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0 Department of Biology, Campus Box 1137, Washington University, St. Louis, Missouri 63130-4899 USA
Received for publication October 22, 1999. Accepted for publication May 25, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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ST = 0.54). This differentiation has probably arisen primarily through random genetic drift (rather than mutation) following recent population divergence.
Key Words: cassava • Manihot esculenta • Manihot pruinosa • microsatellites, origin of domestication • population structure • wild relatives
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), making it the primary source of carbohydrates in sub-Saharan Africa (Cock, 1985
), and the sixth most important crop worldwide (Mann, 1997
). Cassava is grown primarily by subsistence farmers and, despite its global economic importance, has traditionally received less attention by researchers than have temperate crops (Cock, 1985
). As a consequence, many fundamental questions about this "orphan crop" have only been addressed in recent years.
One basic question concerns cassava's origin of domestication. The genus Manihot comprises 98 species spread throughout the Neotropics (Rogers and Appan, 1973
), and in the past, various species from Mexico, Central America, and South America have all been proposed to be cassava's closest wild relatives (reviews by Renvoize, 1972
; Rogers and Appan, 1973
; and Sauer, 1993
; see also Olsen and Schaal, 1999
). Traditionally, no individual species was thought to be morphologically similar enough to cassava to be considered the crop's sole progenitor. Many domestication hypotheses therefore speculated that cassava is a hybrid ("compilospecies") derived from interbreeding Manihot species complexes in Mexico (Rogers, 1965
; Rogers and Appan, 1973
) and/or South America (Rogers, 1963
; Ugent, Pozorski, and Pozorski, 1986
; Sauer, 1993
).
These hybrid origin hypotheses were called into question by Allem (1987, 1994
), who identified wild Manihot populations in Brazil that are morphologically close enough to cassava to be considered conspecific. Wild M. esculenta populations are referred to here as M. esculenta subsp. flabellifolia (Pohl) Ciferri (see Allem, 1994
; Roa et al., 1997
). Differences between cassava and M. esculenta subsp. flabellifolia are restricted almost entirely to traits that would be selected upon during domestication (described below). Several recent studies have examined the relationship between cassava and these wild M. esculenta populations (Fregene et al., 1994
; Roa et al., 1997
; Olsen and Schaal, 1999
), and speculation has persisted that the crop's origins may extend beyond this subspecies to other Manihot species (Fregene et al., 1994
; Roa et al., 1997
).
The most recent assessment of cassava's origin (Olsen and Schaal, 1999
) involved a phylogeographic study of M. esculenta and a potentially hybridizing sympatric species, M. pruinosa Pohl, based on DNA sequence variation in a low-copy nuclear gene (Glyceraldehyde 3-phosphate dehydrogenase). The findings of that study suggested the following: genetic variation in M. esculenta subsp. flabellifolia alone can account for the crop's genetic diversity, with no need to invoke other progenitor species; cassava is most closely related to populations of M. esculenta subsp. flabellifolia occurring along the southern border of the Amazon basin; and the potentially hybridizing species M. pruinosa has apparently not contributed to the germplasm of the crop.
Like most phylogeographic analyses, the study by Olsen and Schaal (1999)
was based on a single locus, providing a single estimate of phylogenetic relationships in the study system. An alternative, complementary approach for addressing the question of cassava's origin would be to examine allele frequency variation at several unlinked loci. Although such data are not amenable to cladistic analyses (as are DNA sequence data), they do provide multiple, independent assessments of genetic relationships. In the present study, we use this multilocus approach to reassess the question of cassava's origin of domestication and the evolutionary relationships among cassava's closest wild relatives. Using five variable microsatellite loci, we analyze the same individuals sampled in the earlier phylogeographic study to test the findings of that study and to further elucidate the genetic structure of cassava's wild relatives. First, do microsatellite allele frequency data confirm that cassava is derived solely from M. esculenta subsp. flabellifolia—and specifically from those populations occurring along the southern border of the Amazon basin? Second, what is the population structure of the crop's wild relatives? In particular, what are the patterns of population differentiation within M. esculenta subsp. flabellifolia and M. pruinosa and is there any evidence of interspecific hybridization between them? This study is one of the first to compare microsatellite data with an intraspecific gene genealogy in an analysis of population structure in a plant species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Manihot esculenta subsp. flabellifolia is found in mesic forest patches (often in ravines) in the transition zone between the cerrado (savanna scrub) vegetation of the Brazilian Shield plateau and the lowland rainforest of the Amazon basin; it grows as a clambering understory shrub or treelet. In Brazil, this taxon occurs along the eastern and southern borders of the Amazon basin, in the states of Tocantins, Goiás, Mato Grosso, Rondônia and Acre. Manihot esculenta subsp. flabellifolia differs from cultivated cassava in root and stem characteristics. In addition to increased root tuberization, the crop also tends to have thickened, knobby stems with short internodes, and swollen leaf scars and stipule bases. This increased resource allocation to the stem probably reflects artificial selection for vegetative propagation in the crop; stem cuttings are the primary means by which cassava is propagated.
