Euphytica 124: 35–45, 2002. 35 © 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Genetic diversity of Brazilian oil palm (Elaeis oleifera H.B.K.) germplasm collected in the Amazon Forest

M.C. Moretzsohn∗, M.A. Ferreira, Z.P.S. Amaral, P.J.A. Coelho, D. Grattapaglia & M.E. Ferreira Laborat´orio de Gen´etica de Plantas, Embrapa Recursos Gen´eticos e Biotecnologia, C.P. 02372, 70849-970, Bras´ılia, D.F., Brazil (∗author for correspondence: Email: [email protected])

Received 18 October 2000; accepted 1 May 2001

Key words: AMOVA, Elaeis spp., genetic diversity, genetic resources, oil palm, RAPD

Summary Elaeis oleifera or ‘caiaué’, a close relative of oil palm (E. guineensis), has some agronomic traits of great interest for the oil palm genetic breeding such as slow growth, oil quality (mostly unsaturated) and disease resistance. An analysis of a Brazilian oil palm germplasm collection was carried out using RAPD (Random Amplified Poly- morphic DNA) markers with the objective of understanding the genetic variation of ‘caiaué’ accessions collected in the Amazon Forest in the last two decades. A sample of 175 accessions obtained along the Amazon River Basin was analyzed and compared to 17 accessions of oil palm from Africa. Ninety-six RAPD markers were used in the analysis, of which fourteen were shown to be specific to oil palm, while twelve were specific to ‘caiaué’. Results showed that the Brazilian ‘caiaué’ accessions studied have moderate levels of genetic diversity as compared to oil palm accessions. The data allowed the establishment of similarity groups for ‘caiaué’ accessions, which is useful for selecting parental plants for population breeding. Cluster analysis showed that, in general, genetic similarities are not correlated to geographical distances, but consistent with geographical dispersal along the Amazon River network. AMOVA showed that most of the genetic variation is found within populations, as expected for an allogamous and long-lived perennial species. The study provides important information to define strategies for future collection expeditions, for germplasm conservation and for the use of E. oleifera in breeding programs.

