Genetic diversity and relationships of cacao (Theobroma cacao L.)

Theor Appl Genet (1998) 96: 621Ð627 ( Springer-Verlag 1998

R. Whitkus á M. de la Cruz á L. Mota-Bravo

A. Go«mez-Pompa

Genetic diversity and relationships of cacao (Theobroma cacao L.)

in southern Mexico

Received: 21 May 1997/ Accepted: 9 October 1997

Abstract Neotropical tree crops are a¤ected by a

combination of biological and human factors that

complicate the study of genetic diversity and crop

evolution. Genetic diversity and relationships among

southern Mexican populations and horticultural collections

of ¹heobroma cacao (chocolate, cocoa, cacao)

are examined in light of the agricultural practices of the

Maya. Collections of cacao were obtained from the

extremes of its geographic range including archeological

sites in southern Mexico where cacao was Þrst

domesticated. Genetic diversity was assayed by 57 informative

random ampliÞed polymorphic DNA

(RAPD) marker loci. A unique sample of the total

diversity found in this study exists in the southern

Mexican populations. These populations are signiÞ-

cantly di¤erent from all other cacao with regards to

their proÞle of RAPD bands, including the ÔcriolloÕ

variety, their morphological and geographical group.

A population of cacao found in a sinkhole (cenote) in

northern Yucatan with genetic a¦nities to populations

in Chiapas suggests the Maya maintained plants far

away from their native habitat. This Þnding concurs

with known agroforestry practices of the Maya. Modern

e¤orts to increase germplasm of tropical tree crops

such as cacao should carefully examine archeological

sites where genetic diversity, either deliberately or by

Communicated by G. E. Hart

R. Whitkus (¥) á A. Go« mez-Pompa

Department of Botany and Plant Sciences, University of California,

Riverside, CA 92521, USA

Fax: (909) 787Ð4437

E-mail: whitkus@moe.ucr.edu

M. de la Cruz

Instituto de Ecologia, UNAM, Apdo. Post. 70-275, C.P. 04510,

Mexico

L. Mota-Bravo

ITESM Campus Morelos, Av. Refoma 182-A, Cuernavaca,

Morelos 62589, Mexico

chance, was collected and maintained by ancient cultures.

Key words Cacao á ¹heobroma cacao á

Genetic diversity á Crop evolution á RAPD

Introduction

The genetic diversity of Neotropical tree crops is

inßuenced by a combination of biological attributes

and human-precipitated changes. Some intrinsic characteristics

of the plants that directly a¤ect genetic variability

are extensive geographic ranges of numerous

species, fairly small populations sizes, and outcrossing

breeding systems. Historical evidence indicates a long

history of management of species by native cultures

(Go«mez-Pompa and Kaus 1990), while the greatest

modern human factor is extensive deforestation of the

region. These factors make it di¦cult to identify wild

progenitors, locate centers of diversity, decipher the

history of the domestication process, and conserve genetic

diversity of important tropical crops.

One of the more important Neotropical tree crops is

¹heobroma cacao L. (chocolate, cacao), ranked 29th in

planted hectares of world crops (N. Ellstrand, personal

communication). Cacao is an understory tree in tropical

rainforests extending from the Amazonian basin of

South America to southern Mexico. Plants from Mesoamerica

(Mexico and Central America) are classiÞed

as ¹. cacao subsp. cacao (Cuatrecasas 1964). Cultivated

forms of the subspecies represent the horticultural

variety ÔcriolloÕ, considered to have been domesticated

by the Maya (or their ancestors) more than 2000

years ago. South American plants are placed in ¹.

cacao subsp. sphaerocarpum (Cuatrecasas 1964), and

its cultivars represent the horticultural variety Ôforastero

Õ. This variety is the basis of most chocolate production

because of its higher yield and greater disease

Table 1 Polymorphism of cacao collections based on 57 polymorphic

RAPD loci (ID groups ) n sample size ) P percentage polymorphic

bands ) º total unique bands)

Collection ID n P U

Chiapas 1 28 12.3 5

Yucatan 2 5 8.8 5

Cultivars 3 48 73.4 11

South America 4 5 33.3 0

Southern Mexico 1&2 33 49.1 12

Cultivars and

South America 3&4 53 77.2 29

resistance. A third horticultural form, ÔtrinitarioÕ, represents

a hybrid derivative between ÔcriolloÕ and Ôforastero

Õ (Cheesman 1944; Kennedy 1995).