Although cassava is interfertile with M. esculenta subsp. flabellifolia (Roa et al., 1997
) and is cultivated throughout the range of the wild subspecies, introgression from the crop is not thought to have significantly altered the genetic structure of the wild subspecies. Manihot esculenta subsp. flabellifolia is found in forest habitats where cassava is not grown, and cassava does not survive well in abandoned fields or as escapes from cultivation (Rogers, 1965
; Allem, 1994
; K. Olsen, personal observation). Moreover, because cassava is propagated almost exclusively by stem cuttings, unintentional spread of the crop by humans would be minimal.
The other species in the study system, M. pruinosa, is a small shrub found in dense cerrado vegetation in the Brazilian states of Tocantins, Goiás, and eastern Mato Grosso. Although this species occurs in a different habitat from that of M. esculenta subsp. flabellifolia, the patchy nature of the cerrado-forest ecotone allows populations of the two species to grow in close proximity. Based on its morphological similarity to M. esculenta, M. pruinosa has been grouped into cassava's "secondary gene pool" of potentially interfertile species (Allem, 1992
); it is the only such species to occur in sympatry with M. esculenta subsp. flabellifolia. Manihot pruinosa is included in the present study to test the hypothesis that this species has contributed to genetic diversity in cassava via hybridization with M. esculenta subsp. flabellifolia.
Sampling and data collection
Populations of wild taxa were sampled along two transects spanning most of the range of M. esculenta subsp. flabellifolia: along the southern border of the Amazon basin (through the states of Mato Grosso, Rondônia, and Acre), and along the eastern border (through the states of Tocantins and Goiás) (Fig. 1). This sampling area overlaps the range of M. pruinosa in the states of Tocantins, Goiás, and southeastern Mato Grosso. Collections were made during the months of November and December in 1996 and 1997. Undisturbed populations of these species usually comprise < 15 individuals, and young leaf tissue from up to ten plants per population was sampled and dried in silica gel. Voucher specimens from each population are housed at the Missouri Botanical Garden (MO), in St. Louis, and at the Centro Internacional de Pesquisa de Recursos Genéticos e Biotecnologia (CEN), in Brasília, Brazil. From these wild taxa, 157 individuals of M. esculenta subsp. flabellifolia, representing 27 populations, and 35 individuals of M. pruinosa, representing six populations, were included in analyses. Genetic diversity within cassava was assessed by sampling 20 landraces from the cassava "world core collection" maintained by the Centro Internacional de Agricultura Tropical, in Cali, Colombia.
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). Fourteen variable microsatellite loci have previously been identified in cassava (Chavarriaga-Aguirre et al., 1998
), and these loci were surveyed for polymorphism in a sample of 12–36 individuals of M. esculenta subsp. flabellifolia and M. pruinosa. Five loci with strong, unambiguous banding patterns were selected for use in the study (GAGG5, GA134, GA16, GA12, and GA140); these loci are all composed of dinucleotide (GA) repeats.
PCR amplifications were carried out in 10-µL volumes, with each reaction containing the following: 10 mmol/L Tris-HCl (pH 9.0), 50 mmol/L KCl, 2.5 mmol/L MgCl2, 0.1% Triton X-100, 0.5 units taq polymerase (Promega), 200 µmol/L each dNTP, 0.2 µmol/L each primer (forward and reverse), 0.02 µmol/L [
32P]-ATP end-labeled forward primer, and 
10 ng genomic DNA. Reaction conditions were the same for all five loci: one cycle of 94°C (2 min); then 30 cycles of 94°C (1 min), 55°C (1 min), 72°C (2 min); and finally one cycle of 72°C (5 min). Following PCR, 2.0 µL of formamide dye were added to each reaction, and reaction tubes were heated to 85°C for 2 min, then snap-chilled in ice water. PCR products were electrophoresed on 6% polyacrylamide gels and autoradiographed. A sequencing ladder of known length was included on gels to facilitate size determination of alleles.
