# Genetic relatedness between cassava (Manihot esculenta Crantz) and M. flabellifolia and M. peruviana based on both RAPD and AFLP markers

C. Colombo, Gérard Second, A. Charrier

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C. Colombo, Gérard Second, A. Charrier. Genetic relatedness between cassava (Manihot esculenta Crantz) and M. flabellifolia and M. peruviana based on both RAPD and AFLP markers. Genetics and Molecular Biology, 2000, 23 (2), pp.417-423. 10.1590/S1415-47572000000200030. hal-02699271

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Genetic relatedness between cassava (*Manihot esculenta* Crantz) and *M. flabellifolia* and *M. peruviana* based on both RAPD and AFLP markers

Carlos Colombo<fnref n="1" />, Gérard Second<fnref n="2" /> and André Charrier<fnref n="3" />

Abstract

The taxonomy of the genus *Manihot* is still uncertain and the genetic origin of cassava (*M. esculenta* Crantz) continues to be controver-
sial. We studied the degree of genetic relatedness between cassava and two naturally occurring species (*M. flabellifolia* and *M. peruviana*)
which are probably involved in the evolution of cassava, using RAPD and AFLP molecular markers. Thirty-three clonal accessions of
cassava of known genetic diversity and 15 accessions of the wild species *M. flabellifolia* and *M. peruviana* were analyzed using 92
polymorphic RAPD bands and 73 polymorphic AFLP bands. The genetic markers were unable to differentiate the two wild species,
which confirms their botanical similarity. Half of the total number of amplified bands were monomorphic in all of the genotypes evaluated.
The mean genetic similarity (Jaccard) between cassava and the species *M. flabellifolia/M. peruviana* was 0.59. A grouping analysis
(neighbor-joining method) with RAPD markers of cultivated cassava, *M. flabellifolia/M. peruviana* and the other wild species located
the genotypes of cassava and *M. flabellifolia/M. peruviana* at one extremity and the three Mexican species (*M. aesculifolia*, *M.
michaelis* and *M. chlorostica*) at the other. An intermediate position between these groups was occupied by two wild species (*M.
glaziovii* and *M. reptans*) native to central and northeastern Brazil. These results are consistent with the hypothesis that the species *M.
flabellifolia* and *M. peruviana* gave rise to the cultivated species.

INTRODUCTION

Natural occurrence of the genus *Manihot* is limited
to the tropical regions of the American continent, between
latitudes 33°N (southern part of the USA) and 33°S (cen-
tral part of northern Argentina). According to Rogers and
Appan (1973), the genus *Manihot* contains 98 species, one
fifth of which are native to North America, while the re-
maining four fifths occur in South America. Harlan (1971)
and Nassar (1978) identified the central region of Brazil
as the main center of diversity for *Manihot* species, fol-
lowed by two other centers in southern Mexico and north-
eastern Brazil. *Manihot* species are classified as sporadi-
cally distributed colonizing perennials commonly found
in semi-arid regions or disturbed zones of humid regions
(Rogers and Appan, 1973). However, natural interspecific
hybridization makes it difficult to determine the true taxo-
nomic limits of the genus (Rogers and Appan, 1973).

The genetic origin of cassava is controversial. Com-
parative studies of reproductive characteristics, botanic ori-
gin and phylogenetic relatedness of cassava with other
*Manihot* species, as well as the history of the domestica-
tion of cassava are still in the early stages. While some re-
searchers believe cassava to be a cultigen that resulted from
hybridization between several natural species (Rogers and
Appan, 1973), others (Allem, 1994) consider cassava as a
true natural species with two subspecies, *M. esculenta*
*flabellifolia* and *M. esculenta peruviana*.