Introduction the species that could be valuable to oil palm breeding, such as: slow growth, oil quality (mostly unsaturated Oil palm, Elaeis guineensis Jacq., is currently one of oil) and disease resistance, including lethal yellowing, the major sources of vegetable oil in the world. The the major problem of oil palm cultivation in America crop ranks second among the world oil production (Bergamin Filho et al., 1998), as well as Fusarium crops and first in the international market of veget- wilt, the major oil palm disease of Africa (Renard et able oils and fats. Oil palm is grown throughout the al., 1980; Hardon et al., 1985). humid tropics and cultivation exploits almost exclus- The cultivation of ‘caiaué’ is still not viable eco- ively the Elaeis guineensis species, whose origin is the nomically, due to its smaller yields as compared to African continent (Zeven, 1964; Hartley, 1977). There the African oil palm. However, since the two species is, however, another species in the genus, E. oleifera, hybridize easily, interspecific hybrids can be obtained found in the Amazon Forest and known in Brazil as with yields around 90% of the commercial oil palm ‘caiaué’. This species has been incorporated into oil yields, but usually showing partial sterility (Amblard palm breeding programs (Meunier, 1975; Hardon et et al., 1995). Oil palm breeding programs that in- al., 1985; Le Guen et al., 1991; Santos, 1991). The in- corporate ‘caiaué’ germplasm have focussed on tests terest in the ‘caiaué’ germplasm is due to some traits of 36 of intraspecific combining ability, interspecific hy- Table 1. Origin and number of ‘caiaue’´ accessions analyzed per Amazonian site, grouped on 6 major regions of the Amazon bridization, and backcross to E. guineensis to restore Forest. Seventeen elite oil palm accessions from Africa were fertility. Some studies have shown ‘interpopulation also studied heterosis’ (Baudouin, 1992) both in E. guineensis (Gascon et al., 1981; Ghesquière, 1985; Baudouin et Amazonian sites Number of plants al., 1995) and in E. oleifera breeding programs (Le analyzed Guen et al., 1991; Amblard et al., 1995). Madeira River Region: ‘Caiaué’ populations are spread throughout the Manicore(Ma)´ 45 humid areas of Central and South America. Extens- Novo Aripuanã (Na) 12 ive collections of ‘caiaué’ germplasm were done in Manaus Region: the Amazon River Basin by EMBRAPA (Empresa Careiro (Ca) 28 Brasileira de Pesquisa Agropecuária, Brazil) and Amazon River Region: IRHO (Institut de Recherches pour les Huiles et Oléa- Amatari (Am) 13 gineux, France), in the early 80s, constituting a valu- Autazes (Au) 11 able source of genetic material. In the Amazon Forest, Maues´ (Mu) 13 ‘caiaué’ populations are usually found near rivers, on Solimões River Region: fertile and well-drained lands, known locally as ‘In- Tefe(Te)´ 06 dian black lands’ (Andrade, 1983; Barcelos, 1986). Anori (An) 04 Tonantins (To) 03 Whether the origin of these populations is wild or Negro River Region: subject of antropic influence is still controversial. Acajatuba (Ac) 09 Amazonian populations of ‘caiaué’ have been ana- Moura (Mo) 11 lyzed by morphological traits (Ooi et al., 1981; Bar- Caracara´ıRegion: celos, 1986), and isoenzyme markers (Ghesquière et BR 174 al., 1987; Moretzsohn, 1995), but little is known about Km 157 (Ba) 03 their variation at the DNA level. The knowledge of Km 365 (Bb) 04 the genetic variation of ‘caiaué’ collections is essen- Km 490 (Bc) 07 tial for their efficient use in breeding programs as well Vila Moderna (Perimetral Norte (Pn)) 06 as to establish collection and conservation strategies. TOTAL 175 Molecular characterization of genetic diversity is a quick and efficient approach that can provide informa- tion for the selection of progenitors of new breeding populations, or the identification of highly variable aué’ germplasm collection and compare it to cultivated populations for conservation purposes, and for the accessions of oil palm; (2) assess genetic relationships development of advanced collection strategies. New between ‘caiaué’ and oil palm germplasm, and (3) breeding populations can be formed by crossing plants identify distinct ‘caiaué’ genotypes for use in breeding with known genetic distances, favoring new allelic programs. combinations. RAPD is a powerful tool for the ana- lysis of genetic variation. RAPD does not require prior genomic information, and is simpler, less costly, and Materials and methods less labor intensive than other DNA marker meth- odologies. RAPD markers have been employed in Plant material the analysis of genetic variation among African ger- mplasm accessions of oil palm (Shah et al., 1994), in The ‘caiaué’ material consisted of 175 accessions attempts to detect somaclonal variants among regen- from 47 populations representing the distribution of erant populations of oil palm (Rival et al., 1998) and, this palm in the Brazilian Amazon Forest. Populations recently, in the identification of markers linked to the were distributed in 15 sites within 6 major regions shell thickness locus of E. guineensis (Moretzsohn et (Figure 1). The number of plants analyzed per site al., 2000). is listed on Table 1. These accessions are maintained The main objectives of the present study were to at the Centro de Pesquisa Agroflorestal da Amazônia use RAPD markers to (1) estimate the extent and Ocidental (CPAA) – EMBRAPA, Brazil. Additionally, organization of genetic diversity of the Brazilian ‘cai- 17 elite oil palm accessions were analyzed in order 37