As in other crops, a small portion of the genetic

variability in cacao has been used in breeding programs,

but e¤orts are underway to increase germplasm

diversity (Lockwood 1985; Kennedy 1995). The search

for additional genetic diversity is concentrated on the

upper Amazonian basin because the region is considered

the center of origin for the species (Cheesman

1944) and has the highest known level of diversity

(Laurent et al. 1993a, 1994; Figueira et al. 1994;

NÕGoran et al. 1994). Mesoamerica is not being intensely

searched since wild forms of cacao are considered

rare or non-existent in the region (Cheesman 1944;

Purseglove 1974). The discovery of ¹. cacao subsp.

cacao occurring naturally in the Lacandon forest of

Chiapas and sinkholes (cenotes) of northern Yucatan,

Mexico (Go«mez-Pompa et al. 1990) suggests that novel

genetic variation may exist within these populations

which could be distinct from South American plants

(Cheesman 1944; Lockwood 1985).

A preliminary analysis of cacao from Chiapas and

Yucatan found that the plants are genetically distinctive

from all other cacao, including the ÔcriolloÕ and

ÔforasteroÕ varieties (de la Cruz et al. 1995a). The lack of

population samples from Mexico prevented a comparison

of the organization of diversity in these collections

with that of the cultivated varieties or South American

native plants. The study presented here compared genetic

diversity in natural populations of cacao from

southern Mexico with cacao from South America and

the horticultural varieties, and re-examined the relationships

of the subspecies and horticultural varieties.

Findings from this study indicate future e¤orts in

collecting natural diversity of cacao should include

Mesoamerica since the region contains a unique source

of genetic variation.

Materials and methods

Plant material was obtained from trees growing in southern Mexico

or in germplasm banks. Collections in southern Mexico were made

in the states of Yucatan, Chiapas, and Tabasco. The Yucatan collection

consists of 5 individuals from a cenote near the village of

Yaxcaba in central Yucatan. Although the surrounding area is

a seasonal tropical forest with alternating wet and dry seasons,

cenotes in this region contain vegetation typical of mesic tropical

forests and a number of plants that were likely cultivated by the

Maya (Go«mez-Pompa et al. 1990). Two naturally occurring populations

were collected in Chiapas, one of 6 individuals near the Maya

ruins at Bonampak and a second of 26 individuals along the Lacantun

River in eastern Chiapas, approximately 120 km NE of Comitan.

The three main cultivars (ÔcriolloÕ, ÔforasteroÕ, and ÔtrinitarioÕ)

were included in this study, with a concentration on criollo since this

variety is considered to be derived from Mesoamerican plants (Cuatrecasas

1964). Criollos were collected from plantations at Chajul in

the state of Chiapas (27 individuals) and near Villahermosa (9

individuals) and Comacalco in Tabasco (2 individuals). Additional

criollos (4 clones) as well as forasteros (6 clones), South American

wilds (6 clones), and trinitarios (4 clones) were obtained from the

USDA germplasm collection in Mayaguez, Puerto Rico (clone identi

Þcation list available upon request).

Collections were organized into a hierarchy (Table 1) that reßected

geographic origin, cultivation status, and relationships suggested

in a previous analysis (de la Cruz et al. 1995a). In the Þrst hierarchical

level, four groups represented the southern Mexican populations

of Chiapas (ID1) and Yucatan (ID2), the cultivars (ID3), and South

American native plants (ID4). The second level in the hierarchy

combined the southern Mexican populations (ID1 and 2) and the

cultivars and South American natives (ID3 and 4).

Leaves of individual trees were placed into plastic bags and stored

on dry ice in the Þeld for transportation and at !80 ¡C in the

laboratory. Total genomic DNA extraction and random ampliÞed

polymorphic DNA (RAPD) band ampliÞcation followed the procedure

of de la Cruz et al. (1995b). Our earlier study of cacao (de la

Cruz et al. 1995a) showed 13 decamer primers (Operon Technologies,

Almeda, Calif.) which give strong, reliable banding patterns.

Data were recorded from pictures

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