Data analysis
Genetic diversity
Genetic polymorphism for each population was assessed by calculating the number of alleles per locus (A), the observed heterozygosity (Ho), and the heterozygosity expected under Hardy-Weinberg equilibrium (HE), using GENEPOP version 3.1c (Raymond and Rousset, 1995
). For each population-locus combination, departure from Hardy-Weinberg expectations was assessed by exact tests (Guo and Thompson, 1992
), with unbiased P values estimated through a Markov chain method (Guo and Thompson, 1992
); a global test across loci and populations was constructed using Fisher's method (Raymond and Rousset, 1995
). In order to specifically test the hypothesis of heterozygote deficiency, the multiscore (U) test of Rousset and Raymond (1995)
was employed. Tests for genotypic linkage disequilibrium among pairs of loci in each population were performed using Fisher's exact tests (Raymond and Rousset, 1995
), with unbiased P values again derived by a Markov chain method. For all tests, the global significance of multiple P values was assessed using a sequential Bonferroni adjustment (Rice, 1989
), with an initial 
level of 0.05.
Population structure
Tests for the presence of population differentiation were made using an unbiased estimated P value for a log-likelihood (G)-based exact test (Goudet et al., 1996
). As a conservative approach, the test for genotypic differentiation was invoked. Genetic differentiation among populations was then quantified for each species individually and for all wild populations together.
Methods for quantifying genetic differentiation from microsatellites are currently debated (Goldstein et al., 1995a, b
; Takezaki and Nei, 1996
; Goldstein and Pollock, 1997
). Some measures have been developed specifically for these markers (e.g., Slatkin, 1995
; Goldstein et al., 1995a, b
), which take into account variation in allele sizes under a stepwise mutation model (SMM) (Ohta and Kimura, 1973
). Under the SMM, alleles of similar size are assumed to be more closely related to each other than those of very different size. SMM-based measures of population differentiation are expected to be most accurate for populations that diverged long enough ago that current genetic differentiation reflects mutations accumulated since divergence (reviewed by Goldstein and Pollock, 1997
). In contrast, traditional measures, which do not assume an underlying mutational model, are thought to be more appropriate for recently diverged populations (Goldstein et al., 1995a, b
; Takezaki and Nei, 1996
; Goldstein and Pollock, 1997
), since genetic differentiation among such populations will reflect the sorting of ancestral variation more than mutational divergence. For purposes of comparison, both types of measures were employed in this study.
Genetic differentiation was quantified using F statistics (Weir and Cockerham, 1984
) and statistics (Michalakis and Excoffier, 1996
) using GENEPOP. The statistics are analogous to F statistics but take into account differences in allele size under the SMM. The distribution of genetic variation in the study system was next quantified by an analysis of molecular variance (AMOVA; Excoffier, Smouse, and Quattro, 1992
), using the program ARLEQUIN (Schneider et al., 1997
). Pairwise genetic distances among populations were again calculated two ways, the first based solely on allele frequencies (Weir and Cockerham, 1984
) and the second incorporating information on variances in allele sizes (Michalakis and Excoffier, 1996
). Genetic variance was assessed at three hierarchical levels in the AMOVAs: within populations, among populations of the same species, and between the two species. Statistical significance of the variance measures was tested by nonparametric permutations (Schneider et al., 1997
).
In order to test for isolation by distance among the wild populations, Mantel's (1967)
permutation procedure in GENEPOP was used. A matrix of pairwise great circle distances among populations was compared to a matrix of pairwise genetic distances (FST and ST), with the Spearman rank correlation coefficient used as the test statistic. Isolation by distance was assessed for M. esculenta subsp. flabellifolia and M. pruinosa populations independently, and then for the study system as a whole. In addition, a multilocus estimate of the effective number of migrants per generation (Nm) was calculated by Slatkin's (1985)
private alleles method, with corrections for sample size as given in Barton and Slatkin (1986)
.