Figure 1. Localities in the Amazon River Basin where ‘caiaue’´ samples were collected. 38 to allow a comparison with the ‘caiaué’ accessions. similarity coefficient. Only polymorphic markers were ‘Caiaué’ plants are identified with the code of the site included in the analysis. Dendrograms were construc- where they were collected (Table 1), followed by a ted using the unweighted pair-group method analysis number ranging from 1 to 192. Oil palm plants are (UPGMA). The frequencies of the RAPD fragments identified with the initials Eg, followed by a random were estimated for each of the six ‘caiaué’ popula- number. tions and for oil palm accessions. The resulting matrix was used to calculate Manhattan distances between all DNA extraction and quantification pairs of populations and oil palm accessions. These analyses were performed using NTSYS-pc software, Total genomic DNA was extracted from freeze dried version 2.0 (Rohlf, 1993). This software was also used adult leaf tissue of the 192 accessions using a modified to perform Mantel test (1967) and to estimate the cor- CTAB protocol (Grattapaglia & Sederoff, 1994). DNA relation (r) between the pairwise Jaccard similarity was quantified comparing the fluorescence intensities matrix and a pairwise matrix of geographic distances. of the ethidium bromide treated samples to those of Mantel statistic was performed for each of 2000 per- a λ DNA standard dilution series in 1% agarose gel mutations of rows and columns in the geographical electrophoresis under UV light. distances matrix to test if the original correlation was stronger than expected by chance alone. RAPD assays Analysis of Molecular Variance (AMOVA) (Ex- coffier et al., 1992) was used to analyze the partition RAPD analyses were carried out according to Ferreira of the total genetic variation between the sampled & Grattapaglia (1998). Amplification reactions were Amazonian regions, between ‘caiaué’ populations performed in a 13 µl solution, containing 10X PCR within regions and within populations. A total of buffer (10 mM Tris-HCl pH 8.3; 50 mM KCl; 1.5 mM 76 RAPD markers (with less than 5% of missing MgCl ); 1 µg/µl purified BSA (New England Bio- 2 data) was used for this analysis. Euclidean distances labs); 200 µM of each dNTP; 0.4 µM 10-base primer were calculated between all pairs of RAPD haplo- (Operon Technologies Inc.); 7.5 ng of genomic DNA types. Significance levels for variance components and 1 unit of Taq DNA polymerase, overlaid with 50 were calculated by non-parametric permutation pro- µl of mineral oil to prevent evaporation. RAPD re- cedures using 10,000 permutations. AMOVA was also actions were performed in an MJ Research PT-100 used to analyze the partition of the genetic variation thermal cycler programmed for 40 cycles of 1 min ◦ ◦ ◦ between two groups: (1) accessions collected in ‘In- at 92 C, 1 min at 35 Cand2minat72 C, and a ◦ dian black lands’ and (2) accessions collected in all final DNA extension cycle at 72 Cfor7min.RAPD other types of soils. These analyses were performed products were analyzed by electrophoresis in 1.5% by using the Arlequin software, version 1.1 (Schneider agarose gels in 1X TBE (0.45M tris; 0.45M boric acid; et al., 1997). The frequencies of the RAPD markers 10 mM EDTA, pH 8.0) and 1.5 µg/µl ethidium brom- were estimated on these two groups separately and the ide. Gels were photographed under UV light using + distance between them was calculated by the Average an MP4 Polaroid camera or an EagleeyeTM video Manhattan coefficient (NTSYS-pc). imaging system (Stratagene). Following a screening of eighty 10-base random primers on a subset of six samples (five ‘caiaué’ and one oil palm accessions), 27 primers were selected Results based on the number of amplified polymorphic frag- ments and on band intensity and used to analyze the Of the 80 primers screened for polymorphism, 51 192 accessions. produced at least one polymorphic fragment, 13 pro- duced only monomorphic bands, and 16 did not yield Data analysis any visible or scorable fragment. Out of the 51 poly- morphic primers, 27 were selected based on the num- PCR amplification products of the 192 accessions ber and quality of the amplified fragments and used for were scored as presence (1) or absence (0) of bands. analysis. A total of 96 selected markers were scored Cases where the presence or absence of DNA bands on the 192 accessions. An example of RAPD amp- was unclear were recorded as missing values. The lification patterns is shown on Figure 2. The size of data matrix was used to calculate Jaccard’s (1908) the amplification products ranged from approximately 39

Figure 2. RAPD patterns generated by primer Y09 (Operon Technologies Inc.). Each lane consists of randomly amplified DNA samples of E. oleifera, except the two identified by asterisks (E. guineensis). Arrows indicate the polymorphic fragments used in the analysis. Last lane is a 1 kb DNA ladder used as molecular size marker.

