universidad de Concepción, CONAF V Region and CONAF Juan Fernández and Dr. P.J. Garnock-Jones for sending plant material of Sophora microphylla from New Zealand.

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*To whom correspondence should be addressed

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