Only a relatively small number of wild species of
the *Manihot* have been systematically collected for study.
According to Hershey (1987), related species with spe-
cific characteristics of interest could be used to produce
improved forms of manioc. For such studies, Allem (1994)
proposed the use of wild species belonging to the primary
genic pool (GP I), as defined by Harlan and Wet (1971).
Determination of the reproductive barriers between re-
lated species of cassava is a highly complex and difficult
task. Several techniques using molecular markers have been
developed recently and are increasingly applied to the
study of plant genetics. Gepts (1993) suggested that mo-
lecular markers provide more useful information than mor-
phological markers for studies on the domestication and
evolution of plants. Random amplified polymorphic DNA
markers (RAPD) were used in studies conducted by
Cisneros and Quiros (1995) on triploid potatoes (*Solanum
chaucha*), by Demeke and Adams (1994) on mustard and
radish, by Sharma *et al.* (1996) on lentils, by Reamon-
Büttner *et al.* (1996) on beets and by Kaga *et al.* (1996)
on the genus *Vigna*. Amplified fragment length polymor-
phism markers (AFLP) have been used in studies of ge-
netic relatedness performed by Hill *et al.* (1996), Cervera
*et al.* (1996) and Sharma *et al.* (1996).

We used RAPD and AFLP molecular markers to in-
vestigate the genetic relatedness between cultivated cas-
sava and two naturally occurring species, *M. flabellifolia*
and *M. peruviana*.

<footnote n="1">¹Instituto Agronômico (IAC). Av. Barão de Itapura, 1481, Caixa Postal 28, 13001-970 Campinas,
São Paulo, Brasil. Send correspondence to C.C. E-mail: ccolombo@cec.iac.br</footnote>

<footnote n="2">²ORSTOM, 911, Av. Agropolis, 34032-Montpellier, France.</footnote>

<footnote n="3">³Ecole Nationale Supérieure Agronomique de Montpellier (ENSAM). 2, Place Viala, 34060-Montpellier, France.</footnote>
# MATERIAL AND METHODS

## Material

One plant of each thirty-three clonal accessions of *M. esculenta* (cultivated cassava), nine genotypes of *M. flabellifolia* and six genotypes of *M. peruviana*, as well as one genotype of each of the naturally occurring species *M. glaziovii*, *M. reptans*, *M. chlorostica*, *M. aesculifolia* and *M. michaelis* were studied (Table I). The cultivated cassavas were divided into two groups. The first group consisted of 13 local cultivars (landraces) collected from the small village of Santa Isabel (located in the central portion of the Rio Negro River in the Amazon region), while the second group contained 20 genotypes of different sources, selected to provide the largest possible genetic diversity (Colombo, 1997).

## DNA isolation and genetic analysis

DNA was isolated from leaves dried for 20 h at 48°C. The dry leaves (0.5 g) were ground in liquid nitrogen and transferred to a 20-ml plastic tube to which 10 ml of extraction buffer (0.1 M Tris HCl, pH 8.0, containing 1.25 M NaCl, 0.02 M EDTA, 4% MATAB (mixed alkyltrimethylammonium bromide) and 1% β-mercaptoethanol (added just before use)) was added. After a 90-min incubation at 65°C, with slow stirring, an equal volume of chloroform/isoamylalcohol (24:1) was added twice and the resulting supernatant transferred to a clean plastic tube. RNAs (100 µl of a 10 mg/ml solution) was added immediately after these extractions and the solution subsequently incubated at 37°C for 30 min. DNA pellets were obtained by addition of 0.8 v of isopropanol. After washing with 70% ethanol, the DNA pellet was vacuum dried and dissolved in 200 µl of TE buffer (10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA). The quality and concentration of the DNA fragments were evaluated by electrophoresis in 0.8% agarose gels.

The RAPD amplification reactions were done in 25 µl as described by Williams *et al.* (1990) and Welsh and McClelland (1990). The amplification products were separated by electrophoresis in 1.8% agarose gels, stained with ethidium bromide and photographed under UV light using Polaroid film. AFLP analysis was done as described by Vos *et al.* (1995). The DNA fragments produced by digestion with the enzymes *Eco*R1 and *Mse*1 were amplified in the presence of radioactive nucleotides ($^{33}$P) and separated on 5% acrylamide gels under denaturing conditions followed by autoradiography.