250 to 2100 base pairs. Considering only the ‘caiaué’ The number of amplified fragments per Amazonian accessions, each selected primer amplified between 1 Region ranged from 63 (Solimões) to 71 (Amazon). and 8 polymorphic bands, with an average of 3.0 poly- Eighty-four polymorphic markers were detected in the morphic markers per primer. All primers produced oil palm accessions studied. identical RAPD patterns for the selected bands on the Among the 175 ‘caiaué’ accessions, the average primer screening step and on the final analysis. Jaccard’s similarity value was 0.684, with the low- The frequencies of 54 RAPD markers in ‘caiaué’ est value (0.294) observed between Ma190 accession, populations from six Amazonian regions and in 17 from Manicoré (Madeira River Region), and Au49, elite oil palm accessions are given in Table 2 (14 from Autazes (Amazon River Region). The highest markers specific to oil palm accessions and 28 other similarity value was 0.974 between accessions Pn141 markers with similar frequencies for all ‘caiaué’ re- and Pn144, both from Perimetral Norte (Caracaraí Re- gions and for oil palm accessions are not shown). gion). An UPGMA dendrogram based on Jaccard’s Most of the RAPD products showed similar frequen- similarity was constructed for the 175 ‘caiaué’ acces- cies of occurrence among ‘caiaué’ regions. There were sions and the 17 elite oil palm trees (Figure 3). Some some exceptions, such as the Y09-400 fragment, for ‘caiaué’ accessions from Autazes and Maués (Amazon example, that was detected with frequencies ranging River) clustered with accessions from other Amazo- from 0.05 to 0.88 in ‘caiaué’ accessions and is absent nian Regions. Nevertheless, geographical clustering is in the oil palm accessions tested. Fourteen markers evident, grouping accessions according to the rivers were specific to African oil palm (L03-1340, N10-270, or sites where the populations were originally collec- N10-850, R04-760, R04-1340, W16-420, Y04-670, ted. In group I, 82% of Careiro accessions (Manaus Y04-780, Y04-820, Y09-510, Y10-920, Y10-1040, Region) clustered with accessions from Negro River Y10-1100, Y15-680) and were detected with frequen- (Moura – 9 out of 11 accessions, and Acajatuba – 7 cies ranging from 0.06 to 1.00. Other eight markers de- out of 9 accessions) and Amatari (10 out of 13 acces- tected in low frequencies (0.02 ≤ p ≤ 0.05) were found sions). Group II is mostly composed of accessions of to be specific to ‘caiaué’ populations of the Amazon the Caracaraí Region along the Perimetral Norte and River Region (G04-1830, R08-430, X09-980), Negro BR-174 roads. About 84% of the accessions collected (K09-750, K09-980, X09-1320), Madeira (X09-740), in the Madeira River (Manicoré and Novo Aripuanã and Manaus (Y04-700). These eight bands were also Regions) form a third group (group III), where the detected in oil palm, but with higher frequencies (0.06 majority of the accessions from the Solimões River ≤ p ≤ 0.94). Twelve bands absent in oil palm trees Region also clustered. In this group, about 70% of were detected in ‘caiaué’ accessions with frequen- the accessions were collected in the so-called ‘Indian cies ranging from 0.03 to 1.00 (K01-980, K09-950, Black Lands’, areas with high fertility, considered to K15-890, L03-1410, L03-1490, L03-1580, R06-1150, be cultivation sites of indian populations. The fourth X09-520, Y09-400, Y11-480, Y11-500, Y11-550). group is composed solely of E. guineensis accessions, 40

Table 2. Frequencies per Amazonian region of RAPD markers in ‘caiaue’´ and oil palm trees