Relationships among populations and taxa
In order to examine genetic relationships among the wild populations, and cassava's relationship to these populations, distance trees were inferred from the allele frequency data. Because of the very close evolutionary relationship between cassava and its wild relatives, distance measures that do not assume an underlying mutational model are probably most appropriate for these analyses (see also Tarr, Conant, and Fleischer, 1998
; Wenburg, Bentzen, and Foote, 1998
; Tessier and Bernatchez, 1999
). The chord distance, DC (Cavalli-Sforza and Edwards, 1967
), was chosen for the present study; DC assumes that differentiation among populations is due solely to random genetic drift. Using the program GENDIST in the PHYLIP computer package (version 3.5c; Felsenstein, 1995), a pairwise chord distance (DC) matrix was constructed, from which a neighbor-joining phenogram was generated with the program NEIGHBOR. For the sake of comparison, a neighbor-joining tree was also constructed using Slatkin's (1995)
RST distance, a SMM-based measure that estimates the fraction of the total variance of allele size occurring among populations. The RST distance matrix was generated in the MICROSAT program (Minch et al., 1996
) and then imported into PHYLIP.
In addition to the two neighbor-joining trees, a maximum likelihood distance tree was constructed in PHYLIP using the program CONTML. This algorithm operates under a Brownian motion model that, like DC, assumes evolution purely by random genetic drift (Felsenstein, 1995). The robustness of the DC and maximum likelihood distance trees was assessed by creating 100 bootstrap replicates of the data set with the SEQBOOT algorithm in PHYLIP, and then generating a majority-rule consensus tree in the CONSENSE program. All distance trees were viewed in TREEVIEW (Page, 1996
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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2140= 232.7, P < 0.0001). Specifically testing for heterozygote deficiency (U test) indicated statistically significant deficits at two loci across all populations, both of which were significant following sequential Bonferroni corrections (GA134 and GA140; Table 1). Thus, there appears to be some evidence for nonrandom mating within populations. Exact tests for genotypic linkage disequilibrium yielded significant deviations for three out of 222 comparisons (P < 0.05), a number comparable to that expected by Type I error alone. None of the tests were significant following sequential Bonferroni adjustments, nor were any comparisons significant for locus pairs across all populations (P > 0.3 for all comparisons). Thus, the five loci may be assumed to be unlinked.
Population structure
Tests for genotypic heterogeneity among the wild populations were highly significant for each of the five loci individually and for all loci combined (P < 0.0005 following sequential Bonferroni adjustment). Quantitative estimates of this genetic differentiation differed between the two wild taxa, with M. esculenta subsp. flabellifolia showing greater interpopulation differentiation than M. pruinosa (multilocus FST = 0.42 and 0.28 for M. esculenta subsp. flabellifolia and M. pruinosa, respectively; Table 2). These measures did not change dramatically when allele sizes were taken into account under the SMM (multilocus ST = 0.50 and 0.21 for M. esculenta subsp. flabellifolia and M. pruinosa, respectively; Table 2). Overall, these measures indicate a moderately high level of interpopulation differentiation. This pattern is also suggested by the corrected private alleles estimate of effective interpopulation gene flow, which was less than 1.0 (Nm = 0.32, 0.45 and 0.37 for M. esculenta subsp. flabellifolia, M. pruinosa and all populations combined, respectively). At the intrapopulation level, FIS and IS values were generally positive (FIS = 0.13, IS = 0.23 across all populations of both species; Table 2), a pattern consistent with the heterozygosity deficits observed in tests of Hardy-Weinberg equilibrium.
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), indicates that only 10.5% of the total genetic variation is apportioned between the two species (53 and 36.5% of the variation occurs within populations and among conspecific populations, respectively; P < 0.0001 for all tests of significance). However, when variance in allele size is taken into consideration under the SMM (Michalakis and Excoffier, 1996
), the proportion of genetic variation distributed between the species increases to 38.7% (with 33.0 and 28.3% of the variation occurring within populations and among conspecific populations, respectively; P < 0.0001 for all tests). Thus, while the proportion of species-specific alleles is low, there appears to be some degree of divergence in the overall sizes of alleles predominating in each species. This interspecific divergence in allele size is most evident at locus GA134, where M. pruinosa alleles tend to be smaller than those of M. esculenta (Fig. 2b), and at locus GA12, where M. pruinosa alleles tend to be larger (Fig. 2d).