The RAPD and AFLP fragments were scored as present (1) or absent (0) for each plant sample. Genetic similarity matrices for these sequence differences were calculated for each sample markers (Jaccard, 1908), and the correlation between the two similarity matrices (RAPD and AFLP) was then obtained as described by Mantel (1967). The genetic diversity of the genotypes was analyzed

by principal coordinate analysis (PCA) (Gower, 1996) and by the means of the hierarchic classification (UPMGA aggregation model). The degree of relatedness between cultivated cassava and the other five naturally occurring species was assessed using only RAPD markers. Dendrograms showing the genetic relatedness between these species were constructed using the neighbor-joining method.

## RESULTS

One hundred and ninety-two amplified RAPD bands (average of 9.2 per primer) were analyzed. Of these, 48% were polymorphic (average of 5.2 polymorphic bands per primer). For AFLPs, the two different enzyme-primer combinations (PK) used produced 73 polymorphic bands (31 bands for the first and 42 bands for the second combination).

The relationship between *M. flabellifolia* and *M. peruviana* was studied using data generated with the RAPD and AFLP markers (91 RAPD and 70 AFLP bands). The mean value of similarity was 0.61 for the 15 individuals (0.57 and 0.63 for *M. flabellifolia* and *M. peruviana*, respectively). *M. flabellifolia* and *M. peruviana* were classified by the UPGMA method (Figure 1). The genotypes of these two species are in the two main branches of the dendrogram (A1 and A2), showing that it was not possible to differentiate these species with the markers used and they are therefore considered here as a single species (Figure 1). Fifty percent of the bands were monomorphic for the genotypes studied. The mean genetic similarity between the cassavas and *M. flabellifolia/M. peruviana* was 0.59 (Figure 1). *M. flabellifolia/M. peruviana* were grouped in branch A, while the cassavas of the World Collection and those from Santa Isabel (Amazon region) were located in branch B. The two groups were separated from each other by a distance of approximately 0.44. A distance of about 0.38 (B1 and B2) differentiated the two groups of cassava (Santa Isabel vs. World Collection) which were more closely related to each other than to the *M. flabellifolia/M. peruviana* species.

The global genetic diversity of the cultivated cassavas and the *M. flabellifolia/M. peruviana* group was determined (Figure 2). The cassavas were separated from the *M. flabellifolia/M. peruviana* accessions by axis 1 (9.4% of the total inertia) while axis 2 (7.1% of the contribution) distinguished the cassavas pertaining to the World Collection from those found in Santa Isabel.