Band Amazon Manaus Madeira Caracara´ı Solimões Negro Oil palm

A05-870 0.85 0.97 0.94 0.95 0.91 1.00 0.20 A05-910 0.73 0.97 0.81 0.84 0.82 0.81 0.13 G04-1830 0.03 0.00 0.00 0.00 0.00 0.00 0.19 K01-980 0.70 0.72 0.68 0.56 0.69 0.68 0.00 K01-1380 0.03 0.00 0.00 0.00 0.00 0.05 1.00 K09-750 0.00 0.00 0.00 0.00 0.00 0.05 0.88 K09-950 1.00 1.00 1.00 1.00 1.00 0.95 0.00 K09-980 0.00 0.00 0.00 0.00 0.00 0.05 0.94 K15-890 0.65 0.93 0.78 0.61 0.69 0.85 0.00 L03-770 0.03 0.14 0.04 0.41 0.25 0.00 0.73 L03-1410 0.22 0.03 0.18 0.11 0.00 0.10 0.00 L03-1490 0.97 0.97 0.98 0.88 1.00 1.00 0.00 L03-1580 0.97 0.97 0.98 0.94 1.00 1.00 0.00 N01-390 0.77 0.81 0.88 0.95 0.91 0.47 0.06 N04-520 0.69 0.42 0.72 0.89 0.73 0.05 0.53 N04-560 0.77 0.81 0.88 0.95 0.91 0.47 0.06 N05-490 0.83 1.00 0.91 1.00 0.62 1.00 0.25 R04-1980 0.00 0.00 0.02 0.00 0.00 0.05 0.76 R06-950 0.97 0.85 0.94 0.95 0.92 0.79 0.21 R06-1150 0.41 0.21 0.36 0.42 0.31 0.39 0.00 R06-1300 0.76 0.85 0.65 0.79 0.69 0.55 0.20 R08-430 0.03 0.00 0.00 0.00 0.00 0.00 0.06 R08-450 0.06 0.00 0.02 0.00 0.00 0.00 0.65 W01-650 0.08 0.04 0.04 0.05 0.08 0.05 0.65 W01-680 0.29 0.11 0.53 0.06 0.42 0.11 0.18 W01-700 0.83 0.72 0.93 0.95 0.77 0.60 0.12 W01-750 0.94 0.85 0.91 0.32 0.70 0.75 0.25 W16-550 0.00 0.00 0.00 0.11 0.00 0.11 0.82 W16-1040 0.94 1.00 1.00 1.00 1.00 1.00 0.19 X06-780 0.92 1.00 0.98 1.00 1.00 1.00 0.24 X06-1100 0.08 0.00 0.02 0.00 0.08 0.00 0.81 X06-1230 0.84 0.86 0.91 0.37 0.77 0.75 0.06 X09-520 0.00 0.07 0.05 0.06 0.08 0.05 0.00 X09-550 0.08 0.34 0.12 0.21 0.15 0.70 0.24 X09-580 0.78 0.69 0.82 0.89 0.58 0.95 0.12 X09-740 0.00 0.00 0.02 0.00 0.00 0.00 0.71 X09-980 0.03 0.00 0.00 0.00 0.00 0.00 0.75 X09-1030 0.58 0.89 0.16 0.95 0.50 0.85 0.62 X09-1120 0.00 0.14 0.00 0.00 0.00 0.05 0.50 X09-1320 0.00 0.00 0.00 0.00 0.00 0.05 0.82 Y01-840 0.62 0.90 0.78 1.00 0.83 0.95 0.29 Y02-310 0.40 0.41 0.96 1.00 0.92 0.84 0.07 Y02-520 0.00 0.03 0.02 0.00 0.00 0.00 0.59 Y04-700 0.00 0.03 0.00 0.00 0.00 0.00 0.88 Y04-890 0.92 0.89 0.50 0.84 0.84 1.00 0.14 Y09-400 0.65 0.31 0.88 0.05 0.31 0.85 0.00 Y09-1030 0.95 0.97 1.00 1.00 0.92 1.00 0.06 Y09-1080 0.44 0.82 0.04 0.62 0.18 0.68 0.94 Y10-330 0.68 0.72 0.43 0.95 0.54 0.80 0.12 Y10-370 0.95 1.00 0.95 0.89 0.92 0.90 0.18 Y11-480 0.00 0.03 0.02 0.11 0.00 0.00 0.00 Y11-500 0.23 0.45 0.15 0.27 0.00 0.14 0.00 Y11-520 0.77 0.59 0.82 0.64 0.86 0.79 0.08 Y11-550 0.10 0.38 0.05 0.09 0.00 0.07 0.00 41