Mantel tests for isolation by distance failed to reject the null hypothesis of no correlation between pairwise genetic differentiation (FST and ST) and geographical distances among populations (P > 0.20 for M. esculenta subsp. flabellifolia populations alone, M. pruinosa populations alone, and all populations combined). This result suggests that on the spatial scale examined, present population differentiation reflects gene flow processes other than limited, recurrent expansion from a restricted ancestral range. Alternatively, failure to detect isolation by distance may be an artifact of the small number of individuals examined per population, which limits the power of the test statistic.
Relationships among populations and taxa
Neighbor-joining and maximum likelihood distance methods were used to examine relationships among wild Manihot populations and cassava accessions. Bootstrap values were generally low on both types of distance trees, with most nodes associated with values of <50% (Fig. 3a, b). Nonetheless, certain well-supported features are shared between the neighbor-joining and maximum likelihood trees and can provide insight into genetic relationships in the study system. Most notable is cassava's placement among southern Amazonian populations of M. esculenta subsp. flabellifolia. Both the DC neighbor-joining tree and the maximum likelihood tree grouped cassava most closely with the population from Sena Madureira, Acre with relatively high bootstrap support (62 and 70%, repectively; Fig. 3a, b). Moreover, on both trees the crop falls within a cluster of populations from Acre, Rondônia and western Mato Grosso (plus one aberrant population, Miranorte, in central Tocantins) (Figs. 1, 3a, b). This relationship was also observed on the RST neighbor-joining tree (not shown), which placed cassava in a cluster with four populations from Rondônia and one from Acre.
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Neither the neighbor-joining trees nor the maximum likelihood tree grouped the M. pruinosa populations into a discrete ("monophyletic") cluster. Given the weak statistical support for these trees, this apparent lack of monophyly per se cannot be taken as evidence for reticulate evolution between M. pruinosa and M. esculenta subsp. flabellifolia. On the other hand, the high proportion of alleles shared between the two species (Fig. 2) does suggest either introgression or shared ancestral polymorphisms between these species. It should be noted that the distance trees in Fig. 3 are unrooted and that relationships among the populations are all relative. Thus, the M. pruinosa populations should not be construed to be derived from M. esculenta subsp. flabellifolia, as would be the implication on a rooted tree.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The crop's genetic variation is a subset of that of its conspecific wild relative (Fig. 2), suggesting that M. esculenta subsp. flabellifolia is cassava's sole wild progenitor. In addition, the microsatellite data confirm that cassava is most closely related to populations of M. esculenta subsp. flabellifolia occurring along the southern border of the Amazon basin (Figs. 1, 3). Thus, this southern ecotone is independently identified as the crop's likely geographical origin of domestication.
These conclusions differ from traditional domestication hypotheses (e.g., Rogers and Appan, 1973
; Sauer, 1993
; Jennings, 1995
) in two fundamental respects: (1) a single wild progenitor is specified, rather than a pool of unidentified hybridizing species, and (2) a single geographical area of domestication is proposed, rather than many independent sites throughout the Neotropics. Traditional domestication scenarios are strongly influenced by Rogers and Appan (1973)
, who, in their monograph of Manihot, emphasized the potential role of interspecific hybridization in the crop's origin (see also Rogers, 1963, 1965
). Many species of Manihot show very high levels of intraspecific morphological variation (Rogers and Appan, 1973
; K. Olsen, personal observation). Rogers and Appan (1973)
suggested that this variability is due to widespread interspecific hybridization in nature. Cultivars of cassava are particularly variable morphologically, and the monographers interpreted this variation as evidence that the crop is likely derived from a number of different species complexes in Mesoamerica and South America. They further argued that the wild populations resembling cassava most closely (i.e., M. esculenta subsp. flabellifolia) are actually feral crop derivatives. Of the "truly" wild Manihot species, they contended that those occurring in Mesoamerica [particularly M. aesculifolia (H.B.K.) Pohl.] are cassava's closest relatives, with many locally occurring species also contributing to the crop wherever it is grown in the Neotropics.