An unrooted tree was constructed to show the genetic relatedness between the species *M. esculenta* and the wild species of *Manihot* (Figure 3). This relationship was based on the RAPD markers of two representative samples of the cultivated species, one representative sample of the *M. flabellifolia/M. peruviana* group and one representative sample of each of the five naturally occurring species of *Manihot*. The two varieties of cassava (SRT1276 and E8220) and the *flabellifolia* genotype (Fla2-10) were
<table><caption>**Table I** - Plant samples used to investigate the genetic relatedness among *Manihot* species.</caption><thead><tr><th></th><th>Code</th><th>Manihot</th><th>Section*</th><th>Origin*</th><th>Source</th></tr></thead><tbody><tr><td>1</td><td>E8228</td><td>esculenta</td><td>Manihot</td><td>-</td><td>Santa Isabel/Amazon State</td></tr><tr><td>2</td><td>E8220</td><td>"</td><td>"</td><td>-</td><td>"</td></tr><tr><td>3</td><td>E8216-1</td><td>"</td><td>"</td><td>-</td><td>"</td></tr><tr><td>4</td><td>E8214-1</td><td>"</td><td>"</td><td>-</td><td>"</td></tr><tr><td>5</td><td>E8215-1</td><td>"</td><td>"</td><td>-</td><td>"</td></tr><tr><td>6</td><td>E8209-1</td><td>"</td><td>"</td><td>-</td><td>"</td></tr><tr><td>7</td><td>E8207-1</td><td>"</td><td>"</td><td>-</td><td>"</td></tr><tr><td>8</td><td>E8203-1</td><td>"</td><td>"</td><td>-</td><td>"</td></tr><tr><td>9</td><td>E8201-1</td><td>"</td><td>"</td><td>-</td><td>"</td></tr><tr><td>10</td><td>E8196-1</td><td>"</td><td>"</td><td>-</td><td>"</td></tr><tr><td>11</td><td>E8192-2</td><td>"</td><td>"</td><td>-</td><td>"</td></tr><tr><td>12</td><td>E8191-1</td><td>"</td><td>"</td><td>-</td><td>"</td></tr><tr><td>13</td><td>E8189-2</td><td>"</td><td>"</td><td>-</td><td>"</td></tr><tr><td>14</td><td>VEN25</td><td>"</td><td>"</td><td>-</td><td>Venezuela</td></tr><tr><td>15</td><td>THAI1</td><td>"</td><td>"</td><td>-</td><td>Thailand</td></tr><tr><td>16</td><td>MEX59</td><td>"</td><td>"</td><td>-</td><td>Mexico</td></tr><tr><td>17</td><td>MAL2</td><td>"</td><td>"</td><td>-</td><td>Malaysia</td></tr><tr><td>18</td><td>ECU82</td><td>"</td><td>"</td><td>-</td><td>Equador</td></tr><tr><td>19</td><td>CUB51</td><td>"</td><td>"</td><td>-</td><td>Cuba</td></tr><tr><td>20</td><td>COL2066</td><td>"</td><td>"</td><td>-</td><td>Colombia</td></tr><tr><td>21</td><td>COL1522</td><td>"</td><td>"</td><td>-</td><td>Colombia</td></tr><tr><td>22</td><td>COL1438</td><td>"</td><td>"</td><td>-</td><td>Colombia</td></tr><tr><td>23</td><td>COL22</td><td>"</td><td>"</td><td>-</td><td>Colombia</td></tr><tr><td>24</td><td>BOL3</td><td>"</td><td>"</td><td>-</td><td>Bolivia</td></tr><tr><td>25</td><td>ARG11</td><td>"</td><td>"</td><td>-</td><td>Argentina</td></tr><tr><td>26</td><td>BGM81</td><td>"</td><td>"</td><td>-</td><td>Brazil (Northeastern region)</td></tr><tr><td>27</td><td>BGM1269</td><td>"</td><td>"</td><td>-</td><td>Brazil (Northeastern region)</td></tr><tr><td>28</td><td>BGM5</td><td>"</td><td>"</td><td>-</td><td>Brazil (Southeastern region)</td></tr><tr><td>29</td><td>BGM243</td><td>"</td><td>"</td><td>-</td><td>Brazil (Northeastern region)</td></tr><tr><td>30</td><td>SRT1316</td><td>"</td><td>"</td><td>-</td><td>Brazil (Northeastern region)</td></tr><tr><td>31</td><td>SRT1276</td><td>"</td><td>"</td><td>-</td><td>Brazil (Northeastern region)</td></tr><tr><td>32</td><td>SRT454</td><td>"</td><td>"</td><td>-</td><td>Brazil (Southeastern region)</td></tr><tr><td>33</td><td>F4113</td><td>"</td><td>"</td><td>-</td><td>Brazil (Southeastern region)</td></tr><tr><td>34</td><td>Fla10-6</td><td>flabellifolia</td><td>Heterophyllae</td><td>South America</td><td>Brazil (Porto Franco, MA)</td></tr><tr><td>35</td><td>Fla8-7</td><td>"</td><td>"</td><td>"</td><td>Brazil (Porto Franco, MA)</td></tr><tr><td>36</td><td>Fla4-14</td><td>"</td><td>"</td><td>"</td><td>Brazil (Goiânia, GO)</td></tr><tr><td>37</td><td>Fla2-8</td><td>"</td><td>"</td><td>"</td><td>Brazil (Comodoro, MT)</td></tr><tr><td>38</td><td>Fla3-7</td><td>"</td><td>"</td><td>"</td><td>Brazil (Rio Branco, AC)</td></tr><tr><td>39</td><td>Fla1-8</td><td>"</td><td>"</td><td>"</td><td>Brazil (Jaru, RO)</td></tr><tr><td>40</td><td>Fla2-10</td><td>"</td><td>"</td><td>"</td><td>Brazil (Vilhena, RO)</td></tr><tr><td>41</td><td>Per3-4</td><td>peruviana</td><td>"</td><td>"</td><td>Brazil (Porto Velho, RO)</td></tr><tr><td>42</td><td>Per1-7</td><td>"</td><td>"</td><td>"</td><td>Brazil (Porto Velho, RO)</td></tr><tr><td>43</td><td>Per1-3</td><td>"</td><td>"</td><td>"</td><td>Brazil (Guajara-Mirim, RO)</td></tr><tr><td>44</td><td>Fla1-10</td><td>flabellifolia</td><td>"</td><td>"</td><td>Brazil (Lambari, MT)</td></tr><tr><td>45</td><td>Per2-6</td><td>peruviana</td><td>"</td><td>"</td><td>Brazil (Lambari, MT)</td></tr><tr><td>46</td><td>Per1-1</td><td>"</td><td>"</td><td>"</td><td>Brazil (Pontes e Lacerda, MT)</td></tr><tr><td>47</td><td>Fla2-11</td><td>flabellifolia</td><td>"</td><td>"</td><td>Brazil (Pontes e Lacerda, MT)</td></tr><tr><td>48</td><td>Fla1-9</td><td>"</td><td>"</td><td>"</td><td>Brazil (Vilhena, RO)</td></tr><tr><td>49</td><td>Per2-4</td><td>peruviana</td><td>"</td><td>"</td><td>Brazil (Cacoal, RO)</td></tr><tr><td>50</td><td>-</td><td>glaziovii</td><td>Glaziovianae</td><td>Brazil central and northeast</td><td>Brazil (Northeast region)</td></tr><tr><td>51</td><td>-</td><td>reptans</td><td>Crotalariaeformes</td><td>Brazil central</td><td>Brazil (Central region)</td></tr><tr><td>52</td><td>-</td><td>chlorostica</td><td>Parvibracteatae</td><td>Mexico</td><td>Mexico</td></tr><tr><td>53</td><td>-</td><td>aesculifolia</td><td>"</td><td>"</td><td>"</td></tr><tr><td>54</td><td>-</td><td>michaelis</td><td>Foetidae</td><td>"</td><td>"</td></tr></tbody></table>