Figure 3. Dendrogram based on the unweighted pair-group method analysis (UPGMA) for the 192 Elaeis spp. accessions. 42 except for one accession from the Caracaraí Region cessions in this (4.5) and other studies (Shah et al., (Bc127). 1994). Moreover, in the present study, the average The average genetic similarity indices between ac- genetic similarity was higher between ‘caiaué’ acces- cessions within groups were 0.747 for Group I, 0.756 sions (0.675) than between oil palm trees (0.472), for Group II, and 0.754 for Group III. A considerably despite differences in sampling size. lower similarity value (0.472) was observed among oil Natural populations of ‘caiaué’ are supposed to ex- palm trees. ist mainly in Central America and in the north of South Genetic relationships between regions are shown America (Suriname, Colombia and the extreme north- in Table 3. Considering only the ‘caiaué’ accessions, northwest of Brazil) (Meunier, 1975; Ghesquiere et the smallest average genetic distance was between al., 1987). This region is believed to be the center of accessions of the Madeira Region and the Solimões origin of E. oleifera, due to its higher morphological Region (0.071), while the highest was between ac- variation (Ooi et al., 1981). The Amazon River Basin cessions of the Negro Region and Solimões Region is considered a center of secondary diversification of (0.126). Higher distances were observed between the E. oleifera (Meunier, 1975; Ghesquière et al., 1987). oil palm accessions and the six ‘caiaué’ regions, with UPGMA analysis showed that, in general, genetic values ranging from 0.445 to 0.470. similarities are not related to geographical distances, The correlation between genetic similarities and but consistent with geographical dispersal along the geographic distances were estimated by the Mantel Amazon River network. Thus, accessions collected test (r = –0.190). The probability of obtaining stronger near the same river (or road) were usually more related correlation than that by chance alone is p = 0.212 to each other than to accessions from other regions, (423 out of 2000 permutations). distributed into three major clusters: Group I (rep- The results of the AMOVA partitioning of genetic resented mostly by accessions collected near Manaus variation of E. oleifera in the Amazon River Basin are city and along the Negro River), Group II (represen- shown in Table 4. There were significant differences ted by the Caracaraí Region accessions) and Group between regions (p< 0.00001) as well as between pop- III (formed mainly by a cluster of accessions collec- ulations within regions (p = 0.001), but most of the ted along the Madeira River). The small value of r total genetic variation was found within populations (–0.190) obtained by the Mantel test corroborated the (81.19%). Only 4.78% of the variation was found results evidenced by the UPGMA analysis (Figure 3), between populations within regions. suggesting that similarity groups among ‘caiaué’ ac- AMOVA was also used to analyze the partition cessions were not related to geographical distances, of the genetic variation between accessions collected but to their distribution in the river network. in ‘Indian black lands’ and accessions collected in Considering the moderate levels of variability all other types of soils. The results showed signific- between the ‘caiaué’ accessions and assuming that ant differences between the two groups (p= 0.00109), populations were recently formed from a small num- but only 3.92% of the total genetic variation was ber of seeds from basically the same origins, dif- found between them (Table 5). As expected, most of ferentiation was probably caused by founder effect. the variation was found within populations (81.70%). Accessions from Caracaraí (Group II), for example, Manhattan distance based on fragment frequencies showed a clear grouping and, on average, the smal- between these two groups was estimated as 0.064. lest distances between accessions within group and the highest distances to accessions collected in all the other regions (except for comparisons between Negro Discussion and Solimões accessions, that showed an average distance of 0.126). Plants from Caracaraí are morpho- Previous studies using isozyme markers have shown logically different from accessions of the other regions the lower level of genetic variation of Brazilian ‘cai- studied (Barcelos, 1986; Ghesquière et al., 1987). aué’ accessions as compared to oil palm (Ghesquière, These populations are characterized by a reduced 1985; Ghesquière et al., 1987; Santos, 1991; Moret- height and are morphologically similar to accessions zsohn, 1995). The present study corroborated these from Suriname. Given their geographic proximity, it is results. The average number of polymorphic RAPD possible that Caracaraí and Suriname populations have markers per selected primer (3.0) found for ‘caiaué’ a common origin (Guesquière et al., 1987). Thus, the is smaller than that observed for African oil palm ac- 43