This "compilospecies" hypothesis is clearly not supported by currently available data. Application of molecular genetic markers in Manihot has shown that M. esculenta (including both cassava and the wild subspecies) is more closely related to South American species than to those from Mesoamerica (Fregene et al., 1994
; Roa, 1997
; Roa et al., 1997
; B. Schaal, unpublished data). In addition, observations that cassava's genetic diversity is a subset of that found in M. esculenta subsp. flabellifolia (see also Roa et al., 1997
; Olsen and Schaal, 1999
) indicate that the former is derived from the latter, not the reverse. Finally, in the absence of supporting evidence, we are skeptical that interspecific hybridization has played a major role in the origin of crop. Reports of natural hybridization in Manihot remain largely anecdotal and are based almost entirely on observations of morphological intermediacy in the field. This criterion is a poor indicator of hybridization (Rieseberg and Ellstrand, 1993
; Rieseberg, 1995
) and is likely to be particularly unreliable in Manihot, given the tremendous phenotypic plasticity that occurs in cassava (Rogers and Fleming, 1973
; Olson, 1997
) and the morphological variability observed in its congeners (Rogers and Appan, 1973
). In artificial crossing studies most species do not hybridize readily with cassava (e.g., Allem, 1992
; Nassar et al., 1995
; Nassar, Carvalho, and Vieira, 1996
), and seeds reported from "successful" crosses are rarely tested for viability (Allem, 1992
), let alone proof of hybrid parentage. Given current data on genetic variation in cassava and its relatives, it is not necessary to invoke multiple hybridization events to explain the crop's origin. In this respect, the domestication of cassava parallels that of maize, which was believed to be of hybrid origin until molecular and other data proved otherwise (Iltis, 1983
; Doebley, Goodman, and Stuber, 1984
; Doebley, Renfroe, and Blanton, 1987
).
The possibility remains that subsequent to domestication from M. esculenta subsp. flabellifolia, cassava's genetic diversity has been influenced by occasional introgression from other Manihot species. There is some evidence to suggest spontaneous hybridization between cultivated cassava and Manihot glaziovii Muell.-Arg., a northeastern Brazilian species formerly cultivated as a source of rubber; hybridization between these species has been reported in Africa, where they are both introduced (Wanyera et al., 1992
). Additional sampling of crop accessions and wild Manihot populations will be useful for assessing the potential influence of post-domestication interspecific introgression on the crop's current genetic diversity.
Site of domestication
The concordance of the multilocus microsatellite data with our earlier assessment of cassava's origin (Olsen and Schaal, 1999
) strongly suggests that the crop arose from southern Amazonian populations of M. esculenta subsp. flabellifolia. If correct, this conclusion places cassava within an area of crop domestication that has also been proposed for the peanut (Arachis hypogaea), two species of chili pepper (Capsicum baccatum and C. pubescens) and jack bean (Canavalia plagiosperma) (reviewed by Piperno and Pearsall, 1998
). Interestingly, archaeological evidence from central Rondônia suggests the very early development of agricultural settlements in this region (e.g., preceramic "black soil" occupation beginning about 4800 BP; Miller, 1992; B. Meggers, personal communication, Smithsonian Institution). Moreover, recent archaeological work in northern Bolivia has revealed evidence of an advanced, prehispanic aquacultural complex in this same area (Erickson, 2000). Taken together, these findings suggest that the southern Amazon basin may have been an important center for the development of lowland Neotropical agriculture. We are hopeful that the present study will encourage further examination of early agriculture and crop domestication within this region.
Our sampling transects covered most of the eastern and southern borders of the Amazon basin. However, it is possible that there are additional wild M. esculenta populations occurring along the northern border of the Amazon basin, in the transition zone into the Guiana highlands of northern South America. Manihot populations from this area have been alternatively classified either as M. esculenta subsp. flabellifolia (Allem, 1994
), or as a distinct species, most notably M. tristis (Rogers and Appan, 1973
; Allem, 1987
; Roa et al., 1997
). In an amplified fragment length polymorphism (AFLP) analysis of relationships among cassava and wild Manihot accessions, Roa et al. (1997)
found that individuals classified as M. tristis were more distantly related to cassava than were wild M. esculenta individuals. This finding suggests that these more northerly populations are probably not cassava's closest wild relatives. More extensive population sampling from the northern Amazonian ecotone would be useful for clarifying cassava's relationship to these populations.
Crop genetic diversity
As a species is brought into domestication, founder events, population bottlenecks, and artificial selection are all expected to reduce the genetic diversity of the crop in relation to its wild progenitor (Ladizinsky, 1985
; Doebley, 1989
; Eyre-Walker et al., 1998). In the present study, alleles observed in cassava constitute 22.1% of those found in M. esculenta subsp. flabellifolia. This proportion is consistent with previous comparisons of cassava and its wild relatives. In our phylogeographic analysis (Olsen and Schaal, 1999
), cassava haplotypes represented 25% of those found in M. esculenta as a whole. Similarly, the AFLP survey of Roa et al. (1997)
showed 38 cassava cultivars to contain 30% of the genetic variation found in 14 wild M. esculenta accessions. When our microsatellite variation is expressed in terms of total heterozygosity (Ht; Nei, 1973
), the crop's genetic diversity is 23% less than that of M. esculenta subsp. flabellifolia (Ht = 0.68 and 0.52 for progenitor and crop, respectively). This amount of decrease falls within the range typically observed in other crop-relative comparisons (Doebley, 1989
; Gepts, 1993
).