cated at the center of gravity of each of the three groups in the principal coordinate analysis (Figure 2). Ninety-one polymorphic RAPD markers with an average size of 993 pb were used in this study. An unrooted tree best represented the grouping of the two cassava genotypes and the three naturally occurring Mexican species (*M. aesculifolia*, *M. michaelis* and *M. chlorostica*) as the outgroup. The wild

Brazilian species *M. glaziovii* and *M. reptans* were more closely related to cassava and *M. flabellifolia/M. peruviana* than the naturally occurring species from Mexico.

The two molecular marker techniques were compared by determining their maximum, minimum and mean Jaccard index values. Genetic diversity within each group was similar for both types of markers. A comparison of the mean

<footnote>*From Rogers and Appan (1973).</footnote>
<figure><img src="image_2.png" /><figcaption>**Figure 1** - Classification of *M. esculenta* (two groups, M for the samples of a world collection and A for samples from the Amazon region), *M. flabellifolia* and *M. peruviana* based on the UPGMA grouping method. The distances (similarity index of Jaccard (1908)) were calculated using the combined RAPD and AFLP data (92 and 73 markers, respectively).</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**Figure 2** - Principal coordinate analysis calculated using the genetic similarities (Jaccard index) of 33 clonal accessions of cultivated cassava (Manihot esculenta) and 15 genotypes of the naturally occurring species *M. flabellifolia* and *M. peruviana*.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 3** - Unrooted tree classification of cassava and other wild species of *Manihot* elaborated by the neighbor-joining method using the similarity coefficients of Jaccard derived from RAPD data.</figcaption></figure>

values of similarity using the *t*-test showed that they were not significantly different. Another comparison between the two types of markers used here was done using Mantel's correlation test (1967). This calculation, which takes into account the similarities between all comparisons of the individuals available, i.e., those associated with RAPD markers on the one hand and those relative to AFLP markers on the other, showed a significant correlation (r = 0.75) between the two molecular markers. According to Lapointe and Legendre (1992), the *r* value is significant with a 1% probability if > 0.5 for more than 12 OTU (operational taxonomic units). With the 2,000 permutations done for this test, the Z-value obtained was 0.0020.

DISCUSSION

The genetic relatedness among *Manihot* species revealed by our results demonstrates that both RAPD and RFLP markers are equally suited as tools to study them and the choice of markers for investigating genetic relatedness must take into account practical factors such as ease of use and costs. Technically, AFLPs are more difficult to handle, although they reveal a higher degree of polymorphism. Thus, Mackill *et al.* (1996) used AFLP and obtained 147 polymorphic bands with eighteen primer-enzyme combinations in 12 japonica and two indica rice cultivars compared with 43 polymorphic bands obtained for RAPD markers (21 random primers tested). Lin *et al.* (1996) compared the polymorphism generated by RAPD (two bands per primer) with that generated by AFLP (12 polymorphic bands for one PK combination) in soybeans. The principle of both RAPD and AFLP markers is the amplification of anonymous DNA sequences of unknown size. One of the main criticisms of the use of RAPD markers for studying genetic relatedness, especially among different species, is that there is no information on the homology of the nucleotide sequence of the same band in dif-

ferent individuals. Sequence homologies were not determined in this study, although others (Williams *et al.*, 1990; Thormann and Osborn, 1992) have reported high levels of sequence homology for RAPD in other genera. For AFLP markers, the problem of homology is less important (Vos *et al.* 1995). Since the two types of molecular marker equally differentiated the species, the diversity between the *Manihot* species studied was evaluated using a combination of the AFLP and RAPD test data. According to Loarce *et al.* (1996), the larger the number of parameters used to compare two individuals genetically, the more accurate the estimate of similarity between them, as illustrated by their study on the genetic relatedness of rice varieties using both RAPD and AFLP markers.

This is the first study to examine the relatedness between cassava and *M. flabellifolia* and *M. peruviana* species using RAPD and AFLP markers simultaneously. *M. flabellifolia* and *M. peruviana* species proved to be so closely related that the markers used were unable to group them separately. From a botanical standpoint, these species are extremely similar. According to Allem (1994), the only morphological difference is the absence in *M. flabellifolia* or presence in *M. peruviana* of short, thin soft hairs on the surface of the reproductive organs. Classifying these two taxa as different species on this basis would not be justified. Furthermore, the geographical region where *M. peruviana* occurs naturally overlaps considerably with that of *M. flabellifolia*, although the range of the first is much smaller. The genetic similarities, the geographical distribution and the botanical characteristics all indicate that *M. peruviana* originated from *M. flabellifolia*.