Table 3. Manhatan’s distance matrix between six geographic origins of E. oleifera and a group of E. guineensis elite palms

Amazon Manaus Madeira Caracara´ı Solimões Negro Oil palm

Amazon 0.000 Manaus 0.087 0.000 Madeira 0.089 0.113 0.000 Caracara´ı 0.124 0.117 0.125 0.000 Solimões 0.098 0.114 0.071 0.116 0.000 Negro 0.100 0.088 0.110 0.108 0.126 0.000 Oil palm 0.460 0.458 0.470 0.454 0.445 0.464 0.000

Table 4. AMOVA of 175 E. oleifera individuals collected in 47 ‘caiaue’´ populations distributed in six geographic regions of Brazilian Amazon Forest

Source of variation Degrees of Sum of Variance % of P-value freedom squares components variation

Between regions 5 126.19 0.72 14.03 0.00001 Within regions Between populations 41 205.83 0.24 4.78 0.0001 Within populations 128 531.60 4.15 81.19 0.00001

differentiation of the accessions from this region and rence of other mechanisms of ‘caiaué’ dispersal in the others was not surprising. the Amazon Forest besides the possible influence of In the Brazilian Amazon Forest, ‘caiaué’ popula- man. Knowledge of the cultural and commercial habits tions usually occur near rivers, on fertile and well- of the Amazonian indian tribes could lead to a bet- drained lands, known locally as ‘Indian black lands’ ter understanding of the origin of ‘caiaué’ populations (Andrade, 1983; Barcelos, 1986). These ‘caiaué’ pop- in the river network and their distribution of genetic ulations are supposed to have been recently formed variability, as well as other possible mechanisms of by influence of indian tribes, according to the fluvial dispersal. network used by them, probably with seeds origin- It was observed that some accessions showed ally collected from natural populations of the Western greater similarity to accessions from other regions than Amazon (Meunier, 1975; Ghesquière et al., 1987). to other accessions within their own collection sites The movement of ‘caiaué’ germplasm between inhab- (Figure 3). It is possible that some ‘caiaué’ popula- ited areas would follow the indian routes and settle- tions could have been formed by a mixture of seeds ments. Therefore, populations where ‘caiaué’ samples from different localities. This was particularly evid- were collected should contain only a fraction of the enced for accessions from Autazes and Maués, that genetic diversity of E. oleifera natural populations. In- are located near the confluence of the rivers and were dians are supposed to use ‘caiaué’ to make fire with scattered on groups I and III in the UPGMA analysis. dried fruits, to make ‘wine’, to use it as natural dye, as This could be explained by the recent formation of mosquito repellent, and to feed domestic animals (E. these populations and germplasm movement in the Barcelos, personal communication). A genetic vari- river network. ability analysis based on RAPD marker frequencies The establishment of similarity groups, jointly and AMOVA, comparing accessions collected in ‘In- with morphologic and agronomic data, can be used in dian black lands’ and accessions from other types of the selection and crossing of superior plants, optim- soils, indicated similar levels of variability between izing the expression of ‘interpopulation heterosis’ and accessions of these two groups (Manhattan distance = allowing a more efficient use of the existing genetic 0.064 and Table 5). These data suggest the occur- variability. Hybrid performance analysis could be im- 44

Table 5. AMOVA for 175 E. oleifera individuals collected in 47 ‘caiaue’´ populations distrib- uted in two groups (‘Indian black lands’ and ‘other types of soil’) of the Brazilian Amazon Forest

Source of variation Degrees of Sum of Variance % of P-value freedom squares components variation