Population structure of wild taxa
Genetic diversity
Heterozygote deficits (Table 1) and positive FIS and IS values (Table 2) suggest some degree of inbreeding in the wild populations. This observation is not surprising, given the life history of these taxa. There is no known genetic self-incompatibility system in Manihot (although inflorescences are metandrous), and the mechanism of seed dispersal (explosively dehiscent pods) does not promote long-distance gene flow. Both of these traits would favor mating among relatives.
Another potential cause of heterozygosity deficits in these populations could be the occurrence of null (nonamplifying) alleles, which can cause heterozygotes to be misscored as homozygous. Two lines of evidence suggest that null alleles are not the cause of the heterozygote deficits observed here. First, heterozygote deficits can be observed at all of the loci examined (although statistical significance across populations is restricted to two loci; Table 1); the occurrence of null alleles at all five loci would be unlikely. Moreover, in an earlier survey of Manihot genetic diversity that included loci GAGG5 and GA12, no evidence for null alleles was found in M. esculenta or in four other Manihot species (Roa, 1997
; see also Chavarriaga-Aguirre et al., 1998
).
Population differentiation
The distribution of alleles and genotypes among the wild populations (Tables 1 and 2) indicates a moderate-to-high level of population differentiation. Estimates of differentiation among populations (FST and ST) are greater for M. esculenta subsp. flabellifolia than for M. pruinosa. This difference probably reflects the more restricted sampling scheme used for the latter species. Manihot pruinosa was collected only in its region of sympatry with M. esculenta subsp. flabellifolia, and only six populations were examined (vs. 27 of M. esculenta subsp. flabellifolia). Another factor that could potentially affect measures of differentiation in the two species is ascertainment bias (e.g., Ellegren, Primmer, and Sheldon, 1995
; Hutter, Schug, and Aquadro, 1998
). When microsatellites are isolated in a particular species, the selected loci tend to have higher levels of polymorphism in that focal species than in related species. The loci used in the present study were originally isolated in cassava (Chavarriaga-Aguirre et al., 1998
), which could result in a bias towards polymorphism in M. esculenta subsp. flabellifolia over M. pruinosa. Such a bias could lead to underestimates of genetic differentiation among M. pruinosa populations. However, observed levels of polymorphism do not suggest that this has occurred. The allelic diversity in M. pruinosa (35 alleles in 35 individuals) is proportionally more than twice that of M. esculenta subsp. flabellifolia (68 alleles in 157 individuals), the opposite of the pattern expected with ascertainment bias.
F and statistics yield similar measures of population structure in this study (Table 2). Thus, taking into account allele size variation under the SMM does not greatly alter estimates of genetic differentiation. These two measures are expected to be similar when population differentiation reflects processes other than mutation; this could occur either under conditions of high interpopulation gene flow, or when populations are recently diverged and differentiation is primarily due to random genetic drift (Slatkin, 1995
; Rousset, 1996
). The latter situation most likely applies here. Morphological and molecular data suggest that M. esculenta and M. pruinosa are very recently diverged species (Allem, 1992
; Olsen and Schaal, 1999
; B. Schaal, unpublished data) within a recently radiated genus (Rogers and Appan, 1973
; reviewed by Olsen and Schaal, 1999
). At the same time, private alleles estimates of gene flow and estimates of interpopulation differentiation (FST and ST) indicate that gene flow is low, not high. Thus, contemporary population structure has probably been shaped largely by random genetic drift. This conclusion is consistent with the large number of alleles shared between M. esculenta and M. pruinosa (Fig. 2), and it further suggests that the measures chosen for distance tree construction (e.g., DC) are appropriate, since they assume evolution solely by random genetic drift.