Our results provide evidence to support the hypothesis of Allem (1994), who identified *M. flabellifolia*/*M. peruviana* as being the wild or naturally occurring forms of cassava. Thirty-four bands (20% of the polymorphic bands) were monomorphic in all of the *M. flabellifolia*/*M. peruviana* and *M. esculenta* genotypes studied. The dis-
tinction between the wild species and cassava was attribut-
able primarily to variations in the frequencies of the poly-
morphic bands of each of the species. No band whatsoever
was found to be specific (100% occurrence) to either *M.
flabellifolia/M. peruviana* or *M. esculenta*.

Other studies have examined the genetic proximity of
species belonging to the genus *Manihot*. Second *et al.*
(1997) reported a high level of similarity between *M.
flabellifolia* and *M. peruviana* based on AFLP markers.
Schaal *et al.* (1997) reached the same conclusion using
RAPD markers and two ITS regions of nuclear ribosomal
DNA. Based on the analysis of DNA extracted from the
chloroplasts of the two species, Fregene *et al.* (1994) clas-
sified *M. flabellifolia* as being very closely related to cas-
sava. These results suggest that *M. flabellifolia/M.
peruviana* and *M. esculenta* share the same origin. *M.
esculenta* may have been domesticated to produce culti-
vated cassava (*M. esculenta* ssp. *esculenta*), while other
specimens of the same species, represented by *M. esculenta*
ssp. *flabellifolia/peruviana*, continued in their natural state.
Contrary to and, at the same time, partly supporting the
theory of Rogers and Appan (1973), who claimed that there
were no wild forms of cassava, the presence of 20 bands in
naturally occurring species but absent from cultivated cas-
sava may indicate the existence of a third species which,
when crossed with *M. flabellifolia/M. peruviana*, would
have given rise to cassava, or which, when crossed with cas-
sava, would have given rise to *M. flabellifolia/M. peru-
viana*. This uncertainty will only be clarified by finding the
third species mentioned above.

Second *et al.* (1997) studied the taxonomy of a large number of *Manihot* species, some of which are little known. Their results, based on 93 AFLP markers, show that other naturally occurring species, such as *flabellifolia*, were genetically very closely related to the varieties of cultivated cassava: *M. procumbens*, *M. fruticulosa*, *M. pentaphylla* and *M. pruinosa*. On the basis of field observations, Nassar (1978) found that the fertility rate of crosses between *M. oligantha* (Pax) Nassar and cassava is about 90%, indicating that the former species can be classified in the primary genic pool, as proposed by Harlan and Wet (1971).

The high degree of relatedness between cassava and
*M. glaziovii* and *M. reptans* emphasizes the tenuous repro-
ductive barriers that exist between these two species and
cassava. Nassar *et al.* (1985) described a population of *M.
reptans* in central Brazil with traces of introgression from
cassava. Similarly, spontaneous hybrids of *M. esculenta* and
*M. glaziovii* were described in Africa by Nichols (1947),
Cours (1951), INEAC (1952) and Lefèvre (1989). Contrary
to our results, Fregene *et al.* (1994) found no difference
between *M. glaziovii* and *M. michaelis*, based on an analy-
sis of DNA extracted from chloroplasts. Likewise, Haysom
*et al.* (1994) used RFLP markers to classify the Mexican
species *M. chlorostica* as the species most closely related
to *M. flabellifolia*, followed by *M. esculenta* and *M.
glaziovii*. The results of these authors corroborate the hy-

pothesis of Rogers and Appan (1973) and refute the idea
that *M. flabellifolia* is a native species of South America.