Between groups 1 24.61 0.20 3.92 0.00109 Within groups Between populations 45 307.41 0.73 14.38 0.00001 Within populations 128 531.61 4.15 81.70 0.00109

plemented between ‘caiaué’ accessions of groups I, Acknowledgements II and III, and between accessions belonging to each group and E. guineensis elite clones. Group II is rep- We acknowledge the logistical support of Embrapa resented by the Caracaraí region accessions. These Amazônia Ocidental (CPAA) for the collection of populations are characterized by a reduced height samples in the Rio Urubu Station, specially to Cley and should be considered immediate candidates for Nunes for his help in the field work. interspecific crosses with E. guineensis accessions. AMOVA showed that most of the genetic variation References is found within populations (81.19%), as expected for an allogamous and long-lived perennial species Amblard, P., J.-M. Noiret, B. Kouamé, F. Potier & B. Adon, (Hamrick & Godt, 1989). Only 4.78% of the ge- 1995. Performances comparées des hybrides interspécifiques et netic variation was found between populations within du matériel commercial E. guineensis. Oléagineux Corps gras Lipides 2: 335–340. regions. The proportion of genetic variation found Andrade, E.B., 1983. Relatório de expedição para coleta de germo- between regions (14.03%) was similar to the values plasma de caiaué (Elaeis oleifera(H.B.K.) Cortés), na Amazônia estimated by isozymes for natural populations of oil brasileira. Manaus, EMBRAPA/CNPSD (atual CPAA). palm in Africa (20.0%) (Ghesquière, 1985) and for Barcelos, E., 1986. Características genético-ecológicas de pop- ulações naturais de caiaué (Elaeis oleifera (H.B.K.) Cortés) ‘caiaué’ Brazilian populations (21.0%) (Ghesquière na Amazônia brasileira. Instituto Nacional de Pesquisas da et al., 1987). Differences were probably due to the Amazônia, Fundação Universidade do Amazonas. Dissertação different techniques used in these studies and to the de mestrado. higher number of plants and markers used on the Baudouin, L., 1992. Utilisation des marqueurs moléculaires pour l’amélioration du palmier à huile. I – Marqueurs protéiques. present study. The knowledge of genetic diversity Oléagineux 47: 681–691. distribution is important to determine strategies for fu- Baudouin, L., T.V. Cao. & A. Gallais, 1995. Analysis of the ge- ture collection expeditions and for the conservation netic effects for several traits in oil palm (Elaeis guineensis of ‘caiaué’ germplasm. The results obtained so far Jacq.) populations. I. Population means. Theor Appl Genet 90: 561–570. indicated that new collection expeditions of ‘caiaué’ Bergamin Filho, A., L. Amorim, F.F. Laranjeira, R.D. Berger germplasm should consider to sample as many indi- & B. Hau, 1998. Análise temporal do amarelecimento fatal viduals as possible within a few populations. Such do dendezeiro como ferramenta para elucidar sua etiologia. Fitopatologia Brasileira 23: 391–396. expeditions should be conducted on regions such as Excoffier, L., P. Smouse & J. Quattro, 1992. Analysis of molecular Negro and Madeira, where higher levels of genetic variance inferred from metric distances among DNA haplo- variation were observed. Simultaneous collection and types: Application to human mitochondrial DNA restriction data. analysis of plants from natural populations of Cent- Genetics 131: 479–491. Ferreira, M.E. & D. Grattapaglia, 1998. Introdução ao uso de ral America and North of South America would allow marcadores moleculares em análise genética. 3a ed. Brasília: a better understanding of the origin of the Brazilian EMBRAPA-CENARGEN. ‘caiaué’ populations, an enrichment of the germplasm Gascon, J.P., J.C. Jacquemard, M. Houssou, D. Boutin, H. Chaillard collections and, certainly, contribute to an efficient & F. Kangafondjo, 1981. La production de semences sélec- tionées de palmier à huile Elaeis guineensis. Oléagineux 36: use of the genetic variability of ‘caiaué’ in breeding 481. programs. Ghesquière, M., 1985. Polymorphisme enzymatique chez le palmier à huile (Elaeis guineensis, Jacq.). II – Variabilité et structure génétique de sept origines de palmiers. Oléagineux 40: 529–540. 45

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