While the incidence of shared alleles between M. esculenta and M. pruinosa is very high, analyses of molecular variance (AMOVAs) indicate some divergence in overall allele sizes between the two species. In other interspecific comparisons using microsatellites, such divergence has been attributed to ascertainment bias (e.g., Ellegren, Primmer, and Sheldon, 1995
), since allele length is positively correlated with polymorphism in microsatellites (Ellegren, Primmer, and Sheldon, 1995
; Goldstein and Pollock, 1997
). In the present study, ascertainment bias would result in alleles of M. pruinosa being consistently shorter than those of M. esculenta subsp. flabellifolia. This pattern is not observed (Fig. 2). Manihot pruinosa alleles are significantly shorter only at one locus (GA134; t test, P < 0.0001) and were significantly longer at two loci (GAGG5 and GA12; t tests, P < 0.0001); there was no significant divergence between species at the other two loci (P > 0.06). Thus, the divergence in allele lengths between these two species is most likely the result of random genetic drift combined with some mutational divergence following speciation.
Relationships among the wild M. esculenta populations
The DC and maximum likelihood trees generally group populations according to the geographical regions in which they occur (Fig. 3). The sole exception is the population from Miranorte, Tocantins, which clusters with cassava's closest wild relatives in the southern Amazon-border region (Figs. 1 and 3). One possible explanation for this grouping is that there has been introgression into this population from cultivated cassava. Cassava and M. esculenta subsp. flabellifolia are interfertile (Roa et al., 1997
), and cassava is occasionally planted in close proximity to M. esculenta subsp. flabellifolia populations (K. Olsen, personal observation). On the other hand, the Miranorte population was sampled in primary forest, and we found no evidence for introgression into this population in our phylogeographic analysis (Olsen and Schaal, 1999
). In that study, six haplotypes were observed in the Miranorte population, five of which were found only in the states of Tocantins and Goiás, and none of which were observed in cassava. Considering the weak statistical support for groupings on the microsatellite distance trees, the placement of the Miranorte population in the southern Amazonian cluster may not be of any real biological significance.
Interspecific relationships
The question of whether there is interspecific hybridization between M. esculenta subsp. flabellifolia and M. pruinosa remains unresolved. The high proportion of alleles shared between these species (Fig. 2) and the intermingling of species on the distance trees (Fig. 3) are consistent with the occurrence of interspecific introgression. On the other hand, these features would be expected for any two recently diverged species, whether there is current hybridization between them or not. Although M. pruinosa is considered to fall within cassava's "secondary gene pool" of potentially interfertile species (Allem, 1992
), this designation is based on morphological similarity rather than evidence of hybridization; the interfertility of the two species has yet to be established in artificial crossing studies. Furthermore, these species are adapted to different habitats, and while populations of the two species can occur in close proximity, no putative hybrids were observed during field collections for this study. Definitive documentation of hybridization between these species would require more thorough sampling of M. pruinosa populations (including those plants outside the range of M. esculenta subsp. flabellifolia), crossing experiments to establish interfertility, and possibly the use of more rapidly evolving genetic markers.
Regardless of whether or not interspecific hybridization occurs, we have little reason to suspect that M. pruinosa has contributed to cassava's germplasm. Cassava is not grouped with M. pruinosa populations on distance trees (Fig. 3); moreover, the populations most closely related to cassava all occur west of the range of M. pruinosa (Figs. 1 and 3), suggesting little contact between M. pruinosa and cassava's presumed progenitors. It is notable that these same patterns were observed in our phylogeographic study (Olsen and Schaal, 1999
): the crop did not share haplotypes with M. pruinosa, and populations containing cassava haplotypes were all west of M. pruinosa populations. Given current information on genetic variation in M. esculenta, M. pruinosa, and other Manihot species (Fregene et al., 1994
; Roa et al., 1997
, Olsen and Schaal, 1999
; B. Schaal, unpublished data), cassava appears to be derived solely from its conspecific wild relative, specifically from those populations occurring along the southern border of the Amazon. This information will be useful for crop improvement efforts (Tanksley and McCouch, 1997
; M. Fregene, personal commumication, Centro Internacional de Agricultura Tropical), for examining the genetics of domestication in this species (Gepts, 1993
; Eyre-Walker et al., 1998), and possibly for setting priorities in preserving wild Manihot populations, many of which are threatened by deforestation.
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FOOTNOTES |
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2 Author for reprint requests, current address: North Carolina State University, Department of Genetics, Campus Box 7614, Raleigh, NC 27695-7614 USA (email: kmolsen{at}unity.ncsu.edu
).
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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