Using only molecular markers, we were able to dem-
onstrate in this work the genetic relatedness between the
subspecies *M. esculenta* and five other naturally occurring
species in the genus, each represented by one genotype.
There was a much greater similarity between *M.
flabellifolia/M. peruviana* and cultivated cassava than be-
tween *M. esculenta* and the other wild species. Among the
five wild species investigated, *M. glaziovii* and *M. reptans*
proved to be more closely related to *M. esculenta* than the
remaining species (*M. aesculifolia*, *M. michaelis* and *M.
chlorostica*). These results are consistent with the geo-
graphical distribution of these species. The three Mexican
species are quite different from the Brazilian species. *M.
glaziovii* occurs in the arid zone of northeastern Brazil, an
important center of genetic diversity for the genus *Manihot*
(Nassar, 1978). This species has been widely used to intro-
duce genes of specific agricultural importance into culti-
vated cassava, especially because of the ease in producing
desired crosses and the possibility of restoring the fertility
of infertile hybrids (Léfèvre, 1989). *M. reptans*, a species
much less used than *M. glaziovii* in genetic studies, is na-
tive to the humid regions of central Brazil, the most impor-
tant center of diversity for *Manihot* (Nassar, 1978). The
three species most distantly related to cultivated cassava
are those native of Mexico, the second most important center
of diversity for *Manihot* (Nassar, 1978).

ACKNOWLEDGMENTS

The authors gratefully acknowledge CAPES for financial
support and fellowships conceded to Carlos Colombo. Publica-
tion supported by FAPESP.

RESUMO

A taxonomia do gênero *Manihot* em grande parte não está resolvida e a origem genética da mandioca (*M. esculenta* Crantz) continua controvertida. Na tentativa de contribuir para elucidar sua história evolutiva, as relações de proximidade genética da mandioca com duas espécies selvagens que provavelmente participaram da sua história evolutiva, *M. flabellifolia* e *M. peruviana*, foram estudadas através de dois tipos de marcadores moleculares, os RAPDs e os AFLPs. Para tanto, foram empregados 33 acessos clonais de mandioca de reconhecida diversidade genética e 15 acessos das espécies selvagens *M. flabellifolia* e *M. peruviana* das regiões central e norte do Brasil, importantes centros de ocorrência natural destas espécies. Noventa e duas bandas polimórficas RAPD e 73 AFLP foram utilizadas para análise dos resultados. Ambos marcadores foram incapazes de diferenciar as duas espécies selvagens utilizadas, confirmando a grande semelhança botânica entre elas. Em relação à mandioca cultivada, os resultados revelaram grande proximidade genética entre estas e as espécies selvagens. Metade do total de bandas amplificadas apresentaram-se monomórficas entre todos os genótipos avaliados. O valor médio de similaridade genética (Jaccard) entre a mandioca e as espécies *M. flabellifolia/M. peruviana* é de 0.59. As relações de proxi-
midade genética entre a mandioca e *M. flabellifolia/M. peruviana*
foram confirmadas quando outras cinco espécies selvagens foram
também incorporadas em relação ao polimorfismo gerado pelos
RAPDs. A análise de agrupamento (neighbor-joining) realizada com
genótipos de mandioca, de *M. flabellifolia/M. peruviana* e das
demais espécies selvagens reuniu numa extremidade os genótipos
de mandioca e *M. flabellifolia/M. peruviana* e na outra extre-
midade três espécies mexicanas (*M. aesculifolia*, *M. michaelis* e
*M. chlorostica*). Entre estes dois grupos se posicionaram outras
duas espécies selvagens cuja ocorrência natural é na região central
e no nordeste brasileiro (*M. glaziovii* e *M. reptans*). Embora não
conclusivos, os resultados apresentados são coerentes com a
hipótese de que as espécies *M. flabellifolia* e *M. peruviana* po-
deriam ter originado a espécie cultivada. No entanto, outras espécies
pouco estudadas (*M. procumbens*, *M. fruticulosa*, *M. pen-
taphylla* e *M. pruinosa*) foram recentemente citadas como
geneticamente muito próximas da mandioca. Assim, um estudo
abordando maior número de espécies e com marcadores mais
apropriados, a exemplo dos microsatélites, merece ser feito.

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(Received October 14, 1999)
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